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*
Department of Biomedical Sciences and Human Oncology, Section of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy; and
Chair of Internal Medicine, University of Foggia Medical School, Foggia, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Basically, ICs consist of HCV, IgG anti-HCV Abs, and IgM molecules directed against IgG (9). Approximately one-third of HCV-infected patients have circulating ICs with cryoprecipitating properties. HCV has indeed been implicated as the possible etiologic factor of mixed cryoglobulinemia (MC), a chronic IC-mediated disease with underlying B cell clonal proliferation (10). Morphologically, type II MC is characterized by bone marrow multifocal lymphoid infiltrates of monoclonal B cells (11). Despite the occurrence of a monoclonal component (IgMk) in the serum of patients with type II MC, their bone marrow B cell proliferation investigated at the DNA level showed to be oligoclonal in nature. This supports the concept that MC is a nonneoplastic disorder, in agreement with its indolent clinical course (12, 13).
Lymphoid aggregates morphologically similar to those found in bone marrow of type II MC patients are detected in the liver of chronically HCV-infected patients (14). Inflammatory infiltrates mainly recruited in the portal areas frequently occur as round aggregates of small lymphoid cells. Immunohistochemical characterization have demonstrated that they mainly consist of B cells surrounded by a T cell zone (15). These follicle-like structures often display a well-formed germinal center and may act as true functional follicular structures (16).
In infectious diseases, B cell activation, differentiation, and proliferation occur in the lymphoid follicles of secondary lymphoid organs, such as regional lymph nodes and spleen, or in the so-called "ectopic" germinal centers found in nonlymphoid organs on abnormal sites, namely rheumatoid synovial membrane (17), thyroid gland (18), choroid (19), or lung (20). The liver may indeed be considered an "ectopic" lymphoid organ, in that B cells bearing Ag-specific receptors are stimulated to proliferate and differentiate into Ab-secreting plasma cells within germinal centers of intraportal lymphoid follicles of HCV-positive patients. This concept is consistent with our previous observations conducted in the liver of these patients with and without MC (21). Molecular analysis of the IgH VDJ region on DNA extracted from the biopsy core showed that B cell clonal expansions occurred in almost 60% of HCV-positive patients without MC and in 90% of those with MC. Analyses of expanded clones also revealed that D genes were frequently mutated, as compared with the known germline segments (22), pointing to the presence of an Ag-driven process in the B cell growth and clonal evolution.
In this paper, we have defined the frequency of intrahepatic B cell clonal expansions of unselected patients with chronic hepatitis C and compared their presence to the intrahepatic viral load. Cloning and sequencing analyses were also performed on intrahepatic portal lymphocyte clusters isolated by microdissection. Results demonstrate that these clusters are often clonally restricted.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Forty-two consecutive patients (23 male, 19 female) aged 34–72
years (mean 53 years) attending the Liver Diseases Unit of the
Department of Internal Medicine and Clinical Oncology of the University
of Bari (Bari, Italy) were studied. All had clinical and serological
evidence of chronic liver disease (see Table I
). Sera obtained at
diagnosis were all positive for anti-HCV Abs by ELISA (HCV 3.0;
Ortho Diagnostic Systems, Raritan, NJ) and by recombinant-based
immunoblot assay (second generation; Ortho Diagnostic Systems) as well
as for HCV RNA. All patients were HIV seronegative and heterosexual,
with no history of i.v. drug abuse. Twenty of them had been transfused
8–15 years before enrollment into this study, whereas in the remaining
22 patients the source of HCV infection remained undefined. None was
positive for serological markers of active hepatitis B virus (HBsAg,
IgM anti-HBc Abs), nor for antinuclear or antismooth muscle
autoantibodies. None had received corticosteroids or IFN therapy before
enrollment. Liver biopsy was performed for diagnostic purposes, and
informed consent in writing was obtained from each patient.
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Serum cryoglobulins were determined as described elsewhere (23), and the monoclonal component in the serum or in the cryoprecipitate was characterized by immunofixation (Paragon; Beckman, Fullerton, CA) and classified according to the criteria described by Brouet et al. (24).
Molecular analysis of B cell clonality
DNA was purified from frozen liver tissue and
circulating or bone marrow-derived lymphocytes by phenol-chloroform
extraction according to standard protocols. Samples from heparinized
blood and bone-marrow aspirates were immediately used for mononuclear
cell isolation by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) gradient
centrifugation. Recovered mononuclear cells (2 x
106 cells), liver tissue bioptic samples (
5
mg), and lymphoid nodules microdissected from portal tracts
of fresh-frozen liver sections were processed for DNA preparation
according to the method previously described (25).
Briefly, samples were digested overnight in a lysis buffer containing 5
µg/ml proteinase K, 1% SDS, 20 mM Tris-HCl (pH 8.0), and 5 mM EDTA
(pH 8.0). DNA was extracted by phenol/chloroform in the
presence of 300 mM sodium acetate (pH 5.2) followed by
precipitation with isopropanol and yeast tRNA (10 mg/ml). The resulting
pellet was washed in 80% ethanol, dried, and resolved in 50 µl
diethylpyrocarbonate-treated water. Spectrophotometry using a Genequant
DNA/RNA calculator (Pharmacia) was used to quantitate DNA.
For each sample, 0.5–1 µg DNA was processed in the PCR analysis for
B cell clonal expansion using two different seminested protocols of
amplification, according to well-established procedures
(26). In the first protocol, the upstream primer was
complementary to the third framework V region (Fr3,
5'-ACACGGC[C/T][G/C]TGTATTACTGT-3') of the IgH gene, whereas in the
second the upstream primer was complementary to the second framework V
region (Fr2,
5'-TGG[A/G]TCCG[C/A]CAG[G/C]C[T/C][T/C]CNGG-3'). In both
protocols the downstream primer was the same and was directed to an
outer conserved region of the IgH J region (5'-TGAGGAGACGGTGACC-3') in
the first round of amplification and to an inner conserved sequence of
the same J region in the second round (5'-GTGACCAGGGTNCCTTGGCCCCAG-3').
DNA was amplified for 30 cycles in the first round and for 20 cycles in
the second. Each cycle consisted of 94°C for 45 s, 50°C for
45 s, and 72°C for 30 s, with an additional extension
interval of 5 min at 72°C after the last cycle. The sensitivity of
the technique was checked by the amplification of serial dilutions of
DNA from clonal B cells admixed with DNA from polyclonal B cells. The
detection threshold was estimated to be 1% (21).
A "cold" nonradioactive PCR was first performed. At the end of the
second round of amplification, 20 µl of the reaction mixture were
analyzed in parallel by electrophoresis (150 V) in 5% (Fr3 protocol)
or 3% (Fr2 protocol) agarose gel (Seaken LE; FMC Bioproduct, Rockland,
ME) in TBE (100 mM Tris, 100 mM boric acid, 2 mM EDTA) buffer, stained
with ethidium bromide, and optically evaluated by UV transillumination.
The clonal pattern detected by nonradiactive PCR was subsequently
confirmed by a radioactive PCR approach, which enhanced the
identification of the number of multiple dominant bands in oligoclonal
B cell expansions. Radioactive PCR was performed using a labeled
nucleotide, i.e., [32P]dATP (Amersham, Little
Chalfont, U.K.) in the second round of amplification in both Fr2 and
Fr3 protocol. The radioactive PCR products were then subjected to a
long run (20–40 cm) electrophoresis on a polyacrylamide gel. Dried
gels were autoradiographed using a 
-max film (Amersham).
For each experiment, a control was included using primers for the
-actin gene, which resulted in a single band of 300 bp. In all
cases, a -actin PCR product was identified by gel electrophoresis,
which controlled for quality, quantity, and presence of potential
contaminants.
A monoclonal B cell expansion was defined as one or two (if both alleles were rearranged) discrete narrow band(s) within the predicted size. Distinction of biclonal monoallelic rearrangement from monoclonal biallelic rearrangements was based on the results of subsequent sequence analyses, in that a nonfunctional rearrangement of one of the two alleles (i.e., one of the two dominants bands) was detected in the case of a monoclonal disorder, whereas both dominant bands were representative of a functional IgH rearrangement in biclonal disorders.
Cloning and sequencing analyses
PCR products amplified from liver DNA were run on agarose gel in
TBE buffer and stained with ethidium bromide according to standard
procedures. Individual bands were excised from the gel, cloned, and
sequenced. Cloning and sequencing were also performed on bone marrow
and circulating lymphocytes obtained from patients with MC. In such
cases, individual bands or the entire smear within the VDJ PCR range
were cut from the gel. DNA was purified using the QIAEX II gel
extraction kit (Qiagen, Hilden, Germany), ligated into a pGem-T cloning
vector (Promega, Madison, WI), and transfected into Escherichia
coli DH5
-competent cells. Transfected cells were plated onto
Luria-Bertani-ampicillin agar plates containing
5-bromo-4-chloro-3-indolyl--D-galactoside and
isopropylthiogalactose. Uncolored colonies were selected at random and
cultured. Plasmid DNA was purified with the Wizard Plus Minipreps DNA
Purification System (Promega).
Sequence reactions were conducted on an ABI Prism 310 Genetic Analyzer (Perkin-Elmer, Foster City, CA). All sequences were confirmed by sequencing in both directions with primers T7 and SP6. At least 10 different clones were sequenced for each dominant band. Sequences were considered to be related if they shared the same complementarity-determining region (CDR)H3, but had differences in the number of point mutations. Where a sequence showed similarity to more than one DH region, all possible DH regions in the CDRH3 were identified, and those that could be assigned without overlap, and with the smallest number of nucleotides in between, were used.
A clonospecific oligoprobe based on the third CDR DNA sequence labeled
at the 5'-end with T4 polynucleotide kinase (Boehringer Mannheim,
Mannheim, Germany) and [
-32P]ATP were used
to detect predominant B cell clones in different areas of the same
liver and in different livers. IgH VDJ PCR products of the liver biopsy
specimens were blotted on nylon membrane (Pall, Portsmouth, U.K.) and
hybridized with the clonospecific oligoprobe at a melting temperature
of 5°C in 5x SSC/7% SDS and washed at the same temperature in 2x
SSC/0.2% SDS. Appropriate controls were included. The reliability of
the method was ensured by repeating the experiments and comparing
recurrent cases with the initial presentation to show identical
bands.
To minimize the risk of contamination, each step of tissue digestion, PCR mixture/preparation/reaction, and electrophoresis was conducted in a separate room using a category II laminar air flow cabinet restricted to PCR use together with separate pipettes dedicated to PCR. Samples referred to different specimens, and each specimen to different areas of the liver were processed in different experiments and in different days. Clone sequencing was processed and confirmed in two independent laboratories.
RT-PCR for HCV RNA
For qualitative detection of HCV RNA sequences, RNA was extracted from serum, mononuclear cells, and liver tissue. cDNA was synthesized using random reverse transcriptase primers. HCV RNA was detected by two-stage PCR using primers from the 5' noncoding region of the HCV genome, as previously described (27).
Genotype of HCV RNA
HCV genotypes were determined by two-stage PCR using universal and type-specific primers from the putative C gene of the HCV genome. The first stage was performed with primers consisting of 5'-TGCCGCGCGAC(TA)AGGAAGACTTC-3' (sense) and 5'-ATGTACCCCATGAGGTCGGCGA-3' (antisense). The second stage was performed with a sense primer consisting of 5'-AGGAAGACTTCCGAGCGGTC-3' and a mixture of four HCV type-specific antisense primers: 5'-TGCCTTGGGGATAGGCTGAC-3' (type 1a), 5'-GAGCCATCCTGCCCACCCCA-3' (type 1b), 5'-CCAAGAGGGACGGGAACCTC-3' (type 2a), and 5'-ACCCTCGTTTCCGTACAGAG-3' (type 2b). The genotype nomenclature proposed by Simmonds (28) was used.
Intrahepatic measurement of HCV RNA
HCV RNA levels were measured by signal amplification with a
branched DNA probe assay (Quantiplex; Chiron, Emeryville, CA) whose
detection limit was 200,000 HCV genome equivalents (Eq)/ml. RNA was
recovered from 5 mg liver tissue specimens for all patients.
Specimens were stored at -80°C without any preservative solution.
Then 0.5 ml of cold guanidine-HCl homogenizing solution (8 M guanidium
thiocyanate, 50 mM Tris-HCl, pH 7.5, 25 mM EDTA, 8% (v/v) 2-ME
containing 3 M sodium acetate, pH 5.2) was added to the frozen tissue
and homogenized with a pellet pestle mixer. Next, 0.025 ml 10%
Sarkosyl was added to each tube and gently mixed. After 5 min, tubes
were centrifuged to sediment particulates. Supernatants (0.5 ml) were
removed and added to tubes containing 0.01 ml of poly(A) (10 mg/ml).
Then 0.25 ml of 100% ethanol was added to each tube and mixed very
thoroughly. Tubes were placed at -20°C overnight and then
centrifuged for 20 min at 4°C. Supernatants were aspirated, and the
pellets were dried down with a Speedvac rotatory vacuum device
(Eppendorf-Netheler-Hinz, Hamburg, Germany). After solubilization in
nuclease-free H2O, HCV RNA was measured.
Duplicate samples were added to the wells in which lysis,
hybridization, capture, and signal amplification occurred. A mixture of
synthetic oligonucleotides, which included probes that mediated capture
and probes that bound to the basic DNA amplifier molecule, hybridized
equally well to the highly conserved 5' noncoding and core regions of
the HCV RNA of all known genotypes, thereby capturing the RNA molecules
onto the surface of a microwell plate and linking the target to
synthetic basic DNA molecules added to the well. Multiple copies of an
alkaline phosphatase-linked synthetic probe hybridized to the
immobilized complex, resulting in amplification of the target
signal. Detection was achieved by incubating the complex with a
chemiluminescent substrate (dioxethane) and measuring the light
emission, which was proportional to the concentration of target nucleic
acid in the specimen.
The standard curve was constructed from a diluted sample from a patient whose serum HCV RNA had been quantitated by comparison with a highly purified RNA transcript covering the first 3200 nucleotides from the 5' end of the HCV genome. Results were expressed in picograms per milliliter by multiplying the conversion factor 0.52 obtained by dividing HCV RNA molecular mass by the number of viral copies/ml (3.13 x 106 g/mol/6.023 x 1023 copies/mol) x 105. Based on the weight of liver processed, results were normalized to picograms HCV RNA per gram of bioptic specimen. When considering bone marrow-derived mononuclear cells and PBMC, picograms were divided by the number of cells and results were expressed as picograms HCV RNA per cell.
Micromanipulation of liver tissue sections
Cryostat liver sections from patients 1 and 10 showing a single band feature in PCR IgH VDJ gene rearrangement were micromanipulated and harvested under an inverted microscope (model IX-50; Olympus, Tokyo, Japan) equipped with x4, x10, and x40 long-distance lenses. Two three-dimensional hydraulic micromanipulators equipped with a joystick (Narishige, Tokyo, Japan), a reflex camera, and a TV monitor were used. Manipulation tools consisted of a reception pipette prepared from glass capillaries (model GD-1; Narishige) processed through a vertical pipette puller (model PB-7; Narishige) and a manipulator knife. Reception pipettes were further ground at a 50° angle on a pipette grinding machine (model EG-40; Narishige) to obtain an opening diameter of 10–15 µm at the capillary tip.
Identified portal tracts were mobilized with the help of the manipulation knife and aspirated into the reception pipette. Each portal tract was expelled under microscopic supervision into a 0.5-ml tube containing cell-lysis buffer and digested overnight at 37°C. Each sample was divided into two aliquots and processed with PCR assay for detection of IgH VDJ gene rearrangements and HCV RNA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. All patients were shown to harbor
productive infection by repeated confirmation of anti-HCV
positivity and HCV viremia. Analysis of IgH VDJ gene rearrangement was
performed on DNA extracted from liver biopsy specimens and compared
with intrahepatic HCV RNA levels on the same specimens. According to
PCR-based criteria, the occurrence of one or two distinct bands,
considered diagnostic of B cell monoclonality, was found in 14 (33.3%)
patients (group 1), whereas oligoclonality, defined as three or more
bands, was displayed by 12 (28.6%) patients (group 2). The presence of
a smear of products suggestive of polyclonality was detected in the
remaining 16 (38.1%) patients (group 3). Group 1 patients showed a lower mean age (50.8 ± 8.5 vs 53.8 ± 3.7 and 59.8 ± 10.2 years, mean ± SD for group 2 and group 3) and a shorter duration of liver disease (5.8 ± 2.5 vs 5.8 ± 4.1 and 8.9 ± 5.5 years; mean ± SD). No significant differences in terms of serum alanine aminotransferase were found (group 1, 94.3 ± 38.0 IU/ml; group 2, 77.8 ± 69.9 IU/ml; group 3, 99.8 ± 78.7 IU/ml, mean ± SD). A similar distribution of superimposed cirrhosis occurred: it was diagnosed in three (21.4%) patients of group 1, two (16.7%) of group 2, and four (25%) of group 3. In addition, the distribution of HCV genotypes showed no significant difference, type 1b being prevalent in each group.
The overall incidence of associated MC and MGUS was significantly
higher in patients with intrahepatic B cell expansion than in those
without (42.3% vs 6.2%, p = 0.01). Type II MC
occurred in five (35.7%) patients of group 1, five (41.7%) of group
2, and none of group 3. IgGk and IgG
MGUS were found in one (8.3%)
patient of group 2 and one (6.3%) of group 3.
Intrahepatic HCV RNA levels were analyzed in relation to intrahepatic B
cell clonal expansion (Fig. 1). Group 1
patients showed significantly higher mean values than group 3
(1106.4 ± 593.5 vs 406.2 ± 354.3 pg/g, mean ± SD,
p = 0.001) and group 2 (677.3 ± 424.3 pg/g;
mean ± SD, p = 0.048). No direct relation was
established between intrahepatic levels of HCV RNA and the occurrence
of type II MC either in group 1 (p = 0.38) or
in group 2 (p = 0.07).
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Both cloning and sequence analyses of the single dominant band showed
that for each patient of group 1, each tissue compartment yielded a
different VDJ sequence, and was therefore dominated by a different B
cell clone (Fig. 2).
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To overcome this critical point, IgH VDJ gene rearrangement and HCV RNA
were assayed on DNA and RNA, respectively, extracted from portal
tract-containing lymphoid aggregates directly isolated from liver
biopsy sections. Fine microdissection of fresh-frozen sections of two
of three patients (1 and 9) showing B cell monoclonality was used to
precisely separate inflammatory cells from the surrounding
contaminating hepatocytes. Lymphoid follicle-like structures were
isolated from the remaining infiltrating mononuclear cells in the
portal tract, as depicted in Fig. 3.
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A further significant aspect of PCR analysis of microdissected samples
was that HCV RNA genomic sequences were successfully amplified in each
inflammatory aggregate (Fig. 4A).
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). To assess
specificity of the results, PCR-derived products hybridized with a
clonospecific oligoprobe using oligonucleotide specific for the
CDRH3 sequences to detect the same B cell clone
originated in patients 1 and 9. A positive hybridization signal was
detected with DNA extracted either from the core liver biopsies or from
microdissected portal tracts in both patients. No hybridization was
detected in the liver samples that lacked D3-22-related genes. (Fig. 4B). To further exclude nonspecific hybridization signals,
both the hybridization protocol and washing temperatures were optimized
in repeated experiments.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Measurement of intrahepatic HCV RNA levels confirms conclusions of other studies, namely that hepatic viral load does not distinguish patients with more severe liver damage from those with higher serum alanine aminotransferase levels, and probably reflects the lack of a direct contribution of ongoing viral replication to the liver cell injury (30, 31). The direct relation between intrahepatic B cell monoclonal feature and viral load suggests that they are closely related events and is consistent with the notion that HCV plays a direct role in stimulating and maintaining in situ expansion of these B cell clones.
With respect to patients associating type II MC, intrahepatic levels of HCV RNA were found to lack a direct correlation with cryoglobulin production, further suggesting that the occurrence of cryoglobulinemia is an independent variable of HCV infection (13).
In the first part of this study, we investigated the B cell repertoire in different biologic compartments in three patients showing a gel electrophoretic monoclonal profile of intrahepatic B cell expansion and concomitant type II MC. Conclusions that emerged when B cell clonal profiles either from peripheral blood or bone marrow were compared in the same patient provided evidence that monoclonal profiles of B cell expansion do occur in each compartment.
Features of monoclonal B cell expansions appeared in the liver just when examined on total DNA extracted from the core biopsy specimen. When analysis was performed on DNA obtained from microdissected portal lymphoid infiltrates directly isolated from the relative cryostat sections of the same liver biopsy, definite oligoclonal pictures were achieved. Multiple IgH bands, whose number likely corresponded to the number of proliferating B cell clones, were consistently demonstrated. This, indeed, likely reflects methodological problems mainly related to the poor efficiency of the PCR assay used for detecting IgH VDJ gene rearrangements. IgH VDJ PCR sensitivity mainly depends on the proportion of polyclonal B cells present in the sample. This issue was clearly demonstrated by the present experiments, in that cloning and sequence analyses showed that the B cell clone obtained from total DNA was included in those present in the portal tract inflammatory aggregates.
It was of interest that the same B cell clonotypes that expanded commonly in different areas of the same liver had identical size and identical nucleotide sequence in the liver samples from the two patients we studied (1 and 9). These results strongly suggest that common B cell clones in HCV-related lesions are likely induced by a relatively restricted number of Ags. Furthermore, it can be emphasized that common clonotypes can be the result of mutation and division of the same type of B cells, revealing the relatively conserved sequence motifs in CDRH3 region.
The presence of identical clones in these samples might have resulted from contamination during DNA processing. However, the isolation of rearranged D genes from a single clone is not a fake due to selection bias during the PCR amplification, as shown by identification of multiple independently rearranged D genes. All samples were processed in duplicate, and the utmost care was taken to prevent contamination (30). Previous limiting dilution experiments suggested that when the starting DNA concentration is very low, there is preferential amplification of some Ig genes so that one or few monoclonal bands may be detected in polyclonal DNA. The microdissection technique by which we have been able to isolate the smallest lymphocytic structural units in the portal tract of liver tissue sections likely increased PCR sensitivity. Negative controls were conducted through the entire DNA extraction and PCR protocol for each sample. Tissue specimens were divided into two separate samples in the early stages of processing as an added precaution to provide back-up samples, and a clonospecific oligoprobe based on common CDRH3 DNA sequence specifically hybridized with amplified PCR products.
The DH regions of our four panels share some structural features with those that display few random mutations compared with germline D sequences, suggesting that they are daughters of a parental cell that divided within the liver. This implies that intrahepatic clones develop by proliferation of a common precursor accompanied by IgV gene somatic diversification. Indeed, the origin of the parental B cell from which the clones arose is not known, but the small number of mutations in D3-22 germline-related clones found in the portal tract clusters suggests that it arose from a naive B cell, whereas those more heavily mutated may have arisen from memory B cells.
Our technique may not have been sensitive enough to detect a single germline gene in the microdissected clusters. Even so, the present findings suggest that there is a marked heterogeneity in H chain gene usage by individual patients. Hypermutation within a germinal center is closely related to Ag-induced B cell proliferation, so it is reasonable to deduce that the response within the liver is also driven by multiple Ags. In this context, of particular interest are the findings that HCV RNA genomic sequences could be amplified in each microdissected intraportal lymphoid cluster in support of the evidence that HCV is directly involved in the pathogenesis of B cell clonal expansions. The constant presence of HCV in the inflammatory cells of portal tracts suggests that viral Ags are necessary for the continuous maintenance of B cell clones. Though no formal proof exists that this reflects direct infection of B lymphocytes, it likely emphasizes the possible attachment of viral particles by means of cell surface molecules, perhaps CD81 determinants, a member of tetraspanin family, which is considered to represent the putative HCV receptor (31). Interactions with components of the B cell receptor complex would switch on a signal complex to sustain B cell activation (32).
If confirmed by future studies using direct Ab characterization, these findings may have substantial pathobiologic implications because it can be assumed that the intrahepatic B cell response is mainly not polyclonal and that selected Ags are preferentially recognized, thus conferring a strong survival advantage on a distinct B cell subset (i.e., RF-positive cells) already implicated in type II MC and HCV-related lymphoproliferation (33).
This study extends and reinforces previous observations in that the clonally restricted B cell response appears to be a hallmark of HCV infection. Although we have no data on "genuine" autoimmune chronic hepatitis, tissue B cell clonal restriction was rarely detected in HBV-related chronic liver disease (15, 21, 22).
The relationships between the natural pathobiology exhibited by the virus and the cellular origin of B cell clonal restriction must be considered in the context of a limited host response to a pathogen capable of undergoing long-term spontaneous mutations that heavily contribute to the viral burden (34). This Ag overload could initially expand a normal B cell response. Saturation of the APC system could also paralyze subsequent recognition of closely related evolving virus populations. If an array of cross-reactive viral epitopes exists, their presence continues to expand the initially derived and "higher affinity" B cell clones (35). In contrast, this mechanism might constitute a trigger for subsequent transforming events. Although the bearing of B cell clone expansion on malignant transformation is not clear, it seems reasonable to envisage a predisposing role.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Franco Dammacco, Department of Biomedical Sciences and Human Oncology, Section of Internal Medicine and Clinical Oncology, University of Bari Medical School, Policlinico, Piazza G. Cesare 11, 70124 Bari, Italy. E-mail address: dimoclin{at}cimedoc.uniba.it
3 Abbreviations used in this paper: HCV, hepatitis C virus; IC, immune complex; MC, mixed cryoglobulinemia; MGUS, monoclonal gammopathy of undetermined significance; Fr, framework; CDR, complementarity-determining region; Eq, equivalent.
Received for publication August 7, 2000. Accepted for publication April 18, 2001.
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X 174). Lanes
17 and 18, Negative and positive controls of
RT-PCR for HCV RNA assay performed on well-characterized HCV-negative
and -positive liver tissues, respectively. B,
Clonospecific oligoprobe hybridization of the PCR products with a
radiolabeled oligoprobe based on the CDRH3 sequences of the
D3-22-related gene segment (5'-GGATTACTCTGATAGTAGTGATTCTACTTCAAT-3').
Under stringent experimental conditions, positive hybridization signals
were detected in entire liver tissue specimens and in portal tracts of
both patients. No signal was seen in lane 10 with a DNA
segment lacking D3-22 gene (IgH VDJ PCR product obtained from DNA of
entire liver tissue sample of patient 10).