Control of Heterologous Simian Immunodeficiency Virus SIVsmE660 Infection by DNA and Protein Coimmunization Regimens Combined with Different Toll-Like-Receptor-4-Based Adjuvants in Macaques.

We developed a method of simultaneous vaccination with DNA and protein resulting in robust and durable cellular and humoral immune responses with efficient dissemination to mucosal sites and protection against simian immunodeficiency virus (SIV) infection. To further optimize the DNA-protein coimmunization regimen, we tested a SIVmac251-based vaccine formulated with either of two Toll-like receptor 4 (TLR4) ligand-based liposomal adjuvant formulations (TLR4 plus TLR7 [TLR4+7] or TLR4 plus QS21 [TLR4+QS21]) in macaques. Although both vaccines induced humoral responses of similar magnitudes, they differed in their functional quality, including broader neutralizing activity and effector functions in the TLR4+7 group. Upon repeated heterologous SIVsmE660 challenge, a trend of delayed viral acquisition was found in vaccinees compared to controls, which reached statistical significance in animals with the TRIM-5α-resistant (TRIM-5α R) allele. Vaccinees were preferentially infected by an SIVsmE660 transmitted/founder virus carrying neutralization-resistant A/K mutations at residues 45 and 47 in Env, demonstrating a strong vaccine-induced sieve effect. In addition, the delay in virus acquisition directly correlated with SIVsmE660-specific neutralizing antibodies. The presence of mucosal V1V2 IgG binding antibodies correlated with a significantly decreased risk of virus acquisition in both TRIM-5α R and TRIM-5α-moderate/sensitive (TRIM-5α M/S) animals, although this vaccine effect was more prominent in animals with the TRIM-5α R allele. These data support the combined contribution of immune responses and genetic background to vaccine efficacy. Humoral responses targeting V2 and SIV-specific T cell responses correlated with viremia control. In conclusion, the combination of DNA and gp120 Env protein vaccine regimens using two different adjuvants induced durable and potent cellular and humoral responses contributing to a lower risk of infection by heterologous SIV challenge.IMPORTANCE An effective AIDS vaccine continues to be of paramount importance for the control of the pandemic, and it has been proven to be an elusive target. Vaccine efficacy trials and macaque challenge studies indicate that protection may be the result of combinations of many parameters. We show that a combination of DNA and protein vaccinations applied at the same time provides rapid and robust cellular and humoral immune responses and evidence for a reduced risk of infection. Vaccine-induced neutralizing antibodies and Env V2-specific antibodies at mucosal sites contribute to the delay of SIVsmE660 acquisition, and genetic makeup (TRIM-5α) affects the effectiveness of the vaccine. These data are important for the design of better vaccines and may also affect other vaccine platforms.

Genetic variants in COL13A1, ADIPOQ and SAMM50, in addition to the PNPLA3 gene, confer susceptibility to elevated transaminase levels in an admixed Mexican population.

Non-alcoholic fatty liver disease (NAFLD) is the accumulation of extra fat in liver cells not caused by alcohol. Elevated transaminase levels are common indicators of liver disease, including NAFLD. Previously, we demonstrated that PNPLA3 (rs738409), LYPLAL1 (rs12137855), PPP1R3B (rs4240624), and GCKR (rs780094) are associated with elevated transaminase levels in overweight/obese Mexican adults. We investigated the association between 288 SNPs identified in genome-wide association studies and risk of elevated transaminase levels in an admixed Mexican-Mestizo sample of 178 cases of NAFLD and 454 healthy controls. The rs2896019, rs12483959, and rs3810622 SNPs in PNPLA3 and rs1227756 in COL13A1 were associated with elevated alanine aminotransferase (ALT, ≥40IU/L). A polygenic risk score (PRS) based on six SNPs in the ADIPOQ, COL13A1, PNPLA3, and SAMM50 genes was also associated with elevated ALT. Individuals carrying 9-12 risk alleles had 65.8% and 48.5% higher ALT and aspartate aminotransferase (AST) levels, respectively, than those with 1-4 risk alleles. The PRS showed the greatest risk of elevated ALT levels, with a higher level of significance than the individual variants. Our findings suggest a significant association between variants in COL13A1, ADIPOQ, SAMM50, and PNPLA3, and risk of NAFLD/elevated transaminase levels in Mexican adults with an admixed ancestry. This is the first study to examine high-density single nucleotide screening for genetic variations in a Mexican-Mestizo population. The extent of the effect of these variations on the development and progression of NAFLD in Latino populations requires further analysis.

MicroRNA-548-3p overexpression inhibits proliferation, migration and invasion in osteoblast-like cells by targeting STAT1 and MAFB.

Osteoporosis is the most common bone disease and a public health issue with increasing prevalence in Mexico. This disease is caused by an imbalance in the bone remodelling process mediated by osteoclast and osteoblast. MicroRNAs have emerged as key players during the differentiation of both types of cells specialized involved in bone metabolism. We found high expression levels of miR-548x-3p in circulating monocytes derived from postmenopausal osteoporotic women. This study aimed to analyse the functional characterization of miR-548x-3p roles in the bone remodelling process. We validated by RT-qPCR, the elevated levels of miR-548x-3p in circulating monocytes derived from osteoporosis women. Through bioinformatics analysis, we identify MAFB and STAT1 as potential target genes for miR-548x-3p. Both genes showed low levels of expression in circulating monocytes derived from osteoporotic women. In addition, we demonstrated the binding of miR-548x-3p to the 3'-UTR of both mRNAs. MiR-548x-3p was overexpressed in osteoblasts-like cell lines decreasing the levels of MAFB and STAT1 mRNA and protein. We found that miR-548x-3p overexpression inhibits the proliferation, migration and invasion of the cell lines evaluated. Our results identified, by the first time, the potential role of miR-548x-3p as a modulator of the bone remodelling process by regulating the expression of MAFB and STAT1.

The Variant rs1784042 of the SIDT2 Gene is Associated with Metabolic Syndrome through Low HDL-c Levels in a Mexican Population.

The Mexican population has one of the highest prevalences of metabolic syndrome (MetS) worldwide. The aim of this study was to investigate the association of single-nucleotide polymorphisms (SNPs) with MetS and its components. First, we performed a pilot Genome-wide association study (GWAS) scan on a sub-sample derived from the Health Workers Cohort Study (HWCS) (n = 411). Based on GWAS results, we selected the rs1784042 and rs17120425 SNPs in the SIDT1 transmembrane family member 2 (SIDT2) gene for replication in the entire cohort (n = 1963), using predesigned TaqMan assays. We observed a prevalence of MetS in the HWCS of 52.6%. The minor allele frequency for the variant rs17120425 was 10% and 29% for the rs1784042. The SNP rs1784042 showed an overall association with MetS (OR = 0.82, p = 0.01) and with low levels of high-density lipoprotein (HDL-c) (odds ratio (OR) = 0.77, p = 0.001). The SNP rs17120425 had a significant association with type 2 diabetes (T2D) risk in the overall population (OR = 1.39, p = 0.033). Our results suggest an association of the rs1784042 and rs17120425 variants with MetS, through different mechanisms in the Mexican population. Further studies in larger samples and other populations are required to validate these findings and the relevance of these SNPs in MetS.

MicroRNA expression in relation with clinical evolution of osteosarcoma.

Osteosarcoma is the most common malignant bone tumor. Early diagnosis remains a major challenge, mainly because of the lack of specific biomarkers. We performed miRNAs expression analysis through qPCR in affected and paired healthy bone derived from osteosarcoma patients. Hierarchical clustering using the top ten miRNAs with differential expression showed two main clusters. One integrated by patients with the presence of metastasis or relapse and the other without these complications. Further pathway enrichment analysis reduced to four main miRNAs, hsa-miR-486-3p, hsa-miR-355-5p, hsa-miR-34a-5p and hsa-miR-1228-3p. Afterwards, we compared patients with and without metastasis, the function enrichment analysis along with review of relevant literature, showed that hsa-miR-93-5p and hsa-miR-28-5p were associated with metastasis development. Our results support the relevance of miRNAs in the pathogenesis of osteosarcoma and contribute with evidence regarding the potential role of miRNAs as potential biomarkers. More studies are needed to define the most informative miRNAs in osteosarcoma patients.

Expression profiles of the Wnt/ß-catenin signaling-related extracellular antagonists during proliferation and differentiation in human osteoblast-like cells.

Bone formation is a dynamic process directed by osteoblast activity. The transition from the proliferation to differentiation stage during osteoblast maturation involves the downregulation of the Wnt/ß-catenin signaling pathway, and extracellular antagonists are important for the regulation of Wnt signaling. However, the expression levels of Wnt antagonists in these stages of human osteoblast maturation have not been fully elucidated. Therefore, the aim of the present study was to investigate the expression levels of extracellular Wnt antagonists during proliferation and differentiation in osteoblast-like cell lines. The results demonstrated an overlap between the differential expression of secreted Frizzled-related protein (SFPR)2, SFRP3, SFRP4 and Dickkopf (DKK) 2 genes during the differentiation stage in the MG-63 and Saos-2 cells. Furthermore, high expression levels of DKK3 in MG-63 cells, Wnt inhibitory factor 1 (WIF1) in Saos-2 cells and DKK4 in hFOB 1.19 cells during the same stage (differentiation), were observed. The upregulated expression levels of Wnt antagonists were also correlated with the high expression of anxin 2 during the differentiation stage. These findings suggested that Wnt-related antagonists could modulate the Wnt/ß-catenin signaling pathway. By contrast, DKK1 was the only gene that was found to be upregulated during the proliferation stage in hFOB 1.19 and Saos-2 cells. To the best of our knowledge, the present study provides, for the first time, the expression profile of Wnt antagonists during the proliferation stage and the initial phases of differentiation in osteoblast-like cell lines. The current results offer a basis to investigate potential targets for bone-related Wnt-signaling modulation in bone metabolism research.

A Multi-Omic Analysis for Low Bone Mineral Density in Postmenopausal Women Suggests a RELATIONSHIP between Diet, Metabolites, and Microbiota.

The effect of microbiota composition and its health on bone tissue is a novel field for research. However, their associations with bone mineral density (BMD) have not been established in postmenopausal women. The present study investigates the relation of diet, the microbiota composition, and the serum metabolic profile in postmenopausal women with normal-BMD or with low-BMD. Ninety-two Mexican postmenopausal women were classified into normal-BMD (n = 34) and low-BMD (n = 58). The V4 hypervariable region was sequenced using the Miseq platform. Serum vitamin D was determined by chemiluminescence immunoassay. Serum concentrations of acyl-carnitines and amino acids were determined by electrospray tandem mass spectrometry. Diet was assessed by a food frequency questionnaire. The low-BMD group had fewer observed species, higher abundance of γ-Proteobacteria, lower consumption of lycopene, and lower concentrations of leucine, valine, and tyrosine compared with the normal-BMD group. These amino acids correlated positively with the abundance of Bacteroides. Lycopene consumption positively correlated with Oscillospira and negatively correlated with Pantoea genus abundance. Finally, the intestinal microbiota of women with vitamin D deficiency was related to Erysipelotrichaceae and Veillonellaceae abundance compared to the vitamin D non-deficient group. Associations mediated by the gut microbiota between diet and circulating metabolites with low-BMD were identified.

Serum Proteomic Analysis Reveals Vitamin D-Binding Protein (VDBP) as a Potential Biomarker for Low Bone Mineral Density in Mexican Postmenopausal Women.

Osteoporosis is a skeletal disease mainly affecting women over 50 years old and it represents a serious public health problem because of the high socioeconomic burden. This disease is characterized by deterioration of bone microarchitecture, low bone mineral density (BMD), and increased risk of fragility fractures. This study aimed to identify serum useful proteins as biomarkers for the diagnosis and/or prognosis of osteoporosis and fracture risk. We collected 446 serum samples from postmenopausal women aged ≥45 years old. Based on the BMD measurement, we classified the participants into three groups: osteoporotic, osteopenic, and normal. In an initial discovery stage, we conducted a proteomic approach using two-dimensional differential gel electrophoresis (2D-DIGE). The peptides into the spots of interest were identified through matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF/TOF). Enzyme-linked immunosorbent assay (ELISA) was performed to validate the proteins of interest. We identified 27 spots of interest when comparing low BMD versus normal BMD postmenopausal women. Based on their relevance in bone metabolism, we analyzed three proteins: ceruloplasmin (CP), gelsolin (GSN), and vitamin D-binding protein (VDBP). Our results demonstrated that low serum VDBP levels correlate with low BMD (osteopenic and osteoporotic). Therefore, VDBP could be considered as a novel, potential, and non-invasive biomarker for the early detection of osteoporosis.

Influence of Genetic and Non-Genetic Risk Factors for Serum Uric Acid Levels and Hyperuricemia in Mexicans.

Risk of hyperuricemia is modified by genetic and environmental factors. Our aim was to identify factors associated with serum uric acid levels and hyperuricemia in Mexicans. A pilot Genome-wide association study GWAS was performed in a subgroup of participants (n = 411) from the Health Workers Cohort Study (HWCS). Single nucleotide polymorphisms (SNPs) associated with serum uric acid levels were validated in all the HWCS participants (n = 1939) and replicated in independent children (n = 1080) and adult (n = 1073) case-control studies. The meta-analysis of the whole HWCS and replication samples identified three SLC2A9 SNPs: rs1014290 (p = 2.3 × 10-64), rs3775948 (p = 8.2 × 10-64) and rs11722228 (p = 1.1 × 10-17); and an ABCG2 missense SNP, rs2231142 (p = 1.0 × 10-18). Among the non-genetic factors identified, the visceral adiposity index, smoking, the metabolic syndrome and its components (waist circumference, blood pressure, glucose and hyperlipidemia) were associated with increased serum uric acid levels and hyperuricemia (p < 0.05). Among the female HWCS participants, the odds ratio for hyperuricemia was 1.24 (95% CI, 1.01-1.53) per unit increase in soft drink consumption. As reported in other studies, our findings indicate that diet, adiposity and genetic variation contribute to the elevated prevalence of hyperuricemia in Mexico.

Serum miRNAs miR-140-3p and miR-23b-3p as potential biomarkers for osteoporosis and osteoporotic fracture in postmenopausal Mexican-Mestizo women.

Osteoporosis is a metabolic bone disorder characterized by low bone mineral density and decreased bone strength, leading to an increased risk of fractures with a consequent increase in morbidity and mortality. The current methods to estimate the fracture risk are very limited. microRNAs (miRNAs) have been considered as good biomarkers for many pathological processes, including osteoporosis. Some circulating miRNAs are associated with regulation of bone formation and differentiation of bone cells. The aim of this study, was to analyze the expression of miRNAs in serum of patients with osteoporosis (n = 20) and healthy controls (n = 20). Expression of 754 miRNAs was analyzed through quantitative real time RT-PCR arrays. Seven miRNAs showed significant differences between groups. The microRNAs miR-23b-3p, miR-140-3p and miR-885-5p were selected based on fold change and p-values (40.5, p = 0.038, 20.7, p = 0.045, and 2.2, p = 0.002; respectively) for validation in independent serum samples from patients with osteopenia (n = 28), osteoporosis (n = 26) and osteoporotic hip fracture (n = 21). After validation, we confirm differences across the groups for miR-23b-3p and miR-140-3p. Our data pointed miR-140-3p and miR-23b-3p as potential biomarkers candidates for osteoporosis in postmenopausal women.

Identification of miR-708-5p in peripheral blood monocytes: Potential marker for postmenopausal osteoporosis in Mexican-Mestizo population.

IMPACT STATEMENT: This is the first study in which hsa-miR-708-5p has been identified in peripheral blood monocytes (osteoclast precursors) and associated with postmenopausal osteoporosis through small RNA-Sequencing, in an Admixed Mexican Mestizo population. By conducting in silico and bioinformatic analyzes, we identified target genes and important signaling pathways involved in bone metabolism pointing hsa-miR-708-5p as a candidate marker for osteoporosis in Mexican population. These approaches provide a landscape of the post-transcriptional regulation, which can be useful for the management of postmenopausal osteoporosis along with the potential use of microRNAs as markers for its early detection.

Circulating miR-215-5p and miR-642a-5p as potential biomarker for diagnosis of osteosarcoma in Mexican population.

Osteosarcoma is the most common malignant bone neoplasia affecting individuals in the second decade of life. The survival rate has not been improved during the last 25 years, in part because of the lack of specific markers. The microRNAs have been identified as important regulators of gene expression, experimental evidence suggests these molecules as key players in cancer development and progression. To identify miRNAs differentially expressed in serum from patients with osteosarcoma compared to healthy donors in Mexican population. Fifteen osteosarcoma patients and fifteen age and sex matched healthy individuals were recruited. Two pools of total RNA extracted from serum per study group were prepared and the miRNA expression profiles were analyzed through TaqMan Low Density Arrays. Validation was carried out through RT-qPCR using individual TaqMan assays for those miRNAs differentially expressed. Fifteen miRNAs were differentially expressed in osteosarcoma patients compared to healthy controls. Overexpression of miR-215-5p and miR-642a-5p was confirmed by validation through RT-qPCR. The expression analysis of miRNAs from serum in osteosarcoma patients revealed differential expression of miR-215-5p and miR-642a-5p. Both microRNAs are potential markers for osteosarcoma diagnosis.

DNA Vaccine-Induced Long-Lasting Cytotoxic T Cells Targeting Conserved Elements of Human Immunodeficiency Virus Gag Are Boosted Upon DNA or Recombinant Modified Vaccinia Ankara Vaccination.

DNA-based vaccines able to induce efficient cytotoxic T-cell responses targeting conserved elements (CE) of human immunodeficiency virus type 1 (HIV-1) Gag have been developed. These CE were selected by stringent conservation, the ability to induce T-cell responses with broad human leukocyte antigen coverage, and the association between recognition of CE epitopes and viral control in HIV-infected individuals. Based on homology to HIV, a simian immunodeficiency virus p27gag CE DNA vaccine has also been developed. This study reports on the durability of the CE-specific T-cell responses induced by HIV and simian immunodeficiency virus CE DNA-based prime/boost vaccine regimens in rhesus macaques, and shows that the initially primed CE-specific T-cell responses were efficiently boosted by a single CE DNA vaccination after the long rest period (up to 2 years). In another cohort of animals, the study shows that a single inoculation with non-replicating recombinant Modified Vaccinia Ankara (rMVA62B) also potently boosted CE-specific responses after around 1.5 years of rest. Both CE DNA and rMVA62B booster vaccinations increased the magnitude and cytotoxicity of the CE-specific responses while maintaining the breadth of CE recognition. Env produced by rMVA62B did not negatively interfere with the recall of the Gag CE responses. rMVA62B could be beneficial to further boosting the immune response to Gag in humans. Vaccine regimens that employ CE DNA as a priming immunogen hold promise for application in HIV prevention and therapy.

Identification of microRNAs in human circulating monocytes of postmenopausal osteoporotic Mexican-Mestizo women: A pilot study.

MicroRNAs (miRNAs or miRs) are a class of short non-coding RNAs that serve an important regulatory role in living organisms. These molecules are associated with multiple biological processes and are potential biomarkers in multiple diseases. The present study aimed to further identify miRNAs that are differentially expressed in circulating monocytes (CMCs) from postmenopausal Mexican-Mestizo women. Microarray analyses of monocytes using Affymetrix miRNA 4.0 and Human Genome U133 Plus 2.0 arrays were performed in 6 normal and 6 osteoporotic women, followed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) validation. The overexpression of miR-1270, miR-548×-3p and miR-8084 were detected in the osteoporosis compared with the normal group according to the microarray analysis; miR-1270, a miRNA with several target genes associated with bone remodeling, was validated by RT-qPCR. Bioinformatics analysis identified that interferon regulatory factor 8 (IRF8) is the most likely target gene of miR-1270, which is associated with osteoclastogenesis. Furthermore, the findings of the present study demonstrate that an upregulation of miR-1270 may reduce the gene expression of IRF8 in CMCs (osteoclast precursors), implicating its potential role in leading to low bone mineral density and contributing to osteoporosis development in postmenopausal women.