Association of Polymorphisms in FSHR, ESR1, and BMP15 with Primary Ovarian Insufficiency and Meta-Analysis.

Primary ovarian insufficiency (POI) can lead to menstrual disturbance, resulting in ovarian dysfunction before age 40. Prevalence of POI is usually less than 1%; however, ethnicity or population characteristics may affect prevalence. POI is a heterogeneous disease that results from abnormalities in immunological and hormonal factors. Genetic factors can also contribute to POI. Here, we examine FSHR, ESR1, and BMP15 polymorphisms in patients with POI, and controls. We examined a hormonal gene that is important for pregnancy, follicle-stimulating hormone receptor (FSHR), as well as estrogen receptor 1 (ESR1), and associated it with FSHR expression, ovulation rate, and bone morphogenetic protein 15 (BMP15). We examined 139 Korean patients under age 40 with POI, and 350 Korean control participants without POI. Genotyping was performed by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and TaqMan assays. Each identified genotype was subjected to statistical analysis to determine the odds ratios (ORs) and 95% confidence intervals (CIs). In combination genotype analyses, FSHR rs6165 A > G combined with ESR1 rs9340799 A > G, AG/GG (OR: 5.693; 95% CI: 1.088-29.792), as well as FSHR rs6166 A > G combined with ESR1 rs9340799 C > T, AG/GG (OR: 5.940; 95% CI: 1.134-31.131), were significantly associated with POI prevalence. Furthermore, an FSHR rs6165 A > G and BMP rs17003221 C > T, AG/CC combination was associated with POI prevalence (OR: 1.874; 95% CI: (1.059-3.316; p-value: 0.031)). In meta-analysis, FSHR rs6165 AA vs. AG + GG is associated with POI (p = 0.0013), and ESR1 rs2234693 AA vs. AG + GG is also associated with POI (p = 0.0101). Here, we compared the genotypes of FSHR, ESR1, and BMP15 in patients with POI, and controls. We found significant differences in genotype combinations between polymorphisms in FSHR and other genes. Through meta-analysis, we found that ESR1 rs9340799 and rs2234693 are associated with POI prevalence, and that BMP15 rs17003221 increases POI risk. These findings help to improve POI diagnosis in Korean women.

Association between TGF-ß/BMP signaling pathway polymorphisms and the risk of primary ovarian insufficiency in Korean women.

BACKGROUND: Primary ovarian insufficiency (POI) is one of the leading female infertility diseases in which ovarian function stops before the age of 40. Reports that POI is associated with transforming growth factor (TGF)-ß/bone morphogenetic protein (BMP) signaling pathway-associated genes (e.g., TGF-ß, and BMP15) have been continuous since publication that the TGF-ß superfamily acts as important regulators for ovary and placenta function in humans. Mechanistically, the secretion of follicle-stimulating hormone, progesterone, and estrogen is affected by the TGF-ß superfamily in granulosa cells, which are involved in the development of theca cells, oocytes, and granulosa cells. OBJECTIVE: This study aimed to identify the association between genes related to the TGF-ß/BMP signaling pathway and the risk of POI pathogenesis. METHODS: Possible associations between six gene polymorphisms and POI susceptibility were examined in 139 patients with POI and 345 control subjects. RESULTS: Allele combination of TGFBR1 rs334348 G > A and TGFBR3 rs1805110G > A exhibited association with decreased POI risk (adjusted odds ratio [AOR] = 0.165; 95% confidence interval [CI] 0.032-0.847; P = 0.031). Also, TGFBR1 rs1590 G > T and rs334348 G > A and TGFBR3 rs1805110 G > A allele combination exhibited association with decreased POI risk (OR = 0.553; 95% CI 0.374-0.816; P = 0.003). CONCLUSION: This study suggests that polymorphisms in the TGF-ß signaling pathway genes can be useful biomarkers for POI diagnosis and treatment.

Genetic Correlation of miRNA Polymorphisms and STAT3 Signaling Pathway with Recurrent Implantation Failure in the Korean Population.

The growing prevalence of in vitro fertilization-embryo transfer procedures has resulted in an increased incidence of recurrent implantation failure (RIF), necessitating focused research in this area. STAT3, a key factor in maternal endometrial remodeling and stromal proliferation, is crucial for successful embryo implantation. While the relationship between STAT3 and RIF has been studied, the impact of single nucleotide polymorphisms (SNPs) in miRNAs, well-characterized gene expression modulators, on STAT3 in RIF cases remains uncharacterized. Here, we investigated 161 RIF patients and 268 healthy control subjects in the Korean population, analyzing the statistical association between miRNA genetic variants and RIF risk. We aimed to determine whether SNPs in specific miRNAs, namely miR-218-2 rs11134527 G>A, miR-34a rs2666433 G>A, miR-34a rs6577555 C>A, and miR-130a rs731384 G>A, were significantly associated with RIF risk. We identified a significant association between miR-34a rs6577555 C>A and RIF prevalence (implantation failure [IF] ≥ 2: adjusted odds ratio [AOR] = 2.264, 95% CI = 1.007-5.092, p = 0.048). These findings suggest that miR-34a rs6577555 C>A may contribute to an increased susceptibility to RIF. However, further investigations are necessary to elucidate the precise mechanisms underlying the role of miR-34a rs6577555 C>A in RIF. This study sheds light on the genetic and molecular factors underlying RIF, offering new avenues for research and potential advancements in the diagnosis and treatment of this complex condition.

Genetic Association between Inflammatory-Related Polymorphism in STAT3, IL-1ß, IL-6, TNF-α and Idiopathic Recurrent Implantation Failure.

Recurrent implantation failure (RIF) is defined as a failure to achieve pregnancy after multiple embryo transfers. Implantation is closely related to inflammatory gradients, and interleukin-1beta (IL-1ß), IL-6, and tumor necrosis factor-alpha (TNF-α) play a key role in maternal and trophoblast inflammation during implantation. Signal transducer and activator of transcription 3 (STAT3) interacts with cytokines and plays a critical role in implantation through involvement in the inflammation of the embryo and placenta. Therefore, we investigated 151 RIF patients and 321 healthy controls in Korea and analyzed the association between the polymorphisms (STAT3 rs1053004, IL-1ß rs16944, IL-6 rs1800796, and TNF-α rs1800629, 1800630) and RIF prevalence. In this paper, we identified that STAT3 rs1053004 (AG, adjusted odds rate [AOR] = 0.623; p = 0.027; GG, AOR = 0.513; p = 0.043; Dominant, AOR = 0.601, p = 0.011), IL-6 rs1800796 (GG, AOR = 2.472; p = 0.032; Recessive, AOR = 2.374, p = 0.037), and TNF-α rs1800629 (GA, AOR = 2.127, p = 0.010, Dominant, AOR = 2.198, p = 0.007) have a significant association with RIF prevalence. This study is the first to investigate the association of each polymorphism with RIF prevalence in Korea and to compare their effect based on their function on inflammation.

Genetic variants of MUC4 are associated with susceptibility to and mortality of colorectal cancer and exhibit synergistic effects with LDL-C levels.

As a disease with high mortality and prevalence rates worldwide, colorectal cancer (CRC) has been thoroughly investigated. Mucins are involved in the induction of CRC and the regulation of intestinal homeostasis but a member of the mucin gene family MUC4 has a controversial role in CRC. MUC4 has been associated with either decreased susceptibility to or a worse prognosis of CRC. In our study, the multifunctional aspects of MUC4 were elucidated by genetic polymorphism analysis in a case-control study of 420 controls and 464 CRC patients. MUC4 rs1104760 A>G polymorphism had a protective effect on CRC risk (AG, AOR = 0.537; GG, AOR = 0.297; dominant model, AOR = 0.493; recessive model, AOR = 0.382) and MUC4 rs2688513 A>G was associated with an increased mortality rate of CRC (5 years, GG, adjusted HR = 6.496; recessive model, adjusted HR = 5.848). In addition, MUC4 rs1104760 A>G showed a high probability of being a potential biomarker for CRC patients with low-density lipoprotein cholesterol (LDL-C) in the risk range while showing a significant synergistic effect with the LDL-C level. This is the first study to indicate a significant association between MUC4 genetic polymorphisms and CRC prevalence, suggesting a functional genetic variant with the LDL-C level, for CRC prevention.

In vitro effects of monophosphoryl lipid A and Poly I:C combination on equine cells.

BACKGROUND: Toll-like receptor (TLR) agonists have been used as adjuvants to modulate immune responses in both animals and humans. OBJECTIVES: The objective of this study was to evaluate the combined effects of the TLR 4 agonist monophosphoryl lipid A (MPL) and the TLR 3 agonist polyinosinic:polycytidylic acid (Poly I:C) on equine peripheral blood mononuclear cells (PBMCs), monocyte-derived dendritic cells (MoDCs), and bone marrow-derived mesenchymal stromal cells (BM-MSCs). METHODS: The PBMCs, MoDCs, and BM-MSCs collected from three mixed breed horses were treated with MPL, Poly I:C, and their combination. The mRNA expression of interferon gamma (IFN-γ), interleukin (IL)-1ß, IL-4, IL-6, IL-8, IL-12p40, tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), and monocyte chemoattractant protein-1 (MCP-1) was determined using real-time polymerase chain reaction. RESULTS: The combination of MPL and Poly I:C significantly upregulated immunomodulatory responses in equine cells/ without cytotoxicity. The combination induced greater mRNA expression of pro-inflammatory cytokines IFN-γ and IL-6 than MPL or Poly I:C stimulation alone in PBMCs. In addition, the combination induced significantly higher mRNA expression of IL-1ß, IL-6, and IL-12p40 in MoDCs, and IL-8, MCP-1, and VEGF in BM-MSCs compared to stimulation with a single TLR agonist. CONCLUSIONS: The combination of MPL and Poly I:C can be used as a potential adjuvant candidate for vaccines to aid in preventing infectious diseases in horses.

Association of Polymorphisms in the Long Non-Coding RNA HOTAIR with Recurrent Pregnancy Loss in a Korean Population.

Recurrent pregnancy loss (RPL) affects 1% to 5% of women, with devastating effects on both reproductive health and psychological well-being. Homeobox (HOX) transcript antisense RNA (HOTAIR) is a long non-coding RNA (lncRNA) produced by HOXC; it plays a major role in invasion and development of ovarian and other cancers. The aim of the present study was to analyze effects of HOTAIR polymorphisms (rs4759314 A>G, rs920778 T>C, rs1899663 G>T, and rs7958904 G>C) on RPL in Korean women. A total of 403 women with RPL and 383 healthy women were selected for this study. Genotyping analysis was performed with the polymerase chain reaction, restriction fragment length polymorphism, and the TaqMan genotyping assay. Clinical characteristics were compared using Student's unpaired t-test and the chi-square test for categorical variables. Logistic regression was performed to evaluate associations between single nucleotide polymorphisms and RPL incidence. In all assays, p < 0.05 was considered significant. HOTAIR polymorphisms rs4759314A>G and rs920778T>C were highly associated with increased risk of RPL, specifically the haplotypes rs4759314A>G/rs1899663G>T (G-T) and rs4759314A>G/rs920778 T>C (G-C). These associations were maintained in haplotypes that contained three polymorphisms (rs4759314 A>G, rs920778 T>C, and rs1899663 G>T) A-C-G, G-T-G, and G-T-T, further indicating that the HOTAIR rs4759314 and rs920778 polymorphisms play significant roles in idiopathic RPL in Korean women.

Study of the Association between VEGF Polymorphisms and the Risk of Coronary Artery Disease in Koreans.

Coronary artery disease (CAD), a leading cause of death worldwide, has a complex etiology comprising both traditional risk factors (type 2 diabetes, dyslipidemia, arterial hypertension, and cigarette smoking) and genetic factors. Vascular endothelial growth factor (VEGF) notably contributes to angiogenesis and endothelial homeostasis. However, little is known about the relationship between CAD and VEGF polymorphisms in Koreans. The aim of this study is to investigate the associations of 2 VEGF promoter region polymorphisms (−1154G>A [rs1570360], −1498T>C [rs833061]) and 4 VEGF 3'-UTR polymorphisms (+936C>T [rs3025039], +1451C>T [rs3025040], +1612G>A [rs10434], and +1725G>A [rs3025053]) with CAD susceptibility in Koreans. We studied 885 subjects: 463 CAD patients and 422 controls. Genotyping was conducted with polymerase chain reaction-restriction fragment length polymorphism analysis and TaqMan allelic discrimination assays, and the genotype frequencies were calculated. We then performed haplotype and genotype combination analyses and measured the associations between VEGF polymorphisms and clinical variables in both the CAD patients and control subjects. We detected statistically significant associations between CAD and certain VEGF allele combinations. In the haplotypes of 5 single-nucleotide polymorphisms, the VEGF allele combination −1154A/+936T was associated with a decreased prevalence of CAD (A-T-T-G-G of VEGF −1154G>A/−1498T>C/+936C>T/+1612G>A/+1725G>A, AOR = 0.077, p = 0.021). In contrast, the VEGF allele combinations −1498T/+1725A and −1498T/+1612A/+1725A were associated with an increased prevalence of CAD (G-T-C-C-A of VEGF −1154G>A/−1498T>C/+936C>T/+1451C>T/+1725G>A, AOR = 1.602, p = 0.047; T-C-C-A-A of VEGF −1498T>C/+936C>T/+1451C>T/+1612G>A/+1725G>A, AOR = 1.582, p = 0.045). Gene−environment combinatorial analysis showed that the combination of the VEGF +1725AA genotype and several clinical factors (e.g., body mass index, hemoglobin A1c, and low-density lipoprotein cholesterol) increased the risk of CAD. Therefore, we suggest that VEGF polymorphisms and clinical factors may impact CAD prevalence.

Prognostic significance of three endothelial nitric oxide synthase (eNOS) polymorphisms and metabolic syndrome (MetS) in patients with colorectal cancer.

BACKGROUND: Polymorphisms of endothelial nitric oxide synthases (eNOS) have been associated with cancer susceptibility. Also, metabolic syndrome is associated with cancer malignancy. However, the effect of eNOS polymorphisms and metabolic syndrome on colorectal cancer (CRC) prognosis remains unclear. OBJECTIVE: To investigated whether three genetic polymorphisms (- 786 T > C rs2070744, 4a4b rs869109213, and 894G > T rs1799983) in the eNOS and metabolic syndrome (MetS) were associated with CRC patient survival. METHODS: We genotyped three polymorphisms of eNOS (- 786 T > C, 4a4b, and 894G > T) in 312 CRC cases from the Korean population by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. RESULTS: Although the three eNOS polymorphisms were not causative of MetS, the TT genotype of the 894G > T polymorphism was associated with a worse survival rate compared with the GG genotype in the CRC group with MetS than in the CRC group without MetS (5-years survival; adjusted HR = 54.777; 95% CI 5.073-591.487 and RFS; adjusted HR = 14.909; 95% CI 1.571-141.528). CONCLUSIONS: The eNOS polymorphisms were not associated with metabolic syndrome prevalence in CRC patients. However, our findings suggest that the eNOS 894G > T polymorphism with MetS was associated with poor clinical outcomes.

Multi-Center, Randomized, Double-Blind, Placebo-Controlled, Exploratory Study to Evaluate the Efficacy and Safety of HAD-B1 for Dose-Finding in EGFR Mutation Positive and Locally Advanced or Metastatic NSCLC Subjects Who Need Afatinib Therapy.

Afatinib is a target anticancer drug of the second-generation EGFR TKI type, showing an advantage in treatment effect compared to conventional chemotherapy. However, patients on EGFR-TKI drugs also usually progress after 9 to 13 months according to secondary resistance. HAD-B1 is composed of drugs that are effective against lung cancer. This study is an exploratory study to evaluate the efficacy and safety between dosage groups by conducting a clinical trial in subjects requiring afatinib drug treatment in non-small cell lung cancer with EGFR mutation positive to determine the optimal dosage for HAD-B1 administration. At the final visit compared to before administration, each change in the disease control rate was measured according to the HAD-B1 doses of the test group 1 (972 mg), the test group 2 (1944 mg), and the control group. The efficacy and safety of HAD-B1 were compared and evaluated through sub-evaluation variables. As a result of the study, there was no statistically significant difference in the disease control rate at 12 weeks after dosing, but complete and partial remission were evaluated as 1 patient each in the test group 1, and none in the other groups. There was no statistically significant difference between groups in the sub-evaluation variable. In addition, there was no problem of safety from taking the test drug. However, the initially planned number of subjects was 66, but the number of enrolled subjects was only 14, which may limit the results of this study.

Association between HOTAIR lncRNA Polymorphisms and Coronary Artery Disease Susceptibility.

Coronary artery disease (CAD), one of the most frequent causes of mortality, is the most common type of cardiovascular disease. This condition is characterized by the accumulation of plaques in the coronary artery, leading to blockage of blood flow to the heart. The main symptom of CAD is chest pain caused by blockage of the coronary artery and shortness of breath. HOX transcript antisense RNA gene (HOTAIR) is a long non-coding RNA which is well-known as an oncogene involved in various cancers, such as lung, breast, colorectal, and gastric cancer. We selected six single nucleotide polymorphisms, rs4759314 A>G, rs1899663 G>T, rs920778 T>C, rs7958904 G>C, rs12826786 C>T, and rs874945 C>T, for genotype frequency analysis and assessed the frequency of HOTAIR gene polymorphisms in 442 CAD patients and 418 randomly selected control subjects. To analyze the differences between these two populations, we performed a Student's t-test, adjusted odds ratio (AOR), 95% confidence intervals (CIs), and ANOVA analysis. According to our baseline characteristic analysis, control subjects and CAD patients were significantly different in hypertension and diabetes mellitus. We also found that the rs4759314 A>G, rs1899663 G>T, and rs12826786 C>T genotypes were strongly associated with CAD susceptibility (AA vs. AG+GG: AOR = 0.608, 95% CI = 0.393-0.940, p = 0.025; GG vs. TT: AOR = 2.276, 95% CI = 1.125-4.607, p = 0.022; CC vs. CT+TT: AOR = 1.366, 95% CI = 1.027-1.818, p = 0.032, respectively). Our data also demonstrated that the genotype of HOTAIR polymorphisms, genotype combination, and haplotype analysis affect disease occurrence. Moreover, these polymorphisms are linked to clinical factors that contribute to disease susceptibility. In conclusion, results from our study suggest that HOTAIR polymorphisms may be useful novel biomarkers for diagnosing CAD.

BCG Cell Wall Skeleton As a Vaccine Adjuvant Protects Both Infant and Old-Aged Mice from Influenza Virus Infection.

Bacillus Calmette-Guerin (BCG) and the cell wall skeleton (CWS) derived from BCG are known to enhance nonspecific immune activation and anti-cancer immunity; however, their roles as a vaccine adjuvant are largely unknown. Here, we report that BCG-CWS acts as a strong immune adjuvant by promoting the protective immune responses in mouse models with influenza vaccination. The different aged mice immunized with inactivated split vaccine with or without BCG-CWS were challenged with an influenza pandemic virus. When protective immune responses were compared, even a single immunization of adult mice with a BCG-CWS-adjuvanted vaccine showed significantly enhanced humoral immune responses with increased IgG1 and IgG2a isotype antibodies. Importantly, the protective effects by the BCG-CWS adjuvant for influenza vaccination upon humoral and cellular immunogenicity were comparable between infants (6 days and 2 weeks old) and aged (20 months old) mice. Moreover, BCG-CWS dramatically augmented vaccine-mediated protective responses, including decreased viral loads, lung damage, and airway resistance, as well as increased mouse survival, amelioration of weight loss, and proinflammatory cytokine expression in all experimental groups including infant, adults, and old aged mice. We further provided the evidence that the BCG-CWS adjuvant effects were mediated through Toll-like receptors (TLR) 2 and TLR4 signaling pathways. Together, these data suggest that BCG-CWS can be promising as a potential influenza vaccine adjuvant in both young and old aged population through TLR2/4-mediated immune-boosting activities.

The 3'-UTR Polymorphisms in the Thymidylate Synthase (TS) Gene Associated with the Risk of Ischemic Stroke and Silent Brain Infarction.

Thymidylate synthase (TS) is a key gene involved in the repair of DNA damage and DNA synthesis that plays an important role in vascular development and recovery. In particular, TS gene polymorphisms play a major role in the progression of vascular disease and cancer metastasis. Therefore, the aim of this study was to investigate the association of three TS polymorphisms (1100T>C [rs699517], 1170A>G [rs2790], and 1494ins/del [rs151264360]) with ischemic stroke and silent brain infarction (SBI) in Koreans. A total of 1299 participants (507 stroke patients, 383 SBI patients, and 409 controls) were enrolled in the study. Genotyping of the three TS polymorphisms was performed by polymerase chain reaction-restriction fragment length polymorphism analysis. To examine the association between TS gene polymorphisms and the diseases, we performed statistical analyses, including multivariable logistic regression and Fisher's exact tests. We found that TS 1100T>C and 1170A>G genotypes were strongly associated with ischemic stroke and SBI susceptibility. More specifically, the TS 1100T>C polymorphism was associated with the likelihood of ischemic stroke (TT vs. CC: AOR = 2.151, 95% CI = 1.275-3.628, P = 0.004) and SBI (TT vs. TC+CC: AOR = 1.443, 95 % CI = 1.009-2.063, P = 0.045). In contrast, the TS 1170A > G polymorphism exhibited lower correlation with the risk of stroke (AA vs. GG: AOR = 0.284, 95% CI = 0.151-0.537, P < 0.0001) and SBI (AA vs. GG: AOR = 0.070, 95% CI = 0.016-0.298, P = 0.0002). Furthermore, we confirmed that the TS 1100T>C polymorphism was synergistic with low folic acid levels (AOR = 6.749, P < 0.0001). Altogether, these results suggest that TS 1100T>C and 1170A > G polymorphisms are associated with the risk of ischemic stroke and SBI, and our study provides the first evidence that 3'-UTR variants in TS are potential biomarkers in ischemic stroke and SBI.

Natural killer cells contribute to enhanced respiratory disease after oil-in-water emulsion adjuvanted vaccination against respiratory syncytial virus and infection.

Respiratory syncytial virus (RSV) infection caused severe acute respiratory disease in children and the elderly. There is no licensed vaccine. It has been a challenging problem to avoid vaccine enhanced respiratory disease in developing a safe and effective RSV vaccine. Here, we investigated the impact of MF59-like oil-in-water emulsion adjuvant Addavax on the vaccine efficacy of inactivated split RSV (sRSV) and the roles of natural killer (NK) cells in enhanced respiratory disease in sRSV vaccinated mice after RSV infection. Addavax-adjuvanted sRSV vaccination induced higher levels of IgG1 isotype antibodies and more effective lung viral clearance upon RSV infection but promoted enhanced respiratory disease of weight loss, pulmonary inflammation, and NK and NK T (NKT) cell infiltrations in the lungs. Antibody treatment depleting NK cells prior to RSV infection resulted in preventing severe weight loss and histopathology, as well as attenuating infiltration of dendritic cell subsets and TNF-α+ T cells in the lungs. This study demonstrated the impacts of oil-in-water emulsion adjuvant on sRSV vaccination and the potential roles of NK and NKT cells in protection and respiratory disease after adjuvanted RSV vaccination and infection in a mouse model.

Association of genetic variants of RNF213 with ischemic stroke risk in Koreans.

BACKGROUND: Large artery disease (LAD), cardioembolism (CE), and small vessel disease (SVD) are well-established causes of ischemic stroke. Although a founder variant of RNF213 has been regarded a genetic susceptibility for Moyamoya disease (MMD) and certain types of intracranial atherosclerotic stenosis (ICAS), correlations between RNF213 variants and ischemic stroke with SVD remain largely unknown. OBJECTIVES: This study aimed to characterize the associations of four RNF213 polymorphisms (4448G>A, 4810G>A, 4863G>A, and 4950G>A) with ischemic stroke subtypes in Koreans. METHODS: Genetic data from 529 stroke patients were analyzed and compared to 424 age- and sex-matched controls. Genetic variants of RNF213, as obtained from the Human Gene Mutation Database, were analyzed in the study subjects using the polymerase chain reaction restriction fragment length polymorphism assay. We investigated four single-nucleotide polymorphisms of RNF213 to elucidate their association with ischemic stroke [LAD, (n = 192), SVD (n = 145) and CE (n = 51)]. RESULTS: The RNF213 4950G>A genotype was observed more frequently in cerebral stroke patients and was more strongly associated with SVD than LAD (P = 0.014). RNF213 4448/4950 in combination with G-A was higher in SVD patients. However, the RNF213 4863/4950 allele combination was associated with increased risk of SVD and LAD. These results confirmed that RNF213 4950GA+AA variants were more frequent in ischemic stroke, especially in SVD, and that RNF213 G-G-G-A and G-G-G-A (4448/4810/4863/4950) haplotype sequences play a role in LAD and CE as well as SVD. CONCLUSIONS: Our data reported that the RNF213 4950G>A genotypes and several RNF213 (4448/4810/4863/4950) haplotypes were associated with ischemic stroke in Koreans.

A Mucosal Adenovirus Prime/Systemic Envelope Boost Vaccine Regimen Elicits Responses in Cervicovaginal and Alveolar Macrophages of Rhesus Macaques Associated With Delayed SIV Acquisition and B Cell Help.

Vaccine strategies targeting the mucosal portal of entry may prevent HIV acquisition and systemic infection. Macrophages in cervicovaginal compartments are one of the first cell types to encounter virus upon vaginal exposure. Their activation can lead to recruitment of additional macrophages and CD4+ T-cells susceptible to viral infection. However, they are also critical in providing early protection against invading pathogens. Therefore, understanding their response to immunization is important for vaccine design. We immunized rhesus macaques twice mucosally with replicating adenovirus (Ad) SIV recombinants, followed by two intramuscular boosts with SIV gp120 protein. Macaques were subsequently challenged intravaginally with repeated low doses of SIVmac251. Using flow cytometry, we evaluated responses of cervicovaginal macrophages (CVM) and alveolar macrophages (AM) in bronchoalveolar lavage as initial immunization was to the upper respiratory tract. The frequency of CVM increased over the course of immunization; however, CCR5 expression significantly decreased. Significantly increased expression of the chemokines CCL3 (p < 0.01), CCL4, CCL5, and CXCL8 (p < 0.0001 for all) on CVM was seen post-1st Ad but their expression significantly decreased post-2nd boost. CD4+ T-cell frequency in the cervical mucosa remained unchanged. CVM FcγRIII expression was significantly increased at all time points post-immunization compared to naïve animals. FcγRIII expression post-2nd Ad positively correlated with the number of challenges needed for infection (r = 0.68; p = 0.0051). Vaccination increased AM FcγRIII expression which post-2nd boost correlated with antibody-dependent phagocytosis. Activation of AMs was evident by increased expression of CD40 and CD80 post-2nd Ad compared to naïve macaques. APRIL expression also significantly increased post-2nd Ad and correlated with B cell frequency in bronchoalveolar lavage (BAL) (r = 0.73; p = 0.0019) and total IgG in BAL-fluid (r = 0.53; p = 0.047). B cells cultured with SIV gp120-stimulated AM supernatant from vaccinated macaques exhibited significant increases in B cell activation markers CD38 and CD69 compared to B cells cultured alone or with AM supernatant from unvaccinated macaques. Overall, the vaccine regimen did not induce recruitment of susceptible cells to the vaginal mucosa but increased CVM FcγRIII expression which correlated with delayed SIV acquisition. Further, immunization induced expression of AM cytokines, including those associated with providing B cell help.

Mucosal-associated invariant T (MAIT) cells provide B-cell help in vaccinated and subsequently SIV-infected Rhesus Macaques.

Mucosal-associated invariant T (MAIT) cells help combat opportunistic infections. Thus, MAIT cells are of interest in HIV/SIV vaccination and infection. We investigated MAIT cell dynamics and function in rhesus macaque blood and bronchoalveolar lavage (BAL) following mucosal adenovirus (Ad)-SIV recombinant priming, intramuscular SIV envelope boosting and infection following repeated low-dose intravaginal SIV exposures. Increased frequencies of blood MAIT cells over the course of vaccination were observed, which were maintained even 12-weeks post-SIV infection. BAL MAIT cells only increased after the first Ad immunization. Vaccination increased MAIT cell levels in blood and BAL expressing the antiviral cytokine IFN-γ and TNF-α and the proliferation marker Ki67. Upon T cell-specific α-CD3, α-CD28 stimulation, MAIT cells showed a greater capacity to secrete cytokines/chemokines associated with help for B cell activation, migration and regulation compared to CD3+MR1- cells. Culture of MAIT cell supernatants with B cells led to greater tissue like memory B cell frequencies. MAIT cell frequencies in blood and BAL correlated with SIV-specific antibody levels in rectal secretions and with SIV-specific tissue resident memory B cells. Overall, SIV vaccination influenced MAIT cell frequency and functionality. The potential for MAIT cells to provide help to B cells was evident during both vaccination and infection.

An SAMT-247 Microbicide Provides Potent Protection against Intravaginal Simian Immunodeficiency Virus Infection of Rhesus Macaques, whereas an Added Vaccine Component Elicits Mixed Outcomes.

Because of microbicide noncompliance and lack of a durable, highly effective vaccine, a combined approach might improve HIV prophylaxis. We tested whether a vaccine-microbicide combination would enhance protection against SIV infection in rhesus macaques. Four macaque groups included vaccine only, vaccine-microbicide, microbicide only, and controls. Vaccine groups were primed twice mucosally with replicating adenovirus type 5 host range mutant SIV env/rev, gag, and nef recombinants and boosted twice i.m. with SIV gp120 proteins in alum. Controls and the microbicide-only group received adenovirus type 5 host range mutant empty vector and alum. The microbicide was SAMT-247, a 2-mercaptobenzamide thioester that targets the viral nucleocapsid protein NCp7, causing zinc ejection and preventing RNA encapsidation. Following vaccination, macaques were challenged intravaginally with repeated weekly low doses of SIVmac251 administered 3 h after application of 0.8% SAMT-247 gel (vaccine-microbicide and microbicide groups) or placebo gel (vaccine-only and control groups). The microbicide-only group exhibited potent protection; 10 of 12 macaques remained uninfected following 15 SIV challenges. The vaccine-only group developed strong mucosal and systemic humoral and cellular immunity but did not exhibit delayed acquisition compared with adjuvant controls. However, the vaccine-microbicide group exhibited significant acquisition delay compared with both control and vaccine-only groups, indicating further exploration of the combination strategy is warranted. Impaired protection in the vaccine-microbicide group compared with the microbicide-only group was not attributed to a vaccine-induced increase in SIV target cells. Possible Ab-dependent enhancement will be further investigated. The potent protection provided by SAMT-247 encourages its movement into human clinical trials.

Differential Effect of Mucosal NKp44+ Innate Lymphoid Cells and Δγ Cells on Simian Immunodeficiency Virus Infection Outcome in Rhesus Macaques.

NK cells are essential for controlling viral infections. We investigated NK cell and innate lymphoid cell (ILC) dynamics and function in rhesus macaque rectal tissue and blood following mucosal priming with replicating adenovirus (Ad)-SIV recombinants, systemic boosting with SIV envelope protein, and subsequent repeated low-dose intravaginal SIV exposures. Mucosal memory-like NK and ILC subsets in rectal and vaginal tissues of chronically infected macaques were also evaluated. No differences in NK cell or ILC frequencies or cytokine production were seen between vaccinated and Ad-empty/alum controls, suggesting responses were due to the Ad-vector and alum vaccine components. Mucosal NKp44+ ILCs increased postvaccination and returned to prelevels postinfection. The vaccine regimen induced mucosal SIV-specific Ab, which mediated Ab-dependent cellular cytotoxicity and was correlated with mucosal NKp44+CD16+ ILCs. Postvaccination NKp44+ and NKp44+IL-17+ ILC frequencies were associated with delayed SIV acquisition and decreased viremia. In chronically SIV-infected animals, NKp44+ ILCs negatively correlated with viral load, further suggesting a protective effect, whereas, NKG2A- NKp44- double-negative ILCs positively correlated with viral load, indicating a pathogenic role. No such associations of circulating NK cells were seen. Δγ NK cells in mucosal tissues of chronically infected animals exhibited impaired cytokine production compared with non-Δγ NK cells but responded to anti-gp120 Ab and Gag peptides, whereas non-Δγ NK cells did not. Mucosal Δγ NKp44+ and Δγ DN cells were similarly associated with protection and disease progression, respectively. Thus, the data suggest NKp44+ ILCs and Δγ cells contribute to SIV infection outcomes. Vaccines that promote mucosal NKp44+ and suppress double-negative ILCs are likely desirable.

A unique combination adjuvant modulates immune responses preventing vaccine-enhanced pulmonary histopathology after a single dose vaccination with fusion protein and challenge with respiratory syncytial virus.

Alum adjuvanted formalin-inactivated respiratory syncytial virus (RSV) vaccination resulted in enhanced respiratory disease in young children upon natural infection. Here, we investigated the adjuvant effects of monophosphoryl lipid A (MPL) and oligodeoxynucleotide CpG (CpG) on vaccine-enhanced respiratory disease after fusion (F) protein prime vaccination and RSV challenge in infant and adult mouse models. Combination CpG + MPL adjuvant in RSV F protein single dose priming of infant and adult age mice was found to promote the induction of IgG2a isotype antibodies and neutralizing activity, and lung viral clearance after challenge. CpG + MPL adjuvanted F protein (Fp) priming of infant and adult age mice was effective in avoiding lung histopathology, in reducing interleukin-4+ CD4 T cells and cellular infiltration of monocytes and neutrophils after RSV challenge. This study suggests that combination CpG and MPL adjuvant in RSV subunit vaccination might contribute to priming protective immune responses and preventing inflammatory RSV disease after infection.