CD4+ T-cell epitopes associated with antibody responses after intravenously and subcutaneously applied human FVIII in humanized hemophilic E17 HLA-DRB1*1501 mice.

Today it is generally accepted that B cells require cognate interactions with CD4(+) T cells to develop high-affinity antibodies against proteins. CD4(+) T cells recognize peptides (epitopes) presented by MHC class II molecules that are expressed on antigen-presenting cells. Structural features of both the MHC class II molecule and the peptide determine the specificity of CD4(+) T cells that can bind to the MHC class II-peptide complex. We used a new humanized hemophilic mouse model to identify FVIII peptides presented by HLA-DRB1*1501. This model carries a knockout of all murine MHC class II molecules and expresses a chimeric murine-human MHC class II complex that contains the peptide-binding sites of the human HLA-DRB1*1501. When mice were treated with human FVIII, the proportion of mice that developed antibodies depended on the application route of FVIII and the activation state of the innate immune system. We identified 8 FVIII peptide regions that contained CD4(+) T-cell epitopes presented by HLA-DRB1*1501 to CD4(+) T cells during immune responses against FVIII. CD4(+) T-cell responses after intravenous and subcutaneous application of FVIII involved the same immunodominant FVIII epitopes. Interestingly, most of the 8 peptide regions contained promiscuous epitopes that bound to several different HLA-DR proteins in in vitro binding assays.

Cardiovascular diseases crossroads: cGAS-STING signaling and disease progression.

It is now widely accepted that inflammation is critical in cardiovascular diseases (CVD). Here, studies are being conducted on how cyclic GMP-AMP synthase (cGAS), a component of innate immunity's DNA-sensing machinery, communicates with the STING receptor, which is involved in activating the immune system's antiviral response. Significantly, a growing body of research in recent years highlights the strong activation of the cGAS-STING signalling pathways in several cardiovascular diseases, such as myocardial infarction, heart failure, and myocarditis. This developing collection of research emphasises these pathways' crucial role in initiating and advancing cardiovascular disease. In this extensive narrative, we explore the role of the cGAS-STING pathway in the development of CVD. We elaborate on the basic mechanisms involved in the onset and progression of CVD. This review explores the most recent developments in the recognition and characterization of cGAS-STING pathway. Additionally, it considers the field's future prospects while examining how cGAS-STING pathway might be altered and its clinical applications for cardiovascular diseases.

Beyond the silence: A comprehensive exploration of long non-coding RNAs as genetic whispers and their essential regulatory functions in cardiovascular disorders.

Long non-coding RNAs (lncRNAs) are RNA molecules that regulate gene expression at several levels, including transcriptional, post-transcriptional, and translational. They have a length of more than 200 nucleotides and cannot code. Many human diseases have been linked to aberrant lncRNA expression, highlighting the need for a better knowledge of disease etiology to drive improvements in diagnostic, prognostic, and therapeutic methods. Cardiovascular diseases (CVDs) are one of the leading causes of death worldwide. LncRNAs play an essential role in the complex process of heart formation, and their abnormalities have been associated with several CVDs. This Review article looks at the roles and relationships of long non-coding RNAs (lncRNAs) in a wide range of CVDs, such as heart failure, myocardial infarction, atherosclerosis, and cardiac hypertrophy. In addition, the review delves into the possible uses of lncRNAs in diagnostics, prognosis, and clinical treatments of cardiovascular diseases. Additionally, it considers the field's future prospects while examining how lncRNAs might be altered and its clinical applications.

Cardiovascular challenges in the era of antiretroviral therapy for AIDS/ HIV: A comprehensive review of research advancements, pathophysiological insights, and future directions.

Cardiovascular disease, particularly coronary heart disease, is becoming more common among those living with HIV. Individuals with HIV face an increased susceptibility to myocardial infarction, also known as a heart attack, as compared to the general population in developed countries. This heightened risk can be attributed mainly to the presence of effective antiretroviral drugs and the resulting longer lifespan. Some cardiac issues linked to non-antiretroviral medications, including myocarditis, endocarditis, cardiomyopathy with dilation, pulmonary hypertension, and oedema of the heart, may affect those not undergoing highly active antiretroviral therapy (ART). Impaired immune function and systemic inflammation are significant contributors to this phenomenon after initiating highly aggressive antiretroviral treatment ART. It is becoming more challenging to determine the best course of treatment for HIV-associated cardiomyopathy due to new research suggesting that protease inhibitors might have a negative impact on the development of HF. Currently, the primary focus of research on ART medications is centered on the cardiovascular adverse effects of nucleoside reverse transcriptase inhibitors and protease inhibitors. This review paper thoroughly evaluates the advancements achieved in cardiovascular disease research and explores the potential implications for prospects. Additionally, it considers the field's future prospects while examining how ART might be altered and its clinical applications.

Stimulation and inhibition of FVIII-specific memory B-cell responses by CpG-B (ODN 1826), a ligand for Toll-like receptor 9.

Factor VIII (FVIII)-specific memory B cells are essential components for regulating anamnestic antibody responses against FVIII in hemophilia A with FVIII inhibitors. We asked how stimulation and inhibition of FVIII-specific memory B cells by low and high concentrations of FVIII, respectively, are affected by concurrent activation of the innate immune system. Using CD138(-) spleen cells from hemophilic mice treated with FVIII to study restimulation and differentiation of memory B cells in vitro, we tested modulating activities of agonists for Toll-like receptors (TLRs) 2, 3, 4, 5, 7, and 9. Ligands for TLR7 and 9 were most effective. They not only amplified FVIII-specific memory responses in the presence of stimulating concentrations of FVIII, but also countered inhibition in the presence of inhibitory concentrations of FVIII. Notably, CpG oligodeoxynucleotide (CpG-ODN), a ligand for TLR9, expressed biphasic effects. It amplified memory responses at low concentrations and inhibited memory responses at high concentrations, both in vitro and in vivo. Both stimulatory and inhibitory activities of CpG-ODN resulted from specific interactions with TLR9. Despite their strong immunomodulatory effects in the presence of FVIII, ligands for TLR induced negligible restimulation in the absence of FVIII in vitro and no restimulation in the absence of FVIII in vivo.

T cell-independent restimulation of FVIII-specific murine memory B cells is facilitated by dendritic cells together with toll-like receptor 7 agonist.

Memory B cells are involved in long-term maintenance of antibody-dependent immunologic disorders. Therefore, it is essential to understand how the restimulation of FVIII-specific memory B cells in hemophilia A with FVIII inhibitors is regulated. We asked whether concurrent activation of the innate immune system by an agonist for toll-like receptor (TLR) 7 is able to facilitate the differentiation of FVIII-specific memory B cells in the absence of T-cell help. TLR7 recognizes single-stranded RNA as contained in RNA viruses such as influenza, Sendai, and Coxsackie B viruses. Our results indicate that highly purified murine memory B cells do not differentiate into FVIII-specific antibody-secreting cells in the presence of FVIII and the TLR7 agonist when cultured in the absence of CD4(+) T cells. However, CD11c(+) dendritic cells facilitate the T cell-independent differentiation of FVIII-specific memory B cells but only in the presence of FVIII and the TLR7 agonist. In contrast to T cell-dependent restimulation, the antibody response after T cell-independent restimulation of FVIII-specific memory B cells is skewed toward IgG2a, an antibody subclass that is efficient in activating the complement system and in inducing Fc-receptor-mediated effector functions, both are required for effective immune responses against pathogens.

Maintenance and break of immune tolerance against human factor VIII in a new transgenic hemophilic mouse model.

Replacement of the missing factor VIII (FVIII) is the current standard of care for patients with hemophilia A. However, the short half-life of FVIII makes frequent treatment necessary. Current efforts focus on the development of longer-acting FVIII concentrates by introducing chemical and genetic modifications to the protein. Any modification of the FVIII protein, however, risks increasing its immunogenic potential to induce neutralizing antibodies (FVIII inhibitors), and this is one of the major complications in current therapy. It would be highly desirable to identify candidates with a high risk for increased immunogenicity before entering clinical development to minimize the risk of exposing patients to such altered FVIII proteins. In the present study, we describe a transgenic mouse line that expresses a human F8 cDNA. This mouse is immunologically tolerant to therapeutic doses of native human FVIII but is able to mount an antibody response when challenged with a modified FVIII protein that possesses altered immunogenic properties. In this situation, immunologic tolerance breaks down and antibodies develop that recognize both the modified and the native human FVIII. The applicability of this new model for preclinical immunogenicity assessment of new FVIII molecules and its potential use for basic research are discussed.

Tolerance to factor VIII in a transgenic mouse expressing human factor VIII cDNA carrying an Arg(593) to Cys substitution.

Inhibitory antibodies develop in approximately 25% of patients with severe hemophilia. A following treatment with factorVIII. In E-16KO or E-17KO mice, in which the factor VIII gene has been inactivated by insertion of a neo cassette, inhibitors develop following administration of factor VIII. Here, we describe the generation of transgenic mice expressing human factor VIII-R593C (huFVIII-R593C). Human factor VIII-R593C cDNA under control of a mouse albumin enhancer/promoter was injected into fertilized oocytes. Analysis of transgenic mice revealed that human factor VIII-R593C was expressed in the liver. Transgenic mice were crossed with factor VIII-deficient mice (E-16KO mice). In plasma of E-16KO mice antibodies were detected after five serial intravenous injections of factor VIII, while plasma of huFVIII-R593C/E-16KO mice did not contain detectable levels of antibodies. No antibody secreting cells were observed in either spleen or bone marrow of huFVIII-R593C/E-16KO mice. Also, factor VIII-specific memory B cells were not observed in the spleen of huFVIII-R593C/E-16KO mice. Analysis of T cell responses revealed that splenocytes derived of E-16KO mice secreted IL-10 and IFN-gamma following restimulation with factor VIII in vitro. In contrast, no factor VIII-specific T cell responses were observed in huFVIII-R593C/E-16KO mice. These results indicate that huFVIII-R593C/E-16KO mice are tolerant to intravenously administered factor VIII. It is anticipated that this model may prove useful for studying immune responses in the context of factor VIII gene therapy.

Single cell analysis of factor VIII-specific T cells in hemophilic mice after treatment with human factor VIII.

A multi-parameter flow-cytometry assay was established suitable for analyzing T-cell-specific cell surface markers (CD3, CD4) together with intracellular cytokines on a single cell level. This assay was used to identify the frequency and the kinetic of different populations of factor VIII (FVIII)-specific CD4+ T cells in hemophilic E-17 mice after treatment with human FVIII. A clear temporal correlation was found between the appearance of FVIII-specific CD4+ T cells in the spleen and the detection of anti-FVIII antibodies in plasma. These cells and antibodies were detectable in all experiments after two doses of FVIII and in a few even after a single dose. The IFN-gamma-producing T cells were the most prominent type of FVIII-specific T cells suggesting Th1-type T cells have an important role in regulating the anti-FVIII immune response in E-17 mice. IL-10-producing T cells were the second most dominant type. They were detectable after two doses of FVIII and increased in frequency after four. Cytokine co-expression studies analyzing IL-10 and IFN-gamma in the same cell indicated that there might be at least two types of IL-10 positive T cells, those cells that produce IL-10 only and in addition cells that produce IL-10 and IFN-gamma. Furthermore, FVIII-specific T cells producing IL-2 were found in all experiments after two doses of FVIII. In a few experiments IL-4-producing T cells were seen but in most experiments they were not detectable. In contrast, IL-4 could be found in supernatants of in vitro restimulated CD8- spleen cells.

Long-term persistence of anti-factor VIII antibody-secreting cells in hemophilic mice after treatment with human factor VIII.

The analysis of anti-factor VIII (FVIII) antibody-secreting cells (ASC) at different anatomic sites provides valuable information about the nature of the anti-FVIII immune response in hemophilic mice after treatment with human FVIII. An Elispot system is described that is suitable for analyzing frequencies and IgG subclasses of anti-FVIII ASC at the single-cell level. Hemophilic mice were treated with four doses of FVIII. Anti-FVIII antibodies in blood as well as anti-FVIII ASC in spleen and bone marrow were analyzed after each dose of FVIII and subsequently up to 22 weeks after termination of the FVIII treatment. Anti-FVIII ASC first appeared in the spleen where they were detectable after two intravenous doses of FVIII. Their appearance correlated with that of anti-FVIII antibodies in blood plasma. Anti-FVIII ASC in bone marrow were detectable after three doses of FVIII and were probably cells that initially formed in the spleen and subsequently migrated to the bone marrow. Whereas the frequency of anti-FVIII ASC in the spleen increased up to the fourth dose of FVIII and declined thereafter, in the bone marrow it remained constant for up to at least 22 weeks after the termination of the FVIII treatment. Titers of anti-FVIII antibodies in blood plasma increased up to the fourth dose of FVIII, then remained high constantly for 14 weeks and decreased but the antibodies were still detectable for up to at least 22 weeks after the fourth dose of FVIII. The IgG-subclass distribution of anti-FVIII ASC was similar in spleen and bone marrow and matched the subclasses of anti-FVIII antibodies in blood plasma indicating that both organs contribute to circulating antibodies in the blood.

High-dose factor VIII inhibits factor VIII-specific memory B cells in hemophilia A with factor VIII inhibitors.

Hemophilia A in its severe form is a life-threatening hemorrhagic disease that is caused by mutations in the factor VIII (FVIII) gene (symbol F8). About 25% of patients who receive replacement therapy develop neutralizing antibodies that inhibit the function of substituted FVIII. Long-term application of high doses of FVIII has evolved as an effective therapy to eradicate the antibodies and to induce long-lasting immune tolerance. Little is known, however, about the immunologic mechanisms that cause the down-modulation of anti-FVIII antibodies by high doses of FVIII. We report that high doses of FVIII inhibit the restimulation of FVIII-specific memory B cells and their differentiation into antibody-secreting plasma cells in vitro and in vivo in a murine model of hemophilia A. The inhibition of memory B-cell responses is irreversible and not mediated by FVIII-specific T cells. Furthermore, it seems to involve the activation of caspases. We conclude that the inhibition of FVIII-specific memory B cells might be an early event in the down-modulation of anti-FVIII antibodies in patients with hemophilia A who receive high doses of FVIII.

Preventing restimulation of memory B cells in hemophilia A: a potential new strategy for the treatment of antibody-dependent immune disorders.

Memory B cells are responsible for the rapidly emerging antibody response after antigen reexposure. The signals required for the restimulation of memory B cells have not been fully explained. We used a murine model of anti-factor VIII (FVIII) antibody responses in hemophilia A to study the requirements for the restimulation of FVIII-specific memory B cells and their differentiation into anti-FVIII antibody-producing cells. We were particularly interested in the significance of activated T cells and costimulatory interactions. Our results indicate that the restimulation of FVIII-specific memory B cells is strictly dependent on interactions with activated T cells. These activated T cells can be specific for either FVIII or third-party antigens. Restimulation by T cells specific for third-party antigens requires the presence of FVIII, indicating that signals induced by B-cell receptor (BCR) triggering and by interactions with activated T cells are important. The blockade of B7-1 or B7-2 as well as the blockade of CD40L inhibits the restimulation and differentiation of FVIII-specific memory B cells in vitro and in vivo. The interference with inducible costimulator-inducible costimulator ligand (ICOS-ICOSL) interactions, however, does not cause any modulation. As expected, the production of anti-FVIII antibodies by plasma cells is not dependent on any of the costimulatory interactions tested.