Amoebiasis (a human intestinal infection affecting 50 million people every year) is caused by the protozoan parasite Entamoeba histolytica. To study the molecular mechanisms underlying human colon invasion by E. histolytica, we have set up an ex vivo human colon model to study the early steps in amoebiasis. Using scanning electron microscopy and histological analyses, we have established that E. histolytica caused the removal of the protective mucus coat during the first two hours of incubation, detached the enterocytes, and then penetrated into the lamina propria by following the crypts of Lieberkühn. Significant cell lysis (determined by the release of lactodehydrogenase) and inflammation (marked by the secretion of pro-inflammatory molecules such as interleukin 1 beta, interferon gamma, interleukin 6, interleukin 8 and tumour necrosis factor) were detected after four hours of incubation. Entamoeba dispar (a closely related non-pathogenic amoeba that also colonizes the human colon) was unable to invade colonic mucosa, lyse cells or induce an inflammatory response. We also examined the behaviour of trophozoites in which genes coding for known virulent factors (such as amoebapores, the Gal/GalNAc lectin and the cysteine protease 5 (CP-A5), which have major roles in cell death, adhesion (to target cells or mucus) and mucus degradation, respectively) were silenced, together with the corresponding tissue responses. Our data revealed that the signalling via the heavy chain Hgl2 or via the light chain Lgl1 of the Gal/GalNAc lectin is not essential to penetrate the human colonic mucosa. In addition, our study demonstrates that E. histolytica silenced for CP-A5 does not penetrate the colonic lamina propria and does not induce the host's pro-inflammatory cytokine secretion. Entamoeba histolytica is the causative agent of amoebiasis, a human disease. Like other enteric infections, the lack of animal models enhances the difficulty of studying the development of amoebiasis. To date, no experimental model has been developed that reproduces the invasive intestinal amoebic lesions seen in human colon. We present the first study that examines, using human colon explants, the early steps of the human colonic barrier invasion by E. histolytica. With this ex vivo integrative model we have investigated both parasite behaviour and the human tissue response. Remarkably, in this model E. histolytica was able to cross and destroy the intestinal barrier evoking a tissue inflammatory response, while E. dispar, a non-pathogenic species, was unable to penetrate nor induce tissue responses. Furthermore, we have explored the role of three virulence factors during the invasive process, using gene-silenced E. histolytica trophozoites, particularly the kinetics of invasion, tissue destruction and induction of an early inflammatory responses. This is, to our knowledge, the first time that their role is highlighted in a complex human system. Our study provides new insights in the molecular mechanisms involved in the early steps of human colon invasion by E. histolytica.

The requirement of cholesterol for internalization of eukaryotic pathogens like protozoa (Leishmaniasis, Malaria and Toxoplasmosis) and the exchange of cholesterol along with other metabolites during reproduction in Schistosomes (helminths) under variable circumstances are poorly understood. In patients infected with some other helminthes, alterations in the lipid profile have been observed. Also, the mechanisms involved in lipid changes especially in membrane proteins related to parasite infections remain uncertain. Present review of literature shows that parasites induce significant changes in lipid parameters, as has been shown in the in vitro study where substitution of serum by lipid/cholesterol in medium and in experimental models (in vivo). Thus changes in lipid profile occur in patients having active infections with most of the parasites. Membrane proteins are probably involved in such reactions. All parasites may be metabolising cholesterol, but the exact relationship with pathogenic mechanism is not clear. So far, studies suggest that there may be some factors or enzymes, which allow the parasite to breakup and consume lipid/cholesterol. Further studies are needed for better understanding of the mechanisms involved in vivo. The present review analysis the various studies till date and the role of cholesterol in pathogenesis of different parasitic infections.

Background: Amoebiasis is a major public health problem in tropical and subtropical countries. Although a number of antiamoebic agents are used for its treatment, yet the susceptibility data on clinical isolates of Entamoeba histolytica and Entamoeba dispar are not available. Therefore, the present study was aimed to assess the in vitro susceptibility of clinical isolates of E. histolytica and E. dispar to metronidazole, chloroquine, emetine and tinidazole. Methods: A total of 45 clinical isolates (15 E. histolytica and 30 E. dispar) were maintained in polyxenic cultures followed by monoxenic cultures. In vitro drug sensitivity (IC₅₀) of clinical isolates and standard reference strain of E. histolytica (HM1: IMSS) was assessed by nitro blue tetrazolium (NBT) reduction assay after exposure to various concentrations of each drug. Results: The results showed that all clinical isolates had a higher IC₅₀ compared to reference strain to all the four drugs. E. histolytica isolates appeared to be more susceptible [IC₅₀ (μm) 13.2,26.3,31.2 and 12.4] compared to E. dispar isolates [IC₅₀(μm) 15.6,28.9,32.8 and 13.2] and the reference strain of E. histolytica [IC₅₀ (μm) 9.5, 15.5, 29.9 and 10.2] to the metronidazole, chloroquine, emetine and tinidazole respectively. Conclusions: The results indicate that till date, Entamoeba isolates in India do not seem to be resistant to the commonly used antiamoebic drugs.

Plasmodium falciparum malaria in India is characterized by high rates of severe disease, with multiple organ dysfunction (MOD)—mainly associated with acute renal failure (ARF)—and increased mortality. The objective of this study is to identify cytokine signatures differentiating severe malaria patients with MOD, cerebral malaria (CM), and cerebral malaria with MOD (CM-MOD) in India. We have previously shown that two cytokines clusters differentiated CM from mild malaria in Maharashtra. Hence, we also aimed to determine if these cytokines could discriminate malaria subphenotypes in Odisha. P. falciparum malaria patients from the SCB Medical College Cuttack in the Odisha state in India were enrolled along with three sets of controls: healthy individuals, patients with sepsis and encephalitis (n = 222). We determined plasma concentrations of pro- and anti-inflammatory cytokines and chemokines for all individuals using a multiplex assay. We then used an ensemble of statistical analytical methods to ascertain whether particular sets of cytokines/chemokines were predictors of severity or signatures of a disease category. Of the 26 cytokines/chemokines tested, 19 increased significantly during malaria and clearly distinguished malaria patients from controls, as well as sepsis and encephalitis patients. High amounts of IL-17, IP-10, and IL-10 predicted MOD, decreased IL-17 and MIP-1α segregated CM-MOD from MOD, and increased IL-12p40 differentiated CM from CM-MOD. Most severe malaria patients with ARF exhibited high levels of IL-17. We report distinct differences in cytokine production correlating with malarial disease severity in Odisha and Maharashtra populations in India. We show that CM, CM-MOD and MOD are clearly distinct malaria-associated pathologies. High amounts of IL-17, IP-10, and IL-10 were predictors of MOD; decreased IL-17 and MIP-1α separated CM-MOD from MOD; and increased IL-12p40 differentiated CM from CM-MOD. Data also suggest that the IL-17 pathway may contribute to malaria pathogenesis via different regulatory mechanisms and may represent an interesting target to mitigate the pathological processes in malaria-associated ARF.

The genetic diversity in Plasmodium falciparum antigens is a major hurdle in developing an effective malaria vaccine. Protective efficacy of the vaccine is dependent on the polymorphic alleles of the vaccine candidate antigens. Therefore, we investigated the genetic diversity of the potential vaccine candidate antigens i.e. msp-1, msp-2, glurp, csp and pfs25 from field isolates of P.falciparum and determined the natural immune response against the synthetic peptide of these antigens. Genotyping was performed using Sanger method and size of alleles, multiplicity of infection, heterogeneity and recombination rate were analyzed. Asexual stage antigens were highly polymorphic with 55 and 50 unique alleles in msp-1 and msp-2 genes, respectively. The MOI for msp-1 and msp-2 were 1.67 and 1.28 respectively. A total 59 genotype was found in glurp gene with 8 types of amino acid repeats in the conserved part of RII repeat region. The number of NANP repeats from 40 to 44 was found among 55% samples in csp gene while pfs25 was found almost conserved with only two amino acid substitution site. The level of genetic diversity in the present study population was very similar to that from Asian countries. A higher IgG response was found in the B-cell epitopes of msp-1 and csp antigens and higher level of antibodies against csp B-cell epitope and glurp antigen were recorded with increasing age groups. Significantly, higher positive responses were observed in the csp antigen among the samples with ≥42 NANP repeats. The present finding showed extensive diversity in the asexual stage antigens.