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Endogenous retroviruses (ERVs) are an integral part of the mammalian genome. The role of immune control of ERVs in general is poorly defined as is their function as anti-cancer immune targets or drivers of autoimmune disease. Here, we generate mouse-strains where Moloney-Murine Leukemia Virus tagged with GFP (ERV-GFP) infected the mouse germline. This enables us to analyze the role of genetic, epigenetic and cell intrinsic restriction factors in ERV activation and control. We identify an autoreactive B cell response against the neo-self/ERV antigen GFP as a key mechanism of ERV control. Hallmarks of this response are spontaneous ERV-GFP + germinal center formation, elevated serum IFN-γ levels and a dependency on Age-associated B cells (ABCs) a subclass of T-bet + memory B cells. Impairment of IgM B cell receptor-signal in nucleic-acid sensing TLR-deficient mice contributes to defective ERV control. Although ERVs are a part of the genome they break immune tolerance, induce immune surveillance against ERV-derived self-antigens and shape the host immune response., (© 2024. The Author(s).)

Immunoglobulin E (IgE) production is tightly regulated at the cellular and genetic levels and is believed to be central to allergy development. At least two cellular pathways exist that lead to systemic anaphylaxis reactions in vivo: IgE-sensitized mast cells and IgG1-sensitized basophils. Passive anaphylaxis, by application of allergen and allergen-specific antibodies in mice, indicates a differential contribution of immunoglobulin isotypes to anaphylaxis. However, analysis of a dynamic immunization-mediated antibody response in anaphylaxis is difficult. Here, we generated IgE knock-in mice (IgE(ki) ), which express the IgE heavy chain instead of IgG1, in order to analyze the contribution of IgG1 and IgE to active anaphylaxis in vivo. IgE(ki) mice display increased IgE production both in vitro and in vivo. The sensitization of IgE(ki) mice by immunization followed by antigen challenge leads to increased anaphylaxis. Homozygous IgE(ki) mice, which lack IgG1 due to the knock-in strategy, are most susceptible to active systemic anaphylaxis. The depletion of basophils demonstrates their importance in IgE-mediated anaphylaxis. Therefore, we propose that an enhanced, antigen-specific, polyclonal IgE response, as is the case in allergic patients, is probably the most efficient way to sensitize basophils to contribute to systemic anaphylaxis in vivo., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The genome of vertebrates contains endogenous retroviruses (ERVs) that are largely nonfunctional relicts of ancestral germline infection by exogenous retroviruses. However, in some mouse strains ERVs are actively involved in disease. Here we report that nucleic acid-recognizing Toll-like receptors 3, 7, and 9 (TLR 3, TLR7, and TLR9) are essential for the control of ERVs. Loss of TLR7 function caused spontaneous retroviral viremia that coincided with the absence of ERV-specific antibodies. Importantly, additional TLR3 and TLR9 deficiency led to acute T cell lymphoblastic leukemia, underscoring a prominent role for TLR3 and TLR9 in surveillance of ERV-induced tumors. Experimental ERV infection induced a TLR3-, TLR7-, and TLR9-dependent group of "acute-phase" genes previously described in HIV and SIV infections. Our study suggests that in addition to their role in innate immunity against exogenous pathogens, nucleic acid-recognizing TLRs contribute to the immune control of activated ERVs and ERV-induced tumors., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Glutathione exists in biological samples in the reduced form (GSH), as its disulfide (GSSG) and as a mixed disulfide (GSSR) with thiols (RSH). GSH is the most abundant low-molecular-mass thiol and plays important roles as a cofactor and as a main constituent of the intracellular redox status. Due to its own sulfhydryl (SH) group, GSH reacts readily with o-phthaldialdehyde (OPA) to form a highly stable and fluorescent isoindole derivative (GSH-OPA), which allows for sensitive and specific quantitative determination of GSH in biological systems by HPLC with fluorescence (FL) detection. In the present article we report on the utility of the novel, strongly disulfide bond-reducing thiol N-acetyl-cysteine ethyl ester (NACET) for the specific quantitative analysis of GSH and GSSG in the cytosol of red blood cells (RBC) as GSH-OPA derivative with FL (excitation/emission 338/458nm) or UV absorbance (338nm) detection. Unlike in aqueous solution, the derivatization of GSH in RBC cytosol yielded two closely related derivatives in the absence of NACET and only the GSH-OPA derivative in the presence of NACET. The HPLC method was optimized and validated for human RBC and applied to measure GSH and GSSG in RBC of healthy subjects. Basal GSH and GSSG concentrations were determined to be 2340+/-350microM and 11.4+/-3.2microM, respectively, in RBC of 12 healthy young volunteers (aged 23-38 years). The method was also applied to study the effects of nitrite on the glutathione status in intact and lysed human RBC. Nitrite at mM-concentrations caused instantaneous and considerable GSSG formation in lysed but much less pronounced in intact RBC. GSH externally added to lysed RBC inhibited nitrite-induced methemoglobin formation. Our findings suggest that nitric oxide/nitrite-related consumption rate of GSH, and presumably that of NADH and NADPH, could be of the order of 600micromol/day in RBC of healthy subjects.

S-Nitrosothiols from low-molecular-mass and high-molecular-mass thiols, including glutathione, albumin and hemoglobin, are endogenous potent vasodilators and inhibitors of platelet aggregation. By utilizing the S-transnitrosation reaction and by using the lipophilic (pK(L) 0.78) and strong nucleophilic synthetic thiol N-acetyl cysteine ethyl ester (NACET) we have developed a GC-MS method for the analysis of S-nitrosothiols and their (15)N- or (2)H-(15)N-labelled analogs as S-nitroso-N-acetyl cysteine ethyl ester (SNACET) and S(15)NACET or d(3)-S(15)NACET derivatives, respectively, after their extraction with ethyl acetate. Injection of ethyl acetate solutions of S-nitrosothiols produced two main reaction products, compound X and compound Y, within the injector in dependence on its temperature. Quantification was performed by selected-ion monitoring of m/z 46 (i.e., [NO(2)](-)) for SNACET and m/z 47 (i.e., [(15)NO(2)](-)) for S(15)NACET/d(3)-S(15)NACET for compound X, and m/z 157 for SNACET and m/z 160 for d(3)-S(15)NACET for compound Y. In this article we describe the development, validation and in vitro and in vivo applications of the method to aqueous buffered solutions, human and rabbit plasma. Given the ester functionality of SNACET/S(15)NACET/d(3)-S(15)NACET, stability studies were performed using metal chelators and esterase inhibitors. The method was found to be suitable for the quantitative determination of various S-nitrosothiols including SNACET externally added to human plasma (0-10microM). Nitrite contamination in ethyl acetate was found to interfere. Our results suggest that the concentration of endogenous S-nitrosothiols in human plasma does not exceed about 200nM in total. Oral administration of S(15)NACET to rabbits (40-63micromol/kg body weight) resulted in formation of ALB-S(15)NO, [(15)N]nitrite and [(15)N]nitrate in plasma.