Correlation of meta 1 expression with culture stage, cell morphology and infectivity in Leishmania (Leishmania) amazonensis promastigotes

The parasitic protozoan Leishmania (Leishmania) amazonensis alternates between mammalian and insect hosts. In the insect host, the parasites proliferate as procyclic promastigotes andthen differentiate into metacyclic infective forms. The meta 1 gene is preferentially expressed during metacyclogenesis. Meta 1 expression profile determination along parasite growth curves revealed that the meta 1 mRNA level peaked at the early stationary phase then decreased to an intermediate level. No correlation was observed between meta 1 expression and infectivity. Conversely, infectivity correlated with the increase of apoptotic cells in the late stationary phase.

Evaluation of two long synthetic merozoite surface protein 2 peptides as malaria vaccine candidates.

Merozoite surface protein 2 (MSP2) is a promising vaccine candidate against Plasmodium falciparum blood stages. A recombinant 3D7 form of MSP2 was a subunit of Combination B, a blood stage vaccine tested in the field in Papua New Guinea. A selective effect in favour of the allelic family not represented by the vaccine argued for a MSP2 vaccine consisting of both dimorphic variants. An alternative approach to recombinant manufacture of vaccines is the production of long synthetic peptides (LSP). LSP exceeding a length of well over 100 amino acids can now be routinely synthesized. Synthetic production of vaccine antigens cuts the often time-consuming steps of protein expression and purification short. This considerably reduces the time for a candidate to reach the phase of clinical trials. Here we present the evaluation of two long synthetic peptides representing both allelic families of MSP2 as potential vaccine candidates. The constructs were well recognized by human immune sera from different locations and different age groups. Furthermore, peptide-specific antibodies in human immune sera were associated with protection from clinical malaria. The synthetic fragments share major antigenic properties with native MSP2. Immunization of mice with these antigens yielded high titre antibody responses and monoclonal antibodies recognized parasite-derived MSP2. Our results justify taking these candidate poly-peptides into further vaccine development.

Gefitinib in Combination With Irradiation With or Without Cisplatin in Patients With Inoperable Stage III Non-Small Cell Lung Cancer: A Phase I Trial.

Purpose : To establish the feasibility and tolerability of gefitinib (ZD1839, Iressa) with radiation (RT) or concurrent chemoradiation (CRT) with cisplatin (CDDP) in patients with advanced non small cell lung cancer (NSCLC).Patients and Methods : In this multicenter Phase I study, 5 patients with unresectable NSCLC received 250 mg gefitinib daily starting 1 week before RT at a dose of 63 Gy (Step 1). After a first safety analysis, 9 patients were treated daily with 250 mg gefitinib plus CRT in the form of RT and weekly CDDP 35 mg/m(2) (Step 2). Gefitinib was maintained for up to 2 years until disease progression or toxicity.Results : Fourteen patients were assessed in the two steps. In Step 1 (five patients were administered only gefitinib and RT), no lung toxicities were seen, and there was no dose-limiting toxicity (DLT). Adverse events were skin and subcutaneous tissue reactions, limited to Grade 1-2. In Step 2, two of nine patients (22.2%) had DLT. One patient suffered from dyspnea and dehydration associated with neutropenic pneumonia, and another showed elevated liver enzymes. In both steps combined, 5 of 14 patients (35.7%) experienced one or more treatment interruptions.Conclusions : Gefitinib (250 mg daily) in combination with RT and CDDP in patients with Stage HI NSCLC is feasible, but CDDP likely enhances toxicity. The impact of gefitinib on survival and disease control as a first-line treatment in combination with RT remains to be determined. (C) 2011 Elsevier Inc.

Progression from high insulin resistance to type 2 diabetes does not entail additional visceral adipose tissue inflammation.

Obesity is associated with a low-grade chronic inflammation state. As a consequence, adipose tissue expresses pro-inflammatory cytokines that propagate inflammatory responses systemically elsewhere, promoting whole-body insulin resistance and consequential islet β-cell exhaustation. Thus, insulin resistance is considered the early stage of type 2 diabetes. However, there is evidence of obese individuals that never develop diabetes indicating that the mechanisms governing the association between the increase of inflammatory factors and type 2 diabetes are much more complex and deserve further investigation. We studied for the first time the differences in insulin signalling and inflammatory pathways in blood and visceral adipose tissue (VAT) of 20 lean healthy donors and 40 equal morbidly obese (MO) patients classified in high insulin resistance (high IR) degree and diabetes state. We studied the changes in proinflammatory markers and lipid content from serum; macrophage infiltration, mRNA expression of inflammatory cytokines and transcription factors, activation of kinases involved in inflammation and expression of insulin signalling molecules in VAT. VAT comparison of these experimental groups revealed that type 2 diabetic-MO subjects exhibit the same pro-inflammatory profile than the high IR-MO patients, characterized by elevated levels of IL-1β, IL-6, TNFα, JNK1/2, ERK1/2, STAT3 and NFκB. Our work rules out the assumption that the inflammation should be increased in obese people with type 2 diabetes compared to high IR obese. These findings indicate that some mechanisms, other than systemic and VAT inflammation must be involved in the development of type 2 diabetes in obesity.

The L162V polymorphism of the PPARA gene is associated with stage C of heart failure.

In human heart failure (HF) peroxisome proliferator-activated receptor alpha (PPAR alpha) is downregulated and consequently, the expression of genes involved in fatty acid oxidation repressed. The L162V (rs1800206) is a functional polymorphism of the human PPAR alpha gene (PPARA). In the present study we have investigated whether this polymorphism is associated with the development of stage C of HF.

Prevalence and Clinical Outcomes for Patients With ALK-Positive Resected Stage I to III Adenocarcinoma: Results From the European Thoracic Oncology Platform Lungscape Project.

PURPOSE: The prevalence of anaplastic lymphoma kinase (ALK) gene fusion (ALK positivity) in early-stage non-small-cell lung cancer (NSCLC) varies by population examined and detection method used. The Lungscape ALK project was designed to address the prevalence and prognostic impact of ALK positivity in resected lung adenocarcinoma in a primarily European population. METHODS: Analysis of ALK status was performed by immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) in tissue sections of 1,281 patients with adenocarcinoma in the European Thoracic Oncology Platform Lungscape iBiobank. Positive patients were matched with negative patients in a 1:2 ratio, both for IHC and for FISH testing. Testing was performed in 16 participating centers, using the same protocol after passing external quality assessment. RESULTS: Positive ALK IHC staining was present in 80 patients (prevalence of 6.2%; 95% CI, 4.9% to 7.6%). Of these, 28 patients were ALK FISH positive, corresponding to a lower bound for the prevalence of FISH positivity of 2.2%. FISH specificity was 100%, and FISH sensitivity was 35.0% (95% CI, 24.7% to 46.5%), with a sensitivity value of 81.3% (95% CI, 63.6% to 92.8%) for IHC 2+/3+ patients. The hazard of death for FISH-positive patients was lower than for IHC-negative patients (P = .022). Multivariable models, adjusted for patient, tumor, and treatment characteristics, and matched cohort analysis confirmed that ALK FISH positivity is a predictor for better overall survival (OS). CONCLUSION: In this large cohort of surgically resected lung adenocarcinomas, the prevalence of ALK positivity was 6.2% using IHC and at least 2.2% using FISH. A screening strategy based on IHC or H-score could be envisaged. ALK positivity (by either IHC or FISH) was related to better OS.

Impact of Prophylactic Cranial Irradiation Timing on Brain Relapse Rates in Patients With Stage IIIB Non-Small-Cell Lung Carcinoma Treated With Two Different Chemoradiotherapy Regimens.

PURPOSE: To retrospectively assess the influence of prophylactic cranial irradiation (PCI) timing on brain relapse rates in patients treated with two different chemoradiotherapy (CRT) regimens for Stage IIIB non-small-cell lung cancer (NSCLC). METHODS AND MATERIALS: A cohort of 134 patients, with Stage IIIB NSCLC in recursive partitioning analysis Group 1, was treated with PCI (30 Gy at 2 Gy/fr) following one of two CRT regimens. Regimen 1 (n = 58) consisted of three cycles of induction chemotherapy (ICT) followed by concurrent CRT (C-CRT). Regimen 2 (n = 76) consisted of immediate C-CRT during thoracic radiotherapy. RESULTS: At a median follow-up of 27.6 months (range, 7.2-40.4), 65 patients were alive. Median, progression-free, and brain metastasis-free survival (BMFS) times for the whole study cohort were 23.4, 15.4, and 23.0 months, respectively. Median survival time and the 3-year survival rate for regimens 1 and 2 were 19.3 vs. 26.1 months (p = 0.001) and 14.4% vs. 34.4% (p < .001), respectively. Median time from the initiation of primary treatment to PCI was 123.2 (range, 97-161) and 63.4 (range, 55-74) days for regimens 1 and 2, respectively (p < 0.001). Overall, 11 (8.2%) patients developed brain metastasis (BM) during the follow-up period: 8 (13.8%) in regimen 1 and 3 (3.9%) in regimen 2 (p = 0.03). Only 3 (2.2%) patients developed BM at the site of first failure, and for 2 of them, it was also the sole site of recurrence. Median BMFS for regimens 1 and 2 were 17.4 (13.5-21.3) vs. 26.0 (22.9-29.1 months), respectively (p < 0.001). CONCLUSION: These results suggest that in Stage IIIB NSCLC patients treated with PCI, lower BM incidence and longer survival rates result from immediate C-CRT rather than ITC-first regimens. This indicates the benefit of earlier PCI use without delay because of induction protocols.

zyg-11and cul-2 promote the metaphase to anaphase transition and M phase exit of meiosis II and prevent polarity establishment in C.elegans

Résumé Les mécanismes qui coordonnent la progression du cycle cellulaire lors de la méiose avec les événements du développement embryonnaire précoce, y compris la formation des axes de polarité embryonnaire, sont peu compris. Dans le zygote du vers Caenorhabditis elegans, les premiers signes de polarité Antéro-Postérieur (A-P) embryonnaire apparaissent après que la méiose soit terminée. La nature des protéines et des mécanismes moléculaires qui cassent la symétrie du zygote n'est pas connue. Nous démontrons que zyg-11 et cul-2 promeuvent la transition métaphase - anaphase et la sortie de la phase M lors de la seconde division méiotique. Nos résultats indiquent que ZYG-11 agit comme unité recrutant le substrat d'une ligase E3 comprennant CUL-2. Nos résultats montrent aussi que le délai de sortie de la phase M dépend de l'accumulation de la Cyclin B, CYB-3. Nous démontrons que dans des embryons zyg-11(RNAi) ou cul-2(RNAi), une polarité inversée est établie lors du délai de méiosis II. Enfin nous montrons que les défauts de cycle cellulaire et ceux de polarité peuvent être séparés. De plus, nous faisons apparaitre que l'établissement d'une polarité inversée pendant le délai de méiose II des embryons zyg-11(RNAi), comme l'établissement de la A-P polarité des embryons sauvage ne semblent pas requérir les microtubules. Nous montrons également les premiers résultats d'un crible deux hybrides ainsi qu'un crible génomique qui vise à identifier des gènes dont l'inactivation augmente ou supprime les défauts de mutants pour le gène zyg-11, afin d'identifier les gènes qui intéragissent avec ZYG-11 pour assumer ses deux fonctions séparables. Par conséquent, nos trouvailles suggèrent un modèle selon lequel ZYG-11 est une sous-unité qui recrute les substrats d'une ligase E3 basée sur CUL-2 qui promeut la progression du cycle cellulaire et empêche l'établissement de la polarité pendant la méiose II, et où le centrosome agit comme la clé qui polarise l'embryon à la fin de la méiose. Summary The mechanisms that couple meiotic cell cycle progression to subsequent developmental events, including specification of embryonic axes, are poorly understood. In the one cell stage embryos of Caenorhabditis elegans, the first signs of Antero-Posterior (A-P) polarity appear after meiosis completion. A centrosome ¬derived component breaks symmetry of the embryo, but the molecular nature of this polarity signal is not known. We established that zyg-11 and cul-2 promote the metaphase to anaphase transition and M phase exit at meiosis II. Our results indicate that ZYG-11 acts as a substrate recruitment subunit of a CUL-2-based E3 ligase. Moreover, we find that the delayed meiosis II exit of embryos lacking zyg-11 is caused by accumulation of the B-type cyclin, CYB-3. We demonstrate that inverted A-P polarity is established during the meiosis II delay in zyg-11(RNAi) and cul¬2(RNAi) embryos. Importantly, we demonstrate that the polarity defects following zyg-11 or cul-2 inactivation can be uncoupled from the cell cycle defects. Furthermore, we found that microtubules appear dispensable for inverted polarity during the meiosis II delay in zyg-11(RNAi) embryos, as well as for A-P polarity during the first mitotic cell cycle in wild-type embryos. We also show the initial results from a comprehensive yeast two hybrid, as well as an RNAi-based functional genomic enhancer and suppressor screen, that may lead to identification of proteins that interact with zyg-11 to ensure the two functions. Our findings suggest a model in which ZYG-11 is a substrate recruitment subunit of an CUL-2-based E3 ligase that promotes cell cycle progression and prevents polarity establishment during meiosis II, and in which the centrosome acts as a cue to polarize the embryo along the AP axis after exit from the meiotic cell cycle.

Strain-transcending Fc-dependent killing of Plasmodium falciparum by merozoite surface protein 2 allele-specific human antibodies.

It is widely accepted that antibody responses against the human parasitic pathogen Plasmodium falciparum protect the host from the rigors of severe malaria and death. However, there is a continuing need for the development of in vitro correlate assays of immune protection. To this end, the capacity of human monoclonal and polyclonal antibodies in eliciting phagocytosis and parasite growth inhibition via Fcγ receptor-dependent mechanisms was explored. In examining the extent to which sequence diversity in merozoite surface protein 2 (MSP2) results in the evasion of antibody responses, an unexpectedly high level of heterologous function was measured for allele-specific human antibodies. The dependence on Fcγ receptors for opsonic phagocytosis and monocyte-mediated antibody-dependent parasite inhibition was demonstrated by the mutation of the Fc domain of monoclonal antibodies against both MSP2 and a novel vaccine candidate, peptide 27 from the gene PFF0165c. The described flow cytometry-based functional assays are expected to be useful for assessing immunity in naturally infected and vaccinated individuals and for prioritizing among blood-stage antigens for inclusion in blood-stage vaccines.

A genome-wide association search for type 2 diabetes genes in African Americans.

African Americans are disproportionately affected by type 2 diabetes (T2DM) yet few studies have examined T2DM using genome-wide association approaches in this ethnicity. The aim of this study was to identify genes associated with T2DM in the African American population. We performed a Genome Wide Association Study (GWAS) using the Affymetrix 6.0 array in 965 African-American cases with T2DM and end-stage renal disease (T2DM-ESRD) and 1029 population-based controls. The most significant SNPs (n = 550 independent loci) were genotyped in a replication cohort and 122 SNPs (n = 98 independent loci) were further tested through genotyping three additional validation cohorts followed by meta-analysis in all five cohorts totaling 3,132 cases and 3,317 controls. Twelve SNPs had evidence of association in the GWAS (P<0.0071), were directionally consistent in the Replication cohort and were associated with T2DM in subjects without nephropathy (P<0.05). Meta-analysis in all cases and controls revealed a single SNP reaching genome-wide significance (P<2.5×10(-8)). SNP rs7560163 (P = 7.0×10(-9), OR (95% CI) = 0.75 (0.67-0.84)) is located intergenically between RND3 and RBM43. Four additional loci (rs7542900, rs4659485, rs2722769 and rs7107217) were associated with T2DM (P<0.05) and reached more nominal levels of significance (P<2.5×10(-5)) in the overall analysis and may represent novel loci that contribute to T2DM. We have identified novel T2DM-susceptibility variants in the African-American population. Notably, T2DM risk was associated with the major allele and implies an interesting genetic architecture in this population. These results suggest that multiple loci underlie T2DM susceptibility in the African-American population and that these loci are distinct from those identified in other ethnic populations.

The development of anemia is associated to poor prognosis in NKF/KDOQI stage 3 chronic kidney disease

BACKGROUND: Anemia is a common condition in CKD that has been identified as a cardiovascular (CV) risk factor in end-stage renal disease, constituting a predictor of low survival. The aim of this study was to define the onset of anemia of renal origin and its association with the evolution of kidney disease and clinical outcomes in stage 3 CKD (CKD-3). METHODS: This epidemiological, prospective, multicenter, 3-year study included 439 CKD-3 patients. The origin of nephropathy and comorbidity (Charlson score: 3.2) were recorded. The clinical characteristics of patients that developed anemia according to EBPG guidelines were compared with those that did not, followed by multivariate logistic regression, Kaplan-Meier curves and ROC curves to investigate factors associated with the development of renal anemia. RESULTS: During the 36-month follow-up period, 50% reached CKD-4 or 5, and approximately 35% were diagnosed with anemia (85% of renal origin). The probability of developing renal anemia was 0.12, 0.20 and 0.25 at 1, 2 and 3 years, respectively. Patients that developed anemia were mainly men (72% anemic vs. 69% non-anemic). The mean age was 68 vs. 65.5 years and baseline proteinuria was 0.94 vs. 0.62 g/24h (anemic vs. non anemic, respectively). Baseline MDRD values were 36 vs. 40 mL/min and albumin 4.1 vs. 4.3 g/dL; reduction in MDRD was greater in those that developed anemia (6.8 vs. 1.6 mL/min/1.73 m2/3 years). These patients progressed earlier to CKD-4 or 5 (18 vs. 28 months), with a higher proportion of hospitalizations (31 vs. 16%), major CV events (16 vs. 7%), and higher mortality (10 vs. 6.6%) than those without anemia. Multivariate logistic regression indicated a significant association between baseline hemoglobin (OR=0.35; 95% CI: 0.24-0.28), glomerular filtration rate (OR=0.96; 95% CI: 0.93-0.99), female (OR=0.19; 95% CI: 0.10-0.40) and the development of renal anemia. CONCLUSIONS: Renal anemia is associated with a more rapid evolution to CKD-4, and a higher risk of CV events and hospitalization in non-dialysis-dependent CKD patients. This suggests that special attention should be paid to anemic CKD-3 patients.

Validation and performance comparison of two carbon isotope ratio methods to control the misuse of androgens in humans.

Carbon isotope ratio of androgens in urine specimens is routinely determined to exclude an abuse of testosterone or testosterone prohormones by athletes. Increasing application of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) in the last years for target and systematic investigations on samples has resulted in the demand for rapid sample throughput as well as high selectivity in the extraction process particularly in the case of conspicuous samples. For that purpose, we present herein the complimentary use of an SPE-based assay and an HPLC fractionation method as a two-stage strategy for the isolation of testosterone metabolites and endogenous reference compounds prior to GC/C/IRMS analyses. Assays validation demonstrated acceptable performance in terms of intermediate precision (range: 0.1-0.4 per thousand) and Bland-Altman analyses revealed no significant bias (0.2 per thousand). For further validation of this two-stage analyses strategy, all the specimens (n=124) collected during a major sport event were processed.

Role of cyclooxygenase-2 in Trypanosoma cruzisurvival in the early stages of parasite host-cell interaction

Chagas disease, caused by the intracellular protozoan Trypanosoma cruzi, is a serious health problem in Latin America. During this parasitic infection, the heart is one of the major organs affected. The pathogenesis of tissue remodelling, particularly regarding cardiomyocyte behaviour after parasite infection and the molecular mechanisms that occur immediately following parasite entry into host cells are not yet completely understood. When cells are infected with T. cruzi, they develop an inflammatory response, in which cyclooxygenase-2 (COX-2) catalyses rate-limiting steps in the arachidonic acid pathway. However, how the parasite interaction modulates COX-2 activity is poorly understood. In this study, the H9c2 cell line was used as our model and we investigated cellular and biochemical aspects during the initial 48 h of parasitic infection. Oscillatory activity of COX-2 was observed, which correlated with the control of the pro-inflammatory environment in infected cells. Interestingly, subcellular trafficking was also verified, correlated with the control of Cox-2 mRNA or the activated COX-2 protein in cells, which is directly connected with the assemble of stress granules structures. Our collective findings suggest that in the very early stage of the T. cruzi-host cell interaction, the parasite is able to modulate the cellular metabolism in order to survives.

Extrapleural Pneumonectomy With Venous Confluence Resection for Stage IVA Thymic Tumors.

We report 4 patients with stage IVA thymic tumors who underwent extrapleural pneumonectomy and thymectomy with venous confluence resection using a temporary percutaneous venous jugular-femoral bypass technique. The superior vena cava was replaced in 2 patients, and the innominate vein was resected in 2 patients. Complete tumor resection was obtained in all patients. There was no 90-day postoperative mortality. One patient died at 6 months postoperatively of an unrelated cause, without recurrent disease, and 3 are alive and disease-free with a follow-up ranging from 19 to 80 months. Extrapleural pneumonectomy can be combined with thymectomy and venous confluence resection for stage IVA thymic tumors.

Role of Age and Sex in the Diagnosis of Early-stage Malignant Melanoma: A Cross-Sectional study.

Age and sex have been identified as predictors of outcome in malignant melanoma (MM). This aim of this multicentre, cross-sectional study was to analyse the role of age and sex as explanatory variables for the diagnosis of thin MM. A total of 2430 patients with MM were recruited. Cases of in situ-T1 MM were more frequent than T2-T4 MM (56.26% vs. 43.74%). Breslow thickness increased throughout decades of life (analysis of variance (ANOVA) p < 0.001), with a weak correlation between Breslow thickness and patient's age (r   = 0.202, p < 0.001). Breslow thickness was significantly less in women (1.79 vs. 2.38 mm, p = 0.0001). Binary logistic regression showed a significant (p < 0.001) odds ratio for age 0-29 years (1.18), and 30-59 years (1.16), and for women (1.09). Age and sex explained 3.64% of the variation observed in Tis-T1 frequency (R2 = 0.0364). Age and sex appear to explain a low percentage of the variation in the early detection of MM.