A rapid and simple method of detection of Blepharisma japonicum using PCR and immobilisation on FTA paper

BACKGROUND: The rapid expansion in the availability of genome and DNA sequence information has opened up new possibilities for the development of methods for detecting free-living protozoa in environmental samples. The protozoan Blepharisma japonicum was used to investigate a rapid and simple detection system based on polymerase chain reaction amplification (PCR) from organisms immobilised on FTA paper. RESULTS: Using primers designed from the α-tubulin genes of Blepharisma, specific and sensitive detection to the equivalent of a single Blepharisma cell could be achieved. Similar detection levels were found using water samples, containing Blepharisma, which were dried onto Whatman FTA paper. CONCLUSION: This system has potential as a sensitive convenient detection system for Blepharisma and could be applied to other protozoan organisms

High frequency detection of Toxoplasma gondii DNA in human neonatal tissue from Libya

Background: Toxoplasma gondii is a parasite that causes significant disease in humans. Toxoplasmosis is normally asymptomatic, unless associated with congenital transmission, or in immunocompromised people. Congenital transmission generally occurs at low frequencies. In this study, we use PCR to investigate possible congenital transmission of T. gondii during pregnancy in a cohort of mothers from Libya. Methods: Two hundred and seventy two pregnant women (producing 276 neonates) were recruited to obtain umbilical cord tissue from their neonates at birth. DNA was extracted from umbilical cord tissue and tested for T. gondii DNA using two specific PCR protocols based on the sag 1 and sag 3 genes. Results: Toxoplasma gondii DNA was detected in the umbilical cord DNA from 27 of the 276 neonates giving a prevalence of 9.9% (95% CI: 6.8-13.9%). Compared with more commonly reported rates of congenital transmission of 0.1% of live births, this is high. There was no association of infection with unsuccessful pregnancy. Conclusions: This study shows a high frequency presence of T. gondii DNA associated with neonatal tissue at birth in this cohort of 276 neonates from Libya. Although PCR cannot detect living parasites, there is the possibility that this indicates a higher than usual frequency of congenital transmission

High prevalence of trypanosomes in European badgers detected using ITS-PCR.

BACKGROUND: Wildlife can be important sources and reservoirs for pathogens. Trypanosome infections are common in many mammalian species, and are pathogenic in some. Molecular detection tools were used to measure trypanosome prevalence in a well-studied population of wild European badgers (Meles meles). FINDINGS: A nested ITS-PCR system, that targeted the ribosomal RNA gene locus, has been widely used to detect pathogenic human and animal trypanosomes in domestic animals in Africa and some wildlife hosts. Samples from a long-term DEFRA funded capture-mark-recapture study of wild badgers at Woodchester Park (Gloucestershire, SW England) were investigated for trypanosome prevalence. A total of 82 badger blood samples were examined by nested ITS-PCR. Twenty-nine of the samples were found to be positive for trypanosomes giving a prevalence of 35.4 % (25.9 % - 46.2 %; 95 % CI). Infection was not found to be linked to badger condition, sex or age. Analysis of DNA sequence data showed the badgers to be infected with Trypanosoma (Megatrypanum) pestanai and phylogenetic analysis showed the Woodchester badger trypanosomes and T. pestanai to cluster in the Megatrypanum clade. CONCLUSIONS: The results show that the ITS Nested PCR is an effective tool for diagnosing trypanosome infection in badgers and suggests that it could be widely used in wildlife species with unknown trypanosomes or mixed infections. The relatively high prevalence observed in these badgers raises the possibility that a significant proportion of UK badgers are naturally infected with trypanosomes

Circulating tumour DNA is a promising biomarker for risk stratification of central chondrosarcoma with IDH1/2 and GNAS mutations

Chondrosarcoma (CS) is a rare tumour type and the most common primary malignant bone cancer in adults. The prognosis, currently based on tumour grade, imaging and anatomical location, is not reliable, and more objective biomarkers are required. We aimed to determine whether the level of circulating tumour DNA (ctDNA) in the blood of CS patients could be used to predict outcome. In this multi-institutional study, we recruited 145 patients with cartilaginous tumours, of which 41 were excluded. ctDNA levels were assessed in 83 of the remaining 104 patients, whose tumours harboured a hotspot mutation in IDH1/2 or GNAS. ctDNA was detected pre-operatively in 31/83 (37%) and in 12/31 (39%) patients postoperatively. We found that detection of ctDNA was more accurate than pathology for identification of high-grade tumours and was associated with a poor prognosis; ctDNA was never associated with CS grade 1/atypical cartilaginous tumours (ACT) in the long bones, in neoplasms sited in the small bones of the hands and feet or in tumours measuring less than 80 mm. Although the results are promising, they are based on a small number of patients, and therefore, introduction of this blood test into clinical practice as a complementary assay to current standard-of-care protocols would allow the assay to be assessed more stringently and developed for a more personalised approach for the treatment of patients with CS

Guanylate-binding Protein 1 (GBP1) contributes to the immunity of human mesenchymal stromal cells against toxoplasma gondii

Mesenchymal stromal cells (MSCs) have recently been shown to play important roles in mammalian host defenses against intracellular pathogens, but the molecular mechanism still needs to be clarified. We confirmed that human MSCs (hMSCs) pre-stimulated with IFN-γ showed a significant and dose-dependent ability to inhibit the growth of two types of Toxoplasma gondii (type I strain RH/GFP or type II strain PLK/RED). However, in contrast to previous reports, the anti-T. gondii activity of hMSCs was not mediated by indoleamine 2,3-dioxygenase (IDO). Genome-wide RNA-seq analysis revealed that IFN-γ increased the expression of the p65 family of guanylate-binding proteins (hGBPs) in hMSCs, especially hGBP1. To analyze the functional role of hGBPs, stable knockdowns of hGBP1, -2, -5 in hMSCs were established using a lentiviral transfection system. hGBP1 knockdown in hMSCs resulted in a significant loss of the anti-T. gondii host defense property, compared with hMSCs infected with non-targetted control sequences. hGBP2 and -5 knockdowns had no effect. Moreover, the hGBP1 accumulation on the parasitophorous vacuole (PV) membranes of IFN-γ-stimulated hMSCs might protect against T. gondii infection. Taken together, our results suggest that hGBP1 plays a pivotal role in anti-T. gondii protection of hMSCs and may shed new light on clarifying the mechanism of host defense properties of hMSCs

Innate resistance to Leishmania amazonensis Infection in rat is dependent on NOS2

Leishmania infection causes diverse clinical manifestations in humans. The disease outcome is complicated by the combination of many host and parasite factors. Inbred mouse strains vary in resistance to Leishmania major but are highly susceptible to Leishmania amazonensis infection. However, rats are highly resistant to L. amazonensis infection due to unknown mechanisms. We use the inducible nitric oxide synthase (Nos2) gene knockout rat model (Nos2−/− rat) to investigate the role of NOS2 against leishmania infection in rats. Our results demonstrated that diversion toward the NOS2 pathway is the key factor explaining the resistance of rats against L. amazonensis infection. Rats deficient in NOS2 are susceptible to L. amazonensis infection even though their immune response to infection is still strong. Moreover, adoptive transfer of NOS2 competent macrophages into Nos2−/− rats significantly reduced disease development and parasite load. Thus, we conclude that the distinct L-arginine metabolism, observed in rat macrophages, is the basis of the strong innate resistance to Leishmania. These data highlight that macrophages from different hosts possess distinctive properties and produce different outcomes in innate immunity to Leishmania infections

The Toxoplasma monocarboxylate transporters are involved in the metabolism within the apicoplast and are linked to parasite survival

The apicoplast is a four-membrane plastid found in the apicomplexans, which harbors biosynthesis and organelle housekeeping activities in the matrix. However, the mechanism driving the flux of metabolites, in and out, remains unknown. Here we used TurboID and genome engineering to identify apicoplast transporters in Toxoplasma gondii. Among the many novel transporters, we show that one pair of apicomplexan monocarboxylate transporters (AMTs) appears to be evolved from the putative host cell that engulfed a red alga. Protein depletion showed that AMT1 and AMT2 are critical for parasite growth. Metabolite analyses supported the notion that AMT1 and AMT2 are associated with biosynthesis of isoprenoids and fatty acids. However, stronger phenotypic defects were observed for AMT2, including in the inability to establish T. gondii parasite virulence in mice. This study clarifies, significantly, the mystery of apicoplast transporter composition and reveals the importance of the pair of AMTs in maintaining the apicoplast activity in apicomplexan

The transplant rejection response involves neutrophil and macrophage adhesion-mediated trogocytosis and is regulated by NFATc3

The anti-foreign tissue (transplant rejection) response, mediated by the immune system, has been the biggest obstacle to successful organ transplantation. There are still many enigmas regarding this process and some aspects of the underlying mechanisms driving the immune response against foreign tissues remain poorly understood. Here, we found that a large number of neutrophils and macrophages were attached to the graft during skin transplantation. Furthermore, both types of cells could autonomously adhere to and damage neonatal rat cardiomyocyte mass (NRCM) in vitro. We have demonstrated that Complement C3 and the receptor CR3 participated in neutrophils/macrophages-mediated adhesion and damage this foreign tissue (NRCM or skin grafts). We have provided direct evidence that the damage to these tissues occurs by a process referred to as trogocytosis, a damage mode that has never previously been reported to directly destroy grafts. We further demonstrated that this process can be regulated by NFAT, in particular, NFATc3. This study not only enriches an understanding of host-donor interaction in transplant rejection, but also provides new avenues for exploring the development of novel immunosuppressive drugs which prevent rejection during transplant therapy