Spontaneous and pokeweed mitogen induced in vitro immunoglobulin and IgM rheumatoid factor production by peripheral blood and synovial fluid mononuclear cells in rheumatoid arthritis

Enzyme-linked immunosorbent assays (ELISA) were used to measure IgG, IgM and IgM rheumatoid factor (IgM RF) production in supernatants of pokeweed mitogen (PWM) stimulated peripheral blood mononuclear cells (PBMNC) of patients with rheumatoid arthritis (RA) and controls. Spontaneous and stimulated IgG and IgM production by RA and controls was comparable, but spontaneous production of IgM RF was only observed in RA and was related to their drug therapy. A significant difference was found between PWM induced IgM RF production in RA and controls and also between patients on "second-line" and on nonsteroidal anti-inflammatory drugs (NSAID). In addition, spontaneous IgM RF production by synovial fluid cells was significantly higher than the paired PBMNC.     

Use of anti-idiotypic antibodies to demonstrate rheumatoid factor producing bone marrow cells in essential mixed cryoglobulinaemia.

Specific cells producing rheumatoid factor (RF) were demonstrated in a bone marrow preparation of a patient with essential mixed cryoglobulinaemia. This was achieved by the use of labelled anti-idiotypic antibodies raised against purified RF from the patient's circulating cryoglobulin. This technique may be applicable to other diseases in which specific antibodies are pathogenetically implicated. The more precise demonstration of specific antibody-producing marrow cells may in addition have therapeutic implications.     

Occult hepatitis C virus infection in type II mixed cryoglobulinaemia

Summary. Mixed cryoglobulinaemia, when not secondary to other well-defined immunological disorders, is commonly associated with hepatitis C virus (HCV) infection. However, a minority of cases lack evidence of HCV infection and are, therefore, defined as 'true essential' mixed cryoglobulinaemias. We thoroughly investigated three such patients to determine the aetiology of this disorder.
Antibodies to HCV (anti-HCV) and HCV RNA, detected by sensitive enzyme-linked immunosorbent and polymerase chain reaction assays in serum and in concentrated cryoglobulins, were repeatedly negative in the three patients. Despite the lack of evidence for HCV infection, two of them were still treated with interferon α -2a assuming unrecognized viral infection. Both patients demonstrated excellent clinical and laboratory responses, but cryoglobulinaemia relapsed after the withdrawal of therapy. At the time of relapse, HCV RNA genomic sequences were detected for the first time in the cryoprecipitates of both patients. In the third case, HCV RNA was demonstrated for the first time during a flare of cryoglobulinaemia coincident with varicella infection. In all three patients anti-HCV antibodies remained negative throughout follow-up.
We conclude that some apparently 'essential' forms of mixed cryoglobulinaemia can be caused by occult HCV infection. Interferon therapy can be taken into consideration in such HCV-negative cases.     

Plasma fibronectin and microvascular damage in essential mixed cryoglobulinaemia

Summary Plasma fibronectin (FN) was measured in 17 patients with essential mixed cryoglobulinaemia (EMC) and in 17 normal subjects by single radial immunodiffusion (RID) and enzyme-linked immunosorbent assay (ELISA). In 9 patients the presence of FN in the cryoprecipitates was also assessed by immunoblotting. In the EMC group, plasma FN levels were significantly lower than in control subjects, using both methods, and FN was constantly demonstrated in EMC cryoprecipitates. Capillaroscopic observation of the capillary bed in skin and bulbar conjunctiva, performed in all cases, showed severe alterations of microcirculation in EMC patients. A negative correlation between plasma FN and capillaroscopic abnormalities of skin capillaries was observed. These data support the hypothesis that plasma fibronectin plays a role in the pathogenesis of systemic vasculitis in EMC.     

Spontaneous and aggregated IgG induced rheumatoid factor producing cells in rheumatoid arthritis

Summary Seropositive rheumatoid arthritis (RA) patients were found to have high numbers of spontaneously occurring cells making rheumatoid factor (RF) reactive with human IgG as measured by a RF plaque forming cell (RF-PFC) assay. There was a significant positive correlation between the number of RF-PFC and both disease activity measured by the sedimentation rate and RF titer measured by the RA latex test. Aggregated IgG and pokeweed mitogen were equally effective stimulators of RF-PFC in cultures of RA peripheral blood mononuclear leukocytes. The rheumatoid ratio of helper (T4): suppressor (T8) T lymphocytes was also significantly increased over the ratio of normal controls, but this ratio did not correlate with the number of RF-PFC. Aggregated IgG or immune complexes may be responsible for stimulating RA RF-PFC in vivo.     

Bone marrow B-cell clonal expansion in type II mixed cryoglobulinaemia: association with nephritis

OBJECTIVES: To investigate the relationship between the pattern of bone marrow (BM) B-cell expansion and the clinical features of mixed cryoglobulinaemia (MC) syndrome. METHODS: Fifty-five patients with type II MC syndrome were analysed. Their median age was 64 yrs (range 24-82), the median disease duration was 6 yrs (range 1-26) and the mean follow-up after BM analysis was 2.65 yrs (s.d. = 1.33). Peripheral neuropathy was present in 33 patients (60%), nephritis in 14 (25.4%), skin ulcers in 14 (25.4%) and lymphoma or atypical lymphoproliferative disorder (LPD) in 17/55 (30.9%). Anti-HCV antibodies were found in 43/55 patients (78.2%). BM B-cell expansion was evaluated by a semi-nested PCR amplification of the V-D-J region of the IgH genes. RESULTS: A clonal B-cell expansion in the BM was found in 33/55 (60%) patients, while a polyclonal pattern in 22/55 (40%). A BM pattern of clonal B-cell expansion increased the risk of nephritis of about 10 times [odds ratio (OR) = 10.11, CI95%1.52-67.31], if compared to a polyclonal pattern. In contrast, the risk of skin ulcers was decreased in BM clonal cases (OR = 0.09, CI95%0.02-0.49). Overt lymphomas did not emerge from patients with BM monoclonal expansion (without clinical or histopathological features of lymphoproliferation; or with LPD) in a short-term, consistent with the finding that monoclonality was associated with nephritis and not with an underlying, not recognized lymphoma. CONCLUSION: BM clonal B-cell expansion is associated with nephritis in MC syndrome. Particular B-cell clones may be preferentially expanded and may play a pathogenic role in MC nephritis.     

Lack of efficacy of Rituximab in a patient with essential mixed cryoglobulinaemia

SIR, Whilst it is encouraging to read the increasing body ofliterature reporting positive results using Rituximab in hepatitisC-associated mixed cryoglobulinaemia (MC) such as the recentarticle by Quartuccio et al. [1], we feel that it is equallyimportant to record treatment failures in this area where theevidence is not yet clear. We describe the case of a woman withessential mixed cryoglobulinaemic (EMC) vasculitis, who failedto respond to treatment with rituximab. A 57-yr-old woman presented with     

Detailed analysis of the E2-IgM complex in hepatitis C-related type II mixed cryoglobulinaemia

Hepatitis C virus (HCV) plays a major role in the induction of type II mixed cryoglobulinaemia (MCII). The role of HCV proteins and virus-host interaction in the pathogenesis of MC remains to be defined. To address this issue, we have characterized, in detail, the monoclonal IgM and the viral component of circulating immune complexes in eight patients with HCV-associated MCII. The proportion of HCV-RNA compartmentalized in the cryoprecipitate (CP) varied greatly (10-80% of total HCV-RNA). The complementary determining region (CDR)3 sequences of monoclonal immunoglobulin M (IgM) VH and VK genes were highly homologous to rheumatoid factor and to antibodies against HCV-E2. Furthermore, the CDR3 sequences in some of our MCII patients were highly similar to those described in HCV-positive patients with non-Hodgkin's lymphoma (NHL). From these results, it appears that, as in the case of NHL, the IgM-rheumatoid factor (RF) production in MCII patients is antigen driven, namely by E2. However, the limited number of mutations in VH and VK genes with respect to the germline and their distribution showed that the B-cell response in these cases was prevented from undergoing affinity maturation. Furthermore, in patients with monoclonal IgM and definite compartmentalization of HCV in either CP or supernatant, a highly homogeneous E2-hypervariable region (HVR)1 sequence distribution was found (90-100% identical clones), a feature of the quasispecies frequently associated with an impaired humoral immune response to HCV. These findings suggest that in patients with HCV-associated MCII, maturation of monoclonal B lymphocytes may be blocked in a primitive stage preventing serious damaging effects because of the auto-reactivity of their secreted immunoglobulins.     

Defects in pokeweed mitogen (PWM) induced immunoglobulin (Ig) synthesis by lymphocytes of patients with juvenile rheumatoid arthritis

In vitro pokeweed mitogen (PWM) induced immunoglobulin (Ig) synthesis by lymphocytes of 33 patients with juvenile rheumatoid arthritis (JRA) was assessed and the results were correlated with clinical characteristics and laboratory measurements of disease activity. Comparisons with results of lymphocytes from adult controls and age related controls, greater than or equal to 12 years, showed reduced responses in patients with JRA. Low responders had significantly higher sedimentation rates (p = .0052) and platelet counts (p = .033), than patients with normal results. No correlations with onset subtype, medications, or age were found. These findings suggest a reduction in in vitro PWM induced B cell differentiation in patients with JRA, which may be related to disease activity.     

Calcitonin inhibits production of immunoglobulins, rheumatoid factor and interleukin-1 by mononuclear cells from patients with rheumatoid arthritis.

OBJECTIVES--Elcatonin (eCT), an eel calcitonin derivative, is shown to considerably improve the clinical signs and symptoms, as well as laboratory data, in patients with rheumatoid arthritis (RA). The therapeutic efficacy of eCT, however, is reduced by preceding and/or concomitant use of corticosteroid. Thus the effects of eCT on the production of immunoglobulins, IgMRF and interleukin-1 (IL-1) by mononuclear cells (MNCs)/monocytes were studied, and compared among patients with RA that received three kinds of treatment and also normal volunteers (NV). METHODS--Ten patients with RA had been treated with a non-steroidal anti-inflammatory drug only (NSAID group), 11 with oral prednisolone (PSL group), and eight with intramuscular eCT (eCT group). MNCs/monocytes from these patients, and also 10 from the NV group, were collected and cultured. IgG, IgA, IgM, IgMRF, IL-1 alpha and IL-1 beta in the supernatants were measured by enzyme-linked immunosorbent assay (ELISA). In the NSAID, PSL and NV groups, eCT was added to the culture medium, and the effects of eCT on production of these substances were studied. RESULTS--Baseline production of IgM, IL-1 alpha and IL-1 beta by MNCs/monocytes in the eCT and NV groups was significantly lower than that in the NSAID group. Furthermore, addition of eCT to the culture medium significantly inhibited the productions of IgG, IgMRF, IL-1 alpha and IL-1 beta by MNCs/monocytes in the NSAID group, whereas production of neither IgG, IgA, IgM, IgMRF nor IL-1 by MNCs/monocytes in the PSL and NV groups was affected by eCT. CONCLUSION--eCT may regulate immune responses through MNC/monocyte function in patients with RA. The present results support our proposal that eCT is an effective agent for the treatment of RA.     

Systemic manifestations and liver disease in patients with chronic hepatitis C and type II or III mixed cryoglobulinaemia

The aim of this study was to evaluate the prevalence of cryoglobulins in patients with chronic hepatitis B and C virus infection and to investigate the association of type II and type III mixed cryoglobulinaemia with systemic manifestations and liver disease stage and outcome in hepatitis C virus (HCV)-positive patients. We analysed the prevalence of cryoglobulinaemia in a cohort of patients with chronic liver disease and compared the systemic manifestations and liver involvement in HCV-positive patients with type II or type III mixed cryoglobulinaemia. The prevalence of serum cryoglobulins was significantly higher in HCV-positive patients than in hepatitis B surface antigen (HBsAg)-positive patients (55.4 vs 20.6%). In HCV-positive patients, stage of liver disease correlated with the prevalence of cryoglobulinaemia. Patients with type II cryoglobulins showed a significantly higher risk of cirrhosis and of extrahepatic manifestations while patients with type III cryoglobulins had a significantly higher prevalence of hepatocellular carcinoma. During follow-up the former had an odds ratio of 11.9 of death from extrahepatic complications while the latter had an odds ratio of 3.4 of dying from hepatic disease. Our study confirms the high frequency of mixed cryoglobulinaemia in patients with chronic hepatitis C virus infection. The presence and type of cryoglobulins seem to be associated with different clinical manifestations and outcome.     

Long-term results of therapy with interferon-alpha for type II essential mixed cryoglobulinemia.

Severe type II essential mixed cryoglobulinemia (EMC) bears a poor prognosis. Treatment with corticosteroids and/or cytotoxic drugs infrequently results in long-term remissions, and is associated with significant toxicity. We conducted a prospective study with interferon (IFN) in 21 patients with severe type II EMC unresponsive to immunosuppressive regimens. They were treated with recombinant IFN-alpha 2a (18 patients) or with natural IFN-beta (three patients), alone, at a dosage of 3 megaunits (MU)/d for 3 months, followed by 3 MU every other day as maintenance. We observed 11 complete remissions, five partial remissions, and five minor responses. Of 16 patients observed for more than 1 year, 11 remained in remission for 14 to 40 months; five of them remained in complete remission for 18 to 40 months after withdrawal of treatment. Four patients discontinued treatment because of side effects. In four patients who relapsed while on maintenance therapy with recombinant IFN-alpha 2a, remission could be reinduced by treatment with natural IFN-alpha. The response rate of 77% achieved in this study prompts the use of IFN-alpha as a first-choice drug for type II EMC.     

Pseudothrombocytosis and pseudoleukocytosis in a case of essential mixed cryoglobulinemia (type II)

Pseudothrombocytosis and pseudoleukocytosis occurred in a patient with essential mixed cryoglobulinemia (EMC) and atypical cutaneous ulcers, when the blood cell counts were estimated by the Model S Plus Coulter Counter. The spurious cell counts were found in serum as well as in plasma and whole blood, so the involvement of fibrinogen in this phenomenon is questioned. When timed serial counts were performed on whole blood at room temperature the highest value of WBCs was detected one hour after sample collection and that of platelets occurred at 6 hours, when leukocytosis had disappeared. Possible explanations for this phenomenon are offered.