A Recombinant Acetylcholine Receptor α1 Subunit Extracellular Domain Is a Promising New Drug Candidate for Treatment Of Myasthenia Gravis.

Background and Aims: Myasthenia gravis (MG) is a T-cell dependent antibody-mediated autoimmune disease in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen, comprising several T and B cell auto-epitopes. We hypothesized that an efficacious drug candidate for antigen-specific therapy in MG should comprise a broad range of these auto-epitopes and be administered in a noninflammatory and tolerogenic context. Methods: We used a soluble mutated form of the extracellular domain of the α1 chain of the AChR (α1-ECDm), which represents the major portion of auto-epitopes involved in MG, and investigated, in a well-characterized rat model of experimental autoimmune myasthenia gravis (EAMG) whether its intravenous administration could safely and efficiently treat the autoimmune disease. Results: We demonstrated that intravenous administration of α1-ECDm abrogates established EAMG, in a dose and time dependent manner, as assessed by clinical symptoms, body weight, and compound muscle action potential (CMAP) decrement. Importantly, the effect was more pronounced compared to drugs representing current standard of care for MG. The protein had a short plasma half-life, most of what could be recovered was sequestered in the liver, kidneys and spleen. Further, we did not observe any signs of toxicity or intolerability in animals treated with α1-ECDm. Conclusion: We conclude that intravenous treatment with α1-ECDm is safe and effective in suppressing EAMG. α1-ECDm is in preclinical development as a promising new drug candidate for MG.

A reduced population of CD103(+)CD11b(+) dendritic cells has a limited impact on oral Salmonella infection.

CD103(+)CD11b(+) dendritic cells (DC) are the major migratory DC subset in the small intestine lamina propria (siLP) and their survival is dependent on the transcription factor interferon regulatory factor 4 (IRF4). Mice with a DC-specific deletion of irf4 (CD11c-cre.Irf4 mice) have reduced mucosal CD103(+)CD11b(+) DC and altered T cell differentiation to protein antigen. The influence of CD103(+)CD11b(+) DC on oral infection with the gastrointestinal pathogen Salmonella, however, is poorly understood and is investigated here. We show that, despite being infected with Salmonella, CD11c-cre.Irf4 mice (called Cre(+) mice) conserve the reduction in CD103(+)CD11b(+) DC observed in naive Cre(+) mice, particularly in the mesenteric lymph nodes (MLN) but also in the siLP at day 3 post infection. Moreover, Salmonella-infected Cre(+) mice have a similar bacterial burden in intestinal tissues (siLP, MLN and Peyer's patches) as well as the spleen compared to infected Cre(-) controls. The T cell compartment, including the frequency of IFN-γ and IL-17-producing T cells, is not altered in intestinal tissues of Salmonella-infected Cre(+) mice relative to infected Cre(-) controls. In addition, no difference between infected Cre(+) and Cre(-) mice was observed in either the concentration of IL-6 or IL-17 in whole tissue lysates of siLP, MLN or Peyer's patches or in the serum concentration of Salmonella-specific IgG and IgM. Overall the data suggest that the reduction of CD103(+)CD11b(+) DC in Cre(+) mice has little if any impact on Salmonella burden in infected tissues or eliciting effector functions important in host survival at later stages of the infection.

Synergy between CD40 and MyD88 Does Not Influence Host Survival to Salmonella Infection.

Previous studies using purified toll-like receptor (TLR) ligands plus agonistic anti-CD40 antibodies showed that TLRs and CD40 can act synergistically on dendritic cells (DCs) to optimize T cell activation and Th1 differentiation. However, a synergistic effect of TLRs and CD40 during bacterial infection is not known. Here, we show that mice lacking the TLR adaptor MyD88 alone, or lacking both MyD88 and CD40 [double knockout (DKO) mice], are compromised in survival to Salmonella infection but have intact recruitment of neutrophils and inflammatory monocytes as well as unaltered abundance of DC subsets and DC activation in infected tissues. In contrast to infected wildtype and CD40(-/-) mice, both MyD88(-/-) mice and DKO mice lack detectable serum IFN-γ and have elevated IL-10. A synergistic effect of TLRs and CD40 was revealed in co-culture experiments where OT-II T cell proliferation was compromised when DKO DCs were pulsed with OVA protein and OVA323-339 peptide, but not with heat-killed Salmonella expressing OVA (HKSOVA), relative to MyD88(-/-) DCs. By contrast, MyD88(-/-) or DKO DCs pulsed with any of the antigens had a similar ability to induce IFN-γ that was lower than WT or CD40(-/-) DCs. DKO DCs pulsed with HKSOVA, but not with OVA or OVA323-339, had increased IL-10 relative to MyD88(-/-) DCs. Finally, HKSOVA-pulsed MyD88(-/-) and DKO DCs had similar and low induction of NFκB-dependent and -independent genes upon co-culture with OT-II cells. Overall, our data revealed that synergistic effects of CD40 and MyD88 do not influence host survival to Salmonella infection or serum levels of IFN-γ or IL-10. However, synergistic effects of MyD88 and CD40 may be apparent on some (IL-10 production) but not all (OT-II proliferation and IFN-γ production) DC functions and depend on the complexity of the antigen. Indeed, synergistic effects observed using purified ligands and well-defined antigens may not necessarily apply when complex antigens, such as live bacteria, challenge the immune system.

Regulation of pregnane-X-receptor, CYP3A and P-glycoprotein genes in the PCB-resistant killifish (Fundulus heteroclitus) population from New Bedford Harbor.

Killifish survive and reproduce in the New Bedford Harbor (NBH) in Massachusetts (MA), USA, a site severely contaminated with polychlorinated biphenyls (PCBs) for decades. Levels of 22 different PCB congeners were analyzed in liver from killifish collected in 2008. Concentrations of dioxin-like PCBs in liver of NBH killifish were ∼400 times higher, and the levels of non-dioxin-like PCBs ∼3000 times higher than in killifish from a reference site, Scorton Creek (SC), MA. The NBH killifish are known to be resistant to the toxicity of dioxin-like compounds and to have a reduced aryl hydrocarbon receptor (AhR) signaling response. Little is known about the responses of these fish to non-dioxin-like PCBs, which are at extraordinarily high levels in NBH fish. In mammals, some non-dioxin-like PCB congeners act through nuclear receptor 1I2, the pregnane-X-receptor (PXR). To explore this pathway in killifish, a PXR cDNA was sequenced and its molecular phylogenetic relationship to other vertebrate PXRs was determined. Killifish were also collected in 2009 from NBH and SC, and after four months in the laboratory they were injected with a single dose of either the dioxin-like PCB 126 (an AhR agonist) or the non-dioxin-like PCB 153 (a mammalian PXR agonist). Gills and liver were sampled three days after injection and transcript levels of genes encoding PXR, cytochrome P450 3A (CYP3A), P-glycoprotein (Pgp), AhR2 and cytochrome P450 1A (CYP1A) were measured by quantitative PCR. As expected, there was little effect of PCB exposure on mRNA expression of AhR2 or CYP1A in liver and gills of NBH fish. In NBH fish, but not in SC fish, there was increased mRNA expression of hepatic PXR, CYP3A and Pgp upon exposure to either of the two PCB congeners. However, basal PXR and Pgp mRNA levels in liver of NBH fish were significantly lower than in SC fish. A different pattern was seen in gills, where there were no differences in basal mRNA expression of these genes between the two populations. In SC fish, but not in NBH fish, there was increased mRNA expression of branchial PXR and CYP3A upon exposure to PCB126 and of CYP3A upon exposure to PCB153. The results suggest a difference between the two populations in non-AhR transcription factor signaling in liver and gills, and that this could involve killifish PXR. It also implies possible cross-regulatory interactions between that factor (presumably PXR) and AhR2 in liver of these fish.

The Gut Microbiota Reduces Colonization of the Mesenteric Lymph Nodes and IL-12-Independent IFN-γ Production During Salmonella Infection.

The intestinal commensal microbiota is essential for many host physiological processes, but its impact on infectious diseases is poorly understood. Here we investigate the influence of the gut microbiota during oral Salmonella infection. We report a higher bacterial burden in mesenteric lymph nodes (MLN) of intragastrically infected germ-free (GF) mice compared to conventionally-raised (CONV-R) animals, despite similar inflammatory phagocyte recruitment. Salmonella penetration into the lamina propria of the small intestine and splenic bacterial burden were not altered in the absence of the microbiota. Intragastrically infected GF mice also displayed a higher frequency of IFN-γ-producing NK, NKT, CD4(+), and CD8(+) T cells in the MLN despite IL-12 levels similar to infected CONV-R mice. However, infecting mice intraperitoneally abrogated the difference in MLN bacterial load and IFN-γ-producing cells observed in intragastrically-infected animals. Moreover, mice treated with antibiotics (ABX) and intragastrically infected with Salmonella had a greater bacterial burden and frequency of IFN-γ-producing cells in the MLN. In ABX mice the number of Salmonella correlated with the frequency of IFN-γ-producing lymphocytes in the MLN, while no such correlation was observed in the MLN of infected GF mice. Overall, the data show that the lack of the microbiota influences pathogen colonization of the MLN, and the increased IFN-γ in the MLN of infected GF mice is not only due to the absence of commensals at the time of infection but the lack of immune signals provided by the microbiota from birth.