Fluorescent Plasmodium berghei sporozoites and pre-erythrocytic stages: a new tool to study mosquito and mammalian host interactions with malaria parasites.

To track malaria parasites for biological studies within the mosquito and mammalian hosts, we constructed a stably transformed clonal line of Plasmodium berghei, PbFluspo, in which sporogonic and pre-erythrocytic liver-stage parasites are autonomously fluorescent. A cassette containing the structural gene for the FACS-adapted green fluorescent protein mutant 2 (GFPmut2), expressed from the 5' and 3' flanking sequences of the circumsporozoite (CS) protein gene, was integrated and expressed at the endogenous CS locus. Recombinant parasites, which bear a wild-type copy of CS, generated highly fluorescent oocysts and sporozoites that invaded mosquito salivary glands and were transmitted normally to rodent hosts. The parasites infected cultured hepatocytes in vitro, where they developed into fluorescent pre-erythrocytic forms. Mammalian cells infected by these parasites can be separated from non-infected cells by fluorescence activated cell sorter (FACS) analysis. These fluorescent insect and mammalian stages of P. berghei should be useful for phenotypic studies in their respective hosts, as well as for identification of new genes expressed in these parasite stages

Early elimination of cyclosporine in kidney transplant recipients receiving sirolimus prevents progression of chronic pathologic allograft lesions

Cyclosporine elimination in a regimen including sirolimus has been shown to be a safe and effective approach to improve graft function. Nevertheless, it is still unknown whether the functional benefit of CyA withdrawal coincides with a subsequent reduction in histologic lesions of chronic damage or development of chronic allograft nephropathy. This consideration would forecast a reduction in the rate of long-term graft loss. We analyzed 114 graft biopsies from a subgroup of 57 patients that had been included in a randomized study to eliminate CyA at 3 months posttransplant from a regimen including sirolimus either in group A CyA + SRL vs group B of SRL with CyA elimination at 3 months. Every patient had two biopsies, one at transplantation and another at 1 year. The biopsy reading was performed in a blinded manner by a central pathologist using the Banff 1997 and the CADI classifications. A significantly lower rate of progression of tubular and interstitial chronic lesions between basal and 1-year biopsies was observed for group B patients. In addition, the incidence of new cases of chronic allograft nephropathy during the first year was significantly lower in the group in which CyA had been eliminated at 3 months posttransplant. We conclude that early elimination of CyA in the first months posttransplant, when SRL is used as the main immunosuppressant, reduces the appearance or worsening of chronic histologic lesions, probably as a consequence of long-term CyA toxicity prevention

Early cyclosporine a withdrawal in kidney-transplant recipients receiving sirolimus prevents progression of chronic pathologic allograft lesions

BACKGROUND: Nephrotoxicity of calcineurin inhibitors (CNIs) is partially responsible for the development of chronic allograft nephropathy (CAN). Sirolimus has demonstrated its potential to substitute for CNIs because it lacks significant nephrotoxicity and shows a short-term immunosuppressive capacity comparable with that of cyclosporine. This results in the maintenance of better renal function when cyclosporine is eliminated, but it has not been demonstrated whether this benefit is associated with an improvement in the pathologic substrate and a reduction in CAN. METHODS: We analyzed pretransplant and 1-year renal-allograft biopsies from 64 patients enrolled in a multicenter trial. Patients received cyclosporine and sirolimus during the first 3 months after transplant and were then randomly assigned to continue with cyclosporine or have it withdrawn. Histologic chronic allograft lesions were compared between groups. RESULTS: The percentage of patients in whom chronic pathologic lesions progressed was lower in the group of cyclosporine elimination. Significant differences were observed in chronic interstitial and tubular lesions (70% vs. 40.9% [P<0.05] and 70% vs. 47.8% [P<0.05], respectively), whereas no differences were observed in acute lesions (subclinical rejection). Prevalence of CAN at 1 year was lower in this group, as was the severity and incidence of new cases (P<0.05). CONCLUSIONS: Early cyclosporine withdrawal associated with sirolimus administration is followed by an improvement in renal function, a reduction in the progression of chronic pathologic allograft lesions, and a lower incidence of new cases and severity of CAN during the first year after transplantation. This benefit may result in better long-term graft outcome

Micro-costing analysis of guideline-based treatment by direct-acting agents: the real-life case of hepatitis C management in Brazil

Background Eradication of hepatitis C virus (HCV) using direct-acting agents (DAA) has been associated with a financial burden to health authorities worldwide. We aimed to evaluate the guideline-based treatment costs by DAAs from the perspective of the Brazilian Ministry of Health (BMoH). Methods The activity based costing method was used to estimate the cost for monitoring/treatment of genotype-1 (GT1) HCV patients by the following strategies: peg-interferon (PEG-IFN)/ribavirin (RBV) for 48 weeks, PEG-IFN/RBV plus boceprevir (BOC) or telaprevir (TEL) for 48 weeks, and sofosbuvir (SOF) plus daclastavir (DCV) or simeprevir (SIM) for 12 weeks. Costs were reported in United States Dollars without (US$) and with adjustment for purchasing power parity (PPP$). Drug costs were collected at the National Database of Health Prices and an overview of the literature was performed to assess effectiveness of SOF/DCV and SOF/SIM regimens in real-world cohorts. Results Treatment costs of GT1-HCV patients were PPP$43,176.28 (US$ 24,020.16) for PEG-IFN/RBV, PPP$71,196.03 (US$ 39,578.23) for PEG-IFN/RBV/BOC and PPP$86,250.33 (US$ 47,946.92) for PEG-IFN/RBV/TEL. Treatment by all-oral interferon-free regimens were the less expensive approach: PPP$19,761.72 (US$ 10,985.90) for SOF/DCV and PPP$21,590.91 (US$ 12,002.75) for SOF/SIM. The overview reported HCV eradication in up to 98% for SOF/DCV and 96% for SOF/SIM. Conclusion Strategies with all oral interferon-free might lead to lower costs for management of GT1-HCV patients compared to IFN-based regimens in Brazil. This occurred mainly because of high discounts over international DAA prices due to negotiation between BMoH and pharmaceutical industries

Profiling of lung microbiota discloses differences in adenocarcinoma and squamous cell carcinoma

The lung is a complex ecosystem of host cells and microbes often disrupted in pathological conditions. Although bacteria have been hypothesized as agents of carcinogenesis, little is known about microbiota profile of the most prevalent cancer subtypes: adenocarcinoma (ADC) and squamous cell carcinoma (SCC). To characterize lung cancer (LC) microbiota a first a screening was performed through a pooled sequencing approach of 16S ribosomal RNA gene (V3-V6) using a total of 103 bronchoalveaolar lavage fluid samples. Then, identified taxa were used to inspect 1009 cases from The Cancer Genome Atlas and to annotate tumor unmapped RNAseq reads. Microbial diversity was analyzed per cancer subtype, history of cigarette smoking and airflow obstruction, among other clinical data. We show that LC microbiota is enriched in Proteobacteria and more diverse in SCC than ADC, particularly in males and heavier smokers. High frequencies of Proteobacteria were found to discriminate a major cluster, further subdivided into well-defined communities’ associated with either ADC or SCC. Here, a SCC subcluster differing from other cases by a worse survival was correlated with several Enterobacteriaceae. Overall, this study provides first evidence for a correlation between lung microbiota and cancer subtype and for its influence on patient life expectancy.We would like to thank all patients for donating their samples and for collaborating in this study. IPATIMUP integrates the i3S Research Unit, which is partially supported by the Portuguese Foundation for Science and Technology (FCT). This work was supported by the Portuguese Foundation for Science and Technology (FCT), financed by the European Social Funds (COMPETE-FEDER) and National Funds through the FCT (projects PEstC/SAU/LA0003/2013 and POCI-01-0145-FEDER-007274, fellowships SFRH/BPD/77646/2011 and SFRH/BPD/120777/2016 to S.G. and P.I.M., respectively, grant PTDC/BEXGMG/0242/2012 to S.S. and by Programa Operacional Regional do Norte (ON.2 – O Novo Norte and Norte 2020), through FEDER funds under the Quadro de Referência Estratégico Nacional (QREN; projects NORTE-07-0162-FEDER-00018 and NORTE-070162-FEDER-000067, and NORTE-01-0145-FEDER-000029)

Methodological considerations in the analysis of fecal glucocorticoid metabolites in tufted capuchins (Cebus apella)

Analysis of fecal glucocorticoid (GC) metabolites has recently become the standard method to monitor adrenocortical activity in primates noninvasively. However, given variation in the production, metabolism, and excretion of GCs across species and even between sexes, there are no standard methods that are universally applicable. In particular, it is important to validate assays intended to measure GC production, test extraction and storage procedures, and consider the time course of GC metabolite excretion relative to the production and circulation of the native hormones. This study examines these four methodological aspects of fecal GC metabolite analysis in tufted capuchins (Cebus apella). Specifically, we conducted an adrenocorticotrophic hormone (ACTH) challenge on one male and one female capuchin to test the validity of four GC enzyme immunoassays (EIAs) and document the time course characterizing GC me- tabolite excretion in this species. In addition, we compare a common field-friendly technique for extracting fecal GC metabolites to an established laboratory extraction methodology and test for effects of storing “field extracts” for up to 1 yr. Results suggest that a corticosterone EIA is most sensitive to changes in GC production, provides reliable measures when extracted according to the field method, and measures GC metabolites which remain highly stable after even 12 mo of storage. Further, the time course of GC metabolite excretion is shorter than that described yet for any primate taxa. These results provide guidelines for studies of GCs in tufted capuchins, and underscore the importance of validating methods for fecal hormone analysis for each species of interest

Determinants of carotid-femoral pulse wave velocity in prepubertal children

BACKGROUND: Pulse wave velocity (PWV) is a noninvasive technique to evaluate arterial stiffness, a dynamic property of the vessels, reflecting their structure and function. Childhood obesity is associated with several cardiovascular comorbidities and to the progression of atherosclerosis. We aimed to compare carotid-femoral PWV between normal weight and overweight/obese prepubertal children and to quantify its association with other cardiovascular risk factors. METHODS: Cross-sectional study of 315 children aged 8-9years. Anthropometrics, 24-h ambulatory blood pressure (BP) and carotid-femoral PWV were measured. Classification of obesity was according to World Health Organization (WHO) body mass index (BMI)-for-age reference values. RESULTS: Compared to normal weight children, overweight and obese children presented significantly higher levels of PWV (4.95 (P25-P75: 4.61-5.23), 5.00 (4.71-5.33), 5.10 (4.82-5.50) m/s, respectively; ptrend<0.001). Significant positive correlations were found between PWV and total cholesterol, LDL cholesterol, triglycerides, fasting insulin and insulin resistance levels (HOMA-IR) and with high-sensitivity C-reactive protein (hs-CRP). In a multivariate linear regression model adjusted for sex, age, height and 24-h systolic blood pressure z-score, the independent determinants of PWV were BMI, HOMA-IR and the absence of dipping. CONCLUSIONS: The association between PWV and the loss of dipping and insulin resistance levels, independently of the BMI, reinforces the contribution of these comorbidities to vascular injury in early life