Molecular and biochemical parasitology最新文献

The scabies mite is known to induce a complicated immune response that involves both innate and long-term adaptive immunity. Many immune effectors and pathways are involved. Th17/Treg balance can influence the complex immune response to scabies. The immunological effectors including IL-17A, as a pro-inflammatory cytokine, and Treg cells, anti-inflammatory regulatory T cells, are essential for preserving cutaneous immunological homeostasis. So, evaluating these immune effectors may help in comprehending the pathophysiology of scabies and facilitate the development of new treatment approaches. This study examined the expression of IL-17A and FoxP3⁺ in the skin and serum of 50 scabies patients and 25 healthy controls. An assessment of their correlation with clinical features was performed. Regarding tissue response, scabietic patients exhibited a significant increase in IL-17A and FoxP3⁺ expression in their epidermis and dermis compared to controls (P<0.001), but the correlation between these factors was not significant in either area (P>0.05). Also, patients showed a significant increase in serum IL-17A levels compared to controls (P<0.001), with a significant association between serum IL-17A levels and lesion severity, but no significant correlation was observed between skin and serum responses (P>0.05). In conclusion, there was increased expression of both IL-17A and FoxP3⁺, with FoxP3⁺ being significantly more abundant than IL^-17A in the skin of scabies patients. Skin FoxP3⁺ up-regulation has been linked to the severity of the condition.

The Leishmania life cycle alternates between promastigotes, found in the sandfly, and amastigotes, found in mammals. When an infected sandfly bites a host, promastigotes are engulfed by phagocytes (i.e., neutrophils, dendritic cells, and macrophages) to establish infection. When these phagocytes die or break down, amastigotes must be re-internalized to survive within the acidic phagolysosome and establish disease. To define host kinase regulators of Leishmania promastigote and amastigote uptake and survival within macrophages, we performed an image-based kinase regression screen using a panel of 38 kinase inhibitors with unique yet overlapping kinase targets. We also targeted inert beads to complement receptor 3 (CR3) or Fcγ receptors (FcR) as controls by coating them with complement/C3bi or IgG respectively. Through this approach, we identified several putative host kinases that regulate receptor-mediated phagocytosis and/or the uptake of L. amazonensis. Findings included kinases previously implicated in Leishmania uptake (such as Src family kinases (SFK), Abl family kinases (ABL1/c-Abl, ABL2/Arg), and spleen tyrosine kinase (SYK)), but we also uncovered many novel kinases. Our methods also predicted host kinases necessary for promastigotes to convert to amastigotes or for amastigotes to survive within macrophages. Overall, our results suggest that the concerted action of multiple interconnected networks of host kinases are needed over the course of Leishmania infection, and that the kinases required for the parasite’s life cycle may differ substantially depending on which receptors are bound and the life cycle stage that is internalized. In addition, using our screen, we identified kinases that appear to preferentially regulate the uptake of parasites over beads, indicating that the methods required for Leishmania to be internalized by macrophages may differ from generalized phagocytic mechanisms. Our findings are intended to be used as a hypothesis generation resource for the broader scientific community studying the roles of kinases in host-pathogen interactions.

Most protocols used to study the dynamics of calcium (Ca²⁺) in the malaria parasite are based on dyes, which are invasive and do not allow discrimination between the signal from the host cell and the parasite. To avoid this pitfall, we have generated a parasite line expressing the genetically encoded calcium sensor GCaMP3. The PfGCaMP3 parasite line is an innovative tool for studying spontaneous intracellular Ca²⁺ oscillations without external markers. Using this parasite line, we demonstrate the occurrence of spontaneous Ca²⁺ oscillations in the ring, trophozoite, and schizont stages in Plasmodium falciparum. Using the Fourier transform to fluorescence intensity data extracted from different experiments, we observe cytosolic Ca²⁺ fluctuations. These spontaneous cytosolic Ca²⁺ oscillations occur in the three intraerythrocytic stages of the parasite, with most oscillations occurring in the ring and trophozoite stages. A control parasite line expressing only a GFP control did not reveal such fluctuations, demonstrating the specificity of the observations. Our results clearly show dynamic, spontaneous Ca²⁺ oscillations during the asexual stage in P. falciparum, independent from external stimuli.

RNA interference (RNAi) on parasitic nematodes has been described as a valuable tool for screening putative targets that could be used as novel drug and/or vaccine candidates. This study aimed to set up a pipeline to identify potential targets using RNAi for vaccine/anti-parasite therapy development against Haemonchus contortus, a blood-feeding abomasal nematode parasite. The available H. contortus sequence data was mined for targets, which were tested for essentiality using RNAi electroporation assays. A total of 56 genes were identified and tested for knockdown using electroporation of first-stage larvae (L1) H. contortus with the target double-stranded RNA. Electroporation of L1 proved to be effective overall; 17 targets had a strong phenotype and significant reduction in alive H. contortus, and another 24 had a moderate phenotype with a significant reduction in larvae development. A total of 28 targets showed a significant reduction in the development of H. contortus larvae to the infective stage (L3) following the RNAi assay. Down-regulation of target transcript levels was evaluated in some targets by semi-quantitative PCR. Four out of five genes tested showed complete knockdown of mRNA levels via semi-quantitative PCR, whereas the knockdown was partial for one. In conclusion, the results indicate that the RNAi pathway is confirmed in H. contortus and that several target genes have the potential to be investigated further as possible vaccine candidates.

Cysteinyl leukotrienes (CysLTs) can induce a disruption of the blood–brain barrier (BBB), and this reaction is mediated by cysteinyl-leukotriene receptors. In this study, we used A. cantonensis-induced eosinophilic meningoencephalitis as a model to investigate whether the CysLT2 receptor involved in the pathogenesis of angiostrongyliasis meningoencephalitis. The present study provides evidence that the CysLT2 receptor antagonist HAMI3379 reduced the number of infiltrated eosinophils and brain edema in eosinophilic meningoencephalitis. Additionally, we found that HAMI3379 significantly decreased the protein levels of M1 polarisation markers (CD80, iNOS, IL-5 and TNF-α), increased the expression of M2 polarisation markers (CD206, IL-10 and TGF-β) both in vivo and in vitro. Matrix metalloproteinase-9, S100B, GFAP, fibronectin, and claudin-5 were markedly lower after HAMI3379 treatment. Therefore, HAMI3379 reduced the BBB dysfunction in angiostrongyliasis meningoencephalitis. We have identified microRNA-155 as a BBB dysfunction marker in eosinophilic meningoencephalitis. The results showed that microRNA-155 was 15-fold upregulated in eosinophilic meningoencephalitis and 20-fold upregulated after HAMI3379 treatment. Our results suggest that CysLT2R may be involved in A. cantonensis-induced brain edema and eosinophilic meningoencephalitis and that down-regulation of CysLT2R could be a novel and potential therapeutic strategy for the treatment of angiostrongyliasis meningoencephalitis.

Entamoeba histolytica, an intestinal parasite of global significance, poses substantial health risks with its associated high morbidity and mortality rates. Despite the current repertoire of molecular tools for the study of gene function in, the regulatory mechanisms governing its pathogenicity remain largely unexplored. This knowledge gap underscores the need to elucidate key genetic determinants orchestrating cellular functions critical to its virulence. Previously, our group generated an avirulent strain, termed UG10, with the same genetic background as the HM1:IMSS strain. UG10 strain, despite showing normal expression levels of well-known virulence factors, was unable to perform in-vitro and in-vivo activities related to amoebic virulence. In this study, we aimed to uncover the genome-wide modifications that rendered the avirulent phenotype of the UG10 strain through whole-genome sequencing. As a complementary approach, we conducted Methylated DNA Immunoprecipitation coupled with sequencing (MeDIP-seq) analysis on both the highly virulent HM1:IMSS strain and the low-virulence UG10 strain to uncover the genome-wide methylation profile. These dual methodologies revealed two aspects of the UG10 avirulent strain. One is the random integration of fragments from the ribosomal gene cluster and tRNA genes, ranging from 120 to 400 bp; and secondly, a clear, enriched methylation profile in the coding and non-coding strand relative to the start codon sequence in genes encoding small GTPases, which is associated with the previously described avirulent phenotype. This study provides the foundation to explore other genetic and epigenetic regulatory circuitries in E. histolytica and novel targets to understand the pathogenic mechanism of this parasite.