V. De Re

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 (NS31250–1334 and IgG Fc345–355). The same phenomenon of crossreactivity has been shown to occur in vivo after immunization of a mouse with the NS31251−1270 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

Hepatitis C virus productive infection in mononuclear cells from patients with cryoglobulinaemia

The relationship between the occurrence of cryoglobulins and hepatitis C virus (HCV) productive infection in peripheral blood and bone marrow‐derived lymphocytes was explored. HCV minus strand RNA, the viral replicative intermediate, was searched for by a polyA+ tract strand‐specific Tth‐based reverse transcriptase–polymerase chain reaction (RT–PCR) in lymphoid cells of 46 patients with acute and chronic infection. The HCV minus strand was demonstrated in RNA extracted from six (13%) and five (11%) peripheral blood and bone marrow‐derived lymphocytes, respectively. The HCV replicating form in lymphoid cells was associated strictly with mixed cryoglobulinaemia (MCG), in that it was found in six of 13 (46%) MCG patients, including two with B cell non‐Hodgkins lymphoma (NHL). No traces of HCV‐negative strand RNA were found in four patients with acute hepatitis C, in 15 with chronic active hepatitis without extrahepatic disorders, in seven with monoclonal gammopathy of undetermined significance, and in seven with B‐NHL without MCG. These results emphasize the direct role of the virus in the pathogenesis of MCG and support the contention that HCV is not specifically lymphotropic, its entry and replication in lymphoid cells being determined largely by selective interactions.

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 Hodgkins disease (HD) and in a single case of non-Hodgkins 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.

Genetic insights into the disease mechanisms of type II mixed cryoglobulinemia induced by hepatitis C virus

The ability of the immune system to distinguish between self and non-self is critical to the functioning of the immune response. A breakdown in these mechanisms can lead to the onset of autoimmune disease. Clinical and molecular data suggest that shared immunogenetic mechanisms lead to the autoimmune process. The most studied part of the autoimmune process is the human leukocyte antigen (HLA) region. Recently, progress has been made in narrowing down HLA cluster classifications based on structural and functional features of HLA alleles. Using this approach we have investigated 175 patients with hepatitis C virus (HCV)-induced type II cryoglobulinemia (MC), and compared them to a control group of 14,923 bone marrow donors. Additionally, we investigated the frequency of HLA homozygosity in the same groups of subjects. Our results provide evidence of a role for DR5 and DQ3 HLA class II clusters and a higher frequency of HLA homozygous leading to the clinical outcome of type II mixed cryoglobulinemic autoimmune disease. The DR5 cluster is characterized by a Glu in beta 9 and its polymorphism is connected with preferred anchors at beta 9 of the binding peptide, while the DQ3 cluster is characterized by Glu B86 and Leu B87, which allows the binding of large hydrophobic amino acids at p1 of the binding peptide. The mechanisms by which variations in HLA lead to autoimmunity remain unknown, although they are likely to be mediated by continuous presentation of HCV epitopes to T cells and a genetic background that limits the effective clearance of HCV. The results presented in this paper have increased our knowledge of the mechanism of autoimmune disease and B-cell lymphoproliferation during HCV infection. The work was performed in accordance with the principles of the 1983 Declaration of Helsinki. There is no conflict of interest.

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-Hodgkins 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<or= 0.001, RR = 2.498), while the contribution of DR1-DQ1 was higher in HCV (+) NHL without MC (P<or= 0.001, RR = 2.519). Thus, cryoglobulinemia clinical manifestation was found to be correlated with the preferential use of HLA DR-DQ combination in HCV-associated NHL. These data provide new insight into HCV-associated lymphoproliferative pathogenesis.

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.

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.

Absence of human parvovirus B19 DNA in myoepithelial sialadenitis of primary Sjögren’s syndrome

Sjogren’s syndrome (SS) is an autoimmune disease that mainly affects exocrine glands and presents as persistent dryness of the mouth and eyes owing to functional impairment of the salivary and lachrymal glands. The histological hallmark is local infiltration of lymphocytes, which play a major role in tissue damage. Although B cells represent a minority of the lymphoid infiltrates in SS tissue, they may undergo polyclonal activation and oligoclonal/monoclonal expansion, which may, in turn, predispose them to a still unidentified B cell neoplastic transformation. The process of B cell activation and expansion is presumably a consequence of a chronic, although at present unidentified, antigenic stimulus that activates specific subsets of B lymphocytes.1,2 This process resembles a germinal centre reaction, in which B cells that express the antigen receptor with …

T cell receptor variable β 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) β 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 Vβ 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 Vβ clonotypic expansions in 23 of 30 (77%) and in 15 of 21 (71%) MC and non‐MC patients, respectively. The blood compartment contained TCR Vβ 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 Vβ repertoire is clonally expanded in HCV‐related MC with features comparable to those found in chronically HCV‐infected patients without MC.

Proteins specifically hyperexpressed in a coeliac disease patient with aberrant T cells

An aberrant T cell population is the basis for diagnosis of refractory coeliac disease and determines the risk of enteropathy‐associated T cell lymphoma. This disease is serious with a poor survival. Pathogenetic mechanisms sustaining aberrant T cell proliferation remain unknown. Recently, alemtuzumab has been proposed as a promising new approach to treat these patients. Only few single cases have been tested at present; nevertheless, in all the cases a clinical improvement was observed. However, whether intraepithelial lymphocytes have been targeted effectively by alemtuzumab is still debated. This study reports, using two‐dimensional difference gel electrophoresis (2D DIGE), hyperexpressed proteins associated specifically with aberrant T cells found in a patient with coeliac disease by comparison of the protein expression of this sample with that of patients with coeliac disease and polyclonal T cells or with control subjects. The data demonstrated a significantly higher expression of IgM, apolipoprotein C‐III and Charcot–Leyden crystal proteins in a duodenal biopsy specimen of the patient with clonal T cells compared with that of other patients. These preliminary results allow hypothesizing different clinical effects of alemtuzumab in patients with coeliac disease and aberrant T cell proliferation, because as well as the probable effect on T cells, alemtuzumab could exert its effect by acting on inflammatory associated CD52+ IgM+ B cells and eosinophil cells, known to produce IgM and Charcot–Leyden crystal proteins, that we demonstrated to be altered in this patient. The results also emphasize the possible association of apolipoprotein with aberrant T cell proliferation.