Bacterial CpG induces hyper-IgM production in CD27(+) memory B cells in primary biliary cirrhosis

BACKGROUND AND AIMS: Sera from patients with primary biliary cirrhosis (PBC) are characterized by the presence of antimitochondrial antibodies and elevated levels of immunoglobulin (Ig) M. We hypothesized that the increase in serum IgM is the result of chronic B-cell activation induced via the Toll-like receptor (TLR) signaling pathway. METHODS: We analyzed peripheral blood mononuclear cells (PBMCs) from patients with PBC and controls following incubation with CpG, a natural ligand for TLR9, and determined the basal and stimulated levels of intracellular IgM, the density of TLR9, and the contribution of specific B-cell subpopulations. RESULTS: Our data demonstrate uniquely that in vitro incubation of PBMCs from PBC with CpG-B, but not CpG-A, led to a markedly high frequency of intracellular IgM-positive B cells, associated with high levels of synthesized IgM and identified to be a function of CD27(+) memory B cells. This memory B-cell subset also expressed higher densities of TLR9 as compared with naive B cells. These results were not due to increased proliferation, as defined by 5-carboxyfluoresein diacetate succinimidyl ester labeling, or an increase in the life span of B cells, as defined by Bcl-2 expression. CONCLUSIONS: These findings for the first time identify a major role for innate immune mechanisms in the induction and persistence of abnormal humoral immune responses in PBC.     

Bovine Neonatal Pancytopenia: is this alloimmune syndrome caused by vaccine-induced alloreactive antibodies?

Bovine Neonatal Pancytopenia (BNP) is a new emerging disease observed since 2007 in Germany and neighbouring countries. The syndrome affects newborn calves and is characterized by pancytopenia, severe bleeding and high lethality. So far, a causative role of infectious or toxic agents has been ruled out. Instead, the syndrome is induced after ingestion of colostrum, the first milk that supplies the calf with maternal antibodies. In analogy to similar diseases in humans it has therefore been postulated that BNP is caused by alloreactive, maternal antibodies. There is a striking association between BNP and a previous vaccination of the respective dams with a particular vaccine against Bovine Virus Diarrhoea (BVD). This association has led to a suspension of the marketing authorisation for the vaccine, by the European Commission. The current study investigates the role of this vaccine in the pathogenesis of BNP. By flow cytometry we were able to demonstrate that sera of BNP dams (dams that gave birth to a BNP calf) harbour alloreactive antibodies binding to surface antigens on bovine leukocytes. A significantly weaker alloreactivity was observed with sera of non-BNP dams that have been vaccinated with the same vaccine but delivered healthy calves. No binding was seen with non-BVD-vaccinated control cows and animals that were vaccinated with other inactivated BVD vaccines so far not associated with BNP. The binding is functionally relevant, because opsonization of bovine leukocytes with alloantibodies led to an elevated cytophagocytosis by bovine macrophages. To test whether the vaccine induces alloreactive antibodies two strategies were employed: Guinea pigs were vaccinated with a panel of commercially available BVD-vaccines. Only the incriminated vaccine induced antibodies binding surface antigens on bovine leukocytes. Additionally, two calves were repeatedly vaccinated with the suspected vaccine and the development of alloreactivity was monitored. In dependence of the number of booster immunizations the induction of alloreactive antibodies could be observed. Finally, by affinity purification we were able to directly demonstrate that BNP associated alloantibodies cross react with the bovine kidney cell line used for vaccine production. Together this provides strong evidence that this particular BVD vaccine has the potential to induce BNP associated alloantibodies.     

流式细胞术检测抗生素最低抑菌浓度

目的　建立一种流式细胞术 (FCM )快速检测抗生素最低抑菌浓度 (MIC)的方法 ,与常规药敏试验结果进行比较 ,探讨其应用价值。方法　取 2 0株肠杆菌科细菌 ,用碘化丙啶 (PI)染色 ,分别检测阴性对照管和MIC浓度抗生素作用管的平均荧光强度 (MFI) ,计算两者比值 ,据此 ,设定流式细胞药敏试验 (FCST)检测MIC的判断值 ,根据设定的判断值对 2 8株临床分离细菌同时进行常规药敏和FCST双盲测定。结果　 2 0株肠杆菌科细菌测得的MFI比值 x为 2 .0 7,CV为 0 .3 1;将MFI大于阴性对照细菌 2倍 (增加 10 0 % )的最低药物浓度定义为该菌的MIC ,2 8株临床菌株FCST所测得的MIC与常规药敏试验相比r=0 .92 ,(P <0 .0 0 1) ,回归方程为 :Y =1.4X -0 .48。结论　FCST与常规药敏试验结果有显著相关性 ,并且具有快速、稳定、准确、便于自动化等优点 ,有着较大的临床应用前景。     

Ethyl pyruvate and ethyl lactate down-regulate the production of pro-inflammatory cytokines and modulate expression of immune receptors

Esters of alpha-oxo-carbonic acids such as ethyl pyruvate (EP) have been demonstrated to exert inhibitory effects on the production of anti-inflammatory cytokines. So far, there is no information about effects, if any, of ethyl lactate (EL), an obviously inactive analogue of EP, on inflammatory immune responses. In the present study, we provide evidence that the anti-inflammatory action of alpha-oxo-carbonic acid esters is mediated by inhibition of glyoxalases (Glo), cytosolic enzymes that catalyse the conversion of alpha-oxo-aldehydes such as methylglyoxal (MGO) into the corresponding alpha-hydroxy acids using glutathione as a cofactor. In vitro enzyme activity measurements revealed the inhibition of human Glo1 by alpha-oxo-carbonic acid esters, whilst alpha-hydroxy-carbonic acid esters such as EL were not inhibitory. In contrast, both EP and EL were shown to suppress the Lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines such as tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6 and IL-8 from human immunocompetent cells, and modulated the expression of the immune receptors HLA-DR, CD14 and CD91 on human monocytes. Here, we show a crossing link between glyoxalases and the immune system. The results described herein introduce glyoxalases as a possible target for therapeutic approaches of immune suppression.     

Cytokine-targeted therapy for the management of solid organ transplant recipients

IntroductionThe number of solid organ transplants completed annually continues to trend upwards each year. Despite this, maintenance immunosuppression available on the market has remained relatively stagnant. Standard triple immunosuppression, composed typically of tacrolimus, mycophenolate, and steroids, lead to many side effects that limit the use of these medications. Tacrolimus, specifically, causes nephrotoxicity that can lead to renal dysfunction requiring a kidney transplant down the road. Alternative therapies for the management of immunosuppression need to be identified to try to mitigate these adverse effects.BodyCytokines are responsible for facilitating T cell differentiation and lead to the activation of inflammatory mediators that can contribute to graft damage and ultimately rejection. IL-4, IL-6, IL-12/23, and IL-15 are attractive targets for medications to try to ameliorate graft rejection. Various cytokine-targeted medications are currently available on the market for the treatment of inflammatory and autoimmune conditions such as rheumatoid arthritis, psoriatic arthritis, Crohn’s, and multiple sclerosis.ConclusionThis article reviews cytokine involvement in alloimmunity and the potential role cytokine-targeted therapy may play in prevention of allograft rejection in solid organ transplant recipients.     

Getting on target: Development of the novel,prozone-resistant,dual antibody rapid test (DART) for the LABScreen single antigen bead (SAB) assay

A major limitation of the single antigen bead (SAB) assay is the so called prozone effect, whereby the detection of high titer complement fixing HLA antibodies is compromised due to complement split product (from C3 and C4 components) deposition and interference with the reporter anti-IgG-PE antibody binding. Strategies to minimize prozone include serum titration or treatment with heat, dithiotreitol (DTT), or ethylenediaminetetraacetic acid (EDTA). While effective, these treatments may compromise HLA antibody binding and detection. Here we describe the Dual Antibody Rapid Test (DART), a modified version of the rapid optimized SAB (ROB) protocol, in which we use an IgG-PE/C3d-PE antibody cocktail to simultaneously detect bead bound IgG and C3d, which allows for detection of HLA antibodies independent of the prozone effect. Twenty prozone positive sera (10 class I and 10 class II), identified by titration, were tested by the ROB protocol, with or without EDTA pre-treatment, using three reporter antibody cocktails: (1) IgG-PE, (2) C3d-PE, or (3) IgG-PE/C3d-PE (DART). Mean fluorescence intensity (MFI) values were then compared. IgG negative (n = 735) vs IgG positive (n = 1185) reactions were identified using a 1000 MFI IgG EDTA cutoff. IgG positive reactions were classified based on ΔMFI (IgG EDTA – IgG) as follows: (1) prozone negative (ΔMFI < 3000; n = 737), (2) slight prozone (ΔMFI 3001–5000; n = 49), (3) moderate prozone (ΔMFI 5001–10,000; n = 93), and (4) marked prozone (ΔMFI > 10,001; n = 306). No C3d deposition was present on IgG negative beads, and the majority of prozone positive specificities (438/448; 98%) fixed complement and were detected with the C3d-PE reporter. Interestingly, C3d-PE MFI was directly proportional to the degree of prozone (mean C3d-PE MFI = 4419.5 ± 1606.3 for slight, 5991.0 ± 2302.7 for moderate, and 12,417.4 ± 2969.9 for marked prozone specificities). Interestingly, EDTA treatment was found to have a negative impact on MFI of up to 15% of prozone negative specificities. Importantly, the DART protocol detected all prozone positive specificities while MFI for prozone negative specificities correlated well with those seen with the IgG-PE reporter alone (R² = 0.97). In conclusion, the DART protocol accurately detects HLA antibodies independent of the prozone effect. Implementation of DART is an easy way to overcome the prozone effect without compromising HLA antibody detection.