Angiogenesis and diabetes: different responses to pro-angiogenic factors in the chorioallantoic membrane assay

Hyperglycemia induces defects in angiogenesis without alteration in the expression of major vascular growth factors in the chicken chorioallantoic membrane (CAM) model. A direct negative effect of hyperglycemia on angiogenesis may participate in failures of "therapeutic angiogenesis" trials. Here, we tested the hypothesis that the response to pro-angiogenic molecules such as angiotensin-converting enzyme (ACE), endothelin-1 (ET-1), and vascular endothelial growth factor-A (VEGF) is altered by hyperglycemia. Transfected (Chinese hamster ovary [CHO] or human embryonic kidney [HEK]) cells overexpressing ACE, ET-1, or VEGF were deposed onto the CAM of hyperglycemic or control embryos. The proangiogenic effect was evaluated 3 d later by angiography and histological analyses. Gene expression in response to these factors was assessed by in situ hybridization. Only VEGF overexpression evoked a proangiogenic response in the CAM from hyperglycemic embryos, upregulating the expression of endogenous VEGF, VEGF-R2, and Tie-2, all of them related to activation of endothelial cells. In conclusion, in a model where hyperglycemia does not alter the major vascular growth factor expression, the negative effect of diabetes on capillary density was overcome only by VEGF overexpression, whereas responses to other vasoactive peptides were practically abolished under hyperglycemic conditions.     

Role of Catalase and Superoxide Dismutase Activities on Oxidative Stress in the Brain of a Phenylketonuria Animal Model and the Effect of Lipoic Acid

Phenylketonuria (PKU) is an inherited metabolic disorder caused by deficiency of phenylalanine hydroxylase which leads to accumulation of phenylalanine and its metabolites in tissues of patients with severe neurological involvement. Recently, many studies in animal models or patients have reported the role of oxidative stress in PKU. In the present work we studied the effect of lipoic acid against oxidative stress in rat brain provoked by an animal model of hyperphenylalaninemia (HPA), induced by repetitive injections of phenylalanine and α-methylphenylalanine (a phenylalanine hydroxylase inhibitor) for 7 days, on some oxidative stress parameters. Lipoic acid prevented alterations on catalase (CAT) and superoxide dismutase (SOD), and the oxidative damage of lipids, proteins, and DNA observed in HPA rats. In addition, lipoic acid diminished reactive species generation compared to HPA group which was positively correlated to SOD/CAT ratio. We also observed that in vitro Phe inhibited CAT activity while phenyllactic and phenylacetic acids stimulated superoxide dismutase activity. These results demonstrate the efficacy of lipoic acid to prevent oxidative stress induced by HPA model in rats. The possible benefits of lipoic acid administration to PKU patients should be considered.     

Morphological study for understanding the sexual pattern in ghost shrimp Callichirus major (Crustacea: Axiidea)

Callichirus major is a species of ghost shrimp that has burrowing habits, building underground galleries in the intertidal region of beaches. This study aimed to analyse some morphological parameters to verify its type of sexual pattern. A sample of 79 individuals, collected at the beach of Corujão, Espírito Santo, Brazil, was used. Data on the external morphology, presence of gonopores, carapace length (CL) and cheliped lengths (Lch) were obtained. In addition, the gonads were processed for histological analysis. The animals were divided into three categories: A, individuals with male characteristics; B, those presenting exclusively female characteristics; C, animals with characteristics of intersex, which presented supernumerary gonopores. Positive allometries were found for Lch versus CL only for category A, characterizing the group's marked heterochely. Histologically, it was possible to observe the presence of oocytes in category A and one case of male tissue in an intersex individual (category C). However, no genital ducts of both sexes were found in that individual. Callichirus major presents controversial sexual characters, which makes it difficult to understand the reproductive parameters and sexual pattern. However, maybe that species can develop a sexual system of hermaphroditism.     

Evaluation of monocyte subsets and markers of activation in leprosy reactions

Understanding host immune pathways associated with tissue damage during reactions are of upmost importance to the development of immune intervention strategies. The participation of monocytes in leprosy reactions was evaluated by determining the frequency of monocyte subsets and the degree of cellular activation through the expression of MHCII and the co-stimulatory molecules CD40, CD80, CD86. Leprosy subjects with or without reactions were included in this cross-sectional study. Peripheral blood mononuclear cell were isolated and stained ex vivo to determine monocyte subsets and the degree of cellular activation by flow cytometry. Intermediate monocytes were increased in leprosy patients with reactions when compared to patients without reactions. Although no difference was detected in the frequency of monocyte subsets between type 1 and 2 reactions, the expression of CD80 was increased in monocytes from patients with type 1 reactions and CD40 was higher in paucibacillary subjects presenting type 1 reactions. Moreover, CD86 and MHC II expression were higher in intermediate monocytes when compared to the other subsets in leprosy reaction types 1 and 2. Intermediate monocyte activation with CD86 and MHCII expression is involved with both type 1 and 2 reactions, whereas CD80 and CD40 expression is related to type 1 reactions.     

Circulating endothelial progenitor cells in kidney transplant patients

Background

Kidney transplantation (RTx) leads to amelioration of endothelial function in patients with advanced renal failure. Endothelial progenitor cells (EPCs) may play a key role in this repair process. The aim of this study was to determine the impact of RTx and immunosuppressive therapy on the number of circulating EPCs.

Methods

We analyzed 52 RTx patients (58±13 years; 33 males, mean ± SD) and 16 age- and gender-matched subjects with normal kidney function (57±17; 10 males). RTx patients received a calcineurin inhibitor (CNI)-based (65%) or a CNI-free therapy (35%) and steroids. EPC number was determined by double positive staining for CD133/VEGFR2 and CD34/VEGFR2 by flow cytometry. Stromal cell-derived factor 1 alpha (SDF-1) levels were assessed by ELISA. Experimentally, to dissociate the impact of RTx from the impact of immunosuppressants, we used the 5/6 nephrectomy model. The animals were treated with a CNI-based or a CNI-free therapy, and EPCs (Sca+cKit+) and CD26+ cells were determined by flow cytometry.

Results

Compared to controls, circulating number of CD34+/VEGFR2+ and CD133+/VEGFR2+ EPCs increased in RTx patients. There were no correlations between EPC levels and statin, erythropoietin or use of renin angiotensin system blockers in our study. Indeed, multivariate analysis showed that SDF-1 – a cytokine responsible for EPC mobilization – is independently associated with the EPC number. 5/6 rats presented decreased EPC counts in comparison to control animals. Immunosuppressive therapy was able to restore normal EPC values in 5/6 rats. These effects on EPC number were associated with reduced number of CD26+ cells, which might be related to consequent accumulation of SDF-1.

Conclusions

We conclude that kidney transplantation and its associated use of immunosuppressive drugs increases the number of circulating EPCs via the manipulation of the CD26/SDF-1 axis. Increased EPC count may be associated to endothelial repair and function in these patients.     

Comparative cytogenetics of three economically important Piper L. species from the Brazilian Amazon

The species Piper hispidinervum, Piper aduncum, and Piper affinis hispidinervum have essential oils with high levels of safrole, dillapiole, and sarisan, respectively. Safrole is important for pharmaceutical and chemical industries, while dillapiole and sarisan are promising compounds to control insects and fungi. These species are very similar morphologically and their taxonomy is controversial. Divergent hypotheses consider P. aduncum and P. hispidinervum either as a single species or as distinct taxa, while P. affinis hispidinervum is inferred to be a natural hybrid or a chemotype of P. hispidinervum. Delimiting the taxonomic boundaries would be helpful for germplasm conservation and breeding programs. This study aimed to undertake a detailed analysis of P. aduncum, P. hispidinervum, and P. affinis hispidinervum karyotype and rDNA sites. Genomic in situ hybridization (GISH) was used to establish genomic homology among species and to test the natural hybridization hypothesis for origin of P. affinis hispidinervum. Karyotype traits were similar for all three species: 2n = 26 small chromosomes, predominantly metacentric. All three species exhibited CMA+ bands on the secondary constriction of chromosome pair 4. A size-heteromorphic 35S rDNA site was co-localized with the CMA+ band. A 5S rDNA site was located in the proximal region of chromosome pair 7. The patterns of genomic hybridization revealed that the repetitive DNA fraction of the species is highly similar in terms of proportion of genome, sequence type, and distribution. Our findings did not allow us to differentiate the three species and point to the importance of deeper genomic studies to elucidate the taxonomic controversy.

     

Cell Therapy Attenuates Cardiac Dysfunction Post Myocardial Infarction: Effect of Timing,Routes of Injection and a Fibrin Scaffold

Background

Cell therapy approaches for biologic cardiac repair hold great promises, although basic fundamental issues remain poorly understood. In the present study we examined the effects of timing and routes of administration of bone marrow cells (BMC) post-myocardial infarction (MI) and the efficacy of an injectable biopolymer scaffold to improve cardiac cell retention and function.

Methodology/Principal Findings

^99mTc-labeled BMC (6×10⁶ cells) were injected by 4 different routes in adult rats: intravenous (IV), left ventricular cavity (LV), left ventricular cavity with temporal aorta occlusion (LV⁺) to mimic coronary injection, and intramyocardial (IM). The injections were performed 1, 2, 3, or 7 days post-MI and cell retention was estimated by γ-emission counting of the organs excised 24 hs after cell injection. IM injection improved cell retention and attenuated cardiac dysfunction, whereas IV, LV or LV* routes were somewhat inefficient (<1%). Cardiac BMC retention was not influenced by timing except for the IM injection that showed greater cell retention at 7 (16%) vs. 1, 2 or 3 (average of 7%) days post-MI. Cardiac cell retention was further improved by an injectable fibrin scaffold at day 3 post-MI (17 vs. 7%), even though morphometric and function parameters evaluated 4 weeks later displayed similar improvements.

Conclusions/Significance

These results show that cells injected post-MI display comparable tissue distribution profile regardless of the route of injection and that there is no time effect for cardiac cell accumulation for injections performed 1 to 3 days post-MI. As expected the IM injection is the most efficient for cardiac cell retention, it can be further improved by co-injection with a fibrin scaffold and it significantly attenuates cardiac dysfunction evaluated 4 weeks post myocardial infarction. These pharmacokinetic data obtained under similar experimental conditions are essential for further development of these novel approaches.     

Cytokines IL-1α, IL-6, and GM-CSF constitutively secreted by oral squamous carcinoma induce down-regulation of CD80 costimulatory molecule expression: restoration by interferon γ

We previously characterized the expression of CD80 in different murine head and neck squamous cell carcinoma (HNSCC) clones derived following tumor progression in the absence of T cell–mediated immunity in severe combined immunodeficient (SCID) mice. We found that HNSCCs that did not express CD80 grew as progressors, while those that expressed CD80 were regressors when grown in immune-competent animals. In the present study, we characterized expression of a repertoire of immunoregulatory cytokines in these HNSCC lines, and found that HNSCCs that express cytokines IL-1, IL-6, and GM-CSF do not express CD80, suggesting the hypothesis that these cytokines may down-modulate expression of CD80. Cytokine-conditioned medium from progressor HNSCC and recombinant IL-1, IL-6, and GM-CSF caused a reduction of CD80 expression in regressor HNSCCs without affecting proliferation. Conversely, the decrease in CD80 expression in progressor HNSCCs could be restored by IFN-, a known inducer of CD80 expression. These data strongly suggest that high levels of cytokines IL-1, IL-6, and GM-CSF expressed by tumor cells can down-regulate CD80 expression in HNSCC, and that IFN- can independently stimulate expression. These data provide evidence for a novel mechanism of cytokine-mediated down-modulation of CD80 during malignant progression of HNSCC that can be restored by IFN-.     

SNUFER: A software for localization and presentation of single nucleotide polymorphisms using a Clustal multiple sequence alignment output file

SNUFER is a software for the automatic localization and generation of tables used for the presentation of single nucleotide polymorphisms (SNPs). After input of a fasta file containing the sequences to be analyzed, a multiple sequence alignment is generated using ClustalW ran inside SNUFER. The ClustalW output file is then used to generate a table which displays the SNPs detected in the aligned sequences and their degree of similarity. This table can be exported to Microsoft Word, Microsoft Excel or as a single text file, permitting further editing for publication. The software was written using Delphi 7 for programming and FireBird 2.0 for sequence database management. It is freely available for noncommercial use and can be downloaded from http://www.heranza.com.br/bioinformatica2.htm.