Defective immune adherence and elimination of hepatitis B surface antigen/antibody complexes in patients with mixed essential cryoglobulinemia type II

Mixed essential cryoglobulinemia type II (monoclonal Ig/polyclonal IgG) is characterized by systemic vasculitis caused by the deposition of circulating immune reactants that include the monoclonal component. Such reactants may include immune complexes (IC) formed from exogenous Ag. IC binding to E C receptor type 1 appears to play a role in transport and buffering of such IC (immune adherence: IA). To define the mechanisms responsible for immune deposition, 7 patients with cryoglobulinemia type II (IgM kappa/polyclonal IgG) and 14 normal volunteers were injected i.v. with hepatitis B surface Ag/antibody complexes. Two minutes after injection, only 19.4% (mean) of the circulating complexes were bound to E in patients as compared with 63.1% in normal subjects. This IA correlated directly with C4 and inversely with the IgM rheumatoid factor (RF) titer. Disappearance of IC was faster in patients (mean elimination rate: 15.7%/min) than in normal subjects (9.3%). In vitro experiments demonstrated that C depletion, interference with IC opsonization by monoclonal IgM RF, and decreased binding of opsonized IC in the presence of monoclonal RF are each associated with decreased IA. These observations suggest that, in patients with cryoglobulinemia type II, monoclonal IgM RF and low C contribute to reducing IA of circulating IC that might be rapidly trapped in tissues, resulting in injury.     

The role of complement in cryoglobulin-induced immune complex glomerulonephritis

Many forms of glomerulonephritis are triggered by Ab localization in the glomerulus, but the mechanisms by which this induces glomerular inflammation are not fully understood. In this study we investigated the role of complement in a mouse model of cryoglobulin-induced immune complex glomerulonephritis. Several complement-deficient mice on a C57BL/6 and BALB/c genetic background were used and compared with strain-matched, wild-type controls. Cryoglobulinemia was induced by i.p. injection of 6-19 hybridoma cells producing an IgG3 cryoglobulin with rheumatoid factor activity against IgG2a of allotype a present in BALB/c, but not C57BL/6, mice. Thus, the cryoprecipitate in C57BL/6 mice consisted of the IgG3 cryoglobulin only (type I cryoglobulinemia) compared with IgG3-IgG2a complexes in BALB/c (type II cryoglobulinemia). The survival of mice was not affected by complement deficiency. Glomerular influx of neutrophils was significantly less in C3-, factor B-, and C5-deficient mice compared with wild-type and C1q-deficient mice. It did not correlate with C3 deposition, but did correlate with the amount of C6 deposited. Deficiency of CD59a, the membrane inhibitor of the membrane attack complex, did not induce an increase in neutrophil infiltration, suggesting that the generation of C5a accounts for the effects observed. There was no apparent difference between cryoglobulinemia types I and II regarding the role of complement. Our results suggest that in this model of cryoglobulin-induced glomerulonephritis the neutrophil influx was mediated by C5 activation with the alternative pathway playing a prominent role in its cleavage. Thus, blocking C5 is a potential therapeutic strategy for preventing renal injury in cryoglobulinemia.     

Absence of auto-antiidiotypic activity between the IgM and IgG fractions of human mixed cryoglobulins

Experimental animal models and observations in humans suggest that levels of Id and auto-anti-Id fluctuate reciprocally after Ag stimulation. In human monoclonal B cell disorders, however, the co-existence of paraprotein Id and its auto-anti-Id has been described in essential mixed cryoglobulinemia and in association with acquired C1 inhibitor deficiency. Because the majority of cryoglobulin IgM possess rheumatoid factor activity and thus bind the Fc region of IgG, we examined potential idiotypic interactions between cryoglobulin IgM and F(ab')2 fragments of autologous cryoglobulin IgG fractions. A rabbit antibody to the pepsin agglutinator site of human F(ab')2 was used as detection reagent. By recognizing epitopes exposed on F(ab')2 after the removal of Fc determinants by pepsin digestion, this reagent eliminates the detection of contaminating intact IgG. In a sensitive assay, we were unable to detect idiotypic interactions between the separated IgM and pepsin-digested IgG fractions of 10 mixed cryoglobulins. On the basis of these results, we suggest that in mixed cryoglobulinemia, the coexistence of paraprotein Id and its auto-anti-Id is unlikely.     

Preliminary survey of Capillaria hepatica (Bancroft, 1893) in Malaysia.

The prevalence of Capillaria hepatica (Bancroft, 1893) infection in a total of 2324 rats trapped from 25 localities in West Malaysia was 15.5%. Infection rates in males (16.0%) and females (15.1%) are similar. A significantly higher percentage of adults (18.1%) than young (7.7%) was infected. Capillaria hepatica infection rates among urban (0.7%) and jungle (0.0%) rats was very low as compared to field rats (17.7%) trapped from agricultural areas such as oil palm estates and rice growing areas. Prevalence of C. hepatica infection in rats is not evenly distributed throughout West Malaysia. There seem to be localised foci of infection. In some areas as many as 77.8% of the adult rats are found to be infected while in other areas the same species of rats are found free of infection.     

Hepatitis C virus-associated mixed cryoglobulinemia. Clinical manifestations, histopathological changes, mechanisms of cryoprecipitation and options of treatment

Chronic hepatitis C virus (HCV) infection is frequently associated with a variety of autoimmune phenomenons. Mixed cryoglobulinemia (MC) appears in up to 50% of chronic HCV-infected patients. Cryoglobulins consist of immunoglobulin complexes precipitating in vitro when cooled below body temperature. In most cases IgM with rheumatoid factor activity is found in cryoprecipitates which could lead to vasculitis induced by the deposition of immnuocomplexes in small vessels. This vasculitis is thought to cause clinical symptoms called Meltzer's triad. This triad is represented by purpura, arthralgia and weakness. One third of patients suffering from HCV-associated mixed cryoglobulinemia are developing typical symptoms during their course of disease. The striking association between HCV infection and MC has conduced to the hypothesis that HCV is of major importance in the production of MC with followed vasculitis. Both hepatrophism and lymphotrophism have been reported for the hepatitis C virus. Infection of B-cells by HCV could probably lead to a bcl-2 translocation and immunoglobulin gene rearrangement which results in clonal lymphoproliferation and in synthesis of monoclonal IgM with rheumatoid factor activity. These IgM form immunocomplexes with IgG in the cold, which are finally responsible for the described vasculitis. Histopathological changes of the liver are dominated by chronic HCV infection. The majority of times mild activity of hepatitis or mild fibrosis could be found. Nevertheless, cirrhosis is more often found in HCV-infected patients suffering from MC compared to patients without MC. Conventional treatment of MC is aimed to reduce circulating immune complexes by immunosupression and plasmapheresis. With the emerging concept of a viral pathogenesis the therapeutic approach has changed during the last decade. Interferon treatment of MC, particularly of HCV-associated MC is well established nowadays.     

A role for complement in antibody-mediated inflammation: C5-deficient DBA/1 mice are resistant to collagen-induced arthritis

Collagen-induced arthritis (CIA) represents an animal model of autoimmune polyarthritis with significant similarities to human rheumatoid arthritis that can be induced upon immunization with native type II collagen. As in rheumatoid arthritis, both cellular and humoral immune mechanisms contribute to disease pathogenesis. Genotypic studies have identified at least six genetic loci contributing to arthritis susceptibility, including the class II MHC. We have examined the mechanism of Ab-mediated inflammation in CIA joints, specifically the role of complement activation, by deriving a line of mice from the highly CIA-susceptible DBA/1LacJ strain that are congenic for deficiency of the C5 complement component. We show that such C5-deficient DBA/1LacJ animals mount normal cellular and humoral immune responses to native type II collagen, with the activation of collagen-specific TNF-alpha-producing T cells in the periphery and substantial intra-articular deposition of complement-fixing IgG Abs. Nevertheless, these C5-deficient mice are highly resistant to the induction of CIA. These data provide evidence for an important role of complement in Ab-triggered inflammation and in the pathogenesis of autoimmune arthritis.     

Monoclonal rheumatoid factor-secreting cells in a patient with mixed cryoglobulinemia. Homogeneity and stability of the idiotypic production and in vitro idiotypic suppression

The potential therapeutic value of anti-idiotypic antibodies during B cell proliferations largely depends on the stability of the target Ig idiotopes. We investigated this stability in a clinical condition of so called nonmalignant monoclonal B cell proliferation, mixed cryoglobulinemia. The idiotypic profile of a single IgM kappa monoclonal auto-antibody with anti-IgG activity (rheumatoid factor (RF] which originated from a patient suffering from a nonmalignant mixed cryoglobulinemia was followed over a period of 3 yr. As judged from the reactivity of a panel of five different mouse monoclonal anti-idiotypic antibodies mapping the RF variable regions, there was no idiotypic change in the serum IgM RF. At a cellular level, in vitro stimulation of the patient's PBL gives rise to IgM kappa auto-antibodies that were shown to bear the same idiotypic determinants as the serum IgM kappa. We then investigated the effects of the anti-idiotypic antibodies on the in vitro IgM kappa production. When stimulated with PWM and in the presence of anti-idiotypic antibodies (10 micrograms/ml), the patient's PBL produced less IgM RF (18 to 62% inhibition). The same inhibition of IgM RF production was observed after EBV infection of the patient's PBL (from 19 to 90% inhibition). In both cases, the remaining IgM RF production was idiotypically indistinguishable from the serum IgM RF. The implications of the idiotypic stability and of the results of in vitro idiotypic manipulation could be important in view of both the understanding of nonmalignant cryoglobulinemia and of the possible therapeutic use of anti-idiotypic antibodies in diseases.     

Specific immunoglobulin response in mice immunized with porins and challenged with Salmonella typhi

Specific antiporin antibody (IgG, IgM, and IgA) response was studied in control, infected, immunized-infected, and immunized mice. The activity of specific IgG immunoglobulins was found to be the highest in immunized and immunized-infected groups in which 87.5% protection had been observed by laboratory potency test in mice; the next-highest activities were those of IgM and IgA immunoglobulins. However, in the infected group the activity of specific IgM and IgG immunoglobulins was almost similar.     

Complement deficiency ameliorates collagen-induced arthritis in mice

Collagen-induced arthritis (CIA) is an experimental animal model of human rheumatoid arthritis being characterized by synovitis and progressive destruction of cartilage and bone. CIA is induced by injection of heterologous or homologous collagen type II in a susceptible murine strain. DBA/1J mice deficient of complement factors C3 (C3(-/-)) and factor B (FB(-/-)) were generated to elucidate the role of the complement system in CIA. When immunized with bovine collagen type II emulsified in CFA, control mice developed severe arthritis and high CII-specific IgG Ab titers. In contrast, the C3(-/-) and FB(-/-) were highly resistant to CIA and displayed decreased CII-specific IgG Ab response. A repeated bovine collagen type II exposure 3 wk after the initial immunization led to an increase in the Ab response in all mice and triggered arthritis also in the complement-deficient mice. Although the arthritic score of the C3(-/-) mice was low, the arthritis in FB(-/-) mice ranked intermediate with regard to C3(-/-) and control mice. We conclude that complement activation by both the classical and the alternative pathway plays a deleterious role in CIA.     

Capillaria hepatica (Nematoda) infections in human-habituated mountain gorillas (Gorilla gorilla beringei) of the Parc National de Volcans, Rwanda

Habituation to humans of free-ranging populations of endangered mountain gorillas (Gorilla gorilla beringei) raised concern of anthropozoonotic transmission of parasitic helminths and protozoans. Examinations of liver tissue of 19 gorillas found dead in the Parc National de Volcans, Rwanda, revealed 10 cases of hepatic nematodiasis due to Capillaria hepatica. Identifiable C. hepatica eggs were present in the liver of 4 gorillas (3 juveniles, 1 adult), and nematode cross-sections were found in 1 juvenile gorilla. Six other adult gorillas had areas of periportal and subcapsular fibrosis with calcified eggs. Histologically, the lesions surrounded by the areas of mild inflammatory reaction were characterized by subcapsular, periportal foci of fibrosis in which were embedded numerous C. hepatica eggs. Control of hepatic capillariasis in the remaining populations of mountain gorillas should be focused on eradication or control of populations of rodent pests (i.e., mice and rats) that sustain the reservoir of C. hepatica in habitats shared by gorillas and humans.     

The potential of Capillaria hepatica to control mouse plagues

The potential of helminths as bio-control agents of mammalian pests has been largely ignored. However, the nematode Capillaria hepatica is currently being examined for its potential to control population outbreaks of house mice in Australia. Grant Singleton and Hamish McCallum discuss laboratory and ecological studies of the parasite and host, and describe the results of two models that explore the interaction between C. hepatica and mouse populations.     

Secretory IgA specific for Toxoplasma gondii

Secretory IgA specific for Toxoplasma gondii was identified in intestinal secretions of mice infected perorally with bradyzoites (encysted in brain) of the Me49 strain of T. gondii by using immunofluorescence microscopy and an ELISA. This activity was absorbed with tachyzoites of T. gondii but not mouse brain. To determine whether increased total amount of intestinal IgA might cause a nonspecific reaction in the ELISA for T. gondii-specific IgA, mice were immunized perorally with cholera toxin. This immunization produced intestinal IgA antibody to cholera toxin and increased the total amount of intestinal IgA, but there was no reactivity of intestinal secretions in the ELISA for T. gondii-specific IgA. Experiments in which ELISA were performed with monoclonal IgA, IgG, or IgM and antisera to IgA, IgG, or IgM demonstrated that the ELISA was specific for each Ig class. In addition, monoclonal IgA competed with the anti-Toxoplasma IgA activity of intestinal secretions obtained from mice infected with T. gondii.     

Effect of interferon-alpha on experimental septal fibrosis of the liver - study with a new model

Interferon-alpha is used in antiviral therapy in humans, mainly for viral hepatitis B and C. An anti-fibrotic effect of interferon has been postulated even in the absence of anti-viral response, which suggests that interferon directly inhibits fibrogenesis. Rats infected with the helminth Capillaria hepatica regularly develop diffuse septal fibrosis of the liver, which terminates in cirrhosis 40 days after inoculation. The aim of this study was to test the anti-fibrotic effect of interferon in this experimental model. Evaluation of fibrosis was made by three separate methods: semi-quantitative histology, computerized morphometry and hydroxyproline measurements. Treatment with interferon-alpha proved to inhibit the development of fibrosis in this model, especially when doses of 500,000 and 800,000 IU were used for 60 days. Besides confirming the anti-fibrotic potential of interferon-alpha on a non-viral new experimental model of hepatic fibrosis, a clear-cut dose-dependent effect was observed.     

布氏田鼠肝毛细线虫感染率与其体重的关系

2005年5月和8月,在内蒙古锡林郭勒北部典型草原调查了肝毛细线虫对布氏田鼠种群的感染特征,分析肝毛细线虫对布氏田鼠的感染率与其性别、年龄、体重及种群密度的关系。结果表明:肝毛细线虫对布氏田鼠感染率没有性别差异,雄鼠与雌鼠的感染率相当;但是与布氏田鼠体重/年龄密切相关:幼鼠的感染率较低,成鼠感染率较高,感染率和平均感染度均随着个体年龄的增长而增高。布氏田鼠达到一定的年龄(或体重)后才可感染肝毛细线虫病,其最低感染体重为24.3 g。布氏田鼠的种群密度对肝毛细线虫的感染率和平均感染度没有明显的影响,但同一样地不同季节感染率不同,本次调查显示,2005年5月份感染率高于8月份群体感染率,同一样地的春季感染率与秋季感染率之间呈现出显著的正相关。     

Hepatitis C virus drives the unconstrained monoclonal expansion of VH1-69-expressing memory B cells in type II cryoglobulinemia: a model of infection-driven lymphomagenesis

Chronic hepatitis C virus infection causes B cell lymphoproliferative disorders that include type II mixed cryoglobulinemia and lymphoma. This virus drives the monoclonal expansion and, occasionally, the malignant transformation of B cells producing a polyreactive natural Ab commonly encoded by the V(H)1-69 variable gene. Owing to their property of producing natural Ab, these cells are reminiscent of murine B-1 and marginal zone B cells. We used anti-Id Abs to track the stages of differentiation and clonal expansion of V(H)1-69(+) cells in patients with type II mixed cryoglobulinemia. By immunophenotyping and cell size analysis, we could define three discrete stages of differentiation of V(H)1-69(+) B cells: naive (small, IgM(high)IgD(high)CD38(+)CD27(-)CD21(high)CD95(-)CD5(-)), "early memory" (medium-sized, IgM(high)IgD(low)CD38(-)CD27(+)CD21(low)CD95(+)CD5(+)), and "late memory" (large-sized, IgM(low)IgD(low-neg)CD38(-)CD27(low)CD21(low-neg)CD5(-)CD95(-)). The B cells expanded in cryoglobulinemia patients have a "memory" phenotype; this fact, together with the evidence for intraclonal variation, suggests that antigenic stimulation by hepatitis C virus causes the unconstrained expansion of activated V(H)1-69(+) B cells. In some cases, these cells replace the entire pool of circulating B cells, although the absolute B cell number remains within normal limits. Absolute monoclonal V(H)1-69(+) B lymphocytosis was seen in three patients with cryoglobulinemia and splenic lymphoma; in two of these patients, expanded cells carried trisomy 3q. The data presented here indicate that the hepatitis C virus-driven clonal expansion of memory B cells producing a V(H)1-69(+) natural Ab escapes control mechanisms and subverts B cell homeostasis. Genetic alterations may provide a further growth advantage leading to an overt lymphoproliferative disorder.     

Hymenolepis citelli, H. diminuta and H. microstoma: immunoglobulin-containing cells in the lamina propria of the mouse gut during primary and secondary infections

The indirect immunofluorescent technique was used to determine the occurrence of IgA, IgM and IgG1 immunoglobulin-containing cells in local intestinal mucosal immune responses to Hymenolepis citelli, H. diminuta and H. microstoma infections in mice. In the intestinal lamina propria of H. citelli and H. diminuta infected mice there was no increase in the mean numbers of immunoglobulin-containing cells when compared with uninfected control mice, but there was in H. microstoma infected mice. The numbers of IgG1- positive cells in both infected and uninfected mice were very small relative to IgA and IgM immunocytes. The distribution of immunocytes in the lamina propria of infected and uninfected mice was essentially similar and the localization of isotypes in duodenal sections showed no immunoglobulins in the villous epithelial cells. There was also no marked difference between primary and secondary infections indicating that immunoglobulin-containing cells play no major role in functional immunity against hymenolepid infections in the mouse. The presence of IgA and IgM was also demonstrated on the tegument of the tapeworms, although the distribution was patchy and more abundant on H. microstoma than on H. diminuta or H. citelli. The time of appearance of both isotypes was latest on H. citelli.     

Age-dependent infectivity of orally transferred juvenile Fasciola hepatica.

Juvenile Fasciola hepatica is infective when administered orally. To determine whether the age of juveniles is a factor in infectivity by oral transfer, experimental mice were challenged orally with immature F. hepatica that had been grown in donor mice for 12, 14, 16, and 18 days. Experimental mice were examined for infections 12 16 days after the oral transfers. The infection success in experimental mice decreased with the age of juveniles. The worm recovery also decreased according to the age of juveniles. None of the juveniles was infective when grown for longer than 11 days. Once infected, orally transferred worms continued to grow. Juvenile age was a significant factor in determining the infectivity of orally transferred juvenile F. hepatica.     

Bead Arrays for Antibody and Complement Profiling Reveal Joint Contribution of Antibody Isotypes to C3 Deposition

The development of antigen arrays has provided researchers with great tools to identify reactivities against self or foreign antigens from body fluids. Yet, these approaches mostly do not address antibody isotypes and their effector functions even though these are key points for a more detailed understanding of disease processes. Here, we present a bead array-based assay for a multiplexed determination of antigen-specific antibody levels in parallel with their properties for complement activation. We measured the deposition of C3 fragments from serum samples to reflect the degree of complement activation via all three complement activation pathways. We utilized the assay on a bead array containing native and citrullinated peptide antigens to investigate the levels of IgG, IgM and IgA autoantibodies along with their complement activating properties in serum samples of 41 rheumatoid arthritis patients and 40 controls. Our analysis revealed significantly higher IgG reactivity against the citrullinated fibrinogen β and filaggrin peptides as well as an IgA reactivity that was exclusive for citrullinated fibrinogen β peptide and C3 deposition in rheumatoid arthritis patients. In addition, we characterized the humoral immune response against the viral EBNA-1 antigen to demonstrate the applicability of this assay beyond autoimmune conditions. We observed that particular buffer compositions were demanded for separate measurement of antibody reactivity and complement activation, as detection of antigen-antibody complexes appeared to be masked due to C3 deposition. We also found that rheumatoid factors of IgM isotype altered C3 deposition and introduced false-positive reactivities against EBNA-1 antigen. In conclusion, the presented bead-based assay setup can be utilized to profile antibody reactivities and immune-complex induced complement activation in a high-throughput manner and could facilitate the understanding and diagnosis of several diseases where complement activation plays role in the pathomechanism.     

Defense mechanisms against primary influenza virus infection in mice. I. The roles of interferon and neutralizing antibodies and thymus dependence of interferon and antibody production.

To investigate the defensive roles and production of interferon and antibodies, C3H/He mice were subjected to various immunosuppressive treatments and infected with influenza virus. In infected normal control mice the pattern of pulmonary viral growth can be divided into three phases. The first phase is characterized by an exponential increase of virus titer, the second by a rapid decrease, and the third by a moderate decrease. At the time of transition from the first phase to the second in pulmonary virus growth, interferon could be detected in the tracheobronchial washings of infected mice, but neutralizing antibodies could not. In infected B cell-deprived mice and infected anti-mu-treated mice, the transition from the first phase to the second occurred without any detectable antibody production, and interferon could be induced in the early stage of infection. However, the pulmonary virus in these mice increased again exponentially until the death of the mice. In infected T cell-deprived mice which could not induce interferon, but produced IgM-neutralizing antibodies, the second phase was not observed after the first phase, but a transient plateau phase could be demonstrated, and then the pulmonary virus increased again exponentially until the death of the mice. In anti-gamma-treated infected mice, pulmonary virus growth and production of interferon and neutralizing antibody were almost similar to those of infected normal control mice except for the absence of IgG neutralizing antibody production. Although anti-alpha-treated infected mice produced interferon and no IgA antibody, the transition from the first exponential increase of pulmonary virus to the second rapid decrease was seen, but then the virus increased exponentially again until the death of the mice. These results suggest that interferon plays an important role in the transition from the first phase to the second, and that T cells are required for interferon induction in mice infected with influenza virus. These data also suggest that IgA antibodies play an important role in the inhibition of virus propagation in the lungs after the disappearance of interferon. Moreover, infected T cell-deprived mice could produce only IgM neutralizing antibodies, but not IgG and IgA antibodies. Therefore, T cells are required for the production of IgG and IgA antibodies and even     

Immunological tolerance to pig-serum partially inhibits the formation of septal fibrosis of the liver in Capillaria hepatica-infected rats

Systhematized septal fibrosis of the liver can be induced in rats either by repeated intraperitoneal injections of pig-serum or by Capillaria hepatica infection. The relationship between these two etiological factors, as far as hepatic fibrosis is concerned, is not known, and present investigation attempts to investigate it. C. hepatica-induced septal fibrosis of the liver was considerably inhibited in rats previously rendered tolerant to pig-serum. Pig-serum-tolerant rats developed antibodies against pig-serum when infected with C. hepatica, but this did not happen when the infection occurred in normal rats. On the other hand, anti-C. hepatica antibodies failed to recognize any epitope in pig-serum, by Western blot. However, no evidence of an immunological cross reactivity was found, at least at the humoral level. Alternatively, cell-mediated mechanisms may be involved, and further investigations are warranted.