Epigenetic profiling of the D4Z4 locus: Optimization of the protocol for studying DNA methylation at single CpG site level.

The alteration of epigenetic modifications, including DNA methylation, can contribute to the etiopathogenesis and progression of many diseases. Among them, facioscapulohumeral dystrophy (FSHD) is a muscular disorder characterized by the loss of repressive epigenetic features affecting the D4Z4 locus (4q35). As a consequence, these alterations are responsible for DNA hypomethylation and a transcriptional-active chromatin conformation change that, in turn, lead to the aberrant expression of DUX4 in muscle cells. In the present study, methylation levels of 29 CpG sites of the DR1 region (within each repeat unit of the D4Z4 macrosatellite) were assessed on 335 subjects by employing primers designed for enhancing the performance of the assay. First, the DR1 original primers were optimized by adding M13 oligonucleotide tails. Moreover, the DR1 reverse primer was replaced with a degenerate one. As a result, the protocol optimization allowed a better sequencing resolution and a more accurate evaluation of DR1 methylation levels. Moreover, the assessment of the repeatability of measurements proved the reliability and robustness of the assay. The optimized protocol emerges as an excellent method to detect methylation levels compatible with FSHD.

Protective Role of Bacterial Alkanesulfonate Monooxygenase under Oxidative Stress.

Bacterial alkane metabolism is associated with a number of cellular stresses, including membrane stress and oxidative stress, and the limited uptake of charged ions such as sulfate. In the present study, the genes ssuD and tauD in Acinetobacter oleivorans DR1 cells, which encode an alkanesulfonate monooxygenase and a taurine dioxygenase, respectively, were found to be responsible for hexadecanesulfonate (C16SO3H) and taurine metabolism, and Cbl was experimentally identified as a potential regulator of ssuD and tauD expression. The expression of ssuD and tauD occurred under sulfate-limited conditions generated during n-hexadecane degradation. Interestingly, expression analysis and knockout experiments suggested that both genes are required to protect cells against oxidative stress, including that generated by n-hexadecane degradation and H2O2 exposure. Measurable levels of intracellular hexadecanesulfonate were also produced during n-hexadecane degradation. Phylogenetic analysis suggested that ssuD and tauD are mainly present in soil-dwelling aerobes within the Betaproteobacteria and Gammaproteobacteria classes, which suggests that they function as controllers of the sulfur cycle and play a protective role against oxidative stress in sulfur-limited conditions.IMPORTANCEssuD and tauD, which play a role in the degradation of organosulfonate, were expressed during n-hexadecane metabolism and oxidative stress conditions in A. oleivorans DR1. Our study confirmed that hexadecanesulfonate was accidentally generated during bacterial n-hexadecane degradation in sulfate-limited conditions. Removal of this by-product by SsuD and TauD must be necessary for bacterial survival under oxidative stress generated during n-hexadecane degradation.

Heterodimer HLA-DM Fused with Constant Fragment of the Heavy Chain of the Human Immunoglobulin Accelerates Influenza Hemagglutinin HA306-318 Loading to HLA-DR1.

Major histocompatibility complex class II (MHC II) plays an important role not only in the adaptive immune responses to foreign pathogens, but also in the development of some autoimmune diseases. Non-classical MHC, HLA-DM is directly involved in MHC II loading with the peptide. To study this process, we synthesized recombinant proteins HLA-DR1 and HLA-DM. α/ß-Chains of DR1 heterodimer contained C-terminal leucine domains of the fos and jun factors, respectively. Each DM chain contained constant fragment of human antibody heavy chain fused via a long linker domain. In addition, DM α-chain carried N165D substitution suppressing potential glycosylation at this site. We observed significant acceleration of DR1 peptide loading with influenza HA306-318 hemagglutinin in the presence of DM, which indicates functionality of recombinant DR1-DM protein couple. Our results can be used to study the presentation of other viral and self-antigens and can become the basis for the development of new drug modeling.

A cyclic peptide accelerates the loading of peptide antigens in major histocompatibility complex class II molecules.

Major histocompatibility complex (MHC)-loading enhancers (MLE) have recently attracted attention because of their ability to enhance the efficacy of peptide immunotherapeutics. As small molecular weight compounds, they influence the loading of peptides in MHC molecules by converting them from a non-receptive to a receptive state. Herein, we report a 14-mer cyclic peptide 1 (CP-1) as a new class of MLE-peptide. This peptide was used to investigate its loading on human leukocyte antigen (HLA)-DR molecules. It was found that CP-1 strongly accelerates peptide-loading on both soluble and cell surface HLA-DR molecules in a dose-dependent manner. The effect was evident for all subsets of HLA-DR tested, including HLA-DRB1*1501, indicating that it acts independently of P1-pocket size, which is the canonical MLE-binding site. Importantly, increased peptide-loading by CP-1 was correlated with improved CD4(+) T cell responses in vitro, while propidium iodide staining indicated low peptide-induced cytotoxicity. Thus, this study revealed a new class of peptide-based enhancers that catalyze peptide-loading by allosteric interactions with MHC molecules. Because of its low cellular cytotoxicity and high MLE activity, it may be useful in stimulating antigen-specific T cell responses for therapeutic purposes.

Hepatocyte nuclear factor 4α regulates the expression of the murine pyruvate carboxylase gene through the HNF4-specific binding motif in its proximal promoter.

Pyruvate carboxylase (PC) is the first regulatory enzyme of gluconeogenesis. Here we report that the proximal promoter of the murine PC gene contains three binding sites for hepatocyte nuclear factor 4α (HNF4α). These sites include the classical direct repeat 1 (DR1) (-386/-374), non-perfect DR1 (-118/-106) and HNF4α-specific binding motif (H4-SBM) (-26/-14). Under basal conditions, mutation of the non-perfect DR1 decreased promoter activity by 50%, whereas mutation of neither the DR1 nor the H4-SBM had any effect. In marked contrast, only mutation of the H4-SBM decreased HNF4α-transactivation of the promoter activity by 65%. EMSA revealed that HNF4α binds to the DR1site and H4-SBM with similar affinity while it binds poorly to the non-perfect DR1. Interestingly, this non-perfect DR1 also coincides with two E-boxes. Mutation of the non-perfect DR1 together with the nearby E-box reduced USF1- but not USF2-transactivation of promoter activity, suggesting that USF1 partly contributes to the basal activity of the promoter. Substitution of the H4-SBM with the DR1 marginally reduced the basal promoter activity but did not eliminate HNF4α-transactivation, suggesting that HNF4α can exert its effect via DR1 within this promoter context. ChIP-assay confirmed that HNF4α is associated with the H4-SBM. Suppression of HNF4α expression in AML12 cells down-regulated PC mRNA and PC protein by 60% and 50%, respectively, confirming that PC is a target of HNF4α. We also propose a model for differential regulation of P1 promoter of PC gene in adipose tissue and liver.

SREBP-2 negatively regulates FXR-dependent transcription of FGF19 in human intestinal cells.

Sterol regulatory element-binding protein-2 (SREBP-2) is a basic helix-loop-helix-leucine zipper transcription factor that positively regulates transcription of target genes involved in cholesterol metabolism. In the present study, we have investigated a possible involvement of SREBP-2 in human intestinal expression of fibroblast growth factor (FGF)19, which is an endocrine hormone involved in the regulation of lipid and glucose metabolism. Overexpression of constitutively active SREBP-2 decreased FGF19 mRNA levels in human colon-derived LS174T cells. In reporter assays, active SREBP-2 overexpression suppressed GW4064/FXR-mediated increase in reporter activities in regions containing the IR-1 motif (+848 to +5200) in the FGF19 gene. The suppressive effect disappeared in reporter activities in the region containing the IR-1 motif when the mutation was introduced into the IR-1 motif. In electrophoretic mobility shift assays, binding of the FXR/retinoid X receptor α heterodimer to the IR-1 motif was attenuated by adding active SREBP-2, but SREBP-2 binding to the IR-1 motif was not observed. In chromatin immunoprecipitation assays, specific binding of FXR to the IR-1-containing region of the FGF19 gene (+3214 to +3404) was increased in LS174T cells by treatment with cholesterol and 25-hydroxycholesterol. Specific binding of SREBP-2 to FXR was observed in glutathione-S-transferase (GST) pull-down assays. These results suggest that SREBP-2 negatively regulates the FXR-mediated transcriptional activation of the FGF19 gene in human intestinal cells.

A targeted single mutation in influenza A virus universal epitope transforms immunogenicity and protective immunity via CD4+ T cell activation.

CD4+ T cells are central to adaptive immunity. Their role in cross-protection in viral infections such as influenza and severe acute respiratory syndrome (SARS) is well documented; however, molecular rules governing T cell receptor (TCR) engagement of peptide-human leukocyte antigen (pHLA) class II are less understood. Here, we exploit an aspect of HLA class II presentation, the peptide-flanking residues (PFRs), to "tune" CD4+ T cell responses within an in vivo model system of influenza. Using a recombinant virus containing targeted substitutions at immunodominant HLA-DR1 epitopes, we demonstrate limited weight loss and improved clinical scores after heterosubtypic re-challenge. We observe enhanced protection linked to lung-derived influenza-specific CD4+ and CD8+ T cells prior to re-infection. Structural analysis of the ternary TCR:pHLA complex identifies that flanking amino acids influence side chains in the core 9-mer peptide, increasing TCR affinity. Augmentation of CD4+ T cell immunity is achievable with a single mutation, representing a strategy to enhance adaptive immunity that is decoupled from vaccine modality.

A Mutated Prostatic Acid Phosphatase (PAP) Peptide-Based Vaccine Induces PAP-Specific CD8+ T Cells with Ex Vivo Cytotoxic Capacities in HHDII/DR1 Transgenic Mice.

BACKGROUND: Current treatments for castrate (hormone)-resistant prostate cancer (CRPC) remain limited and are not curative, with a median survival from diagnosis of 23 months. The PAP-specific Sipuleucel-T vaccine, which was approved by the FDA in 2010, increases the Overall Survival (OS) by 4 months, but is extremely expensive. We have previously shown that a 15 amino accid (AA) PAP sequence-derived peptide could induce strong immune responses and delay the growth of murine TRAMP-C1 prostate tumors. We have now substituted one amino acid and elongated the sequence to include epitopes predicted to bind to several additional HLA haplotypes. Herein, we present the immunological properties of this 42mer-mutated PAP-derived sequence (MutPAP42mer). METHODS: The presence of PAP-135-143 epitope-specific CD8+ T cells in the blood of patients with prostate cancer (PCa) was assessed by flow cytometry using Dextramer™ technology. HHDII/DR1 transgenic mice were immunized with mutated and non-mutated PAP-derived 42mer peptides in the presence of CAF®09 or CpG ODN1826 (TLR-9 agonist) adjuvants. Vaccine-induced immune responses were measured by assessing the proportion and functionality of splenic PAP-specific T cells in vitro. RESULTS: PAP-135-143 epitope-specific CD8+ T cells were detected in the blood of patients with PCa and stimulation of PBMCs from patients with PCa with mutPAP42mer enhanced their capacity to kill human LNCaP PCa target cells expressing PAP. The MutPAP42mer peptide was significantly more immunogenic in HHDII/DR1 mice than the wild type sequence, and immunogenicity was further enhanced when combined with the CAF®09 adjuvant. The vaccine induced secretory (IFNγ and TNFα) and cytotoxic CD8+ T cells and effector memory splenic T cells. CONCLUSIONS: The periphery of patients with PCa exhibits immune responsiveness to the MutPAP42mer peptide and immunization of mice induces/expands T cell-driven, wild-type PAP immunity, and therefore, has the potential to drive protective anti-tumor immunity in patients with PCa.

Targeted long-read sequencing reveals clonally expanded HBV-associated chromosomal translocations in patients with chronic hepatitis B.

Background & Aims: HBV infects over 257 million people worldwide and is associated with the development of hepatocellular carcinoma (HCC). Integration of HBV DNA into the host genome is likely a key driver of HCC oncogenesis. Here, we utilise targeted long-read sequencing to determine the structure of HBV DNA integrations as well as full isoform information of HBV mRNA with more accurate quantification than traditional next generation sequencing platforms. Methods: DNA and RNA were isolated from fresh frozen liver biopsies collected within the GS-US-174-0149 clinical trial. A pan-genotypic panel of biotinylated oligos was developed to enrich for HBV sequences from sheared genomic DNA (∼7 kb) and full-length cDNA libraries from poly-adenylated RNA. Samples were sequenced on the PacBio long-read platform and analysed using a custom bioinformatic pipeline. Results: HBV-targeted long-read DNA sequencing generated high coverage data spanning entire integrations. Strikingly, in 13 of 42 samples (31%) we were able to detect HBV sequences flanked by 2 different chromosomes, indicating a chromosomal translocation associated with HBV integration. Chromosomal translocations were unique to each biopsy sample, suggesting that each originated randomly, and in some cases had evidence of clonal expansion. Using targeted long-read RNA sequencing, we determined that upwards of 95% of all HBV transcripts in patients who are HBeAg-positive originate from cccDNA. In contrast, patients who are HBeAg-negative expressed mostly HBsAg from integrations. Conclusions: Targeted lso-Seq allowed for accurate quantitation of the HBV transcriptome and assignment of transcripts to either cccDNA or integration origins. The existence of multiple unique HBV-associated inter-chromosomal translocations in non-HCC CHB patient liver biopsies suggests a novel mechanism with mutagenic potential that may contribute to progression to HCC. Lay summary: Fresh frozen liver biopsies from patients infected with HBV were subjected to targeted long-read RNA and DNA sequencing. Long-read RNA sequencing captures entire HBV transcripts in a single read, allowing for resolution of overlapping transcripts from the HBV genome. This resolution allowed us to quantify the burden of transcription from integrations vs. cccDNA origin in individual patients. Patients who were HBeAg-positive had a significantly larger fraction of the HBV transcriptome originating from cccDNA compared with those who were HBeAg-negative. Long-read DNA sequencing captured entire integrated HBV sequences including multiple kilobases of flanking host sequence within single reads. This resolution allowed us to describe integration events flanked by 2 different host chromosomes, indicating that integrated HBV DNA are associated with inter-chromosomal translocations. This may lead to significant transcriptional dysregulation and drive progression to HCC.

A Novel HAGE/WT1-ImmunoBody® Vaccine Combination Enhances Anti-Tumour Responses When Compared to Either Vaccine Alone.

Many cancers, including myeloid leukaemia express the cancer testis antigen (CTA) DDX43 (HAGE) and/or the oncogene Wilms' tumour (WT1). Here we demonstrate that HAGE/WT1-ImmunoBody® vaccines derived T-cells can kill ex-vivo human CML cell lines expressing these antigens and significantly delay B16/HHDII+/DR1+/HAGE+/WT1+ tumour growth in the HHDII/DR1 mice and prolonged mouse survival in the prophylactic setting in comparison to non-immunised control mice. We show that immunisation of HHDII/DR1 mice with HAGE- and WT1-ImmunoBody® DNA vaccines in a prime-boost regime in two different flanks induce significant IFN-γ release by splenocytes from treated mice, and a significant level of cytotoxicity against tumour targets expressing HAGE/WT1 in vitro. More importantly, the combined HAGE/WT1 ImmunoBody® vaccine significantly delayed tumour growth in the B16/HHDII+/DR1+/HAGE+/WT1+ tumour model and prolonged mouse survival in the prophylactic setting in comparison to non-immunised control mice. Overall, this work demonstrates that combining both HAGE- and WT1-ImmunoBody® into a single vaccine is better than either vaccine alone. This combination vaccine could be given to patients whose cancer expresses HAGE and WT1 in parallel with existing therapies in order to decrease the chance of disease progression and relapse.

Structure and function prediction of arsenate reductase from Deinococcus indicus DR1.

Arsenic prevalence in the environment impelled many organisms to develop resistance over the course of evolution. Tolerance to arsenic, either as the pentavalent [As(V)] form or the trivalent form [As(III)], by bacteria has been well studied in prokaryotes, and the mechanism of action is well defined. However, in the rod-shaped arsenic tolerant Deinococcus indicus DR1, the key enzyme, arsenate reductase (ArsC) has not been well studied. ArsC of D. indicus belongs to the Grx-linked prokaryotic arsenate reductase family. While it shares homology with the well-studied ArsC of Escherichia coli having a catalytic cysteine (Cys 12) and arginine triad (Arg 60, 94, and 107), the active site of D.indicus ArsC contains four residues Glu 9, Asp 53, Arg 86, and Glu 100, and with complete absence of structurally equivalent residue for crucial Cys 12. Here, we report that the mechanism of action of ArsC of D. indicus is different as a result of convergent evolution and most likely able to detoxify As(V) using a mix of positively- and negatively-charged residues in its active site, unlike the residues of E. coli. This suggests toward the possibility of an alternative mechanism of As (V) degradation in bacteria.

Construction and Screening of an Antigen-Derived Peptide Library Displayed on Yeast Cell Surface for CD4+ T Cell Epitope Identification.

Antigenic peptides (termed T cell epitopes) are assembled with major histocompatibility complex (MHC) molecules and presented on the surface of antigen-presenting cells (APCs) for T cell recognition. T cells engage these peptide-MHCs using T cell receptors (TCRs). Because T cell epitopes determine the specificity of a T cell immune response, their prediction and identification are important steps in developing peptide-based vaccines and immunotherapies. In recent years, a number of computational methods have been developed to predict T cell epitopes by evaluating peptide-MHC binding; however, the success of these methods has been limited for MHC class II (MHCII) due to the structural complexity of MHCII antigen presentation. Moreover, while peptide-MHC binding is a prerequisite for a T cell epitope, it alone is not sufficient. Therefore, T cell epitope identification requires further functional verification of the MHC-binding peptide using professional APCs, which are difficult to isolate, expand, and maintain. To address these issues, we have developed a facile, accurate, and high-throughput method for T cell epitope mapping by screening antigen-derived peptide libraries in complex with MHC protein displayed on yeast cell surface. Here, we use hemagglutinin and influenza A virus X31/A/Aichi/68 as examples to describe the key steps in identification of CD4+ T cell epitopes from a single antigenic protein and the entire genome of a pathogen, respectively. Methods for single-chain peptide MHC vector design, yeast surface display, peptide library generation in Escherichia coli, and functional screening in Saccharomyces cerevisiae are discussed.

Therapeutic vaccination using minimal HPV16 epitopes in a novel MHC-humanized murine HPV tumor model.

Therapeutic vaccination as a treatment option for HPV-induced cancers is actively pursued because the two HPV proteins E6 and E7 represent ideal targets for immunotherapy, as they are non-self and expressed in all tumor stages. MHC-humanized mice are valuable tools for the study of therapeutic cancer vaccines - given the availability of a suitable tumor model. Here, we present for the first time an HPV16 tumor model suitable for fully MHC-humanized A2.DR1 mice, PAP-A2 cells, which in contrast to existing HPV16 tumor models allows the exclusive study of HLA-A2- and DR1-mediated immune responses, without any interfering murine MHC-presented epitopes. We used several HPV16 epitopes that were shown to be presented on human cervical cancer cells by mass spectrometry for therapeutic anti-tumor vaccination in the new tumor model. All epitopes were immunogenic when rendered amphiphilic by incorporation into a molecule containing stearic acids. Prophylactic and therapeutic vaccination experiments with the epitope E7/11-19 demonstrated that effective immune responses could be induced with these vaccination approaches in A2.DR1 mice. Interestingly, the combination of E7/11-19 with other immunogenic HPV16 E6/E7 epitopes caused a reduction of vaccine efficacy, although all tested combinations resulted in a survival benefit. In summary, we present the first HPV16 tumor model for exclusive studies of HLA-A2-mediated anti-HPV tumor immune responses and show anti-tumor efficacy of minimal epitope vaccines.

Generation of human MHC (HLA-A11/DR1) transgenic mice for vaccine evaluation.

The rapid occurrence of emerging infectious diseases demonstrates an urgent need for a new preclinical experimental model that reliably replicates human immune responses. Here, a new homozygous humanized human leukocyte antigen (HLA)-A11/DR1 transgenic mouse (HLA-A11(+/+)/DR01(+/+)/H-2-ß2m(-/-)/IAß(-/-)) was generated by crossing HLA-A11 transgenic (Tg) mice with HLA-A2(+/+)/DR01(+/+)/H-2-ß2m(-/-)/IAß(-/-) mice. The HLA-A11-restricted immune response of this mouse model was then examined. HLA-A11 Tg mice expressing a chimeric major histocompatibility complex (MHC) molecule comprising the α1, α2, and ß2m domains of human HLA-A11 and the α3 transmembrane and cytoplasmic domains of murine H-2D(b) were generated. The correct integration of HLA-A11 and HLA-DR1 into the genome of the HLA-A11/DR1 Tg mice (which lacked the expression of endogenous H-2-I/II molecules) was then confirmed. Immunizing mice with a recombinant HBV vaccine or a recombinant HIV-1 protein resulted in the generation of IFN-γ-producing cytotoxic T lymphocyte (CTL) and antigen-specific antibodies. The HLA-A11-restricted CTL response was directed at HLA immunodominant epitopes. These mice represent a versatile animal model for studying the immunogenicity of HLA CTL epitopes in the absence of a murine MHC response. The established animal model will also be useful for evaluating and optimizing T cell-based vaccines and for studying differences in antigen processing between mice and humans.

Role of farnesoid X receptor and bile acids in alcoholic liver disease.

Alcoholic liver disease (ALD) is one of the major causes of liver morbidity and mortality worldwide. Chronic alcohol consumption leads to development of liver pathogenesis encompassing steatosis, inflammation, fibrosis, cirrhosis, and in extreme cases, hepatocellular carcinoma. Moreover, ALD may also associate with cholestasis. Emerging evidence now suggests that farnesoid X receptor (FXR) and bile acids also play important roles in ALD. In this review, we discuss the effects of alcohol consumption on FXR, bile acids and gut microbiome as well as their impacts on ALD. Moreover, we summarize the findings on FXR, FoxO3a (forkhead box-containing protein class O3a) and PPARα (peroxisome proliferator-activated receptor alpha) in regulation of autophagy-related gene transcription program and liver injury in response to alcohol exposure.

Immunogenicity of HLA-DR1 Restricted Peptides Derived from Leishmania major gp63 Using FVB/N-DR1 Transgenic Mouse Model.

BACKGROUND: Leishmaniasis is a worldwide disease prevalent in tropical and sub-tropical countries. In the present study the immunogenicity of three human HLA-DR1 restricted peptides derived from L. major gp63 protein was evaluated using FVB/N-DR1 transgenic mouse model. METHODS: The immunity generated by three MHC class II - restricted peptides with the sequence of AARLVRLAAAGAAVT (AAR), AAPLVRLAAAGAAVT (AAP) and SRYDQLVTRVVTHE (ASR) derived from L. major gp63 protein were predicted using a web-based software (SYFPEITHI) and tested in FVB/N-DR1 transgenic mice. RESULTS: Immunization of FVB/N-DR1 transgenic mice with one of the three predicted peptides (AAR) resulted in high levels of Th1-type immune response as well as significant levels of IFN-γ detected by Proliferation assay and ELISA. CONCLUSION: The results indicate a high level of immunogenicity for AAR, which can be a potent candidate for peptide vaccine in Leishmania infections.

MH-DAB gene polymorphism and disease resistance to Flavobacterium columnare in grass carp (Ctenopharyngodon idellus).

In this study, the association between MH-DAB gene polymorphism and disease resistance was evaluated by challenging grass carp (Ctenopharyngodon idellus) with Flavobacterium columnare. Eight genotypes and six alleles were found, and named by common nomenclature. The genotypes AA, BB, EE, and DE, and the alleles Ctid-DAB1*0101, Ctid-DAB1*0201 and Ctid-DAB1*0401 were more preponderant in fish. The genotype BB was associated with higher resistance to F. columnare, as well as two alleles Ctid-DAB*0101 and Ctid-DAB*0201. Allele Ctid-DAB*0102 has decreased resistance to F. columnare. The expression of MH-DAB gene was decreased in the liver, kidney, and intestine but not in the spleen, gill, and skin at 2 days post infection (dpi), versus to that in the control group. MH-DAB gene expression was up-regulated in most tissues but remained at normal levels in the intestine at 15 days post infection. Our data suggested that MH-DAB polymorphism can be used as a potential genetic marker for disease resistance breeding of grass carp in the future.

Revealing natural antisense transcripts from Plasmodium vivax isolates: evidence of genome regulation in complicated malaria.

Plasmodium vivax is the most geographically widespread human malaria parasite causing approximately 130-435 million infections annually. It is an economic burden in many parts of the world and poses a public health challenge along with the other Plasmodium sp. The biology of this parasite is less studied and poorly understood, in spite of these facts. Emerging evidence of severe complications due to infections by this parasite provides an impetus to focus research on the same. Investigating the parasite directly from infected patients is the best way to study its biology and pathogenic mechanisms. Gene expression studies of this parasite directly obtained from the patients has provided evidence of gene regulation resulting in varying amount of transcript levels in the different blood stages. The mechanisms regulating gene expression in malaria parasites are not well understood. Discovery of Natural Antisense Transcripts (NATs) in Plasmodium falciparum has suggested that these might play an important role in regulating gene expression. We report here the genome-wide occurrence of NATs in P. vivax parasites from patients with differing clinical symptoms. A total of 1348 NATs against annotated gene loci have been detected using a custom designed microarray with strand specific probes. Majority of NATs identified from this study shows positive correlation with the expression pattern of the sense (S) transcript. Our data also shows condition specific expression patterns of varying S and antisense (AS) transcript levels. Genes with AS transcripts enrich to various biological processes. To our knowledge this is the first report on the presence of NATs from P. vivax obtained from infected patients with different disease complications. The data suggests differential regulation of gene expression in diverse clinical conditions, as shown by differing sense/antisense ratios and would lead to future detailed investigations of gene regulation.

Human leukocyte antigen HLADRB1 determinants susceptibility to gastroesophageal reflux disease / Antígeno leucocitário humano HLADRB1 é determinante de susceptibilidade para a doença do refluxo

ABSTRACT BACKGROUND Gastroesophageal reflux disease (GERD) is characterized by diverse symptoms. There is an evidence for a genetic component to GERD as supported by familial aggregation of this disease. OBJECTIVE To investigate whether certain human leucocyte antigen genes HLA-DRB1 are associated with GERD. METHODS Patients and controls were prospectively recruited from GIT center at Al-Kindy Teaching Hospital (Baghdad-Iraq) between January 2014 and July 2016. Sixty Iraqi Arab Muslims patients with a history of heartburn and dyspepsia compared with 100 Iraqi Arab Muslims controls. All study patients and control groups underwent upper gastrointestinal endoscopic examinations and their serums were analyzed for CagA antibodies Immunoglobulin G (IgG) for H. pylori. HLA-DRB1 genotyping were done to both groups. RESULTS A total of 60 patients with erosive gastritis; GERD (Grade II and III) were evaluated, together with 100 controls. There is a significant increase of H. pylori infection (P=0.0001) in GERD patients than control group. HLA-DRB1* 15:01 was significantly increased in GERD patients in comparison with control group and an increased frequency of HLADRB1*11:01 in control group compared with patients group. CONCLUSION There is an association between HLA-DRB1 *15:01 in GERD patients with H. pylori positive patients.

RESUMO CONTEXTO A doença do refluxo gastroesofágico (DRGE) caracteriza-se por diversos sintomas. Há evidências de um componente genético para a doença de refluxo suportado pela agregação familiar desta doença. OBJETIVO Investigar se certos genes de antígeno de leucócito humano HLA-DRB1 são associados à DRGE. MÉTODOS Pacientes e indivíduos controles foram recrutados prospectivamente do centro GIT no Al-Kindy Hospital (Bagdá-Iraque) entre de 2014 janeiro e julho de 2016. Sessenta pacientes muçulmanos árabes iraquianos com uma história de azia e dispepsia foram comparados com 100 indivíduos controles. Todos os pacientes do estudo e grupos de controle foram submetidos a exames de endoscopia gastrointestinal alta e seus soros foram analisados para anticorpos CagA imunoglobulina G (IgG) para H. pylori. Genotipagem HLA-DRB1 foram feitas para ambos os grupos. RESULTADOS Um total de 60 pacientes com gastrite erosiva; GERD (grau II e III) foram avaliados, em conjunto com 100 controles. Houve aumento significativo de infecção pelo H. pylori (P=0,0001) em pacientes com DRGE em relação ao grupo controle. O HLA-DRB1* 15:01 aumentou significativamente em pacientes com DRGE em comparação com o grupo controle e houve uma maior frequência de HLADRB1* 11:01 no grupo controle em comparação com o grupo de pacientes com DRGE. CONCLUSÃO Há uma associação entre HLA-DRB1* 15:01 em pacientes com DRGE positivos para a infecção por H. pylori.