Seroprevalence of anti-Leptospira antibodies in equines and associated workers-Isolation of Leptospira interrogans serogroup Canicola from equine urine.

To investigate seroprevalence of anti-Leptospira antibodies in equines and associated workers in Uruguay, 891 equine and 150 human sera were drawn; 212 equine urine samples were also taken for culture. Environmental conditions and equine raising or managing practices were recorded in all 72 visited establishments; epidemiological information was obtained from each worker. Microscopic agglutination technique (MAT) was performed with 10 Leptospira strains for equines and 18 for human sera, that were also studied with IgM indirect immunofluorescence (IgM-IIF). Equine titres ≥100 were considered positive, and human sera titres ≥200 suggested probable recent or past infection. Urines were cultured in Ellinghausen-McCullough-Johnson-Harris (EMJH) media; local identification of one obtained isolate with lipL32 PCR, Multiple Locus Variable number tandem repeat Analysis and partial rrs gene sequencing, were completed at Institut Pasteur, Paris. Estimated reactivity was 61.3% for equines, which was higher than the studied bovine national levels (21%) and mainly observed with Icterohaemorrhagiae serogroup (40.3%), Sejroe, Canicola, Pomona or Ballum. Aged animals from slaughterhouses and cattle farms were the most frequently positive. Multiple regression analysis confirmed a significant association between seropositivity and equine age. Only one positive culture could be fully studied, and confirmed to be Leptospira interrogans serogroup Canicola; it was added to the MAT antigen panel and revealed fairly frequent reaction with equine and human sera. Three workers (2%) showed titres = 200 with Icterohaemorrhagiae or Canicola serogroups, without recent clinical manifestations. Their attended equines reacted with the same serogroups, suggesting common source infections or infection transmitted by equines. Three other humans yielded titres = 100, and none of the 150 showed an IgM-IIF-positive result. Equines seem not to be an important origin of regional human leptospirosis, except perhaps during acute animal infection. More culture work is required to study intensity and lapses of leptospiruria, as well as to further identify circulating strains.

Cost-Effectiveness Analysis of Gemtuzumab Ozogamicin for First-Line Treatment of Patients with Cd-33 Positive Acute Myeloid Leukaemia in Spain.

OBJECTIVE: To assess the incremental cost-utility ratio (ICUR) of gemtuzumab ozogamicin (GO) + standard of care (SOC) vs SOC alone for treatment of patients with de novo AML from a Spanish Health Service perspective. METHODS: A cohort state-transition model, with 12 health-states, was used to estimate the lifetime accumulated cost and benefits in terms of quality-adjusted-life-years (QALYs) in AML patients with favourable, intermediate, and unknown cytogenetic profiles. Patient profile was defined based on the ALFA-0701 trial. Therapeutic regimens were defined by 5 haematologists. SOC was assumed to be idarubicin and cytarabine, the combination most used in Spain. QALYs were estimated by applying utilities for the time spent by the cohort in each health-state and utility decrements associated with adverse events (AE). Total cost (€,2020) included drug-acquisition, hematologic stem-cell transplantation, disease management, AE management and end-of-life costs. Unit costs were derived from local databases. All parameters were validated by haematologist. Costs and outcomes were discounted (3%/year). RESULTS: Higher cost/patient (€177,618 vs €151,434) and greater QALYs (5,70 vs 4,62) were obtained with GO+SOC vs SOC. The ICUR was €24,203/QALY gained. CONCLUSION: This simulation suggests that GO + SOC could be a cost-effective option for treatment of patients with de novo AML in first line.

A drought-responsive rice amidohydrolase is the elusive plant guanine deaminase with the potential to modulate the epigenome.

Drought stress in plants causes differential expression of numerous genes. One of these differentially expressed genes in rice is a specific amidohydrolase. We characterized this amidohydrolase gene on the rice chromosome 12 as the first plant guanine deaminase (OsGDA1). The biochemical activity of GDA is known from tea and coffee plants where its catalytic product, xanthine, is the precursor for theine and caffeine. However, no plant gene that is coding for GDA is known so far. Recombinant OsGDA1 converted guanine to xanthine in vitro. Measurement of guanine and xanthine contents in the OsGDA1 knockout (KO) line and in the wild type Tainung 67 rice plants also suggested GDA activity in vivo. The content of cellular xanthine is important because of its catabolic products allantoin, ureides, and urea which play roles in water and nitrogen stress tolerance among others. The identification of OsGDA1 fills a critical gap in the S-adenosyl-methionine (SAM) to xanthine pathway. SAM is converted to S-adenosyl-homocysteine (SAH) and finally to xanthine. SAH is a potent inhibitor of DNA methyltransferases, the reduction of which leads to increased DNA methylation and gene silencing in Arabidopsis. We report that the OsGDA1 KO line exhibited a decrease in SAM, SAH and adenosine and an increase in rice genome methylation. The OsGDA1 protein phylogeny combined with mutational protein destabilization analysis suggested artificial selection for null mutants, which could affect genome methylation as in the KO line. Limited information on genes that may affect epigenetics indirectly requires deeper insights into such a role and effect of purine catabolism and related genetic networks.

[Serogroup C invasive meningococcal disease in the post-vaccine era and vaccine failures]. / Enfermedad meningocócica invasiva por serogrupo C en la era posvacunal y fallos vacunales.

INTRODUCTION: The incidence of serogroup C invasive meningococcal disease (IMD) has decreased since the introduction of systematic vaccination in 2000. The aim of this study is to determine the number of serogroup C IMD cases diagnosed since then and the vaccine failures. PATIENTS AND METHODS: A retrospective analysis was performed on patients diagnosed with IMD by culture or polymerase chain reaction (PCR) in a maternity and childhood hospital in Barcelona between 2001 and 2018. An analysis was made of the number of vaccine doses and the age received, as well as on the medical records and vaccine cards. RESULTS: There were 128 confirmed cases of IMD (7.1 cases/year; 70.3 in <5 years). The serogroup was studied in 125 (97.6%) cases, in which 103 (82.4%) were B, 10 (8%) were C, one (0.8%) was 29E, and one (0.8%) was Y, and only 10 (8%) were not able to be serogrouped. Of the 10 patients with serogroup C, 4 were not vaccinated, and in 3, the course was not complete as regards the number of doses. The other 3 received the complete course according to age and current calendar, and thus were considered vaccine failures. A total of 6 patients died (mortality rate: 4.7%), 5 due to serogroup B (mortality: 4.8%), and one due to serogroup C (mortality: 10%). CONCLUSIONS: Serogroup C only represented 8% of IMD cases in the period studied, with 30% of cases due to this serogroup being vaccine failures.

Seroprevalence of leptospirosis in human groups at risk due to environmental, labor or social conditions.

Leptospirosis is important in Uruguay due to the economic loss caused by the diseases of production animals, mainly bovines, and also due to frequent human infection. We decided to study anti-Leptospira antibodies in the sera of dairy workers, rice laborers, veterinarians, suburban slum dwellers and garbage recyclers. Our aims were to estimate the seroprevalence of infection by Leptospira spp. in these people at risk, the relative importance of the known risk factors associated with infection, and the impact of human infections in each setting. Groups at risk were identified and 35 visits to their locations were made, conducting field surveys and exchange talks for information and education. Simple epidemiological questionnaires were administered and sera samples were taken from 308 persons. The microagglutination Technique (MAT) and the IgM Indirect Immunofluorescence (IIF) assay were employed to detect antibodies. Environmental water samples, canine and equine sera were also examined. More than 45% of human sera were reactive and the studied groups were confirmed to be widely exposed to infection. Female sera were frequently reactive, though most illnesses occur in men, and the most severe cases in elderly males; the emergence and evolution of the disease may strongly depend on the host condition and functions. Animal contact and unsafe water usage were the main identified risk factors to be considered in prevention. Fifty per cent of the studied horses showed a positive MAT reaction. The underdiagnosis of the illness and its long-term symptoms require further study, as well as greater health and social attention efforts.

Enhanced anti-tumor efficacy of checkpoint inhibitors in combination with the histone deacetylase inhibitor Belinostat in a murine hepatocellular carcinoma model.

Immune checkpoint inhibitors are currently tested in different combinations in patients with advanced hepatocellular carcinoma (HCC). Nivolumab, an anti-PD-1 agent, has gained approval in the second-line setting in the USA. Epigenetic drugs have immune-mediated antitumor effects that may improve the activity of immunotherapy agents. Our aim was to study the therapeutic efficacy of checkpoint inhibitors (anti-CTLA-4 and anti-PD-1 antibodies) in combination with the histone deacetylase inhibitor (HDACi) Belinostat. In a subcutaneous Hepa129 murine HCC model, we demonstrated that Belinostat improves the antitumor activity of anti-CTLA-4 but not of anti-PD-1 therapy. This effect correlated with enhanced IFN-γ production by antitumor T-cells and a decrease in regulatory T-cells. Moreover, the combination induced early upregulation of PD-L1 on tumor antigen-presenting cells and late expression of PD-1 on tumor-infiltrating effector T-cells, suggesting the suitability of PD-1 blockade. Indeed, Belinostat combined with the simultaneous blockade of CTLA-4 and PD-1 led to complete tumor rejection. These results provide a rationale for testing Belinostat in combination with checkpoint inhibitors to enhance their therapeutic activity in patients with HCC.

The Toll like receptor 4 ligand cold-inducible RNA-binding protein as vaccination platform against cancer.

Tumor infiltrating lymphocytes have been associated with a better prognostic and with higher response rates in patients treated with checkpoint inhibiting antibodies, suggesting that strategies promoting tumor inflammation may enhance the efficacy of these currently available therapies. Our aim was thus to develop a new vaccination platform based on cold-inducible RNA binding protein (CIRP), an endogenous TLR4 ligand generated during inflammatory processes, and characterize whether it was amenable to combination with checkpoint inhibitors. In vitro, CIRP induced dendritic cell activation, migration and enhanced presentation of CIRP-bound antigens to T-cells. Accordingly, antigen conjugation to CIRP conferred immunogenicity, dependent on immunostimulatory and antigen-targeting capacities of CIRP. When applied in a therapeutic setting, vaccination led to CD8-dependent tumor rejection in several tumor models. Moreover, immunogenicity of this vaccination platform was enhanced not only by combination with additional adjuvants, but also with antibodies blocking PD-1/PD-L1, CTLA-4 and IL-10, immunosuppressive molecules usually present in the tumor environment and also induced by the vaccine. Therefore, priming with a CIRP-based vaccine combined with immune checkpoint-inhibiting antibodies rejected established B16-OVA tumors. Finally, equivalent activation and T-cell stimulatory effects were observed when using CIRP in vitro with human cells, suggesting that CIRP-based vaccination strategies could be a valuable clinical tool to include in combinatorial immunotherapeutic strategies in cancer patients.

Anion exchange chromatography for the determination of 5-methyl-2'-deoxycytidine: application to cisplatin-sensitive and cisplatin-resistant ovarian cancer cell lines.

Epigenetic alterations are increasingly implicated in the initiation and progression of cancer. Genome-wide (global) hypomethylation seems to occur in early neoplasia and is a feature of genomic DNA derived from solid tumour tissues like ovarian cancer. Thus, analytical methods that provide sensitive and quantitative information about cytosine methylation in DNA are currently required. In this work, we compare two different anion-exchange columns for the separation of methylated cytosine from the other DNA nucleotides: a silica-based (Tracer Extrasil SAX) column and a polystyrene/divinyl benzene-based (Mono-Q™) column. Under the optimised conditions, linearity range, precision and detection limits of the developed high-performance liquid chromatography (HPLC) method were evaluated and compared using conventional ultraviolet (UV) absorbance detection at 270 nm. Good separation of the five target nucleotides, including 5-methyl-2'-deoxycytidine monophosphate (5mdCMP) and 2'-deoxycytidine monophosphate (dCMP) was achieved on the Mono-Q™ column with a gradient elution of ammonium acetate buffer (1 M, pH 6.9) at a flow rate of 1 mL min(-1). The coupling of this column to inductively coupled plasma mass spectrometry (ICP-MS) permitted also phosphorous ((31)P) specific detection of the nucleotides. Both detection systems offered adequate analytical performance characteristics, with detection limits of 30 and 40 µg L(-1) for 5mdCMP by HPLC-UV and HPLC-ICP-MS, respectively. However, the latter method allowed the determination of the global DNA methylation level (%) without the need for external calibration. Different genomic DNA samples were analysed including calf thymus DNA and DNA from two human cancer cell lines (adenocarcinoma epithelial A549 and ovarian carcinoma A2780) using the proposed strategy. In the line A2780, the cisplatin-sensitive and cisplatin-resistant variants were analysed, finding no significant differences in the methylation percentage after treatment with cisplatin.

Ontological differences in first compared to third trimester human fetal placental chorionic stem cells.

Human mesenchymal stromal/stem cells (MSC) isolated from fetal tissues hold promise for use in tissue engineering applications and cell-based therapies, but their collection is restricted ethically and technically. In contrast, the placenta is a potential source of readily-obtainable stem cells throughout pregnancy. In fetal tissues, early gestational stem cells are known to have advantageous characteristics over neonatal and adult stem cells. Accordingly, we investigated whether early fetal placental chorionic stem cells (e-CSC) were physiologically superior to their late gestation fetal chorionic counterparts (l-CSC). We showed that e-CSC shared a common phenotype with l-CSC, differentiating down the osteogenic, adipogenic and neurogenic pathways, and containing a subset of cells endogenously expressing NANOG, SOX2, c-MYC, and KLF4, as well as an array of genes expressed in pluripotent stem cells and primordial germ cells, including CD24, NANOG, SSEA4, SSEA3, TRA-1-60, TRA-1-81, STELLA, FRAGILIS, NANOS3, DAZL and SSEA1. However, we showed that e-CSC have characteristics of an earlier state of stemness compared to l-CSC, such as smaller size, faster kinetics, uniquely expressing OCT4A variant 1 and showing higher levels of expression of NANOG, SOX2, c-MYC and KLF4 than l-CSC. Furthermore e-CSC, but not l-CSC, formed embryoid bodies containing cells from the three germ layer lineages. Finally, we showed that e-CSC demonstrate higher tissue repair in vivo; when transplanted in the osteogenesis imperfecta mice, e-CSC, but not l-CSC increased bone quality and plasticity; and when applied to a skin wound, e-CSC, but not l-CSC, accelerated healing compared to controls. Our results provide insight into the ontogeny of the stemness phenotype during fetal development and suggest that the more primitive characteristics of early compared to late gestation fetal chorionic stem cells may be translationally advantageous.

Desarrollo y ensayo de dos procedimientos para la detección rápida de Streptococcus agalactiae en exudados vaginorrectales / Development and testing of two procedures for the quick detection of Streptococcus agalactiae in rectal-vaginal exudates

Introducción: la infección por Estreptococo grupo B (EGB) puede afectar gravemente a la madre y al feto durante la gestación y al recién nacido luego del parto. Actualmente, eldiagnóstico de colonización durante el embarazo se realiza por métodos microbiológicos a partir de exudados vaginorrectales. Objetivo: desarrollar métodos rápidos y de bajo costo para la detección del antígeno grupo específico de EGB en exudados vaginorrectales.Material y método: se utilizaron dos cepas de EGB, una autóctona (IH23) y la cepa de referencia O90R, que solo expresa el polisacárido específico de grupo. Para cada una se preparó un antisuero policlonal que se utilizó para el desarrollo de un test inmunocromatográfico y uno de aglutinación de látex. Como controles se emplearon cultivos bacterianos, polisacáridos purificados de EGB y muestras vaginorrectales. Resultados: los límites de detección obtenidos para la inmunocromatografía fueron de 210 μg/ml y 50 μg/ml para los polisacáridos purificados de sobrenadante y pared, respectivamente, no lográndose detectar antígenos de EGB en las muestras clínicas analizadas. El límite dedetección del látex fue 65 μg/ml frente al polisacárido purificado de sobrenadante de cultivo de IH23 y 6,5 x 107 UFC/ml de IH23. La sensibilidad y especificidad para el látex fue de 30% y90%, respectivamente. Conclusiones: los métodos desarrollados no alcanzaron el límite de detección requerido parasu aplicación en muestras clínicas. Esto concuerda con lo descripto en la bibliografía para ensayos rápidos basados en reacciones antígeno-anticuerpo y muestra la necesidad deagregar pasos previos de extracción y concentración o mejorar la calidad de los reactivos inmunológicos empleados.

Introduction: group B streptococcal infection (GBS) may seriously affect mother and fetuses during pregnancy, and the newborn after delivery. Today, diagnosis of colonization during pregnancy is done by means of microbiological methods of vaginal and rectal exudates. Objective: to develop fast and low cost methods to detect the GBS specific group antigen in vaginal-rectal exudates. Method: we used two EGB strains, one of the (IH23)autochthonous and the reference strain O90R, that only expresses the group specific polysaccharide. We prepareda polyclonal antiserum for each one of them which was used to conduct an immunochromatographic test and alatex agglutination test. We used bacterial culture, EGB purified polysaccharides and vagina,-rectal samples as control. Results: detection limits obtained for the immunochromatographic test were 210 μg/ml and 50 μg/ml for purifiedpolysaccharides and cell wall, respectively, there being no EGB antigens detected in the clinical samples analyzed. Latex detection limit was 65 μg/ml compared to purified polysaccharides of IH23 culture supernatant and 6,5 x 107 UFC/ml of IH23. Sensitivity and specificity for latex was 30% and 90% respectively.Conclusions: the methods used failed to reach the detection limit required for its application in our clinical samples. This agrees with what is described in bibliography about quick tests based on antigen-antibody reactions and indicated the need to add previous extractionand concentration steps or to improve the quality of the immunologic reagents used.

Introdução: a infecção por Estreptococo grupo B (EGB) pode afetar gravemente a mãe e o feto durante a gravidez e o recém nascido imediatamente depois do parto. Atualmente, o diagnóstico de colonização durante a gestação é feita por métodos microbiológicos empregando exsudados vaginorretais.Objetivo: desenvolver métodos rápidos de baixo custo para a detecção do antígeno grupo específico de EGB emexsudados vaginorretais. Material e método: foram utilizadas duas cepas deEGB, uma autóctone (IH23) e a cepa de referência O90R, que expressa somente o polissacarídeo específico do grupo.Para cada uma foi preparado um antisoro policlonal utilizado para o desenvolvimento de um teste imunocromatográfico e um de aglutinação de látex. Foram empregadoscultivos bacterianos, polissacarídeos purificados de EGB e amostras vaginorretais como controles. Resultados: os limites de detecção obtidos para a imunocromatografia foram de 210 μg/ml e 50 μg/ml para ospolissacarídeos purificados de sobrenadante e parede, respectivamente, no sendo possível detectar antígenos de EGB nas amostras clínicas analisadas. O limite dedetecção do látex foi 65 μg/ml quando comparado com o polissacarídeo purificado de sobrenadante de cultivo deIH23 y 6,5 x 107 UFC/ml de IH23. A sensibilidade e especificidade para o látex foi de 30% y 90%, respectivamente. Conclusões: os métodos desenvolvidos não alcançaramo limite de detecção requerido para sua aplicação em amostras clínicas. Estes resultados são similares aos dadosdescritos na bibliografia para ensaios rápidos baseados em reações antígeno-anticorpo e mostram a necessidade de agregar passos prévios de extração econcentração ou melhorar a qualidade dos reativos imunológicos utilizados.