T cell receptor variable ß gene repertoire in liver and peripheral blood lymphocytes of chronically hepatitis C virus-infected patients with and without mixed cryoglobulinaemia.

To characterize the repertoire of T lymphocytes in chronically hepatitis C virus (HCV)-infected patients with and without mixed cryoglobulinaemia (MC). T cell receptor (TCR) variable (V) ß clonalities in portal tracts isolated from liver biopsy sections with a laser capture microdissection technique in 30 HCV-positive MC patients were studied by size spectratyping. Complementarity-determining region 3 (CDR3) profiles of liver-infiltrating lymphocytes (LIL) were also compared with those circulating in the blood. The representative results of TCR Vß by CDR3 were also obtained from liver tissues and peripheral blood lymphocytes (PBL) of 21 chronically HCV-infected patients without MC. LIL were highly restricted, with evidence of TCR Vß clonotypic expansions in 23 of 30 (77%) and in 15 of 21 (71%) MC and non-MC patients, respectively. The blood compartment contained TCR Vß expanded clones in 19 (63%) MC and 12 (57%) non-MC patients. The occurrence of LIL clonalities was detected irrespective of the degree of liver damage or circulating viral load, whereas it correlated positively with higher levels of intrahepatic HCV RNA. These results support the notion that TCR Vß repertoire is clonally expanded in HCV-related MC with features comparable to those found in chronically HCV-infected patients without MC.

A new human leukocyte antigen class I allele: HLA-A*02:374.

HLA-A*02:374 differs from HLA-A*02:01:01 by one amino acid change at codon 112 where G is replaced by V.

Identification and sequence analysis of a novel human leukocyte antigen allele B*51:141.

The newly detected HLA-B*51:141 is distinguished from HLA-B*51:08 by a single-nucleotide exchange at codon 30 where D is replaced by Y.

Identification of a new Patr-B*01 variant, Patr-B*0102, by sequence-based typing in a chimpanzee (Pan troglodytes).

Description of a new non-human primate major histocompatibility complex (MHC)-B (Patr-B) allele.

HLA DR-DQ combination associated with the increased risk of developing human HCV positive non-Hodgkin's lymphoma is related to the type II mixed cryoglobulinemia.

This investigation was focused on the contribution of individual human leukocyte antigen (HLA)-DR and -DQ alleles to the human hepatitis C virus (HCV)(+) non-Hodgkin's lymphoma (NHL), with and without mixed cryoglobulinemia (MC), to study whether individual HLA class II alleles are expressed preferentially or equally in human HCV-specific NHL. For this purpose, peripheral blood mononuclear cells were obtained from two groups of patients with HCV(+) NHL and with or without MC (70 and 71 cases, respectively), and from 4575 blood donors. Eighty-three subjects with HCV infection only, and 118 patients with MC, only without lymphoma, were added as additional control groups. Individual HLA-DR and -DQ alleles were determined using high-resolution sequence-based typing and then data were collected by considering the HLA-DRB1 and DQB1 supertypes on the basis of common structural and functional features, proposed by in silico Bioinformatic studies. From the data, it is evidenced that the DR5-DQ3 HLA combination was strongly associated with the HCV (+) MC (+) NHL group of patients compared with bone marrow donor population (P

A new HLA-A*680106 allele identified in individuals with celiac disease from the Friuli area of northeast Italy.

A novel human leukocyte antigen (HLA)-A*680106 antigen was identified in two Italian individuals by polymerase chain reaction sequencing-based typing.

Fibronectin gene polymorphisms are associated with the development of B-cell lymphoma in type II mixed cryoglobulinemia.

OBJECTIVE: To analyse fibronectin (FN) gene polymorphisms in type II mixed cryoglobulinemic syndrome (MCsn), an immune-complex mediated systemic vasculitis linked to hepatitis C virus (HCV) infection and characterised by rheumatoid factor (RF) positive B-cell proliferation at high risk for the progression into non-Hodgkin's lymphoma (NHL). METHODS: Samples from 74 patients with MCsn (type II serum cryoglobulins and clinical signs of vasculitis) were studied. In all, 58 (78.4%) patients were HCV-positive. In total, 21 (28.4%) patients developed a B-cell NHL during the course of MCsn. A total of 72 patients with HCV-negative and MC-unrelated NHL and 110 healthy blood donors (HBDs) were taken as controls. HaeIIIb and MspI FN gene polymorphisms were analysed by PCR and specific restriction enzyme digestions, following reported procedures. Plasma FN levels were analysed by ELISA, whenever possible. RESULTS: HaeIIIb and MspI allele and genotype frequencies did not differ between MCsn patients and HBDs. Of note, the DD-MspI (OR = 5.99; CI 1.77-20.261, p = 0.0039) and the AA-HaeIIIb (OR = 4.82, CI 1.42-16.39, p = 0.0176) homozygosis appeared significantly associated with the development of B-cell NHL in MCsn patients, with the HaeIIIb A allele possibly conferring an increased risk of NHL in the general population (OR = 1.72, CI 1.128-2.635, p = 0.0133). None of the other MCsn-related clinical manifestations were significantly associated with a particular genetic pattern. No association between FN plasma levels and FN genotypes was found. CONCLUSION: Genotyping for MspI and HaeIIIb FN gene polymorphisms may be clinically relevant to define the risk of lymphoma development in MCsn.

[Fibronectin gene polymorphisms and clinical manifestations of mixed cryoglobulinemic syndrome: increased risk of lymphoma associated to MspI DD and HaeIII AA genotypes]. / Polimorfismi del gene della fibronectina e manifestazioni cliniche della sindrome crioglobulinemica: aumentato rischio di linfoma legato ai genotipi MspI DD e HaeIII AA.

OBJECTIVE: To analyse FN gene polymorphisms in type II mixed cryoglobulinemic syndrome (MCsn), an immune-complex mediated systemic vasculitis linked to hepatitis C virus (HCV) infection and characterized by rheumatoid factor (RF) positive B-cell proliferation at high risk for the progression into non Hogkin's lymphoma (NHL). METHODS: Samples from eighty-one patients, with MCsn (type II serum cryoglobulins and clinical signs of vasculitis were studied. Sixty-five (65/81, 80.3%) patients were HCV-positive. Twenty-one (25.9%) patients had developed a B-cell NHL during the course of MCsn. Seventy-two patients with HCV-negative and MC-unrelated NHL and 110 healthy blood donors (HBDs) were taken as controls. HaeIIIb and MspI FN gene polymorphisms were analysed by ELISA, whenever possible. RESULTS: HaeIIIb and MspI allele and genotypic frequencies did not differ between MCsn patients and HBDs. Of note, the DD-MspI allele and genotype frequencies did not differ between MCsn patients and HBDs. Of note, the DD-MspI (OR = 5.56; DI = 1.67-18.51, p = 0.0046) and the AA-HaeIIIb (OR = 5.54, CI = 1.64- 18.76, p = 0.0066) homozygosis appeared significantly and independently associated with the development of B-cell NHL in MCsn patients, with the HaeIIIbA allele possibly conferring an increased risk of NHL in the general population (OR = 1.72, CI = 1.128-2.635, p = 0.0133). In contrast, the major vasculitic manifestations, such as peripheral neuropathy, skin ulcers and glomerulonephritis tended to be associated with the counterpart MspI C allele. No association between FN plasma levels and FN genotypes was found. CONCLUSION: Genotyping for MspI and HaeIIIb FN gene polymorphisms may be clinically relevant to define the predisposition to the major clinical manifestations in MCsn.

HCV-NS3 and IgG-Fc crossreactive IgM in patients with type II mixed cryoglobulinemia and B-cell clonal proliferations.

We demonstrate that in three cases of MC (two with immunocytoma), the IgM-RF+ component of their cryoprecipitated represents the circulating counterpart of the B-cell receptor (BCR) of the monoclonal overexpanded B-cell population. These IgMs were isolated and used to demonstrate a crossreactivity against both hepatitis C virus (HCV) NS3 antigen and the Fc portion of IgG. Epitopes were identified in a fraction of exemplary samples by using epitope excision approach (NS(31250-1334) and IgG Fc(345-355)). The same phenomenon of crossreactivity has been shown to occur in vivo after immunization of a mouse with the NS3(1251-1270) peptide. To verify if the same reaction was also present in MC samples characterized by an oligo/polyclonal B-cell proliferation, IgM crossreactivity was tested in 14 additional samples. Five out of the 14 were reactive against HCV NS3 and 11 out of 14 were reactive against IgG-Fc peptide. The data support the role of HCV NS3 antigen in a subset of patients with MC, whereas the high frequency of the IgG-Fc epitope suggests that these B cells originate from precursors strongly selected for auto-IgG specificity. We suggest that engagement of specific BCRs by NS3 (or NS3-immunocomplex) antigen could explain the prevalence of IgM cryoglobulins in these patients.

Identification and characterization of an Epstein-Barr virus-specific T-cell response in the pathologic tissue of a patient with Hodgkin's disease.

Several lines of evidence indicate that an impairment of EBV-specific immune responses may contribute to the pathogenesis of Hodgkin's disease (HD). At present, however, it is not clear whether a defective immunity to EBV is a characteristic restricted to EBV-associated HD cases or a more generalized phenomenon, part of the inherent immune deficiency of HD patients. In this study, we have addressed this issue by analyzing EBV-specific responses in infiltrating T lymphocytes (TILs) from one HD biopsy, where the virus was confined to a small proportion of apparently normal lymphocytes. TIL cultures were established using low amounts of recombinant interleukin 2 and in the absence of specific stimulation, conditions that preferentially induce the proliferation of in vivo activated T cells. An EBV-specific cytotoxic component was revealed by the capacity of these TILs to lyse autologous EBV-positive lymphoblastoid cell lines (LCLs) obtained by spontaneous transformation from the lesion but not HLA-mismatched LCLs and autologous phytohemagglutinin blasts. This cytotoxic activity closely resembled that of EBV-specific memory T cells, which may be reactivated from the blood lymphocytes of healthy donors by in vitro stimulation with autologous LCLs. The use of a panel of appropriately HLA-matched B95.8-transformed LCLs as targets in standard 51Cr release assays revealed EBV-specific cytotoxic responses to be restricted mainly through the A11 and B44 HLA alleles with a minor HLA-A26-restricted component. Using autologous fibroblasts infected with recombinant vaccinia viruses expressing the EBV latent antigens, the TIL culture was shown to recognize latent membrane protein 2 and, to a lesser extent, EBV-encoded nuclear antigen 6. In addition, a strong proliferative response was induced by coculture of TILs with autologous but not with allogeneic LCLs or autologous phytohemagglutinin blasts. Six CD4-positive, EBV-specific T-cell clones were isolated by limiting dilution. The study of cytokine mRNA expression, carried out by reverse transcriptase-assisted PCR, revealed that three of these T-cell clones expressed a Th0 phenotype, whereas 1 had a Th2 phenotype. These findings are consistent with the presence in this HD lesion of an ongoing immune response against EBV-carrying cells and suggest that the complex immune deficiency that characterizes HD patients probably does not include a generalized, constitutional defect of EBV-specific T-cell responses.

N-myc activation by proviral insertion in MCF 247-induced murine T-cell lymphomas.

N-myc proto-oncogene rearrangement was found in three out of six AKR murine T-cell lymphomas induced by the highly oncogenic MCF 247 MuLV. Molecular analyses showed that structural modification of the proto-oncogene in all three lymphomas was in the consequence of MCF 247 proviral integration within the gene III exon. All integrated proviruses have the same transcriptional orientation as the N-myc gene. As a consequence of proviral insertion, the N-myc gene becomes transcriptionally active, producing an abnormal mRNA. These findings suggest a possible causative role of such an integrative event in murine T-cell lymphomagenesis.

Frequency of bcl-2/IgH translocation in patients with non-Hodgkin's lymphoma and chronic hepatitis C virus infection.

AIM: It has been previously suggested that t(14;18) translocation of bcl-2 to the immuno-globulin heavy chain (IgH) locus may contribute to pathogenesis of lymphoproliferative disorders related to hepatitis C virus (HCV) infection, including type II mixed cryoglobulinemia (MC). METHODS: In this study, the presence or absence of t(14;18) translocation was determined in tumor biopsy specimens and peripheral blood mononuclear cells (PBMCs) for 48 NHL patients with chronic HCV infection. RESULTS: In tumor biopsy specimens from 32 HCV-positive NHL patients, bcl-2/IgH translocation was detected in 1 of 13 patients with MC syndrome (7.7%) and 3 of 19 patients without MC syndrome (15.8%). In PBMCs from 23 HCV-positive NHL patients, this translocation was observed in 3 of 6 patients with MC syndrome (50%) and 4 of 17 patients without MC syndrome (23.5%). Interestingly, bcl-2/IgH translocation was found in 2 extranodal marginal zone B-cell lymphoma tissues from HCV-infected patients. CONCLUSIONS: However, additional studies are required to better clarify the relationship between this translocation and extranodal marginal zone B-cell lymphoma development. Although the frequency of bcl-2/IgH translocation in PBMCs from patients with chronic HCV infection is higher than that of other NHL patients, this increased translocation rate remains to be elucidated.

Hepatitis C virus-related hepatocellular carcinoma and B-cell lymphoma patients show a different profile of major histocompatibility complex class II alleles.

Comparison of human leukocyte antigen (HLA) frequencies in patients with hepatitis C virus (HCV)-associated hepatocellular carcinoma (HCC) and in patients with HCV-associated non-Hodgkin's lymphoma (NHL) has not been addressed previously. To this aim, we investigated the distribution of HLA class II alleles in two selected groups of HCV-infected patients. Group 1 included 50 patients with HCV-associated NHL; group 2 included 29 patients with HCV-associated HCC. A control group included 144 hospitalized patients without NHL or HCC and who were negative for HCV, hepatitis B virus, and human immunodeficiency virus antibodies. Polymerase chain reaction sequence DRB1 and DQB1 specific-primer methods were used. DRB1*1101/DQB1*0301 haplotype, which mainly favors the spontaneous clearance of HCV infection, was lower in HCC subjects than in controls, whereas HLA-DRB1*1104/DQB1*0301, was higher in NHL patients. These findings suggest different pathogenic pathways in HCC and in NHL development. In patients with HCV-associated HCC, a major protective role of DQB1*0301 allele, rather than DRB1*11, was found, probably because of a better HLA class II-associated virus clearance. By contrast, the same allele as HLA-DRB1*04 showed an increase in HCV-associated NHL. These data suggest that NHL and HCC development may be associated to a different response with respect to chronic HLA class II-restricted antigen presentation (perhaps a switch toward CD4+Th2 response in NHL?) or, alternatively, that these alleles could be in linkage disequilibrium to unrelated gene(s), or are in synergy with other immunomodulatory genes that may confer increased risk for NHL.

A coordinated proto-oncogene expression characterizes MCF 247 murine leukemia virus-induced T-cell lymphomas irrespectively of proviral insertion affecting myc loci.

Proto-oncogene transcriptional activation was analyzed in a group of MCF 247 MuLv-induced T-cell lymphomas to identify transformation-specific gene activations and determine whether the proviral insertion near a myc gene could promote a peculiar mechanism of transformation through a differential proto-oncogene expression pattern. Of the six lymphomas analyzed, three showed the MCF 247 provirus integrated within the N-myc locus, one carried the provirus integrated near c-myc, whereas for the remaining two, no evidence of proviral integrations in any of the known myc loci was obtained. Independently of the integrative events, the pattern of proto-oncogene expression was almost identical in all six lymphomas. These findings seem to rule out the existence of a peculiar pathway of transformation associated with the proviral insertion near a myc locus. Moreover, the transcription pattern observed was qualitatively identical to that displayed by normal thymocytes; only quantitative differences in c- or N-myc, c-myb and Ha-ras were observed. These results suggest that the T-cell proto-oncogene activation program is not qualitatively affected by the transforming event(s).

Genotypic and immunohistological demonstration of the progression of an unusual reactive-like B-cell lymphoproliferative disorder to a high grade diffuse lymphoma.

In the present study the clinical and pathological evolution of a reactive-like B-cell lymphoproliferative disorder with an unusually high content of T cells is described. Immunogenotypic analysis showed that the same phenotypically atypical B-cell clone, characterized by the unusual presence of an immunoglobulin (Ig)K gene rearrangement, with the heavy chain (IgH) gene in germline configuration, was invariantly present in all phases of the disease. The disorder showed an indolent course for a long period of time during which the clonal B-cell population coexisted with an abundant, reactive T-cell component in different locations of the disease. These findings, together with the observation of spontaneous progression and regression phases of the disorder and its responsiveness to corticosteroids, suggest that functional interactions between the B-cell clone and the polyclonal infiltrating T cells probably were involved in the pathogenesis of the disease. After the administration of the antiblastic treatment, a progressive reduction of the reactive T-cell component was observed with the concomitant evolution to a diffuse large cell (immunoblastic) B-cell lymphoma and the appearance of an IgH gene rearrangement. The biological characteristics and the clinical evolution of the case described here are similar to those reported for the so-called "T-cell-rich B-cell lymphomas" (TCRBCLs). These findings suggest that the T-cell-rich pattern may identify a group of B-cell lymphoproliferations with common pathogenetic mechanisms and clinical behavior.

Type 2 Epstein-Barr virus genome and latent membrane protein-1 expression in a T-cell-rich lymphoma of probable B-cell lineage.

In a 79-year-old white woman, a lymphoproliferative disorder that was associated with type 2 Epstein-Barr virus (EBV) infection or reactivation, documented by three subsequent lymph node biopsies, was studied. After an initial phase with features of reactive lymphadenopathy with exhaustion of the follicular germinal centers and depletion of the B-cell lymphoid population, the disease evolved to a T-cell-rich lymphoma in which a clonal cell population of probable B-cell origin was identified. Such clonal cell population harbored the viral genome and expressed EBV latent membrane protein-1 but not EBV nuclear antigen-2. The implications of immunologic interactions between the clonal EBV-infected cells and the reactive T-cell component in the pathogenetic process are discussed.

Genotypic and immunophenotypic characterization of two human light chain-only B-cell non-Hodgkin's lymphomas.

Two atypical human non-Hodgkin's lymphomas (NHLs) that exhibited unusual genotypic and in situ immunophenotypic abnormalities are described. Immunophenotypically, both NHLs lacked surface Ig heavy chains. With the exception of the MB2 B-cell-associated antigen, no B- and T-cell differentiation antigen was detected in case 1. NHL 2 failed to show evidence of clonality by immunohistochemical analysis but revealed the presence of many B-lymphocytes with an abnormal phenotypic profile: CD19+, CD20+, CD22+, kappa-, lambda-, CD9-, CD10-, CD21-, and CD24-. Genotypic analysis indicated that both lymphomas derived from anomalously matured pre-B-cells that had rearranged the lambda or kappa light chain genes but not the Ig heavy chain gene. The neoplastic cells of the two NHLs resemble the light chain-only B-cells recently discovered, following Epstein-Barr virus immortalization, in the human bone marrow. The authors' data confirm, therefore, the existence of the light chain-only B-cells in the human hematopoietic compartment. Moreover, their results emphasize the conclusive role of the immunogenotypic analysis in defining clonality, lineage, and maturation abnormalities of such atypical NHLs.

Head and neck lymphomas associated with human immunodeficiency virus infection.

OBJECTIVE: To focus on clinicopathologic data of non-Hodgkin's lymphomas (NHLs) of the head and neck area (with lymph nodal or extranodal localization) arising in patients with immunodeficiency virus (HIV) infection. PATIENTS: Among 73 evaluable patients for presenting symptoms, of a total of 82 with HIV-related NHLs whose conditions were diagnosed at the Centro di Riferimento Oncologico, Aviano (Italy), between September 1984 and May 1992, 15 (21%) had primary, solitary head and neck (P-HN) lymphoma and 13 (18%) had systemic head and neck (S-HN) lymphoma arising from this region. RESULTS: Ten (67%) of 15 patients with P-HN NHL had stages I and II, whereas all patients with S-HN NHL had stages III and IV. Twenty-seven of 28 patients had extranodal disease at presentation, the principal sites being Waldeyer's ring and soft tissues. There were only high-grade (14 cases) or intermediate-grade (three cases) NHLs, the most frequent histotypes being small noncleaved cell, Burkitt's type, and large-cell immunoblastic. Seven of 11 cases in the miscellaneous group of the working formulation were classified as Ki-1+ anaplastic large-cell lymphoma. By immunophenotypic and genotypic characterization, a B-cell derivation was suggested for 21 of 28 NHLs. After combination chemotherapy with or without radiotherapy, a complete remission was observed in seven (58%) of 12 patients with P-HN lymphoma and in only two patients with S-HN lymphoma. Median survival was 9.8 months for the patients with P-HN lymphoma and 8.3 months for the other patients. Thirteen patients died, the most common causes of death being opportunistic infections (five cases) and progression of lymphoma (four cases). CONCLUSIONS: Most HIV-infected patients with head and neck NHL had severe immunodeficiency, extranodal disease, aggressive histologic findings, and a poor treatment response.

Is the Epstein-Barr virus involved in Hodgkin's disease?

EBV genomes, in clonal episomal form, were detected in 7 out of 17 cases of Hodgkin's disease (HD) and in a single case of non-Hodgkin's lymphoma which occurred in a patient after therapeutic treatment for HD. The experimental data presented imply that a clonal cell population, harboring the EBV genome, must be present in EBV-positive HD. In light of this finding we are attempting to reconsider the abundant literature on this lymphoproliferative disorder, and suggest a reevaluation of the possibility that EBV could be etiologically involved in HD.