Factors Influencing Mortality in Children with Central Nervous System Tumors: A Cohort Study on Clinical Characteristics and Genetic Markers.

Multidrug resistance (MDR) commonly leads to cancer treatment failure because cancer cells often expel chemotherapeutic drugs using ATP-binding cassette (ABC) transporters, which reduce drug levels within the cells. This study investigated the clinical characteristics and single nucleotide variant (SNV) in ABCB1, ABCC1, ABCC2, ABCC4, and ABCG2, and their association with mortality in pediatric patients with central nervous system tumors (CNST). Using TaqMan probes, a real-time polymerase chain reaction genotyped 15 SNPs in 111 samples. Patients were followed up until death or the last follow-up day using the Cox proportional hazards model. An association was found between the rs1045642 (ABCB1) in the recessive model (HR = 2.433, 95% CI 1.098-5.392, p = 0.029), and the ICE scheme in the codominant model (HR = 9.810, 95% CI 2.74-35.06, p ≤ 0.001), dominant model (HR = 6.807, 95% CI 2.87-16.103, p ≤ 0.001), and recessive model (HR = 6.903, 95% CI 2.915-16.544, p = 0.038) significantly increased mortality in this cohort of patients. An association was also observed between the variant rs3114020 (ABCG2) and mortality in the codominant model (HR = 5.35, 95% CI 1.83-15.39, p = 0.002) and the dominant model (HR = 4.421, 95% CI 1.747-11.185, p = 0.002). A significant association between the ICE treatment schedule and increased mortality risk in the codominant model (HR = 6.351, 95% CI 1.831-22.02, p = 0.004, HR = 9.571, 95% CI 2.856-32.07, p ≤ 0.001), dominant model (HR = 6.592, 95% CI 2.669-16.280, p ≤ 0.001), and recessive model (HR = 5.798, 95% CI 2.411-13.940, p ≤ 0.001). The genetic variants rs3114020 in the ABCG2 gene and rs1045642 in the ABCB1 gene and the ICE chemotherapy schedule were associated with an increased mortality risk in this cohort of pediatric patients with CNST.

First characterization of a microsporidial triosephosphate isomerase and the biochemical mechanisms of its inactivation to propose a new druggable target.

The microsporidia are a large group of intracellular parasites with a broad range of hosts, including humans. Encephalitozoon intestinalis is the second microsporidia species most frequently associated with gastrointestinal disease in humans, especially immunocompromised or immunosuppressed individuals, including children and the elderly. The prevalence reported worldwide in these groups ranges from 0 to 60%. Currently, albendazole is most commonly used to treat microsporidiosis caused by Encephalitozoon species. However, the results of treatment are variable, and relapse can occur. Consequently, efforts are being directed toward identifying more effective drugs for treating microsporidiosis, and the study of new molecular targets appears promising. These parasites lack mitochondria, and oxidative phosphorylation therefore does not occur, which suggests the enzymes involved in glycolysis as potential drug targets. Here, we have for the first time characterized the glycolytic enzyme triosephosphate isomerase of E. intestinalis at the functional and structural levels. Our results demonstrate the mechanisms of inactivation of this enzyme by thiol-reactive compounds. The most striking result of this study is the demonstration that established safe drugs such as omeprazole, rabeprazole and sulbutiamine can effectively inactivate this microsporidial enzyme and might be considered as potential drugs for treating this important disease.

Toxocara canis: Analysis of the kinetics of antigen release and antibody production in an in vivo model for the detection of past or present infection.

Worldwide, Toxocara canis is an important zoonotic nematode of public health concern. This soil-transmitted helminth causes visceral larva and ocular larva migrans in paratenic hosts. The detection of T. canis larva migrans is complicated because current immunological tests detect only IgG antibodies, which can cross-react with antigens from other parasites and cannot distinguish between the past and present infection. Analysis of antigen release and antibody production could help improve the detection of larva migrans. Here, we report the kinetics of antigen release, IgM and IgG production in an in vivo model for the detection of past or present infection. We used four groups of seven mice: two groups infected orally with 50 or 100 embryonated eggs, and the other two infected intraperitoneally with 50 or 100 live larvae. We obtained blood samples at 0, 3, 7, and 14days and, then, every two weeks until day 140. Sandwich ELISA and indirect ELISA were performed for antigen capture and the detection of immunoglobulins, respectively. Mice inoculated with larvae developed an immune response faster than those inoculated with eggs. In all groups, antigen capture was positive starting at 3days until 140days post-inoculation (dpi). Detection of immunoglobulins was at 14 or 28dpi in mice inoculated with larvae or eggs, respectively. Negative IgM values were detected at days 98 and 112. The samples remained positive for IgG until the last day of the experiment. Data suggest that in mice inoculated with T canis eggs, some larvae did not hatch, others died or never reached the bloodstream. Based on our model, we propose that there is early infection when only antigens are present, and active larva migrans when antigen and immunoglobulins are detected, implying an immune response of the host against the antigen. Our study offers a view into the parasite-host relationship and enables us to infer if there are live larvae. Additionally, these findings provide a foundation for the diagnosis and differentiation of recent infection and active larva migrans.

Successful capture of Toxocara canis larva antigens from human serum samples.

BACKGROUND: Toxocara canis is a nematode that parasitizes dogs, while humans are paratenic hosts. When humans are infected the migrating larvae damage the liver, lungs and even the nervous system. Larva migrans diagnosis is based on immunological techniques; however, the commercial immunodiagnostic kits detect anti-T. canis antibodies which may cross-react with other parasites, mainly nematodes with extra-intestinal migration. Moreover, antibodies do not necessarily reflect an active infection; so detection and quantification of circulating antigens may provide appropriate and timely information for treatment, which prevents irreversible damage. Here we report the standardization of a monoclonal antibody based antigen capture ELISA to diagnose human toxocariasis without cross-reaction. METHODS: We developed anti-T. canis polyclonal antibodies in rabbits and a monoclonal antibody in mouse which did not cross-react with 15 antigens from several parasites. The sandwich ELISA standardization was performed using sera from mice experimentally infected. We tested the method using 29 positive and 58 negative human sera previously typified with a commercial kit, which detects antibodies. RESULTS: Only 5.0 µg/mL and 10 µg/mL polyclonal antibodies and monoclonal antibody, respectively, were needed in the sandwich ELISA standardization, detecting since 440 pg/mL larva antigens. Nine out of 29 antibody-positive sera were also positive for antigens and no false positive were found. Taking the antibody kit as the reference standard, the sensibility and specificity of the antigen test were 31% and 100%, respectively. CONCLUSIONS: With these tools we established a detection threshold as low as 440 pg/mL antigen. Monoclonal antibody is specific, and did not cross-react with antigens from other parasites. Detection of circulating antigens helps provide appropriate and timely treatment and prevents irreversible damage.

[Microsporidia in pediatric patients with leukemia or limphoma]. / Microsporidiosis en pacientes pediátricos con leucemia o linfoma.

INTRODUCTION: Microsporidia are intracellular micro-organisms, characterized by mature spores with chitin walls and by one extrusive polar tube through which they pour their sporoplasm to the host cells. In immunocompromised patients, Enterocytozoon bieneusi and Encephalitozoon intestinalis produce diarrhea and systemic dissemination. In Mexico there is not information about microsporidia in children with cancer. OBJECTIVE: The aim of this pilot study was to investigate the presence of microsporidia species in pediatric patients with leukemia or lymphoma. MATERIAL AND METHODS: We obtained fecal samples from thirteen patients. The samples were processed to detect microsporidia by both modified Ziehl-Neelsen and clacofluor white stains, DNA was isolated to amplify rRNA specific sequences, to identify E. bieneusi, E. intestinalis, E. cuniculi and E. hellem by DNA polymerase chain reaction (PCR). Other parasites and pathogenic bacteria were also tested. RESULTS: Based on morphologic traits 7/13 samples were found positives to microsporidia and 6/10 by PCR. Was identified E. bieneusi in three patients with leukemia and one with lymphoma, another two children with leukemia were infected with E. intestinalis. Almost all children were high-risk patients and in phase of re-induction, consolidation or with many chemotherapy treatments. All the patients with microspiridia did not present diarrhea at the moment of the sampling; however, in two children with diarrhea it was found Cyclospora cayetanensis. Also we obtained feces from five patients' mothers and microsporidia spores were identified by stain in all of them and by PCR it was diagnosed the species in three of them. CONCLUSION: It was demonstrated that the feces of patients with leukemia or lymphoma had microsporidia, therefore is necessary to know the prevalence of these microorganisms and to analyze their impact in evolution of cancer patients.