Toxoplasma gondii profilin induces NLRP3 activation and IL-1ß production/secretion in THP-1 cells.

Toxoplasma gondii is a highly prevalent protozoan that infects a broad spectrum of warm-blooded animals. Profilin is a critical protein that plays a role in the movement and invasion of T. gondii. In the current study, we assessed how profilin stimulates inflammasomes and how it induces transcription and secretion of IL-1ß. For this purpose, we assessed the level of TLR 2, 4, 5, and 9 expressions in a THP-1 cell line treated with profilin from T. gondii (TgP). In addition, we analyzed the expression levels of various inflammasomes, as well as IL-1ß, and IL-18 in THP-1 cells treated with the NLRP3 inhibitor MCC950. TgP significantly increased the expression of TLR5 but the expression of TLR2, 4, and 9 was not significantly increased. In addition, TgP did not significantly increase the level of inflammasomes after 5 h. Treatment with MCC950 significantly reduced NLRP3 and IL-1ß on both transcription and protein levels. Although the transcription level of NLRP3 was reduced 5 h after treatment with TgP, western blot analysis showed an increase in NLRP3. The western blot and ELISA analysis also showed that TgP increased both pro- and mature IL-1ß. In summary, our study showed that NLRP3 most probably plays a pivotal role in the expression and production levels of IL-1ß during the interaction between TgP and macrophages.

Methylation and Polymorphism in CDH1 Gene Promoter Among Patients with Diffuse Gastric Cancer.

Background: The promoter methylation and single nucleotide polymorphisms (SNPs) affect the transcription activity of cancer-related genes in several cancers including diffuse gastric cancer (DGC). Here we aimed to evaluate the promoter methylation status and the rs16260 at the promoter region of the CDH1 gene in DGC. Methods: This case-control study was performed of 48 formalin-fixed paraffin-embedded (FFPE) blocks of DGC patients and 41 fresh frozen tissue samples of healthy individuals. Methylation status was evaluated using methylation-specific polymerase chain reaction (PCR) and the rs16260 at the promoter region of the CDH1 gene was assessed using PCR and sequencing method. Results: The occurrence of methylation at the promoter region of the CDH1 gene in DGC patients was significantly higher than control samples (P < 0.0001). The methylated status was significantly associated with the poor differentiated histological type of DGC (P = 0.0428). The frequency of AC genotype and the A allele in DGC patients was significantly higher than the control subjects (P = 0.006 and 0.003, respectively). Conclusions: Here we showed that methylation at the CDH1 promoter may contribute to the DGC development, and also the AC genotype was associated with the risk of DGC.

Soluble total antigen derived from Toxoplasma gondii tachyzoites increased the expression levels of NLRP1, NLRP3, NLRC4, AIM2, and the release of mature form of IL1ß, but downregulated the expression of IL1ß and IL18 genes in THP-1cell line.

Toxoplasma gondii (T. gondii) is an intracellular parasitic protozoan infecting homoeothermic animals and about a third of the world's population. Inflammasomes are intracellular multi-protein complex, which are activated by many factors. Inflammasomes are activated during toxoplasmosis; however, there are a lot of obscure aspects. THP-1 monocyte cells were converted to M0 macrophages by PMA and treated by 100 µg/mL soluble total Ag (STAg) derived from T. gondii strain RH for two time points 3 h and 24 h. After total RNA extraction and cDNA synthesis, the expression pattern of NLRP1, NLRP3, NLRC4, AIM2, IL1ß, and IL18 was evaluated by relative real-time PCR. In addition, the cytokine release of IL1ß and TNFα was evaluated in the supernatant of each well. The results showed statistically significant time-dependent overexpression of inflammasomes. NLRP1 and NLRP3 showed the higher and lower expression, respectively, during 3 h and 24 h after exposure. Both IL1ß and IL18 downregulated 3 h after exposure. IL18 presented statistically significant upregulation after 24 h, but IL1ß showed statistically significant downregulation after 24 h. The release of IL1ß increased after 3 h, but it slightly decreased during 24 h after exposure. The concentration of TNFα showed an insignificant decrease compared to control, while it increased during 24 h after exposure. Taken together, this study suggested that T. gondii STAg induces NLRP1 more than NLRP3, NLRC4, and AIM2. Our findings also proposed that T. gondii STAg downregulates the gene expression of IL1ß, but increases the release of this cytokine. It seems that Toxoplasma STAg probably increase the release of IL1ß via activating NLRPs and AIM2 to cleave pro-caspase 1 to caspase 1 that leads to conversion of pro IL1ß to mature IL1ß.

Small-scale risk assessment of transmission of parasites from wastewater treatment plant to downstream vegetable farms.

AIM: The aim of the present study was to simultaneously investigate parasitic contamination of treated wastewater and downstream vegetable farms that are irrigated with treated sewage, during a year. BACKGROUND: (Oo) Cysts and eggs of parasites are resistant to most of routine wastewater treatment process. Irrigation of vegetables farms with either treated wastewater or illegally use of raw wastewaters enhances the risk of contamination with enteric pathogens. METHODS: The treated wastewater samples were taken after chlorination from a wastewater treatment plant located at the south of Tehran. In addition, 60 vegetable samples (5 samples from each farm) were collected from the selected downstream farms that routinely used treated wastewater for irrigation of crops. Parasitological tests were performed using Ziehl-Neelsen, conventional lugol's iodine staining and direct microscopical examination. RESULTS: Parasites including free living larvae, eggs of Toxoascaris leonina, egg of Toxocara sp. Trichuris sp, Trichostrongylus sp and amoeboid trophozoite were seen in 5/12 (41.7%) of vegetable samples gathered during a year. There was no statistically significant correlation between the season and parasitic contamination of the vegetables (P= 1). Furthermore, parasitic contamination was observed in 7/12 (53.8%) of treated wastewater samples. The correlation between season and parasitic contamination of treated wastewater was evaluated that the results showed a higher contamination of treated wastewater in spring and autumn (P<0.05). Fisher's exact test also showed that there was no significant correlation between parasitic contaminations of vegetable samples and treated wastewater according to seasonal change. CONCLUSION: The results showed parasites in both treated wastewater plant and downstream crops farms that suggests the public health importance of the quality of water resources that routinely used for irrigation of vegetable farms.

Molecular and phylogenetic evidences of dispersion of human-infecting microsporidia to vegetable farms via irrigation with treated wastewater: One-year follow up.

BACKGROUND: Human-infecting microsporidia are a group of spore-forming eukaryotic microorganisms that can infect both animals and humans. Recent evidences indicate waterborne transmission of microsporidia spores to human via either drinking water or irrigation of vegetable farms with contaminated water resources. The current study aimed to evaluate the presence of human-infecting microsporidia in treated wastewater (TW) and vegetable farms irrigated with treated wastewater during a year. METHODS: Totally, twelve samples of each treated wastewater and vegetables were collected. In order to recover microsporidia spores, filtration using cellulose nitrate membrane (pore size 0.4 µm) and sedimentation were employed. DNA extraction was performed for all samples and genus/species were characterized using specific primers. In order to characterize genotypes, ITS fragment of E. bieneusi was amplified, sequenced and compared in GenBank database. Phylogenetic tree was employed to analysis the probable correlation between obtained genotypes with those E. bieneusi genotypes, which were previously obtained from human and animals from same region. RESULTS: After nested PCR, expected fragments of E. bieneusi and Encephalitozoon spp were observed among 5/12 (41.7%) and 1/12 (8.33%) of vegetable samples, respectively. From total of 12 TW samples, expected fragments of E. bieneusi and Encephalitozoon spp were amplified among 7/12 (53.8%) and 1/12 (8.33%) of TW samples, respectively. Genotypes D and E were characterized from both TW and vegetables samples. Phylogenetic analysis showed close-relationship between E. bieneusi from TW and vegetable samples with E. bieneusi from animals and humans obtained from the same region. CONCLUSION: Our findings suggested the key role of animals in epidemiology of zoonotic transmission of E. bieneusi. Moreover, our findings revealed the occurrence of human-infecting microsporidia in treated wastewater because of either insufficiency of treatment process or distribution of microsporidia spores in wastewater treatment plant via animals.