Comparative profiling of prenatal cortisol and DHEA-S among pregnant women with poor birth outcome and pregnant women with normal birth outcome.

CONTEXT: Cortisol and dehydroepiandrosterone-sulfate (DHEA-S) are indispensable hormones for normal pregnancy. It is unclear if these hormones, specifically DHEA-S can offer value for predicting poor birth outcome. OBJECTIVE: To compare prenatal cortisol and DHEA-S levels among pregnant women with normal or poor birth outcome. METHODS: Plasma and saliva were collected prospectively from women in second-third trimester of pregnancy. Women with normal birth outcome (NBO) (n = 501) included live birth, no pregnancy complications and ≥2.5 kg infant birth weight. Women with poor birth outcome included adverse birth outcome (ABO) (n = 50) or low birth weight outcome (LBW) (n = 147). Enzyme-linked immunosorbent assay was performed to measure hormone concentrations in plasma and saliva. RESULTS: Circulatory-DHEA-S levels in pregnant women with ABO were higher than women with NBO (p = .043). Among ABO, only stillbirth cases demonstrated significant increase in circulatory-DHEA-S levels (p = .006). Circulatory and salivary cortisol/DHEA-S ratio was lower among women with stillbirth (p = .004) and ABO outcome (p = .043) respectively compared with women with NBO. Consistently, increased odds of ABO were observed in pregnant women with highest circulatory-DHEA-S levels (odds ratio quartile score 1 vs. 4, 2.79, p = .027) and lowest salivary cortisol/DHEA-S ratio (score 4 vs. 2, 2.83, p = .025). Increased odds of stillbirth outcome were observed in pregnant women with highest circulatory-DHEA-S levels (odds ratio quartile score 1 vs. 4, 8.47, p = .046) and lowest circulatory cortisol/DHEA-S ratio (score 4 vs. 1, 4.803, p = .048). Associations remained significant after adjusting for confounders. Women with LBW did not demonstrate significant changes in cortisol or DHEA-S levels. CONCLUSION: Prenatal measurement of DHEA-S or cortisol/DHEA-S ratio may offer significant value for predicting adverse birth, specifically stillbirth outcome.

Comparative Profiling of Salivary Cortisol and Salivary DHEA-S Among Healthy Pregnant and Non-Pregnant Women.

During pregnancy, circulatory cortisol levels increase, remaining steady over the second-third trimester. In contrast, profile of salivary cortisol during pregnancy is debatable, more influenced by factors like time of sample collection in the day. Circulatory DHEA-S decrease by at least 50% over the second-third trimester of pregnancy. However, profile of salivary DHEA-S is unclear. Objective was to determine changes in salivary cortisol and DHEA-S in healthy pregnant women, compared to non-pregnant women during late morning-early afternoon sampling to avoid fluctuations associated with other times. Pregnant women in their second-third trimester prospectively (n=500) and non-pregnant women (n=133) were enrolled in study with informed consent. Live birth outcome with no pregnancy complications and≥2.5 Kg infant birth weight were included. Concentrations of salivary cortisol and DHEA-S were determined through ELISA assays. Compared to non-pregnant women, pregnant women demonstrated significant increases in salivary cortisol [median (interquartile range)=4.2 (5.1) nmol/l vs. 17.2 (13.9) nmol/l, p<0.001] and salivary DHEA-S median (interquartile range)=2.7 (2.9) nmol/l vs. 3.8 (3.2) nmol/l, p<0.001). Consistently, quartile scores representing higher levels of salivary cortisol and DHEA-S concentrations demonstrated significant association with pregnancy. Quartile scores representing higher salivary cortisol/DHEA-S ratio demonstrated significant association with pregnancy. Study suggests the indicated time range of saliva sampling might best parallel the established profile of circulatory cortisol in pregnant women. However, unlike cortisol, study indicates that the salivary DHEA-S profile is distinct from the well-known profile of circulatory DHEA-S during pregnancy. A combinatorial approach involving both salivary and circulatory compartments could provide comprehensive picture of DHEA-S and hypothalamus-pituitary-adrenal axis during pregnancy.

Remarkably Robust Antiviral Immune Response despite Combined Deficiency in Caspase-8 and RIPK3.

Caspase-8 (Casp8)-mediated signaling triggers extrinsic apoptosis while suppressing receptor-interacting protein kinase (RIPK) 3-dependent necroptosis. Although Casp8 is dispensable for the development of innate and adaptive immune compartments in mice, the importance of this proapoptotic protease in the orchestration of immune response to pathogens remains to be fully explored. In this study, Casp8-/-Ripk3-/- C57BL/6 mice show robust innate and adaptive immune responses to the natural mouse pathogen, murine CMV. When young, these mice lack lpr-like lymphoid hyperplasia and accumulation of either B220 + CD3+ or B220-CD3+CD4+ and CD8+ T cells with increased numbers of immature myeloid cells that are evident in older mice. Dendritic cell activation and cytokine production drive both NK and T cell responses to control viral infection in these mice, suggesting that Casp8 is dispensable to the generation of antiviral host defense. Curiously, NK and T cell expansion is amplified, with greater numbers observed by 7 d postinfection compared with either Casp8+/-Ripk3-/- or wild type (Casp8+/+Ripk3+/+ ) littermate controls. Casp8 and RIPK3 are natural targets of virus-encoded cell death suppressors that prevent infected cell apoptosis and necroptosis, respectively. It is clear from the current studies that the initiation of innate immunity and the execution of cytotoxic lymphocyte functions are all preserved despite the absence of Casp8 in responding cells. Thus, Casp8 and RIPK3 signaling is completely dispensable to the generation of immunity against this natural herpesvirus infection, although the pathways driven by these initiators serve as a crucial first line for host defense within virus-infected cells.

RIP1 suppresses innate immune necrotic as well as apoptotic cell death during mammalian parturition.

The pronecrotic kinase, receptor interacting protein (RIP1, also called RIPK1) mediates programmed necrosis and, together with its partner, RIP3 (RIPK3), drives midgestational death of caspase 8 (Casp8)-deficient embryos. RIP1 controls a second vital step in mammalian development immediately after birth, the mechanism of which remains unresolved. Rip1(-/-) mice display perinatal lethality, accompanied by gross immune system abnormalities. Here we show that RIP1 K45A (kinase dead) knockin mice develop normally into adulthood, indicating that development does not require RIP1 kinase activity. In the face of complete RIP1 deficiency, cells develop sensitivity to RIP3-mixed lineage kinase domain-like-mediated necroptosis as well as to Casp8-mediated apoptosis activated by diverse innate immune stimuli (e.g., TNF, IFN, double-stranded RNA). When either RIP3 or Casp8 is disrupted in combination with RIP1, the resulting double knockout mice exhibit slightly prolonged survival over RIP1-deficient animals. Surprisingly, triple knockout mice with combined RIP1, RIP3, and Casp8 deficiency develop into viable and fertile adults, with the capacity to produce normal levels of myeloid and lymphoid lineage cells. Despite the combined deficiency, these mice sustain a functional immune system that responds robustly to viral challenge. A single allele of Rip3 is tolerated in Rip1(-/-)Casp8(-/-)Rip3(+/-) mice, contrasting the need to eliminate both alleles of either Rip1 or Rip3 to rescue midgestational death of Casp8-deficient mice. These observations reveal a vital kinase-independent role for RIP1 in preventing pronecrotic as well as proapoptotic signaling events associated with life-threatening innate immune activation at the time of mammalian parturition.

T cell immunoglobulin and mucin protein-3 (Tim-3)/Galectin-9 interaction regulates influenza A virus-specific humoral and CD8 T-cell responses.

Reactions to pathogens are usually tuned to effect immunity and limit tissue damage. Several host counterinflammatory mechanisms inhibit tissue damage but these may also act to constrain the effectiveness of immunity to acute infections, as we demonstrate in mice acutely infected with influenza A virus (IAV). We show that compared with wild type (WT), galectin-9 knockout (G9KO) mice mounted a more robust acute phase virus-specific CD8 T-cell response as well as higher and more rapid virus-specific serum IgM, IgG, and IgA responses and also cleared virus more rapidly than did WT mice. Blocking galectin-9 signals to Tim-3-expressing cells using a Tim-3 fusion protein resulted in improved immune responses in WT mice. When IAV immune mice were challenged with a heterologous IAV, the secondary IAV-specific CD8 T-cell responses were four- to fivefold higher in G9KO compared with WT mice. Our results indicate that manipulating galectin signals may represent a convenient approach to improve immune responses to some vaccines.

Impact of COVID-19 on women and children and the need for a gendered approach in vaccine development.

The COVID-19 pandemic has imposed unprecedented health and socioeconomic challenges on public health, disrupting it on a global scale. Given that women and children are widely considered the most vulnerable in the times of emergency, whether in war or during a pandemic, the current pandemic has also severely disrupted access to reproductive and child health services. Despite this, data on the effect of the pandemic on pregnant women and newborns remain scarce, and gender-disaggregated indicators of mortality and morbidity are not available. In this context, we suggest the implementation of a gendered approach to ensure the specific needs of women and their newborns are considered during the development of COVID-19 vaccines. Taking into account gender-based biological differences, the inclusion of pregnant and lactating mothers in clinical trials for the development of COVID-19 vaccines is of vital importance.

Ramping up of SARS CoV-2 testing for the diagnosis of COVID-19 to better manage the next phase of pandemic and reduce the mortality in India.

The coronavirus disease 2019 (COVID-19) pandemic is caused by the severe acute respiratory syndrome coronavirus-2, a new member of the Coronavirus family. The virus was first identified in Wuhan, China, where the epidemic originated. The viral genome was sequenced and a real time reverse transcription polymerase chain reaction assay was developed and used for the detection of virus. Different countries took different approaches for the diagnosis of COVID-19. Some countries prioritized extensive testing for COVID-19 at a very early phase of the pandemic whereas other countries took a long time to build the testing capacity and to implement the testing extensively. The assay design formats were available in the public domain and thereby allowing researchers to replicate them to make diagnostic kits. Consequently, several antigen or antibody-based diagnostic tests were also developed for the diagnosis of COVID-19. However, there were some validation and regulatory challenges while bringing these assays into the market. During the course of the pandemic, it became clear that the countries which implemented testing at an early stage of the pandemic were capable of controlling the spread more effectively than those that implemented them at later stages. As several countries implemented a lockdown for controlling the spread of the virus, it is critical to build the testing capability to meet the extensive need of testing while exiting the lockdown. Testing and isolation of positive cases are the most effective ways of preventing the spread of virus and gradually returning life back to normality.

Robust mucosal-homing antibody-secreting B cell responses induced by intramuscular administration of adjuvanted bivalent human norovirus-like particle vaccine.

BACKGROUND: Two major antigenically heterogenous norovirus genogroups (GI and GII) commonly infect humans and are the leading cause of foodborne, viral gastrointestinal infections in adults. METHODS: We assessed B cell responses in participants in a double-blind, placebo-controlled, dose-escalation phase 1 study of the safety and immunogenicity of an intramuscular bivalent norovirus virus-like particle (VLP) vaccine. The vaccine contained a GI.1 VLP (Norwalk) and a consensus GII.4 VLP, representing the two major genotypes that cause human disease, and was administered on days 0 and 28 to healthy adults aged 18-49 years. Four separate cohorts received increasing doses of 5 µg, 15 µg, 50 µg, and 150 µg of each VLP adjuvanted in monophosphoryl lipid A and alum. PBMCs were analyzed for B cell activation and mucosal homing markers (flow cytometry) and VLP-specific and total IgG and IgA Ab-secreting cells (ASCs); and serum titers of VLP-specific IgG, IgA, and Pan-Ig were determined. RESULTS: The vaccine elicited CD27+ CD38+ plasmablasts and high frequencies of ASCs specific for both VLP antigens in the peripheral blood at 7 days after the first dose. The plasmablasts exhibited a mucosal-homing phenotype and included a high proportion of IgA ASCs. Serum antibodies increased as early as 7 days after the first immunization. CONCLUSIONS: The data suggest that a single dose of the IM bivalent norovirus vaccine is effective in activating pre-existing B cell memory. The rapid B cell response and the mucosal homing phenotype of induced ASCs are consistent with anamnestic responses in subjects primed by prior oral norovirus infection. This study is registered at ClinicalTrials.gov Identifier NCT01609257.

B cell response and hemagglutinin stalk-reactive antibody production in different age cohorts following 2009 H1N1 influenza virus vaccination.

The 2009 pandemic H1N1 (pH1N1) influenza virus carried a swine-origin hemagglutinin (HA) that was closely related to the HAs of pre-1947 H1N1 viruses but highly divergent from the HAs of recently circulating H1N1 strains. Consequently, prior exposure to pH1N1-like viruses was mostly limited to individuals over the age of about 60 years. We related age and associated differences in immune history to the B cell response to an inactivated monovalent pH1N1 vaccine given intramuscularly to subjects in three age cohorts: 18 to 32 years, 60 to 69 years, and ≥70 years. The day 0 pH1N1-specific hemagglutination inhibition (HAI) and microneutralization (MN) titers were generally higher in the older cohorts, consistent with greater prevaccination exposure to pH1N1-like viruses. Most subjects in each cohort responded well to vaccination, with early formation of circulating virus-specific antibody (Ab)-secreting cells and ≥4-fold increases in HAI and MN titers. However, the response was strongest in the 18- to 32-year cohort. Circulating levels of HA stalk-reactive Abs were increased after vaccination, especially in the 18- to 32-year cohort, raising the possibility of elevated levels of cross-reactive neutralizing Abs. In the young cohort, an increase in MN activity against the seasonal influenza virus A/Brisbane/59/07 after vaccination was generally associated with an increase in the anti-Brisbane/59/07 HAI titer, suggesting an effect mediated primarily by HA head-reactive rather than stalk-reactive Abs. Our findings support recent proposals that immunization with a relatively novel HA favors the induction of Abs against conserved epitopes. They also emphasize the need to clarify how the level of circulating stalk-reactive Abs relates to resistance to influenza.

Host differences in influenza-specific CD4 T cell and B cell responses are modulated by viral strain and route of immunization.

The antibody response to influenza infection is largely dependent on CD4 T cell help for B cells. Cognate signals and secreted factors provided by CD4 T cells drive B cell activation and regulate antibody isotype switching for optimal antiviral activity. Recently, we analyzed HLA-DR1 transgenic (DR1) mice and C57BL/10 (B10) mice after infection with influenza virus A/New Caledonia/20/99 (NC) and defined epitopes recognized by virus-specific CD4 T cells. Using this information in the current study, we demonstrate that the pattern of secretion of IL-2, IFN-γ, and IL-4 by CD4 T cells activated by NC infection is largely independent of epitope specificity and the magnitude of the epitope-specific response. Interestingly, however, the characteristics of the virus-specific CD4 T cell and the B cell response to NC infection differed in DR1 and B10 mice. The response in B10 mice featured predominantly IFN-γ-secreting CD4 T cells and strong IgG2b/IgG2c production. In contrast, in DR1 mice most CD4 T cells secreted IL-2 and IgG production was IgG1-biased. Infection of DR1 mice with influenza PR8 generated a response that was comparable to that in B10 mice, with predominantly IFN-γ-secreting CD4 T cells and greater numbers of IgG2c than IgG1 antibody-secreting cells. The response to intramuscular vaccination with inactivated NC was similar in DR1 and B10 mice; the majority of CD4 T cells secreted IL-2 and most IgG antibody-secreting cells produced IgG2b or IgG2c. Our findings identify inherent host influences on characteristics of the virus-specific CD4 T cell and B cell responses that are restricted to the lung environment. Furthermore, we show that these host influences are substantially modulated by the type of infecting virus via the early induction of innate factors. Our findings emphasize the importance of immunization strategy for demonstrating inherent host differences in CD4 T cell and B cell responses.

Quantitative analysis of influenza virus-specific B cell memory generated by different routes of inactivated virus vaccination.

We consider both Ab-secreting cell (ASC) and memory B cell (B(Mem)) populations in a quantitative analysis of virus-specific B cell memory generated by intramuscular or intranasal vaccination of mice with inactivated influenza virus. After both forms of vaccination, the memory phase was characterized by localization of ASCs in the bone marrow and dispersion of B(Mem) to organized lymphoid tissues. The stronger IgG response to intramuscular vaccination correlated with larger numbers of IgG ASCs in the bone marrow and IgG B(Mem). IgA production was only prominent in the response to intranasal vaccination and was associated with IgA ASC localization in the lung and IgA B(Mem) formation. Notably, few IgG ASCs or B(Mem) localized in the lung after intramuscular vaccination, in contrast to the situation following influenza pneumonia. Our analysis links the nature of immunization to characteristics of the state of B cell memory that may relate to protective immunity.

Influenza virus variation in susceptibility to inactivation by pomegranate polyphenols is determined by envelope glycoproteins.

Pomegranates have high levels of polyphenols (PPs) and may be a rich source of compounds with antiviral activity. We evaluated the direct anti-influenza activity of three commercially available pomegranate extracts: pomegranate juice (PJ), a concentrated liquid extract (POMxl), and a 93% PP powder extract (POMxp). The acidity of PJ and POMxl solutions contributed to rapid anti-influenza activity, but this was not a factor with POMxp. Studies using POMxp showed that 5min treatment at room temperature with 800µg/ml PPs resulted in at least a 3log reduction in the titers of influenza viruses PR8 (H1N1), X31 (H3N2), and a reassortant H5N1 virus derived from a human isolate. However, the antiviral activity was less against a coronavirus and reassortant H5N1 influenza viruses derived from avian isolates. The loss of influenza infectivity was frequently accompanied by loss of hemagglutinating activity. PP treatment decreased Ab binding to viral surface molecules, suggesting some coating of particles, but this did not always correlate with loss of infectivity. Electron microscopic analysis indicated that viral inactivation by PPs was primarily a consequence of virion structural damage. Our findings demonstrate that the direct anti-influenza activity of pomegranate PPs is substantially modulated by small changes in envelope glycoproteins.