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Volume: 4
Issue: 08
Date: 14-Jun-96

Table of Contents:

I.    LDF: Notes from the IXth Annual LDF Scientific Conference  
      (Part 1 of 3)
II.   About The LymeNet Newsletter


*                  The National Lyme Disease Network                  *
*                         LymeNet Newsletter                          *

IDX#                Volume 4 - Number 08 - 6/14/96
IDX#                            INDEX
IDX#  I.    LDF: Notes from the IXth Annual LDF Scientific Conference  
IDX#        (Part 1 of 3)
IDX#  II.   About The LymeNet Newsletter

I.    LDF: Notes from the IXth Annual LDF Scientific Conference
     (Part 1 of 3)
Sender: Lloyd E. Miller, DVM <[email protected]>

   Notes from the Ninth Annual Scientific Conference Lyme Disease      
    Foundation -- April 19-20, 1996 -- Westin Copley Plaza Hotel,
                     Boston, Massachusetts, USA

                  Prepared by Lloyd E. Miller, DVM
Every effort has been made to report the speaker's statements and to
report suggested drug doses accurately. However, typos do occur and
as information gets passed around or edited mistakes can occur.
The reader is cautioned to not take any medication before checking
the accuracy of name and dose recommendations with the speaker.

Selected abstract statements have been included in the notes.

The notes themselves are from statements made by the speakers.

My editorial comments will be found in brackets like this [COMMENT].

Full abstracts are available electronically from the Lyme/Other
Zoonoses section in the Public Health Forum on Compuserve.
Protective and Disease-modulating Antibody-mediated Immunity to
Borrelia burgdorferi Antigens Expressed in vivo. - Stephen W.
Barthold, Ph.D., D.V.M.

*  We have utilized a well-defined mouse model of Lyme disease, in
  which mice infected by syringe of tick develop heart and joint
  disease that undergo spontaneous resolution and episodic
  recurrence over the course of persistent infection.
*  Spirochetes appear to persist extracellularly within collagenous
  connective tissue, particularly the skin.
*  Spirochetes entering the host do not express OspA, and are
  therefore not vulnerable to OspA immunity.  During infection,
  other proteins are expressed that are probably required for
  tissue invasion.
*  Host humoral, but not cellular, responses directed against in
  vivo-expressed proteins have varying degrees of protective
  activity and are also involved in resolution (and recurrence)
  of disease.

*  Basis for first statement in above abstract excerpt: Bb which are
  in the skin chronically do not produce an inflammatory response.
  They can be cultured and produce disease when injected into
  another mouse.
*  Persistent infection has been seen in most animals tested.
*  Lyme carditis occurs spontaneously and clears without treatment
  and then periodically recurs in humans.  The same occurs in
  laboratory mice.
*  In mice with arthritis, spirochetes can be found.  When the
  arthritis clears no spirochetes can be found.  When the
  arthritis recurs spirochetes can again be found.  Lacking an
  immune response the host can not clear spirochetes and becomes
  severely arthritic.  Bb react differently in different tissues in
  the same host.
*  Patients do not produce antibodies to OspA.  Host adapted
  spirochetes do not express OspA and therefore OspA vaccine will
  not protect. [ See notes further on for differing opinions on this
*  Bb in feeding tick stops production of OspA and increases

  production of OspC.
*  OspC can immunize against challenge but has no effect against
  existing infection.
*  Serum from infected mice would protect naive mice from infection by
  injection of Bb but not from infection by tick bite.
*  Immune serum injected into previously infected mice did not result
  in a decrease of spirochetes in the blood. They were eliminated
  from the joints but not the heart.
*  Immune serum injected soon after allowing tick to feed in naive mice
  did not protect.
*  Bb express different antigens in vivo than in vitro. There are
  probably antigens expressed in vivo that could be important in
  vaccination protection.
*  Transfer of T-lymphocytes from infected mice to naive mice did not
  protect the naive mice from infection.  Thus the third statement
  in the abstract excerpt above.
*  Speculated that Bb may shut down antigen production when it enters
  the host and thus resists detection by the hosts immune system.

Hamster Model of Lyme Borreliosis  -  Ronald F. Schell, Ph.D.

[ COMMENT: This presentation was a continuation of research reported at
this same conference in 1994 and 1995 where it was reported that
hamsters challenged with homologous stains of Bb developed severe
arthritis post vaccination and prior to the development of immunity.
It was also reported that when the protective titer waned arthritis
would also develop on challenge.  This report shows that Bb specific
T-lymphocytes were responsible for the development of the arthritis.
The importance of this is related to the development of safe and
effective vaccination and in the development of medications that may
prevent or treat the arthritis caused by Bb. ]

*  When vaccinated hamsters are depleted of CD4+ T lymphocytes by
  administration of monoclonal antibody GK1.5 and challenged, they
  failed to develop severe destructive arthritis.  
*  Similarly, nonvaccinated hamsters with or without depletion of CD4
  T-lymphocytes failed to develop severe destructive arthritis.
*  Our results also suggest that as more protective antigens are added
  to develop a comprehensive Lyme vaccine, the ability of these
  proteins to induce or elicit adverse effects may increase.

*  He expressed surprise at the extent of the severity of arthritis
  that CD4 lymphocytes influence.
*  CD8 lymphocytes also influenced the arthritis.

Chronic Lyme Disease in the Rhesus Monkey  -  Mario Philipp, Ph.D.

*  Two mechanisms that members of the genus Borrelia use to avoid the
  host's immune response are antigenic variation and residence in
  tissues into which antibodies and lymphocytes have poor access.

*  Possible mechanisms of persistence of Bb and disease:
   1. Poorly immunogenic species - not cleared by the immune system.
   2. Antigenic variability through various genetic mechanisms -
      expressing various antigens over time staying one step ahead of
      the immune system.
   3. Intracellular location - avoids immune system and many
   4. Access to niches - privileged sites - clear precedence =
      syphilis in the brain.  CNS niche is important.
   5. Nucleic acid persistence - blebs continue to produce protein
      but technically speaking no live organisms are present.
*  OspA does have significance in mammalian hosts - most patients on
  Shelter Island with arthritis have strong expression of both OspA
  and OspB. [ This information appears to differ from Barthold's ].

Correlation of Severity of Arthritis with Level of Persistence of
Spirochetes in Murine Lyme Disease  -  Janis Weis, Ph.D.

*  Findings support the model in which the severity of pathology is
  directly related to level of persisting spirochetes in tissues.

*  C3H mice develop more severe disease of the joints and heart than
  BALB/c mice which can be overcome by injecting more Bb into BALB/c
*  Nitrous oxide (NO) is found in high levels in inflammed tissues.  
  NO has antimicrobial properties.  NO is toxic to Bb in vitro.
  Is it involved in host defense against Bb? BALB/c mice produce
  more NO.  When NO production was decreased in both species of mice
  no attenuation of spirochete infection was found.  Conclusion NO is
  not involved in host defenses against Bb in mice and probably other
*  Nitrous oxide is an important defense in intracellular infection.  
  Evidence that it is not involved in defenses against Bb argues
  that Bb may not be an intracellular pathogen.  [ Several speakers
  feel this may be true - others are not yet convinced ].
*  Study of infection in pregnant mice:
   1. Recent (near the time of mating) infection produced 12% fetal
      deaths.  Bb was found in the uterus of mother but not in the

   2. Chronic infection (more than three weeks prior to gestation)
      resulted in fewer fetal deaths and no Bb were found in the
   3. Bb was rare in fetal tissue.  Fetal deaths are associated with
      the mother not the fetus.
   4. Therefore effect on pregnancy low.

Effects of Borrelia burgdorferi on Human B- and T-Cells  -  David W.
Dorward, Ph.D.

*  Since discovering late in 1994 that virulent B. burgdorferi can
  target, invade, and kill primary and cultured human T-and
  B-lymphocytes, we have investigated the mechanics of such
  interactions to help understand the role they may play in the
  onset, development, and persistence of Lyme disease.
*  In vitro co-incubations of low (<8) or high (>30) passage
  spirochetes with SKW 6.4 B-cells or H9 T-cells were used as a model
  to quantitate and study the consequences of interactions on cell
  structure and viability.
*  Attachment and invasion were detectable in all mixtures examined.  
  By 30 minutes, numerous spirochetes were observed either extending
  or detached from host cells, yet surrounded by a layer(s) of
  membrane(s) and cytoplasm, apparently derived from the host cells.  
  Prior to these studies invasion and killing of human lymphocytes
  was unrecognized among bacterial pathogens.
*  These results indicate that attachment and invasion of human

  lymphocytes by Lyme disease spirochetes is active and rapid.
*  These findings also raise the possibility that the spirochetes could
  acquire lymphocytic membranes and surface markers upon or even
  prior to transmission by infected ticks, which would be expected to
  have profound effects on recognition by the immune system.

*  Dramatic photographs taken though the electron microscope showed
  spirochetes being engulfed and extruded from the lymphocytes
*  The spirochete coating itself with lymphocyte membrane inhibits the
  ability of antibodies to recognize and kill them.
*  The process allows avoidance of phagosomal fusion of normal

[ COMMENT: This presentation was really fascinating and very thought
provoking.  If this process occurs in vivo the implications are
immense and provides a very plausible explanation on how Bb avoids
detection and elimination.  Just think what this means if it is a
continuous process not just limited to the time of tick attachment.
Could it explain the resistance to antibiotic elimination too?  Is
the spirochete creating its own privileged site?  Gaining an
understanding of this process may provide important clues for new
preventive and treatment strategies.  This was one of the most
significant presentations. ]

Acquisition and Induction of Enzymes which Degrade the Extracellular
Matrix by Borrelia burgdorferi and other Borrelia Species.  -  Mark
Klempner, M.D.

*  For virtually all bacteria which disseminate from a skin or soft
  tissue inoculation site produce bacterial proteases, which digest
  extracellular matrix proteins, facilitate spreading in the skin
  and subsequent invasion into the lymphatic or vascular
*  We have found that B. burgdorferi lacks these proteases but is able
  to spread from its inoculation site in the skin.  Instead, B.
  burgdorferi has evolved a mechanism for accomplishing this step in
  pathogenesis by utilizing human proteases which are generated at
  the inoculation site and become bound to the bacterial surface.
*  We have also discovered that B. burgdorferi induces the release
  of enzymes that degrade the extracellular matrix from cells in the
  skin and the central nervous system.
*  Utilization of host proteases instead of proteases of microbial
  origin could explain why the immune response to B. burgdorferi
  infection is blunted.

These observations represent a new mechanism for bacterial virulence
which may identify new targets for prevention, diagnosis, and
treatment of Lyme disease.

[ COMMENT: The abstract is very complete.  No notes were necessary.
This presentation also provides significant information on how Bb can
gain entrance to the body.  Such host adaption as reported here
doesn't happen overnight.  Is this further evidence that Bb is an
ancient organism ?]

Why is Chronic Lyme Borreliosis Chronic?  -  Elizabeth Aberer, M.D.

*  Recently, it was shown that the most important cell for antigen
  presentation, the epidermal Langerhans cell (LC), is heavily
  damaged in erythema migrans (EM).
*  In this study, the most prominent immunohistochemical changes were
  seen on the epidermal dendritic cell population.  Our data suggest
  that MHC class II molecules are strongly down regulated on LC not
  only in the early but also in the late stage skin manifestation
  of LB.

*  The numbers of LC cells in EM was reported to be normal and even
  increased in ACA (acrodermatitis chronica migrans) but their
  function was adversely affected.

[ COMMENT: This provides yet another possible mechanism for the
survival (persistence) of Bb. ]

Anti-Borrelial Activity of Serum From Patients with Late Lyme Disease -
Charles Pavia, Ph.D.

*  Separate groups of mice (C3H strain) received intraperitoneal
  injections of 0.5 ml of high titer serum from human patients with
  (i) early (erythema migrans) or
  (ii) late Lyme disease (arthritis).
  Some of these patients were culture-positive; all were seropositive
  and antibiotic-free.
*  Twenty-four hours after passive serum transfer, all of the mice,
  including matched controls given the appropriate normal sera
  lacking borrelia antibodies, were challenged intradermally with
  100,000 organisms of two New York isolates (B31, P103) or of a
  California strain (CA-287)
*  Seven to 10 days after challenge, the mice were sacrificed and
  cultures for the urinary bladder and peripheral blood were
  established in BSK media.  Late Lyme sera fully protected the mice
  against Borrelia challenge infection with all three isolates (based
  on negative cultures of urinary bladder and blood) but, in marked
  contrast, early stage Lyme sera were generally ineffective in

  preventing infection.
*  Immunoblot analysis revealed that the protective properties of late
  Lyme sera were associated with a multi-protein antibody response.  
  This included strong reactivity with the outer surface proteins A
  (31kDa) and B (34 kDa), which was lacking in sera from those with
  early stage disease.
*  These findings show that patients with untreated Lyme disease of  
  long duration can develop a potent anti-borrelial humoral immune
  response which can be protective against infection and possibly
  reinfection. [ This statement appears to be contrary to what has
  been reported by others. ]

*  Bb grow at 32 -34 degrees C - cooler temperatures - maybe this helps
  explain its predilection for skin.
*  In patients with EM seropositivity increases with time. Up to 80% of
  patients have a positive test by one month - depending on the tests
  [ This also differs from seropositivity rates reported by others. ]
*  With early Lyme - will never get all patients to be seropositive for
  diagnostic purposes.
*  The number of Western blot bands reacting increases with time in
  *untreated* patients.
*  Note: the sera that protected the mice from challenge was
  unprotective in the patient as evidenced by the positive cultures
  in some of the patients.

Western Immunoblot for Lyme Disease:  Determination of Sensitivity,
Specificity and Interpretive Criteria Using Commercially Available
Performance Panels  -  Richard C. Tilton, Ph.D.

*  All Western blots are not created equal.
*  Essentially the CDC criteria and testing is no better than others.
  There is no gold standard test.
*  By considering the 83Kda band in early Lyme disease it appears to
  improve the diagnostic value of the Western blot test. Currently
  the CDC does not consider this band important in early Lyme disease.
*  Questioned the appropriateness of accepting Western blot criteria
  edicts without question - i.e. the CDC criteria.

Use of PCR Assays to Monitor the Clearance of B. burgdorferi DNA From
Blood Following Antibiotic Therapy  -  Mark Manak, Ph.D.

*  A PCR approach was used to monitor the effectiveness of antibiotic
  therapy on the persistence of B. burgdorferi sequences in the blood
  of Lyme disease patients.
*  As few as 3-10 copies of the B. burgdorferi DNA in the sample could
  be detected.  Positive PCR results were obtained mainly with the
  buffy coat fraction, although occasional plasma fractions were also
*  Within 1 week of administration of antibiotic treatment 7 of 8
  initially PCR positive patients became PCR negative. The remaining
  patient became PCR negative after 5 weeks of therapy.
*  Four late stage patients under therapy continued to show sporadic
  PCR positive results.  When an alternative antibiotic was
  administered in two of these patients, both became PCR negative
  within one week following the change in therapy.
*  One patient who had been PCR negative while under therapy, became
  PCR positive within 2 weeks of cessation of therapy.
*  These studies demonstrate the usefulness of PCR results in

  monitoring the effectiveness of antibiotic therapy in Lyme disease

[ COMMENT: This was one of the more controversial presentations.  
The PCR tests were run on the buffy coat (white blood cell) fraction
of the blood not on any other fluids or tissues.  No correlation was
made in the presentation between PCR result and clinical symptoms.
There were no controls in this study.  It is my understanding that Bb
will only be found sporadically in the blood stream.  How this
phenomenon relates to this study is unclear.  In discussion after the
presentation I learned that in order to get the first positive PCR in
a patient multiple PCR's were necessary.  At any one time an infected
patient will be PCR negative.  During the discussion period it seemed
to me the presenter was reluctant to correlate the PCR data with the
patient symptom data.  Does the sporadically positive PCR tests in the
four patients imply persistent infection following therapy.  I'm not
sure I can agree with the conclusion of this study, at least not until
the PCR results are correlated with the clinical responses and not

until long term follow up is reported and not without the study of
other fluids and tissues in the appropriate circumstance. ]

PCR in the Diagnosis of Patients with Early and Late Lyme Borreliosis:
Comparison of Methods  -  Bruno L. Schmidt, Ph.D.

*  This presentation described a nested PCR technique developed for
  testing urine and made the conclusion that ; In urine samples from
  patients with Lyme Borreliosis, known to harbour only low numbers
  of spirochetes (<50/ml), the nested PCR is superior in comparison
  to other methods.  In addition, results indicate that primers are
  decisive for sensitivity.

Progress in the Clinical Development of a Lyme Disease Vaccine in the
U.S.A.  -  Francois Meurice, M.D.

*  A double blinded, placebo controlled, dose-range study was
  conducted in 350 healthy adult residents of three New England
  islands on which LD is highly endemic.  An Osp A antibody
  response was detected in >97% of subjects receiving vaccine.
*  A second trial addressed the issue of vaccination of subjects
  previously infected with LD.  The safety and reactogenicity profile
  of the candidate vaccine in this population was similar to the
  previous observations: all doses were well tolerated, although mild
  local reactions were common (mostly soreness at the injection site:  
  40-85%).  Transient systemic reactions were reported by <40% of
  subjects and included headache, fatigue and arthralgia.
  Adverse events did not increase following subsequent injections.

[ COMMENT: This presentation described the complexities of doing
vaccine trials in humans. No efficacy results were presented. ]

Overview of Lyme Disease Vaccine Trials  - John M. Zahradnik, M.D.

*  Studies in dogs with recombinant OspA (rOspA) vaccine showed it to
  be very effective in preventing infection shortly following
  vaccination and one year later.
*  The amount of antigen in the vaccine correlates with the immune
  response.  The study vaccine contains 30 micrograms of rOspA per
  dose.  Two doses a month apart are given for initial vaccination.
*  Side effects reported in the study were joint pain, fatigue and
  headache.  There was little difference between the vaccination and
  placebo group.  Repeat vaccination did not produce any other
*  This presentation did not provide any information about efficacy in
  people.  These trials are still in progress.
*  Efficacy trial is being conducted in 14 centers in individuals over
  18 years of age in good health and considered to be at high risk in
  endemic areas or working in endemic areas.
*  It will be at least two more years before a human vaccine will be
*  Vaccination does not remove the need to take protective measures to

  prevent tick bite.


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