Polyclonal B cell activation in hamsters infected with parasites of the genus Leishmania.

Mesocricetus auratus (golden hamsters) infected with leishmania developed characteristic B cell immune responses that depended on the infecting species of leishmania. Thus, hamsters infected with viscerotropic leishmania (Leishmania donovani) developed antileishmania antibodies and hypergammaglobulinemia due to polyclonal activation of B cells as measured by reverse hemolytic plaque assay. In contrast, dermotropic leishmanias (L. braziliensis braziliensis and L. mexicana amazonensis) stimulated antileishmania antibodies with no increase in either serum immunoglobulin concentration or in the number of antibody-forming cells per spleen. The dermotropic leishmanias were unable to stimulate polyclonal activation even in hamsters in which visceralization had occurred with high splenic parasitization. These findings suggest that species-specific leishmania antigens (or factors) might be the modulators of the altered immune response present in these diseases.     

Prevalence of hepatitis B virus infection among alcoholic patients with liver disease.

The aim of this investigation was to elucidate a possible role of hepatitis B virus (HBV) in the pathogenesis of liver diseases in alcoholics. Two hundred and fifty-three alcoholics with liver disease were admitted to two medical departments in Copenhagen during a 15 months period. Seventy-nine patients (31%) showed serological signs (HBsAg, anti-HBs) of previous or active HBV infection. This is a significantly higher prevalence than found in an age-matched control population. Among the 79 patients with HBV markers, a total of 11 was found to be HBsAg-positive. From these 11 patients liver specimens were available for re-evaluation in nine cases. In only three of these liver biopsies, morphological changes indicating alcohol as the aetiological cause were found. In conclusion, different or concomitant aetiology must be considered in alcoholics with liver disease.     

T-lymphocyte activation pathways in alcoholic liver disease.

Immune system derangement in cirrhotic patients with evidence of malnutrition is a well-recognized characteristic of chronic alcohol abuse. However, in vitro studies on cellular immune function performed with lectin mitogens have produced conflicting results. The recent development of more accurate immunological techniques for studying lymphocyte transformation, that use monoclonal antibodies directed against surface structures (CD3 and CD2) involved in antigen recognition, as well in adhesion functions, prompted us to study discrete in vitro T-cell hypo-responsiveness in a series of alcoholic liver disease (ALD) patients with no evidence of malnutrition or hepatic cirrhosis. The results indicated that the CD2 pathway is markedly defective in ALD T lymphocytes, accompanied by reduced interleukin-2 (IL-2) receptor expression upon in vitro activation. This defect cannot be reversed by the addition of recombinant IL-2 (rIL-2) or rIL-1. Faulty intracellular signal transduction by protein kinase C (PKC) and defective intracellular Ca2+ mobilization may be responsible for the CD2 pathway impairment. The addition of small amounts of phorbol 12-myristate, 13-acetate, but not Ca2+ ionophore A23187, is able to overcome the defect, thereby suggesting a direct PKC involvement. The hypothesis of a direct ethanol effect on transmembrane signal transduction systems is suggested by the demonstration of an expansion of circulating virgin (naive) T cells (CD3+/UCHL1-low) that binds tyrosine phosphatase (CD45RA antigen) on their surface.     

Polyclonal activation of salmonid B lymphocytes

The process of in vitro polyclonal activation of coho salmon (Oncorhynchus kisutch) lymphocytes was examined with respect to the induction of mitogenesis, total immunoglobulin production, and the production of specific antibodies or plaque forming cells. These studies demonstrate that antigen specific stimulation of antibody production is not linked to mitogenic activity, or total immunoglobulin production, while the polyclonal activation of specific antibody production is closely linked to these functions. Stimulation of immunoglobulin production by phytohemagglutinin suggests that this mitogen may not be limited to T cell activation in salmonids or, alternatively, it may induce the production of lymphokines capable of polyclonally activating B cells. Further, fetal calf serum was found to cause production of large amounts of immunoglobulin in vitro without antigenic stimulation.     

Polyclonal B cell activation during the course of Angiostrongylus cantonensis infection

The numbers of IgM antibody forming cells against various heterologous antigens: SRBC, HRBC, TNP and FITC, were found to be elevated in the spleens of rats after infection with Angiostrongylus cantonensis. In these rats, the number of splenic B cells and total IgG and IgM levels in serum also increased spontaneously about 2- to 3-fold of uninfected rats during the course of the infection. These results suggested that A. cantonensis infection may activate B cells polyclonally in the spleens of the infected rats. While, it was clearly observed that the antibody response to SRBC injection was significantly depressed in these rats, compared to those of uninfected rats. The relation between the polyclonal B cell activation and the immunodepression was discussed.     

Abnormalities of B cell activation and immunoregulation in patients with chronic inflammatory liver diseases

The immunoglobulin production capacities of peripheral blood lymphocytes obtained from patients with various chronic inflammatory liver diseases and from normal individuals were compared. Using a reverse hemolytic plaque assay, immunoglobulin-secreting cells (ISC) were counted immediately after isolation (immediate ISC) and again after 6-day, in vitro cultivation without stimulant (spontaneous ISC) or in the presence of pokeweed mitogen, PWM (PWM-induced ISC). An increased number of immediate ISC were observed in patients with chronic active hepatitis (CAH) of the autoimmune type (n = 7) or with CAH type B (n = 32), probably reflecting a defect of the in vivo suppressor cell system as previously demonstrated. In vitro preincubation of cells with 5 x 10(-8) M prednisolone reduced the increase in the number of immediate ISC in patients with CAH of the autoimmune type. On the other hand, lymphocytes obtained from patients with CAH-type NANB (n = 9) and with primary biliary cirrhosis (PBC) (n = 12) showed an impaired capacity to generate ISC upon stimulation with PWM. Spontaneous ISC from patients' lymphocytes were not significantly different from those of normal individuals. Using allogeneic co-cultures with lymphocytes from normal individuals and from patients with CAH NANB hepatitis or primary biliary cirrhosis, we observed no increase in suppressor cell activity. Therefore, the diminished responses to PWM are probably attributable to an alteration in the peripheral helper T-cell compartment.     

Erectile dysfunction in patients with liver disease related to chronic hepatitis B

Background/Aims

Despite sexual function making an important contribution to the quality of life, data on erectile function are relatively scant in patients with chronic liver disease. We evaluated the prevalence of and risk factors for erectile dysfunction (ED) in patients with liver disease related to hepatitis B, especially among those with chronic hepatitis B (CHB) or early-stage cirrhosis.

Methods

In total, 69 patients (35 with CHB and 34 with hepatitis-B-related liver cirrhosis [HBV-LC]) aged 40-59 years were analyzed. Child-Pugh classes of A and B were present in 30 (88.2%) and 4 (11.8%) of the patients with HBV-LC, respectively. The erectile function of the patients was evaluated using the Korean version of IIEF-5.

Results

The prevalence of any ED was 24.6% for all patients, and 8.6% and 41.2% for those with CHB and HBV-LC, respectively (P=0.002). While there was only one (2.9%) CHB patient for each stage of ED, mild, moderate, and severe ED stages were seen in three (8.8%), one (2.9%), and ten (29.4%) of the HBV-LC patients, respectively. Multiple regression analysis identified the type of liver disease (P=0.010), hypertension (P=0.022), score on the Beck Depression Inventory (P =0.044), and the serum albumin level (P=0.014) as significant independent factors for the presence of ED.

Conclusions

The prevalence of ED was significantly higher in patients with early-stage HBV-LC than in those with CHB. Therefore, screening male patients with early viral cirrhosis for ED and providing appropriate support are needed, especially when the cirrhosis is accompanied by hypertension, depression, or a depressed level of serum albumin.     

Immunological studies in patients with alcoholic liver disease: evidence for the in vivo activation of helper T cells and of the monocyte-macrophage system

Immunologic parameters were evaluated in 22 patients with alcoholic liver disease (ALD). Patients with ALD had increased levels of circulating immune complexes (CIC) and serum immunoglobulins, particularly IgA. The classical complement pathway was preferentially activated in CIC-positive patients. There was no increase in total lymphocyte or total T lymphocyte counts, but a significant rise in both the helper/inducer population (OKT4) and helper/suppressor cell ratio (T4/T8) was noted. The presence of interleukin-2 receptors and HLA-DC/DS and HLA-DR antigens suggested in vivo activation of ALD patients' T cells. The rate of differentiation of B cells to plasma cells was high in both pokeweed mitogen-stimulated and unstimulated cultures of ALD patients' B cells, whereas plasma cell generation doubled when patient T lymphocytes were added to enriched normal B cells. The above data support a role for activated helper (T4+) T cells in the immune response initiated by alcoholic hepatitis. Serum angiotensin-converting enzyme levels and lysozyme levels were increased in ALD patients, and cultured adherent mononuclear cells from ALD patients secreted more lysozyme in vitro than normal cells, suggesting the presence of an activated monocyte-macrophage system in ALD.     

Polyclonal activation of human peripheral blood B lymphocytes by Fusobacterium nucleatum.

The present study examined in vitro polyclonal human B-lymphocyte (B-cell) activation (PBA) by Fusobacterium nucleatum. Pokeweed mitogen, a well-studied PBA activator, was included in some experiments for comparison. PBA was determined by the total immunoglobulin A, G, and M concentrations in the culture supernatants as measured by micro-enzyme-linked immunosorbent assay. F. nucleatum, at concentrations between 1 and 10 micrograms/ml, stimulated optimal PBA in monocyte-depleted cultures, whereas the pokeweed mitogen response was optimal in unfractionated, monocyte-containing cultures. Immunoglobulin synthesis occurred primarily between days 6 and 8 after stimulation with F. nucleatum. T lymphocytes enhanced the PBA response to F. nucleatum, particularly at a T- to B-cell ratio of 1:1. Immunoglobulin production was greater in round-bottomed wells than in flat-bottomed wells at lymphocyte concentrations of 200,000 cells per well. The PBA response, however, increased dramatically in flat-bottomed wells containing higher lymphocyte concentrations, suggesting that PBA is enhanced by cell-to-cell contact. A delay in stimulation of the lymphocytes with F. nucleatum resulted in diminished immunoglobulin production. The results provide information on the regulation of in vitro PBA induced by F. nucleatum. The data also suggest that there may be differences in the mechanisms by which F. nucleatum and pokeweed mitogen stimulate PBA.     

Polyclonal activation of B lymphocytes by lipopolysaccharide requires macrophage-derived interleukin-1.

Lipopolysaccharide (LPS) is a potent murine polyclonal B-cell activator which induces cellular proliferation and IgM secretion. The precise role of activated macrophages in the induction of LPS-dependent, B-cell responses has been unclear. Although early reports concluded that the LPS effect occurs independently of other cell types, other studies have suggested that adherent macrophages exert either potentiating or inhibitory effects. In the present study, B-cell mitogenesis and IgM production were measured in primary spleen cell cultures after removing adherent cells by a variety of experimental procedures. B-cell activation by LPS was found to be strictly dependent on the presence of adherent macrophages. Antibody neutralization and cytokine reconstitution studies demonstrated that macrophage-derived interleukin- (IL-1) is a necessary co-factor for LPS-induced polyclonal activation.     

Polyclonal B cell activator associated with alpha-2-macroglobulin in the serum of patients with rheumatoid arthritis

We investigated whether patients with rheumatoid arthritis have a polyclonal B cell activator (PBA) in their serum by using three methods: (1) the ability of any PBA to maintain the surface Ig of rabbit or human B cells in vitro; (2) the induction of blast transformation in human B lymphocyte cultures, and (3) stimulation of nude mouse spleen cells in vitro. All three methods indicated that a PBA is present in the serum of patients with rheumatoid arthritis but not in normal individuals or in patients with arthritis in which autoimmune phenomena have not been demonstrated. The entire PBA activity in rheumatoid arthritis patient serum was found associated with the macroglobulin fraction obtained by Sephadex G-200 chromatography and was precipitated by rabbit anti-human alpha 2-macroglobulin, but not by rabbit anti-Ig antibody. When alpha 2-macroglobulin was purified from patient serum the entire PBA activity was recovered in this fraction. Normal alpha 2-macroglobulin prepared by the same procedure had no PBA activity. Thus, the existence of a PBA associated with alpha 2-macroglobulin was demonstrated in serum of patients with rheumatoid arthritis.     

Serological markers of hepatitis B in patients with alcoholic liver disease: a multi-centre survey

In a study of 195 patients derived from five centres in northern Britain and with histologically confirmed alcoholic liver disease we have found an increased prevalence of serological markers of hepatitis B. This increased prevalence was found in each of the five centres; the overall frequency ranged from 11% sero-positivity in fatty liver, 12% in alcoholic hepatitis and 27% in cirrhosis.     

Cerebrospinal fluid from patients with neurodegenerative and neuroinflammatory diseases: no evidence for rat glial activation in vitro.

To determine the possible contribution of glial cells via oxidative stress/cytokine secretion in the pathogenesis of Parkinson's disease (PD), Alzheimer disease (AD), amyotrophic lateral sclerosis (ALS) or multiple sclerosis (MS) the concentration of nitric oxide (NO) (by the Griess method) and Interleukin-6 (IL-6) (by enzyme-linked immunosorbent assay) were measured in resting rat microglial and astrocytic cell culture supernatants stimulated by cerebrospinal fluid (CSF) (dilution 1:4, 1:10) from patients with the aforementioned diseases. Neither the concentration of NO (optical density at 450 nm: control, 0.036+/-0.006; MS, 0.034+/-0.008; AD, 0.031+/-0.006; PD, 0.02+/-0.01; lipopolysaccharide (LPS), 0.26+/-0.018) nor the amount of IL-6 (ng/ml: control, 0.112+/-0.026; PD, 0.12+/-0.027; MS, 0.123+/-0.008; ALS, 0.137+/-0.01; LPS, 1.81+/-0.11) differed in any disease group from those of unaffected controls. These findings suggest that the stimuli for inflammatory activation of glia are quite localized and not present in sufficient concentrations in the CSF of affected patients.     

Polyclonal human T lymphocyte activation results in the secondary functional activation of the human B lymphocyte.

The polyclonal T lymphocyte activators Con A and PHA were demonstrated to induce secretion of IgM and IgG as well as IgA antibodies in cultures of human peripheral blood lymphocytes. A similar response was seen in one-way mixed lymphocyte cultures and the kinetics, dose-response characteristics and optimal culture conditions are presented. The presence of functional T lymphocytes is a prerequisite for in vitro B lymphocyte activation in response to T lymphocyte mitogens. A narrow dose-response profile is a characteristic for both Con A and PHA and polyclonal B cell activation occurred at what have been previously regarded as suboptimal concentrations of these agents. The use of higher doses of these activators failed to generate immunoglobulin-secreting cells despite the presence of early and optimal levels of DNA synthesis in the cultures.     

Evidence for B cell activation in patients with active rheumatoid arthritis

Peripheral blood lymphocytes and in some cases synovial eluate cells from 51 patients with rheumatoid arthritis (RA), were analysed for the percentages of cells bearing surface light chains (total B cells), IgM and IgD. In addition, their capacity to form rosettes with mouse erythrocytes (mRFC)--a property of a B cell subpopulation--was determined. Activity of the disease was assessed by clinical and laboratory criteria and classified as very active, moderately active and inactive. Normal, age and sex matched individuals and a group of patients with a variety of other rheumatological disorders, were used as control populations. Although there was no significant difference in percentages of total B cells in any of the groups compared with normal controls, there was a small but significant increase in the ratio of cells bearing IgM to those bearing IgD in patients with very active disease. This was paralleled by a significant decrease in the mRFC in this disease activity group. Patients with inactive disease showed no change in their proportions of IgM:IgD, but did show a significant increase in mRFC. These results are discussed in terms of the presence of activated B cells in patients with very active RA.     

Polyclonal B cell activation for accurate analysis of pre‐existing antigen‐specific memory B cells

The enzyme‐linked immunospot (ELISPOT) assay is a widely used tool for enumeration of antigen‐specific memory B cells in several disciplines, such as vaccination, cancer immunotherapy and transplantation. For the accurate estimation of antigen‐specific memory B cell frequencies, a well‐defined B cell activation protocol is pivotal. In this study, we aimed to characterize a polyclonal B cell activation protocol to facilitate optimal monitoring of antigen‐specific memory B cell frequencies. Total, naive and memory B cells were activated polyclonally with an α‐CD40 monoclonal antibody, cytosine–phosphate–guanine (CPG) oligodeoxynucleotide (ODN) 2006, interleukin (IL)‐2, IL‐10 and IL‐21. Polyclonal activation of B cells resulted in equal cell death ratios in naive and memory B cells. When tested in an antigen‐specific system, immunoglobulin (Ig)G spots were detected only in the memory fraction. There was no change in B cell polyclonality due to in‐vitro activation. Our data show that the current polyclonal activation protocol may be used reliably to estimate the frequency of memory B cells in ELISPOT assays.