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Reply to letter by winrow et al.

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LETTERS
Regarding the ankylosing spondylitislKlebsiella1
HLA-B27 problem: more inconclusive proof!
To the Editor:
In response to the recent editorial by Terasaki and
Yu (11, we present more “evidence” of an association
between Klebsiella and ankylosing spondylitis (AS).
Klebsiella strains were obtained from the laboratory
of Dr. A. F. Geczy (Sydney, Australia). Geczy’s group has
shown that, in the presence of complement, rabbit antisera
against the strain K43.BTS 1 specifically lyse lymphocytes
from B27+AS+ individuals, but not from B27-AS+ or
B27+AS- subjects; strain F5, like F77, does not induce
cytotoxic antisera in rabbits (2). Furthermore, Dr. Geczy has
postulated that a protein encoded by a plasmid gene present
in enteric bacteria modifies the HLA-B27 molecule and may
account for the results that are obtained with cytotoxicity
testing (3).
Rapid screening showed that plasmids were present
in both Klebsieflu strains. Using K43.BTS1, 2 plasmids of
approximately 3.5 kb were identified: one expressed resistance to tetracycline, and the other was amplified by chloramphenicol. Whole plasmid preparations were isolated by
standard procedures (4) from large-scale cultures of
K43.BTSl grown while being shaken at 37°C. These cultures
were either supplemented with tetracycline (15 pg/ml) or
amplified with chloramphenicol (170 pg/ml). Tetracyclinesupplemented cultures (BTS1-TET) were harvested in the
log phase; chloramphenicol was added to cultures in the log
phase and harvested after incubation overnight (BTS 1CAP). The plasmids were labeled with 32P by nick translation and used as DNA probes.
Human genomic DNA was isolated from the lymphocytes of B27 + and B27 - individuals with or without AS.
The human DNA was restricted with Eco RI, and both
human and plasmid (as used for probes) DNA were separated by electrophoresis, irradiated with ultraviolet light,
and blotted (5) onto nitrocellulose filters. The blots were
hybridized with Klebsiella plasmid DNA to ask for sequence
homology.
Using BTS1-TET as the probe, there was at least 1
high molecular weight band (>lo kb) that was identifiable in
3 B27 + AS + patients and was absent in 1 B27 - AS - and 1
B27+AS- individual; DNA from all B27+ subjects displayed hybridization at approximately 8 kb. No bands were
seen when BTSI-CAP was used as the probe. This experiment was repeated with the same BTS1-TET probe and a
similar result was obtained. It is possible that this result was
artifactual because hybridization was not strong and stringent washing removed all the hybridized bands. Thus, any
sequence homology between Klebsiella plasmid DNA and
human DNA is weak. When a second probe was prepared,
no homologies occurred at high molecular weight and no
differences were noted using BTSI-TET, BTSl-CAP, or F5
plasmids as probes.
Although this is, again, a “one-off’ observation, we
believe that the use of a more sensitive probe (e.g., specific
restriction fragment of plasmid) would facilitate detection of
453
this high molecular weight material which appears to be
specific for B27 +AS + individuals.
Vivienne R. Winrow, PhD
J. R. Archer, PhD
The London Hospital Medical College
Diane J. Mitchell, PhD
Queen Mary College, University of London
London, UK
1. Terasaki PI, Yu DTY: Regarding the ankylosing spondylitisl
Klebsiella/HLA-B27 problem (editorial). Arthritis Rheum
30~353-354, 1987
2. Seager K, Bashir HV, Geczy AF, Edmonds J, de Vere-Tyndall
A: Evidence for a specific B27-associated cell-surface marker on
lymphocytes of patients with ankylosing spondylitis. Nature
277:68-70, 1979
3. Cameron FH, Russell PJ, Sullivan J, Geczy AF: Is a Klebsiella
plasmid involved in the aetiology of ankylosing spondylitis in
HLA-B27-positive individuals? Mol Immunol 20563-566, 1983
4. Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A
Laboratory Manual. Cold Spring Harbor, New York, Cold
Spring Harbor Laboratory, 1982
5 . Southern EM: Detection of specific sequences among DNA
fragments separated by gel electrophoresis. J Mol Biol 98503517. 1975
Reply to letter by Winrow et al
To the Editor:
Winrow and coworkers’ “one-off ’ demonstration of
hybridization between a plasmid probe derived from Klebsiella K43.BTS1 strain (obtained from this laboratory) and
high molecular weight bands of the genomic DNA from
several HLA-B27 + patients with ankylosing spondylitis
(B27 + AS +) is encouraging, but a number of important
points must be considered.
Since the authors did not include specific details of
their hybridization protocol, it is difficult to comment definitively on their data; however, it is possible that the hybridization signal they originally observed may have been due to
a limited sequence homology ( 5 2 0 nucleotides) whose
specific detection might be profoundly influenced by the
stringent washing conditions which were employed posthybridization. If naturally occurring short-stretch homologies between bacteria and B27+AS+ cells exist, their
detection using such a large plasmid probe (3.6 kb) would be
technically difficult. Such homology could be more readily
demonstrated using a specific oligonucleotide probe of minor
complexity (520 nucleotides).
However, we would suggest that the conclusion by
Winrow et a1 that “any sequence homology between Klebsiella plasmid DNA and human DNA is weak” requires
additional clarification.
Over the years, we have produced extensive immunochemical evidence that a 30,000 molecular weight determinant, called modifying factor (MF), can be isolated from a
wide range of apparently unrelated bacteria and from
B27+AS+ cells. We have proposed that it is the constitu-
LETTERS
454
tive expression of this common cell-surface determinant that
is the basis of the specific cross-reactivity between these
bacteria and B27 + AS + cells. Since MF isolated from either
source is serologically and immunochemically identical, and
since M F is permanently expressed on these bacteria and on
B27 + AS + cells, it is reasonable to suggest that the gene(s)
encoding both molecules would most likely be identical or
very similar (Geczy AF, Alexander K, Bashir HV, Edmonds
JP, Upfold LI, Sullivan JS: Klebsiella and ankylosing
spondylitis: biological and chemical studies. Immunol Rev
70:23-50, 1983, and Upfold LI, Sullivan JS, Prendergast JK,
Geczy AF: HLA-B27: speculations on its role in the pathogenesis of ankylosing spondylitis. Prog Allergy 36: 177-189,
1985). A corollary of this hypothesis is that the sequence
homology between the M F genes from bacteria and
B27 + AS + cells is extensive. Indeed, our own recent findings have demonstrated that such segments can be detected,
but because of the obvious importance of this work and its
implications in the pathogenesis of ankylosing spondylitis,
we would consider it premature to publish these data until all
aspects of this phenomenon have been adequately documented.
John S. Sullivan, PhD
Andrew F. Geczy, DSc
New South Wales Red Cross
Blood Transfusion Service
Sydney, NS W , Australia
John K. Prendergast, PhD
Hbpital St. Louis
Paris, France
Reply to letter by Winrow et al
To the Editor:
Winrow et al provide attractive, but ambiguous,
support to the plasmid hypothesis of Geczy’s group concerning HLA-B27/Klebsiella links in the pathogenesis of ankylosing spondylitis (AS). We have also investigated such a
homology at the genomic DNA level between B27+AS+
patient DNA and a plasmid isolated from the Klebsiella
pneumoniae K43.BTS strain obtained from the laboratory of
Dr. Geczy (Toubert A, Philippon A, Amor B: HLA-B27,
ankylosing spondylitis and Klebsiella pneumoniae: toward a
molecular approach [letter]. J Rheumatol 14:391-393, 1987).
Unfortunately, our results were clearly negative. We have
also tested genomic DNA from 3 B27 AS + subjects and 2
B27 +AS - subjects.
Some technical differences between our work and
that of Winrow et al must be pointed out. First, we isolated
a 3.6-kb plasmid which we considered unique from the
K43.BTS Klehsiella strain. It presented the same pattern of
antibiotic resistance as that described by Dr. Geczy’s group
(Cameron FH, Russell PJ, Sullivan J , Geczy AF: Is a
Klebsiella plasmid involved in the aetiology of ankylosing
spondylitis in HLA-B27-positive individuals? Mol Immunol
20563-566, 1983), being resistant to tetracycline, ampicillin,
and carbenicillin, and sensitive to sulfamethoxazole, trimet hopri m, chloramphenicol, and aminosids.
We have also been able to transfer these antibiotic
+
resistances to the Escherichia coli K12.J53 strain in conjugation experiments. However, this does not rule out the
existence of the 2 plasmids BTS1-TET and BTS1-CAP
isolated by Winrow et al from their K43.BTSl Klebsiella
strain.
Southern blotting techniques were used as they were
described by Winrow et al, except that we used stringent
washing conditions (0.1 x standard saline citrate, 0.1% sodium dodecyl sulfate) to keep only highly homologous
signals.
What appears more troublesome is the difficulty in
reproducing the results from one probe preparation to another. This could account for a highly labile genetic element
and could perhaps explain the discrepancy in the results
obtained by the different investigators. To help in resolving
these questions, the next step will be to get a restriction map
of these plasmids and their DNA sequence to define the
proteins they are coding for. An international workshop to
share material and collaborative studies would certainly be
valuable.
A. Toubert, MD
B. Amor, MD
Hbpital Cochin
Paris. France
Treatment of gold-induced thrombocytopenia with
high-dose gamma globulin
To the Editor:
Thrombocytopenia, which is sometimes life-threatening, is a complication of gold therapy in 1-3% of patients
who receive this treatment for rheumatoid arthritis (RA) (1).
Thrombocytopenia may be a component of gold-induced
bone marrow aplasia or may present as an acute, isolated
event. Acute-onset thrombocytopenia is immune-mediated
and is associated with the formation of platelet-specific
autoantibodies and a shortened platelet survival (2).
Conventional treatment for gold-induced thrombocytopenia has included corticosteroids, splenectomy, chelating
agents (dimercaprol, D-penicillamine), cytotoxic agents,
and, in one reported case, prednisone with high-dose intravenous gamma globulin (3). We report the first case of
gold-induced thrombocytopenia treated with high-dose
gamma globulin alone; the treatment resulted in a rapid
normalization of the platelet count.
The patient was a 62-year-old man with seropositive
RA, carpal tunnel syndrome, coronary artery disease,
chronic obstructive lung disease, and diet-controlled diabetes mellitus. Thirteen weeks prior to presentation, he had
begun treatment with gold (aurothioglucose). Before he
began this therapy, his platelet count was >200,000/mm3.
His only other medication, begun simultaneously with the
gold, was prednisone, 5 mg twice daily.
Seven days after the last injection of gold, the patient
abruptly developed epistaxis and palatal and conjunctival
petechiae. He had received 13 weekly intramuscular injections, totaling 600 mg. On admission to New England
Medical Center, his platelet count was 4,000/mm3, with a
hematocrit value of 44% and a white blood cell count of
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