Intracellular localisation of Borrelia burgdorferi

This list probably not complete – if you find that an important article, that  demonstrate
intracellular Borrelia  is missing in the list, please send me a note at

The histopathology of experimentally infected hamsters with the Lyme disease spirochete, Borrelia burgdorferi.
Duray PH, Johnson RC. Proc Soc Exp Biol Med 1986 Feb; 181(2): 263-9. PMID: 3945634

Seven hamsters, experimentally infected with Borrelia burgdorferi, were examined by both cultural and histological techniques at 1 to 9 months postinfection. Spirochetes were detected in the spleen, kidney, or eye of all animals by culture and in the spleen, kidney, eye, liver, or heart blood of five of seven animals by histological examination. Two animals showed nonspecific hepatic portal lymphocytic infiltration, while five of the hamsters displayed no significant histologic signs of inflammation or granuloma formation in the major organ systems. Synovitis and arthropathy did not occur. All animals showed some degree of follicular lymphoid hyperplasia of the spleen. Spirochetes were predominantly extracellular with a rare organism appearing to be partially within a macrophage.

Penetration of endothelial cell monolayers by Borrelia burgdorferi.
Comstock LE, Thomas DD. Infect Immun 1989 May; 57(5): 1626-8. PMID: 2707862   PDF

The ability of Borrelia burgdorferi, the agent of Lyme disease, to penetrate cultured human umbilical vein endothelial cell monolayers was investigated. After 4 h of coincubation, approximately 7.7% of added bacteria passed through the host cell monolayers. Electron microscopy revealed that the borreliae entered the endothelial cells and suggested that the organisms penetrated the host monolayers primarily by passing through them.

Characterization of Borrelia burgdorferi invasion of cultured endothelial cells.
Comstock LE, Thomas DD. Microb Pathog 1991 Feb; 10(2): 137-48. PMID: 1890951

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.

Intracellular localization of Borrelia burgdorferi within human endothelial cells.
Ma Y, Sturrock A, Weis JJ. Infect Immun 1991 Feb; 59(2): 671-8. PMID: 1987083  PDF

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.

The fate of Borrelia burgdorferi in Mouse Macrophages: Destruction, Survival, Recovery
Montgomery RR, Nathanson MH, Malawista SE. (1992) V Int Conf Lb abs#143. 1992-co143, see also PMID: 8423346 

The macrophage is a known reservoir for a number of infectious agents, and is therefore a likely candidate for a privileged site where B. burgdorferi, the Lyme spirochete, may persist. We have shown that B. burgdorferi can enter macrophages, resulting in one instance in degradation and in another in apparent intracellular persistence. We studied uptake of B. burgdorferi by the mouse macrophage cell lime, J774, by simultaneously labeling infected cells with antibodies to B. burgdorferi and to sequential components of the endocytic pathway: the late endosome and lysosome marker, lysosomal glycoprotein (lgp) 110, or the lysosomal hydrolase, cathepsin L. We examined optical sections (0.5-1.0 ?m in thickness) of these double-labeled macrophages by confocal fluorescence microscopy at multiple time points after infection. We found that only 5 minutes of incubation at 370C were required for nearly 100% of B. burgdorferi 10 enter an lgp-positive compartment, while 60 minutes 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 and examined them using confocal microscopy. Although the large majority of spirochetes within a given cell were dead, we saw occasional live ones up to 24 hours (the longest interval examined) after all extracellular organisms had been lysed in distilled water. Moreover, we can reculture spirochetes from both J774 and primary mouse macrophages (strain C3H) for at least a week after infection (the longest interval examined). Persistence of spirochetes within mouse macrophages provides a possible pathogenetic mechanism for chronic or recurrent Lyme disease in man.

Persistence of Borrelia burgdorferi in Chronic Lyme Disease: Altered Immune Regulation or Evasion into Immunologically privileged sites?
Häupl T, Krause A, Rittig M, Schoerner C, Kalden JR, Simon M, Wallich R, Burmester GR. (1992) V Int Conf Lb abs#149.  1992-co149

Patients with Lyme borreliosis (LB) usually develop a vigorous T cell response against the causative pathogen, but little is known about the antigens recognized. Therefore, T cell responses against whole B. burgdorferi (B.b.), outer surface protein A (OspA) and flagellin from B.b. were studied in patients with LB, patients with inflammatory disorders not related to LB, and normal donors. LB patients of all stages exhibited significantly elevated cellular responses to whole B.b. These were directed in part against OspA and flagellin. However, not all patients responded significantly to these proteins. T cell reactivity did not correlate with the presence of specific antibodies as determined by both Western Blot and immunofluorescence assay. 55% of patients with early LB and 80% of patients with late LB had detectable antibodies against B.b.- Almost all seropositive patients showed serum antibodies against flagellin, but antibodies to OspA were detectable only in 4 late LB sera. In one patient with chronic LB, B.b. could be cultured from tendon material and was visualized by electron microscopy. The organisms were both present between collagen fibers and were associated with fibroblasts showing invaginatet and partially intracellular spirochetes. Interestingly, this patient was seronegative at the time of establishing the B.b. strain, but still had elevated T cell responses to the whole organism and OspA. These data demonstrate that the T cell response determined in vitro does not indicate a protective immune response. This may be due to the acti-vation of a T cell population lacking the appropriate lymphokine profile or to an evasion of the B.b. into immunologically privileged sites.
[see published article: Persistence of Borrelia burgdorferi in ligamentous tissue from a patient with chronic Lyme borreliosis.]

Fibroblasts protect the Lyme disease spirochete, Borrelia burgdorferi, from ceftriaxone in vitro.
Georgilis K, Peacocke M, Klempner MS. J Infect Dis 1992 Aug; 166(2): 440-4. PMID: 1634816  PDF

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.

The fate of Borrelia burgdorferi, the agent for Lyme disease, in mouse macrophages. Destruction, survival, recovery.
Montgomery RR, Nathanson MH, Malawista SE. J Immunol 1993 Feb 1; 150(3): 909-15. PMID: 8423346 - see also (1992) V Int Conf Lb abs#143

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.

Invasion of human skin fibroblasts by the Lyme disease spirochete, Borrelia burgdorferi.
Klempner MS, Noring R, Rogers RA. J Infect Dis 1993 May; 167(5): 1074-8. PMID: 8486939

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.

Electron microscopy of Langerhans cells and Borrelia burgdorferi in Lyme disease patients.
Hulinska D, Bartak P, Hercogova J, Hancil J, Basta J, Schramlova J. Int J Med Microbiol Virol Parasitol Infect Dis 1994 Jan; 280(3): 348-59. PMID: 8167429

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.

Localization of Borrelia burgdorferi in murine Lyme borreliosis by electron microscopy.
Pachner AR, Basta J, Delaney E, Hulinska D. Am J Trop Med Hyg 1995 Feb; 52(2): 128-33. PMID: 7872439

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.

Intracellular morphological events observed by electron microscopy on neutrophil phagocytosis of Borrelia garinii
Hulinska D, Basta J, Murgia R, Cinco M. J Spiro Tick Diseases 1995; 2:82-86.

Electron microscopy was used to present morphological events, which accompany the uptake of Borrelia garinii by polymorphonuclear leukocytes (PMNs) and intracellular events of phagocytosis throughout 2 hours of contact. Phagocytosis of borreliae proceeded very quickly, and seemed to be independent of opsonins. Opsonized borreliae emitted membrane-bound blebs, which were attached and engulfed by PMNs. Evidence of coiling and conventional phagocytosis were reported in the same cell. Coiling phagcytosis was time dependent (number of spirochetes internalized by this mechanism with time increased) but was not the preferential mechanism of engulfment by borreliae. Borreliae internalized by coiling phagocytosis were less morphologically altered, and their flagellae protruded into the host cytoplasms. The spirochetes were found discharged in PMN cytoplasm, and were not surrounded by a phagosomal membrane. This could be one of the possible mechanisms of persistence of Borrelia burgdorferi in the host organism.

Entry of Borrelia burgdorferi into macrophages is end-on and leads to degradation in lysosomes.
Montgomery RR, Malawista SE. Infect Immun 1996 Jul; 64(7): 2867-72. PMID: 8698527   PDF

The Lyme disease spirochete, Borrelia burgdorferi, is ingested rapidly by mouse macrophages in vitro. Spirochetes attach by their ends and become progressively coiled as they move deeper into cells. From the earliest measurements, spirochetes colocalize with a marker of endosomes and lysosomes, and degradation of spirochetes occurs within lysosomes.

Malawista: "We have demonstrated that spirochetes enter macrophages end on, are quickly localized in endosomes and lysosomes, and are degraded in the lysosomes. The clarification of initial events in Bb ingestion by macrophages extends our previous work to the ultrastructural level (27) but does not yet explain the occasionally observed cell-associated persistence."
[This is discussed and an answer implied in The Biology of Parasitic Spirochetes, 1976.]

Eucaryotic cells protect Borrelia burgdorferi from the action of penicillin and ceftriaxone but not from the action of doxycycline and erythromycin.
Brouqui P, Badiaga S, Raoult D. Antimicrob Agents Chemother 1996 Jun; 40(6): 1552-4. PMID: 8726038  PDF

Despite appropriate antibiotic treatment, Lyme disease patients may have relapses or may develop chronic manifestations. The intracellular location of Borrelia burgdorferi suggests that antibiotics that penetrate cells will have greater efficiency. Doxycycline or erythromycin was more effective than penicillin or ceftriaxone in killing B. burgdorferi when the organism was grown in the presence of eucaryotic cells.

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. Hum Pathol 1996 Oct; 27(10): 1025-34. PMID: 8892586

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.

Intracellular persistence of Borrelia burgdorferi in human synovial cells.
Girschick HJ, Huppertz HI, Russmann H, Krenn V, Karch H. Rheumatol Int 1996; 16(3): 125-32. PMID: 8893378

To investigate if Borrelia burgdorferi can persist in resident joint cells, an infection model using cell cultures of human synovial cells was established and compared to the interaction of Borrelia burgdorferi and human macrophages. Borrelia burgdorferi were found attached to the cell surface or folded into the cell membrane of synovial cells analysed by transmission electron and confocal laser scanning microscopy. In contrast to macrophages, morphologically intact Borrelia burgdorferi were found in the cytosol of synovial cells without engulfment by cell membrane folds or phagosomes. Borrelia burgdorferi were isolated from parallel cultures. Treatment with ceftriaxone eradicated extracellular Borrelia burgdorferi, but spirochetes were reisolated after lysis of the synovial cells. Borrelia burgdorferi persisted inside synovial cells for at least 8 weeks. These data suggested that Borrelia burgdorferi might be able to persist within resident joint cells in vivo.

Heterogeneity of Borrelia burgdorferi in the skin.
Aberer E, Kersten A, Klade H, Poitschek C, Jurecka W. Am J Dermatopathol 1996 Dec; 18(6): 571-9. PMID: 8989928

The reliability of various in vitro techniques to identify Borrelia burgdorferi infection is still unsatisfactory. Using a high-power resolution videomicroscope and staining with the borrelia genus-specific monoclonal flagellar antibody H9724, we identified borrelial structures in skin biopsies of erythema chronicum migrans (from which borrelia later was cultured), of acrodermatitis chronica atrophicans, and of morphea. In addition to typical borreliae, we noted stained structures of varying shapes identical to borreliae found in a "borrelia-injected skin" model; identical to agar-embedded borreliae; and identical to cultured borreliae following exposure to hyperimmune sera and/or antibiotics. We conclude that the H9724-reactive structures represent various forms of B. burgdorferi rather than staining artifacts. These "atypical" forms of B. burgdorferi may represent in vivo morphologic variants of this bacterium."

p. 573
Studies with antibiotics revealed similar morphologic changes although the formation of granules of a much larger size (spheroblast-like structures) was obvious (fig. 5a).
Vibrio-like forms associated with granules were visualized in the epidermis (Fig. 3b) or short rods in perineural spaces.
Bizarre, heavily stained borreliae were visualized on serial sections (Fig. 9a-d), intracellularly in a macrophage (Fig 4b.), or in the epidermis (Fig. 3b). Large granules or spherical bodies ("gemmae") 1-3 ,um were detected among collagen fibers (Fig. 5b) comparable to cysts arising after culture experiments (Fig. 5a).
p. 574
The morphological forms of borreliae seen in biopsies were correlated with clinical findings. Seropositive patients showed clumped and agglutinated borreliae in tissue (Fig. 4b), whereas seronegative patients exhibited borreliae colony formation (n=2) (Figs. 7b,8b). Neuralgies arising 6 months after ECM in spite of antibiotic therapy were evident in a seronegative patient who showed perineural rod-like borrelia structures. In ACA samples we identified agglutinated, intertwined spirochetal forms that resembled the clumped, dying borreliae seen in our culture experiments. In areas with inflammatory infiltrates, delicate dispersed, serpentine organisms were seen in degenerating collagen fibers. Also, small granular structures were evident among collagen fibers (Fig. 6b).
In biopsy sections from morphea patients, the number of borreliae was low. Yet, heavily stained intertwined forms and, in one case, clusters of delicate borreliae were seen within collagen fibers (Fig. 2d). Similarly, variant spirochetal forms were present in biopsies of three patients with plaque-like and papular eruptions arising at previous ECM sites. Histologically, two of these biopsies revealed epitheloid cell granulomas: skin sarcoidosis in one and cheilitis granulomatosa in the other patient. The third case represented lymphocytoma of the skin.
p. 576-78
The extracellular location of typical borreliae was not necessarily associated with the presence of an inflammatory infiltrate. Rather, these borrelia forms were seen in seronegative patients with uncomplicated ECM. If the extracellular borreliae were accompanied by an inflammatory infiltrate, the bacteria exhibited a heterogeneous morphology. Heavily stained, clumped, and aggregated borreliae and granules, formed by action of hyperimmune sera, were evident as were degererative changes in the connective tissue.
Whether borreliae can be located intracellularly has been heavily debated. In vitro studies demonstrated that borreliae actively penetrate endothelial cells (31) and fibroblasts, where they apparently evade eradication by antibiotics (32). The presence of borreliae in macrophages and keratinocytes, as shown in our studies and also in Berger's silver staining studies, supports the hitherto unproven concept that borreliae may survive intracellularly (33). Whether borreliae are also present in Langerhans cells could not be elucidated by this technique, although recent data suggest that borreliae invade and selectively damage them, as shown ultrastructurally (2).
The conditions necessary for the development of borrelia granules and their function are still unclear. The granules, which do not typically form under short-term culture conditions, do evolve in solid media (21), as well as in oral spirochetes (20,24), T. pallidum (23), and old acidic cultures of B. burgdorferi (34). Cystic borrelia forms have also been reported in tissue imprints of patients with Alzheimer disease (35). The development of spherical bodies or "gemmae" had been repeatedly observed in meticulous studies of spirochetal organisms over 40 years ago (20,23,24), as well as recently under varying culture conditions (26,36), in skin tissue (1,2), and after exposure to antibiotics (25,37).
Our results lead to several conclusions. First, the videomicroscopy technique described here has made possible the identification of borreliae in situ. Second, the behavior of borreliae within collagen fibers is strongly influenced by immune recognition by the patient. Borreliae may escape immune surveillance by colony formation and masking within collagen, resulting in seroregativity. Furthermore, the bacteria can survive in collagen fibers and cause tissue damage resulting in Iong-standing ACA, even in the presence of anti-Bb antibodies, which are known to kill B. burgdorferi (38). Third, whether the formation of granules or cysts represents a mode of degeneration of borreliae or their persistence is not yet clear. Nevertheless, simply knowing that B. burgdorferi are morphologically diverse may explain the large spectrum of Bb-associated diseases, may indicate a heterogenous immune responses in individuals, and may enhance future immunohistochemical studies of borreliae in animal models."

Invasion and cytopathic killing of human lymphocytes by spirochetes causing Lyme disease.
Dorward DW, Fischer ER, Brooks DM. Clin Infect Dis 1997 Jul; 25 Suppl 1: S2-8. PMID: 9233657

Lyme disease is a persistent low-density spirochetosis caused by Borrelia burgdorferi sensu lato. Although spirochetes causing Lyme disease are highly immunogenic in experimental models, the onset of specific antibody responses to infection is often delayed or undetectable in some patients. The properties and mechanisms mediating such immune avoidance remain obscure. To examine the nature and consequences of interactions between Lyme disease spirochetes and immune effector cells, we coincubated B. burgdorferi with primary and cultured human leukocytes. We found that B. burgdorferi actively attaches to, invades, and kills human B and T lymphocytes. Significant killing began within 1 hour of mixing. Cytopathic effects varied with respect to host cell lineage and the species, viability, and degree of attenuation of the spirochetes. Both spirochetal virulence and lymphocytic susceptibility could be phenotypically selected, thus indicating that both bacterial and host cell factors contribute to such interactions. These results suggest that invasion and lysis of lymphocytes may constitute previously unrecognized factors in Lyme disease and bacterial pathogenesis.

[In vitro study. Beautiful Electron Microscopic pictures of the spirochete attaching the lymfocyte with the tip, invading the lymfocyte and later lysis of the human B- and T- lymfocyte. This maybe implicate that the spirochete is capable of selectively searching out the precursor lymfocytes that carries the antibody-markers of the spirochetes and destroy these, blocking the cloning of the antibody producing cells and hence blocking specific antibody-production forever for instance if memory cells are eradicated?]

The isolation of Borrelia burgdorferi spirochetes from clinical material in cell line cultures.
Tylewska Wierzbanowska S, Chmielewski T. Zentralbl Bakteriol 1997 Oct; 286(3): 363-70. PMID: 9361382

It has been found that B. burgdorferi bacteria multiply in mouse fibroblasts. Mouse fibroblast of the L-929 cell line was inoculated with less than 10 up to 10(4) B. burgdorferi cells and incubated for 2-10 days at 35 degrees C in microaerophilic conditions. Within 2 days, visible growth was observed. The bacteria were present in growth medium and on/in mouse fibroblasts as revealed by the indirect immunofluorescence assay. At the same time, development of vacuolized fibroblastic giant cells was observed. Viable spirochetes were also detected in Eagle's medium from a L-929 fibroblast cell line culture, after approximately 2-5 days of incubation with blood, cerebro-spinal and synovial fluids of Lyme borreliosis patients. The bacteria were present in growth medium and on/in endothelial cells as revealed by the indirect immunofluorescence assay. The establishment of B. burgdorferi culture conditions in cell lines gives us a possibility to isolate the etiological agent of Lyme disease from patient blood, cerebrospinal and synovial fluids at different stages of infection. The high sensitivity of this procedure would be helpful in a proper identification of the infection as well as in the control of treatment effectiveness.

Detection of Borrelia burgdorferi by polymerase chain reaction in synovial membrane, but not in synovial fluid from patients with persisting Lyme arthritis after antibiotic therapy.
Priem S, Burmester GR, Kamradt T, Wolbart K, Rittig MG, Krause A. Ann Rheum Dis 1998 Feb;57(2):118-21. PMID: 9613343   PDF

OBJECTIVES: To identify possible sites of bacterial persistence in patients with treatment resistant Lyme arthritis. It was determined whether Borrelia burgdorferi DNA may be detectable by polymerase chain reaction (PCR) in synovial membrane (SM) when PCR results from synovial fluid (SF) had become negative after antibiotic therapy.
METHODS: Paired SF and SM specimens and urine samples from four patients with ongoing or recurring Lyme arthritis despite previous antibiotic therapy were investigated. A PCR for the detection of B burgdorferi DNA was carried out using primer sets specific for the ospA gene and a p66 gene of B burgdorferi.
RESULTS: In all four cases, PCR with either primer set was negative in SF and urine, but was positive with at least one primer pair in the SM specimens. In all patients arthritis completely resolved after additional antibiotic treatment.
CONCLUSIONS: These data suggest that in patients with treatment resistant Lyme arthritis negative PCR results in SF after antibiotic therapy do not rule out the intraarticular persistence of B burgdorferi DNA. Therefore, in these patients both SF and SM should be analysed for borrelial DNA by PCR as positive results in SM are strongly suggestive of ongoing infection.

COMMENT    4 patients w treatment resistant LA, all seropositive:

1: 50y M, acute arthritis left knee. High Lyme IgG. Doxycycline 200mg/d and diclofenac, and intraarticular dexamethasone. Arthritis persisted. B. burgdorferi DNA in SF, Ceftriaxone 2g/d, 2 weeks. Arthritis improved significantly but recurred after about six weeks. Treated with ceftriaxone again. Although SF PCR became negative, gonarthritis persisted. A popliteal cyst developed and a bursitis of the left elbow occurred. An arthroscopic synovectomy of the left knee and a bursectomy were performed. Ceftriaxone 2g for 28d, doxycyclin2 200 mg for 30 d.

2: 51y F, 2 month history of bilateral gonarthritis, remembered tickbite and EM-like lesion at the right thigh years ago. IgG positive ELISA & Western Blot and B. burgdorferi PCR in SF positive. Doxycycline 200mg/d for 35d without effect. Ceftriaxone 2g/d for 3 wekks w only moderate success. Two mo after treatement gonarthritis persisted. An arthrocentesis and synovial biopsy was performed.

3: 28y F, subtotal arthroscopic synovectomy of the right knee had been performed elsewhere because of a gonarthritis with pronounced synovial proliferation, persisted for 9 months. Positive Lyme serology both IgM & IgG. Ceftriaxone 2g 14 d. treatment discontinued at day 11 because of an allergic rash. Gonarthritis persisted. PCR positive in SF doxycycline 200mg/d for 30d. In addition corticosteroid intraarticularly. However only a temporary improvement of the arthritis was seen. Arthroscopy, SF and SM samples.

4: 43y F, bilateral gonarthritis 7 mo, ELISA & Western Blot high IgG, B. burgdorferi in SF and urine positive. Ceftriaxone 2g 14d without improvement. A few weeks later the patient was seen in another hospital with an acute polyarthritis involving both wrists and several metacarpophalangeal joints and a deterioration of the right gonarthritis. Doxycycline 200mg/d 30d and prednisolone 10-20 mg/d. Again there was no sufficient response. A closed needle arthrocentesis with synovial biopsy of the right knee was performed.

No mentioning of any extraarticular symptoms of Borreliosis?

Patients were evaluated 8 to 10 weeks after antibiotic therapy. All still seropositive and had active arthritis. Urine samples were collected and within one week SF and SM specimens were obtained ...

In none of the urine or SF samples could B. burgdorferi DNA be detected, in contrast SM samples was positive.

Patients 1,2,4: cefotaxime 2g x3 3 weeks, followed by six weeks oral doxycycline or minocycline 200mg/d. Pt. 3: imipenem 1.0g x3 for 2 weeks, doxycycline 200mg six weeks. In all four patients arthritis completely subsided within 4-6 mo and did not recur at a median observation period of 18 mo.

Ultrastructural demonstration of intracellular localization of Borrelia burgdorferi in Lyme arthritis.
Chary-Valckenaere I, Jaulhac B, Champigneulle J, Piemont Y, Mainard D, Pourel J. Br J Rheumatol 1998 Apr;37(4):468-70. PMID: 9619908  PDF
(unfortunately the downloadable file lack part of picture on page 469 and page 470 with the references - have written to Br J Rheum about it – no correction of the error !!)

Abstract NA

“We report here the
first demonstration, to our knowledge, of B. burgdorferi in synovial cells of a Lyme arthritis patient using chromosomal DNA detection and ultrastructural study of the synovium.”

Interaction of Borrelia burgdorferi with Peripheral Blood Fibrocytes, Antigen-Presenting Cells with the Potential for Connective Tissue Targeting.
Grab DJ, Lanners H, Martin LN, Chesney J, Cai C, Adkisson HD, Bucala R. Mol Med 1999 Jan;5(1):46-54. PMID: 10072447

Background: Borrelia Burgdorferi has a predilection for collagenous tissue and can interact with fibronectin and cellular collagens. While the molecular mechanisms of how B. burgdorferi targets connective tissues and causes arthritis are not understood, the spirochetes can bind to a number of different cell types, including fibroblasts. A novel circulating fibroblast-like cell called the peripheral blood fibrocyte has recently been described. Fibrocytes express collagen types I and III as well as fibronectin. Besides playing a role in wound healing, fibrocytes have the potential to target to connective tissue and the functional capacity to recruit, activate, and present antigen to CD4(+) T cells. Materials and Methods: Rhesus monkey fibrocytes were isolated and characterized by flow cytometry. B. burgdorferi were incubated with human or monkey fibrocyte cultures in vitro and the cellular interactions analyzed by light and electron microscopy. The two strains of B. burgdorferi studied included JD1, which is highly pathogenic for monkeys, and M297, which lacks the cell surface OspA and OspB proteins.
Results: In this study, we demonstrate that B. burgdorferi binds to both human and monkey (rhesus) fibrocytes in vitro. This process does not require OspA or OspB. In addition, the spirochetes are not phagocytosed but are taken into deep recesses of the cell membrane, a process that may protect them from the immune system.
Conclusions: This interaction between B. burgdorferi and peripheral blood fibrocytes provides a potential explanation for the targeting of spirochetes to joint connective tissue and may contribute to the inflammatory process in Lyme arthritis.

Persistence of Borrelia garinii and Borrelia afzelii in patients with Lyme arthritis
Hulinska D, Votypka J, Valesova M. Int J Med Microbiol Virol Parasitol Infect Dis 1999 Jul;289(3):301-18. PMID: 10467661

We repeatedly detected DNA of Borrelia garinii or B. afzelii and Borrelia-like structures in the blood, joint fluid or in the synovium of 10 patients with Lyme arthritis by means of the polymerase chain reaction and immunoelectron microscopy at 2-4-month intervals in the course of two years. All samples were analyzed using primers which amplified the 16S rRNA gene sequence of Borrelia burgdorferi sensu lato and nucleotide sequences for the OspA gene. No cross hybridization occurred with DNA from human cells and with DNA from other bacteria. Capture and labelling with monoclonal antibodies of aggregated antigens, membranes and flagellae were evident in the blood of 7 patients, in 4 synovial membranes and 2 synovial fluids. Borreliae were found in blood capillaries, in collagen and in clusters surrounding inflammatory cells in the synovium of patients with recurrent infections who carried IgM and IgG antibodies to OspA and to 83 kDa core protein. After significant improvement for several weeks after treatment, arthritis recurred in six patients. Synoviocyte hyperplasia, inflammatory infiltration and concentric adventitial fibroplasia were seen in the synovium of the patients with persisting borreliae. Only two patients were infected with B. afzelii, the others with B. garinii.

Detection of Borrelia DNA in Circulating Monocytes as Evidence of Persistent Lyme Disease
Wang P, Gartenhaus R, Sood SK, DeVoti J, Singer C, Dorante G, Hilton E. J Spiro Tick Diseases 7(1):16-19, 2000. 2000-js016

We report the detection of Borrelia burgdorferi DNA in circulating monocytes in a 31-year-old female who presented with a flu-like syndrome followed by neurological abnormalities after a trip to Southampton, Long Island, New York. ELISA and Western blot were negative. Lymphocyte proliferation assay to Borrelia burgdorferi was positive. Borrelia burgdorferi DNA was detected in circulating monocytes using a nested polymerase chain reaction (PCR). Treatment with parenteral ceftriaxone resulted in clinical improvement and repeat PCR on monocytes was negative. The use of detecting DNAby PCR from circulating monocytes may be useful in evaluating seronegative patients with a high suspicion of Lyme disease.

Insights from a novel three-dimensional in vitro model of lyme arthritis: standardized analysis of cellular and molecular interactions between Borrelia burgdorferi and synovial explants and fibroblasts.
Franz JK, Fritze O, Rittig M, Keysser G, Priem S, Zacher J, Burmester GR, Krause A. Arthritis Rheum 2001 Jan;44(1):151-62. PMID: 11212153

OBJECTIVE: To develop a novel 3-dimensional (3-D) in vitro model of Lyme arthritis to use in the study of the interactions between Borrelia burgdorferi (Bb) and human synovial host cells with respect to phagocytosis and potential persistence of Bb as well as the induction of proinflammatory cytokines and chemokines. METHODS: Two distinct culture systems, consisting of synovial membrane explants or interactive synovial cells embedded in 3-D fibrin matrices, were chosen. Both systems were artificially infected with Bb, and the interactions between Bb and synovial tissue/cells were studied by histology, immunohistochemistry, and electron microscopy. Functional analyses included the induction/secretion of cytokines by Bb in the model system. RESULTS: Both culture systems proved to be stable and reproducible. The host cells and spirochetes showed high levels of viability and maintained their physiologic shape for >3 weeks. Bb invaded the synovial tissue and the artifical matrix in a time-dependent manner. Host cells were activated by Bb, as indicated by the induction of interleukin-1beta and tumor necrosis factor alpha. Electron microscopic analysis revealed Bb intracellularly within macrophages as well as synovial fibroblasts, suggesting that not only professional phagocytes, but also resident synovial cells are capable of phagocytosing Bb. Most interestingly, the uptake of the spirochetes appeared to cause severe damage of the synovial fibroblasts, since the majority of these cells displayed ultrastructural features of disintegration. CONCLUSION: A novel 3-D in vitro model has been established that allows the study of distinct aspects of Lyme arthritis under conditions that resemble the pathologic condition in humans. This reproducible, standardized model supplements animal studies and conventional 2-D cultures. The disintegration of synovial fibroblasts containing Bb or Bb fragments challenges the concept of an intracellular persistence of Bb and may instead reflect a mechanism that contributes to the inflammatory processes characteristic of Lyme arthritis.