miRNA analysis of prostate cancer by quantitative real time PCR: Comparison between formalin-fixed paraffin embedded and fresh-frozen tissue

Objective: Micro RNA (miRNA) is a class of small noncoding RNA that plays a major role in the regulation of gene expression, which has been related to cancer behavior. The possibility of analyzing miRNA from the archives of pathology laboratories is exciting, as it allows for large retrospective studies. Formalin is the most common fixative used in the surgical pathology routine, and its promotion of nucleic acid degradation is well known. Our aim is to compare miRNA profiles from formalin-fixed paraffin embedded (FFPE) tissues with fresh-frozen prostate cancer tissues. Methods: The expression of 14 miRNAs was determined by quantitative real time polymerase chain reaction (qRT-PCR) in 5 paired fresh-frozen and FFPE tissues, which were representative of prostate carcinoma. Results: There was a very good correlation of the miRNA expression of miR-let7c and miR-32 between the fresh-frozen and FFPE tissues, with Pearson`s correlation coefficients of 0.927 (P = 0.023) and 0.960 (P = 0.010), respectively. For the remaining miRNAs, the correlation was good with Spearman correlation coefficient of 0.638 (P < 0.001). Conclusion: Analysis of miRNAs from routinely processed and stored FFPE prostate tissue is feasible for some miRNAs using qRT-PCR. Further studies should be conducted to confirm the reliability of using stock tissues for miRNA expression determination. (C) 2011 Elsevier Inc. All rights reserved.

International epidemiology of human pre-existing adenovirus (Ad) type-5, type-6, type-26 and type-36 neutralizing antibodies: Correlates of high Ad5 titers and implications for potential HIV vaccine trials

Replication-defective adenoviruses have been utilized as candidate HIV vaccine vectors Few studies have described the international epidemiology of pre-existing immunity to adenoviruses We enrolled 1904 participants in a cross-sectional serological survey at seven sites in Africa, Brazil, and Thailand to assess neutralizing antibodies (NA) for adenovirus types Ad5, Ad6, Ad26 and Ad36 Clinical trial samples were used to assess NA titers from the US and Europe The proportions of participants that were negative were 14 8%(Ad5), 31 5%(Ad6),41 2%(Ad26) and 53.6% (Ad36) Adenovirus NA titers varied by geographic location and were higher in non-US and non-European settings, especially Thailand In multivariate logistic regression analysis, geographic setting (non-US and non-European settings) was statistically significantly associated with having higher Ad5 titers, participants from Thailand had the highest odds of having high Ad5 titers (adjusted OR = 3 53,95% CI 224,557) Regardless of location. titers of Ad5NA were the highest and Ad36 NA were the lowest Coincident Ad5/6 titers were lower than either Ad5 or Ad6 titers alone Understanding pre-existing immunity to candidate vaccine vectors may contribute to the evaluation of vaccines in international populations (C) 2009 Published by Elsevier Ltd

Adipose tissue mesenchymal stem cell expansion in animal serum-free medium supplemented with autologous human platelet lysate

BACKGROUND: Mesenchymal stem cells (MSCs) have been considered for human regenerative therapy applications, and safe culture and expansion protocols are needed especially in the context of interspecies contamination. Human platelet lysate (PL) has been proposed as animal serum substitute during in vitro MSC expansion. In this work, a simplified and efficient method to obtain autologous PL to replace animal serum in cell culture applications is described. STUDY DESIGN AND METHODS: PL obtained by freezing and centrifugation procedures was tested as medium supplement for human adipose mesenchymal stem cell (hASC) culture. Differential proliferation, immunophenotypic changes, and differentiation under PL or fetal bovine serum (FBS) were assessed. RESULTS: In contrast to 10% FBS supplementation, cell population doubling time was significantly lower when hASCs were cultured with the same concentration of PL ( PL 22.9 +/- 1.5 hr vs. FBS 106.7 +/- 6.5 hr, t test, p < 0.05). Furthermore, hASCs maintained with 2.5% PL supplementation also showed satisfactory results. Immunophenotypic analysis revealed no differences between hASCs cultivated with PL or FBS supplementation and both cultures retained the potential to differentiate into adipose cells. These results demonstrate that autologous PL obtained from the same donor can be used as animal serum substitute in hASC culture. CONCLUSIONS: Taken together, evidence is provided that platelets provided by a single donor are sufficient to obtain PL for hASC propagation for clinical-scale applications mitigating the potential untoward side effects associated with the use of animal-derived reagents.

Sleep-related changes in hemodynamic and autonomic regulation in human hypertension

Objectives The present study investigates the hemodynamic and autonomic regulation during sleep-awake transitions and across different sleep cycles in patients with essential hypertension. Methods Nineteen individuals free of sleep apnea (10 normotensive and nine hypertensive matched for age, sex, and body mass index) underwent a standard polysomnography, with simultaneous electrocardiography and beat-to-beat blood pressure monitoring (Portapres). All measurements were determined while awake (before and after sleep), as well as in the beginning and at end of the sleep cycle (first/last cycle of nonrapid and rapid eye movement stages). Results Systolic blood pressure was higher in hypertensives and exhibited a similar reduction to the normotensives ones in initial nonrapid eye movement sleep. This reduction was because of different mechanisms: a significant fall in cardiac output in normotensives, whereas in hypertensives was also dependent of a decrease in peripheral vascular resistance. Hypertensive patients presented lower heart rate variation and attenuated baroreflex sensitivity during sleep but not immediately before and after sleep. Spectral analysis suggested a higher sympathetic activity in the sleep stages in hypertension. Additionally, a progressive sympathetic predominance (final rapid eye movement> initial rapid eye movement and awake period postsleep> awake period presleep) was observed in both groups. Conclusion Hypertension is associated with depressed baroreflex sensitivity and increased sympathetic activation during sleep. The greater sympathetic predominance at the end of night (preceding the morning surge of sympathetic activity) could be implicated in the occurrence of cardiovascular events. J Hypertens 27: 1655-1663 (C) 2009 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

Benefit of antiretroviral therapy on survival of human immunodeficiency virus-infected patients admitted to an intensive care unit

Objective: To evaluate the impact of antiretroviral therapy (ART) and the prognostic factors for in-intensive care unit (ICU) and 6-month mortality in human immunodeficiency virus (HIV)-infected patients. Design: A retrospective cohort study was conducted in patients admitted to the ICU from 1996 through 2006. The follow-up period extended for 6 months after ICU admission. Setting: The ICU of a tertiary-care teaching hospital at the Universidade de Sao Paulo, Brazil. Participants: A total of 278 HIV-infected patients admitted to the ICU were selected. We excluded ICU readmissions (37), ICU admissions who stayed less than 24 hours (44), and patients with unavailable medical charts (36). Outcome Measure: In-ICU and 6-month mortality. Main Results: Multivariate logistic regression analysis and Cox proportional hazards models demonstrated that the variables associated with in-ICU and 6-month mortality were sepsis as the cause of admission (odds ratio [OR] = 3.16 [95% confidence interval [CI] 1.65-6.06]); hazards ratio [HR] = 1.37 [95% Cl 1.01-1.88)), an Acute Physiology and Chronic Health Evaluation 11 score >19 [OR = 2.81 (95% CI 1.57-5.04); HR = 2.18 (95% CI 1.62-2.94)], mechanical ventilation during the first 24 hours [OR = 3.92 (95% CI 2.20-6.96); HR = 2.25 (95% CI 1.65-3.07)], and year of ICU admission [OR = 0.90 (95% CI 0.81-0.99); HR = 0.92 [95% CI 0.87-0.97)]. CD4 T-cell count <50 cells/mm(3) Was only associated with ICU mortality [OR = 2.10 (95% Cl 1.17-3.76)]. The use of ART in the ICU was negatively predictive of 6-month mortality in the Cox model [HR = 0.50 (95% CI 0.35-0.71)], especially if this therapy was introduced during the first 4 days of admission to the ICU [HR = 0.58 (95% CI 0.41-0.83)]. Regarding HIV-infected patients admitted to ICU without using ART, those who have started this treatment during ICU, stay presented a better prognosis when time and potential confounding factors were adjusted for [HR 0.55 (95% CI 0.31-0.98)]. Conclusions: The ICU outcome of HIV-infected patients seems to be dependent not only on acute illness severity, but also on the administration of antiretroviral treatment. (Crit Care Med 2009; 37: 1605-1611)

Genetic Analysis of Age-at-Onset for Cardiovascular Risk Factors in a Brazilian Family Study

Background/Aims: Statistical analysis of age-at-onset involving family data is particularly complicated because there is a correlation pattern that needs to be modeled and also because there are measurements that are censored. In this paper, our main purpose was to evaluate the effect of genetic and shared family environmental factors on age-at-onset of three cardiovascular risk factors: hypertension, diabetes and high cholesterol. Methods: The mixed-effects Cox model proposed by Pankratz et al. [2005] was used to analyze the data from 81 families, involving 1,675 individuals from the village of Baependi, in the state of Minas Gerais, Brazil. Results: The analyses performed showed that the polygenic effect plays a greater role than the shared family environmental effect in explaining the variability of the age-at-onset of hypertension, diabetes and high cholesterol. The model which simultaneously evaluated both effects indicated that there are individuals which may have risk of hypertension due to polygenic effects 130% higher than the overall average risk for the entire sample. For diabetes and high cholesterol the risks of some individuals were 115 and 45%, respectively, higher than the overall average risk for the entire population. Conclusions: Results showed evidence of significant polygenic effects indicating that age-at-onset is a useful trait for gene mapping of the common complex diseases analyzed. In addition, we found that the polygenic random component might absorb the effects of some covariates usually considered in the risk evaluation, such as gender, age and BMI. Copyright (C) 2008 S. Karger AG, Basel

Human visceral leishmaniasis expresses Th1 pattern in situ liver lesions

The architectural and infiltrate pattern of liver human visceral leishmaniasis (HVL) have been systematically classified as typical, fibrogenic or nodular. Despite this histopathological classification, the immune response based on cytokines and cellular phenotypes have never been performed. The aim of this study was to determine the immunophenotypic pattern and cytokine profile of the nodular involvement of the Liver in HVL. We evaluated nine cases of the nodular form of HVL. In situ immune response was studied through cytokine analysis and immunohistochemical study for phenotype markers: IL-1, IL-4, IL-1 0, TNF-alpha, IFN-gamma, CD4+ T cells, CD8+ T cells, CD20, CD68, CD57 and macrophage activation was determined by evaluation of iNOS activity. HVL seems to be related to a better immune response. Amastigotes were rarely found on liver sections. Leishmania antigen expression was also rare and located in the inflammatory nodules. The lower expression of IL-4 and IL-10, moderate expression of TNF-alpha and IFN-gamma demonstrate a panorama of Th1 phenotype. The increased expression of NK cells could help in sustaining this model of response. This pattern of immune response is probably responsible for improvement in the parasite`s clearance from liver tissue and it is a prognostic marker of human visceral leishmaniasis. (C) 2008 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Arginine methylation analysis of the splicing-associated SR protein SFRS9/SRP30C

The human SFRS9/SRp30c belongs to the SR family of splicing regulators. Despite evidence that members of this protein family may be targeted by arginine methylation, this has yet to be experimentally addressed. In this study, we found that SFRS9 is a target for PRMT1-mediated arginine methylation in vitro, and that it is immunoprecipitated from HEK-293 lysates by antibodies that recognize both mono- and dimethylated arginines. We further observed that upon treatment with the methylation inhibitor Adox, the fluorescent EGFP-SFRS9 re-localizes to dot-like structures in the cell nucleus. In subsequent confocal analyses, we found that EGFP-SFRS9 localizes to nucleoli in Adox-treated cells. Our findings indicate the importance of arginine methylation for the subnuclear localization of SFRS9.

Molecular cloning, sequencing, and expression analysis of presenilin cDNA from Schistosoma mansoni

Presenilins (PS) are integral membrane proteins involved, among other functions, in regulated intramembrane proteolysis. In this study, we report the identification and characterization of a complementary DNA from Schistosoma mansoni exhibiting a significant homology to human and nonvertebrate presinilins. S. mansoni contained a 1,485 bp open reading frame encoding a predicted protein of 494 amino acids. Alignment of predicted amino acid sequence of S. mansoni with PS (SmPS) from other species revealed up to 40% similarity shared among the investigated organisms. In addition, phylogenetic analyses demonstrated SmPS being closely related to its orthologues found in Schistosoma japonicum and Caenorhabditis elegans. Expression analysis of SmPS using quantitative real-time PCR revealed that the transcript is up-regulated in the egg stage. We hypothesize that the high level of SmPS in the S. mansoni embryo correlates to an important role during cellular signaling associated to larval development. To our knowledge, this study represents the first attempt to investigate the existence and abundance of PS from a helminth parasite.

Functional association of human Ki-1/57 with pre-mRNA splicing events

The cytoplasmic and nuclear protein Ki- 1 / 57 was first identified in malignant cells from Hodgkin`s lymphoma. Despite studies showing its phosphorylation, arginine methylation, and interaction with several regulatory proteins, the functional role of Ki- 1 / 57 in human cells remains to be determined. Here, we investigated the relationship of Ki- 1 / 57 with RNA functions. Through immunoprecipitation assays, we verified the association of Ki- 1 / 57 with the endogenous splicing proteins hnRNPQ and SFRS9 in HeLa cell extracts. We also found that recombinant Ki- 1 / 57 was able to bind to a poly- U RNA probe in electrophoretic mobility shift assays. In a classic splicing test, we showed that Ki- 1 / 57 can modify the splicing site selection of the adenoviral E1A minigene in a dose- dependent manner. Further confocal and. uorescence microscopy analysis revealed the localization of enhanced green. uorescent protein - Ki- 1 / 57 to nuclear bodies involved in RNA processing and or small nuclear ribonucleoprotein assembly, depending on the cellular methylation status and its N- terminal region. In summary, our findings suggest that Ki- 1 / 57 is probably involved in cellular events related to RNA functions, such as pre- mRNA splicing.

Analysis of Detergent-Insoluble and Whole Cell Lysate Fractions of Resting Neutrophils Using High-Resolution Mass Spectrometry

Neutrophilic granulocytes play a major role in the initiation and resolution of the inflammatory response, and demonstrate significant transcriptional and translational activity. Although much was known about neutrophils prior to the introduction of proteomics, the use of MS-based methodologies has provided an unprecedented tool to confirm and extend previous findings. In the present study, we performed a Gel-LC-MS/MS analysis of neutrophil detergent insoluble and whole cell lysate fractions of resting neutrophils. We achieved a set of identifications through the use of high-resolution mass spectrometry and validation of its data. We identified a total of 1249 proteins with a wide range of intensities from both detergent-insoluble and whole cell lysate fractions, allowing a mapping of proteins such as those involved in intracellular transport (Rab and Sec family proteins) and cell signaling (S100 proteins). These results represent the most comprehensive proteomic characterization of resting human neutrophils to date, and provide important information relevant for further studies of the immune system in health and disease. The methods applied here can be employed to help us understand how neutrophils respond to various physiologic and pathophysiologic conditions and could be extended to protein quantitation after cell activation.

Gene expression profile analysis of primary glioblastomas and non-neoplastic brain tissue: identification of potential target genes by oligonucleotide microarray and real-time quantitative PCR

The prognosis of glioblastomas is still extremely poor and the discovery of novel molecular therapeutic targets can be important to optimize treatment strategies. Gene expression analyses comparing normal and neoplastic tissues have been used to identify genes associated with tumorigenesis and potential therapeutic targets. We have used this approach to identify differentially expressed genes between primary glioblastomas and non-neoplastic brain tissues. We selected 20 overexpressed genes related to cell cycle, cellular movement and growth, proliferation and cell-to-cell signaling and analyzed their expression levels by real time quantitative PCR in cDNA obtained from microdissected fresh tumor tissue from 20 patients with primary glioblastomas and from 10 samples of non-neoplastic white matter tissue. The gene expression levels were significantly higher in glioblastomas than in non-neoplastic white matter in 18 out of 20 genes analyzed: P < 0.00001 for CDKN2C, CKS2, EEF1A1, EMP3, PDPN, BNIP2, CA12, CD34, CDC42EP4, PPIE, SNAI2, GDF15 and MMP23b; and NFIA (P: 0.0001), GPS1 (P: 0.0003), LAMA1 (P: 0.002), STIM1 (P: 0.006), and TASP1 (P: 0.01). Five of these genes are located in contiguous loci at 1p31-36 and 2 at 17q24-25 and 8 of them encode surface membrane proteins. PDPN and CD34 protein expression were evaluated by immunohistochemistry and they showed concordance with the PCR results. The present results indicate the presence of 18 overexpressed genes in human primary glioblastomas that may play a significant role in the pathogenesis of these tumors and that deserve further functional investigation as attractive candidates for new therapeutic targets.

Maternal embryonic leucine zipper kinase transcript abundance correlates with malignancy grade in human astrocytomas

We have performed cDNA microarray analyses to identify gene expression differences between highly invasive glioblastoma multiforme (GBM) and typically benign pilocytic astrocytomas (PA). Despite the significant clinical and pathological differences between the 2 tumor types, only 63 genes were found to exhibit 2-fold or greater overexpression in GBM as compared to PA. Forty percent of these genes are related to the regulation of the cell cycle and mitosis. QT-PCR validation of 6 overexpressed genes: MELK, AUKB, ASPM, PRC1, IL13RA2 and KIAA0101 confirmed at least a 5-fold increase in the average expression levels in GBM. Maternal embryonic leucine zipper kinase (MELK) exhibited the most statistically significant difference. A more detailed investigation of MELK expression was undertaken to study its oncogenic relevance. In the examination of more than 100 tumors of the central nervous system, we found progressively higher expression of MELK with astrocytoma grade and a noteworthy uniformity of high level expression in GBM. Similar level of overexpression was also observed in medulloblastoma. We found neither gene promoter hypomethylation nor amplification to be a factor in MELK expression, but were able to demonstrate that MELK knockdown in malignant astrocytoma cell lines caused a reduction in proliferation and anchorage-independent growth in in vitro assays. Our results indicate that GBM and PA differ by the expression of surprisingly few genes. Among them, MELK correlated with malignancy grade in astrocytomas and represents a therapeutic target for the management of the most frequent brain tumors in adult and children. (C) 2007 Wiley-Liss, Inc.

In silico analysis of microRNAS targeting the HLA-G 3 ` untranslated region alleles and haplotypes

It has been reported that microRNAs (miRNA) may have allele-specific targeting for the 3` untranslated region (3` UTR) of the HLA-G locus. In a previous study, we reported 11 3`UTR haplotypes encompassing the 14-bp insertion/deletion polymorphism and seven SNPs (+3003 T/C, +3010 C/G, +3027 C/A, +3035 C/T, +3142 C/G, +3187A/G,and +3196 C/G), of which only the +3142 C/G SNP has been reported to influence the binding of miRNAs. Using bioinformatics analyses, we identified putative miRNA-binding sites considering the haplotypes encompassing these eight polymorphic sites, and we ranked the lowest free energies that could potentially lead to an mRNA degradation or translational repression. When a specific haplotype or a particular SNP was associated with a miRNA-binding site, we defined a free energy difference of 4 kcal/mol between alleles to classify them energetically distant. The best results were obtained for the miR-513a-5p, miR-518c*, miR-1262 and miR-92a-1*, miR-92a-2*, miR-661, miR-1224-5p, and miR-433 miRNAs, all influencing one or more of the +3003, +3010, +3027, and +3035 SNPs. The miR-2110, miR-93, miR-508-5p, miR-331-5p, miR-616, miR-513b, and miR-589* miRNAs targeted the 14-bp fragment region, and miR-148a, miR-19a*, miR-152, mir-148b,and miR-218-2 also influenced the +3142C/G polymorphism. These results suggest that these miRNAs might play a relevant role on the HLA-G expression pattern. (C) 2009 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.