The regulatory function of Blastocystis spp. on the immune inflammatory response in the gut microbiome.

Blastocystis spp. is a unicellular organism that resides in digestive tract of various vertebrates, with a worldwide distribution and a variable prevalence. For many years, Blastocystis spp. was considered a cyst of a flagellate, a fungus, or a saprophyte yeast of the digestive tract; in 1996, it is placed in the group of stramenopiles (heterokonts). Since its new classification, many questions have arisen around this protist about its role as a pathogen or non-pathogen organism. Recent evidence indicates that Blastocystis spp. participates in the immune inflammatory response in the intestinal microbiome generating an anti-inflammatory response, showing a lower concentration of fecal inflammatory markers in infected human hosts. Here, we review recent findings on the regulatory function of Blastocystis spp. in the immune inflammatory response to comprehend the purpose of Blastocystis spp. in health and disease, defining if Blastocystis spp. is really a pathogen, a commensal or even a mutualist in the human gut microbiome.

Exposure to Parasitic Protists and Helminths Changes the Intestinal Community Structure of Bacterial Communities in a Cohort of Mother-Child Binomials from a Semirural Setting in Mexico.

An estimated 3.5 billion people are colonized by intestinal parasites worldwide. Intestinal parasitic eukaryotes interact not only with the host but also with the intestinal microbiota. In this work, we studied the relationship between the presence of multiple enteric parasites and the community structures of gut bacteria and eukaryotes in an asymptomatic mother-child cohort from a semirural community in Mexico. Fecal samples were collected from 46 mothers and their respective children, with ages ranging from 2 to 20 months. Mothers and infants were found to be multiparasitized by Blastocystis hominis, Entamoeba dispar, Endolimax nana, Chilomastix mesnili, Iodamoeba butshlii, Entamoeba coli, Hymenolepis nana, and Ascaris lumbricoides. Sequencing of bacterial 16S rRNA and eukaryotic 18S rRNA genes showed a significant effect of parasite exposure on bacterial beta-diversity, which explained between 5.2% and 15.0% of the variation of the bacterial community structure in the cohort. Additionally, exposure to parasites was associated with significant changes in the relative abundances of multiple bacterial taxa, characterized by an increase in Clostridiales and decreases in Actinobacteria and Bacteroidales. Parasite exposure was not associated with changes in intestinal eukaryote relative abundances. However, we found several significant positive correlations between intestinal bacteria and eukaryotes, including Oscillospira with Entamoeba coli and Prevotella stercorea with Entamoeba hartmanni, as well as the co-occurrence of the fungus Candida with Bacteroides and Actinomyces, Bifidobacterium, and Prevotella copri and the fungus Pichia with Oscillospira. The parasitic exposure-associated changes in the bacterial community structure suggest effects on microbial metabolic routes, host nutrient uptake abilities, and intestinal immunity regulation in host-parasite interactions. IMPORTANCE The impact of intestinal eukaryotes on the prokaryotic microbiome composition of asymptomatic carriers has not been extensively explored, especially in infants and mothers with multiple parasitic infections. In this work, we studied the relationship between protist and helminth parasite colonization and the intestinal microbiota structure in an asymptomatic population of mother-child binomials from a semirural community in Mexico. We found that the presence of parasitic eukaryotes correlated with changes in the bacterial gut community structure in the intestinal microbiota in an age-dependent way. Parasitic infection was associated with an increase in the relative abundance of the class Clostridia and decreases of Actinobacteria and Bacteroidia. Parasitic infection was not associated with changes in the eukaryote community structure. However, we observed strong positive correlations between bacterial and other eukaryote taxa, identifying novel relationships between prokaryotes and fungi reflecting interkingdom interactions within the human intestine.

Entamoeba histolytica Calreticulin Induces the Expression of Cytokines in Peripheral Blood Mononuclear Cells Isolated From Patients With Amebic Liver Abscess.

Calreticulin (CRT) is a highly conserved protein in the endoplasmic reticulum that plays important roles in the regulation of key cellular functions. Little is known about the participation of E. histolytica CRT (EhCRT) in the processes of pathogenicity or in the modulation of the host immune response. The aim of this study was to evaluate the role of CRT in the proliferation and the cytokine profile in peripheral blood mononuclear cells (PBMCs) from patients with amebic liver abscess (ALA) during the acute phase (AP-ALA) of the disease compared to patients during the resolution phase (R-ALA). The PBMCs from each participant were cocultured with EhCRT and tested by the colorimetric method to evaluate their proliferation index (PI). The supernatants were subjected to an enzyme-linked immunosorbent assay (ELISA) to evaluate the concentration of cytokines. The mean values of all groups were compared using the independent t-test. When the PIs of individuals without diagnosis of liver abscess (NEG) were compared, there were no statistically significant differences in the proliferation of PBMCs between patients with AP-ALA and R-ALA when stimulated with EhCRT or concanavalin A (ConA). However, the levels of interleukins [IL-6, IL-10, granulocyte colony stimulating factor (GCSF), and transforming growth factor ß1 (TGFß1)] were higher in patients with AP-ALA, whereas in patients with R-ALA, higher levels of interferon gamma (IFNγ) were detected. These results suggest that EhCRT acts as a mitogen very similar to the activity of ConA. In addition, EhCRT is an excellent immunogen for the specific activation of PBMCs, inducing the differential expression of ILs depending on the outcome of disease, determining the type of immune response: a Th2 cytokine profile during the acute phase and a Th1 profile during the resolution phase.

Genetic Diversity and Distribution of Blastocystis Subtype 3 in Human Populations, with Special Reference to a Rural Population in Central Mexico.

Blastocystis subtype 3 (ST3) is a parasitic protist found in the digestive tract of symptomatic and asymptomatic humans around the world. While this parasite exhibits a high prevalence in the human population, its true geographic distribution and global genetic diversity are still unknown. This gap in knowledge limits the understanding of the spread mechanisms, epidemiology, and impact that this parasite has on human populations. Herein, we provided new data on the geographical distribution and genetic diversity of Blastocystis ST3 from a rural human population in Mexico. To do so, we collected and targeted the SSU-rDNA region in fecal samples from this population and further compared its genetic diversity and structure with that previously observed in populations of Blastocystis ST3 from other regions of the planet. Our analyses reveled that diversity of Blastocystis ST3 showed a high haplotype diversity and genetic structure to the world level; however, they were low in the Morelos population. The haplotype network revealed a common widespread haplotype from which the others were generated recently. Finally, our results suggested a recent expansion of the diversity of Blastocystis ST3 worldwide.

Genotype B of Killer Cell Immunoglobulin-Like Receptor is Related with Gastric Cancer Lesions.

NK cells are important in innate immunity for their capacity to kill infected or cancer cells. The killer cell immunoglobulin-like receptors (KIR) are a family of polymorphic genes with inhibitory and activating functions. The main driving force for gastric cancer (GC) development is a chronic response, which causes an increase of NK cells in the gastric mucosa. The aim of this work was to study polymorphisms in KIR genes in patients with either GC or non-atrophic gastritis (NAG). We studied 242 patients (130 with NAG and 112 with GC) and contrasted with 146 asymptomatic individuals. We analyzed diversity in the content and localization of KIR genes in the different clinical groups studied. Four activating and one inhibitory genes were associated with GC: 2DS1 (OR 3.41), 2DS3 (OR 4.66), 2DS5 (OR 2.25), 3DS1 (OR 3.35) and 2DL5 (OR 3.6). The following were also found as risk factors for GC: Bx genotype (OR 4.2), Bx-Bx centromere-telomere (OR 2.55), cA01|cB03 (OR 36.39) and tB01|tB01 (OR 7.55) gene content and three B motifs (OR 10.9). Polymorphisms in KIR genes were associated with GC and suggest that mutated NK cells may contribute to GC development by increasing gastric mucosa inflammation, leading to constant tissue damage.

Polymorphisms in HLA-DQ genes, together with age, sex, and Helicobacter pylori infection, as potential biomarkers for the early diagnosis of gastric cancer.

BACKGROUND: Polymorphisms in inflammation-related genes are factors associated with the development of gastroduodenal diseases in Helicobacter pylori-infected individuals. MATERIALS AND METHODS: We aimed to analyze polymorphisms in HLA-DQ, together with other host and H. pylori variables as risk factors for precancerous and cancerous gastric lesions. 1052 individuals were studied, including nonatrophic gastritis (NAG), intestinal metaplasia (IM), gastric cancer (GC) or duodenal ulcer (DU) patients, and healthy volunteers. RESULTS: Patients with alleles DQA*01:01 (OR 0.78), *01:02 (OR 0.29), *01:03 (OR 0.31), and DQB*02:01/02 (OR 0.40) showed a reduced risk for GC. A multivariate logistic regression analyses showed that patients with homozygote genotypes DQA1*03:01 (OR 7.27) and DQA1*04:01 (OR 8.99) and DQB1*05:01:01 (OR 12.04) were at significantly increased risk for GC. Multivariate analyses also demonstrated that age (OR>10.0) and gender (OR>2.0) were variables that influenced significantly the risk for GC, while H. pylori infection (OR>2.5) increased the risk for IM. CONCLUSIONS: We identified HLA-DQ alleles associated with IM and GC, and confirm that age, sex, and H. pylori infection are variables that also influence the risk for disease. The use of multiple markers, HLA-DQ alleles, age, sex, and H. pylori infection may be useful biomarkers for the early diagnosis of patients with IM and GC.

Human Intestinal Microbiota: Interaction Between Parasites and the Host Immune Response.

The human gut is a highly complex ecosystem with an extensive microbial community, and the influence of the intestinal microbiota reaches the entire host organism. For example, the microbiome regulates fat storage, stimulates or renews epithelial cells, and influences the development and maturation of the brain and the immune system. Intestinal microbes can protect against infection by pathogenic bacteria, viruses, fungi and parasites. Hence, the maintenance of homeostasis between the gut microbiota and the rest of the body is crucial for health, with dysbiosis affecting disease. This review focuses on intestinal protozoa, especially those still representing a public health problem in Mexico, and their interactions with the microbiome and the host. The decrease in prevalence of intestinal helminthes in humans left a vacant ecological niche that was quickly occupied by protozoa. Although the mechanisms governing the interaction between intestinal microbiota and protozoa are poorly understood, it is known that the composition of the intestinal bacterial populations modulates the progression of protozoan infection and the outcome of parasitic disease. Most reports on the complex interactions between intestinal bacteria, protozoa and the immune system emphasize the protective role of the microbiota against protozoan infection. Insights into such protection may facilitate the manipulation of microbiota components to prevent and treat intestinal protozoan infections. Here we discuss recent findings about the immunoregulatory effect of intestinal microbiota with regards to intestinal colonization by protozoa, focusing on infections by Entamoeba histolytica, Blastocystis spp, Giardia duodenalis, Toxoplasma gondii and Cryptosporidium parvum. The possible consequences of the microbiota on parasitic, allergic and autoimmune disorders are also considered.

Analysis of heat shock protein 70 gene polymorphisms Mexican patients with idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease of unknown etiology. Genetic variation within different major histocompatibility complex (MHC) loci contributes to the susceptibility to IPF. The effect of 70 kDa heat shock proteins (HSP70) gene polymorphisms in the susceptibility to IPF is unknown. The aim of this study was to explore the association between HSP70 polymorphisms and IPF susceptibility in the Mexican population. METHODS: Four HSP70 single nucleotide polymorphisms (SNPs) were evaluated using real time PCR assays in 168 IPF patients and 205 controls: +2763 C>T of HSPA1L (rs2075800), +2437 of HSP HSPA1L A>G (rs2227956), +190 of HSPA1A G>C (rs1043618) and +1267 of HSPA1B G>A (rs1061581). RESULTS: The analysis of the recessive model revealed a significant decrease in the frequency of the genotype HSPA1B AA (rs1061581) in IPF patients (OR = 0.27, 95 % CI = 0.13-0.57, Pc = 0.0003) when compared to controls. Using a multivariate logistic regression analysis in a codominant model the HSPA1B (rs1061581) GA and AA genotypes were associated with a lower risk of IPF compared with GG (OR = 0.22, 95 % CI = 0.07-0.65; p = 0.006 and OR = 0.17, 95 % CI = 0.07-0.41; p = <0.001). Similarly, HSPA1L (rs2227956) AG genotype (OR = 0.34, 95 % CI = 0.12-0.99; p = 0.04) and the dominant model AG + GG genotypes were also associated with a lower risk of IPF (OR = 0.24, 95 % CI = 0.08-0.67; p = 0.007). In contrast, the HSPA1L (rs2075800) TT genotype was associated with susceptibility to IPF (OR = 2.52, 95 % CI = 1.32-4.81; p = 0.005). CONCLUSION: Our findings indicate that HSPA1B (rs1061581), HSPA1L (rs2227956) and HSPA1 (rs1043618) polymorphisms are associated with a decreased risk of IPF.

Prevalent HLA Class II Alleles in Mexico City Appear to Confer Resistance to the Development of Amebic Liver Abscess.

Amebiasis is an endemic disease and a public health problem throughout Mexico, although the incidence rates of amebic liver abscess (ALA) vary among the geographic regions of the country. Notably, incidence rates are high in the northwestern states (especially Sonora with a rate of 12.57/100,000 inhabitants) compared with the central region (Mexico City with a rate of 0.69/100,000 inhabitants). These data may be related to host genetic factors that are partially responsible for resistance or susceptibility. Therefore, we studied the association of the HLA-DRB1 and HLA-DQB1 alleles with resistance or susceptibility to ALA in two Mexican populations, one each from Mexico City and Sonora. Ninety ALA patients were clinically diagnosed by serology and sonography. Genomic DNA was extracted from peripheral blood mononuclear cells. To establish the genetic identity of both populations, 15 short tandem repeats (STRs) were analyzed with multiplexed PCR, and the allelic frequencies of HLA were studied by PCR-SSO using LUMINEX technology. The allele frequencies obtained were compared to an ethnically matched healthy control group (146 individuals). We observed that both affected populations differed genetically from the control group. We also found interesting trends in the population from Mexico City. HLA-DQB1*02 allele frequencies were higher in ALA patients compared to the control group (0.127 vs 0.047; p= 0.01; pc= NS; OR= 2.9, 95% CI= 1.09-8.3). The less frequent alleles in ALA patients were HLA-DRB1*08 (0.118 vs 0.238 in controls; p= 0.01; pc= NS; OR= 0.42, 95% CI= 0.19-0.87) and HLA-DQB1*04 (0.109 vs 0.214; p= 0.02; pc= NS; OR= 0.40, 95% CI= 0.20-0.94). The haplotype HLA-DRB1*08/-DQB1*04 also demonstrated a protective trend against the development of this disease (0.081 vs. 0.178; p=0.02; pc=NS; OR= 0.40, 95% CI= 0.16-0.93). These trends suggest that the prevalent alleles in the population of Mexico City may be associated with protection against the development of ALA.

Characterization of Single-Nucleotide Polymorphisms in the Tumor Necrosis Factor α Promoter Region and in Lymphotoxin α in Squamous Intraepithelial Lesions, Precursors of Cervical Cancer.

Development of cervical cancer is a long process of abnormal cancerous cell growth in the cervix and is primarily the result of infection with specific high-risk types of human papillomavirus (HPV). The cytokines tumor necrosis factor α (TNFα) and lymphotoxin α (LTA) have an important role in all stages of cervical cancer and have the ability to induce the regression or promote the development of human tumors. Biologically important single-nucleotide polymorphisms (SNPs) occur within the TNFα and LTA genes. Therefore, the purpose of this study was to investigate the SNPs in the TNFα promoter region (-163, -238, -244, -308, -376, -857, -863, and -1031) and in the first intron of LTA (+252) in women with precursor lesions of cervical cancer. Overall, we studied 396 women from Mexico City. A total of 191 patients with HPV infection and precursor cervical lesions were subdivided in two groups: those with low-grade squamous intraepithelial lesions (n = 132) and those with high-grade squamous intraepithelial lesions (n = 59). Women (n = 205) negative for HPV and without cervical lesions were also included in the study. DNA was extracted from peripheral white blood cells and from cervical samples, and detection of biallelic polymorphisms of TNFα and LTA was performed using the polymerase chain reaction-sequence-specific oligonucleotide probe and restriction fragment length polymorphism techniques, respectively. We demonstrated that risk is associated with the genotype G/A (odds ratio = 2.48) and that protection is associated with the genotype G/G of SNP TNFα -376 (odds ratio = 0.37).

Polymorphisms in TNF and HSP-70 show a significant association with gastric cancer and duodenal ulcer.

Tumour Necrosis Factor (TNF) and Heat Shock Protein 70 (HSP70) are important molecules in inflammatory, infectious and tumoral processes. The genes codifying these molecules are polymorphic and certain alleles have been associated with susceptibility to disease. Gastric cancer is associated with an Helicobacter pylori-induced chronic inflammatory response. The aim of this work was to analyze whether polymorphisms in inflammation-related genes are associated with the development of gastric cancer. We studied 447 Mexican adult patients including 228 with non-atrophic gastritis, 98 with intestinal metaplasia, 63 with gastric cancer and 58 with duodenal ulcer, and 132 asymptomatic individuals as well. DNA from peripheral white blood cells was typed for the Single Nucleotide Polymorphisms (SNPs) -308 of TNF-alpha, +252 of TNF-beta, +190 of HSP70-1, +1267 of HSP70-2 and +2437 of HSP70-HOM. Compared with the asymptomatic group, we found a significant association of TNF-beta*A and HSP70-1*C alleles with gastric cancer (OR 5.69 and 3.76, respectively) and HSP70-1*C with duodenal ulcer (OR 3.08). Genotype TNF-beta G/G showed a significant gene-dose effect with gastric cancer (OR 0.09); whereas HSP70-1 C/G showed significant association with both, gastric cancer (OR 13.31) and duodenal ulcer (OR 16.19). Polymorphisms in TNF and HSP70 showed a significant severity-dose-response as risk markers from preneoplastic lesions to gastric cancer in Mexican population, probably because of their association with an intense and sustained inflammatory response.