[847] LACK OF ASSOCIATION OF MIXED CRYOGLOBULINEMIA WITH
TRANSFUSION TRASMITTED VIRUS (TTV).A G Tavoni, A Della
Rossa, F Casula, F Maggi, M Bendinelli, S Bombardieri. Pisa, Italy,
Pisa, Italy.
Mixed Cryoglobulinemia (MC) has been recently correlated with the
hepatothropic virus HCV. Recently, a novel virus (TTV) has been
identified in several patients affected by post-transfusion non-A,
B, and C hepatitis. TTV shows some similarities with parvovirus
such as the single-stranded DNA genome. A possible etiological
role of TTV in cryptogenic fulminant hepatic failure in
susceptible individuals has also been hypothesized. Aim of the
present study was to assess the prevalence of TTV DNA in a series
of patients affected by HCV related MC. An unselected series of 19
patients (7 male and 12 female, age range 52-79) affected by MC
was assessed for the presence of TTV DNA by means of the
polymerase chain reaction technique using primers of Okamoto. Nine
out of nineteen patients resulted positive for the presence of TTV
DNA. Similar results were also obtained in a control population of
220 healthy donors (110/220). We performed a restriction fragment
length polymorphism for genotyping the virus and all the patients
had genotype 1b, except for one who had genotype 2c. We confirmed
these data by sequencing the gene fragments.
In conclusion, the prevalence of TTV DNA in a group of patients
affected by MC is similar to that of the general population and
these results suggest that this new hepatothropic TT virus does
not seem to have a pathogenic role in mixed Cryoglobulinemia.
Disclosure:
Keywords: Cryoglobulinemia; Hepatitis C; Hepatitis B
ACR Poster Session C: Vasculitis I (8:00 AM-9:30 AM)
Presentation Date: Monday, November 15, 1999, Time: 8:00AM, Room:
Hall C
[848] INFECTION OF NEOPLASTIC B CELLS BY HCV IN LOW-GRADE
NON-HODGKIN'S LYMPHOMAS ASSOCIATED WITH TYPE II MIXED
CRYOGLOBULINEMIA.S De Vita, V De Re, D Sansonno, A
Gloghini, F D'Amore, B Pivetta, D Gasparotto, R Dolcetti, V
Zagonel, F Dammacco, A Carbone, G Ferraccioli, M Boiocchi. Udine,
Aviano, Italy.
Introduction. Hepatitis C virus (HCV) has been
pathogenetically linked to type II mixed cryoglobulinemia (MC) and
to overt B-cell non-Hodgkin's lymphoma (NHL) which may complicate
its course. The mechanism by which HCV might favour B-cell
expansion and malignant transformation remain highly speculative,
however. A direct oncogenetic role of HCV by direct B-cell
infection and deregulation has been hypothesized due to the
lymphotropism of the virus, while a role as an exogenous trigger
has been hypothesized in other cases. Previous studies failed to
detect HCV in aggressive NHLs, while results in low-grade B-cell
NHLs were inconclusive. Patients and Methods. We
investigated the possible infection of NHL B cells by means of
sensitive and quantitative PCR on whole RNA from affinity-purified
neoplastic cells, and by HCV-specific in situ studies including
immunohistochemistry, in situ hybridization, and in situ reverse
transcriptase (IS-RT) PCR published methods. B-cell NHL lesions (3
low-grade; 2 aggressive NHLs) from 5 HCV-infected patients, all
HCV-RNA positive in the serum, were studied. In 2 patients,
low-grade NHL had complicated the course of type II MC syndrome. Results.
HCV infection of neoplastic B-cells was detected in 2/5 cases (bone
marrow B-cells), i.e., in the sole low-grade NHLs associated with
MC. CDR3 sequences of such cases showed a high homology to
database human rheumatoid factor requences. HCV infection was
detectable only by PCR and IS-RT-PCR, consistent with a low viral
load. The 3 remaining B-cell NHLs, MC-unrelated, were HCV
uninfected. Conclusions. Infection of B cells by HCV may
represent a pathogenetic mechanism for B-cell lymphomagenesis in
the course of type II MC.
Disclosure:
Keywords: Cryoglobulinemia; Lymphoma; B Cells
ACR Poster Session C: Vasculitis I (8:00 AM-9:30 AM)
Presentation Date: Monday, November 15, 1999, Time: 8:00AM, Room:
Hall C
[849] STUDY OF THE ETIOLOGY, CLINICAL MANIFESTATIONS AND
IMMUNOLOGICAL FEATURES OF 200 PATIENTS WITH CRYOGLOBULINEMIA.M
Ramos-Casals, R Cervera, O Trejo, M Garcia-Carrasco, J Yague, F
Atzeni, J Font, M Ingelmo. Barcelona, Spain.
OBJECTIVE. To describe the etiology and the main clinical and
immunological manifestations of cryoglobulinemia in a large series
of patients from a single center.
METHODS. We investigated 200 consecutive patients who tested
positive for cryoglobulins in our Laboratory of Immunology between
1995 and 1998. Serum cryoglobulins were measured after
centrifugation. Blood samples were obtained and kept at 37°C
for 30 minutes before separation. Serum was prepared by
centrifuging at 37°C for 10 minutes at
2500 rpm. Fresh, centrifuged serum was incubated at 4°C
for 7 days after collection, and examined for cryoprecipitation.
Further characterization of cryoglobulins was performed when
sufficient cryoprecipitate (>5%) was available.
RESULTS. Of 200 patients, 114 were women and 86 men, with a mean
age of 62 years (range, 17 to 91 years). The main diseases
associated with cryoglobulinemia were: viral infections in 165
(83%) patients (HCV infection in 150 patients, HBV infection in 5
patients, HIV infection in 6 patients and other in 4 patients),
systemic autoimmune diseases in 22 (11%) patients (primary
Sjogren's syndrome in 9 patients, systemic lupus erythematosus in
8, systemic vasculitis in 2 and other in 3), hematological
processes in 3 (1%) patients and other diseases in 4 (2%). In only
4 (2%) patients, no associated disease was identified. Further
characterization of cryoglobulins was possible in 50 (25%) cases,
and cryoprecipitates were mixed cryoglobulins (33 type II and 14
type III) and 3 cases were type I cryoglobulins. The most common
clinical manifestations were cutaneous vasculitis in 42 (21%)
patients, articular involvement in 39 (20%), renal involvement in
34 (17%), peripheral neuropathy in 15 (8%) and Raynaud's
phenomenon in 13 (7%). The most frequent immunological features
were positive RF in 64/171 (37%) patients, hypocomplementemia in
83/116 (72%) and ANA in 60/129 (46%).
CONCLUSION. The main cause of cryoglobulinemia in a tertiary
university Hospital was HCV infection, present in 75% of cases,
and the main systemic autoimmune disease associated with
cryoglobulinemia was Sjogren's syndrome. In only 2% of cases, no
associated disease was identified. The classical term "essential"
cryoglobulinemia should be eliminated.
ACR Poster Session C: Vasculitis I (8:00 AM-9:30 AM)
Presentation Date: Monday, November 15, 1999, Time: 8:00AM, Room:
Hall C
[1278] BCL-2 RECOMBINATION AND OVEREXPRESSION IN HCV-RELATED
MIXED CRYOGLOBULINEMIA PATIENTS.A L Zignego, C Ferri, F
Giannelli, A Mazzocca, G Longombardo, G Porciello, P Fadda, M E
Marrocchi, S Moscarella, P Gentilini. Florence, Pisa, Italy.
A pathogenetic role of HCV infection has been definitively
established for mixed cryoglobulinemia (MC), a systemic vasculitis
of small-sized vessels. HCV lymphotropism may be responsible for
clonal B-cell expansion underlying the MC. It has been shown that
immunoglobulin heavy chain gene rearrangement occurs in MC
patients, as well as an overexpression of anti-apoptotic bcl-2
protein. T(14;18) translocation is interpreted as an error during
VDJ gene rearrangement processes in Ig genes leading to bcl-2
overexpression in B-cell. Aim of this study was to evaluate the
occurrence of both T(14;18) and bcl-2 overexpression in B
lymphocytes from HCV+MC pts.
Three groups of age- and sex-matched pts were evaluated: a) 25
HCV-related MC (13 with type II and 12 type III MC; 3 cases
complicated by B-cell lymphoma); b) 25 pts with chronic HCV
infection without MC; c) 25 pts with chronic HBV and/or HDV
infection. Both t(14;18) and bcl-2 expression were evaluated in
peripheral blood mononuclear cells (PBMC) by “nested” bcl-2/JH
PCR and by Western blot analysis and immunoprecipitation.
T(14;18) was detected in 18 (72%), 37 (32%), and 1 (4%) patients
from groups, a, b and c, respectively (p<0.01). All PBMC
samples with t(14;18) overexpressed bcl-2 oncoprotein; these
findings did not correlate with various clinico-serological
parameters, including type II/III MC; however, an higher frequency
of bcl-2 recombination and overexpression was observed in older MC
pts and in pts with more severe liver disease. T(14;18) was
detected in 2/3 MC pts with complicating malignant B-cell lymphoma.
The significantly high frequency of t(14;18) in HCV-related MC,
with and without B-cell lymphoma, suggests that HCV may play a
role in the multistep benign or malignant lymphoproliferation. The
inhibition of B-cell apoptosis by bcl-2 oncogene should represent
the early step of such pathogenetic process. The intimate
mechanisms responsible for HCV-related B-cell proliferation are
unknown; it is possible to hypothesise that interaction between
HCV-E2 and its cell receptor CD81 plays a major role. The
detection of t(14;18) in HCV+ pts may be a predictive factor for
the development of MC and/or other autoimmune-lymphoproliferative
disorders.
Disclosure:
Keywords: bcl-2 Gene; Cryoglobulinemia; Hepatitis C
ACR Concurrent Session: Vasculitis: Pathogenesis and Treatment
(4:00 PM-5:30 PM)
[1964] A MOTIF IN THE HVR1 SEQUENCE OF THE HEPATITIS C VIRUS
GENOME IS ASSOCIATED WITH CRYOGLOBULINEMIA AND SUGGEST A POTENTIAL
ROLE OF THE B-CELL ADHESION GLYCOPROTEINS.L Frangeul, P
Cacoub, P Ghillani, P Hausfater, L Musset, P Opolon, V Thibault, H
Agut, J M Huraux, F Lunel. Paris, France.
Background: Several studies have shown a high frequency of
anti-hepatitis C virus (HCV) antibodies in patients with mixed
cryoglobulinemia. Numerous factors involved in the production of
cryoglobulins (CGs) in HCV-infected patients are still unknown,
the most important clinical factor associated with CGs being the
duration of viral infection. Recent reports have shown differences
in the hyper-variable sequence of the glycoprotein E2 (HVR) of HCV
genome when comparing antibody-bound and -free HCV in patients
with CGs.
Aim: To assess the role of HVR sequence in the production of CGs,
we compared HVR sequences in CGs-positive and CGs-negative HCV
patients.
Methods: The HVR sequences of 13 CGs-positive patients infected by
HCV genotypes 1b, 2 and 3 were compared to the HVR sequences of 5
CGs-negative patients, non-responders to interferon therapy,
infected by HCV genotype 1b, with long duration of hepatitis and
high viremia level. The HVR sequences were analyzed in several
sera from each patient and in cryoprecipitate and supernatant for
CGs-positive patients.
Results: Four amino acid residues located in the C-half part of
HVR1 were significantly associated with the presence of CGs :
glycine at position 398 and 401, serin at position 408 and lysin/arginin
at position 410. This led us to define, in this same region, a
CGs-motif associated with the production of CGs. This motif was
found to be similar to fragments of two human proteins directly
related with lymphoproliferative disorders (CSGP and LFA-1). The
similarity between CGs-motif and the B-cell surface adhesion
glycoproteins might induce an antibody-mediated cross reactivity
against their extracellular domain, which, in turn, would result
into cell activation.
Conclusion: The HVR1 sequences of HCV-infected patients differ
between CGs- positive and -negative patients. Cryoglobulinemia
could be induced by the production of anti-HVR1 antibodies, which
could cross-react with B-cell surface adhesion glycoproteins.