Identifying common and specific microRNAs expressed in peripheral blood mononuclear cell of type 1, type 2, and gestational diabetes mellitus patients

Abstract Background Regardless the regulatory function of microRNAs (miRNA), their differential expression pattern has been used to define miRNA signatures and to disclose disease biomarkers. To address the question of whether patients presenting the different types of diabetes mellitus could be distinguished on the basis of their miRNA and mRNA expression profiling, we obtained peripheral blood mononuclear cell (PBMC) RNAs from 7 type 1 (T1D), 7 type 2 (T2D), and 6 gestational diabetes (GDM) patients, which were hybridized to Agilent miRNA and mRNA microarrays. Data quantification and quality control were obtained using the Feature Extraction software, and data distribution was normalized using quantile function implemented in the Aroma light package. Differentially expressed miRNAs/mRNAs were identified using Rank products, comparing T1DxGDM, T2DxGDM and T1DxT2D. Hierarchical clustering was performed using the average linkage criterion with Pearson uncentered distance as metrics. Results The use of the same microarrays platform permitted the identification of sets of shared or specific miRNAs/mRNA interaction for each type of diabetes. Nine miRNAs (hsa-miR-126, hsa-miR-1307, hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-144, hsa-miR-199a-5p, hsa-miR-27a, hsa-miR-29b, and hsa-miR-342-3p) were shared among T1D, T2D and GDM, and additional specific miRNAs were identified for T1D (20 miRNAs), T2D (14) and GDM (19) patients. ROC curves allowed the identification of specific and relevant (greater AUC values) miRNAs for each type of diabetes, including: i) hsa-miR-1274a, hsa-miR-1274b and hsa-let-7f for T1D; ii) hsa-miR-222, hsa-miR-30e and hsa-miR-140-3p for T2D, and iii) hsa-miR-181a and hsa-miR-1268 for GDM. Many of these miRNAs targeted mRNAs associated with diabetes pathogenesis. Conclusions These results indicate that PBMC can be used as reporter cells to characterize the miRNA expression profiling disclosed by the different diabetes mellitus manifestations. Shared miRNAs may characterize diabetes as a metabolic and inflammatory disorder, whereas specific miRNAs may represent biological markers for each type of diabetes, deserving further attention.

Influence of HLA-DRB-1 alleles on the production of antibody against CSP, MSP-1, AMA-1, and DBP in Brazilian individuals naturally infected with Plasmodium vivax

We evaluated the influence of allelic frequency of the human leukocyte antigen (HLA) -DRB1 on the acquisition of antibody response against malaria sporozoite and merozoite peptides in patients with Plasmodium vivax malaria acquired in endemic areas of Brazil. IgG antibodies were detected by enzyme-linked immunosorbent assay against four peptides of circumsporozoite protein (CSP) (amino, carboxyl, and VK210 and VK247 repeats) and peptides of merozoite surface protein 1 (MSP-1), apical membrane antigen 1 (AMA-1), and Duffy-binding protein (DBP). We found an association between HLA-DR3 and HLA-DR5 alleles and lack of antibody response to CSP amino terminal, as well as an association between HILA-DR3 and the highest antibody response to MSP1 (Pv200L). In conclusion, we suggest a potential regulatory role of the H1A-DRB1 alleles in the production of antibodies to a conserved region of P. vivax CSP and MSP1 in Brazilian population exposed to malaria. (C) 2011 Elsevier B.V. All rights reserved.

Autoimmune regulator (AIRE) contributes to Dectin-1-induced TNF-alpha production and complexes with caspase recruitment domain-containing protein 9 (CARD9), spleen tyrosine kinase (Syk), and Dectin-1

Background: Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome is a complex immunologic disease caused by mutation of the autoimmune regulator (AIRE) gene. Autoimmunity in patients with APECED syndrome has been shown to result from deficiency of AIRE function in transcriptional regulation of thymic peripheral tissue antigens, which leads to defective T-cell negative selection. Candidal susceptibility in patients with APECED syndrome is thought to result from aberrant adaptive immunity. Objective: To determine whether AIRE could function in anticandidal innate immune signaling, we investigated an extrathymic role for AIRE in the immune recognition of beta-glucan through the Dectin-1 pathway, which is required for defense against Candida species. Methods: Innate immune signaling through the Dectin-1 pathway was assessed in both PBMCs from patients with APECED syndrome and a monocytic cell line. Subcellular localization of AIRE was assessed by using confocal microscopy. Results: PBMCs from patients with APECED syndrome had reduced TNF-alpha responses after Dectin-1 ligation but in part used a Raf-1-mediated pathway to preserve function. In the THP-1 human monocytic cell line, reducing AIRE expression resulted in significantly decreased TNF-a release after Dectin-1 ligation. AIRE formed a transient complex with the known Dectin-1 pathway components phosphorylated spleen tyrosine kinase and caspase recruitment domain-containing protein 9 after receptor ligation and localized with Dectin-1 at the cell membrane. Conclusion: AIRE can participate in the Dectin-1 signaling pathway, indicating a novel extrathymic role for AIRE and a defect that likely contributes to fungal susceptibility in patients with APECED syndrome. (J Allergy Clin Immunol 2012;129:464-72.)

Endostatin gene therapy stimulates upregulation of ICAM-1 and VCAM-1 in a metastatic renal cell carcinoma model

One of the greatest challenges in urological oncology is renal cell carcinoma (RCC), which is the third leading cause of death in genitourinary cancers. RCCs are highly vascularized and respond positively to antiangiogenic therapy. Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. In this study, we examined the potential of ES-based antiangiogenic therapy to activate tumor-associated endothelial cells in metastatic RCC (mRCC). Balb/c-bearing Renca cells were treated with NIH/3T3-LendSN or, as a control, with NIH/3T3-LXSN cells. The T-cell subsets and lymphocyte populations of tumors, mediastinal lymph nodes and the spleen were assessed by flow cytometry. The expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was assessed by real-time PCR, flow cytometry and immunohistochemistry analysis. ES gene therapy led to an increase in the percentage of infiltrating CD4-interferon (IFN)-gamma cells (P<0.05), CD8-IFN-gamma cells (P<0.01) and CD49b-tumor necrosis factor-alpha cells (P<0.01). In addition, ES therapy caused an increase at the mRNA level of ICAM-1 (1.4-fold; P<0.01) and VCAM-1 (1.5-fold) (control vs treated group; P<0.001). Through flow cytometry, we found a significant increase in the CD34/ICAM-1 cells (8.1-fold; P<0.001) and CD34/VCAM-1 cells (1.6-fold; P<0.05). ES gene therapy induced a significant increase in both T CD4 and CD8 cells in the lymph nodes and the spleen, suggesting that ES therapy may facilitate cell survival or clonal expansion. CD49b cells were also present in increased quantities in all of these organs. In this study, we demonstrate an antitumor inflammatory effect of ES in an mRCC model, and this effect is mediated by an increase in ICAM-1 and VCAM-1 expression in tumor-associated endothelial cells.

Maternal LAMP/p55gagHIV-1 DNA Immunization Induces In Utero Priming and a Long-Lasting Immune Response in Vaccinated Neonates

Infants born to HIV-infected mothers are at high risk of becoming infected during gestation or the breastfeeding period. A search is thus warranted for vaccine formulations that will prevent mother-to-child HIV transmission. The LAMP/gag DNA chimeric vaccine encodes the HIV-1 p55gag fused to the lysosome-associated membrane protein-1 (LAMP-1) and has been shown to enhance anti-Gag antibody (Ab) and cellular immune responses in adult and neonatal mice; such a vaccine represents a new concept in antigen presentation. In this study, we evaluated the effect of LAMP/gag DNA immunization on neonates either before conception or during pregnancy. LAMP/gag immunization of BALB/c mice before conception by the intradermal route led to the transfer of anti-Gag IgG1 Ab through the placenta and via breastfeeding. Furthermore, there were an increased percentage of CD4+ CD25+ Foxp3+ T cells in the spleens of neonates. When offspring were immunized with LAMP/gag DNA, the anti-Gag Ab response and the Gag-specific IFN-gamma-secreting cells were decreased. Inhibition of anti-Gag Ab production and cellular responses were not observed six months after immunization, indicating that maternal immunization did not interfere with the long-lasting memory response in offspring. Injection of purified IgG in conjunction with LAMP/gag DNA immunization decreased humoral and cytotoxic T-cell responses. LAMP/gag DNA immunization by intradermal injection prior to conception promoted the transfer of Ab, leading to a diminished response to Gag without interfering with the development of anti-Gag T- and B-cell memory. Finally, we assessed responses after one intravenous injection of LAMP/gag DNA during the last five days of pregnancy. The intravenous injection led to in utero immunization. In conclusion, DNA vaccine enconding LAMP-1 with Gag and other HIV-1 antigens should be considered in the development of a protective vaccine for the maternal/fetal and newborn periods.

Purification and characterization of a surfactin-like molecule produced by Bacillus sp. H2O-1 and its antagonistic effect against sulfate reducing bacteria

Abstract Background Bacillus sp. H2O-1, isolated from the connate water of a Brazilian reservoir, produces an antimicrobial substance (denoted as AMS H2O-1) that is active against sulfate reducing bacteria, which are the major bacterial group responsible for biogenic souring and biocorrosion in petroleum reservoirs. Thus, the use of AMS H2O-1 for sulfate reducing bacteria control in the petroleum industry is a promising alternative to chemical biocides. However, prior to the large-scale production of AMS H2O-1 for industrial applications, its chemical structure must be elucidated. This study also analyzed the changes in the wetting properties of different surfaces conditioned with AMS H2O-1 and demonstrated the effect of AMS H2O-1 on sulfate reducing bacteria cells. Results A lipopeptide mixture from AMS H2O-1 was partially purified on a silica gel column and identified via mass spectrometry (ESI-MS). It comprises four major components that range in size from 1007 to 1049 Da. The lipid moiety contains linear and branched β-hydroxy fatty acids that range in length from C13 to C16. The peptide moiety contains seven amino acids identified as Glu-Leu-Leu-Val-Asp-Leu-Leu. Transmission electron microscopy revealed cell membrane alteration of sulfate reducing bacteria after AMS H2O-1 treatment at the minimum inhibitory concentration (5 μg/ml). Cytoplasmic electron dense inclusions were observed in treated cells but not in untreated cells. AMS H2O-1 enhanced the osmosis of sulfate reducing bacteria cells and caused the leakage of the intracellular contents. In addition, contact angle measurements indicated that different surfaces conditioned by AMS H2O-1 were less hydrophobic and more electron-donor than untreated surfaces. Conclusion AMS H2O-1 is a mixture of four surfactin-like homologues, and its biocidal activity and surfactant properties suggest that this compound may be a good candidate for sulfate reducing bacteria control. Thus, it is a potential alternative to the chemical biocides or surface coating agents currently used to prevent SRB growth in petroleum industries.

Expression of the P2X(2) receptor in different classes of ileum myenteric neurons in the female obese ob/ob mouse

AIM: To examine whether the ob/ob mouse model of obesity is accompanied by enteric nervous system abnormalities such as altered motility. METHODS: The study examined the distribution of the P2X(2) receptor (P2X(2)R) in myenteric neurons of female ob/ob mice. Specifically, we used immunohistochemistry to analyze the co-expression of the P2X(2)R with neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), and calretinin (CalR) in neurons of the small intestine myenteric plexus in ob/ob and control female mice. In these sections, we used scanning confocal microscopy to analyze the co-localization of these markers as well as the neuronal density (cm(2)) and area profile (mu m(2)) of P2X(2)R-positive neurons. In addition, enteric neurons were labeled using the nicotinamide adenine dinucleotide (NADH) diaphorase method and analyzed with light microscopy as an alternate means by which to analyze neuronal density and area. RESULTS: In the present study, we observed a 29.6% increase in the body weight of the ob/ob animals (OG) compared to the control group (CG). In addition, the average small intestine area was increased by approximately 29.6% in the OG compared to the CG. Immunoreactivity (IR) for the P2X(2)R, nNOS, ChAT and CaIR was detectable in the myenteric plexus, as well as in the smooth muscle, in both groups. This IR appeared to be mainly cytoplasmic and was also associated with the cell membrane of the myenteric plexus neurons, where it outlined the neuronal cell bodies and their processes. P2X(2)R-IR was observed to co-localize 100% with that for nNOS, ChAT and CaIR in neurons of both groups. In the ob/ob group, however, we observed that the neuronal density (neuron/cm(2)) of P2X(2)R-IR cells was increased by 62% compared to CG, while that of NOS-IR and ChAT-IR neurons was reduced by 49% and 57%, respectively, compared to control mice. The neuronal density of CaIR-IR neurons was not different between the groups. Morphometric studies further demonstrated that the cell body profile area (mu m(2)) of nNOS-IR, ChAT-IR and CaIR-IR neurons was increased by 34%, 20% and 55%, respectively, in the OG compared to controls. Staining for NADH diaphorase activity is widely used to detect alterations in the enteric nervous system; however, our qualitative examination of NADH-diaphorase positive neurons in the nnyenteric ganglia revealed an overall similarity between the two groups. CONCLUSION: We demonstrate increases in P2X(2)R expression and alterations in nNOS, ChAT and CaIR IR in ileal myenteric neurons of female ob/ob mice compared to wild-type controls. (c) 2012 Baishideng. All rights reserved.

Interaction of chitosan and mucin in a biomembrane model environment

Chitosans have been widely exploited in biological applications, including drug delivery and tissue engineering, especially owing to their mucoadhesive properties, but the molecular-level mechanisms for the chitosan action are not known in detail. It is believed that chitosan could affect the mucus by interacting with the proteins mucins, in a process mediated by the cell membrane. In this study we used Langmuir monolayers of dimyristoylphosphatidic acid (DMPA) as simplified membrane models to investigate the interplay between the activity of mucins and chitosan. Surface pressure and surface potential measurements were performed with DMPA monolayers onto which chitosan and/or mucin was adsorbed. We found that the expanding effect from mucin was considerably reduced when chitosan was injected after mucin had been adsorbed on the DMPA monolayer. The results were consistent with the formation of complexes between mucin and chitosan, thus highlighting the importance of electrostatic interactions. Furthermore, chitosan could remove mucin that was co-deposited along with DMPA in Langmuir-Blodgett (LB) films, which could be ascribed to molecular-level interactions between chitosan and mucin inferred from the FTIR spectra of the LB films. In conclusion, the results with Langmuir and LB films suggest that electrostatic interactions are crucial for the mucoadhesive mechanism, which is affected by the complexation between chitosan and mucin. (C) 2012 Elsevier Inc. All rights reserved.

Purification and characterization of Hb 98-114: A novel hemoglobin-derived antimicrobial peptide from the midgut of Rhipicephalus (Boophilus) microplus

The antimicrobial activity of hemoglobin fragments (hemocidins) has been reported in a variety of models. The cattle tick Rhipicephalus (Boophilus) microplus is a blood sucking arthropod from where the first in vivo-generated hemocidin was characterized (Hb 33-61). In the present work we identified a novel antimicrobial peptide from the midgut of fully engorged R. (B.) microplus females, which comprises the amino acids 98-114 of the alpha subunit of bovine hemoglobin, and was designated Hb 98-114. This peptide was active against several yeast and filamentous fungi, although no activity was detected against bacteria up to 50 mu M of the synthetic peptide. Hb 98-114 was capable of permeabilizing Candida albicans cell membrane and had a fungicidal effect against this yeast. Circulardichroism (CD) and nuclear magnetic resonance (NMR) experiments showed that Hb 98-114 has a random conformation in aqueous solution but switches to an alpha-helical conformation in the presence of sodium dodecyl sulfate (SDS). This alpha helix adopts an amphipathic structure which may be the mechanism of cell membrane permeabilization. Importantly, Hb 98-114 may play an important role in defending the tick midgut against fungal pathogens and is the first hemocidin with specific antifungal activity to be characterized. (C) 2012 Elsevier Inc. All rights reserved.

Actions of the Kunitz-type serine protease inhibitor Amblyomin-X on VEGF-A-induced angiogenesis

Amblyomin-X is a Kunitz-type serine protease inhibitor (Kunitz-type SPI) designed from the cDNA library of the Amblyomma cajennense tick, which displays in vivo anti-tumor activities. Here, the mechanisms of actions of Amblyomin-X in vascular endothelial growth factor A (VEGF-A)-induced angiogenesis were characterized. Topical application of Amblyomin-X (10 or 100 ng/10 mu l; each 48 h) inhibited VEGF-A-induced (10 ng/10 mu l; each 48 h) angiogenesis in the dorsal subcutaneous tissue in male Swiss mice. Moreover, similar effect was observed in the VEGF-A-induced angiogenesis in the chicken chorioallantoic membrane (CAM). Additional in vitro assays in t-End cells showed that Amblyomin-X treatment delayed the cell cycle, by maintaining them in G0/G1 phase, and inhibited cell proliferation and adhesion, tube formation and membrane expression of the adhesion molecule platelet-endothelial cell adhesion molecule-1 (PECAM-I), regardless of mRNA synthesis. Together, results herein reveal the role of Kunitz-type SPI on in vivo VEGF-A-induced angiogenesis, by exerting modulatory actions on endothelial cell proliferation and adhesion, especially on membrane expression of PECAM-1. These data provide further mechanisms of actions of Kunitz-type SPI, corroborating their relevance as scientific tools in the design of therapeutic molecules. (C) 2012 Elsevier Ltd. All rights reserved.

Properties and secretory mechanism of Musca domestica digestive chymotrypsin and its relation with Drosophila melanogaster homologs

Musca domestica larvae present two different digestive chymotryptic activities found in the posterior midgut (PMG): one major soluble activity in the lumen and another minor present in cell membrane fractions. Both soluble and membrane-bound chymotryptic activities have different half lives of thermal inactivation (46 degrees C) in the presence and absence of 10 mM Triton X-100, indicating that they are two different molecular species. Purified soluble chymotryptic activity has pH optimum 7.4 and a molecular mass of 28 kDa in SDS-PAGE. It does not cleave short substrates, such as Suc-F-MCA, preferring longer substrates, such as Suc-AAPF-MCA, with a primary specificity (kcat/Km) for Phe rather than Tyr and Leu residues. In-gel activity revealed a unique band against S-AAPF-MCA with the same migration as purified chymotrypsin. One chymotrypsinogen-like sequence (MdChy1) was sequenced, cloned and recombinantly expressed in Escherichia coli (DE3) Star. MdChy1 is expressed in the proximal posterior midgut (PMG1), as seen by RT-PCR. Expression analysis of other chymotrypsin genes revealed genes expressed at the anterior midgut (AMG) and PMG. Western blot of M. domestica midgut tissues using anti-MdChy1 antiserum showed a single band in samples from AMG and PMG, co-migrating with recombinant and purified enzymes. Immunogold labeling corresponding to Mdchy1 was found in small vesicles (thus indicating exocytosis) and in the lumen of AMG and PMG, corroborating the existence of two similar groups of chymotrypsins. Transcriptomes of M. domestica AMG and whole midgut prepared by pyrosequencing disclosed 41 unique sequences of chymotrypsin-like enzymes (19 probably functional), from which MdChy1 is highly expressed. Phylogenetic reconstruction of Drosophila melanogaster and M. domestica chymotrypsin-like sequences revealed that the chymotrypsin genes expanded before the evolutionary separation of Musca and Drosophila. (C) 2012 Elsevier Ltd. All rights reserved.

Anticancer Effects of Synthetic Phosphoethanolamine on Ehrlich Ascites Tumor: An Experimental Study

Background: Antineoplastic phospholipids (ALPs) represent a promising class of drugs with a novel mode of action undergoes rapid turnover in the cell membrane of tumors, interfering with lipid signal transduction, inducing cell death. The aim of this study was to investigate the synthetic phosphoethanolamine (Pho-s) as a new anticancer agent. Materials and Methods: Cell viability and morphology were assessed by (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Hoechst and rhodamine staining. Apoptosis was assessed by Annexin V and propidium iodide (PI) staining, caspase-3 activity, mitochondrial membrane potential (Delta m psi) and cell cycle analysis, combined with evaluation of tumor growth in Ehrlich Ascites Tumor (EAT) bearing mice. Results: We found that Pho-s 2.30 mg/ml induced cytotoxicity in all tumor cell lines studied without affecting normal cells. In vitro studies with EAT cells indicated that Pho-s induced apoptosis, demonstrated by an increase in Annexin-V positive cells, loss of mitochondrial potential (Delta m psi) and increased caspase-3 activity. It was also shown to increase the sub-G(1) apoptotic fraction and inhibit progression to the S phase of the cell cycle. Additionally, antitumor effects on the EAT-bearing mice showed that Pho-s, at a concentration of 35 and 70 mg/kg, inhibited tumor growth and increased the lifespan of animals without causing liver toxicity. Conclusion: These findings suggest that Pho-s is a potential anticancer candidate drug.

Cu(GlyGlyHis) effects on MCF7 cells: Copper uptake, reactive oxygen species generation and membrane topography changes

Elevated levels of copper have been detected in various types of human cancer cells, such as breast cancer cells, and a number of mechanisms have been proposed to explain the action and influence of copper on tumor progress. In this work, we found that stimulating the proliferation of mammary epithelial MCF7 cells with the high-redox-potential copper complex Cu (GlyGlyHis) is associated with the copper-induced intracellular generation of reactive oxygen species (ROS) that induces lipid peroxidation and causes increased roughness of external cell membranes, which leads to the formation of larger cell domes. The results presented herein provide new insights into the molecular link between copper and the proliferation of breast cancer cells and, consequently, into the mechanism by which changes in redox balance and ROS accumulation regulates cell membrane roughness. (C) 2012 Elsevier Inc. All rights reserved.

Adhesion molecules of detrusor muscle cells are influenced by a hypercholesterolemic diet or bladder outlet obstruction in a wistar rat model

Abstract Background Cell adhesion molecules (CAMs) are essential for maintaining tissue integrity by regulating intercellular and cell to extracellular matrix interactions. Cadherins and catenins are CAMs that are located on the cell membrane and are important for adherens junction (AJ) function. This study aims to verify if hypercholesterolemic diet (HCD) or bladder outlet obstruction (BOO) promotes structural bladder wall modifications specific to alterations in the expression of cadherins and catenins in detrusor muscle cells. Methods Forty-five 4-week-old female Wistar rats were divided into the following three groups: group 1 was a control group that was fed a normal diet (ND); group 2 was the BOO model and was fed a ND; and group 3 was a control group that was fed a HCD (1.25% cholesterol). Initially, serum cholesterol, LDL cholesterol and body weight were determined. Four weeks later, groups 1 and 3 underwent a sham operation; whereas group 2 underwent a partial BOO procedure that included a suture tied around the urethra. Six weeks later, all rats had their bladders removed, and previous exams were repeated. The expression levels of N-, P-, and E-cadherin, cadherin-11 and alpha-, beta- and gamma-catenins were evaluated by immunohistochemistry with a semiquantitative analysis. Results Wistar rats fed a HCD (group 3) exhibited a significant increase in LDL cholesterol levels (p=0.041) and body weight (p=0.017) when compared to both groups that were fed a normal diet in a ten-week period. We found higher β- and γ-catenin expression in groups 2 and 3 when compared to group 1 (p = 0.042 and p = 0.044, respectively). We also observed Cadherin-11 overexpression in group 3 when compared to groups 1 and 2 (p = 0.002). Conclusions A HCD in Wistar rats promoted, in addition to higher body weight gain and increased serum LDL cholesterol levels, overexpression of β- and γ-catenin in the detrusor muscle cells. Similar finding was observed in the BOO group. Higher Cadherin-11 expression was observed only in the HCD-treated rats. These findings may be associated with bladder dysfunctions that occur under such situations.