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The digestive system is exposed to severe inflammation as a result of taking some medications that have gastrointestinal side effects. Sixty Swiss-albino male mice were randomly distributed into six groups to treat inflammations of the colon, stomach, and small intestine caused by taking high doses of diclofenac (D), with two novel synthesized compounds, pyrazolo [3,4 d] pyridazine derivatives (Co1 and Co2). Myeloperoxidase enzyme activity was determined in the colon and small intestinal tissues. Serum contents of TNF-α, IL-22, IgG, and IgM were determined by ELISA. Histopathological examinations of the colon, small intestinal, and stomach tissues were microscopically analyzed. TNF-α, IL-22, and TNFSF11 gene expression were measured in the colon, intestinal, and spleen using qRT-PCR. Diclofenac caused surface columnar epithelial cell loss, focal necrosis of the gastric mucosa, inflammatory cell infiltration, and congested blood vessels in the stomach, colon, and small intestinal tissues. Co1 component was found to be better than Co2 component in reducing the focal necrosis of gastric mucosa and improving the histological structures of the stomach, colon, and small intestinal tissues. After 14 days, the activity of the myeloperoxidase enzyme was increased in group D and decreased in groups DCo1, DCo2, Co1, and Co2. Serum concentrations of TNF-α and IgG were increased, while IL-22 and IGM were reduced in the D, DCo1, and DCo2 groups compared with the Co1 and control groups. TNF-α gene was upregulated in the D group and downregulated in the Co1 group, while the IL-22 gene was downregulated in the D group and upregulated in the Co1 group compared with the control group. The CO1 component may be useful in reducing digestive system inflammation., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The purpose of this study was to investigate the effects of dietary aflatoxin B 1 (AFB 1 ) on growth performance and AFB 1 biotransformation, and hepatic oxidative stress, endoplasmic reticulum (ER) stress, apoptosis, and inflammation in northern snakehead (Channa argus). A total of 600 northern snakeheads (7.52 ± 0.02 g) were divided into five groups (three replicates/group) and fed the diets with AFB 1 at concentrations of 0, 50, 100, 200, and 400 ppb for 8 weeks. The results demonstrated that dietary AFB 1 (≥ 200 ppb) reduced FBW, WG, and SGR. 100, 200, and 400 ppb AFB 1 treatment groups significantly decreased the PER, CRP, C3, C4, IgM, and LYS levels in northern snakehead, while FCR was significant increased. Moreover, dietary AFB 1 (100, 200, and 400 ppb) increased cyp1a, cyp1b (except 400 ppb), and cyp3a mRNA expression levels, while reducing the GST enzymatic activity and mRNA expression levels in northern snakehead. Furthermore, AFB 1 (≥ 100 ppb) increased ROS, MDA, and 8-OHdG levels, and grp78, ire1, perk, jnk, chop, and traf2 mRNA expression levels, and decreased SOD, CAT, GSH-Px, and GSH (except 100 ppb) levels and the gene expression levels of cat, gsh-px (except 100 ppb), and Cu/Zn sod. In addition, AFB 1 (100, 200, and 400 ppb) up-regulated the cyt-c, bax, cas-3, and cas-9 mRNA levels in the liver, while down-regulating the bcl-2 expression levels. Meanwhile, the expression levels of nf-κb, tnf-α (except 100 ppb), il-1β, and il-8 in the liver were up-regulated in AFB 1 treatment groups (≥ 100 ppb), while the iκbα mRNA levels were down-regulated. In summary, dietary AFB 1 reduced growth performance and humoral immunity in northern snakehead. Meanwhile, the cyclic occurrence of oxidative stress and ER stress, and induced apoptosis and inflammation, is one of the main reasons for AFB 1 -induced liver injury in the northern snakehead, which will provide valuable information and a fresh perspective for further research into AFB 1 -induced liver injury in fish., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Pyraclostrobin (PYR), a strobilurin fungicide, has been widely used to control fungal diseases, posing potential risk to aquatic organisms. However, the toxic effects of PYR to fish remained largely unknown. In this study, common carp (Cyprinus carpio L.) was exposed to environmentally relevant levels of PYR (0, 0.5 and 5.0 μg/L) for 30 days to assess its chronic toxicity and potential toxicity mechanism. The results showed that long-term exposure to PYR induced hepatopancreas damage as evident by increased in serum transaminase activities (AST and ALT). Moreover, PYR exposure remarkably enhanced the expressions of hsp70 and hsp90, decreased the levels of antioxidant enzymes and biomarkers and promoted the reactive oxygen species (H 2 O 2 and O 2 - ) and MDA contents in carp hepatopancreas. PYR exposure also upregulated apoptosis-related genes (bax, apaf-1, caspase-3 and caspase-9) and reduced anti-apoptosis gene bcl-2 in fish hepatopancreas. Moreover, PYR exposure altered the expressions of inflammatory cytokines (IL-1β, IL-6, TNF-α and TGF-β) in the serum and hepatopancreas and the level of NF-κB p65 in the hepatopancreas. Further research indicated that PYR exposure markedly changed the levels of immune parameters (LYZ, C3, IgM, ACP and AKP) in the serum and/or hepatopancreas, indicating that chronic PYR exposure also has immunotoxicity on fish. Additionally, we found that PYR exposure upregulated p38 and jnk MAPK transcription levels, suggesting that MAPK may be play important role in PYR-induced apoptosis and inflammatory response in the hepatopancreas of common carp. In summary, PYR exposure induced oxidative stress, triggered apoptosis, inflammatory and immune response in common carp, which can help to elucidate the possible toxicity mechanism of PYR in fish., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Arsenic is a toxic heavy metal widely found in the natural environment and has adverse effects on the health of waterfowl and human. Curcumin (CUR), a natural pigment of the golden spice turmeric, exhibits excellent anti-tumor, anti-inflammatory and anti-oxidant activities. But the effects of CUR on duck spleen exposed to arsenic remain largely unknown. In this study, 75 ducks were divided randomly into Control, L-ATO, M-ATO, H-ATO and CUR + H-ATO groups to systematically analyze the underlying role of CUR. The results showed that arsenic trioxide (ATO) led to growth retardation of ducks, hyaline degeneration and sparse cell arrangement on their spleen. And in the ATO-exposed ducks, the levels of immunoglobulins (Ig; IgA, IgG, IgM) in the serum and the expression of autophagy-related genes (Atg5, P62, LC3I, LC3II, LC3II/I, Beclin-1) were significantly upregulated compared with the control ducks. Moreover, ATO also activated NF-κB signal pathway and upregulated the expression of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1β, IL-2, IL-18). Meanwhile, application of CUR alleviated the ATO toxicity with the release of growth inhibition, and the reduced hyaline degeneration and distortion of the spleen capsule. CUR also suppressed ATO-induced NF-κB activation, pro-inflammatory cytokine addition and expression of autophagy-related genes. Overall, these results suggested that CUR might exert a protective effect against ATO-induced immunosuppression in ducks via anti-inflammation and autophagy restoring., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Present investigation was carried out to study toxicological damages of copper exposure and mitigation of its adverse effects with β-glucan administration in IgM + B cells which processes multiple roles similar to macrophages in Nile tilapia (Oreochromis niloticus). IgM + B cells were pretreated with β-glucan (25 μg/mL) for 24 h before exposed to cupric oxide nanoparticles (CuO NPs) or cupric chloride (Cu ions) at the doses of 0, 5, 10, and 20 μg/mL for 24 h, respectively. Our results demonstrated that β-glucan increased reduced glutathione (GSH) to against oxidative damage from CuO NPs and Cu ions exposure in IgM + B cells. The apoptosis process through mitochondrial signaling pathway was depressed in IgM + B cells since the mitochondrial membrane potential (ΔΨm) was protected from copper exposure by β-glucan treatment. Furthermore, the inhibition on phagocytic abilities of IgM + B cells caused by copper exposure could be enhanced with β-glucan treatment via evaluation of microspheres and bioparticles uptake and LPS-induced NO production. Importantly, β-glucan might participate in immunomodulation in IgM + B cells through B cell antigen receptor (BCR) to suppress toxicological effect derived from copper exposure. Taken together, this study provides more information on the toxicological damages in IgM + B cells upon copper exposure and explains the molecular mechanism to reverse adverse effects caused by copper exposure with β-glucan administration., (Copyright © 2022. Published by Elsevier Ltd.)

Death receptor 5 (DR5) is an attractive target for cancer therapy due to its broad upregulated expression in multiple cancers and ability to directly induce apoptosis. Though anti-DR5 IgG antibodies have been evaluated in clinical trials, limited efficacy has been attributed to insufficient receptor crosslinking. IGM-8444 is an engineered, multivalent agonistic IgM antibody with 10 binding sites to DR5 that induces cancer cell apoptosis through efficient DR5 multimerization. IGM-8444 bound to DR5 with high avidity and was substantially more potent than an IgG with the same binding domains. IGM-8444 induced cytotoxicity in a broad panel of solid and hematologic cancer cell lines but did not kill primary human hepatocytes in vitro , a potential toxicity of DR5 agonists. In multiple xenograft tumor models, IGM-8444 monotherapy inhibited tumor growth, with strong and sustained tumor regression observed in a gastric PDX model. When combined with chemotherapy or the BCL-2 inhibitor ABT-199, IGM-8444 exhibited synergistic in vitro tumor cytotoxicity and enhanced in vivo efficacy, without augmenting in vitro hepatotoxicity. These results support the clinical development of IGM-8444 in solid and hematologic malignancies as a monotherapy and in combination with chemotherapy or BCL-2 inhibition., (©2021 The Authors; Published by the American Association for Cancer Research.)

Natural IgM antibodies (NAbs) have been shown to recognize injury-associated neoepitopes and to initiate pathogenic complement activation. The NAb termed C2 binds to a subset of phospholipids displayed on injured cells, and its role(s) in arthritis, as well as the potential therapeutic benefit of a C2 NAb-derived ScFv-containing protein fused to a complement inhibitor, complement receptor-related y (Crry), on joint inflammation are unknown. Our first objective was to functionally test mAb C2 binding to apoptotic cells from the joint and also evaluate its inflammation enhancing capacity in collagen antibody-induced arthritis (CAIA). The second objective was to generate and test the complement inhibitory capacity of C2-Crry fusion protein in the collagen-induced arthritis (CIA) model. The third objective was to demonstrate in vivo targeting of C2-Crry to damaged joints in mice with arthritis. The effect of C2-NAb on CAIA in C57BL/6 mice was examined by inducing a suboptimal disease. The inhibitory effect of C2-Crry in DBA/1J mice with CIA was determined by injecting 2x per week with a single dose of 0.250 mg/mouse. Clinical disease activity (CDA) was examined, and knee joints were fixed for analysis of histopathology, C3 deposition, and macrophage infiltration. In mice with suboptimal CAIA, at day 10 there was a significant ( p < 0.017) 74% increase in the CDA in mice treated with C2 NAb, compared to mice treated with F632 control NAb. In mice with CIA, at day 35 there was a significant 39% ( p < 0.042) decrease in the CDA in mice treated with C2-Crry. Total scores for histopathology were also 50% decreased ( p < 0.0005) in CIA mice treated with C2-Crry. C3 deposition was significantly decreased in the synovium (44%; p < 0.026) and on the surface of cartilage (42%; p < 0.008) in mice treated with C2-Crry compared with PBS treated CIA mice. Furthermore, C2-Crry specifically bound to apoptotic fibroblast-like synoviocytes in vitro , and also localized in the knee joints of arthritic mice as analyzed by in vivo imaging. In summary, NAb C2 enhanced arthritis-related injury, and targeted delivery of C2-Crry to inflamed joints demonstrated disease modifying activity in a mouse model of human inflammatory arthritis., (Copyright © 2020 Banda, Tomlinson, Scheinman, Ho, Ramirez, Mehta, Wang, Vu, Simberg, Kulik and Holers.)

Moraxella catarrhalis is a human-specific commensal of the respiratory tract and an opportunistic pathogen. It is one of the leading cause of otitis media in children and of acute exacerbations in patients with chronic obstructive pulmonary disease, resulting in significant morbidity and economic burden. Vaccines and new immunotherapeutic strategies to treat this emerging pathogen are needed. Complement is a key component of innate immunity that mediates the detection, response, and subsequent elimination of invading pathogens. Many pathogens including M. catarrhalis have evolved complement evasion mechanisms, which include the binding of human complement inhibitors such as C4b-binding protein (C4BP) and Factor H (FH). Inhibiting C4BP and FH acquisition by M. catarrhalis may provide a novel therapeutic avenue to treat infections. To achieve this, we created two chimeric proteins that combined the Moraxella-binding domains of C4BP and FH fused to human immunoglobulin Fcs: C4BP domains 1 and 2 and FH domains 6 and 7 fused to IgM and IgG Fc, respectively. As expected, FH6-7/IgG displaced FH from the bacterial surface while simultaneously activating complement via Fc-C1q interactions, together increasing pathogen elimination. C4BP1-2/IgM also increased serum killing of the bacteria through enhanced complement deposition, but did not displace C4BP from the surface of M. catarrhalis . These Fc fusion proteins could act as anti-infective immunotherapies. Many microbes bind the complement inhibitors C4BP and FH through the same domains as M. catarrhalis , therefore these Fc fusion proteins may be promising candidates as adjunctive therapy against many different drug-resistant pathogens., (Copyright © 2020 Laabei, Colineau, Bettoni, Maziarz, Ermert, Riesbeck, Ram and Blom.)

Treatment of ocular disease is hindered by the presence of the blood-retinal barrier, which restricts access of systemic drugs to the eye. Intravitreal injections bypass this barrier, delivering high concentrations of drug to the targeted tissue. However, the recommended dosing interval for approved biologics is typically 6-12 weeks, and frequent travel to the physician's office poses a substantial burden for elderly patients with poor vision. Real-world data suggest that many patients are under-treated. Here, we investigate IgMs as a novel platform for treating ocular disease. We show that IgMs are well-suited to ocular administration due to moderate viscosity, long ocular exposure, and rapid systemic clearance. The complement-dependent cytotoxicity of IgMs can be readily removed with a P436G mutation, reducing safety liabilities. Furthermore, dodecavalent binding of IgM hexamers can potently activate pathways implicated in the treatment of progressive blindness, including the Tie2 receptor tyrosine kinase signaling pathway for the treatment of diabetic macular edema, or the death receptor 4 tumor necrosis family receptor pathway for the treatment of wet age-related macular degeneration. Collectively, these data demonstrate the promise of IgMs as therapeutic agonists for treating progressive blindness.

Described in several epithelial cancer cells, Tn- (GalNAcα1-O-Ser/Thr) and T- (Galβ3GalNAcα1-O-Ser/Thr) antigens are examples of tumor-associated antigens. Increased expression of Tn- and T-antigens is associated with tumor invasion and metastasis, and patients with high concentration of anti-Tn and anti-T antibodies have a more benign evolution of pathology. Asialofetuin (ASF) and ovine submaxillary mucin (OSM) are two glycoproteins that expose T- and Tn-antigen, respectively. In this work, using ASF or OSM we affinity-purified anti-T and anti-Tn antibodies from normal human plasma and tested their ability to specifically recognize tumor human tissues. Whereas purified anti-T antibodies (purity degree increase of 127-fold, and 22% recovery) were mainly IgG, for purified anti-Tn antibodies (purity degree enhancement of 125-fold, and 26% yield) the IgM fraction was predominant over the IgG one. IgG2 subclass was significantly enriched in both purified antibody samples. Purified antibodies did not bind normal human tissue (0/42), although recognized malignant tissues from different origin such as colon carcinoma (11/77 by anti-Tn; 7/79 by anti-T), breast carcinoma (10/23 by anti-Tn; 7/23 by anti-T), and kidney carcinoma (45/51 by anti-Tn; 42/51 by anti-T). Our results suggest that purified human anti-Tn and anti-T antibodies have a potential as anti-tumor therapeutic agents; restoring their levels in human sera could positively affect the evolution of patients with epithelial tumor pathologies.

Objective: To assess the intraepidermal nerve fiber density in patients diagnosed with fibromyalgia (FM) and to evaluate the role of IgM binding to trisulfated heparin disaccharide (TS-HDS) in these patients., Methods: FM is a poorly understood pain disorder with several proposed pathophysiologic mechanisms. It is characterized by widespread pain, fatigue, and sleep abnormalities. Small fiber neuropathy (SFN) has been proposed as an underlying mechanism, and patients with FM have been shown to have a reduction in the intraepidermal nerve fiber density. An underlying inflammatory process that could be a result of autoimmune phenomena has also been suggested. Non-length-dependent SFN (NLDSFN) has been shown to have a higher incidence of autoimmune disease. Twenty-two patients with established diagnosis of FM underwent skin biopsy at 2 sites; 10 cm above the lateral malleolus and 10 cm above the patella. Serum IgM binding to TS-HDS was assayed using an ELISA method., Results: A total of 5/22 patients had positive TS-HDS antibodies; of these, 4 had NLDSFN (P = 0.0393). Comparison with a control group at Washington University showed no significant difference in percentage with TS-HDS antibodies (P = 0.41). When compared with Washington University database of skin biopsy, there was a trend for an increased percentage of NLDSFN in patients with FM (P = 0.06)., Conclusions: This study further supports the hypothesis that a subgroup of patients with FM has SFN. We suggest a correlation between the presence of NLDSFN and TS-HDS antibodies.

Antibodies are vital components of the adaptive immune system for the recognition and response to foreign antigens. However, some antibodies recognize self-antigens in healthy individuals. These autoreactive antibodies may modulate innate immune functions. IgM natural autoantibodies (IgM-NAAs) are a class of primarily polyreactive immunoglobulins encoded by germline V-gene segments which exhibit low affinity but broad specificity to both foreign and self-antigens. Historically, these autoantibodies were closely associated with autoimmune disease. Nevertheless, not all human autoantibodies are pathogenic and compelling evidence indicates that IgM-NAAs may exert a spectrum of effects from injurious to protective depending upon cellular and molecular context. In this chapter, we review the current state of knowledge regarding the potential physiological and therapeutic roles of IgM-NAAs in different disease conditions such as atherosclerosis, cancer, and autoimmune disease. We also describe the discovery of two reparative IgM-NAAs by our laboratory and delineate their proposed mechanisms of action in central nervous system (CNS) disease.

Objectives: B-cells play a crucial role in the pathogenesis of lupus nephritis. Recently, a separate subset has been discovered characterized by expression of Granzyme B. The aim of this study is to investigate this subset in patients with systemic lupus erythematosus (SLE)., Methods: Isolated PBMCs of SLE-patients (n=30) and healthy controls (n=21) were in vitro stimulated with CPG, IgG+IgM and IL-21. Patients were sub-grouped in patients with and without biopsy proven lupus nephritis. B-cells were analyzed for intracellular Granzyme B expression by flow cytometry., Results: The strongest stimulus for Granzyme B secretion of B-cells was IgG+IgM in presence of IL-21. SLE-patients had a significant decreased percentage of Granzyme B + B-cells in particular SLE-patients with active disease and with lupus nephritis., Conclusions: The frequency of GrB+ producing B-cells is reduced in SLE patients. This may contribute to an imbalanced B-cell regulation towards effector B-cells which might promote the development of lupus nephritis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Heterologous secondary infections are at increased risk of developing dengue hemorrhagic fever (DHF) because of antibody-dependent enhancement (ADE). IgG subclasses can fix and activate complement and bind to Fcɣ receptors. These factors may also play an important role in the development of ADE and thus in the pathogenesis of DHF. The aim of this study was to analyze the indices of anti-dengue IgG subclasses in adult patients with febrile and hemorrhagic dengue in the acute phase. In 2013, 129 patients with dengue fever (DF) and 57 with DHF in Veracruz, Mexico were recruited for this study and anti-dengue IgM and IgG determined by capture ELISA. Anti-dengue IgG subclasses were detected by indirect ELISA. Anti-dengue IgG2 and IgG3 subclasses were detected in patients with dengue. IgG1 increased significantly in the sera of patients with both primary and secondary infections and DHF, but was higher in patients with secondary infections. The IgG4 subclass index was significantly higher in the sera of patients with DHF than in that of those with DF, who were in the early and late acute phase of both primary and secondary infection. In conclusion, indices of subclasses IgG1 and IgG4 were higher in patients with DHF., (© 2017 The Societies and John Wiley & Sons Australia, Ltd.)

Failure of oligodendrocyte precursor cells (OPCs) to differentiate and remyelinate axons is thought to be a major cause of the limited ability of the central nervous system to repair plaques of immune-mediated demyelination in multiple sclerosis (MS). Current therapies for MS aim to lessen the immune response in order to reduce the frequency and severity of attacks, but these existing therapies do not target remyelination or stimulate repair of the damaged tissue. Thus, the promotion of OPC differentiation and remyelination is potentially an important therapeutic goal. Previous studies have shown that a recombinant human-derived monoclonal IgM antibody, designated rHIgM22, promotes remyelination, particularly of the spinal cord in rodent models of demyelination. Here, we examined the effects of rHIgM22 in remyelination in the brain using the mouse model of cuprizone-induced demyelination, which is characterized by spontaneous remyelination. The myelination state of the corpus callosum of cuprizone-fed mice treated with rHIgM22 was examined immediately after the end of the cuprizone diet as well as at different time points during the recovery period with regular food, and compared with that of cuprizone-fed animals treated with either vehicle or human IgM isotype control antibody. Mice fed only regular food were used as controls. We demonstrate that treatment with rHIgM22 accelerated remyelination of the demyelinated corpus callosum. The remyelination-enhancing effects of rHIgM22 were found across different, anatomically distinct regions of the corpus callosum, and followed a spatiotemporal pattern that was similar to that of the spontaneous remyelination process. These enhancing effects were also accompanied by increased differentiation of OPCs into mature oligodendrocytes. Our data indicate strong remyelination-promoting capabilities of rHIgM22 and further support its therapeutic potential in MS., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

IgM is the first antibody to be produced in immune responses and plays an important role in the neutralization of bacteria and viruses. Human IgM is heavily glycosylated, featuring five N-linked glycan sites on the μ chain and one on the J-chain. Glycosylation of IgG is known to modulate the effector functions of Fcγ receptors. In contrast, little is known about the effect of glycosylation on IgM binding to the human Fcμ receptor (hFCMR). In this study, we identify the Cμ4 domain of IgM as the target of hFCMR, and show that binding and internalization of IgM by hFCMR is glycan-independent. We generated a homology-based structure for hFCMR and used molecular dynamic simulations to show how this interaction with IgM may occur. Finally, we reveal an inhibitory function for IgM in the proliferation of T cells.

A single peripheral dose of CNS-binding IgMs promote remyelination and preserve axons in a number of animal models of neurologic disease. A myelin-binding recombinant human IgM (rHIgM22) is presently in a safety trial in MS patients following an acute MS exacerbation. rHIgM22 (directed against oligodendrocytes) or rHIgM12 (directed against neurons) were administered to mice with MOG-induced experimental autoimmune encephalomyelitis (EAE) with study endpoints: clinical deficits and brain and spinal cord pathology. IgMs were administered at a therapeutic dose of 100 μ g intra peritoneal at the time of immunization (day -1, 0, +$1), disease onset (15 days) or peak of the disease (28 days). Disease course was not worsened by either human IgM regardless of the time of treatment. Of note, the human IgM that recognizes a carbohydrate epitope on gangliosides and NCAM, rHIgM12, reduced brain pathology when given at time of immunization or at onset of disease, but did not reduce clinical deficits or spinal cord disease burden. Hence, treatment with rHIgM12 resulted in marked reduction in meningeal inflammation. Data consistent with the hypothesis that in the EAE model this molecule has an immune-modulatory effect. Treatment with an anti-CD4 blocking IgG prevented both clinical course and CNS pathology. This pre-clinical study further supports the safety of therapeutic CNS-binding human IgMs in the presence of autoimmunity and clearly differentiates them from IgGs directed against MOG or aquaporin-4 that worsen neurologic disease.

Objective: In vitro studies suggest that the type I interferon (IFN) signature seen in most lupus patients results from Fcγ receptor-mediated uptake of nucleic acid-containing immune complexes by plasmacytoid dendritic cells and engagement of endosomal Toll-like receptors. The aim of this study was to reexamine the pathogenesis of the IFN signature in vivo., Methods: Lupus was induced in mice by injecting pristane. Some mice were treated with normal immunoglobulin or with cobra venom factor to deplete complement. The IFN signature was evaluated by polymerase chain reaction. The IFN signature also was determined in C4-deficient patients and control subjects., Results: Wild-type C57BL/6 mice with pristane-induced lupus developed a strong IFN signature, which was absent in immunoglobulin-deficient (μMT), C3 -/- , and CD18 -/- mice. Intravenous infusion of normal IgM, but not IgG, restored the IFN signature in μMT mice, and the IFN signature in wild-type mice was inhibited by depleting complement, suggesting that opsonization by IgM and complement is involved in IFN production. Consistent with that possibility, the levels of "natural" IgM antibodies reactive with dead cells were increased in pristane-treated wild-type mice compared with untreated controls, and in vivo phagocytosis of dead cells was impaired in C3-deficient mice. To examine the clinical relevance of these findings, we identified 10 C4-deficient patients with lupus-like disease and compared them with 152 C4-intact patients and 21 healthy controls. In comparison with C4-intact patients, C4-deficient patients had a different clinical/serologic phenotype and lacked the IFN signature., Conclusion: These studies define previously unrecognized roles of natural IgM, complement, and complement receptors in generating the IFN signature in lupus., Competing Interests: Disclosures: The authors declare no competing financial interests, (© 2016, American College of Rheumatology.)

Intravenous immunoglobulin (IVIG), consisting of IgG, is the first-line treatment for Guillain-Barré syndrome and multifocal motor neuropathy. IgG, but neither IgM nor IgA, has been demonstrated in vitro to inhibit complement deposition mediated by anti-ganglioside autoantibodies in sera from patients with both conditions. The objective of this study is to investigate the in vitro effectiveness of IgM and IgA in inhibiting complement deposition to ganglioside/anti-ganglioside antibody complexes. Serum samples were obtained from patients with multifocal motor neuropathy associated with anti-GM1 IgM antibodies, Guillain-Barré syndrome associated with anti-GM1 IgG antibodies and Miller Fisher syndrome associated with anti-GQ1b IgG antibodies. Inhibition of complement deposition using different immunoglobulin preparations was measured by enzyme-linked immunosorbent assay. IgM/A-enriched IVIG and immunoglobulin isotypes (polyclonal IgM and IgA) showed higher potential in inhibiting complement deposition than standard IVIG. Although the safety concerns about the use of IgM and IgA for an immunotherapy still remain, IgM and IgA may serve as an alternative immunotherapy in those anti-ganglioside antibody-mediated neuropathies., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Introduction: Sepsis remains associated with a high mortality rate. Endotoxin has been shown to influence viscoelastic coagulation parameters, thus suggesting a link between endotoxin levels and the altered coagulation phenotype in septic patients. This study evaluated the effects of systemic polyspecific IgM-enriched immunoglobulin (IgM-IVIg) (Pentaglobin® [Biotest, Dreieich, Germany]) on endotoxin activity (EA), inflammatory markers, viscoelastic and conventional coagulation parameters., Methods: Patients with severe sepsis were identified by daily screening in a tertiary, academic, surgical ICU. After the inclusion of 15 patients, the application of IgM-IVIg (5 mg/kg/d over three days) was integrated into the unit's standard operation procedure (SOP) to treat patients with severe sepsis, thereby generating "control" and "IgM-IVIg" groups. EA assays, thrombelastometry (ROTEM®) and impedance aggregometry (Multiplate®) were performed on whole blood. Furthermore, routine laboratory parameters were determined according to unit's standards., Results: Data from 26 patients were included. On day 1, EA was significantly decreased in the IgM-IVIg group following 6 and 12 hours of treatment (0.51 ±0.06 vs. 0.26 ±0.07, p<0.05 and 0.51 ±0.06 vs. 0.25 ±0.04, p<0.05) and differed significantly compared with the control group following 6 hours of treatment (0.26 ±0.07 vs. 0.43 ±0.07, p<0.05). The platelet count was significantly higher in the IgM-IVIg group following four days of IgM-IVIg treatment (200/nl ±43 vs. 87/nl ±20, p<0.05). The fibrinogen concentration was significantly lower in the control group on day 2 (311 mg/dl ±37 vs. 475 mg/dl ±47 (p = 0.015)) and day 4 (307 mg/dl ±35 vs. 420 mg/dl ±16 (p = 0.017)). No differences in thrombelastometric or aggregometric measurements, or inflammatory markers (interleukin-6 (IL-6), leukocyte, lipopolysaccharide binding protein (LBP)) were observed., Conclusion: Treatment with IgM-enriched immunoglobulin attenuates the EA levels in patients with severe sepsis and might have an effect on septic thrombocytopenia and fibrinogen depletion. Viscoelastic, aggregometric or inflammatory parameters were not influenced., Trial Registration: clinicaltrials.gov NCT02444871.

Background: Whereas demyelination underlies early neurological symptoms in multiple sclerosis (MS), axonal damage is considered critical for permanent chronic deficits. Intracerebral infection of susceptible mouse strains with Theiler's murine encephalomyelitis virus (TMEV) results in chronic induced demyelinating disease (TMEV-IDD) with progressive axonal loss and neurologic dysfunction similar to progressive forms of MS. We previously reported that treatment of chronic TMEV-IDD mice with a neurite outgrowth-promoting natural human antibody, HIgM12, improved brainstem NAA concentrations and preserved functional motor activity. In order to translate this antibody toward clinical trial, we generated a fully human recombinant form of HIgM12, rHIgM12, determined the optimal in vivo dose for functional improvement in TMEV-IDD, and evaluated the functional preservation of descending spinal cord axons by retrograde labeling., Findings: SJL/J mice at 45 to 90 days post infection (dpi) were studied. A single intraperitoneal dose of 0.25 mg/kg of rHIgM12 per mouse is sufficient to preserve motor function in TMEV-IDD. The optimal dose was 10 mg/kg. rHIgM12 treatment protected the functional transport in spinal cord axons and led to 40 % more Fluoro-Gold-labeled brainstem neurons in retrograde transport studies. This suggests that axons are not only present but also functionally competent. rHIgM12-treated mice also contained more mid-thoracic (T6) spinal cord axons than controls., Conclusions: This study confirms that a fully human recombinant neurite outgrowth-promoting monoclonal IgM is therapeutic in a model of progressive MS using multiple reparative readouts. The minimum effective dose is similar to that of a remyelination-promoting monoclonal human IgM discovered by our group that is presently in clinical trials for MS.

Immunoglobulins M (IgMs) are gaining increasing attention as biopharmaceuticals since their multivalent mode of binding can give rise to high avidity. Furthermore, IgMs are potent activators of the complement system. However, they are frequently difficult to express recombinantly and can suffer from low conformational stability. Here, the broadly neutralizing anti-HIV-1 antibody 2G12 was class-switched to IgM and then further engineered by introduction of 17 germline residues. The impact of these changes on the structure and conformational stability of the antibody was then assessed using a range of biophysical techniques. We also investigated the effects of the class switch and germline substitutions on the ligand-binding properties of 2G12 and its capacity for HIV-1 neutralization. Our results demonstrate that the introduced germline residues improve the conformational and thermal stability of 2G12-IgM without altering its overall shape and ligand-binding properties. Interestingly, the engineered protein was found to exhibit much lower neutralization potency than its wild-type counterpart, indicating that potent antigen recognition is not solely responsible for IgM-mediated HIV-1 inactivation., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: Anticitrullinated protein antibodies (ACPA) are specifically associated with rheumatoid arthritis (RA) and produced in inflamed synovial membranes where citrullinated fibrin, their antigenic target, is abundant. We showed that immune complexes containing IgG ACPA (ACPA-IC) induce FcγR-mediated tumour necrosis factor (TNF)-α secretion in macrophages. Since IgM rheumatoid factor (RF), an autoantibody directed to the Fc fragment of IgG, is also produced and concentrated in the rheumatoid synovial tissue, we evaluated its influence on macrophage stimulation by ACPA-IC., Methods: With monocyte-derived macrophages from more than 40 healthy individuals and different human IgM cryoglobulins with RF activity, using a previously developed human in vitro model, we evaluated the effect of the incorporation of IgM RF into ACPA-IC., Results: IgM RF induced an important amplification of the TNF-α secretion. This effect was not observed in monocytes and depended on an increase in the number of IgG-engaged FcγR. It extended to the secretion of interleukin (IL)-1β and IL-6, was paralleled by IL-8 secretion and was not associated with overwhelming secretion of IL-10 or IL-1Ra. Moreover, the RF-induced increased proinflammatory bioactivity of the cytokine response to ACPA-IC was confirmed by an enhanced, not entirely TNF-dependent, capacity of the secreted cytokine cocktail to prompt IL-6 secretion by RA synoviocytes., Conclusions: By showing that it can greatly enhance the proinflammatory cytokine response induced in macrophages by the RA-specific ACPA-IC, these results highlight a previously undescribed, FcγR-dependent strong proinflammatory potential of IgM RF. They clarify the pathophysiological link between the presence of ACPA and IgM RF, and RA severity., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Rheumatoid factors (RF) and the disease-specific anti-citrullinated protein autoantibodies (ACPA) coexist in the joints of rheumatoid arthritis (RA) patients where they probably contribute to synovitis. We investigated the influence of IgM and IgA RF on the FcR- and complement-dependent effects of ACPA immune complexes (ACPA-IC). When stimulated by ACPA-IC formed in the presence of IgM RF or IgA RF fractions purified from RA serum pools, M-CSF-generated macrophages skewed their cytokine response toward inflammation, with increases in the TNF-α/IL-10 ratio and in IL-6 and IL-8 secretion, and decreases in the IL-1Ra/IL-1β ratio. In the IgM RF-mediated amplification of the inflammatory response of macrophages, the participation of an IgM receptor was excluded, notably by showing that they did not express any established receptor for IgM. Rather, this amplification depended on the IgM RF-mediated recruitment of more IgG into the ACPA-IC. However, the macrophages expressed FcαRI and blocking its interaction with IgA inhibited the IgA RF-mediated amplification of TNF-α secretion induced by ACPA-IC, showing its major implication in the effects of RF of the IgA class. LPS further amplified the TNF-α response of macrophages to RF-containing ACPA-IC. Lastly, the presence of IgM or IgA RF increased the capacity of ACPA-IC to activate the complement cascade. Therefore, specifically using autoantibodies from RA patients, the strong FcR-mediated or complement-dependent pathogenic potential of IC including both ACPA and IgM or IgA RF was established. Simultaneous FcR triggering by these RF-containing ACPA-IC and TLR4 ligation possibly makes a major contribution to RA synovitis., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Purpose: We compared different immunoglobulin preparations containing IgG (Intraglobin/Intratect) or a mixture of IgG, IgA, and IgM (Pentaglobin) to assess the opsonic and protective efficacy of human immunoglobulin preparations against multiresistent nosocomial pathogens., Materials and Methods: Clinical isolates of E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecalis, Enterococcus faecium, and Staphylococcus aureus were tested by opsonophagocytic assay using immunologobulin preparations at dilutions usually obtained in patients. The target antigens of opsonic antibodies were characterized by opsonophagocytic inhibition assays, and the protective efficacy in vivo was tested in a mouse bacteremia model as previously described., Results: All strains were killed to at least 50% by Pentaglobin. One P. aeruginosa strain was not efficiently killed by Intraglobin (23%) but the other strains were killed by Intraglobin to a similar degree compared to Pentaglobin. Opsonic IgG antibodies against E. faecalis were directed against LTA, while opsonic antibodies in Pentaglobin were primarily directed against other cell wall carbohydrates. In a mouse bacteremia model, Pentaglobin was more protective than Intratect against Staphylococcus aureus, while Intratect reduced colony counts better than normal rabbit serum or saline., Conclusions: All tested human immunoglobulin preparations contain opsonic and protective antibodies against targets present on multiresistant Gram-positive and Gram-negative bacteria. Enrichment of these preparations with IgM increases the protective efficacy against some strains, probably due to antibodies directed against cell wall carbohydrates.

Haemophilic arthropathy (HA) is characterized by chronic proliferative synovitis leading to cartilage destruction and shares some pathological features with rheumatoid arthritis (RA). Apoptosis has been implicated in RA pathogenesis, and an agonistic anti-Fas monoclonal antibody (mAb) was found to induce RA fibroblast-like synoviocyte (FLS) apoptosis and suppress synovial hyperplasia in animal models of RA. The aim of this study was to evaluate the effect of anti-Fas mAb on HA-FLS. FLS were isolated from knee synovial biopsies from six HA patients, six RA patients and six healthy subjects. The expression of Fas in synovial biopsies was investigated by immunohistochemistry. FLS were stimulated with anti-Fas mAb at different concentrations, alone or in combination with tumour necrosis factor-α (TNF-α) and basic fibroblast growth factor (bFGF). Fas expression in FLS was assessed by Western blot. Cell viability was studied with the WST-1 assay. Active caspase-3 levels were measured using ELISA and Western blot. A strong Fas-immunoreactivity was observed in different cells of HA synovium, including FLS, inflammatory cells and endothelial cells. Fas antigen was constitutively overexpressed in cultured HA-FLS. Anti-Fas mAb had a significant cytotoxicity on HA-FLS in a dose-dependent manner, either alone or in combination with TNF-α and bFGF. These cytotoxic effects were due to the ability of anti-Fas to induce HA-FLS apoptosis, as shown by the increased active caspase-3 levels. Anti-Fas mAb exhibited a more pronounced pro-apoptotic effect on HA-FLS than RA-FLS. Fas antigen is highly expressed on HA-FLS and its stimulation by anti-Fas mAb may be an effective strategy to induce HA-FLS apoptosis., (© 2013 John Wiley & Sons Ltd.)

N-acetyl-d-glucosamine (GlcNAc) is one of the components of peptidoglycan, a biopolymer in the bacterial cell wall. We purified a novel GlcNAc-binding protein, designated as fGBP-78, from sera of fugu (Takifugu rubripes). The fGBP-78 is a heteromer of 78- and 25-kDa subunits. Moreover, fGBP-78 exerted remarkable inhibitory effects on the growth of both Gram-positive and Gram-negative bacteria, including ones virulent for marine fish species as well as non-pathogenic Escherichia coli. These results suggest that fGBP-78 contributes to bacterial clearance in fugu. Furthermore, the nanoLC-MS/MS and Western blotting analyses reveal that the 78-kDa subunit is the fugu IgM heavy chain. In addition, the molecular mass of the other subunit (25 kDa) was equal to that of the Ig light chain. Overall, results indicate that fGBP-78 is an IgM molecule presumably acts as a natural antibody. This paper reports a novel function of teleost IgM as a significant suppresser against bacterial growth., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Autoimmune encephalitis associated with IgG antibodies to the N-methyl-d-aspartic acid receptor subunit NR1 (NMDAR) presents with neurological symptoms, such as seizures, and especially psychiatric symptoms, such as hallucinations, psychosis, agitation and anxiety. To date, however, the pathological relevance of IgM NMDAR antibodies remains elusive. Here, we describe clinical, neuroradiological and neurobiological findings of a 28-year-old male presenting with IgM NMDAR antibodies coincident with autoimmune encephalitis characterized by symptoms of bipolar disorder. After repeated steroid treatment, cognitive and psychiatric abnormalities improved and no NMDAR antibody was detectable. Using primary neuronal cultures, we demonstrate that patient's serum containing IgM NMDAR antibodies reduced the detection of NMDAR on neuronal cells and decreased cell survival. Although NMDAR encephalitis with IgG antibodies is increasingly recognized and diagnosed, atypical presentations with NMDAR antibodies with immunoglobulin subclasses other than IgG pose a diagnostic and therapeutic challenge. Further clinical and neurobiological studies are needed to study the pathophysiological relevance of IgM NMDAR antibodies., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

In contrast to other haematological malignancies, targeted immunotherapy has not entered standard treatment regimens for de novo or relapsed multiple myeloma (MM) yet. While a number of IgG-formatted monoclonal antibodies are currently being evaluated in clinical trials in MM, our study aimed to investigate whether the fully human IgM monoclonal antibody PAT-SM6 that targets a tumour-specific variant of the heat shock protein GRP78 might be an attractive candidate for future immunotherapeutic approaches. We here show that GRP78 is stably and consistently expressed on the surface on tumour cells from patients with de novo, but also relapsed MM and that binding of PAT-SM6 to MM cells can specifically exert cytotoxic effects on malignant plasma cells, whereas non-malignant cells are not targeted. We demonstrate that the induction of apoptosis and, to a lesser extent, complement dependent cytotoxicity is the main mode of action of PAT-SM6, whereas antibody dependent cellular cytotoxicity does not appear to contribute to the cytotoxic properties of this antibody. Given the favourable safety profile of PAT-SM6 in monkeys, but also in a recent phase I trial in patients with malignant melanoma, our results form the basis for a planned phase I study in patients with relapsed MM.

Evidence suggests that adjunctive treatment with intravenous immunoglobulin preparations enriched with IgA and IgM reduce mortality in sepsis. The mode of action of polyvalent immunoglobulin is complex, including neutralization of toxins and modulation of complement activation and cytokine formation toward an anti-inflammatory profile. In this study we explored the effect of Pentaglobin, containing IgG, IgA and IgM, on the initial inflammatory reaction as well as on hemodynamics, using a well characterized and standardized porcine model of sepsis. Anesthetized and mechanically ventilated pigs, mean weight 14.9 kg, were allocated into two groups of 8 animals, receiving either Pentaglobin or saline, before sepsis was induced by intravenous Escherichia coli infusion. Five negative controls received saline only. All animals were observed for 4 h under extensive invasive monitoring. Pentaglobin significantly (p < 0.05) attenuated IL-1β formation by 38% at the end of the experiment, and markedly increased (p < 0.05) the formation of IL-10 at 60 min. TNF-α, IL-6, IL-8 and expression of the cell surface marker wCD11R3 were lower in the Pentaglobin group, but the differences were not significant. The serum concentration of LPS was three times higher in the Pentaglobin group (p < 0.005), indicating binding of LPS to Pentaglobin. Complementary in vitro experiments showed a higher binding affinity for IgM and IgA to LPS than for IgG. LPS-induced formation of IL-6 was significantly (p < 0.05) attenuated by Pentaglobin in an in vitro whole blood model. In conclusion, Pentaglobin decreased the key inflammasome IL-1β molecule in an E. coli-model of pigs sepsis., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Matrix molecules convey biochemical and physical guiding signals to neurons in the central nervous system (CNS) and shape the trajectory of neuronal fibers that constitute neural networks. We have developed recombinant human IgMs that bind to epitopes on neural cells, with the aim of treating neurological diseases. Here we test the hypothesis that recombinant human IgMs (rHIgM) can guide neurite outgrowth of CNS neurons. Microcontact printing was employed to pattern rHIgM12 and rHIgM22, antibodies that were bioengineered to have variable regions capable of binding to neurons or oligodendrocytes, respectively. rHIgM12 promoted neuronal attachment and guided outgrowth of neurites from hippocampal neurons. Processes from spinal neurons followed grid patterns of rHIgM12 and formed a physical network. Comparison between rHIgM12 and rHIgM22 suggested the biochemistry that facilitates anchoring the neuronal surfaces is a prerequisite for the function of IgM, and spatial properties cooperate in guiding the assembly of neuronal networks.

Background: Promotion of remyelination is a major goal in treating demyelinating diseases such as multiple sclerosis (MS). The recombinant human monoclonal IgM, rHIgM22, targets myelin and oligodendrocytes (OLs) and promotes remyelination in animal models of MS. It is unclear whether rHIgM22-mediated stimulation of lesion repair is due to promotion of oligodendrocyte progenitor cell (OPC) proliferation and survival, OPC differentiation into myelinating OLs or protection of mature OLs. It is also unknown whether astrocytes or microglia play a functional role in IgM-mediated lesion repair., Methods: We assessed the effect of rHIgM22 on cell proliferation in mixed CNS glial and OPC cultures by tritiated-thymidine uptake and by double-label immunocytochemistry using the proliferation marker, Ki-67. Antibody-mediated signaling events, OPC differentiation and OPC survival were investigated and quantified by Western blots., Results: rHIgM22 stimulates OPC proliferation in mixed glial cultures but not in purified OPCs. There is no proliferative response in astrocytes or microglia. rHIgM22 activates PDGFαR in OPCs in mixed glial cultures. Blocking PDGFR-kinase inhibits rHIgM22-mediated OPC proliferation in mixed glia. We confirm in isolated OPCs that rHIgM22-mediated anti-apoptotic signaling and inhibition of OPC differentiation requires PDGF and FGF-2. We observed no IgM-mediated effect in mature OLs in the absence of PDGF and FGF-2., Conclusion: Stimulation of OPC proliferation by rHIgM22 depends on co-stimulatory astrocytic and/or microglial factors. We demonstrate that rHIgM22-mediated activation of PDGFαR is required for stimulation of OPC proliferation. We propose that rHIgM22 lowers the PDGF threshold required for OPC proliferation and protection, which can result in remyelination of CNS lesions.

We have discovered a role for natural autoantibodies in central nervous system repair, remyelination and axon protection. These natural human antibodies are of the immunoglobulin M (IgM) isotype, and they bind to the surface of neural cells. The epitope of the antibody includes sialic acid because treatment with sialidase disrupts the binding. A fully human recombinant form of one of these IgMs, rHIgM12, has the same properties as the serum-derived IgM. rHIgM12 enhanced polarized axonal outgrowth from primary neurons when presented as a substrate in vitro and improved motor functions in chronically Theiler's virus-infected SJL mice, a model of MS. rHIgM12 bound to neuronal surfaces and induced cholesterol and ganglioside (GM1) clustering, indicating that rHIgM12 functions through a mechanism of axonal membrane stabilization. Our work demonstrates that a natural human neuron-binding IgM can regulate membrane domain dynamics. This antibody has the potential to improve neurologic disease.

Group A beta-hemolytic streptococcus (GABHS) infections are implicated in neuropsychiatric disorders associated with an increased expression of repetitive stereotyped movements. Anti-streptococcus IgG presumably cross-reacts with elements on basal ganglia cells, modifies their function, and triggers symptoms. IgM may play a unique role in precipitating behavioral disturbances since variations in cortico-striatal activity occur in temporal congruity with peak IgM titers during an orchestrated immune response. We discovered in Balb/c mice that single subcutaneous injections of mouse monoclonal IgM antibodies to streptococcus group A bacteria induce marked dose-dependent increases in repetitive stereotyped movements, including head bobbing, sniffing, and intense grooming. Effects were antibody- and antigen-specific: anti-streptococcus IgG stimulated ambulatory activity and vertical activity but not these stereotypies, while anti-KLH IgM reduced activity. We suggest that anti-streptococcus IgM and IgG play unique roles in provoking GABHS-related behavioral disturbances. Paralleling its stereotypy-inducing effects, anti-streptococcus IgM stimulated Fos-like immunoreactivity in regions linked to cortico-striatal projections involved in motor control, including subregions of the caudate, nucleus accumbens, and motor cortex. This is the first evidence that anti-streptococcus IgM antibodies induce in vivo functional changes in these structures. Moreover, there was a striking similarity in the distributions of anti-streptococcus IgM deposits and Fos-like immunoreactivity in these regions. Of further importance, Fcα/μ receptors, which bind IgM, were present- and co-localized with anti-streptococcus IgM in these structures. We suggest that anti-streptococcus IgM-induced alterations of cell activity reflect local actions of IgM that involve Fcα/μ receptors. These findings support the use of anti-streptococcus monoclonal antibody administration in Balb/c mice to model GABHS-related behavioral disturbances and identify underlying mechanisms., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Objective: Gamma-globulin treatment reduces experimental atherosclerosis by modulating immune function; however the effect of IgM on atherosclerosis is not known. We investigated the effect of serum-derived, non-immune polyclonal IgM (Poly-IgM) on atherosclerosis in mice with advanced disease and also assessed its immune-modulatory effects., Methods and Results: Aortic atherosclerosis was assessed in apoE-/- mice fed atherogenic diet starting at 6 weeks of age. In addition, mice were also subjected to perivascular cuff injury to the carotid artery at 25 weeks of age to induce accelerated atherosclerosis. At the time of injury, the mice were treated weekly with a commercially available Poly-IgM (0.4mg/mouse) or PBS for 4 weeks and euthanized at 29 weeks of age. Poly-IgM reduced aortic atherosclerosis, and reduced lesion size in the aortic sinus and injured carotid artery, without significant changes in serum cholesterol levels. Poly-IgM treatment was associated with increased anti-oxLDL IgG titers and a reduction in the % splenic CD4(+) T cells compared to controls. The splenic CD4(+) T cell cultured from the Poly-IgM treated mice had reduced proliferation in vitro compared with controls., Conclusion: Poly-IgM treatment reduced aortic and accelerated carotid atherosclerosis in apoE-/- mice in association with increased anti-oxLDL IgG titers, and reduced number and proliferative function of splenic CD4(+) T cells. Our study identifies a novel athero-protective and immunomodulatory role for non-immune polyclonal IgM., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Autoimmune thrombocytopenic purpura (AITP) is an acquired autoimmune bleeding disorder, characterized by isolated thrombocytopenia because of destruction of auto-antibody-coated platelets by Fc-receptor-mediated phagocytosis. The destruction of autoantibody-sensitized platelets by FcγR-bearing phagocytic cells and the following antigen presentation are considered to play a key role for the pathophysiology of AITP. Although different isotypes of AITP-mediating autoantibodies, e.g. IgG, IgM and IgA, are frequently found in AITP patients, their role in the pathophysiology of AITP remains unclear. Using a flow cytometric monocyte-based phagocytosis assay, we investigated the impact of disease-associated autoantibody isotype in antibody-mediated phagocytosis of platelets. Platelets, labelled with 5-chloromethyl fluorescein diacetate (CMFDA), were incubated with AITP patients' serum characterized by pure IgG or IgM antiplatelet autoantibodies. Labelled platelets were incubated with monocytes. Phagocytosis was defined as the product of percentage of CMFDA-positive monocytes and mean fluorescence intensity of CMFDA. Adherence of platelets to monocytes was quantified by anti-CD61-PerCp in a CMFDA(+) CD14(+) gate. IgG-coated platelets showed a significantly higher phagocytic index than IgM-coated platelets (mean 796 ± 157 versus 539 ± 78, P < 0.01). There were no significant differences regarding platelet adherence to monocytes. The isotype of autoantibodies influences the quantity of in vitro phagocytosis of autologous platelets by monocytes. Therefore, the AITP-mediating autoantibody isotype should be considered more carefully in pathophysiologic models and furthermore in diagnostic, therapeutic and prognostic approaches in AITP., (© 2011 The Authors. Scandinavian Journal of Immunology © 2011 Blackwell Publishing Ltd.)

The inhibition of vaccination by maternal antibodies is a widely observed phenomenon in human and veterinary medicine. Maternal antibodies are known to suppress the B-cell response. This is similar to antibody feedback mechanism studies where passively transferred antibody inhibits the B-cell response against particulate antigens because of epitope masking. In the absence of experimental data addressing the mechanism underlying inhibition by maternal antibodies, it has been suggested that epitope masking explains the inhibition by maternal antibodies, too. Here we report that in the cotton rat model of measles virus (MV) vaccination passively transferred MV-specific immunoglobulin G inhibit B-cell responses through cross-linking of the B-cell receptor with FcγRIIB. The extent of inhibition increases with the number of antibodies engaging FcγRIIB and depends on the Fc region of antibody and its isotype. This inhibition can be partially overcome by injection of MV-specific monoclonal IgM antibody. IgM stimulates the B-cell directly through cross-linking the B-cell receptor via complement protein 3d and antigen to the complement receptor 2 signaling complex. These data demonstrate that maternal antibodies inhibit B-cell responses by interaction with the inhibitory/regulatory FcγRIIB receptor and not through epitope masking.

Objective: We previously generated MRL/lpr mice deficient in activation-induced deaminase (AID) that lack isotype switching and immunoglobulin hypermutation. These mice have high levels of unmutated (germline) autoreactive IgM, yet they experienced an increase in survival and an improvement in lupus nephritis that exceeded that of MRL/lpr mice lacking IgG. The purpose of the present study was to test the hypothesis that high levels of germline autoreactive IgM in these mice confer protection against lupus nephritis., Methods: Autoreactive IgM antibodies of various specificities, including antibodies against double-stranded DNA (dsDNA), from AID-deficient MRL/lpr mice were given to asymptomatic MRL/lpr mice, and the levels of cytokines, proteinuria, immune complex deposition in the kidneys, and glomerulonephritis were examined. Novel AID-deficient MRL/lpr mice that lack any antibodies were generated for comparison to AID-deficient MRL/lpr mice that secrete only IgM., Results: Treatment with IgM anti-dsDNA resulted in a dramatic improvement in lupus nephritis. Other autoreactive IgM antibodies, such as antiphospholipid and anti-Sm, did not alter the pathologic changes. Secretion of proinflammatory cytokines by macrophages and the levels of inflammatory cells and apoptotic debris in the kidneys were lower in mice receiving IgM anti-dsDNA. Protective IgM derived from AID-deficient MRL/lpr mice displayed a distinct B cell repertoire, with a bias toward members of the V(H) 7183 family., Conclusion: IgM anti-dsDNA protected MRL/lpr mice from lupus nephritis, likely by stopping the inflammatory cascade leading to kidney damage. A distinct repertoire of V(H) usage in IgM anti-dsDNA hybridomas from AID-deficient mice suggests that there is enrichment of a dedicated B cell population that secretes unmutated protective IgM in these mice., (Copyright © 2011 by the American College of Rheumatology.)

We recently reported that IgM MAb B6.1, specific for β-1, 2-mannotriose on the cell wall of Candida albicans, is therapeutic to disseminated candidiasis due to C. albicans. In the current study, we examined if MAbB6.1 enhances therapeutic effect of fluconazole (FLC) to the disseminated disease. To assess the combination effect, determination by the kidneys-colony forming unit and survival times were used. Results showed that the therapeutic effect of FLC on mice with disseminated candidiasis was dose-dependent, but a FLC dose at 0.8 mg/kg body weight of mice was ineffective. To determine combination effect, mice treated intraperitoneally with a combination of FLC plus MAb B6.1 at 1 h post-infection - a condition of developing partial therapeutic activity - enhanced survival times beyond the effect by only antibody (p < 0.05). The resulting MST (mean survival times) value from the combination-received mice was almost the same as MST value from 3.2 mg FLC dose-given animals (p < 0.05). Another combination of 1.6 mg FLC dose and B6.1 reduced severity of the disseminated disease at almost the same rate as combination efficacy of 0.8 mg FLC dose plus B6.1. This data indicates that B6.1 acts in concert with FLC and that this combination therapy augments protection, which suggests a possibility of reducing FLC dose. The augmentation response was specific because an irrelevant IgM MAb S9 was not effective to the disseminated disease. Thus, our present studies demonstrate that this combination immunotherapy may be a way of solving the problem of limited antifungal drug choices caused by drug-resistant C. albicans.

Background: CD20 is a cell surface protein exclusively expressed on B cells. It is a clinically validated target for Non-Hodgkin's lymphomas (NHL) and autoimmune diseases. The B cell receptor (BCR) plays an important role for development and proliferation of pre-B and B cells. Physical interaction of CD20 with BCR and components of the BCR signaling cascade has been reported but the consequences are not fully understood., Methodology: In this study we employed antibodies against CD20 and against the BCR to trigger the respective signaling. These antibodies induced very similar expression patterns of up- and down-regulated genes in NHL cell lines indicating that CD20 may play a role in BCR signaling and vice versa. Two of the genes that were rapidly and transiently induced by both stimuli are CCL3 and CCL4. 4 hours after stimulation the concentration of these chemokines in culture medium reaches a maximum. Spleen tyrosine kinase Syk is a cytoplasmic tyrosine kinase and a key component of BCR signaling. Both siRNA mediated silencing of Syk and inhibition by selective small molecule inhibitors impaired CCL3/CCL4 protein induction after treatment with either anti-CD20 or anti-BCR antibodies., Conclusion: Our results suggest that treatment with anti-CD20 antibodies triggers at least partially a BCR activation-like response in NHL cell lines.

We previously reported that the IgM MAb B6.1, specific for β-1,2-mannotriose on the surface of the cell wall of Candida albicans, prevents mice from disseminated candidiasis. The preventive activity of the antibody is mediated by enhanced phagocytosis that caused killing of the fungus and involvement of the complement system. In the present study, MAb B6.1 was tested if the antibody enhances therapeutic efficacy of amphotericin B (Amp B) in a murine model of disseminated candidiasis due to C. albicans. Determination by the kidneys-cfu (colony forming unit) and survival times was used to assess treatment. Mice treated intraperitoneally with MAb B6.1 at 1h post-infection with C. albicans (5 x 10⁵ yeast cells/mouse) developed fewer 28%cfu and prolonged survival rates than negative controls, whereas administration of B6.1 to mice at 2h post-infection was ineffective. Therapeutic effect of Amp B on mice with the disseminated disease was dose-dependent, but dosage of Amp B (0.5mg/kg body weight of mice) was not effective. A combination of MAb B6.1 given at 1h post-infection plus Amp B (0.5mg dose) enhanced survival times beyond the effect due to only antibody (P<0.05). The MST (mean survival times) value resulted from the combination therapy-received mice was as almost the same as MST value from 2mg dose of Amp B-given animals (P<0.05). In case mice given a combination of Amp B (0.5mg dose) plus MAb B6.1 at 2h post-infection - a condition of developing no therapeutic efficacy, the combination also reduced disease severity. All these data indicate that MAb B6.1 acts in concert with Amp B, the antibody enhances therapeutic efficacy of Amp B, and this combination therapy augments protection which implies a possibility of reducing Amp B dose. The augmentation of the response appeared to be specific because an irrelevant IgM carbohydrate-specific MAb was not protective. In conclusion, these studies show that Amp B combined with MAb B6.1 may be an option of solving the problem of limited antifungal drug choices due to drug-resistant C. albicans., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Purpose: Human remyelination promoting IgM mAbs target oligodendrocytes (OLs) and function in animal models of multiple sclerosis (MS). However, their mechanism of action is unknown. This study seeks to identify the cellular mechanism of action of a recombinant human IgM on OL survival., Methods: Binding of rHIgM22 to the surface of rat OLs was studied by co-localization with various markers. RHIgM22-mediated effects on apoptotic signaling in OLs, differentiation markers, and signaling molecules were detected by Western blotting and immunoprecipitation., Results: RHIgM22 co-localized with integrin β3 but not other integrin β-chains in OLs. Downstream of integrin β3 we identified Src family kinase (SFK) Lyn as a key player of rHIgM22-mediated actions in OLs. Lyn immunoprecipitated in a complex together with integrin αvβ3 and PDGFαR. Lyn expression was 9-fold up-regulated and Lyn activation was 3-fold higher inrHIgM22-treated OL cultures compared with controls. RHIgM22 inhibited apoptotic signaling by greater than 10-fold reduction of caspase-3 and capsase-9 cleavage and reduced by 4-fold expression of differentiation markers MBP and MOG in OLs. SFK inhibitors PP2 and SU6656 inhibited Lyn activity and restored caspase-cleavage in OLs. A human IgM that did not promote remyelination and medium wereused as controls., Conclusions: rHIgM22 prevented apoptotic signaling andinhibited OL differentiation by Lyn implying thatIgM-mediated remyelination is due toprotection of OPC and OLs rather than promotion of OPC differentiation., (© 2010 Wiley-Liss, Inc.)

IgM aggregates in biotechnologically produced preparations have been reported, however, in vitro characteristics and in vivo activity of IgM aggregates have not been well studied. We separated two species of the human monoclonal IgM antibody KBPA-101 by size-exclusion chromatography. Molecular weight determination indicated the presence of dipentameric and pentameric forms. We present here the results of a comparative characterization of these IgM species, including in vitro and in vivo effector function against Pseudomonas aeruginosa. Dipentameric (IgM)(2) species were observed to dissociate into pentameric IgM at 37 degrees C, suggesting a dynamic equilibrium, in which the pentameric species is the predominant form. In vitro antigen binding (P. aeruginosa LPS) and IgM-mediated complement-dependent phagocytosis of labeled bacterial cells did not differ significantly between the dipentameric (IgM)(2) and pentameric IgM species. Furthermore, the in vivo efficacy of dipentameric and pentameric IgM in protecting mice from a lethal dose of P. aeruginosa through passive immunization was nearly equivalent. In conclusion, low concentrations of dipentameric (IgM)(2) may contain an additional but equally active component of the principal biological form. The data presented in this work support the conclusion that the pentameric form of IgM directed against the O-polysaccharide moiety of P. aeruginosa serotype IATS-O11 and dipentameric (IgM)(2) are functionally equivalent., (Copyright 2009 Elsevier B.V. All rights reserved.)

Objective: We here determine the role of IgM antibodies against phosphorylcholine (anti-PC) in prediction of cardiovascular disease (CVD) and on macrophage uptake of Oxidized LDL (OxLDL)., Methods: From a screening of 4232 subjects, 60-year-old (2039 men and 2193 women), 211 incident cases of CVD (myocardial infarction, ischemic stroke, or hospitalized angina pectoris) and 633 age- and sex-matched controls were identified through a 5-7 year follow-up. Serum levels of IgM anti-PC was determined by ELISA. Anti-PC was extracted from pooled human IgM and the effect of anti-PC on the uptake of OxLDL was studied by FACScan., Results: Relative risks (RR) with 95% confidence intervals (CI) by quartiles of anti-PC levels with quartile 4 set as the reference value (RR = 1.0) and adjusted for smoking, BMI, type II diabetes, hypercholesterolaemia, and high blood pressure yielded an excess risk for CVD only for those within the lowest quartile of anti-PC values with an RR of 1.37 (CI 0.87-2.16). However, for men stronger associations were noted with increasing multivariately adjusted RRs from quartile 4 to quartile 1. Subjects within quartile 1 (values below 29.7 U/ml) had a significantly increased RR of 1.96 (CI 1.09-3.55). Further adjustments for hsCRP gave essentially the same results. No excess risk was noted for women. Specific anti-PC could be extracted from IgM and these antibodies inhibited macrophage uptake of OxLDL., Conclusions: Low IgM anti-PC could be a novel risk marker for CVD among men. One possible mechanism could be inhibition of uptake of oxLDL in macrophages., (Copyright 2009 Elsevier Ltd. All rights reserved.)

A two-step purification strategy comprising of polyethylene glycol (PEG) precipitation and anion-exchange chromatography was developed for a panel of monoclonal immunoglobulin M (IgM) (pI 5.5-7.7) produced from hybridoma cultures. PEG precipitation was optimized with regards to concentration, pH and mixing. For anion-exchange chromatography, different resins were screened of which Fractogel EMD, a polymer grafted porous resin had the highest capacity. Despite its significantly slower mass transfer, the binding capacity was still higher compared to a convection driven resin (monolith). This purification strategy was successfully demonstrated for all 9 IgMs in the panel. In small scale most antibodies could be purified to >95% purity with the exception of two which gave a lower final purity (46% and 85%). The yield was dependent on the different antibodies ranging from 28% to 84%. Further improvement of recovery and purity was obtained by the digestion of DNA present in the hybridoma supernatant using an endonuclease, benzonase. So far this strategy has been applied for the purification of up to 2l hybridoma supernatants.

In some tissues, the injection of antibodies to the beta(1)-integrins leads to a reduction in interstitial fluid pressure, indicating an active role for the extracellular matrix in tissue pressure regulation. If perturbations of the matrix occur in the periventricular area of the brain, a comparable lowering of interstitial pressures may induce transparenchymal pressure gradients favoring ventricular expansion. To examine this concept, we measured periventricular (parenchymal) and ventricular pressures with a servo-null micropipette system (2-microm tip) in adult Wistar rats before and after anti-integrin antibodies or IgG/IgM isotype controls were injected into a lateral ventricle. In a second group, the animals were kept for 2 wk after similar injections and after euthanization, the brains were removed and assessed for hydrocephalus. In experiments in which antibodies to beta(1)-integrins (n = 10) but not isotype control IgG/IgM (n = 7) were injected, we observed a decline in periventricular pressures relative to the preinjection values. Under similar circumstances, ventricular pressures were elevated (n = 10) and were significantly greater than those in the periventricular interstitium. We estimated ventricular to periventricular pressure gradients of up to 4.3 cmH(2)O. In the chronic preparations, we observed enlarged ventricles in many of the animals that received injections of anti-integrin antibodies (21 of 29 animals; 72%) but not in any animal receiving the isotype controls. We conclude that modulation/disruption of beta(1)-integrin-matrix interactions in the brain generates pressure gradients favoring ventricular expansion, suggesting a novel mechanism for hydrocephalus development.

Aspergillus fumigatus, a ubiquitous fungus, has been reported to cause human diseases like allergic pulmonary aspergillosis, aspergilloma and invasive infection. Limited spectrum and emergence of resistance has become a serious problem with available antifungals. Therefore, an alternative approach is required for successful treatment of mycoses. In the present study, immunogenic protein profile of A. fumigatus cell wall was generated using two-dimensional-gel electrophoresis and three hybridomas producing monoclonal antibodies (MAbs; IgM) were selected after fusion experiments. Of these three MAbs, MAb-7 exhibited potent in vitro inhibitory activity, which was confirmed by MTT assay, fluorescence-activated cell sorter analysis and immuno-fluorescence studies, and the protein was identified as catalase B using MALDI-TOF-MS.

An immunoglobulin-rich fraction has been prepared from ovine blood in our laboratory. We have investigated its antibacterial activity and binding activity to pathogenic whole cell antigens, lipopolysaccharide (LPS) and staphylococcal enterotoxin B. Ovine immunoglobulin concentrate (OIC) comprised about 73 +/- 2% of IgG and 11 +/- 1% of IgM on a protein basis. It inhibited the growth of all 13 strains of pathogens tested, but the inhibitory activity varied according to bacterial strain. The inhibitory activity of OIC was attributed to the high contents of undenatured immunoglobulin present because its inhibitory activity was destroyed by pepsin digestion and heat treatment (65 degrees C for 30 min). OIC bound to all the Gram-positive and Gram-negative pathogens, regardless of cell wall structure. The highest magnitude of crossreactivity to whole cell antigens was against Staphylococcus epidermidis and Shigella soneii strains (p < 0.001). The binding activity of OIC to LPS obtained from Escherichia coli O111:B4 and Salmonella enterica serotype typhimurium was assessed by enzyme-linked immunosorbent assay and lymphoblast K-562 proliferation assay. OIC bound to LPS with a binding activity that was dependent on OIC concentration and saturable, showing typical hyperbolic curves. For toxin-binding activity, an OIC concentration-dependent trend like that for LPS-binding activity was also observed. This preliminary evidence suggests that the OIC used in this study could be a promising supplement for protecting against pathogenic bacteria.

We analyzed the effect on the mouse neuromuscular synapses of a human monoclonal IgM, which binds specifically to gangliosides with the common epitope [GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-]. We focused on the role of the complement. Evoked neurotransmission was partially blocked by IgM both acutely (1 h) and chronically (10 days). Transmission electron microscopy shows important nerve terminal growth and retraction remodelling though axonal injury can be ruled out. Synapses did not show mouse C5b-9 immunofluorescence and were only immunolabelled when human complement was added. Therefore, the IgM-induced synaptic changes occur without complement-mediated membrane attack.

The biological functions of leptin in the human endometrial epithelium were investigated using the human endometrial epithelial cell line, HHUA. Specifically, the effects of leptin on the proliferation and apoptosis of HHUA cells induced by treatment with anti-Fas IgM or anticancer drugs were examined. RT-PCR detected the expression of four leptin receptor isoform mRNAs in the cells and flow cytometric analysis revealed cell surface expression of the leptin receptor molecules. Leptin stimulated HHUA cell proliferation in a dose-dependent manner at concentrations below the normal serum leptin level. Leptin enhanced anti-Fas IgM-mediated growth inhibition and DNA fragmentation, but did not enhance the expression of either Fas antigen or Fas ligand. Moreover, leptin had no effect on anticancer drug-induced apoptosis. Based on these results, leptin at a physiological serum concentration, may regulate the remodeling of the human endometrial epithelium by stimulating cell proliferation and enhancing the Fas-specific intracellular apoptotic signaling pathway.