Gianfranco Ferraccioli

Oligoclonal non-neoplastic B cell expansion is the key feature of type II mixed cryoglobulinemia: clinical and molecular findings do not support a bone marrow pathologic diagnosis of indolent B cell lymphoma.

OBJECTIVE Type II mixed cryoglobulinemia (type II MC) is often characterized by features of indolent B cell lymphoma (IBCL) found on pathologic examination of bone marrow, whereas the clinical evidence does not indicate a neoplastic disorder. To better address the issue of indolent malignant versus nonmalignant bone marrow lymphoproliferation underlying type II MC, molecular analyses of B cell clonality were performed in the present study, in conjunction with clinical and pathologic characterization. METHODS Polymerase chain reaction DNA amplification of immunoglobulin heavy chain genes was performed in bone marrow biopsy specimens obtained from 15 selected patients with type II MC, all infected with hepatitis C virus. Five of them had also developed overt B cell lymphoma (OBCL) during followup. Bone marrow features were consistent with IBCL in 9 of the 15 patients (group 1) and with reactive lymphoplasmacytosis in 6 of the 15 (group 2). RESULTS An oligoclonal B cell expansion was detected in 6 of 9 baseline bone marrow lesions from group 1 patients (biclonal or monoclonal expansion in the remaining 3 cases), and in 6 of 6 from group 2 patients. OBCL was always monoclonal. Selected lesions were analyzed by clonospecific hybridization and by cloning and sequence analysis in patients who had developed OBCL at followup. In 4 of 5 cases, OBCL did not originate from the dominant B cell clones that were overexpanded in the putative neoplastic baseline bone marrow lesions. OBCL clones showed significant homology with rheumatoid factor database sequences. CONCLUSION Based on the present results, as well as on evidence from previous studies of liver lesions, oligoclonal non-neoplastic B cell proliferation in the course of chronic infection-related inflammation appears to be the key feature of type II MC. Of note, molecular evidence from target tissues supports the clinical findings both at the time of type II MC diagnosis and in cases of OBCL complication. Bone marrow pathologic findings resembling those of IBCL should thus be considered in the light of clinical and molecular evidence.

Salivary gland B cell lymphoproliferative disorders in Sjögren's syndrome present a restricted use of antigen receptor gene segments similar to those used by hepatitis C virus-associated non-Hodgkins's lymphomas

Sjögrens syndrome (SS) represents a pathological model of the evolution from polyclonal B lymphocyte activation to oligoclonal / monoclonal B cell expansion, which may culminate in the development of a malignant lymphoproliferative disease. The different phases of this process are usually marked by the appearance of antigen‐driven activated B cell clones, which are commonly IgM‐positiveand with rheumatoid factor (RF) activity. However, the agent(s) able to trigger B cell proliferation is still unknown. A similar pathogenetic mechanism exist in mixed cryoglobulinemia, another autoimmune disease that often evolves to non‐Hodgkins lymphoma (NHL) and in which hepatitis C virus (HCV) infection has been demonstrated to play an etiopathogenetic role. In the present study, we cloned and sequenced the antigen receptor (IgR) variable region genes of SS‐associated monoclonal non‐neoplastic lymphoproliferations and compared them with those of our previous reported HCV‐associatedNHL, to derive clues on the antigen(s) that sustains SS. The results obtained showed remarkable homologies between the antigen combinatory regions of the IgR expressed by both diseases. These homologies concern: a) the specific combinations of heavy and light variable region genes; b) the limited length of complementarity‐determining regions (CDR3); c) the homology with antibodies with RF activity; d)the amino acid sequences of CDR3 in which common somatic mutations are present that possibly determine the antigen‐binding specificity. In conclusion, although there are significant differences between SS and HCV‐associated lymphoproliferative diseases, they share many molecular characteristics, which suggest an immunological cross‐reactivity or molecular mimicry among the agents that underlie these disorders.

B-cell clonality and infection with Helicobacter pylori: implications for development of gastric lymphoma.

BACKGROUND: Although Helicobacter pylori has been implicated in the pathogenesis of gastric mucosa associated lymphoid tissue (MALT) and MALT lymphoma, it is not known how it may trigger these lesions and whether there is an identifiable pre-neoplastic stage. AIMS: To investigate the relation between MALT, H pylori infection, and B-cell clonality (a potential marker of pre-neoplastic lesions). PATIENTS: 141 subjects with simple dyspepsia. METHODS: Gastric biopsy specimens from all patients were examined for MALT and H pylori. Of these, 25 consecutive MALT positive specimens were scored for features of MALT lymphoma and VDJ clonality studied by polymerase chain reaction. RESULTS: Overall, prevalence was 62% for H pylori and 46% for MALT. VDJ clonality was frequent in the sub-group studied (nine of 25), mostly associated with lymphoid follicles (eight of nine or 89%), and with a high scoring for MALT lymphoma. VDJ clonality was equally frequent in patients with and without H pylori (seven of 20 and two of five or 35% and 40% respectively). CONCLUSIONS: B-cell clonality is unexpectedly common in subjects with simple dyspepsia and MALT raising clinical management questions. These findings also suggest that the cascade MALT formation--B-cell clonality--MALT lymphoma may not be uniquely associated with H pylori infection.

Changes in regional cerebral blood flow after a cold hand test in systemic lupus erythematosus patients with Raynaud's syndrome.

The appearance of vasospastic features in the central nervous system (CNS) after a cold stressor test was Investigated through single photon emission computed tomography (SPECT) of regional cerebral blood flow in patients with systemic lupus erythematosus, with and without Raynauds syndrome, and in scleroderma patients. We have shown that Raynauds syndrome may occur in the CNS and that anticardiolipin or lupus anticoagulant positivity may favour perfusion defects.

Lack of HCV infection in malignant cells refutes the hypothesis of a direct transforming action of the virus in the pathogenesis of HCV-associated B-cell NHLs.

Aims and background Preliminary evidence suggests that hepatitis C virus (HCV) might play a pathogenetic role in autoimmune-related, non-malignant B-cell lymphoproliferation, as well as in a subset of B-cell non-Hodgkins lymphomas (NHLs). With regard to the mechanism(s) by which HCV might favor B-cell expansion and malignant transformation, most data support an indirect pathogenetic role of the virus as an exogenous trigger. A direct oncogenetic role of HCV by direct cell infection and deregulation has only been hypothesized on the basis of the lymphotropism of the virus. Methods In this study we investigated the possible HCV infection of NHL B cells by means of sensitive and quantitative polymerase chain reaction (PCR) on affinity-purified neoplastic cells, and by HCV-specific immunohistochemistry and in situ hybridization. Results HCV infection of neoplastic B cells was documented in only three cases, namely the low-grade B-cell NHLs that arose in the course of mixed cryoglobulinemia syndrome (MC). HCV infection, below one viral genome per cell, was detectable only by PCR. All the remaining low-grade (one case) and high-grade B-cell NHLs (two cases) were HCV uninfected. Previous immunoglobulin gene analyses were consistent with an antigen-driven B-cell lymphoproliferation in the studied cases. Conclusions Overall, our data are consistent with an indirect oncogenetic role of HCV in B-cell lymphomagenesis as an exogenous trigger. Infection of B cells by HCV appears possible in some NHL subsets, but the implications remain unknown.

Helicobacter pylori Associated Antigastric Autoantibodies: Role in Sjögren's Syndrome Gastritis

Background.  Previous studies have shown that Helicobacter pylori seroprevalence in Sjögrens syndrome is comparable with that of the general population. However, the origin of the chronic gastropathy associated with this syndrome and the role of local autoimmunity – possibly triggered by bacterial infection – in its pathogenesis remain unclear.

The relevance of VDJ PCR protocols in detecting B-cell clonal expansion in lymphomas and other lymphoproliferative disorders

Aims and background The detection of immunoglobulin heavy chain variable (VH)-diversity (DH)-joining (JH) region gene rearrangement by polymerase chain reaction (VDJ PCR) has been recently proposed as a rapid approach to assess B-cell clonality in lymphoproliferative disorders. The aim of the present study was to determine the efficacy of VDJ PCR in a wide spectrum of lymphoproliferative disorders previously characterized by immunohistochemistry and Southern blot (SB). Methods 83 SB-rearranged B-cell non-Hodgkins lymphomas (NHL) of different histotype, 22 cases of SB-unrearranged classical Hodgkins disease (HD), 18 cases of HIV-related reactive lymphadenopathy, and 4 frankly pre-lymphomatous lesions (MESA) in the course of Sjögrens syndrome were investigated by 2 different VDJ PCR protocols (FR3, FR2). Results The detection rate in NHL was 64% and 71% using the protocols FR3 and FR2, respectively. However, the overall VDJ PCR efficacy increased to 81% by combining the results of both protocols. In addition, differences in the combined, as well as in the single FR3 or FR2 protocol efficacy, were noted in the different NHL subgroups. B-cell clonality was also detected in 4/22 (18%) SB-unrearranged classical HD cases and in 2/18 (11%) reactive lymphadenopathy cases, whereas it was demonstrated in all the MESA lesions, 2 of them being SB-negative. Conclusions VDJ PCR represents a useful and rapid technique to detect B-cell clonality in NHL, although with some differences depending on the NHL histotype and the panel of primers employed. The technique may also be of value to investigate the possible progression of early B-cell clonal expansion into frankly B-cell malignancy and to contribute to the controversy about the clonal lineage origin of the putative HD malignant cells.

Platelet GPIIb/IIIa (P1A1/2) polymorphism in SLE: clinical and laboratory association

It has been reported that the level of anticardiolipin antibodies (aCL) is probably genetically determined, as HLA-DR7 (or HLA-DR4) has been shown to be associated with persistently high levels of aCL.1 No reported data are available about genetic factors implicated in ischaemic or thrombotic events in patients with systemic lupus erythematosus (SLE) without antiphospholipid antibodies (aPL). In this study we examined the frequency of the platelet GPIIb/IIIa-P1A2 polymorphism in a series of patients with SLE. We assessed the relationship between the A2 allele prevalence2 and the clinical and immunological manifestations, as possible predisposing biological factors to major ischaemic manifestations such as central nervous system (CNS) lupus or Raynaud’s phenomenon as defined according to the American College of Rheumatology (ACR) Committee.3 We studied a cohort of 109 patients with SLE attending the rheumatology referral centres of the universities of Udine, Pisa, and Milan, classified according to the ACR criteria. For comparison, 161 patients with rheumatoid arthritis (systemic inflammatory disease), 54 with systemic sclerosis (chronic ischaemic-thrombotic vasculopathy), and 128 healthy blood donors (HBD) recruited from the blood transfusion service were studied. The clinical and laboratory manifestations were carefully examined to detect a possible association between genotypes and symptoms, signs, and immunological characteristics. In particular, we focused on nephritis, vasculitis, …

Response of mononuclear cells to lipopolysaccharide and CpG oligonucleotide stimulation: possible additive effect in rheumatoid inflammation

The relationship between infections and autoimmune inflammation is of paramount importance in all chronic inflammatory diseases.1 The crucial step is the interaction between the Toll-like receptor family and bacterial components, which can lead to activation of phagocytic and dendritic cells.2,3 Whether bacterial components may indeed reactivate autoimmune inflammation is at present still unclear. However, it is well known that oligonucleotides containing unmethylated CpG sequences (CpG-ODN), while stimulating bacterial DNA, might have immunostimulatory activity.3 We have recently presented data suggesting that moderate infections such as upper-lower respiratory or urinary tracts infections may reactivate rheumatoid arthritis (RA) in patients receiving etanercept.4 There is evidence in experimental animals2,3 that Toll-like receptor 4 (activated by lipolysaccharide (LPS)) can induce a cytokine response, whereas Toll-like receptor 9, activated by CpG-ODN, induces mononuclear cell activation in a much more restricted manner. Especially in humans, …