Correlation between Resistin, Tuberculosis and Khat Addiction: A Study from South Western Province of Saudi Arabia.

Tuberculosis(TB) is a disease of global significance, which accounts for a death in every 15 seconds. Recent studies shows TB is rising in certain parts of the world, and Saudi Arabia is one of them. Several factor contribute in predisposing the subjects for infection including but not limited to addiction to various compounds which have immune modulation properties, such as amphetamines and Heroin etc. Khat a plant whose leaves are chewed for its euphoric effect in east Africa and Arabian Peninsula including Saudi Arabia, is considered as mildly addictive, and its principle compound, Cathinone shares structural and functional similarity with amphetamine a known immunomodulator. Tuberculosis being a disease of immune modulation has a varied spectrum of complex interplay of proinflammatory molecules, resistin is one of them. In the present study, we try to explore the trinity of khat addiction, serum resistin level and tuberculosis by correlating the serum resistin level in non khat addicted healthy subjects, khat addicted healthy subjects, and in patients, both khat addicted and non khat addicted, with active tuberculosis. We observed significantly higher resistin level among the apparently healthy khat addicted subjects as compared to non addicted healthy controls. Thereafter, when we compare the resistin levels between khat addicted and non khat addicted TB patients we did not found significant difference between the two groups. However bacillary load was observe to be significantly higher among the khat addicted TB patient as compare to non addicted one. Validation of above results in animal model revealed dose dependant increase in bacillary growth in the Wistar rats treated with khat. Taken together these results suggest the role of khat in immune modulation albeit in the limited frame of resistin level.

Seroepidemiology of Toxoplasma gondii amongst Pregnant Women in Jazan Province, Saudi Arabia.

Background. Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite of worldwide distribution. There is limited information about the seroprevalence of toxoplasmosis in the southern area of Saudi Arabia. The current study was carried out to determine the prevalence of T. gondii in pregnant women in Jazan province. Materials and Methods. The study was conducted between January and June 2013 and included 195 pregnant women, data on sociodemographic and predisposing factors were collected from each participant. Venous blood samples were collected following standard operating procedures. Serological analysis for latent toxoplasmosis (levels of IgG) and active toxoplasmosis (IgM) was done using Enzyme Linked Immunosorbent Assay (ELISA). Results. The overall seroprevalence of T. gondii in the study area was 24.1%. The seroprevalence of anti-Toxoplasma IgG was 20% (39 out of 195), whereas IgM seropositivity was 6.2% (12 out of 195). Only 4 pregnant women tested positive for both IgG and IgM. The highest IgG and IgM seroprevalence was among the study participants aged 35 to 39 years (13.5% and 35.1%, resp.). The seropositivity rate of T. gondii-specific antibodies was higher among pregnant women from the urban areas than those from rural communities (7.4% versus 0% and 21% versus 15.4% for IgM and IgG, resp.). Conclusions. The seroprevalence of T. gondii was high in pregnant woman in Jazan. The prevalence of toxoplasmosis increases with increase of age. Awareness health education program in Jazan needs to be maintained and developed to targeted pregnant women.

Epidemiology and pattern of antibiotic resistance in Helicobacter pylori: scenario from Saudi Arabia.

Helicobacter pylori is recognized as a major cause of gastritis, peptic ulcer, gastric cancer, and mucosa-associated lymphoma. Infection with this gram-negative microaerophile has been treated using combination of antibiotics and proton pump inhibitors for different gastrointestinal diseases. The most commonly used treatment is triple therapy which consists of administration of a proton pump inhibitor, clarithromycin, and amoxicillin. Many factors contribute to treatment failure, but one of the main reasons is development of bacterial antibiotic resistance. The percent prevalence of antibiotic resistance varies among different countries; it appears to be partly determined by the geographic factors and its ability to undergo frequent homologous recombination. The aim of this paper is to review the prevalence of H. pylori infection, association of clinical outcomes with H. pylori genotypes, and current status of antibiotic resistance in H. pylori in Saudi Arabia. It also discusses the different alternative approaches for the treatment of H. pylori using antibiotics. In addition, association of antibiotic resistance with H. pylori virulent genotypes in Saudi population and its underlying resistance mechanism will also be discussed.

Does khat chewing increases the risk of Mycobacterium tuberculosis infection by macrophage immune modulation?

Drug abuse is a serious problem associated with different pathological outcomes including modulating the immune system. Drug abuse is rising in Saudi Arabia and so as TB, a disease of worldwide significance, caused by immunological modulation in the host system. Khat chewing is a common practice in Arabian Peninsula which is now gaining momentum in other parts of the world. It is considered as an addiction. It has been associated with different adverse outcomes such as periodontitis, oral leukoplakia and oral cancer and also has shown to promote apoptotic cell death through cysteine proteases. The active ingredient of khat, cathinone is shown to have immunomodulatory effect. In principle, this leads to enhanced susceptibility to various infections. The present study is designed to delineate the mechanism of immunomodulation produced by khat/cathinone in human/mouse macrophage. Further, this activity will be evaluated both in vivo and in vitro in response to infection with Mycobacterium smegmatis to get an insight if there exists a co relation between the Mycobacterium tuberculosis infection and khat chewing.

Concurrent proinflammatory and apoptotic activity of a Helicobacter pylori protein (HP986) points to its role in chronic persistence.

Helicobacter pylori induces cytokine mediated changes in gastroduodenal pathophysiology, wherein, the activated macrophages at the sub-mucosal space play a central role in mounting innate immune response against the antigens. The bacterium gains niche through persistent inflammation and local immune-suppression causing peptic ulcer disease or chronic gastritis; the latter being a significant risk factor for the development of gastric adenocarcinoma. What favors persistence of H. pylori in the gastric niches is not clearly understood. We report detailed characterization of a functionally unknown gene (HP986), which was detected in patient isolates associated with peptic ulcer and gastric carcinoma. Expression and purification of recombinant HP986 (rHP986) revealed a novel, ∼29 kDa protein in biologically active form which associates with significant levels of humoral immune responses in diseased individuals (p<0.001). Also, it induced significant levels of TNF-α and Interleukin-8 in cultured human macrophages concurrent to the translocation of nuclear transcription factor-κB (NF-κB). Further, the rHP986 induced apoptosis of cultured macrophages through a Fas mediated pathway. Dissection of the underlying signaling mechanism revealed that rHP986 induces both TNFR1 and Fas expression to lead to apoptosis. We further demonstrated interaction of HP986 with TNFR1 through computational and experimental approaches. Independent proinflammatory and apoptotic responses triggered by rHP986 as shown in this study point to its role, possibly as a survival strategy to gain niche through inflammation and to counter the activated macrophages to avoid clearance.

Treatment end point determinants for pulmonary tuberculosis: human resistin as a surrogate biomarker.

Treatment of tuberculosis (TB), which takes one human life every 15 s, globally, requires a prolonged (>6 months) antitubercular treatment (ATT) which, is known to have hepatotoxic side effects. This study was designed to explore the utility of human resistin, a proinflammatory hormone, as a sensitive biomarker to determine TB treatment end points. Patients for pulmonary tuberculosis enrolled under the directly observed treatment, short-course (DOTS) program were followed-up for six months and were monitored by sputum analysis, body weight and ELISA-based serum resistin and C-reactive protein (CRP) levels at 0, 2, 4 and 6 months, along with close family contacts of TB patients and healthy controls. The mean circulating resistin levels were found to be significantly higher (P < 0.001) in patients (n = 48, 25.74 ± 9.45 ng/ml) reporting for the first time for treatment (T0) as compared to healthy subjects (n = 45, 7.18 ± 2.40 ng/ml). Resistin levels in contacts (n = 48, 19.61 ± 7.88 ng/ml) also were found to be significantly (P < 0.001) elevated as compared to healthy controls. Significant increase in body weight after four months (P = 0.006) and at 6 months (P < 0.001) of treatment inversely correlated with resistin levels. Our data suggest resistin could be a surrogate marker for TB treatment in addition to its utility as an early prognostic biomarker for monitoring TB disease onset.

Isocitrate dehydrogenase of Helicobacter pylori potentially induces humoral immune response in subjects with peptic ulcer disease and gastritis.

BACKGROUND: H. pylori causes gastritis and peptic ulcers and is a risk factor for the development of gastric carcinoma. Many of the proteins such as urease, porins, flagellins and toxins such as lipo-polysaccharides have been identified as potential virulence factors which induce proinflammatory reaction. We report immunogenic potentials of isocitrate dehydrogenase (ICD), an important house keeping protein of H. pylori. METHODOLOGY/PRINCIPAL FINDINGS: Amino acid sequences of H. pylori ICD were subjected to in silico analysis for regions with predictably high antigenic indexes. Also, computational modelling of the H. pylori ICD as juxtaposed to the E. coli ICD was carried out to determine levels of structure similarity and the availability of surface exposed motifs, if any. The icd gene was cloned, expressed and purified to a very high homogeneity. Humoral response directed against H. pylori ICD was detected through an enzyme linked immunosorbent assay (ELISA) in 82 human subjects comprising of 58 patients with H. pylori associated gastritis or ulcer disease and 24 asymptomatic healthy controls. The H. pylori ICD elicited potentially high humoral immune response and revealed high antibody titers in sera corresponding to endoscopically-confirmed gastritis and ulcer disease subjects. However, urea-breath-test negative healthy control samples and asymptomatic control samples did not reveal any detectable immune responses. The ELISA for proinflammatory cytokine IL-8 did not exhibit any significant proinflammatory activity of ICD. CONCLUSIONS/SIGNIFICANCE: ICD of H. pylori is an immunogen which interacts with the host immune system subsequent to a possible autolytic-release and thereby significantly elicits humoral responses in individuals with invasive H. pylori infection. However, ICD could not significantly stimulate IL8 induction in a cultured macrophage cell line (THP1) and therefore, may not be a notable proinflammatory agent.

Novel protein antigen (JHP940) from the genomic plasticity region of Helicobacter pylori induces tumor necrosis factor alpha and interleukin-8 secretion by human macrophages.

The plasticity region of the Helicobacter pylori genome comprises strain-specific gene loci. We performed genotyping and functional biology analysis of one such locus (jhp940) that was previously found to be functionally unknown but present in gastric cancer-associated strains from many different countries. We found its geographic prevalence to be independent of cagA presence and disease status. Cloning, expression, and purification of JHP940 revealed a novel, approximately 36-kDa protein in a biologically active form which elicited strong and significant levels of tumor necrosis factor alpha and interleukin-8 in human macrophages. Also, JHP940 was able to induce enhanced translocation of the transcription factor NF-kappaB complex in cultured macrophages. The induction of the proinflammatory cytokines by JHP940, therefore, points to its putative role in chronic gastric inflammation and, possibly, the various other outcomes of H. pylori infection, including gastric cancer.

Microevolution of Helicobacter pylori type IV secretion systems in an ulcer disease patient over a ten-year period.

Helicobacter pylori cagA and vacA genotypes have been used for almost a decade as stable entities to link the severity of gastritis and ulcer disease. We describe here microevolution of the two genomic islands, cag pathogenicity island (cagPAI; 40 kb) and tfs3 (16 kb) from isolates obtained at inclusion (one subclone) and after a 10-year period (two subclones) from a duodenal ulcer patient. Our results indicate microevolution in cagA, cagE, and cag7 genes of the cagPAI and open reading frames G, P, and L in tfs3, which possibly leads to inactivation or pseudogenization of these genes. Interestingly, no significant reduction in the severity of gastroduodenal pathology was found. These results point to an obvious difficulty in correlating the continuously evolving virulence factors such as the cagPAI genes with disease characteristics that appear to remain stable.

Ancestral European roots of Helicobacter pylori in India.

BACKGROUND: The human gastric pathogen Helicobacter pylori is co-evolved with its host and therefore, origins and expansion of multiple populations and sub populations of H. pylori mirror ancient human migrations. Ancestral origins of H. pylori in the vast Indian subcontinent are debatable. It is not clear how different waves of human migrations in South Asia shaped the population structure of H. pylori. We tried to address these issues through mapping genetic origins of present day H. pylori in India and their genomic comparison with hundreds of isolates from different geographic regions. RESULTS: We attempted to dissect genetic identity of strains by multilocus sequence typing (MLST) of the 7 housekeeping genes (atpA, efp, ureI, ppa, mutY, trpC, yphC) and phylogeographic analysis of haplotypes using MEGA and NETWORK software while incorporating DNA sequences and genotyping data of whole cag pathogenicity-islands (cagPAI). The distribution of cagPAI genes within these strains was analyzed by using PCR and the geographic type of cagA phosphorylation motif EPIYA was determined by gene sequencing. All the isolates analyzed revealed European ancestry and belonged to H. pylori sub-population, hpEurope. The cagPAI harbored by Indian strains revealed European features upon PCR based analysis and whole PAI sequencing. CONCLUSION: These observations suggest that H. pylori strains in India share ancestral origins with their European counterparts. Further, non-existence of other sub-populations such as hpAfrica and hpEastAsia, at least in our collection of isolates, suggest that the hpEurope strains enjoyed a special fitness advantage in Indian stomachs to out-compete any endogenous strains. These results also might support hypotheses related to gene flow in India through Indo-Aryans and arrival of Neolithic practices and languages from the Fertile Crescent.

Characterization of hepatic progenitors from human fetal liver using CD34 as a hepatic progenitor marker.

AIM: To enrich putative hepatic progenitors from the developing human fetal liver using CD34 as a marker. METHODS: Aborted fetuses of 13-20 wk were used for the isolation of liver cells. The cells were labeled with anti CD34; a marker used for isolating progenitor population and the cells were sorted using magnetic cell sorting. The positive fractions of cells were assessed for specific hepatic markers. Further, these cells were cultured in vitro for long term investigation. RESULTS: Flow cytometric and immunocytochemical analysis for alphafetoprotein (AFP) showed that the majority of the enriched CD34 positive cells were positive for AFP. Furthermore, these enriched cells proliferated in the long term and maintained hepatic characteristics in in vitro culture. CONCLUSION: The study shows that aborted human fetal liver is a potential source for isolation of hepatic progenitors for clinical applications. The study also demonstrates that CD34 can be a good marker for the enrichment of progenitor populations.

genoBASE pylori: a genotype search tool and database of the human gastric pathogen Helicobacter pylori.

Helicobacter pylori is the pathogenic bacterium linked to gastric and duodenal ulcers and gastric carcinoma. Genomic diversity of the organism has enabled new insights into its population biology through comparative genomics. genoBASE pylori is an online databank of several virulence-linked and phylogenetic markers of H. pylori strains obtained from different human populations. This knowledgebase is built upon a relational database management system which is connected to visualize the presence of known, pathogenicity markers such as the co-ordinates within the cag pathogenicity island (cagPAI), the cagA gene and motifs surrounding it, the vacA allotypes and the oipA gene frame status, together with genotypic details in the form of DNA profiling traces and candidate gene sequences for individual strains. This flexible search tool allows inter-laboratory comparison of DNA fingerprinting data in the form of fluorescent amplified fragment length polymorphism (FAFLP), enterobacterial repetitive intergenic consensus (ERIC) and repetitive extragenic palindromic (REP) signature profiles. Besides this, the database also displays diversity of strains based on nucleotide sequences of several house keeping genes and two membrane proteins. Being the first of its kind, genoBASE pylori is expected to be a helpful online tool in strengthening the concept of 'geographic genomics' and will be useful to molecular epidemiologists, clinical laboratory scientists and those interested in diagnostic development for H. pylori. The database can be accessed through its website (http://www.cdfd.org.in/amplibase/HP).

Genomes of Helicobacter pylori from native Peruvians suggest admixture of ancestral and modern lineages and reveal a western type cag-pathogenicity island.

BACKGROUND: Helicobacter pylori is presumed to be co-evolved with its human host and is a highly diverse gastric pathogen at genetic levels. Ancient origins of H. pylori in the New World are still debatable. It is not clear how different waves of human migrations in South America contributed to the evolution of strain diversity of H. pylori. The objective of our 'phylogeographic' study was to gain fresh insights into these issues through mapping genetic origins of H. pylori of native Peruvians (of Amerindian ancestry) and their genomic comparison with isolates from Spain, and Japan. RESULTS: For this purpose, we attempted to dissect genetic identity of strains by fluorescent amplified fragment length polymorphism (FAFLP) analysis, multilocus sequence typing (MLST) of the 7 housekeeping genes (atpA, efp, ureI, ppa, mutY, trpC, yphC) and the sequence analyses of the babB adhesin and oipA genes. The whole cag pathogenicity-island (cagPAI) from these strains was analyzed using PCR and the geographic type of cagA phosphorylation motif EPIYA was determined by gene sequencing. We observed that while European genotype (hp-Europe) predominates in native Peruvian strains, approximately 20% of these strains represent a sub-population with an Amerindian ancestry (hsp-Amerind). All of these strains however, irrespective of their ancestral affiliation harbored a complete, 'western' type cagPAI and the motifs surrounding it. This indicates a possible acquisition of cagPAI by the hsp-Amerind strains from the European strains, during decades of co-colonization. CONCLUSION: Our observations suggest presence of ancestral H. pylori (hsp-Amerind) in Peruvian Amerindians which possibly managed to survive and compete against the Spanish strains that arrived to the New World about 500 years ago. We suggest that this might have happened after native Peruvian H. pylori strains acquired cagPAI sequences, either by new acquisition in cag-negative strains or by recombination in cag positive Amerindian strains.