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I International Immunology i , dxab111, https://doi.org/10.1093/intimm/dxab111 After this paper was accepted, the authors realized that the data in the middle and bottom histograms of Fig. 3C and the right-hand flow cytometry plot of Fig. [Extracted from the article]

IgD, first demonstrated in humans, has been unequivocally shown to exist in primates and rodents. In addition to IgM a second unique membrane isotype, generally considered to be IgD, has been demonstrated in a number of other species, including dogs and chickens. Because of its assumed widespread presence, it is widely accepted that IgD is phylogenetically conserved and therefore functionally important in B cell maturation. In the present paper, we could not demonstrate IgD on bovine B cells derived from peripheral blood, lymph nodes, spleen and fetal spleen by precipitation with anti-light chain antibodies. This lack of detectable IgD was confirmed in peripheral blood B cells of sheep, and raises questions on the requirement for IgD in cell differentiation and Ig secretion. At present it is not clear whether cattle (and sheep) are an exception in this context. Reports of the presence of IgD in different species are largely based on the assumption that non-IgM surface Ig is most likely IgD. Our data question this extrapolation and stress the need for further isotype characterization of the surface Ig in different species. Lack of surface IgD has been observed in human and mouse B-1 cells, most of which express the surface marker CD5. The possibility that all bovine B cells belong to the B-1 lineage is discussed. [ABSTRACT FROM AUTHOR]

Background We previously found two distinct passenger dendritic cell (DC) subsets in the rat liver that played a central role in the liver transplant rejection. In addition, a tolerance-inducing protocol, donor-specific transfusion (DST), triggered systemic polytopical production of depleting alloantibodies to donor class I MHC (MHCI) antigen (DST-antibodies). Methods We examined the role of DST-antibodies in the trafficking of graft DC subsets and the alloresponses in a rat model. We also examined an anti-donor class II MHC (MHCII) antibody that recognizes donor DCs more selectively. Results Preoperative transfer of DST-antibodies or DST pretreatment eliminated all passenger leukocytes, including both DC subsets and depleted the sessile DCs in the graft to ~20% of control. The CD172a+CD11b/c+ immunogenic subset was almost abolished. The intrahost direct or semi-direct allorecognition pathway was successfully blocked, leading to a significant suppression of the CD8+ T-cell response in the recipient lymphoid organs and the graft with delayed graft rejection. Anti-donor MHCII antibody had similar effects without temporary graft damage. Although DST pretreatment had a priming effect on the proliferative response of recipient regulatory T cells, DST-primed sera and the anti-donor MHCII antibody did not. Conclusion DST-antibodies and anti-donor MHCII antibodies could suppress the CD8+ T-cell-mediated liver transplant rejection by depleting donor immunogenic DCs, blocking the direct or semi-direct pathways of allorecognition. Donor MHCII-specific antibodies may be applicable as a selective suppressant of anti-donor immunity for clinical liver transplantation without the cellular damage of donor MHCII– graft cells and recipient cells. [ABSTRACT FROM AUTHOR]

NK cells are functionally controlled by the killer immunoglobulin-like receptor (KIR) family that comprises inhibitory (iKIR) and activating (aKIR) members. Genetic association studies suggest that donors expressing aKIRs next to iKIRs will be superior donors in the setting of hematopoietic stem cell transplantation of patients with leukemia. However, contrary evidence states that aKIR expression may be irrelevant or even detrimental. Using a complex methodology incorporating KIR-Q-PCR, double fluorescence and viSNE analysis, we characterized subset distribution patterns and functionality in haplotype A donors which lack aKIRs and haplotype B donors that express a variety of B-specific genes. Here, we show that the alloreactive KIR2DS1+ NK cell subset in HLA-C1/C2 donors is highly responsive towards C2-expressing targets but quantitatively small and as such does not significantly contribute to cytotoxicity. Thus, we fail to find a direct link between haplotype allocation status and NK cell cytotoxicity at least in HLA-C1/C2 heterozygous donors. [ABSTRACT FROM AUTHOR]

Monoclonal antibody therapy is a new treatment strategy for many types of diseases including cancers and autoimmune diseases, realizing a high efficacy and tolerability. In multiple sclerosis (MS) and neuromyelitis optica (NMO) spectrum disorders, several monoclonal antibodies have been suggested to decrease the incidence of clinical relapse and the disease activity. In MS, anti-α4 integrin (natalizumab), anti-CD52 (alemtuzumab), anti-CD25 (daclizumab) and anti-CD20 (ocrelizumab) have been shown to effectively reduce the relapses in randomized controlled trials and have been approved by the Food and Drug Administration. Specifically, ocrelizumab is the first drug that has shown significant suppression of brain volume loss and suppression of chronic disability progression. In NMO, though there have yet to be any approved monoclonal antibodies, rituximab, anti-complement C5 (eculizumab), anti-IL-6 receptor (tocilizumab), anti-CD19 (inebilizumab) and non-pathogenic anti-aquaporin 4 (aquaporumab) have been suggested to be effective, and some of these are now under clinical trials. Aquaporumab is a non-pathogenic recombinant human monoclonal antibody that competitively inhibits the binding of the pathogenic auto-antibody against aquaporin 4 in NMO patients; thus, it is expected to be highly disease specific with less non-specific adverse events. Some of these monoclonal antibodies in MS and NMO are known to cause several notable adverse events. Natalizumab and rituximab increase the risk of progressive multifocal leukoencephalopathy. Eculizumab increases the risk of meningococcal infection. Tocilizumab is known to cause intestinal diverticulitis that can cause intestinal perforation. In this review, we summarize the characteristics of, evidence for and notable adverse events of each monoclonal antibody in MS and NMO. [ABSTRACT FROM AUTHOR]

One of the characteristic features of asthma is chronic airway inflammation typically with eosinophil infiltration. Most asthmatics can be treated successfully with conventional treatment appropriate for their severity, but in some severe cases, asthma cannot be well controlled even with thorough treatment and this condition is known as 'refractory asthma'. To overcome severe refractory asthma, a new therapeutic strategy with biologics has been developed based on the knowledge of molecular mechanisms of airway inflammation in asthma, induced by the condition of high Th2-type responses and activation of eosinophils as well as allergic reactions. Humanized anti-human IgE antibody (anti-IgE; omalizumab) was the first biological preparation approved for treating asthma. Based on clinical evidence, treatment with anti-IgE (anti-IgE therapy) has been accepted as a new therapeutic approach for severe allergic asthma in adults since 2009 and in children since 2012 and has been shown to have ~60% efficacy. More recently, a humanized anti-IL-5 antibody (anti-IL-5; mepolizumab) was launched in June 2016 and has attracted great interest due to its potential effects. Several clinical studies are also ongoing to evaluate the biological preparations targeting IL-5 receptor α (IL-5Rα), IL-4 receptor α (IL-4Rα), which is shared by IL-4 and IL-13, thymic stromal lymphopoietin (TSLP) and IL-33. The new strategy with biologics targeting eosinophilic airway inflammation might open a new array for us to overcome severe refractory asthma in the future. [ABSTRACT FROM AUTHOR]

One of the major causes of disorders of the immune response in patients undergoing hemodialysis (HD) is weaker activity of their helper T lymphocytes (Th cells), mainly reduced proliferative capacity associated with decreased expression of key surface antigens. Since cooperation between Th and B lymphocytes is essential for B cell function, changes in Th cell phenotype and ability to proliferate or produce cytokines could directly translate into an impaired humoral response. Therefore, we investigated the T cell-dependent activity of B cells in HD patients focusing mainly on their proliferative kinetics, susceptibility to apoptosis and the ability to produce antibodies. Since our previous studies have shown the beneficial effects of recombinant human erythropoietin (rhEPO) on T lymphocytes, we also investigated the in vivo and in vitro influence of rhEPO on B cells. Our results show that B lymphocytes of HD patients, especially of those who are not treated with rhEPO, have reduced proliferative capacity in vitro, reflected in low number of cell divisions, decreased percentage of proliferating cells and an increased susceptibility to apoptosis. They are also characterized by impaired ability to produce immunoglobulins. We have found no significant changes in the expression of key antigens of B lymphocytes with the exception of IL-10R. Furthermore, we demonstrated a time- and health status-dependent impact of rhEPO on patient's B cells. Our results show possible mechanisms responsible for the deficiency of humoral responses in HD patients which, at least partially, can be modulated through the supplementation with rhEPO. [ABSTRACT FROM AUTHOR]

The mechanisms of maintenance of adequate numbers of B lymphocytes and of protective levels of immunoglobulins in the absence of antigenic (re)stimulation remain not fully understood. Meanwhile, our results presented here show that both peripheral blood naive and memory B cells can be activated strongly and non-specifically (in a mitogen-like fashion) in 5-day in vitro cultures of anti-CD3- or concanavalin A (Con A)-stimulated peripheral blood mononuclear cells of healthy people. This polyclonal, bystander activation of the B cells includes multiple divisions of most of them (assessed here by the flow cytometric technique of dividing cell tracking) and significant antibody [immunoglobulin M (IgM) and IgG] secretion. Observed proliferation of the CD19+ B cells depends on contact with stimulated T helper (Th) cells (via CD40-CD40L interaction) and on the response of B cells to secreted interleukins IL-5, IL-10 and IL-4, and is correlated with the levels of these Th-derived molecules, while it does not involve the ligation of the BCR/CD19 complex. We suggest that the effect might reflect the situation occurring in vivo as the homeostatic proliferation of otherwise non-stimulated, peripheral B lymphocytes, providing an always ready pool for efficient antibody production to any new (or cognate) antigen challenge. [ABSTRACT FROM AUTHOR]

The titre and epitope specificity of anti-tTG antibodies correlate with duodenal pathology in coeliac diseaseCoeliac disease (CD) is characterized by several markers, including anti-transglutaminase auto-antibodies (tTGAb) directed against multiple epitopes of the gliadin protein. We aimed to investigate the correlation among CD duodenal lesions, tTGAb titres and the immunoreactivity against tTG constructs. A total of 345 CD patients (209 females, 136 males, overall median age: 7.3 years) were tested for full-length (fl) tTGAb with a fluid-phase radioimmunoassay. Out of the total, 231 patients were also tested for immunoreactivity against tTG fragments (F1: a.a. 227–687 and F2: a.a. 473–687). Patients were classified according to diffuse (D), patchy (P) or bulb (B) histological lesions. All sera were found fltTGAb positive. Patients with D, P and B lesions had a mean Ab index of 0.84±0.39, 0.57±0.39 and 0.45±0.24, respectively. Mean tTGAb titre varied between D and localized (P+B) patients (0.84±0.39 versus 0.52±0.34, P < 0.0001). Overall, 86.1% of patients were F1 auto-antibody (F1Ab) positive (D: 89%, P: 75%, B: 40%; D versus P+B: P = 0.004) and 49% of patients were F2 auto-antibody (F2Ab) positive (D: 53%, P: 19%, B: 10%; D versus P+B: P = 0.0006). Of the D patients 50.7% showed combined F1Ab-F2Ab (D versus P+B: P = 0.001), whereas 60% of B patients were negative for both F1Ab and F2Ab (B versus D: P < 0.0001). Coeliac-specific tTGAb immunoreactivity correlates with the grading and extension of histological duodenal lesions in CD patients at diagnosis. The immunoreactivity against single and combined tTG fragments is significantly higher in patients with D lesions. This is the first evidence of a distinct coeliac-specific immunoreactivity in patients with different duodenal involvement. [ABSTRACT FROM AUTHOR]

HIV-1 integrase (IN) catalyzes integration of a DNA copy of the viral genome into the host genome. In contrast to canonical proteases (trypsin, chymotrypsin and proteinase K), IgGs and IgMs isolated from HIV-infected patients by affinity chromatography on immobilized IN specifically hydrolyzed only IN but not many other tested intact globular proteins. The sites of IN cleavage determined by MALDI mass spectrometry were localized mainly within seven known immunodominant regions of IN. Thin layer chromatography analysis has shown that the abzymes (Abzs) could also cleave 17 to 22-mer oligopeptides (OPs) corresponding to the immunodominant regions of IN sequence with a much higher rate than non-specific long peptides or three- and tetrapeptides of various sequence. Therefore, a prolonged incubation of IN with AIDS IgGs and IgMs having high catalytic activity usually produces many OPs of different length. Since anti-IN IgGs and IgMs can efficiently hydrolyze IN, a positive role of the Abzs in counteracting the infection is possible. [ABSTRACT FROM AUTHOR]

Fc receptor-like A (FCRLA) is an unusual member of the extended Fc receptor family. FCRLA has homology to receptors for the Fc portion of Ig (FCR) and to other FCRL proteins. However, unlike these other family representatives, which are typically transmembrane receptors with extracellular ligand-binding domains, FCRLA has no predicted transmembrane domain or N-linked glycosylation sites and is an intracellular protein. We show by confocal microscopy and biochemical assays that FCRLA is a soluble resident endoplasmic reticulum (ER) protein, but it does not possess the amino acid sequence KDEL as an ER retention motif in its C-terminus. Using a series of deletion mutants, we found that its ER retention is most likely mediated by the amino terminal partial Ig-like domain. We have identified ER-localized Ig as the FCRLA ligand. FCRLA is unique among the large family of Fc receptors, in that it is capable of associating with multiple Ig isotypes, IgM, IgG and IgA. Among hemopoietic cells, FCRLA expression is restricted to the B lineage and is most abundant in germinal center B lymphocytes. The studies reported here demonstrate that FCRLA is more broadly expressed among human B lineage cells than originally reported; it is found at significant levels in resting blood B cells and at varying levels in all B-cell subsets in tonsil. [ABSTRACT FROM AUTHOR]

We previously reported that minocycline treatment of allergic asthmatic patients had oral steroid sparing effects and improved their clinical status and that minocycline suppressed in vitro induction of IgE responses by their PBMC. The effect of minocycline on human or animal IgE responses in vivo has not been studied. Allergic asthmatics (serum IgE: 505 ± 535 IU ml−1) were given minocycline (150 mg po to 250 mg po BID) as add-on therapy to standard care for up to 10 months; control subjects (IgE: 405 ± 472 IU ml−1) received standard care (n = 6 per group). Serum immunoglobulin (IgM, IgG, IgE and IgA) levels were determined monthly (Nephelometry, Unicap Total IgE Fluoroenzyme immunoassay). BALB/c mice (n = 6 per group) were injected intraperitoneally with benzylpenicilloyl14-Keyhole limpet hemocyanin (BPO14-KLH) in alum on days 0, 21 and 42, fed with minocycline or doxycycline (10–100 mg kg−1) on day 44 and numbers of BPO-specific IgG1, IgE and IgA antibody-forming cell (AFC) in mesenteric LN and spleen and serum immunoglobulin levels were determined on days 46–70 (enzyme-linked immunosorbent spot assay, ELISA). The ability of minocycline or doxycycline to suppress in vitro induction of murine memory IgE responses also was investigated. [ABSTRACT FROM PUBLISHER]

Japanese scientists were involved in pioneering work on therapeutic antisera and have made huge contributions to the characterization of the antibody molecules that are responsible for this and many other biological activities, as well as working back to understand the B cells that produce these Igs. This review emphasizes the role of Japanese immunologists in this field, starting with their work in developing antisera and studying the structure of Igs. It describes the molecular mechanisms that generate the enormous antibody repertoire and regulate B-cell development and signaling. It also details the importance of the germinal center in generating B-cell memory and the terminal differentiation of B cells as antibody-secreting plasma cells. [ABSTRACT FROM PUBLISHER]

Intravenous immunoglobulins (IVIg) preparations can be beneficial therapeutic agents for the treatment of tumor metastases as has been shown in both human and animal studies. Operating mechanisms have not yet been completely elucidated. Some of the mechanisms proposed entail the stimulation of the production of IL-12, a cytokine that exhibits anti-angiogenic activities, as well as inhibition of endothelial cells proliferation and vascular endothelial growth factor (VEGF) secretion. The aim of the present study was to investigate whether in an IVIg preparation there are natural antibodies directed against VEGF with the potential to affect angiogenesis. Using both sandwich and direct ELISA assays, IVIg was found to specifically recognize and bind VEGF in a dose-dependent manner. The binding specificity was confirmed by inhibition of IVIg binding to VEGF by VEGF as an inhibitor, as shown by ELISA and immunoblot. A mouse hind limb ischemia model was employed to evaluate the in vivo IVIg-induced inhibition of angiogenesis. IVIg was found to exhibit inhibitory effect on VEGF-mediated blood perfusion in the ischemic limb. The present study shows a presence of anti-VEGF fraction in IVIg preparation. [ABSTRACT FROM PUBLISHER]

The humoral response to the gastrointestinal (GI) flora was analyzed in secretory Ig (sIg)-deficient polymeric IgR (pIgR)−/− mice and otherwise congenic C57BL/6 mice. While both strains carried an ileal flora of similar size and composition, increased bacterial translocation to mesenteric lymph node was demonstrated in pIgR−/− mice. Serum IgA was greatly increased in pIgR−/− mice compared with C57BL/6 mice and reacted with commensal organisms and food. Serum IgG levels in pIgR−/− mice were increased to 6-fold above that of C57BL/6 mice and included specificities that bound to selected flora antigens. The enhanced recognition of flora antigens in pIgR−/− mice was explored using ovalbumin (OVA)-specific CD4+ T cells and feeding of low concentrations of OVA. Increased proliferation of transgenic T cells was observed in pIgR−/− mice, relative to C57BL/6 mice, suggesting elevated net uptake of protein antigens from the GI tract in the absence of sIg. These studies suggest that there is increased recognition of GI flora antigens by systemic antibodies in pIgR−/− mice, most probably as a result of increased access of antigens from the GI flora to the systemic immune compartment, and support the hypothesis that a major function of the secretory immune system is to return environmental antigens to mucosal surfaces. [ABSTRACT FROM AUTHOR]

The majority of maturing T lymphocytes that recognize self-antigens is eliminated in the thymus upon exposure to their target antigens. This physiological process of negative selection requires that tissue-specific antigens be expressed by thymic cells, a phenomenon that has been well studied in experimental animals. Here, we have examined the expression in human thymi of four retinal antigens, that are capable of inducing autoimmune ocular disease retinal S-antigen (S-Ag), recoverin, RPE65 and inter-photoreceptor retinoid-binding protein (IRBP)], as well as four melanocyte-specific antigens, two of which are used as targets for melanoma immunotherapy [gp100, melanoma antigen recognized by T cells 1, tyrosinase-related protein (TRP)-1 and TRP-2]. Using reverse transcription (RT)PCR, we found that all thymic samples from the 18 donors expressed mRNA transcripts of most or all the eight tested tissue antigens. Yet, the expression of the transcripts varied remarkably among the individual thymic samples. In addition, S-Ag, RPE65 and IRBP were detected by immunostaining in rare cells in sections of human thymi by antibodies against these proteins. Quantitative real-time RTPCR analysis revealed that the retinal antigen transcripts in the human thymus are present at trace levels, that are lower by approximately five orders of magnitude than those in the retina. Our observations thus support the notions that thymic expression is a common feature for all tissue-specific antigens and that the levels of expression play a role in determining the susceptibility to autoimmunity against these molecules. [ABSTRACT FROM AUTHOR]

MUC1 mucin is expressed by normal human epithelial cells and is overproduced in underglycosylated form by malignant epithelial cells. A number of anticancer immunotherapeutic strategies are being designed with the goal of inducing humoral and cellular immune responses against MUC1 mucin. Newly synthesized MUC1 mucin is also expressed on polyclonally stimulated human T cells. An immunoregulatory role has been postulated for MUC1 mucin expressed by activated T cells. We now show that several MUC1 peptide and glycopeptide epitope specific antibodies bind to activated T cells and inhibit their proliferation. Inhibition by antibody B27.29 could be reversed by glycopeptide haptens specific for the antibody. Intact antibody B27.29 and its divalent F(ab')2 fragment inhibited the proliferation of T cells undergoing T cell activation but the monovalent Fab' fragment did not, indicating that cross-linking of the MUC1 antigen on T cells is required for the inhibitory effect. MUC1 expression on activated T cells was increased in the presence of IL-12 but was not affected by IFN-?, IL-2, IL-4, IL-5, IL-10, IL-13 or TNF-a. Treatment of T cells inhibited by monoclonal antibody (MAb) B27.29 with either IL-2 or costimulatory anti-CD28 antibody restored proliferation to a level equivalent to that of control cultures. These results provide further support for the hypothesis that the expression of MUC1 on the activated T cell surface is associated with the regulation of T cell responses. [ABSTRACT FROM AUTHOR]

Earlier studies have demonstrated an over-expression of 9-O-acetylated sialoglycoconjugates (9-OAcSGs) on lymphoblasts, concomitant with high titers of anti-9-OAcSGs in childhood acute lymphoblastic leukemia (ALL). The present study was aimed to evaluate whether this high induction of anti-9-OAcSGs at disease presentation contributes toward immune surveillance. Accordingly, anti-9-OAcSGs were affinity purified from sera of ALL patients and normal individuals, and their specificity toward the glycotope having terminal 9-O-acetylated sialic acid-linked subterminal N-acetyl galactosamine (GalNAc) in a26 manner (9-OAcSAa26GalNAc) was established by hemagglutination assay, flow cytometry and confocal microscopy. Subclass distribution of anti-9-OAcSGs revealed a predominance of IgG2 in ALL. Analysis of glycosylation of anti-9-OAcSGs purified from sera of ALL patients (IgGALL) and normal individuals (IgGN) by digoxigenin glycan enzyme assay, fluorimetric estimation, gasliquid chromatography and lectin-binding assays demonstrated that disease-specific antibodies differ in content and nature as compared with normal controls. Enhanced amount of 9-OAcSA-specific IgG2 induced in ALL was unable to trigger activation of Fc?R, the complement cascade and cell-mediated cytotoxicity, although its glycotope-binding ability remains unaffected. Interestingly, only IgG1N emerged as the potent mediator of cell-mediated cytotoxicity, complement fixation and activator of effector cells through Fc?R. In ALL, the observed subclass switching of anti-9-OAcSGs to IgG2, alteration in their glycosylation profile along with impairment of a few Fc-glycosylation-sensitive effector functions hints toward a disbalanced homeostasis, thereby evading the host defense. These findings justify further evaluation of the mechanism for functional unresponsiveness of antibodies and production of 9-OAcSA-specific chimeric antibodies with normal Fc domain for therapeutic applications. [ABSTRACT FROM AUTHOR]

Defects of early complement components (C1, C4 and C2) are associated with the development of systemic lupus erythematosus (SLE). The availability of complement knockout mice has increased our knowledge on the role of complement in the regulation of adaptive immunity. An impaired removal of apoptotic bodies, a disturbed clearance of IgG immune complexes (ICs) and an insufficient B-cell regulation via complement receptors CD21/CD35 have been discussed as explanations for the induction of autoimmunity; however, a unifying hypothesis for the loss of B-cell tolerance in the absence of C1 or C4 is still lacking. Using IgM-containing ICs, we observed a significant accumulation of antigen within the splenic marginal zone (MZ) of C4-deficient mice but not in C3-deficient or complement receptors CD21/CD35-deficient mice. The targeting of antigen toward the MZ restored adaptive immunity (antibody response and class switch) in C4-deficient animals. A new explanation for the association of SLE and complement C4 deficiency would be that in the absence of C4, natural antibodies (IgM type) localize more self-antigen toward the MZ so that the auto-antibody response is increased and autoimmune disease ensues. As such, an inadequate localization of self-antigens might be responsible for the annulment of peripheral B-cell tolerance in the absence of C4. [ABSTRACT FROM AUTHOR]

There is considerable evidence that multiple sclerosis (MS) is an immune-mediated disease characterized by infiltration of inflammatory cells into the CNS and demyelination. Several myelin proteins may be encephalitogenic, including myelin basic protein, proteolipid protein and myelin oligodendrocyte glycoprotein (MOG), the latter being expressed on the external layer of myelin sheaths and hence accessible to antibody attack. We investigated MOG autoreactivity in serum and cerebrospinal fluid (CSF) by ELISA, employing the recombinant extracellular domain of MOG as antigen. We tested serum samples from 262 MS patients (175 relapsing-remitting, 43 primary progressive and 44 secondary progressive), 131 patients with other neurological diseases (OND) and 307 healthy controls. No patients or controls were receiving immunomodulating treatments. We found anti-MOG antibodies in the serum of 13.7% MS patients, mainly in those with secondary progressive MS (25%), in 13.7% of OND patients and in 6.2% of controls. We found a direct correlation (R2 = 0.6, P = 0.002) between disease severity and anti-MOG titer only in patients with primary and secondary progressive MS. Anti-MOG antibodies were present in the CSF of 11.4% MS patients and 18.9% OND patients. Intrathecal synthesis of anti-MOG antibodies was demonstrated in four (4.5%) of MS patients and no OND patients. Anti-MOG antibodies are not specific for MS; however, they may characterize a subset of MS patients and this may be revealed by serial assays in relation to changing disease phase. [ABSTRACT FROM AUTHOR]

Growing evidence suggests that anti-Hsp60 antibodies might be involved in the pathogenesis of different autoimmune diseases. Attempts were reported also on the characterization of epitope specificity of anti-Hsp60 antibodies in different infectious and autoimmune diseases. However, there is a lack of data on the occurrence and epitope specificity of anti-Hsp60 antibodies in the healthy human antibody repertoire. Therefore, the aim of our study was to demonstrate the presence of anti-Hsp60 antibodies and to investigate the epitope specificity of these antibodies using a large set of synthetic 10mer peptides covering regions of Hsp60 with high probability of antigenicity. Here we report the identification of several linear epitopes using serum Ig (IVIG) and sera from healthy subjects by ELISA assay. We have identified two epitopes 'specific' for human Hsp60 and two different epitopes 'specific' for Hsp65. In addition, six epitopes were cross-reactive in nature, detected on both proteins. The presence of these 'specific' epitopes may explain the differences in epitope structure between Hsp60 and Hsp65 observed earlier in patients with cardiovascular disease. The binding of the IVIG preparation to Hsp60 epitopes might indicate that anti-Hsp60 autoantibodies are present in the healthy human natural autoantibody repertoire. [ABSTRACT FROM AUTHOR]

Single-cell PCR analyses of expressed Ig H and L chain sequences presented here show that certain rearrangements occur repeatedly and account for a major segment of the well-studied repertoire of B-1 cell autoantibodies that mediate the lysis of bromelain-treated mouse erythrocytes, i.e. antibodies reactive with phosphatldyicholine (PtC). We repeatedly isolated at least 10 different types of VH region rearrangements, involving three distinct germline genes, among FACS-sorted PtC-binding B-1 cells from three strains of mice (C57BL/6J, BALB/c and C.B-17). The predominant rearrangement, VH11-DSP-JH1 (VH11 type 1), has been previously found in anti-PtC hybridomas in several studies. We show that within each of six mice from two strains (C57BL/6J and BALB/c), unique instances of IgH/IgL pairing arose either from different B cell progenitors prior to IgH rearrangement or from pre-B cells which expanded after IgH rearrangement but prior to IgL rearrangement. Together with other recurrent rearrangements described here, our findings demonstrate that clonal expansion of mature B cells cannot account for all repeated rearrangements. As suggested by initial studies of dominant idiotype expression, these findings confirm that clonal expansion is only one of the mechanisms contributing to the establishment of recurrent rearrangements. [ABSTRACT FROM AUTHOR]

Ligation of surface IgM on B cells responding to lipopolysaccharide (LPS) suppresses terminal differentiation and high-rate Ig secretion with no effect on proliferation. As shown here, different B cell populations show characteristic mean values of ligand concentration required for 50% inhibition, with Gaussian distributions of sensitivity to IgM receptor ligation that reflect cellular heterogeneity of 'al-or-none' inhibitions in single cells. Differential sensitivity of B cell populations to IgM ligation seems to be locally determined by the cellular environment and unrelated to the 'maturity' of the responding cells. Thus, while long-lived peritoneal B cells are 3- to 5-fold more resistant than splenic B cells, there is no difference in sensitivity between short- and long-lived B cells in the spleen. Furthermore, while B cells in bone marrow and spleen differ in sensitivity by two orders of magnitude, B cells differentiated in vitro from bone marrow pre-B cells are as resistant as splenic B cells. Moreover, bone marrow cell culture supernatants restore a high level of sensitivity in such cell populations. Differential sensitivity to IgM receptor ligation is reproduced by multivalent nominal antigen, in cell populations that show identical dose-response inhibition curves to direct activation of protein kinase C by phorbol esters. We conclude that the level of sensitivity to IgM ligation is independent of the life span or maturity of the B cell, but differentially regulated in vivo by putative tissue factors. [ABSTRACT FROM AUTHOR]

Human thymus contains a small population of B cells, a fraction of which expresses CD2 and/or CD5, as revealed by FACS analysis. Human thymic B cells enriched on CD19 panning revealed a constant co-expression of CD19 and Ig light chains. In the absence of detectable B precursors, these cells had reached at least the stage of IgM+ immature B lymphocytes. Analysis of Ig transcripts by PCR amplification first revealed a strong bias in favor of the VH4 and sequencing of 45 VH4-D-J cDNA clones isolated from two thymuses indicated that this thymic repertoire significantly differed from that of peripheral blood lymphocytes. Most genes of the VH4 family were used with various D-J combinations and a relatively high frequency of somatic mutations. This repertoire thus appears clearly selected in the thymus. This is of particular interest since the VH4 gene family is frequently encountered in autoimmunity a situation in which the number of thymic B cells is largely increased. They could play a major role in the control of tolerance in situ. [ABSTRACT FROM AUTHOR]

B cell development is a complex process involving interactions between B cell precursors, stroma, and known and unknown ligands and cytokines. In order to more fully understand the requirements for Ig in that development we have created transgenic mice that carry a gamma 2b transgene and express it early in B cell development. Previously it was believed that these B cells arrested in their development prior to the pro- to pre-B cell transition. We show here that in conventional gamma 2b mice, B cell development actually arrests later, at the pre-B cell stage. This shows for the first time that a constant region different from mu can allow signaling through the pre-B cell receptor, but cannot promote complete development. The pro- and pre-B cells in the conventional gamma 2b transgenics are not fully functional since they cannot grow in IL-7 without stromal cells. This is a novel phenotype, separating development from stroma independence. The few, mature B cells that do develop in these mice express both mu and gamma 2b simultaneously, and are CD5+. Expression of a Bcl-2 transgene allows survival of gamma 2b transgenic immature B cells, but does not promote full maturation, indicating that normally mu provides both an anti-apoptotic signal and a differentiation signal. One line of gamma 2b mice, the C line, does not have this phenotype. B cell development is accelerated in this unconventional line, and the developing B cells have a very different phenotype from both normal mice and conventional gamma 2b mouse lines, but are very similar to mu transgenics. Mature B cells are largely CD5-, gamma 2b-only expressing. This unique phenotype is apparently due to the activation in B cell precursors of a gene at the insertion site of the transgene, circumventing the need for mu. Comparison of conventional gamma 2b transgenics with the C line and mu transgenics reveals the multiple signals required throughout B cell development. [ABSTRACT FROM AUTHOR]

The article discusses several reports published within the issue including "Lineage-Restriction Pathways During Haematopoiesis," "Ligation of CD16 Enhances Antibody Responses," and "IL-23 Induces T-Cell-Dependent and -Independent Antifungal Responses."

The serum of patients with many autoimmune (AI) diseases contains small fractions of antibodies possessing both DNase and RNase activities. It was shown that immunization of rabbits with DNA, RNA, DNase I and RNase leads to production of antibodies with DNase and RNase activities. It is not known whether anti-idiotypic antibodies against DNase II can possess DNase or RNase activity. Electrophoretically and immunologically homogeneous polyclonal IgGs (pIgGs) from the sera of rabbits immunized with DNase II were obtained by sequential chromatography of the serum proteins on Protein A-Sepharose and gel filtration. It was shown for the first time that immunization of healthy rabbits with bovine DNase II produces IgGs with intrinsic DNase and RNase activities. IgGs from rabbits immunized with BSA or non-immunized animals were catalytically inactive. It was shown that ∼10% of the total IgG DNase and RNase activities belong to anti-idiotypic antibodies to DNase II (∼0.1% of total pIgGs), while 90% of the activities did not interact with Sepharose bearing antibodies against DNase II and might be antibodies to nucleic acids bound to DNase II. Affinity chromatography on DNA-cellulose using elution of antibodies with different concentration of NaCl and an acidic buffer separated catalytic IgGs into five antibody subfractions, three of which hydrolyzed RNA faster than DNA while two subfractions demonstrated only DNase activity. Our data suggest that a fraction of abzymes from AI patients hydrolyzing both DNA and RNA can contain subfraction of antibodies against DNase II. [ABSTRACT FROM AUTHOR]

The sera of patients with autoimmune (AI) diseases contain antibodies with DNase and RNase activities. We have shown for the first time that immunization of healthy rabbits with RNase A conjugated with BSA produces a better immune response than immunization with pure RNase and induced IgGs with RNase and DNase activities, which were intrinsic properties of IgGs, while polyclonal IgGs (pIgGs) from non-immunized rabbits and animals immunized with BSA were catalytically inactive. It was shown that 74–85% of the total IgG DNase and RNase activities belongs to anti-idiotypic antibodies to RNase A (0.6–0.8% of total pIgGs), while 15–26% of the activities cannot interact with Sepharose-bearing antibodies against RNase A and may be antibodies to nucleic acids bound to RNase. Affinity chromatography on DNA–cellulose separated catalytic IgGs into several antibody subfractions demonstrating only DNase or RNase activity or hydrolyzing RNA faster than DNA. Our data suggest that a fraction of abzymes (or catalytically active antibodies) from AI patients hydrolyzing both DNA and RNA may be antibodies against RNase or its complexes with other proteins. [ABSTRACT FROM AUTHOR]

Carbohydrate antigens such as glycolipids and glycoproteins are over-expressed in a variety of cancers and have therefore been identified as ideal candidates for tumour vaccines. Detection of anti-carbohydrate antibodies is associated with a good prognosis in cancer patients. However, generation of an efficient adaptive immune response has been hampered by the low immunogenicity of carbohydrates due to tolerance. Here, we describe a method by which tumour-rejecting antibodies directed against carbohydrates can be elicited in two different melanoma mouse models. Thus, using the murine melanoma B16F10 over-expressing Fas ligand (FasL), we have generated mAbs against cancer carbohydrate antigens expressed by the melanoma. Importantly, passive transfer of mAbs resulted in rejection of melanoma in vivo. Their protective effect in vivo was dependent on FcR and in vitro antibody-dependent cellular phagocytosis. They were also able to delay tumour growth when injected after the tumour was established. FasL-expressing tumours as an adjuvant are a novel way to generate anti-carbohydrate antibodies able to reject tumours in vivo. [ABSTRACT FROM AUTHOR]