Search

The aim of this study was to assess the effect of lipodystrophy (LD) associated to metabolic syndrome (MS) on oxidative stress and inflammation in a cohort of 243 HIV-infected patients with MS, all of them under three different antiretroviral regimens. We collected immunovirological, biochemical and metabolic data, as well as anthropometric measurements. In addition, cardiovascular risk was also assessed by means of Atherogenic Index of Plasma (API) and Framingham Risk Score. The MS-LD patient set was characterized by a lower initial lymphocyte CD4 count and CD4/CD8 ratio and a higher initial viral load than the group without LD. We also found worse lipidic and glycaemic profiles (with lower HDL-cholesterol and higher triglyceride and glucose levels) in the MS-LD group. BMI, systolic blood pressure and Framingham score were significantly increased compared to MS-Non LD. In addition, patients with MS and LD had significantly higher levels of carbonylated proteins, lipid peroxidation, IL-6 and IL-8, as well as a significant decrease in the levels of leptin, adiponectin and antioxidant activities of catalase, super oxide dismutase and glutathione associated enzymes. In MS-LD HIV-1 patients, a significant negative correlation was found between Framingham Risk Score and the antioxidant biomarkers, however a positive association was found between API and protein-C reactive and carbonylated proteins. Segregating by ART, the above-mentioned conditions were worse within the MS-LD group whose treatment contained protease inhibitors, such as lopinavir. In conclusion, HIV-1 infected patients treated for at least six months, especially with regimens including PIs, showed a worsening of inflammatory process and oxidative stress.

BACKGROUND Breast cancer (BC) is the most prevalent cancer in women. While usually detected when localized, invasive procedures are still required for diagnosis. METHODS Herein, we developed a novel ultrasensitive pipeline to detect circulating tumour DNA (ctDNA) in a series of 75 plasma samples from localized BC patients prior to any medical intervention. We first performed a tumour-informed analysis to correlate the mutations found in tumour tissue and plasma. Disregarding the tumour data next, we developed an approach to detect tumour mutations in plasma. RESULTS We observed a mutation concordance between tumour and plasma of 29.50% with a sensitivity down to 0.03% in mutant allele frequency (AF). We detected mutations in 33.78% of the samples, identifying 8 patients with plasma-only mutations. Altogether, we determined a specificity of 86.36% and a positive predictive value of 88.46% for BC detection. We demonstrated an association between higher ctDNA median AF and higher tumour grade, multiple plasma mutations with likelihood of relapse and more frequent TP53 plasma mutations in hormone receptor-negative tumours. CONCLUSIONS Overall, we have developed a unique ultra-sensitive sequencing workflow with a technology not previously employed in early BC, paving the way for its application in BC screening.

In this chapter, we describe the possible link between gut microbiota, melatonin, and breast cancer disease. It is widely described that changes in melatonin production due to circadian disruption is one of the causes of breast cancer. In addition, recently it is described that dysbiosis caused by changes in the gut microbiota composition could be as well constitute an important factor to induce breast cancer. The dysbiosis process, in turn, induces the stimulation of kynurenine pathway, leading to reduced circulating melatonin levels. Therefore, in this chapter we deep into the relationship between circadian disruption, dysbiosis, and breast cancer disease. This constitutes an important step in the therapeutic approach and prevention of this pathology.

Colorectal cancer (CRC) is the third most common cancer and the second cause of cancer death worldwide. Several factors have been postulated to be involved in CRC pathophysiology, including heritable and environmental factors, which are the latest to be closely associated with nutritional habits, physical activity, obesity, and the gut microbiota. The latter may also play a key role in CRC prognosis and derived complications in patients undergoing surgery. This is a single-center, open, controlled, randomized clinical trial, in patients with scheduled surgical intervention for CRC. The primary objective is to assess whether a pre-surgical nutritional intervention, based on a high-fiber diet rich in polyunsaturated fatty acids (PUFAs), can reduce disturbances of the gut microbiota composition and, consequently, the rate of post-surgical complications in patients with CRC. Patients will be randomized in a 1:1 ratio after receiving a diagnosis of CRC. In the control arm, patients will receive standard nutritional recommendations, while patients in the intervention arm will be advised to follow a high-fiber diet rich in PUFAs before surgery. Participants will be followed up for one year to evaluate the overall rate of postsurgical complications, recurrences of CRC, response to adjuvant therapy, and overall/disease-free survival.

Melatonin is a molecule with different antitumor actions in breast cancer and has been described as an inhibitor of vascular endothelial growth factor (VEGF). Despite the recognition of the key role exerted by VEGF in tumor angiogenesis, limitations arise when developing models to test new antiangiogenic molecules. Thus, the aim of this study was to develop rapid, economic, high capacity and easy handling angiogenesis assays to test the antiangiogenic effects of melatonin and demonstrate its most effective dose to neutralize and interfere with the angiogenic sprouting effect induced by VEGF and MCF-7. To perform this, 3D endothelial cell (HUVEC) spheroids and a chicken embryo chorioallantoic membrane (CAM) assay were used. The results showed that VEGF and MCF-7 were able to stimulate the sprouting of the new vessels in 3D endothelial spheroids and the CAM assay, and that melatonin had an inhibitory effect on angiogenesis. Specifically, as the 1 mM pharmacological dose was the only effective dose able to inhibit the formation of ramifications around the alginate in the CAM assay model, this inhibition was shown to occur in a dose-dependent manner. Taken together, these techniques represent novel tools for the development of antiangiogenic molecules such as melatonin, with possible implications for the therapy of breast cancer. Funding: This work was funded in part by PE-0106–2019 from the Consejería de Salud de la Junta de Andalucía, C19047–2018 from Fundación Unicaja and UMA18-FEDERJA-042 from UMA-FEDER & ALIANZA MIXTA ANDALUCÍA-ROCHE. Alicia González González is a recipient of a postdoctoral grant Margarita Salas (RMS-08) from European Union-NextGenerationEU, Spanish Ministry of Universities and Recovery Transformation and Resilience Plan, through a call from University of Cantabria. Aurora Laborda Illanes is a recipient of a predoctoral grant, PFIS-ISCIII (FI19–00112), co-funded by the Fondo Social Europeo (FSE). Lidia Sanchez Alcoholado is a recipient of a postdoctoral grant (RH-0026–2021) from the Consejería de Salud y Familia (co-funded by the Fondo Europeo de Andalucía 2014–2020, Andalucía, Spain). Daniel Castellano Castillo is a recipient of a postdoctoral grant Sara Borrell (CD21/00164) from Instituto de Salud Carlos III.

[Abstract] Ischemic heart disease (IHD) and type-2 diabetes mellitus (T2DM) remain major health problems worldwide and commonly coexist in individuals. Gut microbial metabolites, such as trimethylamine N-oxide (TMAO) and short-chain fatty acids (SCFAs), have been linked to cardiovascular and metabolic diseases. Previous studies have reported dysbiosis in the gut microbiota of these patients and the prebiotic effects of some components of the Mediterranean diet. Essential oil emulsions of savory (Satureja hortensis), parsley (Petroselinum crispum) and rosemary (Rosmarinus officinalis) were assessed as nutraceuticals and prebiotics in IHD and T2DM. Humanized mice harboring gut microbiota derived from that of patients with IHD and T2DM were supplemented with L-carnitine and orally treated with essential oil emulsions for 40 days. We assessed the effects on gut microbiota composition and abundance, microbial metabolites and plasma markers of cardiovascular disease, inflammation and oxidative stress. Our results showed that essential oil emulsions in mice supplemented with L-carnitine have prebiotic effects on beneficial commensal bacteria, mainly Lactobacillus genus. There was a decrease in plasma TMAO and an increase in fecal SCFAs levels in mice treated with parsley and rosemary essential oils. Thrombomodulin levels were increased in mice treated with savory and parsley essential oils. While mice treated with parsley and rosemary essential oils showed a decrease in plasma cytokines (INFɣ, TNFα, IL-12p70 and IL-22); savory essential oil was associated with increased levels of chemokines (CXCL1, CCL2 and CCL11). Finally, there was a decrease in protein carbonyls and pentosidine according to the essential oil emulsion. These results suggest that changes in the gut microbiota induced by essential oils of parsley, savory and rosemary as prebiotics could differentially regulate cardiovascular and metabolic factors, which highlights the potential of these nutraceuticals for reducing IHD risk in patients affected by T2DM. Junta de Andalucía; PI-0170-2018 Instituto de Salud Carlos III; PT20/00101 Junta de Andalucía; RH-0078-2021 Instituto de Salud Carlos III; CPII19/00022 Instituto de Salud Carlos III; FI20/00227

Gut microbiome (GM) and its either pro-tumorigenic or anti-tumorigenic role is intriguing and constitutes an evolving landscape in translational oncology. It has been suggested that these microorganisms may be involved in carcinogenesis, cancer treatment response and resistance, as well as predisposition to adverse effects. In melanoma patients, one of the most immunogenic cancers, immune checkpoint inhibitors (ICI) and MAPK-targeted therapy—BRAF/MEK inhibitors—have revolutionized prognosis, and the study of the microbiome as a modulating factor is thus appealing. Although BRAF/MEK inhibitors constitute one of the main backbones of treatment in melanoma, little is known about their impact on GM and how this might correlate with immune re-induction. On the contrary, ICI and their relationship to GM has become an interesting field of research due to the already-known impact of immunotherapy in modulating the immune system. Immune reprogramming in the tumor microenvironment has been established as one of the main targets of microbiome, since it can induce immunosuppressive phenotypes, promote inflammatory responses or conduct anti-tumor responses. As a result, ongoing clinical trials are evaluating the role of fecal microbiota transplant (FMT), as well as the impact of using dietary supplements, antibiotics and probiotics in the prediction of response to therapy. In this review, we provide an overview of GM’s link to cancer, its relationship with the immune system and how this may impact response to treatments in melanoma patients. We also discuss insights about novel therapeutic approaches including FMT, changes in diet and use of probiotics, prebiotics and symbiotics. Finally, we hypothesize on the possible pathways through which GM may impact anti-tumor efficacy in melanoma patients treated with targeted therapy, an appealing subject of which little is known.

Colorectal cancer (CRC) is the third most frequent malignancy and the second cause of cancer death worldwide. Several factors have been postulated to be involved in CRC pathophysiology, including physical inactivity, unhealthy dietary habits, obesity, and the gut microbiota. Emerging data suggest that the microbiome may play a key role in CRC prognosis and derived complications in patients undergoing colorectal surgery. On the other hand, dietary intervention has been demonstrated to be able to induce significant changes in the gut microbiota and related metabolites in different conditions; therefore, the manipulation of gut microbiota through dietary intervention may constitute a useful approach to improve perioperative dysbiosis and post-surgical outcomes in patients with CRC. In this article, we review the role of the gut microbiota in CRC surgery complications and the potential therapeutic modulation of gut microbiome through nutritional intervention in patients with CRC undergoing surgery.

The exposome, defined as the cumulative measure of external exposures and associated biological responses throughout the lifespan, has emerged in recent years as a cornerstone in biomedical sciences. Metabolomics stands out here as one of the most powerful tools for investigating the interplay between the genetic background, exogenous, and endogenous factors within human health. However, to address the complexity of the exposome, novel methods are needed to characterize the human metabolome. In this work, we have optimized and validated a multianalyte metabolomics platform for large-scale quantitative exposome research in plasma and urine samples, based on the use of simple extraction methods and high-throughput metabolomic fingerprinting. The methodology enables, for the first time, the simultaneous characterization of the endogenous metabolome, food-related metabolites, pharmaceuticals, household chemicals, environmental pollutants, and microbiota derivatives, comprising more than 1000 metabolites in total. This comprehensive and quantitative investigation of the exposome is achieved in short run times, through simple extraction methods requiring small-sample volumes, and using integrated quality control procedures for ensuring data quality. This metabolomics approach was satisfactorily validated in terms of linearity, recovery, matrix effects, specificity, limits of quantification, intraday and interday precision, and carryover. Furthermore, the clinical potential of the methodology was demonstrated in a dietary intervention trial as a case study. In summary, this study describes the optimization, validation, and application of a multimetabolite platform for comprehensive and quantitative metabolomics-based exposome research with great utility in large-scale epidemiological studies.

Obesity is considered an important factor that increases the risk of colorectal cancer (CRC). So far, the association of gut microbiota with both obesity and cancer has been described independently. Nevertheless, a specific obesity-related microbial profile linked to CRC development has not been identified. The aim of this study was to determine the gut microbiota composition in fecal samples from CRC patients with (OB-CRC) and without obesity (L-CRC) compared to the microbiota profile present in non-obese healthy controls (L-HC), in order to unravel the possible relationship between gut microbiota and microbial-derived metabolite trimethylamine N-oxide (TMAO), the inflammatory status, and the intestinal permeability in the context of obesity-associated CRC. The presence of obesity does not induce significant changes in the diversity and richness of intestinal bacteria of CRC patients. Nevertheless, OB-CRC patients display a specific gut microbiota profile characterized by a reduction in butyrate-producing bacteria and an overabundance of opportunistic pathogens, which in turn could be responsible, at least in part, for the higher levels of proinflammatory cytokine IL-1&beta, the deleterious bacterial metabolite TMAO, and gut permeability found in these patients. These results suggest a possible role of obesity-related gut microbiota in the development of CRC, which could give new clues for the design of new diagnostic tools for CRC prevention.

Simple Summary In this review we discuss the recent knowledge about the role of breast and gut microbiome in the pathogenesis of breast cancer. We examine the proposed mechanisms of interaction between breast tumors and the microbiome. We focus on the role of the microbiome in: (i) the development and maintenance of estrogen metabolism through bacterial beta-glucuronidase enzymes (ii) the regulation of the host´s immune system and tumor immunity by Treg lymphocyte proliferation through bacterial metabolites such as butyrate and propionate (SCFAs), (iii) the induction of chronic inflammation, (iv) the response and/or resistance to treatments and (v) the epigenetic reprogramming. Moreover, we also discuss that diet, probiotics and prebiotics could exert important anticarcinogenic effects in breast cancer that could indicate their employment as adjuvants in standard-of-care breast cancer treatments. Overall, these findings could give new insights for building up novel strategies for breast cancer prevention and treatment. Abstract In breast cancer (BC) the employment of sequencing technologies for metagenomic analyses has allowed not only the description of the overall metagenomic landscape but also the specific microbial changes and their functional implications. Most of the available data suggest that BC is related to bacterial dysbiosis in both the gut microenvironment and breast tissue. It is hypothesized that changes in the composition and functions of several breast and gut bacterial taxa may contribute to BC development and progression through several pathways. One of the most prominent roles of gut microbiota is the regulation of steroid-hormone metabolism, such as estrogens, a component playing an important role as risk factor in BC development, especially in postmenopausal women. On the other hand, breast and gut resident microbiota are the link in the reciprocal interactions between cancer cells and their local environment, since microbiota are capable of modulating mucosal and systemic immune responses. Several in vivo and in vitro studies show remarkable evidence that diet, probiotics and prebiotics could exert important anticarcinogenic effects in BC. Moreover, gut microbiota have an important role in the metabolism of chemotherapeutic drugs and in the activity of immunogenic chemotherapies since they are a potential dominant mediator in the response to cancer therapy. Then, the microbiome impact in BC is multi-factorial, and the gut and breast tissue bacteria population could be important in regulating the local immune system, in tumor formation and progression and in therapy response and/or resistance.

Obesity and metabolic syndrome are among the most prevalent health problems in developed countries. The impairment of adipose tissue (AT) function is partially responsible for the aetiology of these conditions. Epigenetics refers to several processes that add modifications to either the DNA or chromatin architectural proteins (histones). These processes can regulate gene expression, chromatin compaction and DNA repair. Epigenetics includes mechanisms by which the cell can adapt the cellular response to the environmental conditions. Here, we review the role of epigenetics in the onset of obesity and related metabolic disorders, with special focus on AT. We highlight the importance of nutrients and lifestyle in the regulation of the epigenetic mechanisms and how they can impact on AT plasticity and function in obesity and metabolic diseases. Thus, the epigenetic landscape emerges as a fine-tune regulator of the cellular responses according to the energetic, metabolic and physiological conditions of the cell. Alterations in metabolic pathways deregulated during obesity and metabolic syndrome could in part explain the disturbances in the epigenetic marks of the AT in these disorders. The understanding of how this epigenetic deregulation may affect AT biology and function could lead to new therapeutic approaches based on epigenetic strategies.

Colorectal cancer (CRC) is the third most common cancer worldwide and the leading cause of cancer-related deaths. Recently, several studies have demonstrated that gut microbiota can alter CRC susceptibility and progression by modulating mechanisms such as inflammation and DNA damage, and by producing metabolites involved in tumor progression or suppression. Dysbiosis of gut microbiota has been observed in patients with CRC, with a decrease in commensal bacterial species (butyrate-producing bacteria) and an enrichment of detrimental bacterial populations (pro-inflammatory opportunistic pathogens). CRC is characterized by altered production of bacterial metabolites directly involved in cancer metabolism including short-chain fatty acids and polyamines. Emerging evidence suggests that diet has an important impact on the risk of CRC development. The intake of high-fiber diets and the supplementation of diet with polyunsaturated fatty acids, polyphenols and probiotics, which are known to regulate gut microbiota, could be not only a potential mechanism for the reduction of CRC risk in a primary prevention setting, but may also be important to enhance the response to cancer therapy when used as adjuvant to conventional treatment for CRC. Therefore, a personalized modulation of the pattern of gut microbiome by diet may be a promising approach to prevent the development and progression of CRC and to improve the efficacy of antitumoral therapy.

OBJECTIVE Type 1 diabetes is associated with compositional differences in gut microbiota. To date, no microbiome studies have been performed in maturity-onset diabetes of the young 2 (MODY2), a monogenic cause of diabetes. Gut microbiota of type 1 diabetes, MODY2, and healthy control subjects was compared. RESEARCH DESIGN AND METHODS This was a case-control study in 15 children with type 1 diabetes, 15 children with MODY2, and 13 healthy children. Metabolic control and potential factors modifying gut microbiota were controlled. Microbiome composition was determined by 16S rRNA pyrosequencing. RESULTS Compared with healthy control subjects, type 1 diabetes was associated with a significantly lower microbiota diversity, a significantly higher relative abundance of Bacteroides, Ruminococcus, Veillonella, Blautia, and Streptococcus genera, and a lower relative abundance of Bifidobacterium, Roseburia, Faecalibacterium, and Lachnospira. Children with MODY2 showed a significantly higher Prevotella abundance and a lower Ruminococcus and Bacteroides abundance. Proinflammatory cytokines and lipopolysaccharides were increased in type 1 diabetes, and gut permeability (determined by zonulin levels) was significantly increased in type 1 diabetes and MODY2. The PICRUSt analysis found an increment of genes related to lipid and amino acid metabolism, ABC transport, lipopolysaccharide biosynthesis, arachidonic acid metabolism, antigen processing and presentation, and chemokine signaling pathways in type 1 diabetes. CONCLUSIONS Gut microbiota in type 1 diabetes differs at taxonomic and functional levels not only in comparison with healthy subjects but fundamentally with regard to a model of nonautoimmune diabetes. Future longitudinal studies should be aimed at evaluating if the modulation of gut microbiota in patients with a high risk of type 1 diabetes could modify the natural history of this autoimmune disease.

Context The urinary tract, previously considered a sterile body niche, has emerged as the host of an array of bacteria in healthy individuals, revolutionizing the urology research field. Objective To review the literature on microbiome implications in the urinary tract and the usefulness of probiotics/prebiotics and diet as treatment for urologic disorders. Evidence acquisition A systematic review was conducted using PubMed and Medline from inception until July 2016. The initial search identified 1419 studies and 89 were included in this systematic review. Evidence synthesis Specific bacterial communities have been found in the healthy urinary tract. Changes in this microbiome have been observed in certain urologic disorders such as urinary incontinence, urologic cancers, interstitial cystitis, neurogenic bladder dysfunction, sexually transmitted infections, and chronic prostatitis/chronic pelvic pain syndrome. The role of probiotics, prebiotics, and diet as treatment or preventive agents for urologic disorders requires further investigation. Conclusions There is a microbiome associated with the healthy urinary tract that can change in urologic disorders. This represents a propitious context to identify new diagnostic, prognostic, and predictive microbiome-based biomarkers that could be used in clinical urology practice. In addition, probiotics, prebiotics, and diet modifications appear to represent an opportunity to regulate the urinary microbiome. Patient summary We review the urinary microbiome of healthy individuals and its changes in relation to urinary disorders. The question to resolve is how we can modulate the microbiome to improve urinary tract health.

Emerging evidence has suggested that dysbiosis of the gut microbiota may influence the drug efficacy of colorectal cancer (CRC) patients during cancer treatment by modulating drug metabolism and the host immune response. Moreover, gut microbiota can produce metabolites that may influence tumor proliferation and therapy responsiveness. In this study we have investigated the potential contribution of the gut microbiota and microbial-derived metabolites such as short chain fatty acids and polyamines to neoadjuvant radiochemotherapy (RCT) outcome in CRC patients. First, we established a profile for healthy gut microbiota by comparing the microbial diversity and composition between CRC patients and healthy controls. Second, our metagenomic analysis revealed that the gut microbiota composition of CRC patients was relatively stable over treatment time with neoadjuvant RCT. Nevertheless, treated patients who achieved clinical benefits from RTC (responders, R) had significantly higher microbial diversity and richness compared to non-responder patients (NR). Importantly, the fecal microbiota of the R was enriched in butyrate-producing bacteria and had significantly higher levels of acetic, butyric, isobutyric, and hexanoic acids than NR. In addition, NR patients exhibited higher serum levels of spermine and acetyl polyamines (oncometabolites related to CRC) as well as zonulin (gut permeability marker), and their gut microbiota was abundant in pro-inflammatory species. Finally, we identified a baseline consortium of five bacterial species that could potentially predict CRC treatment outcome. Overall, our results suggest that the gut microbiota may have an important role in the response to cancer therapies in CRC patients.

Circulating tumor DNA (ctDNA) has emerged as a non-invasive &ldquo, liquid biopsy&rdquo, for early breast cancer diagnosis. We evaluated the suitability of ctDNA analysis in the diagnosis of early breast cancer after mammography findings, comparing PIK3CA and TP53 mutations between tumor biopsies and pre-biopsy circulating DNA. Matched plasma and frozen fresh tissue biopsies from patients with Breast Imaging-Reporting and Data System (BIRADS) 4c/5 mammography findings and subsequent diagnosis of primary breast cancer were analyzed using NGS TruSeq Custom Amplicon Low Input Panel (Illumina) and plasma SafeSEQ (Sysmex Inostics). The same plasma and tumor mutations were observed in eight of 29 patients (27.6%) with four in TP53 and five in PIK3CA mutations. Sequencing analysis also revealed four additional ctDNA mutations (three in TP53 and one in PIK3CA) previously not identified in three patients tissue biopsy. One of these patients had mutations in both genes. Age, tumor grade and size, immunohistochemical (IHC) subtype, BIRADS category, and lymph node positivity were significantly associated with the detectability of these blood tumor-derived mutations. In conclusion, ctDNA analysis could be used in early breast cancer diagnosis, providing critical clinical information to improve patient diagnosis.

Circulating tumor DNA (ctDNA) has emerged as a non-invasive “liquid biopsy” for early breast cancer diagnosis. We evaluated the suitability of ctDNA analysis in the diagnosis of early breast cancer after mammography findings, comparing PIK3CA and TP53 mutations between tumor biopsies and pre-biopsy circulating DNA. Matched plasma and frozen fresh tissue biopsies from patients with Breast Imaging-Reporting and Data System (BIRADS) 4c/5 mammography findings and subsequent diagnosis of primary breast cancer were analyzed using NGS TruSeq Custom Amplicon Low Input Panel (Illumina) and plasma SafeSEQ (Sysmex Inostics). The same plasma and tumor mutations were observed in eight of 29 patients (27.6%) with four in TP53 and five in PIK3CA mutations. Sequencing analysis also revealed four additional ctDNA mutations (three in TP53 and one in PIK3CA) previously not identified in three patients tissue biopsy. One of these patients had mutations in both genes. Age, tumor grade and size, immunohistochemical (IHC) subtype, BIRADS category, and lymph node positivity were significantly associated with the detectability of these blood tumor-derived mutations. In conclusion, ctDNA analysis could be used in early breast cancer diagnosis, providing critical clinical information to improve patient diagnosis.

Introduction: Obesity has been associated with increased risk of presenting hypogonadism. Free testosterone (FT) is the fraction of testosterone that carries out the biological function of testosterone, and is determined from total testosterone (TT) and sex-hormone binding globulin (SHBG) levels. We aimed to study the SHBG polymorphism rs1799941 in a cohort of young non-diabetic obese males to unravel the possible implication of this polymorphism in obesity-related hypogonadism. Methodology: 212 young (&lt, 45 years) non-diabetic obese (BMI &ge, 30 kg/m2) males participated in this study. Subjects were classified according to TT and FT levels in: Eugonadal (n = 55, TT &gt, 3.5 ng/mL and FT &ge, 70 pg/mL, EuG), normal FT hypogonadism (n = 40, TT &lt, 3.5 and FT &ge, normal FT HG) and hypogonadism (n = 117, TT &lt, 3.5 ng/mL and TL &lt, HG). The SHBG rs1799941 polymorphism (GG/GA/AA) was analyzed using the Taqman Open Array (Applied biosystem). Results: The rs1799941 frequencies were different among the groups. Higher proportion of the allele (A) was found in HG, compared to EuG and normal FT HG. Among the genotypes, the rare homozygous (AA) were found in the normal FT HG group and higher levels of serum SHBG and lower of FT were observed. The presence of the allele A was related (according to lineal regression models) to an increased of SHBG levels ((GA) &beta, = 3.28, (AA) &beta, = 12.45) and a decreased of FT levels ((GA) &beta, = &minus, 9.19, 18.52). The presence of the allele (A) increased the risk of presenting HG compared to normal FT HG (OR = 2.54). Conclusions: The rs1799941 of the SHBG gene can partially determine the presence of obesity-related hypogonadism in young non-diabetic males and whether these subjects have normal FT HG.

IntroductionAdipose tissue is considered an important metabolic tissue, in charge of energy storage as well as being able to act in systemic homeostasis and inflammation. Epigenetics involves a series of factors that are important for gene regulation or for chromatin structure, mostly DNA methylation and histone-tail modifications, which can be modified by environmental conditions (nutrition, lifestyle, smoking…). Since metabolic diseases like obesity and diabetes are closely related to lifestyle and nutrition, epigenetic deregulation could play an important role in the onset of these diseases and vice versa. However, little is known about histone marks in human adipose tissue. In a previous work, we developed a protocol for chromatin immunoprecipitation (ChIP) of frozen human adipose tissue. By using this method, this study investigates, for the first time, the H3K4 trimethylation (H3K4me3) mark (open chromatin) on the promoter of several factors involved in adipogenesis, lipid metabolism and inflammation in visceral adipose tissue (VAT) from human subjects with different degrees of body mass index (BMI) and metabolic disease.MethodologyVAT was collected and frozen at -80°C. 100 mg VAT samples were fixed in 0.5% formaldehyde and homogenized. After sonication, the sheared chromatin was immune-precipitated with an anti-H3K4me3 antibody linked to magnetic beads and purified. H3K4me3 enrichment was analyzed by qPCR for LEP, LPL, SREBF2, SCD1, PPARG, IL6, TNF and E2F1 promoters. mRNA extraction on the same samples was performed to quantify gene expression of these genes.ResultsH3K4me3 was enriched at the promoter of E2F1, LPL, SREBF2, SCD1, PPARG and IL6 in lean normoglycemic compared to morbid obese subjects with prediabetes. Accordingly H3K4me3 mark enrichment at E2F1, LPL, SREBF2, SCD1, PPARG and IL6 promoters was positively correlated with the BMI and the HOMA-IR. Regression analysis showed a strong relationship between the BMI with H3K4me3 at the promoter of E2F1 and LPL, and with mRNA levels of LEP and SCD. In the case of HOMA-IR, the regression analysis showed associations with H3K4me3 enrichment at the promoter of SCD1 and IL6, and with the mRNA of LEP and SCD1. Moreover H3K4me3 at the E2F1 promoter was positively associated to E2F1 mRNA levels.ConclusionsH3K4me3 enrichment in the promoter of LEP, LPL, SREBF2, SCD1, PPARG, IL6, TNF and E2F1 is directly associated with increasing BMI and metabolic deterioration. The H3k4me3 mark could be regulating E3F1 mRNA levels in adipose tissue, while no associations between the promoter enrichment of this mark and mRNA levels existed for the other genes studied.

Obesity-associated hypoandrogenemia is increasing in parallel to the obesity epidemic. The prevalence of hypoandrogenemia in nondiabetic young men with obesity is not known. This study aimed to evaluate the prevalence of hypoandrogenemia and associated risk factors in this population.This cross-sectional study included 266 nondiabetic men 50 years of age with obesity who were referred from primary care. Total testosterone (high-performance liquid chromatography mass spectrometry), sex hormone-binding globulin, free testosterone (FT), luteinizing hormone (LH), high-sensitivity C-reactive protein, and homeostatic model assessment of insulin resistance were determined. Body composition and erectile function were also assessed. Hypoandrogenemia was defined as FT level 70 pg/mL.Subnormal FT concentrations were found in 25.6% of participants. Hypoandrogenemia prevalence was different along the BMI continuum, being 75% in individuals with BMI ≥ 50 kg/mIn a primary care-based cohort of nondiabetic young men with obesity, hypoandrogenemia was a very prevalent finding and was directly associated with adiposity. Obesity, age, and reduced LH levels were independent risk factors associated with hypoandrogenemia. Further prospective studies are needed to evaluate the long-term consequences of hypoandrogenemia in this population.

Endometrial cancer is one of the most common gynaecological malignancies worldwide. Histologically, two types of endometrial cancer with morphological and molecular differences and also therapeutic implications have been identified. Type I endometrial cancer has an endometrioid morphology and is estrogen-dependent, while Type II appears with non-endometrioid differentiation and follows an estrogen-unrelated pathway. Understanding the molecular biology and genetics of endometrial cancer is crucial for its prognosis and the development of novel therapies for its treatment. However, until now, scant attention has been paid to environmental components like the microbiome. Recently, due to emerging evidence that the uterus is not a sterile cavity, some studies have begun to investigate the composition of the endometrial microbiome and its role in endometrial cancer. In this review, we summarize the current state of this line of investigation, focusing on the relationship between gut and endometrial microbiome and inflammation, estrogen metabolism, and different endometrial cancer therapies.

Simple Summary The relationship between melatonin and breast cancer has been widely described. On the other hand, in recent years, an imbalance in the composition of the intestinal bacterial population has been linked as another possible trigger for this disease. Given that changes in the gut microbiota have been observed to stimulate the kinurenine pathway, reducing circulating melatonin levels, in this review, we summarize the relationship between circadian disruption and breast cancer, as well as the connection with dysbiosis as possible causing this pathology due to a series of changes that lead to an increase in circulating estrogen levels. Abstract In this review we summarize a possible connection between gut microbiota, melatonin production, and breast cancer. An imbalance in gut bacterial population composition (dysbiosis), or changes in the production of melatonin (circadian disruption) alters estrogen levels. On the one hand, this may be due to the bacterial composition of estrobolome, since bacteria with β-glucuronidase activity favour estrogens in a deconjugated state, which may ultimately lead to pathologies, including breast cancer. On the other hand, it has been shown that these changes in intestinal microbiota stimulate the kynurenine pathway, moving tryptophan away from the melatonergic pathway, thereby reducing circulating melatonin levels. Due to the fact that melatonin has antiestrogenic properties, it affects active and inactive estrogen levels. These changes increase the risk of developing breast cancer. Additionally, melatonin stimulates the differentiation of preadipocytes into adipocytes, which have low estrogen levels due to the fact that adipocytes do not express aromatase. Consequently, melatonin also reduces the risk of breast cancer. However, more studies are needed to determine the relationship between microbiota, melatonin, and breast cancer, in addition to clinical trials to confirm the sensitizing effects of melatonin to chemotherapy and radiotherapy, and its ability to ameliorate or prevent the side effects of these therapies.

The aims of this study were to explore intestinal microbial composition and functionality in primary Sj&ouml, gren&rsquo, s syndrome (pSS) and to relate these findings to inflammation, permeability and the transcription factor Forkhead box protein P3 (FOXP3) gene expression in peripheral blood. The study included 19 pSS patients and 19 healthy controls matched for age, sex, and body mass index. Fecal bacterial DNA was extracted and analyzed by 16S rRNA sequencing using an Ion S5 platform followed by a bioinformatics analysis using Quantitative Insights into Microbial Ecology (QIIME II) and Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). Our data suggest that the gut microbiota of pSS patients differs at both the taxonomic and functional levels with respect to healthy controls. The gut microbiota profile of our pSS patients was characterized by a lower diversity and richness and with Bacteroidetes dominating at the phylum level. The pSS patients had less beneficial or commensal butyrate-producing bacteria and a higher proportion of opportunistic pathogens with proinflammatory activity, which may impair intestinal barrier function and therefore contribute to inflammatory processes associated with pSS by increasing the production of proinflammatory cytokines and decreasing the release of the anti-inflammatory cytokine IL-10 and the peripheral FOXP3 mRNA expression, implicated in the development and function of regulatory T cells (Treg) cells. Further studies are needed to better understand the real impact of dysbiosis on the course of pSS and to conceive preventive or therapeutic strategies to counteract microbiome-driven inflammation.

Metabolic syndrome (MetS) has been postulated to increase the risk for type 2 diabetes, cardiovascular disease and cancer. Adipose tissue (AT) plays an important role in metabolic homeostasis, and AT dysfunction has an active role in metabolic diseases. MetS is closely related to lifestyle and environmental factors. Epigenetics has emerged as an interesting landscape to evaluate the possible interconnection between AT and metabolic disease, since it can be modulated by environmental factors and metabolic status. The aim of this study was to determine whether MetS has an impact on the global DNA methylation pattern and the DNA methylation of several genes related to adipogenesis (PPARG, PPARA), lipid metabolism (RXRA, SREBF2, SREBF1, SCD, LPL, LXRb), and inflammation (LRP1 C3, LEP and TNF) in visceral adipose tissue. LPL and TNF DNA methylation values were significantly different in the control-case comparisons, with higher and lower methylation respectively in the MetS group. Negative correlations were found between global DNA methylation (measured by LINE-1 methylation levels) and the metabolic deterioration and glucose levels. There were associations among variables of MetS, BMI, and HOMA-IR with DNA methylation at several CpG positions for the studied genes. In particular, there was a strong positive association between serum triglyceride levels (TG) with PPARA and LPL methylation levels. TNF methylation was negatively associated with the metabolic worsening and could be an important factor in preventing MetS occurrence according to logistic regression analysis. Therefore, global DNA methylation and methylation at specific genes related to adipogenesis, lipid metabolism and inflammation are related to the etiology of MetS and might explain in part some of the features associated to metabolic disorders.

Epigenetic marks, and especially DNA methylation, are becoming an important factor in obesity, which could help to explain its etiology and associated comorbidities. Adipose tissue, now considered as an important endocrine organ, produces complement system factors. Complement component 3 (C3) turns out to be an important protein in metabolic disorders, via either inflammation or the C3 subproduct acylation stimulating protein (ASP) which directly stimulates lipid storage. In this study, we analyze C3 DNA methylation in adipose tissue from subjects with a different grade of obesity. Adipose tissue samples were collected from subjects with a different degree of obesity determined by their body mass index (BMI) as: Overweight subjects (BMI ≥ 25 and

BACKGROUNDDNA methylation is one of the epigenetic mechanisms that regulate gene expression. DNA methylation may be modified by environmental and nutritional factors. Thus, epigenetics could potentially provide a mechanism to explain the etiology of metabolic disorders, such as metabolic syndrome (MetS). The aim of this study was to analyze the level of DNA methylation of several lipoprotein lipase (LPL)-promoter-CpG dinucleotides in a CpG island region and relate this to the gene and protein expression levels in human visceral adipose tissue (VAT) from individuals with and without MetS.METHODSVAT samples were collected from laparoscopic surgical patients without and with MetS, and levels of LPL mRNA, LPL protein, and LPL DNA methylation were measured by qPCR, western blot, and pyrosequencing. Biochemical and anthropometric variables were analyzed. Individuals included in a subset underwent a dietary fat challenge test, and levels of postprandial triglycerides were determined.RESULTSWe found higher levels of DNA methylation in MetS patients but lower gene expression and protein levels. There was a negative association between LPL methylation and LPL gene expression. We found a positive association between LPL methylation status and abnormalities of the metabolic profile and basal and postprandial triglycerides, whereas LPL gene expression was negatively associated with these abnormalities.CONCLUSIONSWe demonstrate that LPL methylation may be influenced by the degree of metabolic disturbances and could be involved in triglyceride metabolism, promoting hypertriglyceridemia and subsequent associated disorders, such as MetS.

Gut microbiota composition has been reported as a factor linking host metabolism with the development of cardiovascular diseases (CVD) and intestinal immunity. Such gut microbiota has been shown to aggravate CVD by contributing to the production of trimethylamine N-oxide (TMAO), which is a pro-atherogenic compound. Treg cells expressing the transcription factor Forkhead box protein P3 (FoxP3) play an essential role in the regulation of immune responses to commensal microbiota and have an atheroprotective role. However, the aim of this study was to analyze the role of gut microbiota on cardio-metabolic parameters and immunity in coronary artery disease (CAD) patients with and without type-2 diabetes mellitus (DM2). The study included 16 coronary CAD-DM2 patients, and 16 age, sex, and BMI matched CAD patients without DM2 (CAD-NDM2). Fecal bacterial DNA was extracted and analyzed by sequencing in a GS Junior 454 platform followed by a bioinformatic analysis (QIIME and PICRUSt). The present study indicated that the diversity and composition of gut microbiota were different between the CAD-DM2 and CAD-NDM2 patients. The abundance of phylum Bacteroidetes was lower, whereas the phyla Firmicutes and Proteobacteria were higher in CAD-DM2 patients than those in the CAD-NDM2 group. CAD-DM2 patients had significantly less beneficial or commensal bacteria (such as Faecalibacterium prausnitzii and Bacteroides fragilis) and more opportunistic pathogens (such as Enterobacteriaceae, Streptococcus, and Desulfovibrio). Additionally, CAD-DM2 patients had significantly higher levels of plasma zonulin, TMAO, and IL-1B and significantly lower levels of IL-10 and FOXP3 mRNA expression than CAD-NDM2. Moreover, in the CAD-MD2 group, the increase in Enterobacteriaceae and the decrease in Faecalibacterium prausnitzii were significantly associated with the increase in serum TMAO levels, while the decrease in the abundance of Bacteroides fragilis was associated with the reduction in the FOXP3 mRNA expression, implicated in the development and function of Treg cells. These results suggest that the presence of DM2 is related to an impaired regulation of the immune system in CAD patients, mediated in part by the gut microbiota composition and functionality and the production and effects of their gut microbiota derived molecules.

The biological properties of dietary polyphenols are greatly dependent on their bioavailability that, in turn, is largely influenced by their degree of polymerization. The gut microbiota play a key role in modulating the production, bioavailability and, thus, the biological activities of phenolic metabolites, particularly after the intake of food containing high-molecular-weight polyphenols. In addition, evidence is emerging on the activity of dietary polyphenols on the modulation of the colonic microbial population composition or activity. However, although the great range of health-promoting activities of dietary polyphenols has been widely investigated, their effect on the modulation of the gut ecology and the two-way relationship “polyphenols ↔ microbiota” are still poorly understood.Only a few studies have examined the impact of dietary polyphenols on the human gut microbiota, and most were focused on single polyphenol molecules and selected bacterial populations. This review focuses on the reciprocal interactions between the gut microbiota and polyphenols, the mechanisms of action and the consequences of these interactions on human health.

Alterations of gut microbiome have been proposed to play a role in metabolic disease, but the major determinants of microbiota composition remain ill defined. Nutritional and sex hormone challenges, especially during early development, have been shown to permanently alter adult female phenotype and contribute to metabolic disturbances. In this study, we implemented large-scale microbiome analyses to fecal samples from groups of female rats sequentially subjected to various obesogenic manipulations, including sex hormone perturbations by means of neonatal androgenization or adult ovariectomy (OVX), as a model of menopause, to establish whether these phenomena are related to changes in gut microbiota. Basic metabolic profiles concerning glucose/insulin homeostasis were also explored. The effects of the sex hormonal perturbations, either developmentally (androgenization) or in adulthood (OVX), clearly outshone the impact of nutritional interventions, especially concerning the gut microbiota profile. Notably, we observed a lower diversity in the androgenized group, with the highest Firmicutes to Bacteroidetes ratio, supporting the occurrence of durable alterations in gut microbiota composition, even in adulthood. Moreover, the elimination of adult ovarian secretions by OVX affected the richness of gut microbiota. Our data are the first to document the durable impact of sex steroid manipulations, and particularly early androgenization, on gut microbiota composition. Such dysbiosis is likely to contribute to the metabolic perturbations of conditions of obesity linked to gonadal dysfunction in the female.

Background: Intermittent hypoxia (IH), a hallmark of obstructive sleep apnea (OSA), can change gut microbial community. However, whether these IH-induced changes are reversible after restoring normal breathing is unknown. The aim of this work was to investigate whether gut microbiota composition and circulating endotoxemia could be recovered after a post-IH normoxic period in a mouse model of OSA. Methods: Ten mice were subjected to IH (40s 21% O2-20s 5% O2), 6h/day for 6 weeks and a control group of 10 mice were breathing room air. After the IH or normoxic exposure, both groups of mice were subjected to normoxia (room air). Microbiome composition of fecal samples was determined by 16S rRNA pyrosequencing. Bioinformatic analysis was performed by Quantitative Insights into Microbial Ecology. Plasmatic LPS was measured by endotoxin assay. Results: After normoxic recovery, the IH group had a significant decrease in the abundance of Bacteroidetes and a significant increase of Firmicutes and Deferribacteres compared to controls. Circulating LPS concentration was considerably higher in the IH group. Moreover, the IH group showed a negative and significant correlation between the abundance of Lactobacillus and Ruminococcus and significant positive correlations between the abundance of Mucispirillum and Desulfovibrio and the plasma LPS levels, respectively. Conclusion: The results obtained suggest that in OSA patients the benefits of CPAP on potential alterations in gut microbiota may require long term treatment.

Submitted for publication July 31, 2 2016; accepted for publication Janu Discordant phenotypes, metabolically healthy obese and unhealthy normal-weight individuals, are always interesting to provide important insights into the mechanistic link between adipose tissue dysfunction and associatedmetabolic alterations. Macrophages can release factors that impair the proper activity of the adipose tissue. Thus, studying subcutaneous and visceral adipose tissues, we investigated for the first time the differences inmonocyte/macrophage infiltration, inflammation, and adipogenesis of normal-weight subjects who differed in their degree of metabolic syndrome. The study included 92 normal-weight subjects who differed in their degree of metabolic syndrome. Their anthropometric and biochemical parameters were measured. RNA from subcutaneous and visceral adipose tissues was isolated, and mRNA expression of monocyte/macrophage infiltration (CD68, CD33, ITGAM, CD163, EMR-1, CD206,MerTK, CD64, ITGAX), inflammation (IL-6, tumor necrosis factor alpha [TNFa], IL-10, IL-1b, CCL2, CCL3), and adipogenic and lipogenic capacity markers (PPARgamma, FABP4)weremeasured. Taken together, our data provide evidence of a different degree of macrophage infiltration between the adipose tissues, with a higher monocyte/macrophage infiltration in subcutaneous adipose tissue in metabolically unhealthy normal-weight subjects, whereas visceral adipose tissue remained almost unaffected. An increased macrophage infiltration of adipose tissue and its consequences, such as a decrease in adipogenesis function, may explain why both the obese and normal-weight subjects can develop metabolic diseases or remain healthy. (Translational Research 2016;-:1–12)

STUDY OBJECTIVES Intermittent hypoxia (IH) mimicking obstructive sleep apnea (OSA) significantly modifies gut microbiota in mice. However, whether these IH-induced gut microbiome changes are reversible after restoring normal oxygenation (the equivalent of effective OSA therapy) is unknown. The aim of this study was to investigate gut microbiota composition and circulating endotoxemia after a post-IH normoxic period in a mouse model of OSA. METHODS Ten mice were subjected to IH (40 sec 21% O2-20 sec 5% O2) for 6 h/day for 6 w and 10 mice breathing normoxic air (NM) were used as controls. After exposures, both groups were subjected to 6 w in normoxia. Microbiome composition of fecal samples was determined by 16S ribosomal RNA (rRNA) pyrosequencing. Bioinformatic analysis was performed by Quantitative Insights into Microbial Ecology. Plasma lipopolysaccharide (LPS) levels were measured by endotoxin assay. RESULTS After normoxic recovery, the Chao and Shannon indices of each group suggested similar bacterial richness and diversity. 16S rRNA pyrosequencing analysis showed that IH-exposed mice had a significant decrease in the abundance of Bacteroidetes and a significant increase of Firmicutes and Deferribacteres compared to the NM group. After normoxic recovery, circulating LPS concentrations were higher in the IH group (P < 0.009). Moreover, the IH group showed a negative and significant correlation between the abundance of Lactobacillus and Ruminococcus and significant positive correlations between the abundance of Mucispirillum and Desulfovibrio and plasma LPS levels, respectively. CONCLUSIONS Even after prolonged normoxic recovery after IH exposures, gut microbiota and circulating endotoxemia remain negatively altered, suggesting that potential benefits of OSA treatment for reversing OSA-induced changes in gut microbiota may either require a longer period or alternative interventions.

Background: Few studies have investigated the effect of dietary polyphenols on the complex human gut microbiota, and they focused mainly on single polyphenol molecules and select bacterial populations. Objective: The objective was to evaluate the effect of a moderate intake of red wine polyphenols on select gut microbial groups implicated in host health benefits. Design: Ten healthy male volunteers underwent a randomized, crossover, controlled intervention study. After a washout period, all of the subjects received red wine, the equivalent amount of dealcoholized red wine, or gin for 20 d each. Total fecal DNA was submitted to polymerase chain reaction (PCR)‐denaturing gradient gel electrophoresis and real-time quantitative PCR to monitor and quantify changes in fecal microbiota. Several biochemical markers were measured. Results: The dominant bacterial composition did not remain constant over the different intake periods. Compared with baseline, the daily consumption of red wine polyphenol for 4 wk significantly increased the number of Enterococcus, Prevotella, Bacteroides, Bifidobacterium, Bacteroides uniformis, Eggerthella lenta, and Blautia coccoides‐Eubacterium rectale groups (P , 0.05). In parallel, systolic and diastolic blood pressures and triglyceride, total cholesterol, HDL cholesterol, and C-reactive protein concentrations decreased significantly (P , 0.05). Moreover, changes in cholesterol and C-reactive protein concentrations were linked to changes in the bifidobacteria number. Conclusion: This study showed that red wine consumption can significantly modulate the growth of select gut microbiota in humans, which suggests possible prebiotic benefits associated with the inclusion of red wine polyphenols in the diet. This trial was registered at controlled-trials.com as ISRCTN88720134. Am J Clin Nutr 2012;95:1323‐34.

Objectives Apolipoprotein C-III (APOC3) is a component of triglyceride rich lipoproteins, and SstI polymorphism has been associated with hypertriglyceridemia. Apolipoprotein A-I (APOA1) is the major component of HDL and MspI polymorphism has been associated with APOA1 and HDL-C levels. Thus, we study the influence of these polymorphisms in the postprandial response in metabolic syndrome (MS). Design and methods 73 MS patients and 21 healthy subjects underwent a fat overload, with measurements of their fasting and postprandial lipid profile. The APOC3 SstI and the APOA1 MspI polymorphisms were genotyped. Results No significant differences were found in the lipid profile with respect to the MspI genotype. Patients with the S2S2 APOC3 genotype had significantly higher fasting and postprandial triglyceride levels and postprandial APOC3 and chylomicron-triglyceride levels compared with the other SstI APOC3 genotypes. Conclusions Homozygosity for the minor allele of the APOC3 SstI polymorphism was associated to a worse postprandial response in MS patients.

Study Design. Case-control study for assessing a diagnostic test. Objective. The aim of this study was to analyze the diagnostic yield of a multiplex real-time polymerase chain reaction (PCR) assay in the differential diagnosis of tuberculous vertebral osteomyelitis (TVO) and brucellar vertebral osteomyelitis (BVO). Summary of Background Data. Vertebral osteomyelitis (VO) is one of commonest osteoarticular complications of tuberculosis and brucellosis. However, the very similar clinical, radiologic, and histologic characteristics of these entities mean that diagnosis requires etiological confirmation, but conventional microbiologic methods have important limitations. Methods. Fifteen vertebral samples from patients with TVO or BVO and 9 from pyogenic and nontuberculous mycobacteria VO were studied by multiplex PCR and conventional microbiologic techniques. To identify Brucella DNA, we used a fragment of 207 bp from the conserved region of the gene coding for an immunogenic membrane protein of 31 kDa of B. abortus (BCSP31) and for Mycobacterium tuberculosis complex, a fragment of 164 bp from the intergenic region SenX3-RegX3. Results. The histopathologic findings were inconclusive in 4 of 14 cases (28.6%) with TVO or BVO and cultures were positive in 11 of 15 cases (73.3%). Multiplex PCR correctly identified 14 of the 15 samples from patients with TVO and BVO and was negative in all the control samples. Thus, the overall sensitivity and specificity of the multiplex PCR were 93.3% and 90%, respectively, with an accuracy of 92% (95% CI, 81.4%–100%). Conclusion. These results suggest that multiplex real-time PCR is far more sensitive than conventional cultures, and this, together with its speed, makes this technique a very practical approach for the differential diagnosis between TVO and BVO.

Molecules involved in antigen processing (LMP) and peptide transport (TAP) are coded by polymorphic genes. This polymorphism may influence the peptide antigen selection process and play a role in the pathogenesis of human brucellosis. We studied the polymorphism of the antigen processing and transport genes (LMP and TAP) in 61 patients with human brucellosis and 102 controls from southern Spain. We found no differences in the frequencies of the LMP and TAP genotypes between the patients and the controls. Study of the patients with and without focal or complicated forms showed a significant increase in the TAP2A/TAP2F genotype in those with focal forms compared with those without focal forms (16% vs 0%, p = 0.02), though this difference lost its significance after correction for the number of comparisons. This study suggests that larger studies will be needed to confirm or rule out the possible association of the TAP2A/TAP2F genotype or other possible associations with focal forms of brucellosis.