Therapeutic potential of co-signaling receptor modulation in hepatitis B.

Reversing CD8+ T cell dysfunction is crucial in treating chronic hepatitis B virus (HBV) infection, yet specific molecular targets remain unclear. Our study analyzed co-signaling receptors during hepatocellular priming and traced the trajectory and fate of dysfunctional HBV-specific CD8+ T cells. Early on, these cells upregulate PD-1, CTLA-4, LAG-3, OX40, 4-1BB, and ICOS. While blocking co-inhibitory receptors had minimal effect, activating 4-1BB and OX40 converted them into antiviral effectors. Prolonged stimulation led to a self-renewing, long-lived, heterogeneous population with a unique transcriptional profile. This includes dysfunctional progenitor/stem-like (TSL) cells and two distinct dysfunctional tissue-resident memory (TRM) populations. While 4-1BB expression is ubiquitously maintained, OX40 expression is limited to TSL. In chronic settings, only 4-1BB stimulation conferred antiviral activity. In HBeAg+ chronic patients, 4-1BB activation showed the highest potential to rejuvenate dysfunctional CD8+ T cells. Targeting all dysfunctional T cells, rather than only stem-like precursors, holds promise for treating chronic HBV infection.

Influence of the Electrode Array Design on Incidence of Vertigo Symptoms and Vestibular Function After Cochlear Implantation.

PURPOSE: To evaluate if a specific type of cochlear implant (CI) electrode array (EA) reveals higher rates/prevalence of vestibular symptoms and to characterize their respective relationship to intracochlear position and objective vestibular function. METHODS: This retrospective study included 71 cochlear implantations in patients older than 18 years. The electrode position within the cochlea, electrode insertion angle, and cochlear coverage were determined from postoperative multiplanar reconstructed cone-beam computed tomography scans. All device manufacturers were represented. Data related to preoperative and postoperative PTA as well as vestibular symptoms in the preoperative and postoperative stages were collected from the patient's records. RESULTS: Twelve of the 71 (16.9%) CI patients experienced vertigo symptoms in the early postoperative period. In 5 (7.0%) patients, the vertigo complaints lasted until the time of the first activation (5-6 weeks postoperative). Postoperative onset of vestibular symptoms was more often seen in patients receiving lateral wall (LW)/straight EAs (19%) compared to perimodiolar/precurved EAs (7%), but this was only a trend and no statistical significance was observed. Moreover, preoperative pathologic caloric responses (CRs) better predicted the postoperative onset of vestibular symptoms. CONCLUSION: The preoperative consideration of a complicated CI-induced vertigo is important in the counseling particularly of elderly patients. We identified some risk factors for post-CI vertigo that should be considered in the patient's counseling: preoperative pathologic CRs, the extent of surgical trauma, and possibly the use of an LW EA, regardless of the length.

TLR7 Signaling Shapes and Maintains Antibody Diversity Upon Virus-Like Particle Immunization.

Virus-like particles (VLPs) are used in different marketed vaccines and are able to induce potent antibody responses. The innate pattern recognition receptors TLR7/8 recognize single stranded (ss) RNA naturally packaged into some VLPs and have been shown to enhance the production of IgG antibodies upon immunization. Here we demonstrate that, upon immunization with RNA-loaded bacteriophage-derived VLP Qß, TLR7 signaling accelerates germinal center formation, promotes affinity/avidity maturation of VLP-specific IgG and isotype switching to IgG2b/2c. These findings extrapolated to antigens displayed on Qß; as Fel d 1, the major cat allergen, chemically attached to Qß also induced higher affinity/avidity IgG2b/2c antibodies in a TLR7-dependent fashion. Chimeric mice lacking TLR7-expression exclusively in B cells demonstrated that the enhanced IgG responses were driven by a B cell intrinsic mechanism. Importantly, deep sequencing of the BCR repertoire of antigen-specific B cells demonstrated higher diversity in mice with TLR7 signaling in B cells, suggesting that TLR7-signaling drives BCR repertoire development and diversity. Furthermore, the current data demonstrate that high levels of clonal diversity are reached early in the response and maintained by TLR7 signaling. In conclusion, TLR7 signaling enhances levels and quality of IgG antibodies, and this finding has major implications for vaccine design.

Virus-Specific Secondary Plasma Cells Produce Elevated Levels of High-Avidity Antibodies but Are Functionally Short Lived.

Most vaccines aim at inducing durable antibody responses and are designed to elicit strong B cell activation and plasma cell (PC) formation. Here we report characteristics of a recently described secondary PC population that rapidly originates from memory B cells (MBCs) upon challenge with virus-like particles (VLPs). Upon secondary antigen challenge, all VLP-specific MBCs proliferated and terminally differentiated to secondary PCs or died, as they could not undergo multiple rounds of re-stimulation. Secondary PCs lived in bone marrow and secondary lymphoid organs and exhibited increased production of antibodies with much higher avidity compared to primary PCs, supplying a swift wave of high avidity antibodies early after antigen recall. Unexpectedly, however, secondary PCs were functionally short-lived and most of them could not be retrieved in lymphoid organs and ceased to produce antibodies. Nevertheless, secondary PCs are an early source of high avidity antibodies and induction of long-lived MBCs with the capacity to rapidly differentiate to secondary PCs may therefore be an underestimated possibility to induce durable protection by vaccination.

RNA and Toll-Like Receptor 7 License the Generation of Superior Secondary Plasma Cells at Multiple Levels in a B Cell Intrinsic Fashion.

Secondary plasma cells (PCs) originate from memory B cells and produce increased levels of antibodies with higher affinity compared to PCs generated during primary responses. Here we demonstrate that virus-like particles (VLPs) only induce secondary PCs in the presence of toll-like receptor (TLR) 7 and if they are loaded with RNA. Furthermore, adoptive transfer experiments demonstrate that RNA and TLR7 signaling are required for secondary PC generation, both at the level of memory B cell as well as PC differentiation. TLR7-signaling occurred in a B cell intrinsic manner as TLR7-deficient B cells in an otherwise TLR7-competent environment failed to differentiate into secondary PCs. Therefore, RNA inside VLPs is essential for the generation of memory B cells, which are competent to differentiate to secondary PCs and for the differentiation of secondary PCs themselves. While we have not tested all other TLR or non-TLR adjuvants with our VLPs, these data have obvious implications for vaccine design, as RNA packaged into VLPs is a simple way to enhance induction of memory B cells capable of generating secondary PCs.

The thioredoxin-1 and glutathione/glutaredoxin-1 systems redundantly fuel murine B-cell development and responses.

Antioxidant systems maintain cellular redox homeostasis. The thioredoxin-1 (Trx1) and the glutathione (GSH)/glutaredoxin-1 (Grx1) systems are key players in preserving cytosolic redox balance. In fact, T lymphocytes critically rely on reducing equivalents from the Trx1 system for DNA biosynthesis during metabolic reprogramming upon activation. We here show that the Trx1 system is also indispensable for development and functionality of marginal zone (MZ) B cells and B1 cells in mice. In contrast, development of conventional B cells, follicular B-cell homeostasis, germinal center reactions, and antibody responses are redundantly sustained by both antioxidant pathways. Proliferating B2 cells lacking Txnrd1 have increased glutathione (GSH) levels and upregulated cytosolic Grx1, which is barely detectable in expanding thymocytes. These results suggest that the redox capacity driving proliferation is more robust and flexible in B cells than in T cells, which may have profound implications for the therapy of B and T-cell neoplasms.

Stat3-mediated alterations in lysosomal membrane protein composition.

Lysosome function is essential in cellular homeostasis. In addition to its recycling role, the lysosome has recently been recognized as a cellular signaling hub. We have shown in mammary epithelial cells, both in vivo and in vitro, that signal transducer and activator of transcription 3 (Stat3) modulates lysosome biogenesis and can promote the release of lysosomal proteases that culminates in cell death. To further investigate the impact of Stat3 on lysosomal function, we conducted a proteomic screen of changes in lysosomal membrane protein components induced by Stat3 using an iron nanoparticle enrichment strategy. Our results show that Stat3 activation not only elevates the levels of known membrane proteins but results in the appearance of unexpected factors, including cell surface proteins such as annexins and flotillins. These data suggest that Stat3 may coordinately regulate endocytosis, intracellular trafficking, and lysosome biogenesis to drive lysosome-mediated cell death in mammary epithelial cells. The methodologies described in this study also provide significant improvements to current techniques used for the purification and analysis of the lysosomal proteome.

Delivering adjuvants and antigens in separate nanoparticles eliminates the need of physical linkage for effective vaccination.

DNA rich in unmethylated CG motifs (CpGs) engage Toll-Like Receptor 9 (TLR-9) in endosomes and are well described stimulators of the innate and adaptive immune system. CpGs therefore can efficiently improve vaccines' immunogenicity. Packaging CpGs into nanoparticles, in particular into virus-like particles (VLPs), improves the pharmacological characteristics of CpGs as the protein shell protects them from DNAse activity and delivers the oligomers to the endosomal compartments of professional antigen presenting cells (APCs). The current consensus in packaging and delivering CpGs in VLP-based vaccines is that both adjuvants and antigens should be kept in close proximity (i.e. physically linked) to ensure delivery of antigens and adjuvants to the same APCs. In the current study, we harness the draining properties of the lymphatic system and show that also non-linked VLPs are efficiently co-delivered to the same APCs in lymph nodes. Specifically, we have shown that CpGs can be packaged in one VLP and mixed with another VLP displaying the antigen prior to administration in vivo. Both VLPs efficiently reached the same draining lymph node where they were taken up and processed by the same APCs, namely dendritic cells and macrophages. This resulted in induction of specific CTLs producing cytokines and killing target cells in vivo at levels seen when using VLPs containing both CpGs and chemically conjugated antigen. Thus, delivery of antigens and adjuvants in separate nanoparticles eliminates the need of physical conjugation and thus can be beneficial when designing precision medicine VLP-based vaccines or help to re-formulate existing VLP vaccines not naturally carrying immunostimulatory sequences.

Membrane estrogen receptors: their role in blood pressure regulation and cardiovascular disease.

Hypertension (HTN) is a leading risk factor for cardiovascular disease (CVD) and continues to affect millions of people in industrialized nations. The increasing prevalence of HTN is closely related to the growing prevalence of obesity. Despite heightened awareness of the disease, a significant percentage of patients are uncontrolled and are at higher risk of heart failure, stroke, and chronic kidney disease. Evidence of the cardiovascular protective role of estrogen in pre-menopausal females has brought attention to estrogen receptor activation as a treatment strategy for HTN. Estrogen promotes vasodilation and decreases inflammation and atherosclerosis. It also controls blood pressure via modulation of the activity of the renin-angiotensin-aldosterone system. The effects of estrogen on the vasculature are partly mediated via membrane receptors. Membrane estrogen receptor α and G-protein-coupled GPER-1 have been studied extensively in the vasculature. This review will describe the available evidence supporting the role of estrogen membrane receptors in blood pressure control and CVD.

Renin inhibition and AT(1)R blockade improve metabolic signaling, oxidant stress and myocardial tissue remodeling.

OBJECTIVE: Strategies that block angiotensin II actions on its angiotensin type 1 receptor or inhibit actions of aldosterone have been shown to reduce myocardial hypertrophy and interstitial fibrosis in states of insulin resistance. Thereby, we sought to determine if combination of direct renin inhibition with angiotensin type 1 receptor blockade in vivo, through greater reductions in systolic blood pressure (SBP) and aldosterone would attenuate left ventricular hypertrophy and interstitial fibrosis to a greater extent than either intervention alone. MATERIALS/METHODS: We utilized the transgenic Ren2 rat which manifests increased tissue expression of murine renin which, in turn, results in increased renin-angiotensin system activity, aldosterone secretion and insulin resistance. Ren2 rats were treated with aliskiren, valsartan, the combination (aliskiren+valsartan), or vehicle for 21 days. RESULTS: Compared to Sprague-Dawley controls, Ren2 rats displayed increased systolic blood pressure, elevated serum aldosterone levels, cardiac tissue hypertrophy, interstitial fibrosis and ultrastructural remodeling. These biochemical and functional alterations were accompanied by increases in the NADPH oxidase subunit Nox2 and 3-nitrotyrosine content along with increases in mammalian target of rapamycin and reductions in protein kinase B phosphorylation. Combination therapy contributed to greater reductions in systolic blood pressure and serum aldosterone but did not result in greater improvement in metabolic signaling or markers of oxidative stress, fibrosis or hypertrophy beyond either intervention alone. CONCLUSIONS: Thereby, our data suggest that the greater impact of combination therapy on reductions in aldosterone does not translate into greater reductions in myocardial fibrosis or hypertrophy in this transgenic model of tissue renin overexpression.