Poly (A)(+) Transcriptome Assessment of ERBB2-Induced Alterations in Breast Cell Lines

We report the first quantitative and qualitative analysis of the poly (A)(+) transcriptome of two human mammary cell lines, differentially expressing (human epidermal growth factor receptor) an oncogene over-expressed in approximately 25% of human breast tumors. Full-length cDNA populations from the two cell lines were digested enzymatically, individually tagged according to a customized method for library construction, and simultaneously sequenced by the use of the Titanium 454-Roche-platform. Comprehensive bioinformatics analysis followed by experimental validation confirmed novel genes, splicing variants, single nucleotide polymorphisms, and gene fusions indicated by RNA-seq data from both samples. Moreover, comparative analysis showed enrichment in alternative events, especially in the exon usage category, in ERBB2 over-expressing cells, data indicating regulation of alternative splicing mediated by the oncogene. Alterations in expression levels of genes, such as LOX, ATP5L, GALNT3, and MME revealed by large-scale sequencing were confirmed between cell lines as well as in tumor specimens with different ERBB2 backgrounds. This approach was shown to be suitable for structural, quantitative, and qualitative assessment of complex transcriptomes and revealed new events mediated by ERBB2 overexpression, in addition to potential molecular targets for breast cancer that are driven by this oncogene.

Shear Stress Induces Nitric Oxide-Mediated Vascular Endothelial Growth Factor Production in Human Adipose Tissue Mesenchymal Stem Cells

It has been demonstrated that human adipose tissue-derived mesenchymal stem cells (hASCs) enhance vascular density in ischemic tissues, suggesting that they can differentiate into vascular cells or release angiogenic factors that may stimulate neoangiogenesis. Moreover, there is evidence that shear stress (SS) may activate proliferation and differentiation of embryonic and endothelial precursor stem cells into endothelial cells (ECs). In this work, we investigated the effect of laminar SS in promoting differentiation of hASCs into ECs. SS (10 dyn/cm(2) up to 96 h), produced by a cone plate system, failed to induce EC markers (CD31, vWF, Flk-1) on hASC assayed by RT-PCR and flow cytometry. In contrast, there was a cumulative production of nitric oxide (determined by Griess Reaction) and vascular endothelial growth factor (VEGF; by ELISA) up to 96 h of SS stimulation ( NO(2)(-) in nmol/10(4) cells: static: 0.20 +/- 0.03; SS: 1.78 +/- 0.38, n = 6; VEGF in pg/10(4) cells: static: 191.31 +/- v35.29; SS: 372.80 +/- 46.74, n = 6, P < 0.05). Interestingly, the VEGF production was abrogated by 5 mM N(G)-L-nitro-arginine methyl ester (L-NAME) treatment (VEGF in pg/10(4) cells: SS: 378.80 +/- 46.74, n = 6; SS + L-NAME: 205.84 +/- 91.66, n = 4, P < 0.05). The results indicate that even though SS failed to induce EC surface markers in hASC under the tested conditions, it stimulated NO-dependent VEGF production.

Expression of eight genes of nuclear factor-kappa B pathway in multiple myeloma using bone marrow aspirates obtained at diagnosis

Purpose: To evaluate the expression of NF-kappa B pathway genes in total bone marrow samples obtained from MM at diagnosis using real-time quantitative PCR and to evaluate its possible correlation with disease clinical features and survival. Material and methods: Expression of eight genes related to NF-kappa B pathway (NFKB1, IKB, RANK, RANKL, OPG, IL6, VCAM1 and ICAM1) were studied in 53 bone marrow samples from newly diagnosed MM patients and in seven normal controls, using the Taqman system. Genes were considered overexpressed when tumor expression level was at least four times higher than that observed in normal samples. Results: The percentages of overexpression of the eight genes were: NFKB1 0%, IKB 22.6%, RANK 15.1%, RANKL 31.3%, OPG 7.5%, IL6 39.6%, VCAM1 10% and ICAM1 26%. We found association between IL6 expression level and International Staging System (ISS) (p = 0.01), meaning that MM patients with high ISS scores have more chance of overexpression of IL6. The mean value of ICAM1 relative expression was also associated with the ISS score (p = 0.02). Regarding OS, cases with IL6 overexpression present worse evolution than cases with IL6 normal expression (p = 0.04). Conclusion: We demonstrated that total bone marrow aspirates can be used as a source of material for gene expression studies in MM. In this context, we confirmed that IL6 overexpression was significantly associated with worse survival and we described that it is associated with high ISS scores. Also, ICAM1 was overexpressed in 26% of cases and its level was associated with ISS scores.

Vascular Endothelial Growth Factor Haplotypes Associated with Childhood Obesity

Expansion of adipose tissue in obesity is associated with angiogenesis and adipose tissue mass depends on neovascularization. Vascular endothelial growth factor (VEGF) is the main angiogenic factor in the adipose tissue, and VEGF expression is tightly regulated at both transcriptional and translational levels. However, no previous study has tested the hypothesis that genetic polymorphisms in the VEGF gene could affect susceptibility to obesity. To test this hypothesis, we compared the distribution of genotypes and haplotypes including three VEGF genetic polymorphisms in obese children and adolescents with those found in healthy controls. We studied 172 healthy children and adolescents and 113 obese children and adolescents. Genotypes of three clinically relevant VEGF polymorphisms in the promoter region (C-2578A, G-1154A, and G-634C) of the VEGF gene were determined by TaqMan allele discrimination assay and real-time polymerase chain reaction. VEGF haplotypes were inferred using Haplo. stats and PHASE 2.1 programs. We found no differences in the distributions of VEGF genotypes and alleles (p > 0.05). However, the CAG haplotype was more frequent in the obese group than in the control group (4% versus 0%, respectively, in white subjects; p = 0.008; odds ratio 10.148 (95% confidence interval: 1.098-93.788). Our findings suggest that VEGF haplotypes affect susceptibility to obesity in children and adolescents.

Interethnic Differences in the Distribution of Clinically Relevant Vascular Endothelial Growth Factor Genetic Polymorphisms

Vascular endothelial growth factor (VEGF) is a homodimeric glycoprotein produced mostly in endothelial cells and its transcription is regulated by a variety of growth factors and cytokines. VEGF plays many relevant roles, and three functional polymorphisms in the promoter region of the VEGF gene (C-2578A, G-1154A, and G-634C) have been associated with disease conditions. Although some studies suggest that interethnic differences exist in the distribution of these variants, no previous study has examined this hypothesis in admixed populations. We examined the distribution of these three clinically relevant VEGF single-nucleotide polymorphisms in 175 white and 185 black subjects. We have also estimated the haplotype distribution and assessed associations between these variants. Although the A-2578 and A-1154 variants were more common in whites (39% and 29%, respectively) than in blacks (29% and 16%, respectively; both p < 0.05), no significant interethnic differences were found with regards to the G-634C polymorphism. While the haplotype including the C-2578, G-1154, and G-634 variants was the most common in both ethnic groups, it was more common in blacks than in whites (p < 0.05). The haplotype including the C-2578, A-1154, and G-634 alleles and the haplotype including the C-2578, A-1154, and C-634 alleles were more common in whites than in blacks (both p < 0.05). These results show marked interethnic differences in the distribution of genetic variants of VEGF that may explain, at least in part, interethnic disparities in the susceptibility to cardiovascular diseases.

HLA-G 14-bp Insertion/Deletion Polymorphism Is a Risk Factor for HTLV-1 Infection

About 95% of HTLV-1 infected patients remain asymptomatic throughout life, and the risk factors associated with the development of related diseases, such as HAM/TSP and ATL, are not fully understood. The human leukocyte antigen-G molecule (HLA-G), a nonclassical HLA class I molecule encoded by MHC, is expressed in several pathological conditions, including viral infection, and is related to immunosuppressive effects that allow the virus-infected cells to escape the antiviral defense of the host. The 14-bp insertion/deletion polymorphism of exon 8 HLA-G gene influences the stability of the transcripts and could be related to HTLV-1-infected cell protection and to the increase of proviral load. The present study analyzed by conventional PCR the 14-bp insertion/deletion polymorphism of exon 8 HLA-G gene in 150 unrelated healthy subjects, 82 HTLV-1 infected patients with symptoms (33 ATL and 49 HAM), and 56 asymptomatic HTLV-1 infected patients (HAC). In addition, the proviral load was determined by quantitative real-time PCR in all infected groups and correlated with 14-bp insertion/deletion genotypes. The heterozygote genotype frequencies were significantly higher in HAM, in the symptomatic group, and in infected patients compared to control (p < 0.05). The proviral load was higher in the symptomatic group than the HAC group (p < 0.0005). The comparison of proviral load and genotypes showed that -14-bp/-14-bp genotype had a higher proviral load than +14-bp/-14-bp and +14-bp/+14-bp genotypes. Although HLA-G 14-bp polymorphism does not appear to be associated

Vascular Endothelial Growth Factor Genetic Polymorphisms and Haplotypes in Women with Migraine

Vascular endothelial growth factor (VEGF) production is regulated by growth factors and inflammatory cytokines, and VEGF plays a role in migraine. We examined for the first time whether three functional polymorphisms in the promoter region of VEGF gene (C(-2578)A, G(-1154A), and G(-634C)) and VEGF haplotypes are associated with migraine. We studied 114 healthy women without migraine and 175 women with migraine (129 without aura, and 46 with aura). We found no differences in the distributions of VEGF genotypes and alleles (p > 0.05). However, the CAC haplotype was more frequent in controls than in migraine patients, and the AGC haplotype was more frequent in patients with migraine with aura than in controls (both p < 0.05). These findings suggest that VEGF haplotypes affect susceptibility to migraine.

Research Smad2 and Smad6 as predictors of overall survival in oral squamous cell carcinoma patients

Background: To test if the expression of Smad1-8 mRNAs were predictive of survival in patients with oral squamous cell carcinoma (SCC). Patients and Methods: We analyzed, prospectively, the expression of Smad1-8, by means of Ribonuclease Protection Assay in 48 primary, operable, oral SCC. In addition, 21 larynx, 10 oropharynx and 4 hypopharynx SCC and 65 matched adjacent mucosa, available for study, were also included. For survival analysis, patients were categorized as positive or negative for each Smad, according to median mRNA expression. We also performed real-time quantitative PCR (QRTPCR) to asses the pattern of TGF beta 1, TGF beta 2, TGF beta 3 in oral SCC. Results: Our results showed that Smad2 and Smad6 mRNA expression were both associated with survival in Oral SCC patients. Cox Multivariate analysis revealed that Smad6 positivity and Smad2 negativity were both predictive of good prognosis for oral SCC patients, independent of lymph nodal status (P = 0.003 and P = 0.029, respectively). In addition, simultaneously Smad2(-) and Smad6(+) oral SCC group of patients did not reach median overall survival (mOS) whereas the mOS of Smad2(+)/Smad6(-) subgroup was 11.6 months (P = 0.004, univariate analysis). Regarding to TGF beta isoforms, we found that Smad2 mRNA and TGF beta 1 mRNA were inversely correlated (p = 0.05, R = -0.33), and that seven of the eight TGF beta 1(+) patients were Smad2(-). In larynx SCC, Smad7(-) patients did not reach mOS whereas mOS of Smad7(+) patients were only 7.0 months (P = 0.04). No other correlations were found among Smad expression, clinico-pathological characteristics and survival in oral, larynx, hypopharynx, oropharynx or the entire head and neck SCC population. Conclusion: Smad6 together with Smad2 may be prognostic factors, independent of nodal status in oral SCC after curative resection. The underlying mechanism which involves aberrant TGF beta signaling should be better clarified in the future.

Cross talk Initiated by Endothelial Cells Enhances Migration and Inhibits Anoikis of Squamous Cell Carcinoma Cells through STAT3/Akt/ERK Signaling

It is well known that cancer cells secrete angiogenic factors to recruit and sustain tumor vascular networks. However, little is known about the effect of endothelial cell-secreted factors on the phenotype and behavior of tumor cells. The hypothesis underlying this study is that endothelial cells initiate signaling pathways that enhance tumor cell survival and migration. Here, we observed that soluble mediators from primary human dermal microvascular endothelial cells induce phosphorylation of signal transducer and activator of transcription 3 (STAT3), Akt, and extracellular signal-regulated kinase (ERK) in a panel of head and neck squamous cell carcinoma (HNSCC) cells (OSCC-3, UM-SCC-1, UM-SCC-17B, UM-SCC-74A). Gene expression analysis demonstrated that interleukin-6 (IL-6), interleukin-8 (CXCL8), and epidermal growth factor (EGF) are upregulated in endothelial cells cocultured with HNSCC. Blockade of endothelial cell-derived IL-6, CXCL8, or EGF by gene silencing or neutralizing antibodies inhibited phosphorylation of STAT3, Akt, and ERK in tumor cells, respectively. Notably, activation of STAT3, Akt, and ERK by endothelial cells enhanced migration and inhibited anoikis of tumor cells. We have previously demonstrated that Bcl-2 is upregulated in tumor microvessels in patients with HNSCC. Here, we observed that Bcl-2 signaling induces expression of IL-6, CXCL8, and EGF, providing a mechanism for the upregulation of these cytokines in tumor-associated endothelial cells. This study expands the contribution of endothelial cells to the pathobiology of tumor cells. It unveils a new mechanism in which endothelial cells function as initiators of molecular crosstalks that enhance survival and migration of tumor cells.

Human Dental Pulp Cells: A New Source of Cell Therapy in a Mouse Model of Compressive Spinal Cord Injury

Strategies aimed at improving spinal cord regeneration after trauma are still challenging neurologists and neuroscientists throughout the world. Many cell-based therapies have been tested, with limited success in terms of functional outcome. In this study, we investigated the effects of human dental pulp cells (HDPCs) in a mouse model of compressive spinal cord injury (SCI). These cells present some advantages, such as the ease of the extraction process, and expression of trophic factors and embryonic markers from both ecto-mesenchymal and mesenchymal components. Young adult female C57/BL6 mice were subjected to laminectomy at T9 and compression of the spinal cord with a vascular clip for 1 min. The cells were transplanted 7 days or 28 days after the lesion, in order to compare the recovery when treatment is applied in a subacute or chronic phase. We performed quantitative analyses of white-matter preservation, trophic-factor expression and quantification, and ultrastructural and functional analysis. Our results for the HDPC-transplanted animals showed better white-matter preservation than the DMEM groups, higher levels of trophic-factor expression in the tissue, better tissue organization, and the presence of many axons being myelinated by either Schwann cells or oligodendrocytes, in addition to the presence of some healthy-appearing intact neurons with synapse contacts on their cell bodies. We also demonstrated that HDPCs were able to express some glial markers such as GFAP and S-100. The functional analysis also showed locomotor improvement in these animals. Based on these findings, we propose that HDPCs may be feasible candidates for therapeutic intervention after SCI and central nervous system disorders in humans.

Retention of progenitor cell phenotype in otospheres from guinea pig and mouse cochlea

Background: Culturing otospheres from dissociated organ of Corti is an appropriate starting point aiming at the development of cell therapy for hair cell loss. Although guinea pigs have been widely used as an excellent experimental model for studying the biology of the inner ear, the mouse cochlea has been more suitable for yielding otospheres in vitro. The aim of this study was to compare conditions and outcomes of otosphere suspension cultures from dissociated organ of Corti of either mouse or guinea pig at postnatal day three (P3), and to evaluate the guinea pig as a potential cochlea donor for preclinical cell therapy. Methods: Organs of Corti were surgically isolated from P3 guinea pig or mouse cochlea, dissociated and cultivated under non-adherent conditions. Cultures were maintained in serum-free DMEM:F12 medium, supplemented with epidermal growth factor (EGF) plus either basic fibroblast growth factor (bFGF) or transforming growth factor alpha (TGF alpha). Immunofluorescence assays were conducted for phenotype characterization. Results: The TGF alpha group presented a number of spheres significantly higher than the bFGF group. Although mouse cultures yielded more cells per sphere than guinea pig cultures, sox2 and nestin distributed similarly in otosphere cells from both organisms. We present evidence that otospheres retain properties of inner ear progenitor cells such as self-renewal, proliferation, and differentiation into hair cells or supporting cells. Conclusions: Dissociated guinea pig cochlea produced otospheres in vitro, expressing sox2 and nestin similarly to mouse otospheres. Our data is supporting evidence for the presence of inner ear progenitor cells in the postnatal guinea pig. However, there is limited viability for these cells in neonatal guinea pig cochlea when compared to the differentiation potential observed for the mouse organ of Corti at the same developmental stage.

The Spleen CD4(+) T Cell Response to Blood-Stage Plasmodium chabaudi Malaria Develops in Two Phases Characterized by Different Properties

The pivotal role of spleen CD4(+) T cells in the development of both malaria pathogenesis and protective immunity makes necessary a profound comprehension of the mechanisms involved in their activation and regulation during Plasmodium infection. Herein, we examined in detail the behaviour of non-conventional and conventional splenic CD4(+) T cells during P. chabaudi malaria. We took advantage of the fact that a great proportion of CD4(+) T cells generated in CD1d(-/-) mice are I-A(b)-restricted (conventional cells), while their counterparts in I-Ab(-/-) mice are restricted by CD1d and other class IB major histocompatibility complex (MHC) molecules (non-conventional cells). We found that conventional CD4(+) T cells are the main protagonists of the immune response to infection, which develops in two consecutive phases concomitant with acute and chronic parasitaemias. The early phase of the conventional CD4(+) T cell response is intense and short lasting, rapidly providing large amounts of proinflammatory cytokines and helping follicular and marginal zone B cells to secrete polyclonal immunoglobulin. Both TNF-alpha and IFN-gamma production depend mostly on conventional CD4(+) T cells. IFN-gamma is produced simultaneously by non-conventional and conventional CD4(+) T cells. The early phase of the response finishes after a week of infection, with the elimination of a large proportion of CD4(+) T cells, which then gives opportunity to the development of acquired immunity. Unexpectedly, the major contribution of CD1d-restricted CD4(+) T cells occurs at the beginning of the second phase of the response, but not earlier, helping both IFN-gamma and parasite-specific antibody production. We concluded that conventional CD4(+) T cells have a central role from the onset of P. chabaudi malaria, acting in parallel with non-conventional CD4(+) T cells as a link between innate and acquired immunity. This study contributes to the understanding of malaria immunology and opens a perspective for future studies designed to decipher the molecular mechanisms behind immune responses to Plasmodium infection.

Impaired Innate Immunity in Tlr4(-/-) Mice but Preserved CD8(+) T Cell Responses against Trypanosoma cruzi in Tlr4-, Tlr2-, Tlr9- or Myd88-Deficient Mice

The murine model of T. cruzi infection has provided compelling evidence that development of host resistance against intracellular protozoans critically depends on the activation of members of the Toll-like receptor (TLR) family via the MyD88 adaptor molecule. However, the possibility that TLR/MyD88 signaling pathways also control the induction of immunoprotective CD8(+) T cell-mediated effector functions has not been investigated to date. We addressed this question by measuring the frequencies of IFN-gamma secreting CD8(+) T cells specific for H-2K(b)-restricted immunodominant peptides as well as the in vivo Ag-specific cytotoxic response in infected animals that are deficient either in TLR2, TLR4, TLR9 or MyD88 signaling pathways. Strikingly, we found that T. cruzi-infected Tlr2(-/-), Tlr4(-/-), Tlr9(-/-) or Myd88(-/-) mice generated both specific cytotoxic responses and IFN-gamma secreting CD8(+) T cells at levels comparable to WT mice, although the frequency of IFN-gamma(+)CD4(+) cells was diminished in infected Myd88(-/-) mice. We also analyzed the efficiency of TLR4-driven immune responses against T. cruzi using TLR4-deficient mice on the C57BL genetic background (B6 and B10). Our studies demonstrated that TLR4 signaling is required for optimal production of IFN-gamma, TNF-alpha and nitric oxide (NO) in the spleen of infected animals and, as a consequence, Tlr4(-/-) mice display higher parasitemia levels. Collectively, our results indicate that TLR4, as well as previously shown for TLR2, TLR9 and MyD88, contributes to the innate immune response and, consequently, resistance in the acute phase of infection, although each of these pathways is not individually essential for the generation of class I-restricted responses against T. cruzi.

Enteric cell proliferation in newborn lambs fed bovine and ovine colostrum

The objective of this study was to investigate immunoglobulin G (IgG) and total serum protein (TP) acquisition in newborn Santa Ines lambs fed Holstein bovine or Santa Ines ovine colostrum as well as the cell proliferation rate in the animals` intestine epithelium. At 0 h and 6 h of life, 12 newborn lambs received 250 mL of bovine 1st milking colostrum (BC) and another 12 animals received 250 mL of ovine 1st milking colostrum (OC). Blood samples were collected at 0, 6, 24. and 72 h of life. Six animals were randomly slaughtered just after birth, without colostrum intake. The other animals were randomly slaughtered at 24 and 72 h. The IgG serum concentration at 6, 24 and 72 h were significantly higher for BC, 16.32 +/- 6.19; 33.80 +/- 5.68 and 27.95 +/- 5.46 mg/mL respectively, compared with OC, 11.31 +/- 6.08, 21.02 +/- 6.53 and 19.88 +/- 7.31 mg/mL BC showed higher (P < 0.05) TP values (7.29 +/- 0.87 and 6.89 +/- 0.30 g/100 mL) at 24 and 72 h in relation to OC (5.73 +/- 1.35 and 5.69 +/- 0.57 g/100 mL). At birth, the animals showed 32.52%, 45.47% and 30.60% cells in division for the duodenum, jejunum and ileum, respectively. At 24 h, the OC animals showed lower (P < 0.0001) mitotic cell percentage in the duodenum (42.12%) and ileum (35.66%) in relation to the BC animals, 46.44% and 39.74%, respectively. At 72 h, a lower (P < 0.0001) rate of proliferation was observed in the duodenum crypts of the OC animals (36.28%) compared with BC (43.18%). The results indicate that this lacteal secretion can accelerate the epithelium renovation process and can be used as an alternative source of IgG for newborn lambs. (C) 2009 Elsevier B.V. All rights reserved.

Effects of Protein-Energy Malnutrition on NF-KappaB Signalling in Murine Peritoneal Macrophages

Protein-energy malnutrition (PEM) is an important public health problem affecting millions of people worldwide. PEM decreases resistance to infection, impairing a number of physiological processes. In unstimulated cells, NF-kappa B is kept from binding to its consensus sequence by the inhibitor I kappa B alpha, which retains NF-kappa B in the cytoplasm. Upon various signals, such as lipopolysaccharide (LPS), I kappa B alpha is rapidly degraded and NF-kappa B is induced to translocate into the nucleus, where it activates expression of various genes that participate in the inflammatory response, including those involved in the synthesis of TNF-alpha. TRAF-6 is a cytoplasmic adapter protein that links the stimulatory signal from Toll like receptor-4 to NF-kappa B. The aim of this study was to evaluate the effect of malnutrition on induction of TNF-a by LPS in murine peritoneal macrophages. We evaluated peritoneal cellularity, the expression of MyD88, TRAF-6, IKK, I kappa B alpha and NF-kappa B, NF-kappa B activation and TNF-alpha mRNA and protein synthesis inmacrophages. Two-month-old male BALB/Cmice were submitted to PEM with a low-protein diet that contained 2% protein, compared to 12% protein in the control diet. When the experimental group had lost about 20% of the original body weight, it was used in the subsequent experiments. Malnourished animals presented anemia, leucopenia and severe reduction in peritoneal cavity cellularity. TNF-a mRNA and protein levels of macrophages stimulated with LPS were significantly lower in malnourished animals. PEM also decreased TRAF-6 expression and NF-kappa B activation after LPS stimulation. These results led us to conclude that PEM changes NF-kappa B signalling pathway in macrophages to LPS stimulus.