Dorwar et al: "Invasion & Cytopahtic Killing......." (v long)

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Rita Stanley

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Jan 12, 1998, 3:00:00 AM1/12/98
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Manyt thanks to MK and Adrianna in their help ingetting this article out to
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Rita
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Invasion and Cytopathic Killing of Human Lymphocytes by Spirochetes Causing
Lyme Disease

David W. Dorward, Elizabeth R Fischer, and Diane M. Brooks

Clinical Infectious Diseases 1997;25(Suppl 1):S2-8

From the National Institute of Allergy and Infectious Diseases. Rocky
Mountain Laboratories, Hamilton. Montana

ABSTRACT: 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 I 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
Iymphocytic 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.

BACKGROUND: Following its discovery as the agent of Lyme disease [1],
Borrelia burgdorferi sensu lato was subdivided into several genospecies,
including the known human pathogens B. burgdorferi sensu stricto
(hereinafter B. burgdorferi), Borrelia garinii, and Borrelia afzelii
(formerly group VS461) [2, 3]. Human infection by these tick-borne agents
progresses slowly from localized dermatologic involvement to a persistent
low-density multisystemic spirochetosis. Because spirochetes causing Lyme
disease can establish chronic infections in otherwise healthy and
immunocompetent individuals, many investigators believe that these bacteria
can occupy immune-privileged niches or otherwise modify and evade immune
responses. Although the mechanisms of immune evasion and modifications are
not fully understood interactions between B. burgdorferi and several types
of mammalian cells and factors have been described.

Previous studies have shown that B. burgdorferi can attach to a variety of
mammalian cells [4-6], invade fibroblasts [4], bind host proteins onto their
surfaces [7-10], and alter secretion of host cytokines [11-14] and
antibodies [15]. Invasion of fibroblasts is relatively benign, involving
intracellular penetration and possible long-term intracellular survival [4].
Invasion of endothelial cells with nonlytic escape was reported [6], but
this finding was later disputed [7]. It is believed that the intracellular
survival of B. burgdorferi may function in chemotherapeutic resistance and
interference with immune clearance [4,6, 16].

In a recent study by Schwan and co-workers [17], it was also reported that
antigenic phase changes involving surface-exposed lipoproteins may occur.
Using infected ticks, these researchers found that upregulation of the
expression of B. burgdorferi outer-surface protein C occurred while ticks
fed on mammalian blood. Temperature was believed to mediate this change.
Furthermore, they reported a concurrent decrease in the apparent expression
of the known protective imrnunogen outer-surface protein A on spirochetal
surfaces.

Cell-surface binding of host proteins such as naive IgM antibodies [8],
urokinase, and fibrinogen [9, IO] may also interfere with immune clearance
by providing immunologic "camouflage" and by facilitating migration of the
spirochetes through interstitial spaces. Induction of inflammatory cytokines
[11-13] and mitogenesis of polyclonal B cells [14, 15] suggest that Lyme
disease spirochetes can manipulate and modify the immune response to
infection. However, current understanding of direct interactions between
spirochetes and lymphocytes is limited. In this study, we examined
interactions between Borrelia species and human leukocytes and assessed
cytopathic effects. We found that B. burgdorferi and B. garinli specifically
attached to, invaded, and killed significant proportions of human B and T
cells.

METHODS
Unless otherwise specified, all reagents were obtained from the Sigma
Chemical Company in St. Louis. The spirochetes and eukaryotic cells used in
this study are described in table 1. All spirochetes were cultured in
modified Barbour-StoennerKelly medium as previously described [18]. Human
cell lines were obtained from the American Type Culture Collection
(Rockville, MD) and propagated according to their instructions. Media for
tissue cultures and certified fetal bovine serum were provided by Life
Technologies (Gaithersburg, MD). Primary human mononuclear cells were
obtained from laboratory volun-teers and prepared by centrifugation in
lymphocyte separation medium (Organon Teknika, Durham, NC) according to the
manufacturer's recommendations. Primary B or T cells were purified from
mononuclear cell preparations by using anti B cell (CDI9) or anti T cell
(CD4 and CD8) immunomagnetic beads (Dynal, A. S., Oslo), respectively.
Eukaryotic cells were quantified by counting in Petroff-Hausser counting
chambers. The number of spirochetes was estimated by means of ab-sorbance at
6OO nm as previously described [19].

Coincubation mixtures typically contained 2 X I,000,000 host cells and 2 X
100,000,000 spirochetes per milliliter of medium. Experimental parameters
that were varied included the species, concentration, and in vitro passage
number of spirochetes; the coincubation period; and the number of sequential
"reinfections" with a single Iymphocytic population or a single spirochetal
popula-tion. Low and high passages were defined as less than eight or more
than 3O in vitro passages, respectively.

For some experiments, B cells resistant to attachment and killing by Lyme
disease spirochetes were enriched by mixing with fresh low-passage B.
burgdorferi every 24 hours for three consecutive days. Similarly,
spirochetes with affinity for B cells were enriched by using differential
centrifugation to recover B cells from coincubation mixtures maintained for
1.5 hours. Cell pellets containing cell-associated spirochetes were washed
in and finally resuspended in Barbour-Stoenner-Kelly medium. Host cell
viability was assessed microscopically by trypan blue exclusion or by flow
cytometric analysis with use of propidium iodide staining [2O].

Attachment and invasion by spirochetes were monitored by light and electron
microscopy. Light microscopy was per-formed on wet mounts by using a Nikon
FXA photomicroscope equipped with a Nomarski differential interference
contrast condenser (Nikon, Tokyo). Digital micrographs were recorded by
means of a Dage-MTI CCD 72 camera and a DSP2OOO image processor (Dage-MTI,
Michigan City, IN).

For scanning electron microscopy, lymphocytes, bacteria, or coincubation
mixtures were concentrated by centrifugation at 1,OOOg for 3 minutes in a
microfuge. Pellets were gently resus-pended and washed once in Tyrode's
buffer (pH, 7.2) [21], and 7O microL of cell suspension was allowed to
settle onto coverslips previously coated with O.O1% poly-L-lysine in water.
After S minutes, much of the buffer was removed, and the cells were fixed in
place by adding 5O microL of 2.5% glutaraldehyde m O.2 M cacodylate (pH,
7.2). Such coverslips were processed further by standard procedures [22].
Samples were examined with a Hitachi S-45OO field emission scanning electron
microscope (Hitachi, Tokyo) operated at 5 kV.
For transmission electron microscopy, equivalent samples were collected by
centrifugation and washed as above. Cell pellets were fixed with 2.5%
glutaraldehyde and 4%p-formal-dehyde in O. 1 M cacodylate (pH, 7.2)
containing O. 1 M sucrose. Pellets were postfixed in 1% OsO4, dehydrated,
embedded in Spurr's resin, and prepared for thin sectioning according to
standard procedures [23]. Sections were examined with a Phil-ips CM1O
transmission electron microscope (Philips Electron optics, Eindhoven, The
Netherlands) operated at 8O kV.

Host cells and coincubation mixtures were prepared for
flu-orescence-activated cell sorting according to previously re-ported
procedures [2O]. Primary leukocyte lineage was deter-mined by flow
cytometric analysis of mixed mononuclear cells
labeled with fluorescein isothiocyanate or phycoerythrin conju-gates of
monoclonal antibodies to CD19 (pan B cells), CD5 (pan T cells and activated
B cells), CD14 (monocytes), or CDI5 (granulocytes) cell-surface markers
according to the manufacturer's instructions (Immunotech, Westbrook, ME).

NOTE. For this study, low and high passages were defimed as less them eight
and more than 30 m vitro passages, respectively. Spirochetal isolates were
not cloned for them experiments. Enriched B. burgdorferi refers to
spirochetes that were recovered in association with SKW 6.4 cells after
three sequential cycles of a 90-minute coincubation period and differential
centrifiugation. Enriched B cells were lymphocytes selected by survival
after three sequential 24-hour coincubation periods with a lOO-fold excess
of low-passage B. burgdorferi spirochetes. ATCC = American Type Culture
Collection; NA = not applicable; ND = not determined.

RESULTS
To examine the interactions between Lyme disease spiro-chetes and primary
and cultured human Iymphocytes, Borrelia species and host cells were mixed
at a multiplicity of infection of IOO; the mixtures were incubated for
varying periods, and changes to the bacteria and cells were assessed by
light and electron microscopy (figure 1). The micrographs in figure I are
representative of structures and events that were observed repeatedly in
such preparations.

Adherence to and invasion of Iymphocytes occurred within 1-2 hours of
coincubation. As previously reported for other cell lines [4-7], light
microscopy with use of a differential interference contrast condenser showed
that low-passage B. burgdorferi strain Sh-2-82 attached (via the tips) to
Iympho-cytic surfaces (figure la). Scanning electron microscopy showed
adherent spirochetes on >9O% of both cultured and primary B and T cells
(data not shown). Host cell penetration appeared to occur at sites of
endocytotic pits (figure lb).

Spirochetes protruding into invaginations, consistent with endo-cytotic
pits, were also observed by transmission electron micros-copy of thin
sections (data not shown). No structural perturbations were observed on
adherent spirochetes. In contrast, evidence of surface penetration by
low-passage B. burgdorferi corresponded with loss of filopodia and other
surface projections on Iymphocytes.

Transmission electron microscopy revealed intracellular spi-rochetes
contained within vacuoles (figure Ic). No evidence of Iysosomal fusion with
such vacuoles was observed. Video microscopy demonstrated marked motility of
spirochetes con-fined within vacuoles of invaded SKW 6.4 cells (data not
shown). However, we found no spirochetes that were clearly free within the
cytosol of intact cells. Numerous Iymphocytes with disrupted cell membranes
were evident in coincubation mixtures with low-passage B. burgdorferi
(figure Id). No sig-nificant cytopathic effects were observed with
Iymphocytes incubated with high-passage bacteria (data not shown) or
unin-fected Iymphocytes (figure le).

The lineages of primary human mononuclear cells that were killed by B.
burgdorferi were identified by flow cytometry. Averaging the findings for
two independent experiments in-volving coincubation mixtures with
low-passage B. burgdorferi and mixed mononuclear cells that were maintained
for 2 hours showed that 53.O% of CDI9+ B cells and 21.O% of CDS+ T cells
were permeable by propidium iodide, compared with 8.3% and 2.8%,
respectively, in uninfected mononuclear cell preparations. An average of
IO.4% of CDI9+ B cells and 13.3% of CDS+ T cells were permeable in
coincubation mixtures with high-passage B. burgdorferi. Dual fluorescence
between propidium iodide and either CD14+ monocytes or CDIS+ gran-ulocytes
was not significantly greater than background levels observed in uninfected
cells.

The kinetics of killing of purified primary Iymphocytes, as assessed by
trypan blue exclusion, are shown in figure 2. Pri-mary B cells and T cells
were fractionated by using either antibodies to CDI9 immunomagnetic beads or
pooled antibod-ies to both CD4 and CD8 immunomagnetic beads. Flow cytom-etry
showed that >99% of cells in each B or T cell fraction expressed either CDI9
or CD5 cell-surface markers, respec-tively (data not shown). Low-passage B.
burgdorferi killed proportions of both classes of Iymphocytes. Spirochetes
ap-peared to kill B cells more rapidly than T cells. The percentages of dead
Iymphocytes in mixtures containing high-passage spi-rochetes were not
significantly greater than those found in uninfected control Iymphocytes.

The effects of coincubation on cultured SKW 6.4 (Burkitt's Iymphoma) cell
lines are shown in figure 3. Up to 4O% of SKW 6.4 cells were Iysed after
coincubation with low-passage B. burgdorferi. Killing of Iymphocytes peaked
during the first day of coincubation. Killing was reduced at infection
ratios of IO:I and 1:1 and was insignificant at lower ratios (data not
shown). No significant killing occurred in mixtures of H9 cells and
low-passage spirochetes (not shown), mixtures of SKW 6.4 cells and
high-passage B. burgdorferi, or mixtures of SKW 6.4 cells and low-passage
Borrelia hermsii, an agent of tick-borne relapsing fever. Reduced killing at
later times reflected continued growth by SKW 6.4 cells that survived the
initial infection and exhibited significant resistance to subsequent
re-infection (see below).
By sequentially reinfecting B cells with low-passage B. burg-dorferi and,
conversely, sequentially enriching for attached or intracellular spirochetes
by differential centrifugation, we ob-tained populations of both SKW 6.4
cells that resisted attach-ment and killing and Lyme disease spirochetes
that exhibited enhanced virulence against SKW 6.4 cells (figure 3, right).
In coincubations of enriched spirochetes and enriched Iympho-cytes, the
spirochetes appeared to overcome much of the resis-tance exhibited by
Iymphocytes.

The qualitative results of coincubation and killing by spiro-chetes are
summarized in table 2. Only coincubations with low-passage or enriched Lyme
disease spirochetes resulted in significant killing of Iymphocytes. Although
killing occurred with B. burgdorferi and B. garinii (IP9O), killing was not
ob-served in coincubations with either B. hermsii or an infectious isolate
of B. afzelii (ACAI). It was also notable that primary T cells and H9 cells
differed in terms of susceptibility. Prelimi-nary experiments suggest that
the rates of initial attachment to Iymphocytes and subsequent killing of
Iymphocytes may correlate with the expression of a cell-surface integrin (D.
W. Dorward and E. R. Fischer, unpublished data).

DISCUSSION

Figure 1. Micrographs reveal-ing attachment to and invasion of Iymphocytes
by Borrelia burgdorf-eri. Cultured SKW 6.4 cells and primary human
peripheral B cells were coincubated with virulent or attenuated B.
burgdorferi for I hour; the mixtures were then pre-pared for and examined by
light or electron microscopy. The micro-graphs are representative of each
susceptible host cell population. a: Light microscopy revealed attach-ment
of spirochetal tips to SKW 6.4 cells; attached spirochetes re-mained highly
motile yet anchored to host cells. b: Scanning electron microscopy revealed
that adherent spirochetes penetrated Iympho-cytes through endocytotic pits;
penetrated Iymphocytes exhibited a noticeable loss of filopodia and other
surface projections. c: Transmission electron microscopy showed that
intracellular spiro-chetes were retained within vacu-oles; no fusion of
Iysosomes to en-docytotic vacuoles was observed. d: Continued coincubation
with virulent spirochetes resulted in nu-merous Iymphocytes with dis-rupted
cell membranes. e: No such cytopathic changes were observed with uninfected
control Iympho-cytes. No such cytopathic changes were observed with
Iymphocytes incubated with Borrelia hermsii or attenuated B. burgdorferi
(not shown). Bars = I micrometer.

Our results indicate that Lyme disease spirochetes can selec-tively attack
and kill purified peripheral human Iymphocytes and SKW 6.4 B cells. Although
internalization of Chlamydiatrachomatis into vacuoles by certain Iymphocytic
cell lines has been reported, chlamydiae neither killed nor grew within host
Iymphocytes [24]. We could find no previous reports of aggres-sive, invasive
cytopathology in Iymphocytes caused by bacteria. Interactions between
Iymphocytes and B. burgdorferi in-volved tip-directed attachment. It was
unclear whether attach-ment was solely initiated at the tip or whether sites
of adherence
on the spirochetes could migrate to the tips after attachment. Invasion
progressed through endocytotic pits into vacuoles.
Invaded Iymphocytes exhibited dramatic morphological changes such as loss of
surface projections and disruption of the cell membrane. Although
supernatants from cultures of low-passage B. burgdorferi and heat-killed
spirochetes do not appear to kill Iymphocytes in vitro (D. W. Dorward and E.
R. Fischer, unpublished data), it is unclear whether cellular inva-sion is a
prerequisite for the cytopathology observed in this study. Furthermore,
because of the severe cytopathic effects observed, we could not rule out the
possibility that spirochetes may also penetrate host cell cytosol. Clearly,
the dynamics and mechanisms of attachment to, invasion of, and killing of
Iymphocytes warrant further investigation.

Flow cytometry of mixed mononuclear cell preparations showed that B.
burgdorferi killed significant numbers of cells expressing CDS and CDI9, but
not CD14 or CD15, cell-surface markers. These markers are characteristic for
pan T cells and activated B cells, pan B cells, monocytes, and granulocytes,
respectively. Possible pathogenic effects in subsets of these cells have yet
to be determined. The finding that neither mono-cytes nor granulocytes were
susceptible is consistent with pre-vious reports that B. burgdorferi is
effectively internalized and destroyed by phagocytes [25, 26]. In kinetic
experiments with purified primary Iymphocytes, we found that CDI9+ B cells
were killed significantly faster than CD4+ and CD8+ T cells. Whether
differing rates of killing by spirochetes reflected dif-ferences in
expression of factors mediating attachment or inter-nalization, procedural
manipulations, or other factors remains to be determined. However, variable
Iymphocytic susceptibility and passage-dependent spirochetal virulence
indicated that the process of attachment, invasion, and killing involves
both Iymphocytic and bacterial factors.

Coincubation experiments with SKW 6.4 B cells demon-strated that spirochetal
virulence and Iymphocytic resistance could be phenotypically selected. The
nature of these factors has not been determined. However, since killing was
not evi-dent in coincubations with attenuated spirochetes, the process of
attachment to, invasion of, and Iysis of Iymphocytes appar-ently involves
factors that are nonessential for growth in vitro.

Differences in killing of Iymphocytes by different species of Borrelia were
noted in this study. Although both the B. hermsii isolate (tick-borne
relapsing fever agent) and the B. afzelii iso-late (Lyme borreliosis agent)
that were used were infectious in mice [23], neither isolate caused an
increase in the number of killed SKW 6.4 cells. B. hermsii, like other
relapsing fever agents, can repeatedly reach levels of 10 to the 6-7 per
milliliter of peripheral blood in patients [27]. At those levels, if
Iympho-cytes were susceptible to killing by spirochetes causing relaps-ing
fever, severe immune deficiencies in patients could be ex-pected. Such
consequences are inconsistent with typical clinical manifestations. We also
found differences in killing of Iympho-cytes by Lyme disease spirochetes.
Further investigation with use of additional isolates should elucidate
whether such differ-ences correlate with observed predilections of B.
afzelii for dermatologic manifestations and of B. burgdorferi and B.
gari-nii for disseminated disease [28].

It is currently unknown whether invasion and killing of Iym-phocytes occur
during natural infections. However, although it is unlikely that the low
numbers of Lyme disease spirochetes in infected mammals would cause
significant Iymphopenia or noticeable immune deficiencies, the ability to
invade Iympho-cytes could provide an effective niche for avoiding immune
detection and clearance. Humoral immunity is protective in laboratory
animals [29]; in contrast, humoral responses are typically delayed in
patients. In vitro spirochete-leukocyte in-teractions may provide an
effective model for understanding the delayed immune response in humans when
they are infected by Lyme disease spirochetes.

Acknowledgments

The authors thank Stanley Falkow and Ted Hackstadt for critical review of
this manuscript, Tom Schwan and Alan Barbour for providing bacterial
strains, Ted Hackstadt for video microscopy, and Robert Evans and Gary
Hettrick for graphic support.

References

1. Burgdorfer W. Barbour AG, Hayes SF, et al. Lyme disease—a tick-borne
spirochetosis? Science 1982;216:1317-9.
2. Baranton G. Postic D, Saint Girons 1, et al. Delineation of Borrelia
burg-dorferi sensu stricto, Borrelia garinii sp. nov., and group VS 461
associ-ated with Lyme borreliosis. Int J Syst Bacteriol 1992;42:378-83.
3. Godffoid E, Ben Messaoud A, Poliszczak A, Lobet Y. Bollen A. Assign-ment
of Borrelia burgdorferi strains G2S and VS461 to the Borrelia garinii and
Borrelfa afzelii genospecies, respectively: a comparison of ospA protein
sequences. DNA Seq l99S;5:2SI-4.
4. Georgilis K, Peacocke M, Klempner MS. Fibroblasts protect the Lyme
disease spirochete, Borrelia burgdorferi, ffom ceftriaxone in vitro. J
Infect Dis 1992;166:44O-4.
5. Coburn J. Leong J. Erban J. Integrin alpha llb beta 3 mediates binding of
the Lyme disease agent Borrelia burgdorferi to human platelds: Proc Natl
Acad Sci USA 1993;9O:7O58-63.
6. Comstock LE, Thomas DD. Pendration of endothelial cell monolayers by
Borrelia burgdorferi. Infect Immun 1989;S7:1626-8.
7. Szczepanski A, Furie MB, Benach JL, Lane BP, Fleit HB. Interaction
between Borrelia burgdorferi and endothelium in vitro. J Clin Invest
199O;8S:1637-47.
8. Dorward DW, Huguenel ED, Davis G. Garon CF. Interactions between
extracellular Borrelia burgdorferi proteins and non-Borrelia-directed
immunoglobulin M antibodies. Infect Immun 1992;6O:638-44.
9. Hu LT, Perides G. Noring R. Klempner MS. Binding of human plasmino-gen to
Borrelia burgdorferi. Infect Immun l99S;63:3491-6.
IO. Fuchs H. Wallich R. Simon MM, Kramer MD. The outer surface protein A of
the spirochete Borrelia burgdorferi is a plasmin (ogen) receptor. Proc Natl
Acad Sci USA 1994;91:12S94-8.
11. Beck G, Benach JL, Habicht GS. Isolation of interleukin I ffom joint
fluids of patients with Lyme disease. J Rheumatol 1989;16:8OO-6.
12. Habicht GS, Katona Ll, Benach JL. Cytokines and the pathogenesis of
neuroborreliosis: Borrelia burgdorferi induces glioma cells to secrete
interleukin-6. J Infect Dis 1991;164:S68-74.
13. Tai KF, Ma Y, Weis J. Nommal B-lymphocytes and mononuclear cells respond
to the mitogenic and cytokine-stimulatory effects of Borrelia burgdorferi
and its lipoprotein ospA. Infect Immun 1994;62:S2O-8.
14. Yang L, May Y, Schoenfeld R, et al. Evidence for B-lymphocyte mitogen
activity in Borrelta burgdorferi-infected mice. Infect Immun 1992;6O:
3O33-41.
I 5. Whitmire WE, Garon CF. Specific and non-specific response of murine B
cells to membrane blebs of Borrelia burgdorferi. Infect Immun 1993; 61:
146O-7.
16. Isberg RR, Tran Van Nhieu G. Two mammalian cell intemalization
strate-gies used by bacteria. Annu Rev Genet 1994;27:39S-422.
17. Schwan TG, Piesman J, Golde WT, Dolan MC, Rosa PA. Induction of an outer
surface protein on Borrelia burgdorferi during tick feeding. Proc Natl Acad
Sci USA 199S;92:29O9-13.
18. Pollack RJ, Telford SR, Spielman A. Standardization of medium for
cultur-ing Lyme disease spirochetes. J Clin Microbiol 1993;31:12SI-S.
19. Schwan TG, Burgdorfer W. Antigenic changes of Borrelia burgdorferi as a
result of in vitro cultivation. J Infect Dis 1987; IS6:8S2-3.
2O. Spangrude GJ, Brooks DM. Mouse strain variability in the expression of
the hematopoietic stem cell antigen LY-6Y/E by bone marrow cells. Blood
1993;82:3327-32.
21. Cuns JH. Cell separation methods in hematology. New York: Academic
Press, 197O.
22. Dey S. Basu TS, Boyde A, et al. Basic biological preparation tech-niques
for SEM. In: Robards AW, Wilson AJ, eds. Procedures in electron microscopy.
Vol. 1. New York: Wiley, 1993:11:O.I-II: 4.17.
23. Bowers B. Caceci VA, Coetzee J. et al. Basic biological preparation
techniques for TEM. In: Robards AW, Wilson AJ, eds. Procedures in electron
microscopy. Vol 1. New Yor}: Wiley, 1993:S:O.I-S: 9.1O.
24. Bard JA, Levid D. Binding, ingestion, and multiplication of Chlamydia
trachomatis (L2 serovar) in human leuLocyte lines. Infect Immun 1985;
SO:93S-7.
25. Kenefick KB, Lederer JA, Schell RF, Czuprynski CJ. Borrelia burgdorferi
stimulates release of mterleukin-l activity from bovine peripheral blood
monocytes. Infect Immun 1992;6O:363O-4.
26. Rinig MG, Haupl T. Krause A, et al. Borrelia burgdorferi-induced
ultra-structural alterations in human phagocytes: a clue to pathogenicity? J
Pathol 1994;173:269-82.
27. Barbour A, Hayes SE. Biology of Borrelia sp. Microbiol Rev 1986;SO:
381 -4OO.
28. van Dam AP, Kuiper H. Vos K, et al. Different genospecies of Borrelia
burgdorferi are associated with distinct clinical manifestations of Lyme
borreliosis. Clin Infect Dis 1993;17:7O8-17.
29. Fikrig E, Barthold SW, Kantor FS, Flavell RA. Long term protection of
mice from Lyme disease by vaccination with ospA. Infect Immun 1992;
6O:773 -7.

This work was described in part in an abstract for the 54th annual meeting
of the Microscopy society of America held in Minneapolis on I l - l s August

Reprints or correspondence: Dr. David w. Dorward, National Institute of
Allergy and Infectious Diseases, Rocky Mountain Laboratories, 9O3 South 4th
Sheet, Hamilton, Montana 5984O.

This article is in the public domain.


LymeFightr

unread,
Jan 13, 1998, 3:00:00 AM1/13/98
to

At those levels, if Iympho-cytes were susceptible to killing by spirochetes
causing relaps-ing fever, severe immune deficiencies in patients could be
ex-pected.

Such consequences are inconsistent with typical clinical manifestations. We
also
found differences in killing of Iympho-cytes by Lyme disease spirochetes.

Further investigation with use of additional isolates should elucidate
whether such differ-ences correlate with observed predilections of B.
afzelii for dermatologic manifestations and of B. burgdorferi and B.
gari-nii for disseminated disease [28].

It is currently unknown whether invasion and killing of Iym-phocytes occur
during natural infections. However, although it is unlikely that the low
numbers of Lyme disease spirochetes in infected mammals would cause
significant Iymphopenia or noticeable immune deficiencies, the ability to
invade Iympho-cytes could provide an effective niche for avoiding immune
detection and clearance. Humoral immunity is protective in laboratory
animals [29]; in contrast, humoral responses are typically delayed in
patients. In vitro spirochete-leukocyte in-teractions may provide an
effective model for understanding the delayed immune response in humans when
they are infected by Lyme disease spirochetes.


This is a great piece Rita, thank you for locating it. Do you think the essence
is that 2-4 weeks of antibotics might not do the trick after all? I am so
grateful
there really are real researchers out there.
Marleen


Rita Stanley

unread,
Jan 13, 1998, 3:00:00 AM1/13/98
to

LymeFightr wrote in message
<19980113021...@ladder01.news.aol.com>...


>>
>
>This is a great piece Rita, thank you for locating it. Do you think the
essence
>is that 2-4 weeks of antibotics might not do the trick after all? I am so
>grateful
>there really are real researchers out there.
>Marleen


Well, let me put it this way: if Bb infection REALLY was eradicated in a
month of antibiotic treatment, why bother doing all this research dealing
with "chemotherapeutic resistance and interference with immune clearance (4,
6, 16)"?

And when Lenny wants to cite he, sure as manure, ain't gonna cite this
stuff. He probably thinks it's driven by us in the counterculture.

Rita

Phyllis Mervine

unread,
Jan 13, 1998, 3:00:00 AM1/13/98
to

This is directed to the person who asked for PROOF that Bb spirochetes
are not numerous enough in the human body to seriously deplete our
immune cells in the manner described in Dorward et al's in vitro
experiment:

Human peripheral blood smears contain 10 million or so lymphocytes per
ml (what's a few million either way?...). In contrast, Bb is almost
never (if ever) observed in human blood smears. Furthermore, with
perhaps one curious exception, a poster presentation at last year's LDF
meeting, labs are rarely able to culture Bb from 10 ml blood samples.
This failure to obtain blood cultures (skin punches and CSF are more
reliable samples) occurs despite several cloning studies that suggest
single spirochetes can be propagated in culture. Therefore, it is
expected that peripheral blood contains less than 1 spirochete per 10
ml.

Bottom line is: If Lyme disease involved bacteremia at anywhere near 10
million per ml, diagnosis would be a five minute blood smear. And
culture confirmation of infection would be nearly 100% sensitive and
specific.

If this isn't PROOF enough, what would be?

need...@juno.com

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Jan 14, 1998, 3:00:00 AM1/14/98
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Yes... isn't that one of the main problems we have? They can't find
the little creature?
barbara

In article , Phyllis says...

Phyllis Mervine

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Jan 16, 1998, 3:00:00 AM1/16/98
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Richard H. Clark wrote:

> >
> > In article , Phyllis says...
> > >
> >
> > >Bottom line is: If Lyme disease involved bacteremia at anywhere near 10
> > >million per ml, diagnosis would be a five minute blood smear. And
> > >culture confirmation of infection would be nearly 100% sensitive and
> > >specific.

.
>
> In short, blood is NOT the place to look for 'em. Period.
> Stick a needle in a numb area. Massage the surrounding area
> while sucking out what little blood you can. That'll loosen
> them up into the fluids while you suck them out. You'll
> have your gazillions per ml...

You have personal experience with this procedure?

Richard H. Clark

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Jan 17, 1998, 3:00:00 AM1/17/98
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need...@juno.com wrote:
>
> Yes... isn't that one of the main problems we have? They can't find
> the little creature?
> barbara
>
> In article , Phyllis says...
> >
>
> >Bottom line is: If Lyme disease involved bacteremia at anywhere near 10
> >million per ml, diagnosis would be a five minute blood smear. And
> >culture confirmation of infection would be nearly 100% sensitive and
> >specific.

Except that it ain't IN the bloodstream. It has adapted to stay
out of the bloodstream for a very obvious reason... it gets killed.

One of the reasons it has flagella (feet, flippers) is so that
it can be mobile. It hides. It's lurks in tissues just a bit away
from the bloodstream. The best place to look for it, by far, is
in tissues the patient complains are "numb". This is where the
toxin concentrations are high enough to go beyond pain and completely
disable to nerves in the area. High concentration of toxin, high
concentration of bacteria, plain and simple. Painful parts contain
LESS toxin than numb parts, and hence are not a great place to
look either.

In short, blood is NOT the place to look for 'em. Period.
Stick a needle in a numb area. Massage the surrounding area
while sucking out what little blood you can. That'll loosen
them up into the fluids while you suck them out. You'll
have your gazillions per ml...


Richard H. Clark

David Bartholomew

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Jan 19, 1998, 3:00:00 AM1/19/98
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You know what's funny. I was flipping through histology from
autopsies of MS and Alzheimer's from 55 years ago... really
spicy brain slices. In one picture I counted twenty spirochetes and in
the other four. If you don't LOOK you just don't see! Or, conversely,
you see what you are familiar with. Selective perception is a
learned shared phenomenon. If you've been drilled to observe
changes in tissue pathology, that is what you look for, and that
is what you find. Basically, when you've go a hole in your head
big enough to drive a truck through, then you've got a problem.
"Generations of worshipping at the shrine of the spectacular
has produced..." (Stokes talking about lack of attention to detail
of the doctors and fledgling professors that followed him because
of ther predeliction for the SPECTACULAR).

Dave


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