Serological based monitoring of a cohort of patients with chronic Chagas disease treated with benznidazole in a highly endemic area of northern Argentina

This study aimed to evaluate well-documented diagnostic antigens, named B13, 1F8 and JL7 recombinant proteins, as potential markers of seroconversion in treated chagasic patients. Prospective study, involving 203 patients treated with benznidazole, was conducted from endemic areas of northern Argentina. Follow-up was possible in 107 out of them and blood samples were taken for serology and PCR assays before and 2, 3, 6, 12, 24 and 36 months after treatment initiation. Reactivity against Trypanosoma cruzi lysate and recombinant antigens was measured by ELISA. The rate of decrease of antibody titers showed nonlinear kinetics with an abrupt drop within the first three months after initiation of treatment for all studied antigens, followed by a plateau displaying a low decay until the end of follow-up. At this point, anti-B13, anti-1F8 and anti-JL7 titers were relatively close to the cut-off line, while anti-T. cruzi antibodies still remained positive. At baseline, 60.8% (45/74) of analysed patients tested positive for parasite DNA by PCR and during the follow-up period in 34 out of 45 positive samples (75.5%) could not be detected T. cruzi DNA. Our results suggest that these antigens might be useful as early markers for monitoring antiparasitic treatment in chronic Chagas disease.

Factores asociados a insulinemia en población general adolescente.

Objectives: To identify factors that correlate with insulin values and to examine its independent associations among adolescents. Methods: A cross-sectional population-based study was conducted among adolescents aged 12-16,9 years old. A multi-stage stratified cluster random sampling method was employed. Anthropometric measurements and nutritional survey were performed, and fasting blood samples for insulin were obtained. Statistics: Multiple lineal regression. Results: 379 adolescents were included. Mean age was 14.08 ± 1.30 years. Factors associated with higher fasting insulin levels were puberty [ 4.55 (95% IC 0.42-8.69)], abdominal obesity [ 6.11 (95% IC 3.93-8.29)] and to be born small for gestational age (SGA) [ 7.45 (95% IC 2.47-12.44)]. It was observed a negative association between the regular intake of olive oil at home and insulin values [ -4.14 (95% IC -7.31- -0.98)]. Conclusions: Abdominal obesity and SGA were factors associated with higher fasting insulin values. In contrast, the regular intake of olive oil at home was an independent protective factor.

Evaluation of two HbA1c point-of-care analyzers.

BACKGROUND Measurement of HbA1c is the most important parameter to assess glycemic control in diabetic patients. Different point-of-care devices for HbA1c are available. The aim of this study was to evaluate two point-of-care testing (POCT) analyzers (DCA Vantage from Siemens and Afinion from Axis-Shield). We studied the bias and precision as well as interference from carbamylated hemoglobin. METHODS Bias of the POCT analyzers was obtained by measuring 53 blood samples from diabetic patients with a wide range of HbA1c, 4%-14% (20-130 mmol/mol), and comparing the results with those obtained by the laboratory method: HPLC HA 8160 Menarini. Precision was performed by 20 successive determinations of two samples with low 4.2% (22 mmol/mol) and high 9.5% (80 mmol/mol) HbA1c values. The possible interference from carbamylated hemoglobin was studied using 25 samples from patients with chronic renal failure. RESULTS The means of the differences between measurements performed by each POCT analyzer and the laboratory method (95% confidence interval) were: 0.28% (p<0.005) (0.10-0.44) for DCA and 0.27% (p<0.001) (0.19-0.35) for Afinion. Correlation coefficients were: r=0.973 for DCA, and r=0.991 for Afinion. The mean bias observed by using samples from chronic renal failure patients were 0.2 (range -0.4, 0.4) for DCA and 0.2 (-0.2, 0.5) for Afinion. Imprecision results were: CV=3.1% (high HbA1c) and 2.97% (low HbA1c) for DCA, CV=1.95% (high HbA1c) and 2.66% (low HbA1c) for Afinion. CONCLUSIONS Both POCT analyzers for HbA1c show good correlation with the laboratory method and acceptable precision.

Immune responses to JC virus in patients with multiple sclerosis treated with natalizumab: a cross-sectional and longitudinal study.

BACKGROUND: Natalizumab is used to prevent relapses and progression of disability in patients with multiple sclerosis but has been associated with progressive multifocal leukoencephalopathy (PML). We aimed to better understand the associations between JC virus, which causes PML, and natalizumab treatment. METHODS: We prospectively assessed patients with multiple sclerosis who started treatment with natalizumab. Blood and urine samples were tested for the presence of JC virus DNA with quantitative real-time PCR before treatment and at regular intervals after treatment onset for up to 18 months. At the same timepoints, by use of proliferation and enzyme-linked immunospot assays, the cellular immune responses against JC virus, Epstein-Barr virus, cytomegalovirus, myelin oligodendrocyte glycoprotein, and myelin oligodendrocyte basic protein (MOBP) were assessed. Humoral immune response specific to JC virus was assessed with an enzyme immunoassay. The same experiments were done on blood samples from patients with multiple sclerosis before and 10 months after the start of interferon beta treatment. FINDINGS: We assessed 24 patients with multiple sclerosis who received natalizumab and 16 who received interferon beta. In patients treated with natalizumab, JC virus DNA was not detected in the blood at any timepoint. However, JC virus DNA was present in the urine of six patients and in most of these patients the concentrations of JC virus DNA were stable over time. Compared with pretreatment values, the cellular immune response was increased to cytomegalovirus at 6 months, to JC virus at 1, 9, and 12 months, and to Epstein-Barr virus and MOBP at 12 months. Humoral responses remained stable. There were no increases in cellular immune responses specific to the viruses or myelin proteins in the 16 patients treated with interferon beta. INTERPRETATION: Natalizumab increases cellular immune responses specific to viruses and myelin proteins in the peripheral blood after 1 year, without evidence of viral reactivation. FUNDING: Swiss National Foundation, Swiss Society for Multiple Sclerosis, and Biogen Dompé.

Protocol for the modeling the epidemiologic transition study: a longitudinal observational study of energy balance and change in body weight, diabetes and cardiovascular disease risk.

ABSTRACT: BACKGROUND: The prevalence of obesity has increased in societies of all socio-cultural backgrounds. To date, guidelines set forward to prevent obesity have universally emphasized optimal levels of physical activity. However there are few empirical data to support the assertion that low levels of energy expenditure in activity is a causal factor in the current obesity epidemic are very limited. METHODS: The Modeling the Epidemiologic Transition Study (METS) is a cohort study designed to assess the association between physical activity levels and relative weight, weight gain and diabetes and cardiovascular disease risk in five population-based samples at different stages of economic development. Twenty-five hundred young adults, ages 25-45, were enrolled in the study; 500 from sites in Ghana, South Africa, Seychelles, Jamaica and the United States. At baseline, physical activity levels were assessed using accelerometry and a questionnaire in all participants and by doubly labeled water in a subsample of 75 per site. We assessed dietary intake using two separate 24-h recalls, body composition using bioelectrical impedance analysis, and health history, social and economic indicators by questionnaire. Blood pressure was measured and blood samples collected for measurement of lipids, glucose, insulin and adipokines. Full examination including physical activity using accelerometry, anthropometric data and fasting glucose will take place at 12 and 24 months. The distribution of the main variables and the associations between physical activity, independent of energy intake, glucose metabolism and anthropometric measures will be assessed using cross-section and longitudinal analysis within and between sites. DISCUSSION: METS will provide insight on the relative contribution of physical activity and diet to excess weight, age-related weight gain and incident glucose impairment in five populations' samples of young adults at different stages of economic development. These data should be useful for the development of empirically-based public health policy aimed at the prevention of obesity and associated chronic diseases.

Circulating insulin-like growth factor-I in pregnancy and maternal risk of breast cancer.

BACKGROUND: Elevated serum concentrations of insulin-like growth factor (IGF)-1 have been associated with increased risk of breast cancer. Previously, we reported a similar association in samples obtained during pregnancy. The current study was conducted to further characterize the association of IGF-1 during pregnancy with maternal breast cancer risk. METHODS: A case-control study was nested within the Finnish Maternity Cohort. The study was limited to primiparous women less than 40 years of age, who donated blood samples during early (median, 12 weeks) pregnancy and delivered a single child at term. Seven hundred and nineteen women with invasive breast cancer were eligible. Two controls (n = 1,434) were matched to each case on age and date at blood donation. Serum IGF-1 concentration was measured using an Immulite 2000 analyzer. Conditional logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI).RESULTS: No significant associations were observed between serum IGF-1 concentrations and breast cancer risk in both the overall analysis (OR 1.08 (95% CI 0.80-1.47) and in analyses stratified by histological subtype, lag-time to cancer diagnosis, age at pregnancy or age at diagnosis.CONCLUSIONS: There was no association between IGF-1 and maternal breast cancer risk during early pregnancy in this large nested case-control study.Impact:Serum IGF-1 concentrations during early pregnancy may not be related to maternal risk of breast cancer.

Quantitative sequence analysis of FBN1 premature termination codons provides evidence for incomplete NMD in leukocytes.

We improved, evaluated, and used Sanger sequencing for quantification of single nucleotide polymorphism (SNP) variants in transcripts and gDNA samples. This improved assay resulted in highly reproducible relative allele frequencies (e.g., for a heterozygous gDNA 50.0+/-1.4%, and for a missense mutation-bearing transcript 46.9+/-3.7%) with a lower detection limit of 3-9%. It provided excellent accuracy and linear correlation between expected and observed relative allele frequencies. This sequencing assay, which can also be used for the quantification of copy number variations (CNVs), methylations, mosaicisms, and DNA pools, enabled us to analyze transcripts of the FBN1 gene in fibroblasts and blood samples of patients with suspected Marfan syndrome not only qualitatively but also quantitatively. We report a total of 18 novel and 19 known FBN1 sequence variants leading to a premature termination codon (PTC), 26 of which we analyzed by quantitative sequencing both at gDNA and cDNA levels. The relative amounts of PTC-containing FBN1 transcripts in fresh and PAXgene-stabilized blood samples were significantly higher (33.0+/-3.9% to 80.0+/-7.2%) than those detected in affected fibroblasts with inhibition of nonsense-mediated mRNA decay (NMD) (11.0+/-2.1% to 25.0+/-1.8%), whereas in fibroblasts without NMD inhibition no mutant alleles could be detected. These results provide evidence for incomplete NMD in leukocytes and have particular importance for RNA-based analyses not only in FBN1 but also in other genes.

Development of a cost-effective method for platelet-rich plasma (PRP) preparation for topical wound healing.

Platelet-rich plasma (PRP) is a volume of plasma fraction of autologous blood having platelet concentrations above baseline whole-blood values due to processing and concentration. PRP is used in various surgical fields to enhance soft-tissue and bone healing by delivering supra-physiological concentrations of autologous platelets at the site of tissue damage. These preparations may provide a good cellular source of various growth factors and cytokines, and modulate tissue response to injury. Common clinically available materials for blood preparations combined with a two-step centrifugation protocol at 280g each, to ensure cellular component integrity, provided platelet preparations which were concentrated 2-3 fold over total blood values. Costs were shown to be lower than those of other methods which require specific equipment and high-cost disposables, while safety and traceability can be increased. PRP can be used for the treatment of wounds of all types including burns and also of split-thickness skin graft donor sites, which are frequently used in burn management. The procedure can be standardized and is easy to adapt in clinical settings with minimal infrastructure, thus enabling large numbers of patients to benefit from a form of cellular therapy.

Increased EBV Reactivation in the Cerebrospinal Fluid of Patients with Early Multiple Sclerosis

Epstein-Barr virus (EBV) has been consistently associated with multiple sclerosis (MS), but whether this virus is a trigger of MS remains undetermined. Recently, EBV-infected B cells recognized by activated CD8_ T cells have been detected in the meninges of autopsied MS patients. In addition, a strong EBV-specific CD8_ T cell response in the blood of patients with MS of recent onset was reported. Here, to further explore the putative relationship between MS and EBV, we assessed the EBV-specific cellular and humoral immune responses in the blood and the cerebrospinal fluid (CSF) of patients with early MS or other neurological diseases, separated into inflammatory (IOND) and non-inflammatory (NIOND) groups. The MS non-associated neurotropic herpesvirus cytomegalovirus (CMV) served as a control. Fifty-eight study subjects were enrolled, including 44 patients (13 with early MS (onset of MS less than one year prior to the assay), 15 with IOND and 16 with NIOND) in the immunological arm of the study. The cellular immune response was investigated using a functional CFSE cytotoxic T lymphocyte (CTL) assay performed with short-term cultured EBV- or CMVspecific effector T cells from the CSF and the blood. The humoral immune response specific for these two viruses was also examined in both the blood and the CSF. The recruitment of a given virusspecific antibody in the CSF as compared to the blood was expressed as antibody indexes (AI). We found that, in the CSF of early MS patients, there was an enrichment in EBV-, but not CMV-specific, CD8_ CTL as compared to the CSF of IOND (P_ 0.003) and NIOND patients (P_0.0009), as well as compared to paired blood samples (P_0.005). Additionally, relative viral capsid antigen (VCA)-, but not EBV encoded nuclear antigen 1 (EBNA1)- or CMV-specific, AI were increased in the CSF of early MS as compared to IOND (P_0.002) or NIOND patients (P_0.008) and correlated with the EBVspecific CD8_ CTL responses in the CSF (rs_0.54, P_0.001). Fourteen additional patients were enrolled in the virological arm of the study: using semi-nested PCR, EBV-encoded nuclear RNA1 (EBER1)-a transcript expressed during all stages of EBV infection-was detected in the CSF of 2/4 early MS, but only 1/6 IOND and 0/4 NIOND patients. Altogether, our data suggest that a reactivation of EBV, but not CMV, is taking place in the central nervous system of patients with MS of recent onset. These data significantly strengthen the link between EBV and MS and may indicate a triggering role of EBV in this disease. This work was supported by grants from the Swiss National Foundation and from the Swiss Society for Multiple Sclerosis.

Combined impact of negative lifestyle factors on cardiovascular risk in children: a randomized prospective study.

PURPOSE: Negative lifestyle factors are known to be associated with increased cardiovascular risk (CVR) in children, but research on their combined impact on a general population of children is sparse. Therefore, we aimed to quantify the combined impact of easily assessable negative lifestyle factors on the CVR scores of randomly selected children after 4 years. METHODS: Of the 540 randomly selected 6- to 13-year-old children, 502 children participated in a baseline health assessment, and 64% were assessed again after 4 years. Measures included anthropometry, fasting blood samples, and a health assessment questionnaire. Participants scored one point for each negative lifestyle factor at baseline: overweight; physical inactivity; high media consumption; little outdoor time; skipping breakfast; and having a parent who has ever smoked, is inactive, or overweight. A CVR score at follow-up was constructed by averaging sex- and age-related z-scores of waist circumference, blood pressure, glucose, inverted high-density lipoprotein, and triglycerides. RESULTS: The age-, sex-, pubertal stage-, and social class-adjusted probabilities (95% confidence interval) for being in the highest CVR score tertile at follow-up for children who had at most one (n = 48), two (n = 64), three (n = 56), four (n = 41), or five or more (n = 14) risky lifestyle factors were 15.4% (8.9-25.3), 24.3% (17.4-32.8), 36.0% (28.6-44.2), 49.8% (38.6-61.0), and 63.5% (47.2-77.2), respectively. CONCLUSIONS: Even in childhood, an accumulation of negative lifestyle factors is associated with higher CVR scores after 4 years. These negative lifestyle factors are easy to assess in clinical practice and allow early detection and prevention of CVR in childhood.

Positive EtG findings in hair as a result of a cosmetic treatment.

In a case of a driving ability assessment, hair analysis for ethyl glucuronide (EtG) was requested by the authorities. The person concerned denied alcohol consumption and did not present any clinical sign of alcoholism. However, EtG was found in concentrations of up to 910pg/mg in hair from different sampling dates suggesting an excessive drinking behavior. The person declared to use a hair lotion on a regularly base. To evaluate a possible effect of the hair lotion, prospective blood and urine controls as well as hair sampling of scalp and pubic hair were performed. The traditional clinical biomarkers of ethanol consumption, CDT and GGT, were inconspicuous in three blood samples taken. EtG was not detected in all collected urine samples. The hair lotion was transmitted to our laboratory. The ethanol concentration in this lotion was determined with 35g/L. The EtG immunoassay gave a positive result indicating EtG, which could be confirmed by GC-MS/MS-NCI. In a follow-up experiment the lotion was applied to the hair of a volunteer over a period of six weeks. After this treatment, EtG could be measured in the hair at a concentration of 72pg/mg suggesting chronic and excessive alcohol consumption. Overnight incubation of EtG free hair in the lotion yielded an EtG concentration of 140pg/mg. In the present case, the positive EtG hair findings could be interpreted as the result of an EtG containing hair care product. To our knowledge, the existence of such a product has not yet been reported, and it is exceptionally unusual to find EtG in cosmetics. Therefore, external sources for hair contamination should always be taken into account when unusual cosmetic treatment is mentioned. In those cases, it is recommended to analyze the hair product for a possible contamination with EtG. The analysis of body hair can help to reveal problems occurring from cosmetic treatment of head hair. As a consequence, the assessment of drinking behavior should be based on more than one diagnostic parameter.

Innate immune response to poxvirus vectors

Summary : A large body of evidence indicates that the innate immune system plays a key role in host response to viral infection. Recently, Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), and NOD-like receptor receptors (NLRs) have emerged as key innate immune sensors of microbial products, eliciting intracellular signaling and leading to the production of chemokines, cytokines and interferons (IFNs) that shape innate immune responses and coordinate the development of adaptive immunity. Poxviruses are currently developed as vaccines vectors for infectious diseases such as HIV, tuberculosis and malaria. Modified vaccinia virus Ankara (MVA) and New York vaccinia virus (NWAC) are attenuated, replication deficient strains of poxvirus. The mechanisms underlying innate immune responses to MVA and NYVAC are poorly characterized. Thus, the objectives of the project were to determine the innate immune profile stimulated by poxviruses in innate immune cells and to evaluate the impact of modifications in the viral genome on MVA and NYVAC immunogenicity. MVA stimulated the production of abundant amounts of chemokines and IFNß but low levels of cytokines by human macrophages. In contrast, NYVAC weakly stimulated the production of all mediators. Interestingly, MVA and NYVAC strongly stimulated innate immune responses in vivo and in human whole blood, suggesting that a soluble factors}, possibly a complement component, was required for optimal activation of innate immune cells by poxviruses. Modified MVA and NYVAC produced by single or multiple deletions of viral genes targeting crucial pathways of host innate immunity, and mutant poxviruses with limited replication capacity, increased the production of pro-inflammatory molecules by human whole blood. Gene expression profiling in human macrophages confirmed the increased immunologic stimulatory capacity of modified poxviruses. The pathways activated by MVA and NYVAC in innate immune cells were described by analysing the response of knockdown or shRNA transduced macrophages with impaired expression of TLRs and their adaptors (MyD8$ and TRIF), RLRs (RIG-I, MDA-5 and the adaptor IPS-1) and the NALP3 inflammasome composed óf the NLR NALP3, caspase-1 and ASC. These experiments revealed a critical role for TLR2-TLR6-MyD88 in the production of tFNß-independent chemokines and of MDA-5-IPS-1 in the production of IFNß and IFNßdependent chemokines. The transcription of the iL1b gene encoding for the IL-1ß cytokine was initiated through TLR2-MyD88, whereas the maturation and the secretion of IL-1ß were controlled by the NALP3 inflammasome. Finally, we analyzed the role of macrophage migration inhibitory factor (MIF), a mediator of inflammation and innate immune responses, in MVA infection. We observed that MVA infection increased MIF production by innate immune cells and that MIF deficiency impaired macrophage and dendritic cell responses (ie migration, maturation, cytokine and IFN production) to MVA infection in vitro and in vivo. Moreover, MIF-deficiency resulted in delayed anti-MVA specific antibody production in mice immunized with the virus. In conclusion, we demonstrate. that poxviruses can be modified genetically to improve their immunogenicity. We also report the first comprehensive analysis of poxvirus sensing by innate immune cells, showing that the TLR, RLR and NLR pathways play specific and coordinated roles in regulating cytokine, chemokine and IFN response to poxvirus infection. Finally, we show that MIF is an integral host component involved in innate and adaptive immune responses to MVA infection. The present findings provide important information relevant to the study of the pathogenesis of poxvirus infections and allow a better understanding of the immunogenic potential of vaccine vectors, which is required for the development of optimized modìfied pox-vaccine vectors.

Evaluation of oxidative stress parameters and metabolic activities of nurses working day and night shifts

The aim of this study was to evaluate the oxidative stress and metabolic activities of nurses working day and night shifts. Intensive care unit (ICU) (n=70) and ordinary service (OS) nurses (n=70) were enrolled in the study. Just before and the end of the shifts, blood samples were obtained to measure the participants' oxidative stress parameters. Metabolic activities were analyzed using the SenseWear Armband. Oxidative stress parameters were increased at the end of the shifts for all OS and ICU nurses compared to the beginning of the shifts. Compared to the OS nurses, the ICU nurses' TAS, TOS, and OSI levels were not significantly different at the end of the day and night shifts. The metabolic activities of the OS and ICU nurses were found to be similar. As a result, the OS and ICU nurses' oxidative stress parameters and metabolic activities were not different, and all of the nurses experienced similar effects from both the day and night shifts.

Polymorphisms of the " macrophage migration inhibitory factor " gene in infectious diseases

Summary The proinflammatory cytokine macrophage migration inhibitory factor (MIF) has emerged as a central mediator of inflammation and innate immune defense against infections. MIF has been shown to play an important role in the pathogenesis of infectious diseases like sepsis, tuberculosis and autoimmune inflammatory diseases, such as arthritis, inflammatory bowel disease and asthma. Two functional polymorphisms of the MIF gene promoter, a five to eight CATT repeat microsatellite at position -794 and a G/C SNP at position -173, have been associated with increased susceptibility to or severity of autoimmune inflammatory diseases like arthritis, colitis and atopy. The aim of this thesis was to define whether, and if so by which mechanisms, MIF gene polymorphisms influence the susceptibility to or the outcome of one of the most severe and one of the most prevalent infectious diseases: meningococcal sepsis and tuberculosis, respectively. The results of the comparison between 1106 patients suffering from severe meningococcal infections and 434 healthy volunteers showed that carriers of the CATT5-5 genotype were protected from meningococcemia. A transmission disequilibrium test involving 106 families confirmed this association. At baseline and after stimulation with Neisseria meningitidis, the CATT5 MIF promoter drove lower transcriptional activity than the CATT6 or CATT7 alleles in human monocytic cells and whole blood of CATT5-5 healthy individuals tended to produce less MIF than whole blood of CATT6-6 individuals. Beyond, we describe several new MIF gene polymorphisms in Africans. Genotyping the CATT microsatellite and the -173*G/C SNP revealed great genetic diversity in six African ethnic groups. Comparing 471 African tuberculosis cases and 932 matched healthy controls, we observed ethnicity dependent associations of the -173*G/C and the CATT5-8 with susceptibility to or severity of tuberculosis, but confirmation in larger cohorts ìs needed. In conclusion, we report that homozygous carriage of a low expression allele of the MIF gene protects from meningococcal disease. These results support the concept that analyses of MIF genotypes in patients with sepsis may help to classify patients into risk categories and to identify those patients who may benefit from anti-MIF therapeutic strategies.

Activity assays and immunoassays for plasma Renin and prorenin: information provided and precautions necessary for accurate measurement.

BACKGROUND: Measurement of plasma renin is important for the clinical assessment of hypertensive patients. The most common methods for measuring plasma renin are the plasma renin activity (PRA) assay and the renin immunoassay. The clinical application of renin inhibitor therapy has thrown into focus the differences in information provided by activity assays and immunoassays for renin and prorenin measurement and has drawn attention to the need for precautions to ensure their accurate measurement. CONTENT: Renin activity assays and immunoassays provide related but different information. Whereas activity assays measure only active renin, immunoassays measure both active and inhibited renin. Particular care must be taken in the collection and processing of blood samples and in the performance of these assays to avoid errors in renin measurement. Both activity assays and immunoassays are susceptible to renin overestimation due to prorenin activation. In addition, activity assays performed with peptidase inhibitors may overestimate the degree of inhibition of PRA by renin inhibitor therapy. Moreover, immunoassays may overestimate the reactive increase in plasma renin concentration in response to renin inhibitor therapy, owing to the inhibitor promoting conversion of prorenin to an open conformation that is recognized by renin immunoassays. CONCLUSIONS: The successful application of renin assays to patient care requires that the clinician and the clinical chemist understand the information provided by these assays and of the precautions necessary to ensure their accuracy.