Intracellular
localisation of Borrelia burgdorferi
This
list may robably not complete – if you find that an important article,
that demonstrate
intracellular Borrelia is
missing in the list, please send a note to kroun@ulmar.dk
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
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
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.
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
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.
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. 1995-js082
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.
Excerpts:
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. ……
B. burgdorferi has been reported in neutrophil phagosomes in vitro
(2, 30) and in an intracellular vacuole in Langerhans cells from the skin of
Lyme disease patients (17); intravacuolar destinations are also documented for
other spirochetes, including T. pallidum (24, 34). We have localized
spirochetes within macrophage lysosomes and demonstrated their concomitant
degradation within lysosomes by ultrastructural analysis. Thus, our studies
lend no support to an unknown nonlysosomal mechanism that has been invoked for
degradation of spirochetes within macrophages (31).
It remains for further exploration to explain the puzzling coexistence in vivo
of intact extracellular spirochetes and phagocytes in chronically infected
tissues. Spirochetes may evade phagocytic clearance by varying recognizable
outer surface proteins, as we have recently demonstrated (26), or by
eliminating them from their surfaces entirely, as has been observed in mutant
spirochetes in vitro (33). Many other infectious microorganisms employ these
effective strategies, including Borrelia hermsii, the agent of relapsing
fever (4).
Alternatively, or in addition, Lyme infection may impede macrophage function
via the action of cytokines, resulting in reduced phagocytic clearance of
infectious agents, as is seen, for
example, in leishmaniasis (1).
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
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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.
Excepts:
The intracellular residence of a bacterium offers protection from both host
defense mechanisms and antibiotics. B.
burgdorferi isolates adhere to, invade, and
survive in human endothelial cells (1, 4, 10). In vivo B.
burgdorferi has been observed in cardiac myocytes of experimentally
infected mice (5). In our study, while penicillin G and ceftriaxone appeared to
be very efficient in axenic medium, they lost their efficiencies when B.
burgdorferi was cultivated in the presence of cells, confirming the
protective effects of cells from the actions of ceftriaxone and penicillin (3).
When B. burgdorferi was cultivated in the presence of cells, the cell
count was reduced dramatically by erythromycin and doxycycline, indicating
their superiority in this situation.
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."
Excerpts:
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 µm 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 if, for instance, 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.
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, but no
correction of the error !!)
Abstract NA
Excerpts:
“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.” …
Although no B. burgdorferi DNA was detected in SF or CSF, it was
detected in SM removed 12 days after the onset of arthritis in the knees. …
Several electron-dense filamentous structures, some
of which were over 2 mm long and 250 nm wide, were detected in the cytoplasm of
fibroblast-like cells of the connective tissue (Fig. 1) and in one
macrophage-like synoviocyte (Fig. 2). Dense and fibrillar at the
centre, and enclosed by a thick dense envelope separated from the thin outlying
membrane by an electrolucent space, they resembled B. burgdorferi.
…
Experimental studies have shown that the spirochaete can penetrate human
resident synovial cells and survive there despite exposure to ceftriaxone,
which eradicates only extracellular pathogens [10]. The
present case suggests that B. burgdorferi may also be able to shelter in
synovial cells after an incomplete course of antibiotic therapy.
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.
[This work
actually demonstrates the formation of cystic forms of borrelia in fig 4.
Culture of Kupffer cells, that had been washed 3 times with Ca- and Mg- free
Hanks balanced salt solution, for 3 weeks at 34 degr. Celcius in BSKII medium
may not be adequate to make L-forms revert to mobile spirochetes again? Phillips et al [PMID: 9861561]
were only able to culture the cystic forms of borrelia if tap water was used
for the culture media instead of distilled water, Ca and Mg may be needed in
the growth media if L-forms are to re-convert to spirochaetal form again?]