Tissue alkalosis in cold-ischemia time.

The control of pre-analytical-factors in human biospecimens collected for health research is currently required. Only two previous reports using post-mortem brain samples have tried to address the impact of cold-ischemia on tissue pH. Here we report pH variations according to time (third-order polynomial model) in mice for liver, kidney and lung samples. Tissue alkalosis in cold-ischemia time may be an underlying mechanism of gene expression changes. Therefore, tissue-pH regulation after organ removal may minimize biological stress in human tissue samples.

Investigation on the association between thyroid tumorigeneses and herpesviruses.

Herpesviruses have been associated with various human malignancies and with thyroid autoimmunity. Aiming to investigate the presence of these viruses in thyroid nodules, we analyzed serum and thyroid tissue from 183 patients (83 benign and 100 malignant thyroid nodules). We also obtained 104 normal thyroid tissues extracted from the contralateral lobe of these patients. We used ELISA to screen the serology of all patients and a real-time quantitative PCR to analyze thyroid tissue viral load in antibody-positive patients. In addition, the presence of herpesviruses was tested by histological analysis in 20 EBV-positive tissues using the expression of LMP-1 by immunohistochemistry (IHC) and EBER by in situ hybridization (ISH). There was no evidence of HSV-2 or CMV DNA, but we found EBV DNA sequences in 29 (16%) thyroid tissue samples. We also found 7 positive EBV cases out of 104 normal tissues. Viral load was higher in tumors than in their respective normal tissues (p = 0.0002). ISH analysis revealed EBER expression in 11 out of 20 (52%) EBV-positive tissues, mostly in malignant cases (8/11, 73%). The presence of high EBV copy numbers in thyroid tumors and the expression of EBER only in malignant cases suggest an association between EBV and thyroid malignancies. However, we did not find any association between the presence of EBV and/or its viral load and any clinical or pathological tumor feature. Further studies aiming to clarify the mechanisms of EBV infection in thyroid cells are necessary to support a possible role in the development of thyroid cancer.

Odorant receptor genes are expressed in olfactory neuroblastoma.

Olfactory neuroblastoma (ONB) is a malignant tumor found in the human nasal cavity. These tumors are rare and poorly characterized at the molecular level. In this study, we asked whether olfactory-specific genes are expressed in ONBs by using reverse-transcriptase-polymerase chain reaction. We found that the olfactory marker protein and the RIC-8B genes, which are specifically expressed in mature olfactory neurons, are expressed in ONBs. Importantly, we also found that ONBs express a large variety of odorant receptor genes, representative of different odorant receptor gene subfamilies. Our results show that the ONBs express genes that are normally expressed in mature olfactory neurons and indicate that they are derived from progenitor or immature cells in the olfactory epithelium and not from a clonal expansion of a single or few mature olfactory neurons.

Mesenchymal stem cells from umbilical cord blood: parameters for isolation, characterization and adipogenic differentiation.

Isolation of mesenchymal stem cells (MSCs) from umbilical cord blood (UCB) from full-term deliveries is a laborious, time-consuming process that results in a low yield of cells. In this study we identified parameters that can be helpful for a successful isolation of UCB-MSCs. According to our findings, chances for a well succeeded isolation of these cells are higher when MSCs were isolated from UCB collected from normal full-term pregnancies that did not last over 37 weeks. Besides the duration of pregnancy, blood volume and storage period of the UCB should also be considered for a successful isolation of these cells. Here, we found that the ideal blood volume collected should be above 80 mL and the period of storage should not exceed 6 h. We characterized UCB-MSCs by morphologic, immunophenotypic, protein/gene expression and by adipogenic differentiation potential. Isolated UCB-MSCs showed fibroblast-like morphology and the capacity of differentiating into adipocyte-like cells. Looking for markers of the undifferentiated status of UCB-MSCs, we analyzed the UCB-MSCs' protein expression profile along different time periods of the differentiation process into adipocyte-like cells. Our results showed that there is a decrease in the expression of the markers CD73, CD90, and CD105 that correlates to the degree of differentiation of UCB-MSCs We suggest that CD90 can be used as a mark to follow the differentiation commitment degree of MSCs. Microarray results showed an up-regulation of genes related to the adipogenesis process and to redox metabolism in the adipocyte-like differentiated MSCs. Our study provides information on a group of parameters that may help with successful isolation and consequently with characterization of the differentiated/undifferentiated status of UCB-MSCs, which will be useful to monitor the differentiation commitment of UCB-MSC and further facilitate the application of those cells in stem-cell therapy.

Effect of oral sirolimus therapy on inflammatory biomarkers following coronary stenting.

We studied the effect of oral sirolimus, administered to prevent and treat in-stent restenosis (ISR), on the variation of serum levels of inflammatory markers following coronary stenting with bare metal stents. The mean age of the patients was 56 +/- 13 years, 65% were males and all had clinically manifested ischemia. Serum levels of high sensitivity C-reactive protein (hs-CRP) concentration were determined by chemiluminescence and serum levels of all other biomarkers by ELISA. One group of patients at high risk for ISR received a loading oral dose of 15 mg sirolimus and 5 mg daily thereafter for 28 days after stenting (SIR-G). A control group (CONT-G) was submitted to stenting without sirolimus therapy. The increase in hs-CRP concentration was highest at 24 h after stenting in both groups. A significant difference between SIR-G and CONT-G was observed at 4 weeks (-1.50 +/- 5.0 vs -0.19 +/- 0.4, P = 0.008) and lost significance 1 month after sirolimus discontinuation (-1.73 +/- 4.3 vs -0.01 +/- 0.7, P = 0.0975). A continuous fall in MMP-9 concentration was observed in SIR-G, with the greatest reduction at 4 weeks (-352.9 +/- 455 vs +395.2 +/- 377, P = 0.0004), while a positive variation was noted 4 weeks after sirolimus discontinuation (227 +/- 708 vs 406.2 +/- 472.1, P = 0.0958). SIR-G exhibited a higher increase in P-selectin after sirolimus discontinuation at week 8 (46.1 +/- 67.9 vs 5.8 +/- 23.7, P = 0.0025). These findings suggest that the anti-restenotic actions of systemic sirolimus include anti-proliferative effects and modulation of the inflammatory response with inhibition of adhesion molecule expression.

Effect of oral sirolimus therapy on inflammatory biomarkers following coronary stenting

We studied the effect of oral sirolimus, administered to prevent and treat in-stent restenosis (ISR), on the variation of serum levels of inflammatory markers following coronary stenting with bare metal stents. The mean age of the patients was 56 ± 13 years, 65 percent were males and all had clinically manifested ischemia. Serum levels of high sensitivity C-reactive protein (hs-CRP) concentration were determined by chemiluminescence and serum levels of all other biomarkers by ELISA. One group of patients at high risk for ISR received a loading oral dose of 15 mg sirolimus and 5 mg daily thereafter for 28 days after stenting (SIR-G). A control group (CONT-G) was submitted to stenting without sirolimus therapy. The increase in hs-CRP concentration was highest at 24 h after stenting in both groups. A significant difference between SIR-G and CONT-G was observed at 4 weeks (-1.50 ± 5.0 vs -0.19 ± 0.4, P = 0.008) and lost significance 1 month after sirolimus discontinuation (-1.73 ± 4.3 vs -0.01 ± 0.7, P = 0.0975). A continuous fall in MMP-9 concentration was observed in SIR-G, with the greatest reduction at 4 weeks (-352.9 ± 455 vs +395.2 ± 377, P = 0.0004), while a positive variation was noted 4 weeks after sirolimus discontinuation (227 ± 708 vs 406.2 ± 472.1, P = 0.0958). SIR-G exhibited a higher increase in P-selectin after sirolimus discontinuation at week 8 (46.1 ± 67.9 vs 5.8 ± 23.7, P = 0.0025). These findings suggest that the anti-restenotic actions of systemic sirolimus include anti-proliferative effects and modulation of the inflammatory response with inhibition of adhesion molecule expression.

Gene expression profiling of clinical stages II and III breast cancer.

Clinical stage (CS) is an established indicator of breast cancer outcome. In the present study, a cDNA microarray platform containing 692 genes was used to identify molecular differences between CSII and CSIII disease. Tumor samples were collected from patients with CSII or CSIII breast cancer, and normal breast tissue was collected from women without invasive cancer. Seventy-eight genes were deregulated in CSIII tumors and 22 in CSII tumors when compared to normal tissue, and 20 of them were differentially expressed in both CSII and CSIII tumors. In addition, 58 genes were specifically altered in CSIII and expression of 6 of them was tested by real time RT-PCR in another cohort of patients with CSII or CSIII breast cancer and in women without cancer. Among these genes, MAX, KRT15 and S100A14, but not APOBEC3G or KRT19, were differentially expressed on both CSIII and CSII tumors as compared to normal tissue. Increased HMOX1 levels were detected only in CSIII tumors and may represent a molecular marker of this stage. A clear difference in gene expression pattern occurs at the normal-to-cancer transition; however, most of the differentially expressed genes are deregulated in tumors of both CS (II and III) compared to normal breast tissue.

Gene expression profiling of clinical stages II and III breast cancer

Clinical stage (CS) is an established indicator of breast cancer outcome. In the present study, a cDNA microarray platform containing 692 genes was used to identify molecular differences between CSII and CSIII disease. Tumor samples were collected from patients with CSII or CSIII breast cancer, and normal breast tissue was collected from women without invasive cancer. Seventy-eight genes were deregulated in CSIII tumors and 22 in CSII tumors when compared to normal tissue, and 20 of them were differentially expressed in both CSII and CSIII tumors. In addition, 58 genes were specifically altered in CSIII and expression of 6 of them was tested by real time RT-PCR in another cohort of patients with CSII or CSIII breast cancer and in women without cancer. Among these genes, MAX, KRT15 and S100A14, but not APOBEC3G or KRT19, were differentially expressed on both CSIII and CSII tumors as compared to normal tissue. Increased HMOX1 levels were detected only in CSIII tumors and may represent a molecular marker of this stage. A clear difference in gene expression pattern occurs at the normal-to-cancer transition; however, most of the differentially expressed genes are deregulated in tumors of both CS (II and III) compared to normal breast tissue.

Escherichia coli lipopolysaccharide inhibits renin activity in human mesangial cells.

Hyperactivation of systemic renin-angiotensin system (RAS) during sepsis is well documented. However, the behavior of intrarenal RAS in the context of endotoxemia is yet to be defined. The present study evaluates the direct effect of Escherichia coli lipopolysaccharide (LPS) on immortalized human mesangial cell (HMC) RAS. Quiescent HMC were incubated with vehicle or LPS (1-100 microg/ml), and levels of angiotensin I and II (Ang I and II) and their metabolites were analyzed by high-performance liquid chromatography. In addition, angiotensin-converting enzyme (ACE) and renin activity were also investigated. Cell lysate and extracellular medium levels of Ang II were rapidly reduced (1 h) in a time- and concentration-dependent manner, reaching a significant -9 fold-change (P<0.001) after 3 h of LPS incubation. Similar results were obtained for Ang I levels (-3 fold-change, P<0.001). We ruled out Ang I and II degradation, as levels of their metabolic fragments were also significantly decreased by LPS. ACE activity was slightly increased following LPS incubation. On the other hand, renin activity was significantly inhibited, as Ang I concentration elevation following exogenous angiotensinogen administration was blunted by LPS (-60% vs vehicle, P<0.001). Renin and angiotensinogen protein levels were not affected by LPS according to Western blot analysis. Taken together, these data demonstrate for the first time that LPS significantly downregulates HMC RAS through inhibition of renin or renin-like activity. These findings are potentially related to the development of and/or recovery from acute renal failure in the context of sepsis.

Phyllanthus niruri normalizes elevated urinary calcium levels in calcium stone forming (CSF) patients.

Phyllanthus niruri is a plant used for years in Brazil to treat urinary calculi. We prospectively evaluated the effect of P. niruri intake on 24 h urinary biochemical parameters in an attempt to assess its in vivo effect in calcium stone forming (CSF) patients. A total of 69 CSF patients (39 males and 30 females, 38+/-8 years old) were randomized to take either P. niruri ( n=33) (450 mg capsules, td) or placebo ( n=36) for 3 months. Blood calcium, uric acid, citrate, magnesium, oxalate, sodium and potassium were determined at baseline and at the end of the study. A subset analysis was made in patients classified according to the presence of metabolic abnormalities (hypercalciuria, hyperuricosuria, hyperoxaluria, hypocitraturia and hypomagnesiuria). Overall, there were no significant differences in the mean values of urinary parameters between the urine samples before and after P. niruri intake, except for a slight reduction in mean urinary magnesium after P. niruri, which was within the normal range. However, in the subset analysis, we observed that P. niruri induced a significant reduction in the mean urinary calcium in hypercalciuric patients (4.8+/-1.0 vs 3.4+/-1.1 mg/kg/24 h, P<0.05). In this short-term follow-up, no significant differences in calculi voiding and/or pain relief between the groups taking P. niruri or the placebo were detected. Our data suggest that P. niruri intake reduces urinary calcium based on the analysis of a subset of patients presenting with hypercalciuria. Larger trials including primary hypercalciuric stone formers should be performed in order to confirm these findings and to determine the possible clinical consequences of urinary calcium reduction during P. niruri administration.

Mechanisms involved in calcium oxalate endocytosis by Madin-Darby canine kidney cells.

Calcium oxalate (CaOx) crystals adhere to and are internalized by tubular renal cells and it seems that this interaction is related (positively or negatively) to the appearance of urinary calculi. The present study analyzes a series of mechanisms possibly involved in CaOx uptake by Madin-Darby canine kidney (MDCK) cells. CaOx crystals were added to MDCK cell cultures and endocytosis was evaluated by polarized light microscopy. This process was inhibited by an increase in intracellular calcium by means of ionomycin (100 nM; N = 6; 43.9% inhibition; P<0.001) or thapsigargin (1 microM; N = 6; 33. 3% inhibition; P<0.005) administration, and via blockade of cytoskeleton assembly by the addition of colchicine (10 microM; N = 8; 46.1% inhibition; P<0.001) or cytochalasin B (10 microM; N = 8; 34.2% inhibition; P<0.001). Furthermore, CaOx uptake was reduced when the activity of protein kinase C was inhibited by staurosporine (10 nM; N = 6; 44% inhibition; P<0.01), or that of cyclo-oxygenase by indomethacin (3 microM; N = 12; 17.2% inhibition; P<0.05); however, the uptake was unaffected by modulation of potassium channel activity with glibenclamide (3 microM; N = 6), tetraethylammonium (1 mM; N = 6) or cromakalim (1 microM; N = 6). Taken together, these data indicate that the process of CaOx internalization by renal tubular cells is similar to the endocytosis reported for other systems. These findings may be relevant to cellular phenomena involved in early stages of the formation of renal stones.

Mechanisms involved in calcium oxalate endocytosis by Madin-Darby canine kidney cells

Calcium oxalate (CaOx) crystals adhere to and are internalized by tubular renal cells and it seems that this interaction is related (positively or negatively) to the appearance of urinary calculi. The present study analyzes a series of mechanisms possibly involved in CaOx uptake by Madin-Darby canine kidney (MDCK) cells. CaOx crystals were added to MDCK cell cultures and endocytosis was evaluated by polarized light microscopy. This process was inhibited by an increase in intracellular calcium by means of ionomycin (100 nM; N = 6; 43.9 percent inhibition; P<0.001) or thapsigargin (1 µM; N = 6; 33.3 percent inhibition; P<0.005) administration, and via blockade of cytoskeleton assembly by the addition of colchicine (10 µM; N = 8; 46.1 percent inhibition; P<0.001) or cytochalasin B (10 µM; N = 8; 34.2 percent inhibition; P<0.001). Furthermore, CaOx uptake was reduced when the activity of protein kinase C was inhibited by staurosporine (10 nM; N = 6; 44 percent inhibition; P<0.01), or that of cyclo-oxygenase by indomethacin (3 µM; N = 12; 17.2 percent inhibition; P<0.05); however, the uptake was unaffected by modulation of potassium channel activity with glibenclamide (3 µM; N = 6), tetraethylammonium (1 mM; N = 6) or cromakalim (1 µM; N = 6). Taken together, these data indicate that the process of CaOx internalization by renal tubular cells is similar to the endocytosis reported for other systems. These findings may be relevant to cellular phenomena involved in early stages of the formation of renal stones

Effects of kinins upon cytosolic calcium concentrations in mouse mesangial cells.

Bradykinin (BK) induces increases in cytosolic calcium concentration [Ca++]i in several cell lines. Because the role of BK in the renal system, particularly in mesangial cell (MC), is not clear, we investigated the effects of kinins on [Ca++]i in mouse-immortalized MC. [Ca++]i was evaluated by spectrofluorometry and expressed as a ratio between the obtained and basal [Ca++]i. BK (0.1 microM) induced a non-sustained increase in [Ca++]i (4.70 +/- 0.27; N = 28). A similar effect was observed with the B2 receptor agonist, Tyr8-BK (0.1 microM, 3.34 +/- 0.48; N = 7), while B1 receptor agonists, des-Arg10-Kallidin (Kal) (1 microM, N = 11) and des-Arg9-BK (1 microM, N = 8), exhibited only discrete responses (1.45 +/- 0.08 and 1.12 +/- 0.04, respectively). Cross-desensitization was seen between BK and Tyr8-BK, but not between BK and des-Arg10-Kal. The BK response was decreased (5.09 +/- 0.30, N = 6 to 1.57 +/- 0.12, N = 7, P < 0.001) by the B2 receptor antagonist HOE 140 (0.1 microM, 15 min), while the B1 receptor antagonist des-Arg9-[Leu8]-BK (1 microM, 15 min) had no effect on BK or des-Arg10-Kal actions. Incubation of cells with Escherichia coli lipopolysaccharide (100 microg/ml, 24 h) alone or in association with tumor necrosis factor-alpha (TNF-alpha) (10 ng/ml, N = 6) did not enhance B1 agonist responses. BK was inhibited by repeated cell washouts in zero Ca++ solution (2.04 +/- 0.19, N = 6 P < 0.001), and the residual response was almost abolished by thapsigargin (Thaps) a sarcoplasmic reticulum (SR) calcium-ATPase inhibitor (1 microM) (1.18 +/- 0.08, N = 5 P < 0.001). Additionally, BK was not inhibited by verapamil (50 microM), nifedipine (30 microM), Ni++ (300 microM) or La (10 microM). In conclusion, BK induces [Ca++]i in mouse MC mainly by B2 receptor activation. B1 receptors have a minor role in this phenomenon even in the presence of known B1 receptor synthesis inducers. Finally, BK mobilizes extracellular calcium sources and, to a lesser extent, intracellular Thaps-sensitive calcium stores. The ion channels involved in calcium influx remain to be detected.

Phyllanthus niruri inhibits calcium oxalate endocytosis by renal tubular cells: its role in urolithiasis.

We investigated the in vitro effect of an aqueous extract of Phyllanthus niruri L. on a model of CaOx crystal endocytosis by Madin-Darby canine kidney cells. The extract exhibited a potent and effective non-concentration-dependent inhibitory effect on the CaOx crystal internalization. This response was present even at very high (pathologic) CaOx concentrations and no P. niruri L.-induced toxic effect could be detected. Biochemical analysis of culture media containing P. niruri L. did not provide any clues for the elucidation of the cellular pathways affected by this natural product. Although further studies are necessary for a better understanding of the role of P. niruri L. in urolithiasis, our findings show that this natural product could be an attractive alternative for the treatment of urinary stones.

O papel das citocinas na disfunção ventricular avançada / The role of cytokines in advanced ventricualar dysfunction

A insuficiência cardíaca congestiva foi definida por muito tempo como uma síndrome envolvendo profundas alteraçöes hemodinâmicas e neuro-hormonais. Nos últimos anos, uma reação inflamatória exuberante tem sido observada na presença de disfunção ventricular sistólica severa, tanto em modelos experimentais como em ensaios clínicos. Essa resposta inflamatória envolve a ativaçäo e a liberaçäo de uma série de substâncias peptídicas denominadas citocinas. Essas substâncias parecem ter papel relevante tanto na gênese como na manutencçäo do processo de insuficiência ventricular, e podem ser utilizadas inclusive como marcadores prognósticos da doença. O presente artigo apresenta, de forma resumida, os principais estudos clínicos nessa área, destacando a correlaçäo entre determinadas citocinas e variáveis clínico-laboratoriais já descritas em portadores de insuficiência cardíaca congestiva avançada.

Analysis of the mechanisms underlying the contractile response induced by the hydroalcoholic extract of Phyllanthus urinaria in the guinea-pig urinary bladder in-vitro.

The hydroalcoholic extract of Phyllanthus urinaria (Euphorbiaceae), substance P and substance P methyl ester all caused graded contractions in the guinea-pig urinary bladder. Responses to hydroalcoholic extract and substance P were markedly inhibited in calcium-free Krebs solution, this effect being reversed by reintroduction of calcium in the medium. The contraction in response to hydroalcoholic extract was unaffected by atropine, propranolol, prazosin, yohimbine, tetrodotoxin, w-conotoxin, nicardipine, HOE 140, guanethidine, staurosporine, phorbol ester or indomethacin, excluding the involvement of nervous mediated responses, or action via cholinergic, adrenergic, kinins, cyclo-oxygenase metabolites, protein kinase C or activation of L or N-type calcium channels. The selective NK1 tachykinin antagonist (FK 888), but not NK2 (SR 48968) antagonized substance P-induced contraction, but both drugs failed to effect Phyllanthus urinaria-induced contraction. Prolonged desensitization of guinea pig urinary bladder with capsaicin (10 microM) or preincubation of guinea-pig urinary bladder with capsazepine did not affect contraction caused by hydroalcoholic extract. Ruthenium red almost completely abolished capsaicin-induced contraction, but had no effect on hydroalcoholic extract-mediated contraction. Substance P and the hydroalcoholic extract caused marked potentiation of the twitch response in the preparations field stimulated. The facilitatory effect of substance P, but not that of hydroalcoholic extract, was prevented by the NK1 (FK 888), but not by NK2 (SR 48968) antagonist. We concluded that contraction induced by hydroalcoholic extract of Phyllanthus urinaria in the guinea pig urinary bladder involves direct action on smooth muscle and relies on the mobilization of extracellular calcium influx unrelated to activation of L- and N-type calcium channels or activation of protein kinase C mechanisms. In addition contraction caused by the hydroalcoholic extract of Phyllanthus urinaria in guinea-pig urinary bladder does not involve the activation of tachykinin or vanilloid receptors.

Mechanisms involved in the contractile responses of kinins in rat portal vein rings: mediation by B1 and B2 receptors.

This study investigates the mechanisms involved in kinin-induced contractions in rings of rat portal vein (RPV). Bradykinin (BK), Lys-BK, Met-Lys-BK, Tyr8-BK (TBK) and des-Arg9-BK (DABK) all caused graded contractions in RPV, with the following order of potency (EC50, nanomolar): Met-Lys-BK (0.3) > Lys-BK (0.5) > BK (0.9) > TBK (2.3) >> DABK (46.0). The potency of DABK and maximal contractions for DABK and BK, but not for TBK or NE, increased as a function of in vitro incubation period, reaching the maximum at 4.5 hr. Cycloheximide (a protein synthesis inhibitor, 70 microM), incubated for 4.5 hr, inhibited almost completely the CRCs for DABK and blocked the latter phase of CRCs for BK, not altering contractions induced by U46619 (9,11-dideoxy-9 alpha, 11 alpha-methano-epoxy prostaglandin F2 alpha) (a thromboxane A2/prostaglandin H2-mimetic). Incubation of RPV with D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]-BK (HOE 140, a selective B2 receptor antagonist, 0.01-100 nM), caused a parallel rightward displacement of the BK and TBK concentration-response curves (CRCs). Schild plots were linear, yielding pA2 values of 11.4 and 9.3, respectively. The slope for HOE 140 against TBK-induced contractions did not differ from unity (1.23 +/- 0.21), whereas against BK was significantly lesser than unity (0.72 +/- 0.20). The CRCs induced by DABK were not affected by HOE 140 (100 nM). In addition, the CRCs for DABK at 4.5 hr were shifted to the right in a parallel form in the presence of des-Arg9-[Leu8]-BK (a selective B1-receptor antagonist, 1 microM), yielding a pA2 value of 6.7.(ABSTRACT TRUNCATED AT 250 WORDS)