Home -- Literature -- Long-Term Inflammation in Lyme Borreliosis

Long-Term Inflammation in Lyme Borreliosis

A Medline-Literature Survey by Joachim Gruber

Some of this literature has been used in my overview paper and drafts:
A Tentative Tnterpretation of Lyme Flare Cycles and a Corresponding Therapy,
Evaluation of the Long-Term Inflammation in Neuroborreliosis and
Lyme Disease: Statistical Evaluation of a Symptom Log and an Empirical Theory of Flare Cycles
Date: February 3, 1999


Honegr K, Hulinska D, Dostal V, Gebousky P, Hankova E, Horacek J, Vyslouzil L, Havlasova J.

Infekcni klinika, Fakultni nemocnice, Hradec Kralove.

[Persistence of Borrelia burgdorferi sensu lato in patients with Lyme borreliosis]

[Article in Czech]
from:Epidemiol Mikrobiol Imunol. 2001 Feb;50(1):10-6.
In 18 patients with Lyme borreliosis the authors proved the persistence of Borrelia burgdorferi sensu lato by detection of the causal agent by immune electron microscopy or of its DNA by PCR in plasma or cerebrospinal fluid after an interval of 4-68 months.
  • Clinical manifestations common in Lyme borreliosis were present in only half the patients,
  • in the remainder non-specific symptoms were found.
  • In nine subjects with confirmed Borrelia burgdorferi sensu lato in the cerebrospinal fluid the cytological and biochemical finding was normal.
  • Examination of antibodies by the ELISA method was negative
    • in 7 of 18 patients during the first examination and
    • in 12 of 18 during the second examination.
  • In all negative examinations the specific antibodies were assessed by the Western blot or ELISA method after liberation from the immunocomplexes.
In the authors' opinion it is advisable to examine repeatedly plasma and other biological material from potentially affected organs by PCR and subjects with persisting or relapsing complaints after the acute form of Lyme borreliosis as well as to examine cerebrospinal fluid in case on non-specific symptoms and concurrent pathic EEG or MR findings.
PMID: 11233667 [PubMed - indexed for MEDLINE]

see also:
Hunfeld KP, Ruzic-Sabljic E, Norris DE, Kraiczy P, Strle F, In Vitro Susceptibility Testing of Borrelia burgdorferi Sensu Lato Isolates Cultured from Patients with Erythema Migrans before and after Antimicrobial Chemotherapy, Antimicrob Agents Chemother. 2005 Apr;49(4):1294-301.
In summary, our study provides compelling evidence that, although rare, survival of B. burgdorferi sensu lato can occur in antibiotically treated individuals with EM after antimicrobial chemotherapy. Spirochete persistence in these patients was not caused by increasing MICs or MBCs for B. burgdorferi sensu lato. Instead, our findings corroborate those of Hansen et al. (Hansen, K., A. Hovmark, A. M. Lebech, K. Lebech, I. Olsson, L. Halkier-Sorensen, L., E. Olsson, and E. Asbrink. 1992. Roxithromycin in Lyme borreliosis: discrepant results of an in vitro and in vivo animal susceptibility study and a clinical trial in patients with erythema migrans. Acta Dermato-Venereol. 72:297-300) and Pfister et al. (Pfister, H. W., V. Preac-Mursic, B. Wilske, E. Schielke, F. Sorgel, and K. M. Einhaupl. 1991. Randomized comparison of ceftriaxone and cefotaxime in Lyme neuroborreliosis. J. Infect. Dis. 163:311-318.) in relapsed patients with early LB, demonstrating that isolates cultured after the conclusion of roxithromycin and ceftriaxone therapy remain fully susceptible to these agents in vitro. These findings, however, do not rule out phenotypic resistance mechanisms similar to those assumed to cause relapse in syphilis and leptospirosis (Panconesi, E., G. Zuccati, and A. Cantini. 1981. Treatment of syphilis: a short critical review. Sex. Transm. Dis. 8:321-325, 37, Straubinger, R. K., B. A. Summers, Y. F. Chang, and M. J. G. Appel. 1997. Persistence of Borrelia burgdorferi in experimentally infected dogs after antibiotic treatment. J. Clin. Microbiol. 35:111-116).

P.K. Coyle

Department of Neurology, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794

Borrelia Burgdorferi Infection: Clinical Diagnostic Techniques
Properties of the Bacterial Agent

from: Immunol Invest 1997 Jan;26(1&2):117-128
... Borrelia burgdorferi (Bb) is believed to be an extracellular organism. Spirochetes readily adhere to and penetrate endothelial cells. In fact they can adhere to a number of different cell types, including glial cells, with attachement mediated by surface-exposed proteoglycans [Isaacs RD, J Clin Invest 93, 809-819 (1994)]. In vitro Bb invades fibroblasts to cause intracellular infection [Georgilis et al. 1992]. The issue of whether Bb in vivo causes intracellular infection is an important one that has therapeutic implications. Although numbers are small, there have been documented late isolations of Bb from skin, cerebrospinal fluid (CSF), synovial tissue, flexor retinaculum, eye, myocardium, blood and synovial fluid weeks to years after initial infection despite evidence for a host immune response [Klempner et al. 1993]. The ability of viable spirochetes to persist inside a cell, protected from extracellular antibiotics, is directly pertinent to the question of chronic infection. ...

Harry Goldhagen, MS, Julie Rawlings, MPH

Fighting Back - How B burgdorferi Persists: Persistence Is a Virtue

from: Medscape coverage of 14th International Scientific Conference on Lyme Disease & Other Tick-Borne Disorders (in cache)
That spirochetes tend to persist in the human body has been demonstrated in both syphilis, caused by Treponema pallidum, and Lyme disease, caused by Borrelia burgdorferi. What accounts for this ability to evade or suppress an effective immune response? According to Charles Pavia, PhD of the New York College of Osteopathic Medicine, New York Institute of Technology, Old Westbury, New York, there are at least 6 potential explanations:
  • antigenic variation (this is seen with the Borrelia species that cause tick-borne relapsing fever) or differential expression of antigens (especially the outer surface proteins; with B burgdorferi, only OspC is expressed during mammalian infection)
  • production of an outer protective coat (e.g.capsule, as seen with T pallidum)
  • atypical forms (e.g.cyst-like variants)
  • incomplete immune response (e.g.insufficient antibody , T-cell , or phagocytic response)
  • deranged host immune response (e.g.host-, tick-, or spirochete-derived immunosuppressive factors)
  • other evasive factors (e.g.motility)










Reactivation and immune evasion of Borrelia infection

Consortium leader: Professor SVEN BERGSTRÖM

Department of Molecular Biology, Umea University, SE-901 87 Umea, Sweden, Tel.: +46 (0)90 7856726, sven.Bergstrom@molbiol.umu.se

Other project leader of the consortium:

Professor Matti Viljanen

Department of Medical Microbiology, University of Turku, Kiinamyllynkatu 13, FIN-20520 Turku, Finland, Tel.: +358-2-3337330, email: matti.viljanen@utu.fi , homepage: http://www.utu.fi/research/tic/viljanen/

Doctoral students of the consortium

- Christer Larsson, Department of Molecular Biology, Umeï University, christer.larsson@molbiol.umu.se
- Marie Andersson, Department of Molecular Biology, Umeï University, marie.andersson@molbiol.umu.se
- Pär Comstedt, Department of Molecular Biology, Umeï University, par.comstedt@molbiol.umu.se
- Heta Yrjänäinen, Department of Medical Microbiology, University of Turku, heta.yrjanainen@utu.fi
- Pauliina Hartiala, Department of Medical Microbiology, University of Turku, pipaha@utu.fi
- Ulla Ahlmen, Department of Medical Microbiology, University of Turku, hunahl@utu.fi
-Jemiina Neuvonen, Department of Medical Microbiology, University of Turku, jemiina.neuvonen@utu.fi
- Jenni Pelkonen, Bioinformatician, Department of Medical Microbiology, University of Turku

Other researchers of the consortium

- Betty Guo, Department of Molecular Biology, Umeï University, bettu.guo@molbiol.umu.se
- Annika Nordstrand, Department of Molecular Biology, Umeï University, annika.nordstrand@molbiol.umu.se
- Jukka Hytžnen, Department of Medical Microbiology, University of Turku, jukhyt@utu.fi
- Markus Penttinen, Department of Medical Microbiology, University of Turku, markus.penttinen@utu.fi
- Jarmo Oksi, Department of Medicine, University of Turku, jarmo.oksi@utu.fi
- Helena Tuominen-Gustafsson, Department of Medical Microbiology, University of Turku, helena.tuominen-gustafsson@utu.fi

Key words
Borreliosis, immune defense, immune evasion, and reactivation

The aim of this project is to gain knowledge of the interactions between Borrelia spirochetes and the host during infection. We are using both the Lyme borreliosis and the relapsing fever borreliosis spirochetes as model organisms. We are investigating how the Borrelia spirochetes can circumvent the immunological defence, how they spread form the infectious focus, reach various sites in the mammalian body, and how the spirochetes can live in these tissues at a dormant state, reactivate, return to the circulatory system and cause acute disease again. We also aim to characterize and define the components involved in the interactions between Borrelia and human cells, including the cells of the innate and adaptive immunity.

The current status of the project is presented below according to the questions and aims of the original research program

Adhesion of Borrelia to endothelium is mediated by specific integrin binding.
This project will further extended to identify outer membrane proteins, i.e. P66, P13, OspA-C etc in pore formation, adhesion to host cells, and tissue tropism. Structural characteristics of some of these proteins are known and will be used to design inhibitory compounds for the binding and interaction process. Additionally, the Borrelia species that causes relapsing fever binds and aggregates erythrocytes as a possible additional mechanism for evasion of the immune system. A potential adhesin involved in this aggregation has been identified. Pore forming assays and aggregation assays will be used to measure the inhibitory effects of test compounds chemical inhibitors that block the function of Borrelia species. Thus, we have demonstrated in earlier studies that some species of relapsing fever Borrelia adhere to erythrocytes, causing the formation of erythrocyte rosettes. This aggregate of Borrelia and red blood cells may protect the spirochetes and contribute to the delayed immune response seen in rosette-forming strains compared non-rosette-forming strains. Mice infected with a rosette-forming strain exhibited more severe pathology and reduced blood flow compared to mice infected with a non-rosette-forming strain. ¾Therefore, adhesins and receptors involved in this interaction would lead to possible therapeutic tools against relapsing fever. We have now identified a 27 kDa Borrelia protein that binds to a component of human erythrocyte membranes. We are in the process of purifying this protein in order to capture the erythrocyte receptor by affinity chromatography and subsequently identify it by mass spectrometry. A Borrelia library will be constructed and screened using the purified receptor.

Borrelia use host proteases to spread from the infection focus to blood, and to invade distant organs.
The initial studies concerning this part were published in 2001 (Nordstrand A., et al. "Delayed kidney and brain invasion by Borrelia crocidurae in plasminogen knock-out mice". Infect Immun 2001, 69: 5832-5839). Further protease evaluation is described below, where B. crocidurae, B. hermsii and B. duttoni are investigated in relation to invasion capability and characteristics. A murine model has been established to test Borrelia-mediated activation of host proteases and modulation of the immune responses in the induction of abortion associated with relapsing fever borrelisosis. Immune cell subsets and the role of proteases are being investigated (Andersson M, Larsson C, Bergström S and Nordstrand A. Invasion, inflammation and host proteases in a murine model of Borrelia-induced complications during pregnancy. 2005 Manuscript in preparation). So far matrix metalloproteases and plasmin have been tested. However, no upregulation have been documented so far, although other proteases will be tested.

Borrelia can evade the first line defense and direct the later specific immune response by interfering with the function of neutrophils and dendritic cells.
We have previously shown that contact with B. burgdorferi induces maturation and IL-8 production of immature dendritic cells (DCs) in a similar manner as contact with LPS, a known maturation inducer. These observations suggest that the interplay between borreliae and DCs is similar to the interplay of DCs with other microbes. However, gene expression studies are essential to investigate in more detail the possibility that borreliae are somehow manipulate DCs to their benefit. We have now completed the microarray experiments where we determined the gene expression profiles of borrelia-stimulated and -unstimulated DCs, and compared the borrelia-induced changes in DC gene expression to the effects of LPS. Changes in gene expression were analysed in four time points, and each time point was done in triplicate resulting in 36 microarray hybridizations with two technical repeats. A computer algorithm has been set up to finalise the array results. As a result, we have identified the differential expression of up to several hundred genes, many of which are important regulators of immune response. Confirmatory experiments with quantitative PCR and protein arrays are underway.

We have also studied the role of borrelial outer surface proteins (Osps) in the phagocytosis of borrelia by neutrophils. In theses studies, we have used B. burgdorferi strains B31 and B313, which is a mutant of B31 lacking OspA and OspB surface proteins, among others. Flow cytometry and Baclight bacterial viability assays have been used to assess the amount and viability of bacteria ingested by neutrophils. We have found that B31 is phagocytized more efficiently than B313. Thus, OspA and/or OspB seem to play a role in the phagocytosis of Borrelia by neutrophils, but this has to be confirmed by using an OspAB complemented B313 strain, which we have just cloned. In addition, we have expanded our experiments concerning Borrelia-neutrophil interaction into a new direction by setting up assays where neutrophil phagocytosis of Borrelia is manipulated with antibodies and other reagents interfering with neutrophil receptors and signal transduction.

Toll-like receptor expression is modulated in immune cells as an effect of spirochete invasion. The Borrelia-TLR interaction is central for clinical outcome.
Organ tissues available have been evaluated by immunohistochemistry (included in PhD project, M Andersson). B. crocidurae, B. hermsii and B. duttoni are evaluated regarding invasion characteristics. Preliminary results indicate differential ability of the species to invade as well as by the invaded spirochetes to trigger an inflammatory response in situ. This will be further investigated by Real-Time PCR methodology.

Severe symptoms are associated with a certain type of T-helper response. Manipulation of the Th1/Th2 ratio and attenuation of inflammatory responses may be used to prevent severe manifestations of borreliosis.

We are currently preparing a manuscript on one part of this project, where we describe the immune response in brain during the early process leading to neuroborreliosis. We report herein on a macrophage-dominated response, with IL-10, IL-15 and IFNg as the most important cytokines. Interestingly following IL-10 increase, the inflammation subsides to a low level, but does not result in eradication of the bacterium from the tissue. These results indicate that during infection IL-10 down regulates the intense macrophage dominated response, but this may occur at the expense of complete eradication of the bacteria (Nordstrand A, Anderssojn M, Shamaei-Tousi A, Jansson A and Bergstržm S. In situ immune response in brain and other organs at the early stage of murine neuroborreliosis. 2005 Manuscript in preparation).

Whether this mechanism reflects the initial explanation to bacterial persistence or not remains to be addressed, as does the role of IL-15, a cytokine that so far has not been investigated during borreliosis. This will be further investigated by Real-Time PCR methodology.

As a new approach, we have investigated the effects of treatment with anti-TNF-a antibody and/or antibiotics on the development and persistence of borrelia arthritis in mice. The results suggest that the administration of anti- TNF-a with or without antibiotics does not lead to amelioration of arthritis. However, during these experiments we have observed that anti- TNF-a given after ceftriaxone treatment leads to activation of a latent form of borrelial infection. Characterization of the cellular and molecular biology of this latent form of borrelial infection is currently underway.

We have also analysed the effect of immunomodulator Linomide on borrelia arthritis in mice. Linomide caused a mild reduction in joint swelling of borrelia infected mice, but there was no decrease in lymphocyte infiltration in Linomide-treated animals.

Borrelia may, by crossing very tight barriers, such as the blood-testis and blood-brain barriers, invade organs and use these as reservoirs for an extensive length of time. Reactivation of infection can occur from these sites.
Results indicate that B. duttoni are able to reside in the immune privileged organ brain an extensive time after their disappearance from blood, and can be reactivated to appear in blood by immunosuppresing conditions. This appears to be a unique feature of this species. The result from this work is expected to result in submission of a manuscript during 2005. Interestingly, we have found that the RF Borrelia species tested in this project has a great ability to pass the blood-placenta barrier. As many as 70% of the foetus are infection positive d18 after plug formation if infected at day 9 indicating a effective mechanism to also pass this important physical barrier to an immune privileged site.

Suhonen J, Komi J, Soukka J, Lassila O, Viljanen MK. Interactionbetween Borrelia burgdorferi and immature human dendritic cells. Scand J Immunol 2003;58:67-75.

Mƒkinen J, Vuorinen I, He Q, Oksi J, Peltomaa M, MarjamƒkiM, Viljanen MK. Prevalence of Granulocytic Ehrlichia and Borreliaburgdorferisensu lato in Ixodes ricinus ticks collected from Southwestern Finland and from Vormsi Island in Estonia. APMIS 2003;111:355-362.

Ekerfelt E, Jarefors S, Tynngïrd N, Hedlund M, Sander B, Bergström S, Forsberg P, and Enerudh J. Phenotypes indicating cytolytic properties of Borrelia-specific interferon-gsecreting cells in chronic Lyme neuroborreliosis. J.Neuroimmunol 2003. 145:115-126.

Widhe M, Jarefors S, Ekerfelt C, Vrethem M, Bergström S, Forsberg P and Ernerudh J. Borrelia specific IFN-gand IL-4 secretion in CSF and blood during the course of Human Lymeborreliosis: relation to clinical outcome. J Inf Dis 2004, 189: 1881-1891.

Östberg Y., Carrol JM, Pinne M, Rosa P and Bergström S. Pleiotropic effects of inactivating acarboxyl-terminal protease, CtpA, in Borrelia burgdorferi.J.Bacteriol. 2004 186: 2074-2084

Pinne M, Östberg Y, Comstedt P, and Bergström S. Molecular analysis of the channel-forming protein P13and its paralog family 48 from different Lyme disease Borrelia species. Microbiology. 2004, 150: 549-559

Östberg Y, Bunikis I, Bergström S and Johansson J ¾The etiological agent of Lyme disease, Borrelia burgdorferi, appears to contain only a few small RNA molecules. J Bacteriol. 2004 186:8472-8477.

Suhonen Juha. "The role of neutrophils and dendritic cellsin Lyme borreliosis". Thesis, University of Turku, 2003.

Folia Biol (Praha). 2003;49(1):40-8.

Interaction of Borrelia burgdorferi sensu lato with Epstein-Barr virus in lymphoblastoid cells.

Hulinska D, Roubalova K, Schramlova J.

National Reference Laboratory on Borreliosis, Electron Microscopy, National Institute of Public Health, Prague, Czech Republic.

Since the possibility of interruption of latent EBV infection has been suggested by the induction of the lytic virus cycle with chemical substances, other viruses, and by immunosuppression, we hypothesized that the same effect might happen in B. burgdorferi sensu lato infection as happens in Lyme disease patients with positive serology for both agents. We have observed EBV replication in lymphoblastoid cells after superinfection with B. garinii and B. afzelii strains after 1 and 4 h of their interaction. We found that viral and borrelial antigens persisted in the lymphoblasts for 3 and 4 days. Morphological and functional transformation of both agents facilitate their transfer to daughter cells. Association with lymphoblasts and internalization of B. garinii by tube phagocytosis increased replication of viruses more successfully than B. afzelii and chemical inductors. Demonstration of such findings must be interpreted cautiously, but may prove a mixed borrelial and viral cause of severe neurological disease.

PMID: 12630667 [PubMed - indexed for MEDLINE]

Dissertation (Dr. rer. nat.), Universität Konstanz, 2003.

Immunomodulation and new therapeutic strategies in Lyme borreliosis

(im Cache)

Isabel Diterich

If infection with Borrelia burgdorferi is not treated adequately with antibiotics in an early stage, it may lead to Lyme borreliosis (LB), a chronic multisystemic disorder which is difficult to cure. In some cases the pathogen survives in spite of antibiotic treatments. It is challenging to understand why Borrelia are often not eradicated, although being recognized by the host's immune defense and occasionally inducing a strong inflammatory reaction. Thus, it remains an area of debate how this pathogen persists in human tissues.

This question was addressed in the present thesis, examining possible immune evasion mechanisms of Borrelia.

  • Blood cells from patients suffering from persistent LB released significantly lower levels of pro-inflammatory cytokines (TNFa and IFNg) in response to either Borrelia lysate or to lipopolysaccharide (LPS) in comparison to cells from healthy volunteers.
  • In blood from healthy volunteers Borrelia lysate led to strong production of anti-inflammatory IL-10 and G-CSF, while inducing only low levels of proinflammatory IFNg, compared to LPS.
  • Similar to endotoxin tolerance, different Borrelia preparations desensitized human blood monocytes on re-stimulation with either stimulus.
  • Borrelia-specific stimuli induced cross-tolerance towards heterelogous stimuli such as lipopolysaccharid (LPS) and lipoteichoic acid (LTA) in human monocytes.
  • Toll-like receptor (TLR) 2 but not TLR4 was required for Borrelia-induced tolerance and cross-tolerance, as shown in experiments with knock-out mice.
  • PBMC tolerized by Borrelia lysate exhibited reduced TLR2-mRNA levels. Further, IL-10 was identified as a key mediator involved in tolerance-induction by Borrelia lysate.
  • Combination of Filgrastim treatment with Ceftriaxone in a late stage LB-patient who failed standard antibiotic therapy led to successful eradication of the pathogen and complete regression of symptoms.
  • The mouse model of Borrelia infection was set up and characterized in order to study the therapeutic effects of Filgrastim in vivo.
  • Treating immunocompetent and immunodeficient SCID mice with Filgrastim, as an immunosupportive therapy of LB did not attenuate the characteristic ankle swelling induced by Borrelia infection.
  • Regular application of Filgrastim led to an enhanced elimination of Borrelia from various organs in SCID mice. In immunocompetent mice this effect was less pronounced.
In summary, we propose that Borrelia modulate the host's immune system in order to evade clearance in the immunologically competent host. Tolerance could represent the mechanism inhibiting host response thereby enabling survival and persistence of the pathogen. Promising results were obtained testing a novel treatment strategy for late stage LB, a combination of Filgrastim as an immunosupportive therapy with antibiotics. The respective clinical trial based on these findings was recently started.

Infection and Immunity 2001;69(2):687-694

Diterich I, Härter L, Hassler D (2), Wendel A and Hartung T

Biochemical Pharmacology, Department of Biology, University of Konstanz, D-78457 Konstanz, except (2): Untere Hofstatt 3, D-76703 Kraichtal, Germany

Modulation of Cytokine Release in Ex Vivo-Stimulated Blood from Borreliosis Patients

In lipopolysaccharide-stimulated blood from 71 late-stage borreliosis patients, the ex vivo cytokine release capacity of tumor necrosis factor alpha (TNF-) and gamma interferon (IFN-) was reduced to 28% ± 5% and to 31% ± 5% (P 0.001), respectively, compared to that of 24 healthy controls. White blood cell counts were normal in both groups. To investigate direct interactions between the pathogen and the immune cells, blood from healthy controls was exposed in vitro to live or heat-killed Borrelia or to Borrelia lysate. Compared to the pattern induced by bacterial endotoxins, a reduced release of TNF- and IFN- and an enhanced secretion of interleukin-10 and granulocyte colony-stimulating factor was found. In blood from 10 borreliosis patients stimulated with Borrelia lysate, TNF- formation was decreased to 31% ± 14% and IFN- formation was decreased to 8% ± 3% (P 0.001) compared to the cytokine response of blood from healthy controls (n = 24). We propose to consider anti-inflammatory changes in the blood cytokine response capacity elicited by Borrelia as a condition that might favor the persistence of the spirochete.

* Corresponding author. Mailing address: Biochemical Pharmacology, University of Konstanz, 78457 Konstanz, Germany. Phone: 49 7531 88 4116. Fax: 49 7531 88 4117. E-mail: Thomas Hartung.

Semin Neurol 1997 Mar;17(1):63-8

Immunologic mechanisms in Lyme neuroborreliosis: the potential role of autoimmunity and molecular mimicry.

Sigal LH

Department of Medicine and Disease Center, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick 08903-0019, USA.

Most of the clinical manifestations of Lyme disease are due to the local presence of the causative agent, Borrelia burgdorferi, in the affected tissues. However, the precise means of tissue damage are not well understood and there is no proof that the organism, live or dead, is always present. An understanding of the complex interaction between the organism, the immune response elicited by the organism, and the host can explain manifestations of the disease and persistence of symptoms and signs after the antibiotic-induced death of the organism. It is possible that dead spirochetes, or fragments thereof may persist and act as a focus of ongoing inflammation. Different immunogenetic types may predispose to different immunologic responses, with distinct clinical outcomes. Vascular changes induced by the infection, either by local infection or the effects of cytokines on the vessel wall, may underlie tissue pathology. Finally, the immune response to B. burgdorferi may elicit the production of antibodies capable of recognizing and damaging or modifying normal host tissues. Only by establishing the mechanisms causing tissue damage in Lyme disease can rational therapeutic strategies be developed. Only by understanding these mechanisms can physicians and patients interpret clinical responses to therapy and accurately appreciate the clinical prognosis.

Publication Types:

PMID: 9166962, UI: 97309547

Infect Immun 1997 May;65(5):1722-8

A monoclonal antibody to Borrelia burgdorferi flagellin modifies neuroblastoma cell neuritogenesis in vitro: a possible role for autoimmunity in the neuropathy of Lyme disease.

Sigal LH, Williams S

Department of Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick 08903, USA.

Although Borrelia burgdorferi is found at the site of many manifestations of Lyme disease, local infection may not explain all features of the disease. Previous work has demonstrated that the organism's flagellin cross-reacts with a component of human peripheral nerve axon, heat shock protein 60. The cross-reacting epitope is identified by a single anti-B. burgdorferi flagellin monoclonal antibody, H9724. We now report that the spontaneous and peptide growth factor-stimulated in vitro neuritogenesis of SK-N-SH neuroblastoma cells and other neural tumor cell lines is suppressed by H9724. In contrast, changes induced by exposure of these cells to optimal and suboptimal concentrations of cyclic AMP, phorbol ester, or retinoic acid are not affected by H9724. H9724 does not decrease cell viability or the ability of the cells to anchor to the culture plate or extracellular matrix and does not block nerve growth factor binding to the cells. These findings are compatible with the premise that antiaxonal antibodies formed during the immune response to B. burgdorferi flagellin might modify axonal function in vivo and play a role in the pathogenesis of neurologic features of Lyme disease. A humoral immune response predicated on molecular mimicry could explain persistent or ongoing neurologic dysfunction occurring after elimination of the organism by appropriate antibiotic therapy.

PMID: 0009125553, UI: 97270470

Proc Natl Acad Sci U S A 1995 Oct 24;92(22):10398-402

Similar antigenic surfaces, rather than sequence homology, dictate T-cell epitope molecular mimicry.

Quaratino S; Thorpe CJ; Travers PJ; Londei M

United Kingdom.

ABSTRACT: Molecular mimicry, normally defined by the level of primary-sequence similarities between self and foreign antigens, has been considered a key element in the pathogenesis of autoimmunity. Here we describe an example of molecular mimicry

  1. 2 intervening peptides did not stimulate the T-cell clone, even though they share 9 amino acids with the stimulatory peptides.
  2. Molecular modeling of major histocompatibility complex class II-peptide complexes suggests that both of the recognized peptides generate similar antigenic surfaces, although these are composed of different sets of amino acids.
  3. The molecular modeling of a peptide shifted one residue from the stimulatory peptide, which was recognized in the context of the same HLA molecule by another T-cell clone, generated a completely different antigenic surface.
  4. Functional studies using truncated peptides confirmed that the anchor residues of the two "mimicking" epitopes in the HLA groove differ.
Our results show, for two natural epitopes, how molecular mimicry can occur and suggest that studies of potential antigenic surfaces, rather than sequence similarity, are necessary for analyzing suspected peptide mimicry.


PMID: 7479792 UI: 96036092

Enferm Infecc Microbiol Clin 1997 Feb;15(2):73-6

[Induction of interleukin-6 by Borrelia burgdorferi in different cell cultures]. [Article in Spanish]

Ponte C, Gadea I, Vallejo JC, Soriano F

Departamento Microbiologia Medica, Fundacion Jimenez Diaz, Madrid.

BACKGROUND: To determine variability, if exists, in interleukin-6 (IL-6) response by different cell cultures stimulated by Borrelia burgdorferi. METHODS: Five human embryonic diploid fibroblast cell lines (three of them of lung origin) and four human synovial cell cultures were used. Induction of IL-6 was made with B. burgdorferi B31 culture and E. coli lipopolysaccharide (LPS). IL-6 was determined by a commercial EIA (Coaliza IL-6). RESULTS: All cell cultures produced IL-6 under basal conditions and responded after adding E. coli LPS. The IL-6 production after B. burgdorferi stimulation was higher than that induced by LPS with three out the four synovial cell cultures and lowest with lung fibroblasts. CONCLUSIONS: The ability of B. burgdorferi to stimulate IL-6 production depends on the nature and origin of cell cultures. This variability and the fact that the most reactive cell cultures were those of synovial precedence may correlated with some clinical aspects of the Lyme disease.

PMID: 9101750, UI: 97222641

Semin Neurol 1997 Mar;17(1):57-62

TNF Elevated in Lyme: Mechanisms of injury in Lyme neuroborreliosis.

Garcia-Monco JC, Benach JL.

Department of Neurology, Hospital de Galdacano, Vizcaya, Spain.

Neurologic injury in infection with Borrelia burgdorferi

Although less likely, there is the possibility that autoreactive mechanisms could have a role in the development of some manifestations of neuroborreliosis.

PMID: 9166961 [PubMed - indexed for MEDLINE]

Microbiol Immunol 1997;41(5):427-430

Levels of endogenous interleukin-1, interleukin-6, and tumor necrosis factor in congenic mice infected with Borrelia garinii.

Isogai H, Kimura K, Hayashi S, Kubota T, Fujii N, Nishikawa T, Kotake S, Isogai E

Institute of Animal Experimentation, Sapporo Medical University, Hokkaido, Japan.

This study describes the levels of interleukin-1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha) and interleukin-6 (IL-6) in the sera and parenchymal organs of various congenic mouse strains infected with Borrelia garinii. A significant elevation of inflammatory cytokine levels was found in the organs of C3H/HeN (H-2k) and B10.BR (H-2k) mice but not in those of BALB/c mice (H-2d). Focally produced cytokines can contribute to antimicrobial defense against these organisms. High levels of IL-1 alpha were observed in the sera of C3H/HeN, B10.BR and B10 (H-2b) mice infected with B. garinii and they were associated with the presence of spirochetes in the skin. Thus, susceptible mice demonstrated a stronger cytokine response than resistant mice. This study presents in vivo evidence that B. garinii infection affects the immunopathogenesis of Lyme disease.

PMID: 9194042, UI: 97337300

Microb Pathog 1994 Apr;16(4):261-7

Borrelia burgdorferi decreases hyaluronan synthesis but increases IL-6 production by fibroblasts.

Jones NC, Germain A, Riley KE, Bautista C, Taylor W, Wells AF

University of South Florida, College of Medicine, Division of Rheumatology, Tampa 33620.

Despite the prevalence of clinical data on human Lyme disease, little is known about the immunopathologic effects of the causative organism on the host. We studied the effect of Borrelia burgdorferi on hyaluronan (hyaluronic acid, HYA) production and the effect on interleukin-6 (IL-6) synthesis by cultured fibroblasts. The cell line employed in this study produced an average of 1406 ng of hyaluronan/ml within 48 h. Using both a morphological staining protocol and a quantitative radiometric assay, we noted that in the presence of a low dose of Borrelia (9.4 x 10(5) cells/ml) the hyaluronan production decreased to an average of 1008 ng/ml, a significant difference (p < 0.05) from the amount of hyaluronan produced by the cells alone. The reduction was even more significant (p < 0.01) when a higher dose of Borrelia (9.4 x 10(6) cells/ml) was used giving an average hyaluronan concentration of 682 ng/ml. In contrast, we found that Borrelia stimulated the cells to produce IL-6 from a baseline of 293 pg/ml to a maximal value of 842 pg/ml (p < 0.01). The spirochetes had no significant effect on cell viability, nor were we able to demonstrate invasion of the cells by the bacteria. Both a decrease in hyaluronan and an increase in IL-6 may correlate with the pathogenicity of Lyme disease in man.

PMID: 7968455, UI: 95058096

Prostaglandins 1994 Jan;47(1):41-54

Increased PGE2 from human monocytes isolated in the luteal phase of the menstrual cycle. Implications for immunity?

Leslie CA, Dubey DP

ENRM Veterans Administration Hospital, Bedford, MA 01730.

The reproductive hormones are implicated in the well documented sexual dimorphism in cellular and immune responses. Prostaglandins (PGs) are mediators of the immune response with their concentration and relative amounts being pivotal to their impact. In resident peritoneal macrophages isolated from mice we had previously noted that the cells from females synthesized significantly more PG than males. In these experiments we investigated whether PG metabolism in the human monocyte was influenced by gender and by stage of the menstrual cycle. Monocytes isolated from the female and activated in vitro with LPS produced on average significantly more PG into the medium than the males. Among females, significantly more PG was found in the medium from cells isolated during the luteal phase of the cycle than during the early follicular phase. It was also in this luteal phase in which the female differed substantially from males. We suggest that the in vivo hormonal changes associated with the menstrual cycle modulate monocyte synthesis of PG and other immune modulators such as IL-1. This could be a key to understanding differences in vulnerability between males and females as well as within phases of the cycle, to immune and inflammatory insult.

PMID: 8140261, UI: 94188536

J Infect Dis 1991 Sep;164(3):568-74

Cytokines and the pathogenesis of neuroborreliosis: Borrelia burgdorferi induces glioma cells to secrete interleukin-6.

Habicht GS, Katona LI, Benach JL

Department of Pathology, State University of New York, Stony Brook 11794-8691.

Lyme disease is a multisystemic disease caused by a tickborne spirochete, Borrelia burgdorferi. Neuroborreliosis is characterized by intrathecal production of antibodies specific for the spirochete. This suggests that spirochetal infection of the central nervous system produces conditions that support the maturation of B lymphocytes to immunoglobulin-secreting cells. Interleukin 6 (IL-6) stimulates B cell differentiation into antibody-secreting cells. The present study was undertaken to determine whether B. burgdorferi can stimulate cells of central nervous system origin to secrete IL-6. C6 rat glioma cells cultured with spirochetes induced secretion of IL-6 activity. Peak stimulation was achieved at 24 h with 25 spirochetes per glioma cell. Glioma cells were also stimulated to produce IL-6 by interleukin 1 and tumor necrosis factor. That very few spirochetes are found in Lyme disease patients suggests that biologic amplification factors derived from the organism or the host, or both, are responsible for the pathogenesis of this disease. IL-6 can now be added to the growing list of such factors.

PMID: 1908002, UI: 91332484

Zentralbl Bakteriol Mikrobiol Hyg [A] 1986 Dec;263(1-2):133-6

A role for interleukin-1 in the pathogenesis of Lyme disease.

Beck G, Habicht GS, Benach JL, Coleman JL, Lysik RM, O'Brien RF

Interleukin-1 (IL-1) is the major immunoregulatory molecule produced by macrophages in response to a variety of environmental insults including chemicals, phagocytosis, bacteria, and bacterial products. Macrophages stimulated by Borrelia burgdorferi produced large quantities of IL-1 when spirochetes were added to macrophages at a ratio of 10 spirochetes per macrophage. The release of IL-1 was dose dependent: a single spirochete per macrophage was sufficient to produce significant quantities of IL-1. Spirochetal lipopolysaccharide was not required for this activity in that polymyxin B in the spirochete-macrophage culture had no effect on IL-1 production. Normal murine fibroblasts cultured with this IL-1 were shown to have an increased rate of DNA synthesis and an increase in secreted collagenase. IL-1 was found in joint fluids from Lyme disease patients. When IL-1 was injected intradermally into the backs of rabbits, the injection sites became indurated, erythematous, and warm to the touch after 4 hrs and annular lesions much like those of erythema chronicum migrans were seen in some animals after 24 hrs. B. burgdorferi is a powerful inducer for IL-1 in vitro, and it is reasonable to presume that it acts similarly in Lyme disease patients. Our results suggest that IL-1 in turn, may play a role in many of the clinical manifestations of Lyme disease.

PMID: 3495083, UI: 87208529

Infect Immun 1997 Apr;65(4):1217-22

Published erratum appears in Infect Immun 1997 Jun;65(6):2508

Production of interleukin-8 (IL-8) by cultured endothelial cells in response to Borrelia burgdorferi occurs independently of secreted [corrected] IL-1 and tumor necrosis factor alpha and is required for subsequent transendothelial migration of neutrophils.

Burns MJ, Sellati TJ, Teng EI, Furie MB

Department of Pathology, School of Medicine, State University of New York at Stony Brook 11794, USA. mburns@path.som.sunysb.edu

Previous studies have shown that Borrelia burgdorferi, the spirochetal agent of Lyme disease, promotes inflammation by stimulating endothelial cells to upregulate adhesion molecules for leukocytes and to produce a soluble agent that is chemotactic for neutrophils. We determined that interleukin-8 (IL-8) was the chemotactic agent for neutrophils present in conditioned media from cultured human umbilical vein endothelial cells stimulated with B. burgdorferi. As few as one spirochete per endothelial cell stimulated production of IL-8 within 8 h of coincubation. When 10 spirochetes per endothelial cell were added, IL-8 was detected after 4 h of coculture. Production of IL-8 continued in a linear fashion for at least 24 h. Neutralizing antibodies against IL-8 reduced migration of neutrophils across spirochete-stimulated endothelial monolayers by 93%. In contrast, pretreatment of neutrophils with antagonists of platelet-activating factor did not inhibit migration. Increases in production of IL-8 and expression of the adhesion molecule E-selectin by endothelial cells in response to B. burgdorferi were not inhibited by IL-1 receptor antagonist or a neutralizing monoclonal antibody directed against tumor necrosis factor alpha, used either alone or in combination. These results suggest that activation of endothelium by B. burgdorferi is not mediated through the autocrine action of secreted IL-1 or tumor necrosis factor alpha. Rather, it appears that B. burgdorferi must stimulate endothelium either by a direct signaling mechanism or by induction of a novel host-derived proinflammatory cytokine.

PMID: 9119454, UI: 97230290

Infect Immun 1996 Aug;64(8):3180-7

Outer surface lipoproteins of Borrelia burgdorferi activate vascular endothelium in vitro.

Sellati TJ, Abrescia LD, Radolf JD, Furie MB

Department of Pathology, School of Medicine, State University of New York at Stony Brook 11794-8691, USA.

Previously, we reported that activation of vascular endothelium by the Lyme disease pathogen Borrelia burgdorferi results in enhanced expression of endothelial cell adhesion molecules and promotion of the transendothelial migration of neutrophils in vitro. To investigate the role of spirochetal lipoproteins in this process, we assessed the ability of a synthetic lipohexapeptide corresponding to the N terminus of B. burgdorferi outer surface protein A (OspA) to activate human umbilical vein endothelial cells (HUVEC). Using a whole-cell enzyme-linked immunosorbent assay, we demonstrated that OspA lipopeptide activated endothelium in a dose-dependent fashion, as measured by upregulation of E-selectin. Near-maximal stimulation was achieved with 100 micromolar lipopeptide. In addition, the lipopeptide increased expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1). Similar results were obtained with 25 nM native OspA or lipidated recombinant OspA or OspB. Incubation of HUVEC with nonlipidated OspA peptide, nonlipidated recombinant OspA or OspB, or tripalmitoyl-S-glyceryl-cysteine had little or no effect on expression of these adhesion molecules. A mutant strain of B. burgdorferi that lacked OspA and OspB upregulated expression of E-selectin to the same degree as its wild-type counterpart, indicating that other spirochetal components also possess the ability to activate endothelium. Conditioned medium from HUVEC incubated with OspA lipopeptide or lipidated recombinant OspA induced chemotaxis of neutrophils in Boyden chamber assays, whereas the OspA preparations alone were devoid of chemotactic activity. When HUVEC grown on connective tissue substrates were treated with OspA lipopeptide, subsequently added neutrophils migrated across the endothelial monolayers. These results implicate the outer surface lipoproteins of B. burgdorferi as potential effector molecules in the promotion of a host inflammatory response.

PMID: 8757851, UI: 96333357

Infect Immun 1998 Oct;66(10):4875-83

Borrelia burgdorferi and interleukin-1 promote the transendothelial migration of monocytes In vitro by different mechanisms.

Burns MJ, Furie MB

Department of Pathology, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794, USA.

A prominent feature of Lyme disease is the perivascular accumulation of mononuclear leukocytes. Incubation of human umbilical vein endothelial cells (HUVEC) cultured on amniotic tissue with either interleukin-1 (IL-1) or Borrelia burgdorferi, the spirochetal agent of Lyme disease, increased the rate at which human monocytes migrated across the endothelial monolayers. Very late antigen 4 (VLA-4) and CD11/CD18 integrins mediated migration of monocytes across HUVEC exposed to either B. burgdorferi or IL-1 in similar manners. Neutralizing antibodies to the chemokine monocyte chemoattractant protein 1 (MCP-1) inhibited the migration of monocytes across unstimulated, IL-1-treated, or B. burgdorferi-stimulated HUVEC by 91% +/- 3%, 65% +/- 2%, or 25% +/- 22%, respectively. Stimulation of HUVEC with B. burgdorferi also promoted a 6-fold +/- 2-fold increase in the migration of human CD4(+) T lymphocytes. Although MCP-1 played only a limited role in the migration of monocytes across B. burgdorferi-treated HUVEC, migration of CD4(+) T lymphocytes across HUVEC exposed to spirochetes was highly dependent on this chemokine. The anti-inflammatory cytokine IL-10 reduced both migration of monocytes and endothelial production of MCP-1 in response to B. burgdorferi by approximately 50%, yet IL-10 inhibited neither migration nor secretion of MCP-1 when HUVEC were stimulated with IL-1. Our results suggest that activation of endothelium by B. burgdorferi may contribute to formation of the chronic inflammatory infiltrates associated with Lyme disease. The transendothelial migration of monocytes that is induced by B. burgdorferi is significantly less dependent on MCP-1 than is migration induced by IL-1. Selective inhibition by IL-10 further indicates that B. burgdorferi and IL-1 employ distinct mechanisms to activate endothelial cells.

PMID: 9746592, UI: 98427157

J Immunol 1996 Nov 15;157(10):4584-90

Borrelia burgdorferi outer membrane protein A induces nuclear translocation of nuclear factor-kappa B and inflammatory activation in human endothelial cells.

Wooten RM, Modur VR, McIntyre TM, Weis JJ

Department of Pathology, University of Utah Health Sciences Center, Salt Lake City 84132, USA.

Lyme disease is caused by infection with Borrelia burgdorferi, and is characterized by bacterial persistence and inflammation in a number of host tissues. B. burgdorferi outer surface lipoproteins possess cytokine stimulatory properties that may be responsible for localized inflammation. B. burgdorferi presence is correlated with severity of disease, and the pathology of many tissues, particularly the arthritic joint, is consistent with localized cytokine production. Spirochete invasion of tissues requires interaction with and penetration of vascular endothelium, suggesting endothelial cells may participate in the inflammation of Lyme disease. In this study, outer surface protein A (OspA), a model B. burgdorferi lipoprotein, was found to be a potent stimulant of nuclear factor-kappa B (NF-kappa B) nuclear translocation in human endothelial cells, resulting in nuclear levels similar to those seen in response to known inflammatory mediators. Only the lipid-modified OspA had activity, and activity was not due to contamination with LPS. Nuclear NF-kappa B was detectable within 15 min, suggesting that OspA directly mediates NF-kappa B nuclear translocation. OspA also rapidly up-regulated endothelial cell production of several proteins whose transcription is dependent on NF-kappa B: the cytokine IL-6; the chemokine IL-8; and the adhesion molecules E-selectin, VCAM-1, and ICAM-1. The adhesion molecules were functional, as demonstrated by enhanced binding of neutrophils to OspA-stimulated endothelial monolayers. These data suggest that OspA may initiate synthesis of many proteins essential for localized inflammation via the direct activation of NF-kappa B-dependent transcription. These observations suggest that the interaction of B. burgdorferi lipoproteins with the endothelium may directly induce the inflammation responsible for the symptoms of Lyme disease.

PMID: 8906837, UI: 97064218

Infect Immun 1993 Sep;61(9):3843-53

Borrelia burgdorferi outer surface lipoproteins OspA and OspB possess B-cell mitogenic and cytokine-stimulatory properties.

Ma Y, Weis JJ

Department of Pathology, University of Utah School of Medicine, Salt Lake City 84132.

Sonicated Borrelia burgdorferi was previously reported to possess both B-cell mitogenic and interleukin-6 (IL-6) stimulatory activities. In this report, two outer surface lipoproteins, OspA and OspB, were purified from B. burgdorferi and assessed for the presence of these functions. OspA was purified from two strains, an OspB-deficient variant of HB19 and N40, while OspB was purified from the N40 strain. All lipoprotein preparations were free of endotoxin contamination, and polymyxin B failed to inhibit responses, indicating that media contamination was not contributing to biological assays. All three preparations were able to stimulate proliferation of mononuclear cells from naive C3H/HeJ and BALB/c mice. Depletion experiments indicated that the responding cells were B lymphocytes and not T lymphocytes. Purified OspA and OspB stimulated immunoglobulin M production by splenocyte cultures from naive mice, a property also previously attributed to sonicated B. burgdorferi. OspA and OspB also stimulated the production of IL-6 and tumor necrosis factor alpha by bone marrow-derived macrophages from BALB/c and C3H/HeJ mice. Cytokine production was enhanced by the presence of gamma interferon in the cultures, indicating that the magnitude of responses to these lipoproteins may be modulated by cytokines in the microenvironment of infected tissues. Human endothelial cells produced IL-6 when incubated with OspA and OspB, indicating that non-hematopoietic lineage cells can respond to the lipoproteins. Purified OspA and OspB had approximately equal activity, with responses detected in the range of 10 ng of lipoprotein per ml to 1 microgram of lipoprotein per ml. Comparison with published dose responses for lipoproteins purified from Escherichia coli indicates that OspA and OspB purified from B. burgdorferi are much more potent. The high potency of the B. burgdorferi lipoproteins and the ability of the spirochete to invade tissues and persist argue that they could be important in the localized events contributing to the pathology of Lyme disease.

PMID: 8359905, UI: 93366445

presented at the10th ANNUAL INT. CONF. ON LYME BORRELIOSIS, National Institue of Health, BETHESDA, MD. APRIL 28-30, 1997.

The Lyme Urine Antigen Test (LUAT) during antibiotic therapy in women with recurrent menstrual cycles

Barkley M., Harris N, Szantyr B

Univ. Calif. Davis, Davis, CA, IGeneX, Inc.Reference Laboratory, Palo Alto, CA and Lincoln, MA



Multiple urine samples

while antibiotics were being administered. The portion of the menstrual cycle chosen for study included Samples were collected in and immediately transferred to BD Urine Vacutainers with preservative. Samples were stored at -76oC. LUATs were performed on each sample (N=285). Urinary metabolites of progesterone were measured by ELISA throughout 3 IR's selected at random.


The case history is particularly interesting because

Menstrual cycle stage was significantly correlated with the level of Bb antigen detected in urine. Urines with a positive LUAT (P<0.05) (N=56) were divided into The results of this clinical study also demonstrate that collection of urine for LUAT determinations should include multiple samples obtained on the to optimize detection of urinary Bb antigen in women with recurrent menstrual cycles.

Neurology 1989 Aug;39(8):1118-20

Latent Lyme neuroborreliosis: presence of Borrelia burgdorferi in the cerebrospinal fluid without concurrent inflammatory signs.

Pfister HW, Preac-Mursic V, Wilske B, Einhaupl KM, Weinberger K

Neurologische Klinik, Klinikum Grosshadern, University of Munich, Federal Republic of Germany.

Borrelia burgdorferi, the etiologic agent of Lyme borreliosis, was isolated from the CSF of a patient with elevated serum IgG antibody titers against B burgdorferi and a history of multiple tick bites. The absence of concurrent inflammatory signs of CSF as well as intrathecal antibody production indicates a phase of latent Lyme neuroborreliosis in which no tissue infection or reaction has yet occurred. Bilateral tinnitus was the only clinical symptom in this patient. The persistence of the bilateral tinnitus after antibiotic therapy did not support a causal relationship between this symptom and the borrelial infection.

PMID: 2668788, UI: 89344421

Infect Immun 1998 Apr;66(4):1408-1412

Elastase is the only human neutrophil granule protein that alone is responsible for in vitro killing of Borrelia burgdorferi.

Garcia R, Gusmani L, Murgia R, Guarnaccia C, Cinco M, Rottini G

International Centre for Genetic Engineering and Biotechnology, Trieste, Italy. garcia@icgeb.trieste.it

Phagocytosis of Borrelia burgdorferi by human polymorphonuclear leukocytes triggers oxygen-dependent and -independent mechanisms of potentially cidal outcome. Nevertheless, no factor or process has yet been singled out as being borreliacidal. We have studied the B. burgdorferi-killing ability of the myeloperoxidase-H2O2-chloride system and that of primary and secondary granule components in an in vitro assay. We found that neither secondary granule acid extracts nor the chlorinating system could kill these microorganisms, while primary granule extracts were effective. The Borrelia-killing factor was purified to homogeneity and demonstrated to be elastase. Its cidal activity was found to be independent of its proteolytic activity.

PMID: 9529060, UI: 98187909

Infect Immun 1999 Jan;67(1):173-81

The immunoglobulin (IgG) antibody response to OspA and OspB correlates with severe and prolonged Lyme arthritis and the IgG response to P35 correlates with mild and brief arthritis.

Akin E, McHugh GL, Flavell RA, Fikrig E, Steere AC

Division of Rheumatology/Immunology, Tufts University School of Medicine, New England Medical Center, Tupper Research Institute, Boston, Massachusetts 02111, USA.

In an effort to implicate immune responses to specific Borrelia burgdorferi proteins that may have a role in chronic Lyme arthritis, we studied the natural history of the antibody response to B. burgdorferi in serial serum samples from 25 patients monitored throughout the course of Lyme disease. In these patients, the immunoglobulin G (IgM) and IgG antibody responses to 10 recombinant B. burgdorferi proteins, determined during early infection, early arthritis, and maximal arthritis, were correlated with the severity and duration of maximal arthritis. The earliest responses were usually to outer surface protein C (OspC), P35, P37, and P41; reactivity with OspE, OspF, P39, and P93 often developed weeks later; and months to years later, 64% of patients had responses to OspA and OspB. During early infection and early arthritis, the levels of IgG antibody to P35 correlated inversely with the subsequent severity or duration of maximal arthritis. In contrast, during periods of maximal arthritis, the levels of IgG antibody to OspA and OspB, especially to a C-terminal epitope of OspA, correlated directly with the severity and duration of arthritis. Thus, the higher the IgG antibody response to P35 earlier in the infection, the milder and briefer the subsequent arthritis, whereas during maximal arthritis, the higher the IgG response to OspA and OspB, the more severe and prolonged the arthritis.

PMID: 9864212, UI: 99081739

Lancet 1990 Feb 10;335(8685):312-315

Sequestration of antibody to Borrelia burgdorferi in immune complexes in seronegative Lyme disease.

Schutzer SE, Coyle PK, Belman AL, Golightly MG, Drulle J

Department of Medicine, University of Medicine and Dentistry-New Jersey Medical School, Newark 07103.

To find out whether apparent seronegativity in patients strongly suspected of having Lyme disease can be due to sequestration of antibodies in immune complexes, such complexes were isolated and tested for antibody to Borrelia burgdorferi. In a blinded analysis the antibody was detected

These findings were confirmed by western blot, which also showed that immune complex dissociation liberated Complexed B burgdorferi antibody was also found in Apparent B burgdorferi seronegativity in serum immune complexes may thus be due to sequestration of antibody in immune complexes.

PMID: 1967770, UI: 90135740

Ann Neurol 1990 Dec;28(6):739-744

Cerebrospinal fluid immune complexes in patients exposed to Borrelia burgdorferi: detection of Borrelia-specific and -nonspecific complexes.

Coyle PK, Schutzer SE, Belman AL, Krupp LB, Golightly MG

Department of Neurology, State University of New York, Stony Brook 11794.

We analyzed cerebrospinal fluid (CSF) from 32 patients with neurological symptoms and evidence of Borrelia burgdorferi infection

  1. CSF immune complexes were found in 22 (69%) of 32 patients;
  2. in 18, there was sufficient sample to isolate immune complexes.
    • By enzyme-linked immunosorbent assay, isolated immune complexes from 10 of these 18 patients contained antibody specific for B. burgdorferi antigens.
    • The isotypes were IgG (n = 8), IgM (n = 3), and IgA (n = 2).
    • By immunoblot, these antibodies were directed against B. burgdorferi 41-kDa antigen and occasionally against the 33- and 17-kDa antigens.
  3. Anti-B. burgdorferi IgM
    • was present in patients with acute neurological symptoms,
    • was predominantly complexed rather than free, and
    • decreased with clinical recovery in the one serial study.
  4. 3 patients were nonreactive for free CSF antibodies, but had complexed antibodies to the organism.

The preliminary finding of specific B. burgdorferi components in immune complexes in CSF suggests an active process triggered by the organism, even in the absence of other CSF abnormalities.

PMID: 2285261, UI: 91136153

J Clin Invest 1994 Jul;94(1):454-457

Early and specific antibody response to OspA in Lyme Disease.

Schutzer SE, Coyle PK, Dunn JJ, Luft BJ, Brunner M

Department of Medicine, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark 07103.

Borrelia burgdorferi (Bb), the cause of Lyme disease, has appeared not to evoke a detectable specific antibody response in humans until long after infection. This delayed response has been a biologic puzzle and has hampered early diagnosis.

To investigate our hypothesis that specific antibody to OspA we analyzed serum samples from patients with concurrent erythema migrans (EM). This is the earliest sign of Lyme disease and occurs in 60-70% of patients, generally 4-14 d after infection. We used less conventional but more sensitive methods: Antibody specificity was confirmed with recombinant OspA.

  1. Specific complexed antibody to
    • whole Bb and
    • recombinant OspA
    was detected in 10 of 11 of the EM patients compared to 0 of 20 endemic area controls.
  2. IgM was the predominant isotype to OspA in these EM patients.
    • Free IgM to OspA was found in half the EM cases.
    • IgM to OspA was also detected in 10 of 10 European patients with EM who also had reactive T cells to recombinant OspA.
In conclusion a specific antibody response to OspA occurs early in Lyme disease. This is likely to have diagnostic implications.

PMID: 8040289, UI: 94314934

J Clin Microbiol 1994 Apr;32(4):1011-7

Use of a hybrid protein consisting of the variable region of the Borrelia burgdorferi flagellin and part of the 83-kDa protein as antigen for serodiagnosis of Lyme disease.

Rasiah C, Rauer S, Gassmann GS, Vogt A

Institut fur Medizinische Mikrobiologie und Hygiene, Freiburg, Germany.

A hybrid protein consisting of the variable region of the Borrelia burgdorferi flagellin (an 18-kDa fragment) and a 59-kDa fragment (lacking the N-terminal part) of the 83-kDa protein has been constructed by genetic engineering. It was expressed as a nonfusion protein of an apparent molecular weight of 77,000 in Escherichia coli. The suitability of this new antigen for the diagnosis of Lyme disease was tested by immunoblotting; for comparison, the recombinant variable region of the flagellin, the 18-kDa fragment (p18), and the whole recombinant 83-kDa protein (p83), both expressed in E. coli, were used. A total of 120 serum samples from various stages of Lyme disease, which were positive in two serological assays, a passive hemagglutination assay and an indirect immunofluorescence assay, were tested. By indirect immunofluorescence, 74 samples were positive for immunoglobulin G (IgG) antibodies and 72 were positive for IgM antibodies. Of these serum samples, 69 of 74 (93%) contained IgG antibodies against p18 and/or p83, and IgG antibodies were detected by the hybrid protein in 67 (90%) samples. IgM antibodies against p18 and/or p83 were detected in 60 of 72 (83%) serum samples, and 57 (79%) serum samples were reactive with the hybrid protein. Twenty serum samples of patients with a history of syphilis and 40 serum samples, negative in routine B. burgdorferi serology, were tested as controls. The hybrid protein, made up of specific epitopes of an early (p18) and late (p83) antigen, is recognized by almost the same number of patient serum samples as the individual antigens.

PMID: 8027303, UI: 94299775

Med Microbiol Immunol (Berl) 1995 May;184(1):23-32

Molecular and immunological characterization of the p83/100 protein of various Borrelia burgdorferi sensu lato strains.

Rossler D, Eiffert H, Jauris-Heipke S, Lehnert G, Preac-Mursic V, Teepe J, Schlott T, Soutschek E, Wilske B

Max von Pettenkofer Institut fur Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians Universitat München, Germany.

The complete coding regions of the chromosomally encoded p83/100 protein of four Borrelia garinii strains and one Borrelia burgdorferi sensu stricto strain have been amplified by the polymerase chain reaction (PCR), cloned and sequenced. From alignment studies with the deduced amino acid sequences presented here, and five other published p83/100 sequences, the most heterologous region of the p83/100 molecule was identified to be located between amino acid position 390-540.

To study the structure of this heterogeneous region, and internal fragment of the p83/100 genes from 11 additional B. burgdorferi sensu lato strains was amplified by PCR. The PCR products were analyzed by DNA sequencing and restriction enzyme analysis. These internal p83/100 fragments varied in size and sequence.

Phenotypic analysis with four p83/100-specific monoclonal antibodies revealed four distinct reactivity patterns.

  1. Antibody L100 1B4 recognized a common epitope of B. burgdorferi sensu stricto and B. afzelii.
  2. Antibodies L100 17D3 and L100 18B4 were reactive with an epitope shared by strains of all three species.
  3. The broadest reactivity was shown by L100 18B4 which, in contrast to L100 17D3, additionally recognized the relapsing fever borreliae B. turicatae and B. hermsii.
  4. L100 8B8 detected a subgroup of the B. burgdorferi sensu stricto strains.

Since comparison of the p83/100 molecule with sequences from protein databases showed similarities with characteristics of eukaryotic cell structures, the p83/100 might mimic these structures and may, therefore, be involved in the immune escape mechanism of the pathogenic agent of Lyme disease.

PMID: 8538575, UI: 96149106

J Clin Microbiol 1995 Oct;33(10):2596-600

Establishment of enzyme-linked immunosorbent assay using purified recombinant 83-kilodalton antigen of Borrelia burgdorferi sensu stricto and Borrelia afzelii for serodiagnosis of Lyme disease.

Rauer S, Kayser M, Neubert U, Rasiah C, Vogt A

Institut fur Medizinische Mikrobiologie und Hygiene, Albert-Ludwigs-Universitat, Freiburg, Germany.

The 83-kDa antigen of Borrelia burgdorferi was expressed as a recombinant protein in Escherichia coli and purified for use in an enzyme-linked immunosorbent assay (p83-ELISA). Antibodies to the 83-kDa antigen of both the immunoglobulin G (IgG) and IgM isotypes could be detected in all stages of Lyme disease. Sensitivity varied, depending on the clinical stage of illness. In early stages, as defined for 118 patients with erythema migrans, it was found to be 20% (24 of 118 patients: 7 with IgM, 16 with IgG, and 1 with IgM and IgG). Of the patients with late-stage Lyme arthritis and acrodermatitis chronica atrophicans, 94% (16 of 17:2 with IgM and IgG and 14 with IgG) and 86% (36 of 42:2 with IgG and IgM and 34 with IgG) revealed positive results in the p83-ELISA, respectively. p83 displays sequence heterogeneity according to the genomospecies, but when the reactions of serum specimens from acrodermatitis chronica atrophicans patients and arthritis patients with p83 derived from representative strains of B. burgdorferi sensu stricto and Borrelia afzelii in ELISAs were compared, no differences in specificity and sensitivity were seen. When 82 serum specimens from healthy controls were tested, none had IgG and only 3 (4%) had IgM antibodies, indicating a high specificity. Positive reactions with antibodies against Treponema pallidum (1 of 37 patients; IgG) and Epstein-Barr virus (1 of 44 patients; IgM) and with autoantibodies of various specificities (1 of 53 patients; IgG) were seen with < 3% of the serum samples te... high speficicity for B. burgdorferi. 2+ 13% for IgM antibodies, the IgM p83-ELISA provided little diagnostic information for Lyme disease, whereas the IgG p83-ELISA appears to be a suitable test for serodiagnosis of advanced-stage Lyme disease.

Publication Types:

PMID: 8567889, UI: 96087193


Academy of Finland Communications, April 18, 2006

Even antibiotics can't always stop the bacterium: Borrelia can hide in the human body for years

Matti Viljanen

University of Turku, Finland, tel. +358 2 3337330, Microbes and Man Research Programme, matti.viljanen@utu.fi,
Programme Director, Soile Juuti National Public Health Institute, P.O. Box 95, FIN-70701 Kuopio, Finland, tel. +358 40 565 1529, soile.juuti@ktl.fi.

Transmitted by tick bites, the Borrelia bacterium can hide in the human body for up to several years in spite of antibiotic treatment. The patient's symptoms may be so vague that it is extremely difficult to make the connection. The research team under Professor Matti Viljanen have now developed a mouse model that can be used to locate the hidden Borrelia bacterium and to target treatment more accurately. Professor Viljanen and his team are working in a joint Finnish-Swedish research consortium under the Microbes and Man research programme that is jointly funded by the Academy of Finland and the Swedish Foundation for Strategic Research...

Project: Reactivation and immune evasion of Borrelia infection

Consortium leader: Professor SVEN BERGSTROM, Department of Molecular Biology, Umea University, SE-901 87 Umea, Sweden, Tel.: +46 (0)90 7856726, sven.bergstrom@molbiol.umu.se

Partner of the consortium: Professor Matti Viljanen, Department of Medical Microbiology, University of Turku, Kiinamyllynkatu 13, FIN-20520 Turku, Finland, Tel.: +358-2-3337330, email: matti.viljanen@utu.fi, homepage: http://www.utu.fi/research/tic/viljanen/

See also: Reactivation and immune evasion of Borrelia infection

Practical Gastroenterology, 2006 April

"Bell's Palsy of the Gut" and Other GI Manifestations of Lyme and Associated Diseases
(in Cache)

V. T. Sherr

From the Abstract:
Bell's palsy signifies paralysis of facial muscles related to inflammation of the associated seventh Cranial Nerve. Physicians may not realize that this syndrome is caused by the spirochetal agent of Lyme disease until proven otherwise. ... In every case of Bell's, doctors need to carefully investigate by history, physical, and laboratory work every shred of evidence that might suggest the presence of cryptic tertiary Lyme, a serious multisystem, gut and neuro-brain infection even though about half of fully diagnosed patients have no evidence whatsoever of having had a tick-bite. ...

Gastrointestinal Lyme disease may cause gut paralysis and a wide range of diverse GI symptoms with the underlying etiology likewise missed by physicians. Borrelia burgdorferi, the microbial agent often behind unexplained GI symptoms„along with numerous other pathogens also contained in tick saliva„influences health and vitality of the gastrointestinal tract from oral cavity to anus. Disruptions caused by GI borreliosis (Lyme) may include, amongst many others, distortions of taste, failure of other neural functions that supply the entire GI tract„paralysis or partial paralysis of the tongue, gag reflex, esophagus, stomach and nearby organs, small and/or large intestines ("ileus"), bowel pseudo-obstruction, intestinal spasms, excitability of gut muscles, inflammation of lumen lining tissues, spirochetal hepatitis, possibly cholecystitis, dysbiosis, jejunal or ileal incompetence with resultant small intestine bacterial overgrowth (SIBO), megacolon, encopresis and rectal muscle cramping (proctalgia fugax). ...

gallbladder: a niche for Borrelia burgdorferi

... Surgery to remove her diseased gallbladder was scheduled. Treatment (doxycycline) for suspected but unproven persistent Lyme was begun. The family physician asked that biopsy specimens of the removed gall bladder be tested in a reference laboratory specializing in tick-borne diseases (Medical Diagnostic Laboratories, L.L.C, 2439 Kuser Road, Hamilton, NJ 08690 USA. http://www.mdlab.com/html/testing/available_tests.html#tick). The resultant PCR test on her gall bladder tissue was positive for DNA of the causative Bb spirochete of Lyme disease. This PCR biopsy confirmation of a seronegative patient's Lyme diagnosis illustrates that, while Western Blot and PCR blood sample testing, especially for active late stage LYD, may not show a positive antibody response, a tissue PCR analysis may confirm the diagnosis, even when the patient has previously been treated. PCR's done on blood are less satisfactory since Bb prefers an in-tissue environment. ...

The Lyme Times, 2000 Spring;28: 86-88

Demonstrating the intracellularity of Borrelia burgdorferi: a compilation

H. Smith

Localization of Borrelia Burgdorferi in the Peripheral and Central Nervous System of the Non-human Primate Model of Lyme Disease

D.Cadavid1, A.R. Pachner1, T. O'Neill2

1Georgetown University, 2Armed Forces Institute of Pathology, Washington D.C.

Lyme disease is a multisystemic disease caused by the spirochete Borrelia burgdorferi. About 15% of patients develop neurologic complications including meningitis, cranial neuritis, peripheral neuropathy, and myopathy, for which the pathogenesis is poorly understood. The purpose of this study was to determine the localization and approximate number of organisms in the central (CNS) and peripheral nervous system (PNS) and other organs during experimental infection.

For this we used 2 immunocompetent and 2 steroid-immunosupressed Maccaca mulatta infected intradermally with 106 spirochetes of the N40Br strain of B. burgdorferi sacrificed 10 weeks after inoculation. 2 non-infected animals were used as controls.

  1. culture and/or PCR and/or passive transfer positive (p<0.05)
    • 11/25 CSF specimens from immunosupressed animals were culture and/or PCR and/or passive transfer positive compared with
    • 1/29 from the immunocompetent animals,
  2. PCR-ELISA revealed the presence of B. burgdorferi DNA
    • with the 2 immunosupressed animals:
      • in the dura mater,
      • in the brain stem,
      • in the spinal cord,
      • in the peripheral nerves and
      • in the muscles
    • with 1 of the 2 immunocompetent animals:
      • in the spinal cord,
      • in the muscles,
      • in the nerves.
  3. Immunohistochemistry (IHC) with a mouse anti B. burgdorferi polyclonal antibody revealed the presence of spirochetes
    • in the collagen tissue supporting the plexus and peripheral nerves (epineurium), and the muscles (epimysium),
    • in the adventitia of the aorta, and
    • in the leptomeninges of the spinal cord
    in immunosupressed but not in immunocompetent animals.
  4. The load of spirochetes by IHC was higher in
    • the epineurium (4x105 /cm3) and
    • the epimysium (6x104/cm3) and lower in the aorta and the spinal cord (1x103 /cm3).
    • In no case did we find evidence of B. burgdorferi in the parenchyma of the brain or spinal cord or in the nerves.

This is the first demonstration of the localization of B. burgdorferi in the CNS.

Lab Invest. 2000 Jul;80(7):1043-54.
Related Articles

Localization of Borrelia burgdorferi in the nervous system and other organs in a nonhuman primate model of lyme disease.

Cadavid D, O'Neill T, Schaefer H, Pachner AR

Department of Neuroscience, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark 07103, USA.

Lyme borreliosis is caused by infection with the spirochete Borrelia burgdorferi. Nonhuman primates inoculated with the N40 strain of B. burgdorferi develop infection of multiple tissues, including the central (CNS) and peripheral nervous system. In immunocompetent nonhuman primates, spirochetes are present in low numbers in tissues. For this reason, it has been difficult to study their localization and changes in expression of surface proteins.

To further investigate this, we


were obtained at necropsy 4 months later.

  1. The spirochetal tissue load was first studied by polymerase chain reaction (PCR)-ELISA of the outer surface protein A (ospA) gene.
  2. Immunohistochemistry was used
    • to study the localization and numbers of spirochetes in tissues and the expression of spirochetal proteins and
    • to characterize the inflammatory response.
  3. Hematoxylin and eosin and trichrome stains were used to study inflammation and tissue injury.

The results showed that the number of spirochetes was significantly higher in immunosuppressed animals.

  1. B. burgdorferi in the CNS localized to the
  2. Outside of the CNS, B. burgdorferi localized to
    • endoneurium and
    • connective tissues of
      • peripheral nerves,
      • skeletal muscle,
      • heart,
      • aorta, and
      • bladder.
  3. Although ospA, ospB, ospC, and flagellin were present at the time of inoculation, only flagellin was expressed by spirochetes in tissues 4 months later.
  4. Significant inflammation occurred only in the heart, and only immunosuppressed animals had cardiac fiber degeneration and necrosis.
  5. Plasma cells were abundant in inflammatory foci of steroid-treated animals.

We concluded that B. burgdorferi has a tropism for the meninges in the CNS and for connective tissues elsewhere in the body.

PMID: 10908149 [PubMed - indexed for MEDLINE]

J Basic Microbiol 1989;29(2):73-83

Ultrastructure of Borrelia burgdorferi in tissues of patients with Lyme disease.

Hulinska D, Jirous J, Valesova M, Herzogova J

Institute of Hygiene and Epidemiology, Prague, Czechoslovakia.

Spirochetal organisms were sought in 18 skin and 4 synovial membrane specimens obtained by biopsy from 22 Lyme disease patients. The presence of spirochetes in body tissues was histologically demonstrated in one patient with lymphadenosis benigna cutis, one patient with acrodermatitis chronica atrophicans and in one patient with active arthritis. The organisms were 5-30 microns long and 0.12-0.25 microns thick, had 8 or 11 flagella arising from both ends of the body, and their ultrastructure was analogous to that of cultured Borrelia burgdorferi strains. They were located intra- or perivascularly, or in the collagenous connective tissue of the skin and synovium. This implies that Lyme spirochetes may have a potential to survive in body tissues and cause injury to blood vessels.

PMID: 2709313, UI: 89216523

Am J Trop Med Hyg 1995 Feb;52(2):128-33

Localization of Borrelia burgdorferi in murine Lyme borreliosis by electron microscopy.

Pachner AR, Basta J, Delaney E, Hulinska D

National Institute of Public Health, Department of Electron Microscopy, Prague, Czech Republic.

Lyme borreliosis is a newly recognized systemic infection with protean clinical manifestations. Because the localization of the causative spirochete (Borrelia burgdorferi) in infected tissues is unknown, we used electron microscopy to find spirochetes in the hearts of chronically infected mice. There were three predominant locations for the spirochete in the hearts. In mice infected for one month or less, the spirochetes were mostly in or around blood vessels. They were either in the lumen or in the perivascular space. Mice infected for more than one month had B. burgdorferi in cardiac myocytes as well, often with clear spaces around them. The third area in which spirochetes were common was collagen fibers; the borreliae were wrapped around fibers with their long axis parallel to the fibers. The number of spirochetes was relatively low, but there was no appreciable decrease in numbers of spirochetes with increasing time postinfection. Inflammatory infiltrates were primarily in the endocardium and pericardium, but spirochetes were generally not in or near areas of inflammation. These data are consistent with previously published information that have identified the heart as a site of chronic infection and inflammation in the mouse. The studies extend our understanding of the behavior of the spirochete in vivo by identifying common locations of B. burgdorferi and by noting the disparity between infection and inflammation.

PMID: 7872439, UI: 95177294

J Pathol 1994 Jul;173(3):269-82

Borrelia burgdorferi-induced ultrastructural alterations in human phagocytes: a clue to pathogenicity?

Rittig MG, Haupl T, Krause A, Kressel M, Groscurth P, Burmester GR

Department of Anatomy I, University of Erlangen, Germany.

A chronic infection with the spirochaete Borrelia burgdorferi typically results in a multistage, multisystem illness. Thus, Lyme borreliosis may provide an interesting model to study the pathomechanisms of microbial persistence. In the present investigation, human peripheral blood monocytes, polymorphonuclear leukocytes, and synovial macrophages were incubated with B. burgdorferi and examined by light and electron microscopy. It was found that incubation with the spirochaetes induced distinct features in the phagocytes. Features which may be related to the pathogenesis of Lyme disease included the segmental uptake of spirochaetes with leaky lysosomes, the invagination of large membrane areas, the extra-lysosomal degradation of internalized B. burgdorferi cells and, finally, the formation of mononuclear syncytial cells and homotypic cell clusters. Features of unknown relevance were the occurrence of two types of cytoplasmic inclusion bodies and exocytic vesicles. These novel findings suggest that reactive alterations of the phagocytes may contribute to the pathogenesis of Lyme borreliosis, which could help to focus future histopathological studies. Moreover, these results may provide new insights into the pathogenesis of other infectious diseases characterized similarly by microbial persistence.

Arthritis Rheum 1993 Nov;36(11):1621-6

Persistence of Borrelia burgdorferi in ligamentous tissue from a patient with chronic Lyme borreliosis.

Häupl T, Hahn G, Rittig M, Krause A, Schoerner C, Schonherr U, Kalden JR, Burmester GR, thomas.haeupl@rz.hu-berlin.de

Department of Medicine III, University of Erlangen-Nuremberg, Germany.

OBJECTIVE. To document the persistence of Borrelia burgdorferi in ligamentous tissue samples obtained from a woman with chronic Lyme borreliosis.
METHODS. Spirochetes were isolated from samples of ligamentous tissue, and the spirochetes were characterized antigenetically and by molecular biology techniques. The ligamentous tissue was examined by electron microscopy. Humoral and cellular immune responses were analyzed.
RESULTS. Choroiditis was the first recognized manifestation of Lyme disease in this patient. Despite antibiotic therapy, there was progression to a chronic stage, with multisystem manifestations. The initially significant immune system activation was followed by a loss of the specific humoral immune response and a decrease in the cellular immune response to B burgdorferi over the course of the disease. "Trigger finger" developed, and a portion of the flexor retinaculum obtained at surgery was cultured. Viable spirochetes were identified. Ultramorphologically, the spirochetes were situated between collagen fibers and along fibroblasts, some of which were deeply invaginated by these organisms. The cultured bacteria were identified as B burgdorferi by reactions with specific immune sera and monoclonal antibodies, and by polymerase chain reaction amplification and Southern blot hybridization techniques.
CONCLUSION. To our knowledge, this is the first report of the isolation of B burgdorferi from ligamentous tissue. This suggests that tendon tissues serve as a specific site of spirochete residence in human hosts.

Hum Pathol 1996 Oct;27(10):1025-34

Ultrastructural demonstration of spirochetal antigens in synovial fluid and synovial membrane in chronic Lyme disease: possible factors contributing to persistence of organisms.

Nanagara R, Duray PH, Schumacher HR Jr

Allergy-Immunology-Rheumatology Division, Department of Medicine, Faculty of Medicine, KhonKaen University, Thailand.

To perform the first systematic electronmicroscopic (EM) and immunoelectron microscopy (IEM) study of the pathological changes and the evidence of spirochete presence in synovial membranes and synovial fluid (SF) cells of patients with chronic Lyme arthritis. EM examination was performed on four synovial membrane and eight SF cell samples from eight patients with chronic Lyme disease. Spirochetal antigens in the samples were sought by IEM using monoclonal antibody to Borrelia burgdorferi outer surface protein A (OspA) as the immunoprobe. Prominent ultrastructural findings were surface fibrin-like material, thickened synovial lining cell layer and signs of vascular injury. Borrelia-like structures were identified in all four synovial membranes and in two of eight SF cell samples. The presence of spirochetal antigens was confirmed by IEM in all four samples studied (one synovial membrane and three SF cell samples). OspA labelling was in perivascular areas, deep synovial stroma among collagen bundles, and in vacuoles of fibroblasts in synovial membranes; and in cytophagosomes of mononuclear cells in SF cell samples. Electron microscopy adds further evidence for persistence of spirochetal antigens in the joint in chronic Lyme disease. Locations of spirochetes or spirochetal antigens both intracellulary and extracellulary in deep synovial connective tissue as reported here suggest sites at which spirochaetes may elude host immune response and antibiotic treatment.

PMID: 8892586, UI: 97047745

J Infect Dis 1993 May;167(5):1074-81

Invasion of human skin fibroblasts by the Lyme disease spirochete, Borrelia burgdorferi.

Klempner MS, Noring R, Rogers RA

The ability of Borrelia burgdorferi to attach to and invade human fibroblasts was investigated by scanning electron and confocal microscopy. By scanning electron microscopy, B. burgdorferi were tightly adherent to fibroblast monolayers after 24-48 h but were eliminated from the cell surface by treatment with ceftriaxone (1 microgram/mL) for 5 days. Despite the absence of visible spirochetes on the cell surface after antibiotic treatment, viable B. burgdorferi were isolated from lysates of the fibroblast monolayers. B. burgdorferi were observed in the perinuclear region within human fibroblasts by laser scanning confocal microscopy. Intracellular spirochetes specifically labeled with monoclonal anti-flagellin antibody were also identified by fluorescent laser scanning confocal microscopy. These observations suggest that B. burgdorferi can adhere to, penetrate, and invade human fibroblasts in organisms that remain viable.

PMID: 8486939, UI: 93253286

J Infect Dis 1992 Aug;166(2):440-4

Fibroblasts protect the Lyme disease spirochete, Borrelia burgdorferi, from ceftriaxone in vitro.

Georgilis K, Peacocke M, Klempner MS

Department of Medicine, New England Medical Center, Boston, Massachusetts.

The Lyme disease spirochete, Borrelia burgdorferi, can be recovered long after initial infection, even from antibiotic-treated patients, indicating that it resists eradication by host defense mechanisms and antibiotics. Since B. burgdorferi first infects skin, the possible protective effect of skin fibroblasts from an antibiotic commonly used to treat Lyme disease, ceftriaxone, was examined. Human foreskin fibroblasts protected B. burgdorferi from the lethal action of a 2-day exposure to ceftriaxone at 1 microgram/mL, 10-20 x MBC. In the absence of fibroblasts, organisms did not survive. Spirochetes were not protected from ceftriaxone by glutaraldehyde-fixed fibroblasts or fibroblast lysate, suggesting that a living cell was required. The ability of the organism to survive in the presence of fibroblasts was not related to its infectivity. Fibroblasts protected B. burgdorferi for at least 14 days of exposure to ceftriaxone. Mouse keratinocytes, HEp-2 cells, and Vero cells but not Caco-2 cells showed the same protective effect. Thus, several eukaryotic cell types provide the Lyme disease spirochete with a protective environment contributing to its long-term survival.

PMID: 1634816, UI: 92340959

Infect Immun 1991 Feb;59(2):671-8

Intracellular localization of Borrelia burgdorferi within human endothelial cells.

Ma Y, Sturrock A, Weis JJ

Department of Pathology, University of Utah School of Medicine, Salt Lake City 84132.

The later stages of infection by the Lyme disease pathogen, Borrelia burgdorferi, are characterized by the persistence of the organism in individuals possessing a strong anti-Borrelia immune response. This suggests that the organism is sequestered in a tissue protected from the immune system of the host or there is a reservoir of the organism residing within the cells of the host. In this report, the ability of B. burgdorferi to gain entrance into human umbilical vein endothelial cells was explored as a model for invasion. Incubation of B. burgdorferi with human umbilical vein endothelial cells at ratios ranging from 200:1 to 5,000:1 resulted in the intracellular localization of 10 to 25% of B. burgdorferi in 24 h. The intracellular location of the spirochetes was demonstrated by the incorporation of radiolabeled B. burgdorferi into a trypsin-resistant compartment and was confirmed by double-immunofluorescence staining which differentiated intracellular from extracellular organisms. Actin-containing microfilaments were required for the intracellular localization, indicating that the host cell participates in the internalization process. Activation of endothelial cells by agents known to increase the expression of several adhesion molecules had no effect on the interaction of B. burgdorferi with the endothelial monolayer. This indicates that the endothelial receptor for B. burgdorferi is constitutively expressed and that internalization is not dependent upon adhesion molecules whose expression is induced by inflammatory mediators. The demonstration of B. burgdorferi within endothelial cells suggest that intracellular localization may be a potential mechanism by which the organism escapes from the immune response of the host and may contribute to persistence of the organism during the later stages of Lyme disease.

PMID: 1987083, UI: 91100043

Microb Pathog 1991 Feb;10(2):137-48

Characterization of Borrelia burgdorferi invasion of cultured endothelial cells.

Comstock LE, Thomas DD

Department of Microbiology and Immunology, Wake Forest University Medical Center, Winston-Salem, North Carolina 27103.

Borrelia burgdorferi can adhere to cultured endothelial cells and penetrate through cell monolayers by passing through intercellular tight junctions and through the host cell cytoplasm. Borrelia burgdorferi strains which were isolated from different sources and areas of the U.S. all demonstrated similar invasive capabilities. Bacterial penetration from the apical to the basal surface of the monolayer was 20 times more efficient than from the basal to the apical surface. Borreliae which were non-viable as a result of either heat treatment or ultraviolet (UV) irradiation showed reduced association with the endothelial cell monolayer and loss of invasive capabilities. Borreliae were able to invade when protein synthesis was inhibited with streptomycin or chloramphenicol. When assays were conducted at 4 degrees C, bacterial penetration of the monolayer was completely inhibited. Treatment of borreliae with proteases affecting outer surface proteins greatly reduced cell association and bacterial invasion.

PMID: 1890951, UI: 91367119

J Clin Neuroophthalmol 1993 Sep;13(3):155-61; discussion 162

First isolation of Borrelia burgdorferi from an iris biopsy.

Preac-Mursic V, Pfister HW, Spiegel H, Burk R, Wilske B, Reinhardt S, Bohmer R

Max-von-Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilian-Universität München, Germany.

The persistence of Borrelia burgdorferi in six patients is described. Borrelia burgdorferi has been cultivated from iris biopsy, skin biopsy, and cerebrospinal fluid also after antibiotic therapy for Lyme borreliosis. Lyme Serology: IgG antibodies to B. burgdorferi were positive, IgM negative in four patients&; in two patients both IgM and IgG were negative. Antibiotic therapy may abrogate the antibody response to the infection as shown by our results. Patients may have subclinical or clinical disease without diagnostic antibody titers. Persistence of B. burgdorferi cannot be excluded when the serum is negative for antibodies against it.

PMID: 8106639, UI: 94149159

Infection 1990 Nov-Dec;18(6):332-41

Persistence of Borrelia burgdorferi and histopathological alterations in experimentally infected animals. A comparison with histopathological findings in human Lyme disease.

Preac Mursic V, Patsouris E, Wilske B, Reinhardt S, Gross B, Mehraein P

Max-von-Pettenkofer-Institut, München, Germany.

Gerbils appear to be susceptible to infection by human isolates of Borrelia burgdorferi; we obtained 100% infection. Isolation of the B. burgdorferi from different organs six months post infection causes a generalized infection thus demonstrating that borreliae persist in these animals for a long period. Spirochetemia was present for 14 days, apparently in two intervals. The Borrelia burgdorferi specific antibody titers increased with time after infection thus indicating the persistence of spirochetes. The intraperitoneal inoculation of the B. burgdorferi to six gerbils of groups A and B induced significant histopathologic changes in most of the major organ systems and their surrounding adipose and fibrous connective tissues. The infiltrates consisted mainly of lymphocytes and histiocytes. Various numbers of plasma cells, eosinophils and high numbers of mast cells were also present. Three further animals which served as controls displayed no histological signs of inflammation in any organ system. No significant differences were noted between the histopathological findings seen in the animals of groups A and B (infected with cells from subcultures no. 25 and with no. 5, respectively). The persistence of B. burgdorferi and the high number of organs involved with slight to severe signs of inflammation in this series can be compared to persistence and to the multiorgan involvement seen in human Lyme disease. Thus gerbils can serve as suitable experimental animals to study the pathogenesis of Lyme disease and the extent of organ damage caused by B. burgdorferi.

PMID: 2076905, UI: 91169644

Infection 1989 Nov-Dec;17(6):355-9

Survival of Borrelia burgdorferi in antibiotically treated patients with Lyme borreliosis.

Preac-Mursic V, Weber K, Pfister HW, Wilske B, Gross B, Baumann A, Prokop J

Neurologische Klinik Grosshadern, München, FR Germany.

The persistence of Borrelia burgdorferi in patients treated with antibiotics is described. The diagnosis of Lyme disease is based on clinical symptoms, epidemiology and specific IgG and IgM antibody titers to B. burgdorferi in serum. Antibiotic therapy may abrogate the antibody response to the infection as shown in our patients. B. burgdorferi may persist as shown by positive culture in MKP-medium; patients may have subclinical or clinical disease without diagnostic antibody titers to B. burgdorferi. We conclude that early stage of the disease as well as chronic Lyme disease with persistence of B. burgdorferi after antibiotic therapy cannot be excluded when the serum is negative for antibodies against B. burgdorferi.

PMID: 2613324, UI: 90129322

Am J Pathol 1985 Jan;118(1):26-34

Lyme arthritis. Spirochetes found in synovial microangiopathic lesions.

Johnston YE, Duray PH, Steere AC, Kashgarian M, Buza J, Malawista SE, Askenase PW

In 17 patients with Lyme disease, synovial specimens, obtained by synovectomy or needle biopsy, showed nonspecific villous hypertrophy, synovial cell hyperplasia, prominent microvasculature, lymphoplasmacellular infiltration, and sometimes lymphoid follicles. The larger surgically obtained specimens also showed striking deposition of fibrin in synovial stroma and a form of endarteritis obliterans. In 2 patients, spirochetes were seen in and around blood vessels by the Dieterle silver stain. Compared with 55 cases of other synovial disease, obliterative microvascular lesions were seen only in Lyme synovia, but marked stromal deposition of fibrin seemed nonspecific. These findings imply that the Lyme spirochete may survive for years in affected synovium and may be directly responsible for the microvascular injury.

PMID: 3966535, UI: 85094500

Antimicrob Agents Chemother 1996 Nov;40(11):2632-2636

In vivo activities of ceftriaxone and vancomycin against Borrelia spp. in the mouse brain and other sites.

Kazragis RJ, Dever LL, Jorgensen JH, Barbour AG

Department of Medicine (Infectious Diseases), University of Texas Health Science Center at San Antonio 78284, USA.

Borrelia burgdorferi, the agent of Lyme disease, and B. turicatae, a neurotropic agent of relapsing fever, are susceptible to vancomycin in vitro, with an MIC of 0.5 microgram/ml. To determine the activity of vancomycin in vivo, particularly in the brain, we infected adult immunocompetent BALB/c and immunodeficient CB-17 scid mice with B. burgdorferi or B. turicatae. The mice were then treated with vancomycin, ceftriaxone as a positive control, or normal saline as a negative control. The effectiveness of treatment was assessed by cultures of blood and brain and other tissues. Ceftriaxone at a dose of 25 mg/kg of body weight administered every 12 h for 7 to 10 days eliminated cultivable B. burgdorferi or B. turicatae from all BALB/c or scid mice in the study. Vancomycin at 30 mg/kg administered every 12 h was effective in eliminating infection from immunodeficient mice if treatment was started within 3 days of the onset of infection. If treatment with vancomycin was delayed for 7 days or more, vancomycin failed to eradicate infection with B. burgdorferi or B. turicatae from immunodeficient mice. The failure of vancomycin in eradicating established infections in immunodeficient mice was associated with the persistence of viable spirochetes in the brain during antibiotic treatment.

PMID: 8913478, UI: 97070552

J Immunol 1993 Feb 1;150(3):909-15

The fate of Borrelia burgdorferi, the agent for Lyme disease, in mouse macrophages. Destruction, survival, recovery.

Montgomery RR, Nathanson MH, Malawista SE

Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06510.

The macrophage is a known reservoir for a number of infectious agents, and is therefore a likely candidate site for persistence of Borrelia burgdorferi, the Lyme spirochete. We report that unopsonized B. burgdorferi enter macrophages rapidly, resulting mainly in degradation but occasionally in apparent intracellular persistence. We studied uptake of spirochetes by macrophages by simultaneously labeling infected cells with antibodies to B. burgdorferi and with sequential components of the endocytic pathway, and we examined optical sections (0.5-1.0 micron in thickness) of these cells by confocal fluorescence microscopy at multiple time points after infection. We found that only 5 min of incubation at 37 degrees C were required for nearly 100% of B. burgdorferi to enter a lysosomal glycoprotein-positive compartment, whereas 60 min were required for 90% of the spirochetes to appear in a cathepsin L-positive compartment under the same conditions. We also labeled infected living cells with acridine orange to distinguish live from killed intracellular organisms. Although the large majority of spirochetes within a given cell were dead, we saw occasional live ones up to 24 h (the longest interval examined) after all extracellular organisms had been lysed in distilled water. Moreover, we can reculture spirochetes from macrophages after infection. Persistence of spirochetes within macrophages provides a possible pathogenetic mechanism for chronic or recurrent Lyme disease in man.

PMID: 8423346, UI: 93139523

Infect Immun 2001, Mar;69(3):1739-46.

Coiling phagocytosis of Borrelia burgdorferi by primary human macrophages is controlled by CDC42Hs and Rac1 and involves recruitment of Wiskott-Aldrich syndrome protein and Arp2/3 complex

Linder S, Heimerl C, Fingerle V, Aepfelbacher M, Wilske B

Institut fur Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Ludwig-Maximilians-Universitat, 80336 Munich, Germany.

Lyme borreliosis is a multisystemic disorder primarily affecting the skin, nervous system, and joints. It is caused by the spirochete Borrelia burgdorferi sensu lato and is transmitted via ticks of the Ixodidae family. Persistence of borreliae within macrophages has been implicated in the often chronic history of borreliosis. The uptake of B. burgdorferi by professional phagocytes occurs predominantly by coiling phagocytosis, a host cell-driven process in which single pseudopods wrap around and engulf the spirochetes. In the present study, we investigated the molecular machinery and the signal transduction pathways controlling the formation of these unique uptake structures. We found that the phagocytosis of borreliae by primary human macrophages is accompanied by the formation of f-actin-rich structures, which in their morphological organization correspond well to the earlier described coiling pseudopods. Further experiments revealed that Wiskott-Aldrich Syndrome protein and Arp2/3 complex, major regulators of actin polymerization, are also recruited to these sites of actin accumulation. In addition, inhibition of an upstream regulator of Wiskott-Aldrich Syndrome protein, the Rho-family GTPase CDC42Hs, greatly inhibited the occurrence of borrelia-induced phagocytic uptake structures. Inhibition of Rac1, another Rho family GTPase, had a less-pronounced inhibitory effect, while blocking of Rho activity showed no discernible influence. These results suggest that basic mechanisms of actin polymerization that control other types of phagocytosis are also functional in the formation of the morphologically unique uptake structures in coiling phagocytosis. Our findings should enhance the understanding of the infection process of B. burgdorferi and contribute to devising new strategies for countering Lyme disease.


Infect Immun. 2004 Jun;72(6):3138-46

Borrelia burgdorferi binds to, invades, and colonizes native type I collagen lattices.

Zambrano MC, Beklemisheva AA, Bryksin AV, Newman SA2, Cabello FC*.

Department of Microbiology and Immunology, 2 Department of Cell Biology and Anatomy, New York Medical College, Valhalla, New York 105952 *Corresponding author. Mailing address: Department of Microbiology and Immunology, New York Medical College, Basic Science Building, Valhalla, NY 10595-1690. Phone: (914) 594-4182. Fax: (914) 594-4176. E-mail: cabello@nymc.edu.

Borrelia burgdorferi binds strongly to the extracellular matrix and cells of the connective tissue, a binding apparently mediated by specific proteins and proteoglycans. We investigated the interactions between B. burgdorferi cells and intact type I collagen using hydrated lattices that reproduce features of in vivo collagen matrices. B. burgdorferi cells of several strains adhered avidly to these acellular matrices by a mechanism that was not mediated by decorin or other proteoglycans. Moreover, following adhesion to these matrices, B. burgdorferi grew and formed microcolonies. The collagen used in these studies was confirmed to lack decorin by immunoblot analysis; B. burgdorferi cells lacking the decorin adhesin bound readily to intact collagen matrices. B. burgdorferi also bound to collagen lattices that incorporated enzymes that degraded glycosaminoglycan chains in any residual proteoglycans. Binding of the bacteria to intact collagen was nonetheless specific, as bacteria did not bind agar and showed only minimal binding to bovine serum albumin, gelatin, pepsinized type I collagen, and intact collagen that had been misassembled under nonphysiological pH and ionic-strength conditions. Proteinase K treatment of B. burgdorferi cells decreased the binding, as did a lack of flagella, suggesting that surface-exposed proteins and motility may be involved in the ability of B. burgdorferi to interact with intact collagen matrices. The high efficiency of binding of B. burgdorferi strains to intact collagen matrices permits replacement of the commonly used isotopic binding assay with visual fluorescent microscopic assays and will facilitate future studies of these interactions.

In summary, we have demonstrated that B. burgdorferi strains bind matrices of type I collagen independently of the presence of decorin and other proteoglycans and of the decorin binding protein on the surfaces of B. burgdorferi cells. We have developed a simple assay to detect the interactions of B. burgdorferi with collagen matrices and generated preliminary evidence that surface receptors, in conjunction with flagellum-mediated motility, may be involved in this interaction. The interactions of B. burgdorferi cells with collagen are a potentially important factor in the ability of the bacterium to target, disseminate within, and colonize host tissues. ...

The notion that burrowing is involved in the B. burgdorferi-collagen interaction gains support from our observation that strain MC-1, which lacks flagella, binds less well than the strains expressing flagella and displaying normal motility, especially on prolonged incubation...

American Journal of Pathology. 2004;165:977-985

Protective Niche for Borrelia burgdorferi to Evade Humoral Immunity.

Liang FT, Brown EL, Wang T, Iozzo RV and Fikrig E

Department of Internal Medicine, Section of Rheumatology, Yale University School of Medicine, New Haven, Connecticut; the Center for Extracellular Matrix Biology, Texas A&M University System Health Science Center, Albert B. Alkek Institute of Biosciences and Technology, Houston, Texas; and the Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania

The Lyme disease spirochete, Borrelia burgdorferi, is an extracellular microbe that causes persistent infection despite the development of strong immune responses against the bacterium. B. burgdorferi expresses several ligand-binding lipoproteins, including the decorin-binding proteins (Dbps) A and B, which may mediate attachment to decorin, a major component of the host extracellular matrix during murine infection. We show that B. burgdorferi was better protected in the joints and skin, two tissues with a higher decorin expression, than in the urinary bladder and heart, two tissues with a lower decorin expression, during chronic infection of wild-type mice. Targeted disruption of decorin alone completely abolished the protective niche in chronically infected decorin-deficient mice but did not affect the spirochete burden during early infection. The nature of protection appeared to be specific because the spirochetes with higher outer surface protein C expression were not protected while the protective niche seemed to favor the spirochetes with a higher dbpA expression during chronic infection. These data suggest that spirochetal DbpA may interact with host decorin during infection and such interactions could be a mechanism that B. burgdorferi uses to evade humoral immunity and establish chronic infection

Int J Med Microbiol Virol Parasitol Infect Dis 1994 Jan;280(3):348-59

Electron microscopy of Langerhans cells and Borrelia burgdorferi in Lyme disease patients.

Hulinska D, Bartak P, Hercogova J, Hancil J, Basta J, Schramlova J

Institute of Public Health, Prague, Czech Republic.

To investigate dermal and epidermal involvement in the presence of Borrelia burgdorferi and to analyze the role of Langerhans cells and keratinocytes, 14 cases of erythema chronicum migrans and two controls were studied by means of electron microscopy, using negative staining and sectioning techniques. Using immunoelectron microscopy and histochemistry, positive results for B. burgdorferi were disclosed in 5 cases of erythema chronicum migrans and 3 cases of neuroborreliosis which were confirmed by cultivation. We cultured 4 stains of B. burgdorferi from the skin, 1 from blood and 2 from cerebrospinal fluid in BSK medium. Near to the centre of erythema chronicum migrans with focal necrosis were both a dissolved basal membrane and keratinocyte desmosomes surrounding damaged B. burgdorferi cells in the epidermis. Markedly oedematous keratinocytes and Langerhans cells with B. burgdorferi were released into lymphocyte infiltrates. At the periphery of all erythema chronicum migrans lesions, keratinocytes were well preserved while all dendritic cells seemed to be vacuolated. Above foci of B. burgdorferi located perivascular or among collagen fibers, Langerhans cells were frequent and more granulated. The possible role of Langerhans cells in the identification and elimination of B. burgdorferi is discussed.

PMID: 8167429, UI: 94220807

J Immunol 1997 Apr 1;158(7):3285-92

Borrelia burgdorferi activates nuclear factor-kappa B and is a potent inducer of chemokine and adhesion molecule gene expression in endothelial cells and fibroblasts.

Ebnet K, Brown KD, Siebenlist UK, Simon MM, Shaw S

Experimental Immunology Branch, National Cancer Institute, Bethesda, MD 20892, USA. ebnetk@uni-muenster.de

Lyme disease, caused by the tick-borne spirochete Borrelia burgdorferi, is a systemic infection with preponderance for the skin, joints, heart, and nervous system. Inflammatory lesions of target organs are characterized by the presence of spirochetes and inflammatory leukocytes. We have analyzed the potential of B. burgdorferi to induce gene expression of chemokines and adhesion molecules in human endothelial cells, keratinocytes, and fibroblasts. We find induction of the chemokines RANTES (regulated upon activation, normal T cells expressed and secreted), monocyte chemoattractant protein-1, IL-8, gro-alpha, IFN-inducible protein-10, and mig (monokine induced by gamma-IFN), and of the adhesion molecules E-selectin, ICAM-1, and VCAM-1 in endothelial cells and induction of the same chemokines and ICAM-1 in fibroblasts. This is mediated by the lipid moiety of the outer surface lipoprotein A. Induction of chemokine and adhesion molecule genes by B. burgdorferi occurs rapidly and does not require new protein synthesis. Induction is blocked by inhibitors of nuclear factor (NF)-kappa B. We also find that B. burgdorferi induces nuclear translocation of NF-kappa B and a transient increase in the expression of its inhibitor I kappa B-alpha. These findings indicate that B. burgdorferi is a potent inducer of molecules required for leukocyte recruitment to inflammatory foci, and the data suggest that this biologic activity is due to the ability of the spirochetes to activate the pleiotropic transcription factor NF-kappa B.

PMID: 9120285, UI: 97240772

Ann N Y Acad Sci 1996 Oct 25;797:107-17

Regulation of chemokine gene expression in human endothelial cells by proinflammatory cytokines and Borrelia burgdorferi.

Ebnet K, Simon MM, Shaw S

Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.

Chemokines play a central role in the process of leukocyte recruitment to tissues. By their chemotactic activity they guide leukocytes to the site of infection/injury. Chemokines have been suggested to trigger firm adhesion of leukocytes to activated endothelial cells as well as the subsequent diapedesis. For these functions, chemokines produced by EC are particularly well suited. Our experiments with proinflammatory stimuli demonstrate that chemokines are induced in EC by a variety of stimuli including inflammatory cytokines and bacterial structures such as LPS and preparations of B. burgdorferi. The induction of chemokines by all of these agents occurs rapidly and does not require new protein synthesis. Two chemokines, MCP-1 and IL-8, respond to very low doses (0.1-1 U/ml) of proinflammatory cytokines which is important at the beginning of an immune response when soluble inflammatory mediators might still be limiting. The chemokines RANTES, IP-10, and mig show synergistic induction by low doses (1 U/ml) of several inflammatory mediators, which again is important when only limiting amounts of inflammatory stimuli are present. The upregulation of six chemokine genes as well as genes encoding adhesion molecules in two cell types, EC and fibroblasts, by B. burgdorferi suggests that chemokines might play a central role in the regulation of spirochete-induced inflammatory responses and the subsequent immune responses. Recent evidence suggests that T cells with pathogenic potential contribute to chronic inflammation at the late stage of Lyme disease. Therefore, the use of therapeutic agents that block chemokine activity might be useful in treating chronic Lyme arthritis.

PMID: 8993355, UI: 97146499

Infect Immun 1998 May;66(5):1946-52

Integrins alpha(beta3) and alpha5beta1 mediate attachment of lyme disease spirochetes to human cells.

Coburn J, Magoun L, Bodary SC, Leong JM

Division of Rheumatology and Immunology, Tufts-New England Medical Center, Boston, Massachusetts 02111, USA. jcoburn_bor@opal.tufts.edu

Borrelia burgdorferi (sensu lato), the agent of Lyme disease, is able to cause chronic, multisystemic infections in human and animal hosts. Attachment of the spirochete to host cells is likely to be important for the colonization of diverse tissues. The platelet-specific integrin alpha(IIb)beta3 was previously identified as a receptor for all three species of Lyme disease spirochetes (B. burgdorferi sensu stricto, B. garinii, and B. afzelii). Here we show that B. burgdorferi also recognizes the widely expressed integrins alpha(v)beta3 and alpha5beta1, known as the vitronectin and fibronectin receptors, respectively. Three representatives of each species of Lyme disease spirochete were tested for the ability to bind to purified alpha(v)beta3 and alpha5beta1. All of the strains tested bound to at least one integrin. Binding to one integrin was not always predictive of binding to other integrins, and several different integrin preference profiles were identified. Attachment of the infectious B. burgdorferi strain N40 to purified alpha(v)beta3 and alpha5beta1 was inhibited by RGD peptides and the appropriate receptor-specific antibodies. Binding to alpha(v)beta3 was also shown by using a transfected cell line that expresses this receptor but not alpha(IIb)beta3. Attachment of B. burgdorferi N40 to human erythroleukemia cells and to human saphenous vein endothelial cells was mediated by both alpha5beta1 and alpha(v)beta3. Our results show that multiple integrins mediate attachment of Lyme disease spirochetes to host cells.

PMID: 9573074, UI: 98234018

Infect Immun 1998 Mar;66(3):994-9

Different classes of proteoglycans contribute to the attachment of Borrelia burgdorferi to cultured endothelial and brain cells.

Leong JM, Wang H, Magoun L, Field JA, Morrissey PE, Robbins D, Tatro JB, Coburn J, Parveen N

Department of Molecular Genetics and Microbiology, University of Massachusetts Medical Center, Worcester 01655, USA. john.leong@banyan.ummed.edu

The Lyme disease spirochete, Borrelia burgdorferi, infects multiple tissues, such as the heart, joint, skin, and nervous system and has been shown to recognize heparan sulfate and dermatan sulfate proteoglycans. In this study, we examined the contribution of different classes of proteoglycans to the attachment of the infectious B. burgdorferi strain N40 to several immortalized cell lines and primary cultured cells, including endothelial cells and brain cells. Bacterial attachment was inhibited by exogenous proteoglycans or by treatment of host cells with inhibitors of proteoglycan synthesis or sulfation, indicating that proteoglycans play a critical role in bacterial binding to diverse cell types. Binding to primary bovine capillary endothelial cells or a human endothelial cell line was also inhibited by digestion with heparinase or heparitinase but not with chondroitinase ABC. In contrast, binding to glial cell-enriched brain cell cultures or to a neuronal cell line was inhibited by all three lyases. Binding of strain N40 to immobilized heparin could be completely inhibited by dermatan sulfate, and conversely, binding to dermatan sulfate could be completely blocked by heparin. As measured by 50% inhibitory dose, heparin was a better inhibitor of binding than dermatan sulfate, regardless of whether the substrate was heparin or dermatan sulfate. These results are consistent with the hypotheses that the species of proteoglycans recognized by B. burgdorferi vary with cell type and that bacterial recognition of different proteoglycans is mediated by the same bacterial molecule(s).

PMID: 9488387, UI: 98147711

Infect Immun 1995 Nov;63(11):4439-47

Borrelia burgdorferi upregulates expression of adhesion molecules on endothelial cells and promotes transendothelial migration of neutrophils in vitro.

Sellati TJ, Burns MJ, Ficazzola MA, Furie MB

Department of Pathology, School of Medicine, State University of New York at Stony Brook 11794-8691, USA.

The accumulation of leukocytic infiltrates in perivascular tissues is a key step in the pathogenesis of Lyme disease, a chronic inflammatory disorder caused by Borrelia burgdorferi. During an inflammatory response, endothelial cell adhesion molecules mediate the attachment of circulating leukocytes to the blood vessel wall and their subsequent extravasation into perivascular tissues. Using cultured human umbilical vein endothelial cells (HUVEC) in a whole-cell enzyme-linked immunosorbent assay, we demonstrated that B. burgdorferi activated endothelium in a dose- and time-dependent fashion as measured by upregulation of the adhesion molecules E-selectin, vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1). As few as one spirochete per endothelial cell stimulated increased expression of these molecules. Expression of E-selectin peaked after spirochetes and HUVEC were coincubated for 4 h and returned to near-basal levels by 24 h. In contrast, expression of VCAM-1 and ICAM-1 peaked at 12 h and remained elevated at 24 h. HUVEC monolayers cultured on acellular amniotic tissue were used to investigate the consequences of endothelial cell activation by spirochetes. After incubation of HUVEC-amnion cultures with B. burgdorferi, subsequently added neutrophils migrated across the endothelial monolayers. This process was mediated by E-selectin and by CD11/CD18 leukocytic integrins. The extent of migration depended on both the number of spirochetes used to stimulate the HUVEC and the length of the coincubation period. These results raise the possibility that B. burgdorferi induces a host inflammatory response and accompanying perivascular damage through activation of vascular endothelium.

PMID: 7591083, UI: 96029742

Infect Immun 1995 Jul;63(7):2478-84

Borrelia burgdorferi binds plasminogen, resulting in enhanced penetration of endothelial monolayers.

Coleman JL, Sellati TJ, Testa JE, Kew RR, Furie MB, Benach JL

State of New York Department of Health, Stony Brook, USA.

Several strains of Borrelia burgdorferi and Borrelia hermsii can bind human Lys-plasminogen specifically. Affinity blots using 125I-labeled plasminogen showed that numerous polypeptides of all the strains and species tested could bind via lysine residues to the plasminogen molecule since binding could be completely inhibited by the lysine analog epsilon-aminocaproic acid. Binding analysis using 125I-labeled plasminogen on live intact organisms showed that the organisms possess two binding sites for plasminogen: a high-affinity site with a Kd of 24 +/- 12 pM and 106 +/- 14 binding sites per spirochete and a low-affinity site with a Kd of 20 +/- 4 nM and 2,683 +/- 36 binding sites per spirochete. Indirect immunofluorescence and confocal microscopy showed a generalized but punctate pattern of plasminogen binding to the spirochete surface. Exogenously provided urokinase-type plasminogen activator converted B. burgdorferi surface-bound plasminogen to enzymatically active plasmin as demonstrated by the breakdown of the chromogenic plasmin substrate S2251. Plasmin-coated organisms showed an enhanced ability to penetrate endothelial cell monolayers grown on connective tissue substrates compared to untreated controls (P < 0.001). This functional assay demonstrated that enzymatically active plasmin on the surface of spirochetes can lead to greater invasion of tissues.

PMID: 7790059, UI: 95310000

J Infect Dis 1995;171:1258-65

Binding of human plasminogen and urokinase-type plasminogen activator to the Lyme disease spirochete, Borrelia burgdorferi.

Klempner MS, Noring R, Epstein MP, McCloud B, Hu R, Limentani SA, Rogers RA

Department of Medicine, New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts, USA.

Many bacteria that spread in the skin produce enzymes that digest extracellular matrix components. Borrelia burgdorferi spreads from a skin inoculation site to form the characteristic erythema migrans skin lesion. It was determined that B. burgdorferi does not produce collagenase, elastase, hyaluronidase, or other enzymes that digest extracellular matrix components. However, B. burgdorferi bound human plasmin, plasminogen (Pgn), and urokinase-type plasminogen activator (uPA). When spirochetes were sequentially incubated with Pgn and uPA, bioactive plasmin was generated on the surface of B. burgdorferi. B. burgdorferi did not produce an endogenous Pgn activator. Fluorochrome-conjugated uPA and Pgn colocalized to the terminus of the spirochete. In a mouse model, uPA-treated B. burgdorferi were more infectious than control spirochetes. Binding of host uPA and Pgn to form a bioactive extracellular matrix protease on B. burgdorferi represents a mechanism that could facilitate dissemination and localization of spirochetes to sites of vascular injury.

UI: 95271064

Cell Adhes Commun 1994 Jun;2(2):145-57

Borrelia burgdorferi upregulates the adhesion molecules E-selectin, P-selectin, ICAM-1 and VCAM-1 on mouse endothelioma cells in vitro.

Boggemeyer E, Stehle T, Schaible UE, Hahne M, Vestweber D, Simon MM

Max-Planck-Institut fur Immunbiologie, Freiburg, Germany.

In order to obtain more information on processes leading to Borrelia burgdorferi-induced inflammation in the host, we have developed an in vitro model to study the upregulation of cell surface expression of adhesion molecules on endothelial cells by spirochetes. A mouse endothelioma cell line, derived from brain capillaries, bEnd3, was used as indicator population. bEnd3 cells were incubated with preparations of viable, inactivated or sonicated spirochetes and the expression of E-selectin, P-selectin, ICAM-1 and VCAM-1 was monitored by immunocytochemistry and quantified by cell surface ELISA. We show that all three spirochetal preparations are able to upregulate cell surface expression of E-selectin, P-selectin, ICAM-1 and VCAM-1 on bEnd 3 cells in a dose-dependent manner. The kinetics of cell surface expression of the individual adhesion molecules in the presence of Borrelia burgdorferi showed maxima at about 50 h of incubation or later; this was distinct from results obtained with sonicated-preparations of Escherichia coli bacteria or with enterobacterial LPS where peak expression was observed between 4 h and 16 h. The fact that Borrelia burgdorferi does not contain conventional LPS suggests that the mode of induction of adhesion molecules on endothelial cells is influenced by the phenotype of bacteria. At the peak of spirochete-induced cell surface expression of adhesion molecules (approximately 50 h), bEnd3 cells were found to bind cells of a VLA-4+ B lymphoma line (L1-2) much more efficiently than untreated control cells. The binding of L1-2 cells to presensitized bEnd3 cells was significantly inhibited (more than 75%) in the presence of monoclonal antibodies to both VLA-4 and its endothelial counterreceptor VCAM-1. These findings demonstrate that Borrelia burgdorferi organisms are able to induce functionally active adhesion molecules on endothelial cells in vitro and suggest that E-selectin, P-selectin, ICAM-1 and VCAM-1 play an important role in the pathogenesis of spirochetal infection.

PMID: 7521760, UI: 94363407

Cell Adhes Commun 1994 Dec;2(6):465-79

Published erratum appears in Cell Adhes Commun 1995 May;3(2):following 177

Expression of endothelial cell adhesion molecules in joints and heart during Borrelia burgdorferi infection of mice.

Schaible UE, Vestweber D, Butcher EG, Stehle T, Simon MM

Max-Planck-Institut fur Immunbiologie, Freiburg, Germany.

The expression of adhesion molecules on endothelia was examined during chronic arthritis and carditis in SCID and immunocompetent susceptible AKR/N mice infected with Borrelia burgdorferi (B. burgdorferi). All stages of disease were associated with the upregulation or new expression of ICAM-1 and P-selectin and of VCAM-1 and E-selectin, respectively, on blood vessels of affected joint tissues of SCID and AKR/N mice as well as on heart tissue of SCID mice but not in other tissues. Moreover, ICAM-1 was also found on infiltrating mononuclear cells. The overall staining intensity for each of the four adhesion molecules on individual tissue sections of joint and heart increased with time of infection and was associated with the presence of spirochetes in the tissue. In addition it is shown that in both mouse strains inflammation of joints but not heart is accompanied by vascular proliferation. Synovial but not heart tissues of infected SCID mice were found to express both peripheral- (PNAd) and mucosal (MAdCAM-1) lymph node high endothelia venule associated vascular addressins as detected by mAb Meca-79 and Meca-367, respectively, but only at later stages of the disease and only on newly generated small venules. However, neither of the two addressins were evident in synovial lesions of AKR/N mice. Together the data suggest that the concomittant induction of ICAM-1, VCAM-1, E-selectin and P-selectin in lesions of infected mice provide a means for enhanced cellular infiltration into affected organs and that the regulation of these structures is conserved in the absence of a functional immune system. Furthermore, the differential induction of vascular proliferation in joint and heart tissues as well as the restricted expression patterns of vascular addressins indicate that the pathogenetic processes induced by B. burgdorferi are distinct for joint and heart.

PMID: 7538017, UI: 95261709

Infect Immun 1993 Feb;61(2):423-31

Published erratum appears in Infect Immun 1993 Apr;61(4):1599

A monoclonal antibody to OspA inhibits association of Borrelia burgdorferi with human endothelial cells.

Comstock LE, Fikrig E, Shoberg RJ, Flavell RA, Thomas DD

Department of Microbiology and Immunology, Bowman Gray School of Medicine, Wake Forest University Medical Center, Winston-Salem, North Carolina 27103.

Previously, it has been shown that polyclonal antibodies to Borrelia burgdorferi and some monoclonal antibodies (MAbs) to borrelia major surface proteins caused inhibition of adherence of the bacteria to cultured human umbilical vein endothelial (HUVE) cells. In this study, fragment antigen binding (Fab) molecules generated from the immunoglobulin G fraction of rabbit anti-recombinant OspA serum were found to inhibit the adherence of B. burgdorferi to HUVE cells by 73%. Subsequently, MAbs were generated for use in determining whether or how B. burgdorferi outer surface proteins (Osps) A and/or B are involved in mediating attachment to, and/or invasion of, HUVE cells by B. burgdorferi. Twenty-two MAbs were generated to borrelial proteins with apparent molecular masses (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of 19, 31 (OspA), 34 (OspB), and 35 kDa. Fab molecules from one anti-OspA MAb, 9B3D, demonstrated an inhibitory effect on bacterial association with HUVE cells. None of the other MAbs, including the other anti-OspA MAbs, showed an inhibitory effect on cell association of greater than 5%. This effect of Fab 9B3D was concentration dependent and plateaued at approximately 6 micrograms of Fab per ml (nearly 80% inhibition of the bacterial association with the monolayer). Penetration assays and cell association experiments performed by using immunofluorescence also suggested that the inhibitory action of 9B3D occurs at the level of adherence. MAb 9B3D recognized the OspA of every North American strain tested (n = 19) but only 3 [corrected] of 20 strains from western Europe, Russia, and Japan, suggesting that the North American strains and strains from other parts of the world may use different molecules and/or different OspA epitopes to interact with endothelial cells. Immunoblots of Escherichia coli expressing different OspA fusion peptides suggested that the 9B3D epitope resides in the carboxy-terminal half of OspA. MAb 9B3D promises to be a valuable tool for elucidating the domain or domains of OspA involved in the endothelial cell cytadherence of North American strains of B. burgdorferi.

PMID: 7678585, UI: 93138760

Med Microbiol Immunol (Berl) 1994 Dec;183(6):325-41

Taxonomic classification of 29 Borrelia burgdorferi strains isolated from patients with Lyme borreliosis: a comparison of five different phenotypic and genotypic typing schemes.

Lebech AM, Hansen K, Wilske B, Theisen M

Department of Infection-Immunology, Statens Seruminstitut, Copenhagen, Denmark.

Twenty-nine European and North American Borrelia burgdorferi strains isolated from patients with Lyme borreliosis, were investigated by restriction fragment length polymorphism (RFLP) of two phylogenetically highly conserved chromosomal genes encoding flagellin (fla) and the p60 common antigen (CA), as well as of the plasmid-borne outer surface protein A (ospA) gene. RFLP of the ospA, fla and CA gene revealed five, two and four distinct subspecies-specific patterns, respectively. RFLP classification of the B. burgdorferi strains was compared with four different classification schemes proposed by others: (i) molecular mass profile of OspA and OspB (Adam et al. [1]);(ii) OspA serotyping (Wilske et al. [34]);(iii) genomic fingerprinting on the central region of the B. burgdorferi fla gene (Picken [24]) and (iv) 16S rRNA signature nucleotide analysis (Marconi and Garon [19]). Results obtained with the different methods correlated highly. All strains classified as B. burgdorferi sensu stricto and B. afzelii could be unequivocally identified as one distinct group by all five typing methods. B. garinii isolates, however, were more heterogeneous and according to RFLP of the CA and ospA gene fell into either two or three subgroups. The agreement of the different approaches supports the recent concept that B. burgdorferi sensu lato strains should be delineated to three genomic groups and that B. burgdorferi sensu lato is clonal. All 12 US strains were B. burgdorferi sensu stricto, whereas the 17 European isolates belonged to any of three genospecies. Among European B. burgdorferi isolates there was an association between B. burgforferi genospecies and the clinical manifestation of Lyme borreliosis. B. afzelii strains were found to predominate in 11 skin isolates (75%), whereas all 6 cerebrospinal fluid isolates from patients with neuroborreliosis were B. garinii. These findings support the concept of a strain-dependent organotropism of B. burgdorferi.

PMID: 7541107, UI: 95319379

Infect. Immun.1997 Nov; 65(11):4384-4388

Borrelia burgdorferi induces chemokines in human monocytes

H Sprenger, A Krause, A Kaufmann, S Priem, D Fabian, GR Burmester, D Gemsa and MG Rittig

Institute of Immunology, Philipps University Marburg, Germany.

Lyme disease is clinically and histologically characterized by strong inflammatory reactions that contrast the paucity of spirochetes at lesional sites, indicating that borreliae induce mechanisms that amplify the inflammatory response. To reveal the underlying mechanisms of chemoattraction and activation of responding leukocytes, we investigated the induction of chemokines in human monocytes exposed to Borrelia burgdorferi by a dose-response and kinetic analysis. Lipopolysaccharide (LPS) derived from Escherichia coli was used as a positive control stimulus. The release of the CXC chemokines interleukin-8 (IL-8) and GRO-alpha and the CC chemokines MIP-1alpha, MCP-1, and RANTES was determined by specific enzyme-linked immunosorbent assays, and the corresponding gene expression patterns were determined by Northern blot analysis. The results showed a rapid and strong borrelia-inducible gene expression which was followed by the release of chemokines with peak levels after 12 to 16 h. Spirochetes and LPS were comparably effective in stimulating IL-8, GRO-alpha, MCP- 1, and RANTES expression, whereas MIP-1alpha production preceded and exceeded chemokine levels induced by LPS. Unlike other bacteria, the spirochetes themselves did not bear or release factors with intrinsic chemotactic activity for monocytes or neutrophils. Thus, B. burgdorferi appears to be a strong inducer of chemokines which may, by the attraction and activation of phagocytic leukocytes, significantly contribute to inflammation and tissue damage observed in Lyme disease.

Infect Immun 1998 Sep;66(9):4557-9

E and P selectins are not required for resistance to severe murine lyme arthritis.

Seiler KP, Ma Y, Weis JH, Frenette PS, Hynes RO, Wagner DD, Weis JJ

Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah, USA.

Borrelia burgdorferi-induced arthritis in mice is characterized by tendonitis, synovitis, and inflammatory-cell infiltrate, predominantly of neutrophils. Because genetic deficiency in E and P selectins results in delayed recruitment of neutrophils to sites of inflammation, mice with this deficiency were tested for their response to infection with B. burgdorferi. E and P selectins were not required for the control of B. burgdorferi numbers, nor did deficiency in E and P selectins result in alteration of arthritis severity.

PMID: 9712820, UI: 98380417


J Clin Invest 1991 Jul;88(1):82-92

A flagella-less mutant of Borrelia burgdorferi. Structural, molecular, and in vitro functional characterization.

Sadziene A, Thomas DD, Bundoc VG, Holt SC, Barbour AG

Department of Medicine, University of Texas Health Science Center, San Antonio 78284.

A nonmotile mutant of Borrelia burgdorferi, the etiologic agent of Lyme disease, was isolated and characterized. The mutant was compared with the wild-type predecessor as well as with a motile back-revertant of the same genetic background. The mutant lacked, by morphologic, biochemical, and immunologic criteria, the major structural protein of flagella, flagellin. This mutation was not associated with major DNA rearrangements or with failure of transcription. An apparent consequence of a loss of flagella was reduced ability to penetrate human endothelial cell layers in vitro. In another assessment of functional significance, the flagella-less mutant was equal if not superior to flagella-bearing, isogenic isolates when examined in an enzyme-linked immunosorbent assay for anti-B. burgdorferi antibodies in the sera of Lyme disease patients. These studies of a mutant, the first among pathogenic Borrelia spp. to be characterized, indicate that the flagellum and motility it confers play a role in B. burgdorferi's invasion of human tissues. A flagella-less B. burgdorferi may be useful as the basis of a more specific immunoassay and a vaccine for protection against Lyme disease.

PMID: 2056133, UI: 91277311

Infect Immun 1998 Aug;66(8):3689-97

Kinetics and in vivo induction of genetic variation of vlsE in Borrelia burgdorferi.

Zhang JR, Norris SJ

Department of Pathology and Laboratory Medicine and Department of Microbiology and Molecular Genetics, University of Texas Medical School at Houston, Houston, Texas 77030, USA.

The Lyme disease agent, Borrelia burgdorferi, is able to persistently infect humans and animals for months or years in the presence of an active immune response. It is not known how the organisms survive immune attack in the mammalian host. vlsE, a gene localized near one end of linear plasmid lp28-1 and encoding a surface-exposed lipoprotein in B. burgdorferi B31, was shown recently to undergo extensive genetic and antigenic variation within 28 days of initial infection in C3H/HeN mice. In this study, we examined the kinetics of vlsE sequence variation in C3H/HeN mice at 4, 7, 14, 21, and 28 days and at 7 and 12 months postinfection. Sequence changes were detected by PCR amplification and sequence analysis as early as 4 days postinfection and accumulated progressively in both C3H/HeN and CB-17 severe combined immunodeficient (SCID) mice throughout the course of infection. The sequence changes were consistent with sequential recombination of segments from multiple silent vls cassette sites into the vlsE expression site. No vlsE sequence changes were detected in organisms cultured in vitro for up to 84 days. These results indicate that vlsE recombination is induced by a factor(s) present in the mammalian host, independent of adaptive immune responses. The possible inducing conditions appear to be present in various tissue sites because isolates from multiple tissues showed similar degrees of sequence variation. The rate of accumulation of predicted amino acid changes was higher in the immunologically intact C3H/HeN mice than in SCID mice, a finding consistent with immune selection of VlsE variants.

Res Microbiol 1992 Jul-Aug;143(6):583-96

Antigenic variation and strain heterogeneity in Borrelia spp.

Wilske B, Barbour AG, Bergstrom S, Burman N, Restrepo BI, Rosa PA, Schwan T, Soutschek E, Wallich R

Max von Pettenkofer Institut fur Hygiene and Medizinische Mikrobiologie, Universitat München, Germany.

Antigenic variation and strain heterogeneity have been demonstrated for the pathogenic Borrelia species, i. e. B. burgdorferi and the relapsing fever borreliae. In relapsing fever, new borrelia serotypes emerge at a high rate spontaneously, a mechanism that is caused by DNA rearrangements on linear plasmid translocating genes coding for variable major proteins from previous silent to expression sites (i. e. from inner sites to telomeric sites of the plasmid). As a result of this variation, the borreliae escape the immune response of the host, thus leading to the relapse phenomenon. In B. burgdorferi, which is the causative agent of the multisystem disorder Lyme borreliosis, there is also a growing body of findings that antigenic variation is involved in pathogenesis of the disease. Phenotypic variation of strains in vitro concerns the size and the amount of surface-associated proteins (OspA, OspB and pC). There are indications that OspA and OspB truncations are due to deletions within the ospAB operon caused by recombination events, and that OspA/OspB-less mutants lack the 49-kb plasmid that bears the ospAB operon. With the increasing number of isolates obtained from various geographic and biological sources, it became apparent that B. burgdorferi is immunologically and genetically more heterogeneous, as previously believed. The major outer surface proteins OspA and OspB (which have been efficient antigens in vaccine studies) are heterogeneous at a genetic level. The same degree of genetic non-identity was observed for the pC protein. Other proteins like flagellin and the highly specific immunodominant p100 range protein show a lower degree of non-identity. Recombinant OspA, pC, p100 range protein and flagellin have been hyperexpressed in E. coli and these proteins are immunologically reactive. This allows further research for development of vaccines and diagnostic tools. B. burgdorferi isolates have been investigated with genotyping (DNA hybridization, PCR and 16S rRNA analysis) as well as serotyping by various authors. Comparison of the different methods has shown good agreement when the same strains have been investigated. No correlation could be found between different phenotypic and genotypic groups with respect to the ability to cause arthritis in SCID mice. A serotyping system based on immunological differences in OspA detected by a panel of monoclonal antibodies has been proposed. Serotyping a large number of B. burgdorferi isolates has shown a striking predominance of the OspA serotype 2 among European isolates from human skin, in contrast to isolates from ticks or CSF.

Publication Types:

PMID: 1475519, UI: 93117533


Pol Merkuriusz Lek. 2005 Jan;18(103):115-9.

[Atypical forms of Borrelia burgdorferi-clinical consequences]

Zajkowska J, Hermanowska-Szpakowicz T, Rubel J.

Klinika Chorob Zakaznych i Neuroinfekcji Akademii Medycznej w Bialymstoku.

Borrelia burgdorferi utilizes a variety of mechanisms to counteract eradication by its host and establish chronic infection. We discuss several of these mechanisms, including plasmid encoded genes, morphologic variants, cysts formation, colonies formation, antigenic variation, and resistance to iron deprivation. These mechanisms, as well as the possible survival of Borrelia burgdorferi in forms with low metabolic activity, may explain relapsing Lyme disease, and may, also account for the difficulties with eradicating this pathogen.

PMID: 15859564 [PubMed - in process]

Infect Immun. 2001 Jun;69(6):3685-91.

Complement evasion by Borrelia burgdorferi: serum-resistant strains promote C3b inactivation.

Alitalo A, Meri T, Ramo L, Jokiranta TS, Heikkila T, Seppala IJ, Oksi J, Viljanen M, Meri S.

Department of Bacteriology and Immunology, Haartman Institute and HUCH Laboratory Diagnostics, FIN-00014 University of Helsinki, Finland.

The most characteristic features of the Lyme disease pathogens, the Borrelia burgdorferi sensu lato (s.l.) group, are their ability to invade tissues and to circumvent the immune defenses of the host for extended periods of time, despite elevated levels of borrelia-specific antibodies in serum and other body fluids. Our aim in the present study was to determine whether B. burgdorferi is able to interfere with complement (C) at the level of C3 by accelerating C3b inactivation and thus to inhibit the amplification of the C cascade. Strains belonging to different genospecies (Borrelia garinii, B. burgdorferi sensu stricto, and Borrelia afzelii) were compared for their sensitivities to normal human serum and abilities to promote factor I-mediated C3b degradation. B. burgdorferi sensu stricto and B. afzelii strains were found to be serum resistant. When the spirochetes were incubated with radiolabeled C3b, factor I-mediated degradation of C3b was observed in the presence of C-resistant B. afzelii (n = 3) and B. burgdorferi sensu stricto (n = 1) strains but not in the presence of C-sensitive B. garinii (n = 7) strains or control bacteria (Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis). Immunoblotting and radioligand binding analyses showed that the C-resistant strains had the capacity to acquire the C inhibitors factor H and factor H-like protein 1 (FHL-1) from growth medium and human serum. A novel surface protein with an apparent molecular mass of 35 kDa was found to preferentially bind to the N terminus region of factor H. Thus, the serum-resistant B. burgdorferi s.l. strains can circumvent C attack by binding the C inhibitors factor H and FHL-1 to their surfaces and promoting factor I-mediated C3b degradation.

PMID: 11349031 [PubMed - indexed for MEDLINE]

Infection and Immunity, October 2004, p. 5759-5767, Vol. 72, No. 10

Borrelia burgdorferi Changes Its Surface Antigenic Expression in Response to Host Immune Responses.

Liang FT 1, Yan J1, Mbow ML2, Sviat SL3, Gilmore RD3, Mamula M1, Fikrig E 3(*)

1 Section of Rheumatology, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut,
2 Centocor, Inc., Malvern, Pennsylvania,
3 Bacterial Zoonoses Branch, Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado
* Corresponding author. Mailing address: Section of Rheumatology, Department of Internal Medicine, Yale University School of Medicine, S525A, 300 Cedar St., New Haven, CT 06520-8031. Phone: (203) 785-2453. Fax: (203) 785-7053. E-mail: erol.fikrig@yale.edu.

The Lyme disease spirochete, Borrelia burgdorferi, causes persistent mammalian infection despite the development of vigorous immune responses against the pathogen. To examine spirochetal phenotypes that dominate in the hostile immune environment, the mRNA transcripts of four prototypic surface lipoproteins, decorin-binding protein A (DbpA), outer surface protein C (OspC), BBF01, and VlsE, were analyzed by quantitative reverse transcription-PCR under various immune conditions. We demonstrate that B. burgdorferi changes its surface antigenic expression in response to immune attack. dbpA expression was unchanged while the spirochetes decreased ospC expression by 446 times and increased BBF01 and vlsE expression up to 20 and 32 times, respectively, under the influence of immune pressure generated in immunocompetent mice during infection. This change in antigenic expression could be induced by passively immunizing infected severe combined immunodeficiency mice with specific Borrelia antisera or OspC antibody and appears to allow B. burgdorferi to resist immune attack.

The Journal of Experimental Medicine2002 February,195(4),415-422

An Immune Evasion Mechanism for Spirochetal Persistence in Lyme Borreliosis

Liang FT, Jacobs MB, Bowers LC, Philipp MT

Department of Parasitology, Tulane Regional Primate Research Center, Tulane University Health Sciences Center, Covington, LA 70433
Address correspondence to Mario T. Philipp, Tulane Regional Primate Research Center, Tulane University Health Sciences Center, 18703 Three Rivers Rd., Covington, LA 70433. Phone: 985-871-6221; Fax: 985-871-6390; E-mail: philipp@tpc.tulane.edu

view this abstract on the The Journal of Experimental Medicine website

Borrelia burgdorferi, the Lyme disease spirochete, persistently infects mammalian hosts despite the development of strong humoral responses directed against the pathogen. Here we describe a novel mechanism of immune evasion by B. burgdorferi. In immunocompetent mice, spirochetes that did not express ospC (the outer-surface protein C gene) were selected within 17 d after inoculation, concomitantly with the emergence of anti-OspC antibody. Spirochetes with no detectable OspC transcript that were isolated from immunocompetent mice reexpressed ospC after they were either cultured in vitro or transplanted to naive immunocompetent mice, but not in OspC-immunized mice. B. burgdorferi persistently expressed ospC in severe combined immune-deficient (SCID) mice. Passive immunization of B. burgdorferiinfected SCID mice with an anti-OspC monoclonal antibody selectively eliminated ospC -expressing spirochetes but did not clear the infection. OspC-expressing spirochetes reappeared in SCID mice after the anti-OspC antibody was eliminated. We submit that selection of surface-antigen nonexpressers is an immune evasion mechanism that contributes to spirochetal persistence.

C Rockefeller University Press, 0022-1007/2002/2/415/ $5.00

Infect Immun 2001 Jun;69(6):4146-53

Distinct regulatory pathways control expression of Borrelia burgdorferi infection-associated OspC and Erp surface proteins.

Babb K, El-Hage N, Miller JC, Carroll JA, Stevenson B

Department of Microbiology and Immunology, University of Kentucky College of Medicine, Lexington 40536-0298, USA

Deciphering the mechanisms by which Borrelia burgdorferi controls the synthesis of proteins associated with mammalian infection will be an important step toward understanding the pathogenic properties of Lyme disease-causing bacteria. We present results of studies indicating that B. burgdorferi senses a wide variety of environmental stimuli, including soluble chemicals, which enables it to independently control synthesis of the Erp and OspC proteins.

Regulation of OspC and Erp expression appears to occur at the level of transcription. In this regard, we observed that one or more DNA-binding proteins interact specifically with erp promoter DNA but not with the ospC promoter.

PMID: 11349090 [PubMed - indexed for MEDLINE]

Antimicrob Agents Chemother 1992 Aug;36(8):1788-1790

In vitro susceptibilities of Borrelia burgdorferi to five oral cephalosporins and ceftriaxone.

Agger WA, Callister SM, Jobe DA

Microbiology Research Laboratory, Gundersen Medical Foundation, Las Crosse, Wisconsin 54601.

We determined the in vitro susceptibilities of eight Borrelia burgdorferi isolates to five oral cephalosporins. MICs for B. burgdorferi 297 were 23 micrograms/ml (cephalexin), 45 micrograms/ml (cefadroxil), 91 micrograms/ml (cefaclor), 0.13 microgram/ml (cefuroxime), 0.8 microgram/ml (cefixime), and 0.02 microgram/ml (ceftriaxone). When B. burgdorferi isolates were exposed to concentrations twice the MIC of cefuroxime, cefixime, or ceftriaxone, at least 72 h of incubation was required to kill 99% of the organisms.

PMID: 1416868, UI: 93037309

Infection 1996 Jan-Feb;24(1):9-16

Kill kinetics of Borrelia burgdorferi and bacterial findings in relation to the treatment of Lyme borreliosis.

Preac Mursic V, Marget W, Busch U, Pleterski Rigler D, Hagl S

Max v. Pettenkofer Institut, Ludwig-Maximilians-Universitat München, Germany.

For a better understanding of the persistence of Borrelia burgdorferi sensu lato (s. l. ) after antibiotic therapy the kinetics of killing B. burgdorferi s. l. under amoxicillin, doxycycline, cefotaxime, ceftriaxone, azithromycin and penicillin G were determined. The killing effect was investigated in MKP medium and human serum during a 72 h exposure to antibiotics. Twenty clinical isolates were used, including ten strains of Borrelia afzelii and ten strains of Borrelia garinii. The results show that the kinetics of killing borreliae differ from antibiotic to antibiotic. The killing rate of a given antibiotic is less dependent on the concentration of the antibiotic than on the reaction time. Furthermore, the data show that the strains of B. afzelii and B. garinii have a different reaction to antibiotics used in the treatment of Lyme borreliosis and that different reactions to given antibiotics also exist within one species. The B. garinii strains appear to be more sensitive to antibiotics used in therapy. Furthermore, the persistence of B. burgdorferi s. l. and clinical recurrences in patients despite seemingly adequate antibiotic treatment is described. The patients had clinical disease with or without diagnostic antibody titers to B. burgdorferi. Published erratum appears in Infection 1996 Mar-Apr;24(2):169

PMID: 8852456, UI: 97005145

Med Microbiol Immunol (Berl) 1995 May;184(1):23-32

Molecular and immunological characterization of the p83/100 protein of various Borrelia burgdorferi sensu lato strains.

Rossler D, Eiffert H, Jauris-Heipke S, Lehnert G, Preac-Mursic V, Teepe J, Schlott T, Soutschek E, Wilske B

Max von Pettenkofer Institut fur Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians Universitat München, Germany.

The complete coding regions of the chromosomally encoded p83/100 protein of four Borrelia garinii strains and one Borrelia burgdorferi sensu stricto strain have been amplified by the polymerase chain reaction (PCR), cloned and sequenced. From alignment studies with the deduced amino acid sequences presented here, and five other published p83/100 sequences, the most heterologous region of the p83/100 molecule was identified to be located between amino acid position 390-540. To study the structure of this heterogeneous region, and internal fragment of the p83/100 genes from 11 additional B. burgdorferi sensu lato strains was amplified by PCR. The PCR products were analyzed by DNA sequencing and restriction enzyme analysis. These internal p83/100 fragments varied in size and sequence. Cluster analysis of internal p83/100 fragments, as well as restriction enzyme analysis, revealed three major groups in accordance with grouping into the three species causing Lyme disease. Strains within the same species (six B. burgdorferi sensu stricto and six B. afzelii strains) showed similar p83/100 partial structures. Nevertheless, nine B. garinii strains showed more sequence variations and could be further divided into two major subgroups. One group is represented by OspA serotype 4 strains, the other more heterogeneous group is represented by OspA serotypes 3, 5, 6 and 7 strains. Phenotypic analysis with four p83/100-specific monoclonal antibodies revealed four distinct reactivity patterns. Antibody L100 1B4 recognized a common epitope of B. burgdorferi sensu stricto and B. afzelii. Antibodies L100 17D3 and L100 18B4 were reactive with an epitope shared by strains of all three species. The broadest reactivity was shown by L100 18B4 which, in contrast to L100 17D3, additionally recognized the relapsing fever borreliae B. turicatae and B. hermsii. L100 8B8 detected a subgroup of the B. burgdorferi sensu stricto strains. Since comparison of the p83/100 molecule with sequences from protein databases showed similarities with characteristics of eukaryotic cell structures, the p83/100 might mimic these structures and may, therefore, be involved in the immune escape mechanism of the pathogenic agent of Lyme disease.

PMID: 8538575, UI: 96149106

Ann N Y Acad Sci 1988;539:126-43

Antigenic variability of Borrelia burgdorferi.

Wilske B, Preac-Mursic V, Schierz G, Kuhbeck R, Barbour AG, Kramer M

Max von Pettenkofer Institute, University of Munich, Federal Republic of Germany.

Borrelia burgdorferi strains (six isolates from North America and 28 isolates from Europe) were analyzed by physicochemical and immunological methods. By SDS-PAGE, all Borrelia burgdorferi strains tested had two major proteins with constant molecular weights of 60 and 41 kDa and one, two, or three variable low molecular weight proteins (OspA = 30-32 kDa, OspB = 34-36 kDa, pC = 21-22 kDa). All combinations--except OspB alone or OspB/pC--were observed. Borrelia burgdorferi strains were different from relapsing fever borreliae by strong reactivity with OspA- and/or pC-specific polyclonal antibodies, whereas relapsing fever borreliae were only weakly reactive. Among 25 Borrelia burgdorferi isolates, seven different serotypes of Borrelia burgdorferi were defined according to their reactivity in the Western blot with three monoclonal OspA-specific antibodies (H5332, H3TS, and LA5), four OspA- or OspB-specific polyclonal antibodies, and 12 polyclonal antibodies against whole borreliae. Antigenic differences between European CSF and skin isolates were observed, all skin isolates (n = 11) belonging to serotype 2 in contrast to only two out of seven CSF isolates. CSF isolates were antigenically heterogenous (serotypes 1, 2, 3, 4, and 5). Serotypes 6 and 7 were represented by two tick isolates, and the other European tick isolates are not yet fully characterized. Antigenic differences between European and North American strains may play a role in differences in the clinical picture of Lyme borreliosis.

PMID: 2461135, UI: 89048785

Zentralbl Bakteriol Mikrobiol Hyg [A] 1986 Dec;263(1-2):92-102

Immunochemical and immunological analysis of European Borrelia burgdorferi strains.

Wilske B, Preac-Mursic V, Schierz G, Busch KV

Borrelia burgdorferi strains (n = 23), isolated from patients (n = 8) and ticks (n = 14), were analyzed by SDS PAGE and Western blotting with monoclonal antibodies and polyclonal sera (rabbit immune serum and sera from patients). Testing the 23 strains by SDS PAGE 9 different patterns of major protein bands were observed. In contrast to US strains some of our strains showed only weak or negative reactivity with the OspA specific monoclonal antibody H5332. Analysis with polyclonal sera gave further support that the OspA proteins of our strains have specific epitopes (recognized by patients sera) besides common ones (recognized by rabbit immune serum). Fifteen of the strains had another major protein in the 22K range without detectable antigenic variability. Patients with erythema migrans and lymphocytic meningitis had a good IgM- and IgG-immunoresponse to this protein. In contrast antibodies to the OspA were very rarely observed, probably due to the antigenetic heterogeneity of the causative agent on the one hand but also due to low response of the human immune system to the OspA on the other hand.

PMID: 2437739, UI: 87208564

Infection 1996 Mar-Apr;24(2):208-12

Immunological and molecular variability of OspA and OspC. Implications for Borrelia vaccine development.

Wilske B, Busch U, Fingerle V, Jauris-Heipke S, Preac Mursic V, Rossler D, Will G

Max-von-Pettenkofer Institut fur Hygiene und Medizinische Mikrobiologie, München, Germany.

Borrelia burgdorferi sensu lato, the etiological agent of Lyme borreliosis is considerably heterogeneous in Europe. Since the outer surface proteins OspA and OspC are the most promising candidates for a Borrelia vaccine the immunological heterogeneity of these proteins was investigated. By immunological analysis with monoclonal antibodies and sequence analysis of PCR amplified OspA and OspC at least seven and 16 different types, respectively, were found. Whereas skin isolates (n = 68) were quite homogeneous (84% belonged to OspA-serotype 2 or Borrelia afzelii), isolates from human cerebrospinal fluid and from ticks (n = 43 and n = 90 respectively) were highly heterogeneous in their OspA-serotypes with prevalence of the Borrelia garinii associated types (about 70%). OspA-type 4 was often found among isolates from cerebrospinal fluid (28%). In ticks type 4 OspA has not been detected by culture so far. However, as reported in a previous study, type 4 OspA could be detected in ticks by the highly sensitive PCR technique.

Publication Types:

PMID: 8740124, UI: 96311594

Med Microbiol Immunol (Berl) 1996 Feb;184(4):195-201

Diversity of OspA and OspC among cerebrospinal fluid isolates of Borrelia burgdorferi sensu lato from patients with neuroborreliosis in Germany.

Wilske B, Busch U, Eiffert H, Fingerle V, Pfister HW, Rossler D, Preac-Mursic V

Max-von-Pettenkofer-Institut fur Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universitat München, Germany.

Neuroborreliosis is the most frequent manifestation of the second stage of Lyme borreliosis in Europe. However, only few isolates from the cerebrospinal fluid (CSF) have been characterized with controversial results. A large panel of 36 CSF isolates isolated over a 10-year period in Munich has now been analyzed for their OspA and OspC type, resulting in at least eight different types, respectively. Representatives of the different types cultivated from CSF in Munich have also been isolated from other geographical regions in Europe from CSF or ticks, suggesting a widespread distribution of pathogenic strains. A certain OspA type (type 4) was frequently observed in adults but rarely in children or ticks. Since OspA and OspC are the most promising candidates for a Borrelia vaccine, the considerable heterogeneity found among CSF isolates has important implications for development of a vaccine in Europe.

PMID: 8811652, UI: 96407599

Med Microbiol Immunol (Berl) 1996 Feb;184(4):181-4

Characterization of Borrelia burgdorferi sensu lato strains isolated from Ixodes ricinus in Mecklenburg-Vorpommern, Germany.

Lottmann H, Wilske B, Herrmann H

Institut fur Medizinische Mikrobiologie, Ernst-Moritz-Arndt-Universitat Greifswald, Germany.

Epidemiological studies in Mecklenburg-Vorpommern have shown a high prevalence of Borrelia burgdorferi-infected ticks. A total of 17 B. burgdorferi sensu lato strains were isolated from ticks and investigated by Western blots (immunoblot) with eight monoclonal anti-bodies against different epitopes of the outer surface protein A (OspA). Except for one, all strains could be classified using this system. The majority of strains belonged to the B. garinii-associated OspA serotypes 3, 5 and 6. Three isolates were classified as OspA serotype 2 (B. afzelii). B. burgdorferi sensu stricto strains (OspA serotype 1) as well as B. garinii-associated OspA serotype 4 were not present.

PMID: 8811650, UI: 96407597

J Clin Microbiol 1996 Feb;34(2):364-8

Molecular polymorphism of the lyme disease agent Borrelia garinii in northern Europe is influenced by a novel enzootic Borrelia focus in the North Atlantic.

Bunikis J, Olsen B, Fingerle V, Bonnedahl J, Wilske B, Bergstrom S

Department of Microbiology, Umea University, Sweden.

Lyme disease Borrelia species are distributed in temperate areas of North America and Eurasia. To elucidate the distribution of borreliae in subarctic regions, strains isolated from Ixodes ricinus and Ixodes uriae ticks found on islands in the northern Atlantic and Baltic Sea were molecularly characterized. All isolates were verified as Borrelia garinii by 16S rRNA gene analysis and immunoblotting with monoclonal antibodies specific for the outer surface proteins A and C. Three ribotypes (RTs) of B. garinii were delineated. I. ricinus complex-associated RT1 was phenotypically most heterogeneous. Two newly identified ribotypes were shared by different tick species and conformed to two established OspA serotypes. RT2 was restricted to the islands in the northern Baltic Sea, whereas RT3 was recovered also from ticks found in the North Atlantic. In conclusion, molecular polymorphism of the studied borrelia isolates suggests a complex enzootic potential of B. garinii in northern Europe and implies a novel, seabird tick I. uriae-associated enzootic focus of Lyme disease borreliae in the North Atlantic.

PMID: 8789017, UI: 96381004

Med Microbiol Immunol (Berl) 1995 Aug;184(2):73-80

Sequence analysis of ospA genes shows homogeneity within Borrelia burgdorferi sensu stricto and Borrelia afzelii strains but reveals major subgroups within the Borrelia garinii species.

Will G, Jauris-Heipke S, Schwab E, Busch U, Rossler D, Soutschek E, Wilske B, Preac-Mursic V

Abteilung Serologie, Max-von-Pettenkofer-Institut fur Hygiene und Medizinische Mikrobiologie der Ludwig-Maximilians-Universitat München, Germany.

The genes coding for the outer surface protein A (OspA) of 19 different Borrelia burgdorferi strains belonging to the seven OspA-serotypes 1-7, previously described [Wilske et al. (1993) J Clin Microbiol, 31: 340-350], have been investigated. B. burgdorferi sensu lato strains were chosen from various biological sources (ticks, human skin and cerebrospinal fluid) as well as different geographical origins (Germany, Slovenia, Austria, United States). The open reading frames of all ospA genes consist of 819-825 nucleotides corresponding to proteins of approximately 30 kDa. The ospA sequences obtained in this study and previous published studies were compared with the results from OspA serotyping with monoclonal antibodies. The classification into the seven OspA serotypes could be confirmed on a genetic basis (ospA genotypes 1-7) for all strains analyzed so far (n = 29). In addition, one strain without OspA expression could be assigned to ospA genotype 2. Genetic stability could be proven for the ospA gene of B. burgdorferi strain PWudI after inocculation and reisolation from a gerbil. However, we found evidence for intragenic recombination by cluster analysis of ospA sequence data. Accordance of ospA genotype 1 strains with B. burgdorferi sensu stricto and ospA genotype 2 strains with B. afzelii, as well as the ospA genotype strains 3-7 with B. garinii was confirmed by pulsed-field gel electrophoresis of MluI-digested genomic DNA. B. garinii is not only more heterogeneous in respect to the OspA-encoding genes, but shows moreover major subgroups formed by genotypes 4, 5 and 6 and genotypes 3 and 7, respectively.

PMID: 7500914, UI: 96085967

Infect Immun 1995 Apr;63(4):1573-1580

Borrelia burgdorferi mutant lacking Osp: biological and immunological characterization.

Sadziene A, Thomas DD, Barbour AG

Department of Microbiology and Medicine, University of Texas Health Science Center at San Antonio 78284.

All Borrelia burgdorferi sensu lato isolates characterized to date have one or a combination of several major outer surface proteins (Osps). Mutants of B. burgdorferi lacking Osps were selected with polyclonal or monoclonal antibodies at a frequency of 10(-6) to 10(-5). One mutant that lacked OspA, -B, -C, and -D was further characterized. It was distinguished from the OspA+B+ cells by its (i) autoaggregation and slower growth rate, (ii) decreased plating efficiency on solid medium, (iii) serum and complement sensitivity, and (iv) diminished capacity to adhere to human umbilical vein endothelial cells. The Osp-less mutant was unable to evoke a detectable immune response after intradermal live cell immunization even though mutant survived in mouse skin for the same duration as wild-type cells. Polyclonal mouse serum raised against Osp-less cells inhibited growth of the mutant but not of wild-type cells, an indication that other antigens are present on the surface of the Osp-less mutant. Two types of monoclonal antibodies (MAbs) with growth-inhibiting properties for mutant cells were identified. The first type bound to a 13-kDa surface protein of B. burgdorferi sensu stricto and of B. afzelii. The MIC of the Fab fragment of one MAb of this type was 0. 2 micrograms/ml. The second type of MAb to the Osp-less mutant did not bind to B. burgdorferi components by Western blotting (immunoblotting) but did not bind to unfixed, viable cells in immunofluorescence and growth inhibition assays. These studies revealed possible functions Osp proteins in borrelias, specifically serum resistance, and indicated that in the absence of Osp proteins, other antigens are expressed or become accessible at the cell surface.

PMID: 7890424, UI: 95197293

J Infect Dis 1998 Feb;177(2):395-400

Immune evasion by tickborne and host-adapted Borrelia burgdorferi.

de Silva AM, Fikrig E, Hodzic E, Kantor FS, Telford SR 3rd, Barthold SW

Center for Comparative Medicine, School of Medicine, University of California, Davis 95616, USA.

Immune sera from mice infected with the Lyme disease spirochete, Borrelia burgdorferi, have strong biologic activity against spirochetes cultured in vitro. Recent studies with rodents and ticks infected with B. burgdorferi indicate that spirochetes undergo major changes in protein expression as they adapt to the diverse environments encountered by a vectorborne pathogen. The purpose of this study was to explore the susceptibility of three different adaptive forms of B. burgdorferi (in vitro cultured, host-derived, and tickborne) to immune sera. Passive transfer of immune sera protected mice when they were challenged with spirochetes cultured in vitro. Immune sera did not protect mice from tickborne spirochetes or spirochetes derived from infected mice. These results indicate that spirochetes that have adapted within either the feeding tick or host are relatively invulnerable to the protective effects of immune sera, unlike spirochetes grown in vitro, which are highly susceptible.

PMID: 9466527, UI: 98126003

Microbiology 1997 Dec;143( Pt 12):3819-3825

An unusual illegitimate recombination occurs in the linear-plasmid-encoded outer-surface protein A gene of Borrelia afzelii.

Wang J, Masuzawa T, Li M, Yanagihara Y

Department of Microbiology, School of Pharmaceutical Sciences, University of Shizuoka, Japan. gp1018@mail. p. u-shizuoka-ken. ac. jp

In this study, we describe an unusual illegitimate recombination in the linear-plasmid-encoded outer-surface protein A gene of Borrelia afzelii. A 96 bp DNA segment was deleted from the ospA structural gene of B. afzelii strain R9. The nature of the rearrangement suggested that it arose by a strand slippage mechanism, which was stimulated by a 18-mer palindromic sequence and 5-mer short direct repeats at both termini of the deleted DNA. The deleted sequence could form a complex hairpin structure suggesting that it may have played important roles in pausing of replication and slippaging of the nascent strand across the replication fork. In addition, the mutant strain was isolated from a chronic Lyme disease patient, implying that the variation mechanism may have been used by the borrelial strain to avoid host immune elimination.

PMID: 9421906, UI: 98083737

J Infect Dis 1991;163:150-5

Infectivity of Borrelia burgdorferi correlates with resistance to elimination by phagocytic cells.

Georgilis K, Steere AC, Klempner MS

Department of Medicine, Tufts University School of Medicine, New England Medical Center Hospitals, Boston, Massachusetts 02111.

The Lyme disease spirochete, Borrelia burgdorferi, causes a disseminated infection in vivo, implying resistance to clearance by phagocytic cells. Because B. burgdorferi loses its infectivity after in vitro cultivation, the relationships between serial passaging of the organism in vitro, its susceptibility to elimination by phagocytes, and its infectivity were examined. When three different high-passage strains were incubated for 4 h at 37 degrees C with peripheral blood mononuclear cells, macrophages, or polymorphonuclear neutrophils, 45%-67% of the organisms were eliminated. In contrast, two low-passage strains were resistant to elimination by phagocytes, and only 5%-6% of the organisms were removed after 4 h. All five strains equally stimulated the neutrophil oxidative burst, indicating that evasion of phagocytes was not a result of avoidance of recognition by these cells. The two low-passage strains were infective when injected into mice, whereas the three high-passage ones were not. These observations indicate that infectivity of the Lyme disease spirochete correlates with resistance to elimination by phagocytic cells.

UI: 91079640


Med Hypotheses. 1999 Apr;52(4):293-6. Related Articles
Click here to read
Microbes and sequestered substances as mechanisms for disease: Bartonella and L-forms as common global etiological agents.

Sood FH, Khatpe DS, Chaudhari MS, Phatak VD.


The pathogenicity of microbes may be determined by substances sequestered from blood and bound to their constituent lipid. The brain may not perceive substances sequestered by microbes, to interfere with control to maintain normal levels. Pathological conditions can be induced as organisms exposed to antimicrobial substances/conditions and/or deprived of nutrients essential to cell wall synthesis, disintegrate to free lipid-bound compounds and produce L-forms that can deplete nutrients as they revert to bacteria. Microbes may act as active carriers for the continuing interaction of sequestered substances. Changes in the molecular structure of substances effected during sequesteration could cause them to be seen as substances 'synthesized' by an organism. In media that contain substances to inhibit 'contaminants', L-forms can be seen as mycoplasma. Elementary bodies of L-forms with a specific substance or tissue affinity may be seen as 'receptors'. Bartonella are global agents for disease--pleomorphic organisms (description suits Proteus)--and they can be seen as 'contaminants'.

PMID: 10465665 [PubMed - indexed for MEDLINE]

Med Hypotheses. 1997 Jun;48(6):511-5. Related Articles

Bartonellosis and human immunodeficiency disease (AIDS): L-forms as persisters, activating factors, and mechanism of disease.

Sood FH, Phatak VD, Chaudhari MS.

Bartonella, genus Proteus, can cause immunodepressive disease. The organisms, in parasitized red blood cells, may invade the brain and every other system and space in the human body. Bartonella henselae is proposed to have a role in the pathogenesis of acquired immunodeficiency syndrome (AIDS) encephalopathy. Bartonella bacilliformis produces two known toxins that can induce spasm and angiomatosis, respectively, and manifest as diseases associated with symptomatic AIDS. The skin lesions of bartonellosis may be mistaken clinically and histologically for Kaposi's sarcoma. Bacteria of the genus Proteus produce L-forms: their elementary bodies may be mistaken for what are called the 'human immunodeficiency viruses' (HIV). Antibiotics, especially penicillin, induce bacteria to produce L-forms. Air pollution and high sugar, salt and fat diets are factors that may increase the lipid content of microbes that produce toxins and L-forms that may persist or revert to bacterial form.

Publication Types:
PMID: 9247895 [PubMed - indexed for MEDLINE]

Med Hypotheses. 1994 Sep;43(3):135-7. Related Articles

Bartonella bacilliformis or a similar organism and cardiovascular disease.

Sood FH, Chaudhari MS.

Red Cross Blood Bank, Pune, India.

Bartonella bacilliformis invades the endothelial lining of the cardiovascular system. Damage to the red blood cells and white blood cells, the effects of the toxins, invasion of the brain and electrical charges induced by the organism so interfering with normal electrical stimulation of the heart may explain many of the features of cardiovascular disease (1-5).

PMID: 7815963 [PubMed - indexed for MEDLINE]

Microbiology 2000 Jan;146 (Pt 1):119-27

Serum-starvation-induced changes in protein synthesis and morphology of Borrelia burgdorferi.

Alban PS, Johnson PW, Nelson DR

Department of Biochemistry, Microbiology, and Molecular Genetics, University of Rhode Island, Kingston 02881, USA.

[Medline record in process]

It has been demonstrated previously that motile Borrelia burgdorferi cells transform into non-motile cyst-forms when incubated for several weeks in BSKII (a complex medium) lacking rabbit serum. B. burgdorferi cells cannot synthesize fatty acids de novo and serum is thought to provide a source of fatty acids and lipids. When B. burgdorferi cells were serum-starved in defined RPMI medium, -90% of the cells formed spherical cysts within 48 h. Cyst formation was inhibited by tetracycline. Cyst opening and recovery of vegetative cells was rapidly induced by the addition of either BSKII or rabbit serum. The percentage of viable cells recovered from cysts ranged from 2.9% to 52-5%. Viability was inversely proportional to cyst age. Protein synthesis by B. burgdorferi during serum starvation was examined by labelling cells with Tran35S-Label and analysing the labelled proteins by two-dimensional gel electrophoresis and fluorography. The synthesis of over 20 proteins was induced during serum starvation. Western blots of proteins from vegetative cells and cysts probed with sera from either B. burgdorferi-infected humans or monkeys revealed that several cyst proteins were antigenic. These data suggest that cells of B. burgdorferi, although possessing a small genome and extremely limited biosynthetic capabilities, rapidly respond to conditions of serum starvation by inducing changes in protein synthesis and cell morphology. This study may help explain how cells of B. burgdorferi can survive periods of nutrient deprivation in different hosts and host tissues.

PMID: 10658658, UI: 20121745

Infection 1998 Nov-Dec;26(6):364-7

A proposal for the reliable culture of Borrelia burgdorferi from patients with chronic Lyme disease, even from those previously aggressively treated.

Phillips SE, Mattman LH, Hulinska D, Moayad H

Greenwich Hospital, CT 06830, USA.

[Medline record in process]

Since culture of Borrelia burgdorferi from patients with chronic Lyme disease has been an extraordinarily rare event, clarification of the nature of the illness and proving its etiology as infectious have been difficult. A method for reliably and reproducibly culturing B. burgdorferi from the blood of patients with chronic Lyme disease was therefore sought by making a controlled blood culture trial studying 47 patients with chronic Lyme disease. All had relapsed after long-term oral and intravenous antibiotics. 23 patients with other chronic illness formed the control group. Positive cultures were confirmed by fluorescent antibody immuno-electron microscopy using monoclonal antibody directed against Osp A, and Osp A PCR. 43/47 patients (91%) cultured positive. 23/23 controls (100%) cultured negative. Although persistent infection has been, to date, strongly suggested in chronic Lyme disease by positive PCR and antigen capture, there are major problems with these tests. This new method for culturing B. burgdorferi from patients with chronic Lyme disease certainly defines the nature of the illness and establishes that it is of chronic infectious etiology. This discovery should help to reestablish the gold standard in laboratory diagnosis of Lyme disease.

PMID: 9861561, UI: 99078554

APMIS 1999 Jun;107(6):566-76

An in vitro study of the susceptibility of mobile and cystic forms of Borrelia burgdorferi to metronidazole.

Brorson O, Brorson SH

Department of Microbiology, Vestfold Sentralsykehus, Tonsberg, Norway.

The aim of this study was to examine the susceptibility of

forms of Borrelia burgdorferi to metronidazole.

Because B. burgdorferi is a microaerobic bacterium like Helicobacter pylori, metronidazole (MZ) was chosen in the susceptibility test.

  1. normal mobile spirochetes: For both microaerobic and aerobic incubation the normal mobile spirochetes were resistant to this antibiotic with an MBC > or = 512 microg/ml. Conversion of mobile spirochetes to cystic forms was not observed when they were incubated with MZ.
  2. the biologically active cystic forms
    • When they were incubated under microaerobic conditions, the biologically active cystic forms had an MBC > or = 4 microg/ml, but
    • the MBC was > or = 32 microg/ml with aerobic incubation at 37 degrees C.
  3. Staining with acridine orange (AO), dark field microscopy (DFM), and transmission electron microscopy (TEM) revealed that the contents of the cysts were degraded when the concentration of MZ was > or = MBC. Some cysts were also ruptured.
  4. When incubated with a sufficient concentration of MZ, core structures did not develop inside the cysts, and AO revealed less RNA in the cysts.
Our observations may help efforts to treat resistant infections caused by B. burgdorferi with a combination of MZ and other antibiotics in order to eradicate both cystic and mobile forms of B. burgdorferi.

PMID: 10379684, UI: 99306352

APMIS 1998 Dec;106(12):1131-41

A rapid method for generating cystic forms of Borrelia burgdorferi, and their reversal to mobile spirochetes.

Brorson O, Brorson SH

Department of Microbiology, Vestfold Sentralsykehus, Tonsberg, Norway.

Mobile Borrelia burgdorferi were transferred to distilled water (10(6) per ml). The cultures were observed by dark field microscopy (DFM), interference contrast microscopy (ICM) and transmission electron microscopy (TEM). 95% of the spirochetes were converted to cysts after 1 min, and after 4 h no normal mobile borreliae were observed. When transferred to growth medium (BSK-H), the cysts became smaller and more irregular, and were filled with organic substances. After 1 day, 1-5 thin structures sprouted from the cysts. They continued to grow in both length and thickness until they attained a normal spirochetal structure. Finally, these new-born spirochetes detached from the cysts, by which time their mobility had become normal. The present method for producing large amounts of cystic forms of B. burgdorferi is well suited for further studies of this unique microbe.

PMID: 10052721, UI: 99160086

Infection 1998 May-Jun;26(3):144-50

In vitro conversion of Borrelia burgdorferi to cystic forms in spinal fluid, and transformation to mobile spirochetes by incubation in BSK-H medium.

Brorson O, Brorson SH

Dept. of Microbiology, Vestfold Sentralsykehus, Tonsberg.

The purpose of this study was to examine the structural alterations of Borrelia burgdorferi when exposed to spinal fluid. Normal, mobile spirochetes were inoculated into spinal fluid, and the spirochetes were converted to cysts (spheroplast L-forms) after 1-24 h. When these cystic forms were transferred to a rich BSK-H medium, the cysts were converted back to normal, mobile spirochetes after incubation for 9 to 17 days. The cultures were examined by dark field microscopy (DFM), interference contrast microscopy (ICM) and transmission electron microscopy (TEM). When neuroborreliosis is suspected, it is necessary to realize that B. burgdorferi can be present in a cystic form, and these cysts have to be recognized by microscopy. This study may also explain why cultivation of spinal fluid often is negative with respect to B. burgdorferi.

PMID: 9646104, UI: 98310072

Infection 1997 Jul-Aug;25(4):240-6

Transformation of cystic forms of Borrelia burgdorferi to normal, mobile spirochetes.

Brorson O, Brorson SH

Dept. of Microbiology, Ulleval University Hospital, Oslo, Norway.

The purpose of this study was to evaluate the behaviour of Borrelia burgdorferi under controlled conditions. The occurrence of cystic forms of Borrelia burgdorferi in vitro was noted, and these cysts were able to be transformed to normal, mobile spirochetes. B. burgdorferi was cultivated in a commercial culture medium without serum. The spirochetes multiplied only slowly in this medium, and transformation to encysted forms was observed after 1 week. When these cysts were transferred to the same culture medium with rabbit serum, the encysted forms developed into regular, mobile spirochetes after 6 weeks, and their regeneration time was normal. Examination of these cysts in the transmission electron microscope revealed transverse fission inside the cysts. It is probable that similar phenomena may occur in vivo under conditions unfavourable for spirochetes. These observations may help to explain why diagnosis and treatment of B. burgdorferi infections in humans can be difficult.

PMID: 9266264, UI: 97411286

Medline-Search for

borrelia AND (MIC OR MBC) AND Brorson

Brorsons' research on antibiotics against cystic forms.

Infection 1996 May-Jun;24(3):218-26

Formation and cultivation of Borrelia burgdorferi spheroplast-L-form variants.

Mursic VP, Wanner G, Reinhardt S, Wilske B, Busch U, Marget W

Max von Pettenkofer-Institut, Ludwig-Maximilians-Universitat München, Germany.

As clinical persistence of Borrelia burgdorferi in patients with active Lyme borreliosis occurs despite obviously adequate antibiotic therapy, in vitro investigations of morphological variants and atypical forms of B. burgdorferi were undertaken. In an attempt to learn more about the variation of B. burgdorferi and the role of atypical forms in Lyme borreliosis, borreliae isolated from antibiotically treated and untreated patients with the clinical diagnosis of definite and probable Lyme borreliosis and from patient specimens contaminated with bacteria were investigated. Furthermore, the degeneration of the isolates during exposure to penicillin G in vitro was analysed. Morphological analysis by darkfield microscopy and scanning electron microscopy revealed diverse alterations. Persisters isolated from a great number of patients (60-80%) after treatment with antibiotics had an atypical form. The morphological alterations in culture with penicillin G developed gradually and increased with duration of incubation. Pleomorphism, the presence of elongated forms and spherical structures, the inability of cells to replicate, the long period of adaptation to growth in MKP-medium and the mycoplasma-like colonies after growth in solid medium (PMR agar) suggest that B. burgdorferi produce spheroplast-L-form variants. With regard to the polyphasic course of Lyme borreliosis, these forms without cell walls can be a possible reason why Borrelia survive in the organism for a long time (probably with all beta-lactam antibiotics) [corrected] and the cell-wall-dependent antibody titers disappear and emerge after reversion. Published erratum appears in Infection 1996 Jul-Aug;24(4):335

PMID: 8811359, UI: 96407306

Antimicrob Agents Chemother 2008 Mar 3;

Persistence of Borrelia burgdorferi following antibiotic treatment in mice.

Hodzic E, Feng S, Holden K, Freet KJ, Barthold SW.

Center for Comparative Medicine, Schools of Medicine and Veterinary Medicine, University of California at Davis, One Shields Avenue, Davis, CA 95616.

The effectiveness of antibiotic treatment was examined in a mouse model of Lyme borreliosis. Mice were treated with ceftriaxone or saline for one month, commencing during the early (3 weeks) or chronic (4 months) stages of infection with Borrelia burgdorferi. Tissues from mice were tested for infection by culture, polymerase chain reaction (PCR), xenodiagnosis, and transplantation of allografts at 1 and 3 months after completion of treatment. In addition, tissues were examined for spirochetes by immunohistochemistry. In contrast to saline-treated mice, mice treated with antibiotic were consistently culture-negative, but tissues from some of the mice remained PCR-positive, and spirochetes could be visualized in collagen-rich tissues. Furthermore, when some of the antibiotic treated mice were fed upon by Ixodes scapularis ticks (xenodiagnosis), spirochetes were acquired by the ticks, based upon PCR, and ticks from those cohorts transmitted spirochetes to na•ve SCID mice, which became PCR-positive, but culture-negative. Results indicated that following antibiotic treatment, mice remained infected with non-dividing but infectious spirochetes, particularly when antibiotic treatment was commenced during the chronic stage of infection.

PMID: 18316520 [PubMed - as supplied by publisher]

Antimicrob Agents Chemother 1995 May;39(5):1127-33

Effects of penicillin, ceftriaxone, and doxycycline on morphology of Borrelia burgdorferi.

Kersten A, Poitschek C, Rauch S, Aberer E

Department of Dermatology, University of Vienna, Austria.

Antibiotic therapy with penicillin, doxycycline, and ceftriaxone has proven to be effective for the treatment of Lyme borreliosis. In some patients, however, it was noticed that borreliae can survival in the tissues in spite of seemingly adequate therapy. For a better understanding of this phenomenon, we investigated the different modes of degeneration of Borrelia burgdorferi suspensions during a 96-h exposure to various antibiotics. By dark-field microscopy and ultrastructural investigations, increasing blebbing and the gradual formation of granular and cystic structures could be followed during the exposure time. Although antibiotic concentrations at the MIC at which 90% of organisms are inhibited after 72 h were 80% or even greater, motile organisms were still present after incubation with penicillin and doxycycline but not after incubation with ceftriaxone. By transmission electron microscopy, intact spirochetal parts, mostly situated in cysts, were seen up to 96 h after exposure with all three antibiotics tested. According to experiences from studies with other spirochetes it is suggested that encysted borreliae, granules, and the remaining blebs might be responsible for the ongoing antigenic stimulus leading to complaints of chronic Lyme borreliosis.

PMID: 7625800, UI: 95351754

Infection 1994 Nov;22(6):401-406

Ultrastructure of Borrelia burgdorferi after exposure to benzylpenicillin.

Schaller M, Neubert U

Dermatologische Klinik, Ludwig-Maximilians-Universitat, München, Germany.

The aim of this study was to investigate the morphological changes of Borrelia burgdorferi associated with penicillin treatment. An isolate of B. burgdorferi from an erythema migrans lesion was cultivated in BSK II medium and exposed to increasing concentrations (0. 0625 mg/l-2 mg/l) of penicillin G for 5 days. The in vitro minimal inhibitory concentration (MIC) was determined to be 0. 5 mg/l by broth dilution method. The morphological structures of untreated spirochetes, as well as their characteristic ultrastructural changes when exposed to penicillin, were observed by electron microscopy. The following alterations were discovered: (i) Numerous outer sheath blebs at a penicillin concentration of 0. 0625 mg/l. (ii) A characteristic irregular waveform of the borrelial cells and complete loss of the outer sheath at a penicillin concentration of 0. 125 mg/l. (iii) The presence of "spheroplasts" at the same concentration. (iv) Structural changes of the protoplasmic cylinder complex which showed an irregular pattern at a penicillin concentration of 0. 125 mg/l. (v) Disruption of the protoplasmic cylinder complex into several parts at penicillin concentrations of 0. 25 mg/l and 0. 5 mg/l. (vi) Severe cytolysis at penicillin concentrations of 1 mg/l and 2 mg/l.

PMID: 7698837, UI: 95213112

The address of this page is http://www.lymenet.de/literatur/niches.htm.
The home page of this website is http://www.lymenet.de.
Send your comments to Joachim Gruber.
version: March 7, 2008