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1.
Cell Death Dis ; 4: e808, 2013 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-24052076

RESUMO

The importance of tissue transglutaminase (TG2) in angiogenesis is unclear and contradictory. Here we show that inhibition of extracellular TG2 protein crosslinking or downregulation of TG2 expression leads to inhibition of angiogenesis in cell culture, the aorta ring assay and in vivo models. In a human umbilical vein endothelial cell (HUVEC) co-culture model, inhibition of extracellular TG2 activity can halt the progression of angiogenesis, even when introduced after tubule formation has commenced and after addition of excess vascular endothelial growth factor (VEGF). In both cases, this leads to a significant reduction in tubule branching. Knockdown of TG2 by short hairpin (shRNA) results in inhibition of HUVEC migration and tubule formation, which can be restored by add back of wt TG2, but not by the transamidation-defective but GTP-binding mutant W241A. TG2 inhibition results in inhibition of fibronectin deposition in HUVEC monocultures with a parallel reduction in matrix-bound VEGFA, leading to a reduction in phosphorylated VEGF receptor 2 (VEGFR2) at Tyr¹²¹4 and its downstream effectors Akt and ERK1/2, and importantly its association with ß1 integrin. We propose a mechanism for the involvement of matrix-bound VEGFA in angiogenesis that is dependent on extracellular TG2-related activity.


Assuntos
Matriz Extracelular/metabolismo , Espaço Extracelular/enzimologia , Proteínas de Ligação ao GTP/metabolismo , Neovascularização Patológica/enzimologia , Transglutaminases/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Embrião de Galinha , Reagentes de Ligações Cruzadas/metabolismo , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Matriz Extracelular/efeitos dos fármacos , Feminino , Fibronectinas/metabolismo , Proteínas de Ligação ao GTP/antagonistas & inibidores , Técnicas de Silenciamento de Genes , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Técnicas In Vitro , Camundongos , Neovascularização Patológica/patologia , Proteína 2 Glutamina gama-Glutamiltransferase , Ratos , Transdução de Sinais/efeitos dos fármacos , Transglutaminases/antagonistas & inibidores
2.
Matrix Biol ; 32(5): 277-87, 2013 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-23369837

RESUMO

Chronic kidney disease (CKD) is characterised by the pathological accumulation of extracellular matrix (ECM) proteins leading to progressive kidney scarring via glomerular and tubular basement membrane expansion. Increased ECM synthesis and deposition, coupled with reduced ECM breakdown contribute to the elevated ECM level in CKD. Previous pre-clinical studies have demonstrated that increased plasmin activity has a beneficial effect in the protein overload model of CKD. As plasmin activation is downregulated by the action of the thrombin activated fibrinolytic inhibitor (TAFI), we tested the hypothesis that inhibition of TAFI might increase plasmin activity and reduce ECM accumulation in an in vitro model of glucose induced ECM expansion. Treatment of NRK52E tubular epithelial cells with increasing concentrations of glucose resulted in a 40% increase in TAFI activity, a 38% reduction in plasmin activity and a subsequent increase in ECM accumulation. In this model system, application of the previously reported TAFI inhibitor UK-396082 [(2S)-5-amino-2-[(1-n-propyl-1H-imidazol-4-yl)methyl]pentanoic acid] caused a reduction in TAFI activity, increased plasmin activity and induced a parallel decrease in ECM levels. In contrast, RNAi knockdown of plasmin resulted in an increase in ECM levels. The data presented here indicate that high glucose induces TAFI activity, inhibiting plasmin activation which results in elevated ECM levels in tubular epithelial cells. The results support the hypothesis that UK-396082 is able to reduce TAFI activity, normalising plasmin activity and preventing excess ECM accumulation suggesting that TAFI inhibition may have potential as an anti-scarring strategy in CKD.


Assuntos
Aminoácidos/farmacologia , Carboxipeptidase B2/genética , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Imidazóis/farmacologia , Túbulos Renais Proximais/efeitos dos fármacos , Animais , Carboxipeptidase B2/antagonistas & inibidores , Carboxipeptidase B2/metabolismo , Linhagem Celular , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Matriz Extracelular/enzimologia , Matriz Extracelular/patologia , Fibrinolisina/antagonistas & inibidores , Fibrinolisina/genética , Fibrinolisina/metabolismo , Fibrinólise/efeitos dos fármacos , Fibrinólise/genética , Expressão Gênica/efeitos dos fármacos , Glucose/efeitos adversos , Humanos , Túbulos Renais Proximais/enzimologia , Túbulos Renais Proximais/patologia , Modelos Biológicos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/enzimologia , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , Trombina/genética , Trombina/metabolismo
3.
Nephron Exp Nephrol ; 120(3): e91-102, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22613868

RESUMO

BACKGROUND: Progressive chronic kidney disease is often associated with albuminuria and renal fibrosis linked to the accumulation of myofibroblasts producing extracellular matrix. Renal myofibroblasts are derived from a number of cells including tubular epithelial cells (TECs) through epithelial mesenchymal transformation (EMT). This study explores the hypothesis that exposure of TECs to albumin induces EMT. METHODS: Normal rat TECs (NRK52E) were exposed in culture to de-lipidated bovine serum albumin (dBSA; 10 mg/ml) for 2, 4 and 6 days. Binding/uptake of fluoresceined albumin by PTCs was evaluated. Transformation into myofibroblasts was assessed by light and electron microscopy, immunofluorescence and Western blotting for α-smooth muscle actin (α-SMA), E-cadherin and transforming growth factor-ß1 (TGF-ß1). We also investigated the expression of fibroblast-specific protein-1 (FSP-1) and collagens I, III and IV. TGF-ß1 biological activity, mRNA and protein were measured. A neutralising anti-TGF-ß1 antibody was used to analyse the role of TGF-ß1 in albumin-induced EMT. RESULTS: Exposure of TECs to dBSA led to binding/uptake of albumin as well as fibroblastic morphological changes. Incubation of TECs with dBSA caused a reduction of TEC marker E-cadherin (ANOVA p = 0.0002) and de novo expression of fibroblast markers α-SMA and FSP-1 (ANOVA p = 0.0001) in a time-dependent manner. It also increased expression and activity of TGF-ß1. Neutralisation of TGF-ß1 significantly reduced EMT (p < 0.01). CONCLUSION: This study demonstrates that in vitro, albumin induces the transformation of TECs into cells with myofibroblast characteristics; a process that may be TGF-ß1 dependent.


Assuntos
Células Epiteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Miofibroblastos/efeitos dos fármacos , Soroalbumina Bovina/farmacologia , Actinas/metabolismo , Animais , Anticorpos Neutralizantes/farmacologia , Northern Blotting , Western Blotting , Caderinas/metabolismo , Bovinos , Linhagem Celular , Colágeno/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Lipídeos/química , Microscopia Eletrônica , Microscopia de Fluorescência , Músculo Liso/química , Miofibroblastos/metabolismo , Miofibroblastos/ultraestrutura , Ratos , Proteína A4 de Ligação a Cálcio da Família S100 , Proteínas S100/metabolismo , Soroalbumina Bovina/química , Soroalbumina Bovina/farmacocinética , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/imunologia , Fator de Crescimento Transformador beta1/metabolismo
4.
Nephron Exp Nephrol ; 108(1): e1-e10, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18087173

RESUMO

BACKGROUND: Stem cell factor (SCF) has been implicated in many disease processes characterized by tissue remodelling and fibrosis. The growth factor (SCF) was evaluated in a rat model of nephrotoxic serum nephritis (NTN), characterized by early inflammation followed by later tissue fibrosis. METHODS: NTN was induced in male Wistar Kyoto rats using rabbit anti-rat glomerular basement membrane antibodies. Animals were sacrificed at days 7, 15, 30 and 45 (n = 4-10 per group). Rats' kidneys were immunostained for ED1 as marker of inflammation, CD34, SCF, c-kit, mast cell tryptase and markers of fibrosis; collagens III and IV and alpha-SMA. Changes in SCF protein and mRNA content were evaluated by Western blotting and Northern blotting, respectively. RESULTS: In the NTN kidney, levels of immunoreactive SCF and SCF receptor (c-kit) were significantly higher in glomerular, tubular and interstitial compartments. Mast cells were barely detectable in NTN and control rat sections. Double immunostaining showed the co-localization of SCF with alpha-SMA and of the SCF receptor with CD34 and ED1 positive cells. Immunostainable SCF protein in each of the 3 compartments, glomerular, tubular and interstitial, showed a positive linear correlation with serum creatinine, proteinuria, glomerulosclerosis score and interstitial fibrosis scores. Using multivariate analysis, immunostainable tubular SCF was a predictor of glomerular sclerosis and immunostainable glomerular SCF predicted tubular atrophy. Increased SCF immunostain was not a consequence of altered transcription as there was a fall in SCF mRNA determined by Northern blotting. Western blotting of NTN kidney homogenates revealed two bands for SCF, a 43-kDa band which decreased, and a 19-kDa band which increased throughout the study. CONCLUSION: These results highlight the potential role of SCF and its receptor in the remodelling process of the NTN kidney. Upregulation of SCF may involve a translational mechanism, with the soluble SCF protein KL-S1 (19 kDa) being derived from the transmembrane SCF protein KL-1 (43 kD) by proteolytic cleavage. The immunohistochemical staining of few CD34+ cells in NTN kidneys warrants further evaluation of the nature of these cells in the context of the inflammatory as well as the fibrotic processes.


Assuntos
Modelos Animais de Doenças , Glomerulonefrite/sangue , Fator de Células-Tronco/sangue , Animais , Glomerulonefrite/genética , Glomerulonefrite/patologia , Masculino , Proteinúria/sangue , Proteinúria/genética , Proteinúria/patologia , Proteínas Proto-Oncogênicas c-kit/biossíntese , Proteínas Proto-Oncogênicas c-kit/sangue , Proteínas Proto-Oncogênicas c-kit/genética , Ratos , Ratos Endogâmicos WKY , Fator de Células-Tronco/biossíntese , Fator de Células-Tronco/genética
5.
Cell Death Differ ; 13(9): 1442-53, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16294209

RESUMO

Administration of active TG2 to two different in vitro angiogenesis assays resulted in the accumulation of a complex extracellular matrix (ECM) leading to the suppression of endothelial tube formation without causing cell death. Matrix accumulation was accompanied by a decreased rate of ECM turnover, with increased resistance to matrix metalloproteinase-1. Intratumor injection of TG2 into mice bearing CT26 colon carcinoma tumors demonstrated a reduction in tumor growth, and in some cases tumor regression. In TG2 knockout mice, tumor progression was increased and survival rate reduced compared to wild-type mice. In wild-type mice, an increased presence of TG2 was detectable in the host tissue around the tumor. Analysis of CT26 tumors injected with TG2 revealed fibrotic-like tissue containing increased collagen, TG2-mediated crosslink and reduced organized vasculature. TG2-mediated modulation of cell behavior via changes in the ECM may provide a new approach to solid tumor therapy.


Assuntos
Matriz Extracelular/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Neovascularização Patológica/patologia , Transglutaminases/metabolismo , Animais , Morte Celular , Técnicas de Cocultura , Colágeno/biossíntese , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Endotélio Vascular/enzimologia , Endotélio Vascular/patologia , Matriz Extracelular/efeitos dos fármacos , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/farmacologia , Humanos , Metaloproteinase 1 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Transplante de Neoplasias , Neovascularização Patológica/enzimologia , Proteína 2 Glutamina gama-Glutamiltransferase , Ratos , Transglutaminases/genética , Transglutaminases/farmacologia , Transplante Heterólogo
7.
Nephron Clin Pract ; 97(3): c108-17, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15292688

RESUMO

INTRODUCTION: Diabetic nephropathy (DN) is the leading cause of chronic kidney failure, however the mechanisms underlying the characteristic expansion of the extracellular matrix (ECM) in diabetic kidneys remain controversial and unclear. In non-diabetic kidney scarring the protein crosslinking enzyme tissue transglutaminase (tTg) has been implicated in this process by the formation of increased epsilon-(gamma-glutamyl)lysine bonds between ECM components in both experimental and human disease. Studies in db+/db+ diabetic mice and in streptozotocin-treated rats have suggested a similar mechanism, although the relevance of this to human disease has not been addressed. METHODS: We have undertaken a retrospective analysis of renal biopsies from 16 DN patients with type 2 diabetes mellitus using an immunohistochemical and immunofluorescence approach, with tTg and epsilon-(gamma-glutamyl)lysine crosslink quantified by confocal microscopy. RESULTS: Immunofluorescent analysis of human biopsies (confocal microscopy) showed increases in levels of tTg (+1,266%, p < 0.001) and epsilon-(gamma-glutamyl)lysine (+486%, p < 0.001) in kidneys with DN compared to normal. Changes were predominantly in the extracellular periglomerular and peritubular areas. tTg staining correlated with epsilon-(gamma-glutamyl)lysine (r = 0.615, p < 0.01) and renal scarring (Masson's trichrome, r = 0.728, p < 0.001). Significant changes in epsilon-(gamma-glutamyl)lysine were also noted intracellularly in some (< or =5%) tubular epithelial cells. This is consistent with cells undergoing a novel transglutaminase-mediated cell death process in response to Ca2+ influx and subsequent activation of intracellular tTg. CONCLUSION: Changes in tTg and epsilon-(gamma-glutamyl)lysine occur in human DN. Cellular export of tTg may therefore be a factor in the perpetuation of DN by crosslinking and stabilisation of the ECM, while intracellular activation may lead to cell death contributing towards tubular atrophy.


Assuntos
Nefropatias Diabéticas/metabolismo , Dipeptídeos/análise , Proteínas de Ligação ao GTP/metabolismo , Rim/enzimologia , Transglutaminases/metabolismo , Adolescente , Adulto , Biópsia , Crioultramicrotomia , Nefropatias Diabéticas/enzimologia , Nefropatias Diabéticas/patologia , Dipeptídeos/química , Indução Enzimática , Matriz Extracelular/metabolismo , Espaço Extracelular/enzimologia , Feminino , Humanos , Rim/química , Rim/patologia , Masculino , Microscopia Confocal , Microscopia de Fluorescência , Inclusão em Parafina , Proteína 2 Glutamina gama-Glutamiltransferase , Estudos Retrospectivos , Solubilidade
9.
Kidney Int ; 60(5): 1765-76, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11703594

RESUMO

BACKGROUND: Caspase-3 is a member of the caspase enzyme family, having a central role in the execution of apoptosis. However, the significance of Caspase-3 in the inappropriate and excessive apoptosis that contributes to the progression of non-immune-mediated renal scarring has not been established. METHODS: Kidneys from sham-operated and subtotal nephrectomized (SNx) rats were harvested on days 7, 15, 30, 60, 90 and 120 post-surgery. These were analyzed for apoptosis (in situ end labeling of DNA, light and electron microscopy), Caspase-3 activity (fluorometric substrate cleavage assay), protein and mRNA (Western and Northern blotting), as well as distribution (immunohistochemistry), inflammation (ED-1 immunohistochemistry) and fibrosis (Masson's Trichrome staining). RESULTS: Apoptosis, inflammation and fibrosis gradually increased in glomeruli, tubules and interstitium of SNx rats. Caspase-3 was mainly located in damaged tubules, but also was found in some glomerular and interstitial cells. Little or no staining was noted in sham-operated kidneys. In SNx kidneys, Caspase-3 activity was significantly increased from day 30 and peaked on day 120 (2.5-fold). This resulted from increases in the 17 and 24 kD active protein subunits. The 32 kD precursor was increased at all time points (1861% on day 120, P < 0.01). Caspase-3 changes were transcription-dependent with the 2.7 kb caspase-3 mRNA significantly increased at all time points (287% on day 120). Caspase-3 activity was a better predictor of apoptosis (Std beta coefficient = 0.347, P < 0.05) than Caspase-3 proteins or mRNA; however, Caspase-3 at all levels correlated with apoptosis, inflammation and fibrosis (all P < 0.01). CONCLUSIONS: Up-regulation of apoptosis in remnant kidneys is likely to be Caspase-3-dependent as it is associated with increases in Caspase-3 at the activity, protein and mRNA levels. Therefore, Caspase-3 is a potential therapeutic target for the modification of renal cell apoptosis and subsequently renal fibrosis.


Assuntos
Apoptose , Caspases/metabolismo , Glomerulonefrite/patologia , Animais , Caspase 3 , Caspases/genética , Doença Crônica , Fibrose , Inflamação/diagnóstico , Masculino , RNA Mensageiro/análise , Ratos , Ratos Wistar
10.
J Endocrinol ; 169(2): 409-15, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-11312157

RESUMO

GH treatment during critical illness and sepsis may increase mortality. A family of negative regulators of cytokine signalling, the suppressors of cytokine signalling (SOCS), have been characterised. SOCS provide a mechanism for cross-talk between the cytokine receptors, including GH. Here, we have investigated the impact of nutrition and GH treatment on GH receptor, SOCS1, SOCS-2, SOCS-3 and cytokine-inducible SH2-containing protein (CIS) hepatic mRNA expression in a rat model of sepsis, caecal ligation and puncture (CLP). Four groups of rats were studied: control (food given ad libitum, n=7), CLP only (n=8), CLP and total parenteral nutrition (TPN) (n=9), and CLP, TPN and GH (n=10). CLP rats underwent surgery and 18 h later received saline or TPN or TPN+GH for 6 h before they were killed. Serum IGF-I levels were lower in all CLP groups (P<0.001). The combination of TPN and GH treatment increased IGF-I levels compared with the saline-treated CLP rats (P<0.01), but IGF-I levels remained lower than control animals (P<0.001). GH receptor and GH-binding protein expression in liver was reduced in animals subjected to CLP and was unaffected by nutrition or GH treatment. Hepatic SOCS-1 was detectable in normal rats, induced in all CLP animals but was unaffected by nutrition and GH. Hepatic SOCS-2 expression was difficult to detect in normal and CLP rats but was greatly induced in CLP rats treated with GH. Hepatic SOCS-3 expression was only just detectable in the control group but was elevated in all CLP groups and unaffected by nutrition and GH. Hepatic CIS expression was difficult to detect in normal rats, was not induced by CLP but was induced by both nutrition and GH. In conclusion, CLP induced low IGF-I levels associated with increased expression of SOCS-1 and SOCS-3, both of which are known to inhibit GH receptor signalling. GH induced SOCS-2 and CIS in the CLP rat despite resistance with respect to IGF-I generation, and parenteral feeding induced CIS in the CLP rat. Thus, there is potential for a complex interaction between GH and cytokine signalling at the level of SOCS expression whereby the inflammatory response may alter GH signalling and GH may influence the inflammatory response.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Citocinas/metabolismo , Proteínas de Ligação a DNA , Hormônio do Crescimento/farmacologia , Fígado/metabolismo , Nutrição Parenteral Total , Proteínas Repressoras , Sepse/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transativadores , Fatores de Transcrição , Análise de Variância , Animais , Northern Blotting , Proteínas de Transporte/análise , Proteínas de Transporte/genética , Hormônio do Crescimento/metabolismo , Fator de Crescimento Insulin-Like I/análise , Fígado/química , Masculino , Proteínas/análise , Proteínas/genética , RNA Mensageiro/análise , Ratos , Ratos Wistar , Receptores da Somatotropina/genética , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Proteína 1 Supressora da Sinalização de Citocina , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina
11.
Leukemia ; 14(3): 412-8, 2000 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10720135

RESUMO

Acute myeloid leukemia (AML) cells are malignant counterparts of normal myeloid pathway progenitors. Myeloid progenitors differentiate into professional antigen presenting cells (APC) under the essential influence of GM-CSF along with additional cytokines. Twelve cases of human AML were tested for ability to be differentiated toward a professional APC phenotype in short-term culture with addition of GM-CSF and the following recombinant proteins: TNFalpha, IL-4, CD40 ligand, Flt3 ligand and SCF. Significant upregulation of CD80 (B7-1) and enhancement of alloantigen presentation was seen with the addition of GM-CSF and TNFalpha alone or with additional cytokines. The combination of GM-CSF and TNFalpha, either alone or in combination with an additional cytokine, resulted in enhancing alloantigen presentation by at least two-fold over the media control group in 10/12 patients studied, and resulted in CD80 expression of greater than 15% in 11/12 patients studied. In AML cultures with GM-CSF and TNFalpha, coexpression of CD80 and either CD34 or an aberrant surface marker (CD56) was seen. In one case, sorted CD80, cells retained a characteristic cytogenetic marker and CD34 expression, proving their derivation from an AML precursor. These studies verify other reports of in vitro differentiation of human AML precursors into enhanced APC, suggesting that this phenomenon could be utilized for immunotherapy strategies aimed at enhancing presentation of leukemia antigens to T cells.


Assuntos
Apresentação de Antígeno/efeitos dos fármacos , Citocinas/farmacologia , Leucemia Mieloide/imunologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Doença Aguda , Adulto , Antígenos CD34/biossíntese , Antígenos CD34/genética , Antígeno B7-1/biossíntese , Antígeno B7-1/genética , Ligante de CD40 , Antígeno CD56/biossíntese , Antígeno CD56/genética , Diferenciação Celular/efeitos dos fármacos , Criança , Sinergismo Farmacológico , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Hibridização in Situ Fluorescente , Interleucina-4/farmacologia , Isoantígenos/imunologia , Leucemia Mieloide/patologia , Ativação Linfocitária , Glicoproteínas de Membrana/farmacologia , Proteínas de Membrana/farmacologia , Células-Tronco Neoplásicas/imunologia , Fator de Células-Tronco/farmacologia , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologia
12.
J Obstet Gynecol Neonatal Nurs ; 28(5): 480-5, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10507673

RESUMO

A home visit program for breastfeeding education and intervention was developed to provide support for mothers and infants at risk for breastfeeding failure. This program was developed to supplement the Early Discharge Program for mothers and newborns who were discharged within 24 hours of delivery. A breastfeeding assessment tool was developed for use in the hospital and during the early discharge home visit. Home visits are provided by a registered nurse with mother-infant assessment skills and competence to provide breastfeeding education, problem management, emotional support, and referrals to lactation consultants or physicians as needed. The response from patients and physicians has been positive. As the demand for services grew, the program was modified to include other mothers and infants (e.g., those delivering via cesarean and high-risk, preterm, and borderline preterm infants in need of breastfeeding support). The readmission rate for breastfeeding infants who receive a home visit is lower than for infants who do not receive a home visit. Patient satisfaction surveys have been positive.


Assuntos
Aleitamento Materno , Serviços de Assistência Domiciliar , Mães/educação , Desenvolvimento de Programas , Apoio Social , Feminino , Humanos , Recém-Nascido , Avaliação de Programas e Projetos de Saúde , Fatores de Risco
13.
Biochem J ; 331 ( Pt 1): 105-12, 1998 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-9512467

RESUMO

Treatment of the hamster fibrosarcoma cell lines (Met B, D and E) and BHK-21 hamster fibroblast cells with the glucocorticoid dexamethasone led to a powerful dose-dependent mRNA-synthesis-dependent increase in transglutaminase activity, which can be correlated with dexamethasone-responsive receptor numbers in each cell line. Increasing the number of dexamethasone-responsive receptors by transfection of cells with the HG1 glucocorticoid receptor protein caused an increase in transglutaminase activity that was proportional to the level of transfected receptor. In all experiments the levels of the tissue transglutaminase-mediated detergent-insoluble bodies was found to be comparable with increases in transglutaminase activity. Despite an increase in detergent-insoluble body formation, an increase in apoptosis as measured by DNA fragmentation was not found. Incubation of cells with the non-toxic competitive transglutaminase substrate fluorescein cadaverine led to the incorporation of this fluorescent amine into cellular proteins when cells were damaged after exposure to trypsin during cell passage. These cross-linked proteins containing fluorescein cadaverine were shown to be present in the detergent-insoluble bodies, indicating that the origin of these bodies is via activation of tissue transglutaminase after cell damage by trypsinization rather than apoptosis per se, since Met B cells expressing the bcl-2 cDNA were not protected from detergent-insoluble body formation. We describe a novel mechanism of cell death related to tissue transglutaminase expression and cell damage.


Assuntos
Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Dexametasona/farmacologia , Fibrossarcoma/enzimologia , Fibrossarcoma/patologia , Neoplasias Experimentais/enzimologia , Neoplasias Experimentais/patologia , Receptores de Glucocorticoides/metabolismo , Transglutaminases/metabolismo , Animais , Cricetinae , Indução Enzimática/efeitos dos fármacos , Células Tumorais Cultivadas
14.
J Clin Invest ; 99(12): 2950-60, 1997 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-9185519

RESUMO

Tissue transglutaminase is a calcium-dependent enzyme that catalyzes the cross-linking of polypeptide chains, including those of extracellular matrix (ECM) proteins, through the formation of epsilon-(gamma-glutamyl) lysine bonds. This crosslinking leads to the formation of protein polymers that are highly resistant to degradation. As a consequence, the enzyme has been implicated in the deposition of ECM protein in fibrotic diseases such as pulmonary fibrosis and atherosclerosis. In this study, we have investigated the involvement of tissue transglutaminase in the development of kidney fibrosis in adult male Wistar rats submitted to subtotal nephrectomy (SNx). Groups of six rats were killed on days 7, 30, 90, and 120 after SNx. As previously described, these rats developed progressive glomerulosclerosis and tubulo-interstitial fibrosis. The tissue level of epsilon-(gamma-glutamyl) lysine cross-link (as determined by exhaustive proteolytic digestion followed by cation exchange chromatography) increased from 3.47+/- 0.94 (mean+/-SEM) in controls to 13.24+/-1.43 nmol/g protein 90 d after SNx, P

Assuntos
Modelos Animais de Doenças , Rim/patologia , Nefrectomia , Transglutaminases/metabolismo , Animais , Reagentes de Ligações Cruzadas , Citoplasma/química , DNA/metabolismo , Dipeptídeos/análise , Dipeptídeos/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Espaço Extracelular/química , Fibrose , Imunofluorescência , Imuno-Histoquímica , Glomérulos Renais/patologia , Túbulos Renais/patologia , Masculino , Ratos , Ratos Wistar
15.
Mech Ageing Dev ; 89(1): 21-43, 1996 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-8819104

RESUMO

Aged mice that have undergone long-term caloric-restriction (CR) have improved health and enhanced longevity in comparison to aged mice that are ad libitum-fed (AL). However, caloric-restriction does not benefit the impaired wound healing of aged mice. To test the hypothesis that CR mice have the capacity for enhanced wound repair, but require a short-term period of additional nutrient intake to show this advantage, we assessed wound healing in CR mice that had been refed (RF) an ad libitum diet for 4 weeks prior to wounding. Two strains of AL young (Y AL) (4-6 months), AL middle-aged (M AL) (15-17 months), and three different, matched cohorts of old mice (O) (30-33 months): O AL, O CR, and O RF were studied. Two full-thickness 4 mm diameter punch biopsy skin wounds were created on the dorsum of each mouse. Animals were sacrificed and wounds were harvested at 1,2,3,5, and 7 days post-wounding. Repair of wounds was slower in O AL and O CR mice compared to Y AL and M AL animals. In contrast, the O RF mice healed similarly to that of the Y AL and M AL mice, as assessed by measures of wound area and histologic criteria. O RF mice demonstrated enhanced synthesis of type I collagen mRNA in comparison to O AL and O CR mice. A greater number of endothelial cells and fibroblasts at the wound edge of the O RF mice exhibited replication in vivo as measured by uptake of BrdU. O RF mice had higher levels of insulin-like binding protein 3 (IGFBP-3). Furthermore, fibroblasts derived from the explant of the punch biopsy of O CR mouse skin revealed enhanced proliferation and contraction in vitro, in comparison to fibroblasts from the O AL mice. In conclusion, O RF mice demonstrate an enhanced capacity to undergo wound repair in comparison to O AL mice. This effect appears to be mediated, in part, by enhanced cell proliferation, contraction, and collagen biosynthesis. In addition, short-term refeeding induced an increase in the serum level of IGFBP-3, the major binding protein for IGF-1. These data confirm that cells from O CR animals have a preserved proliferative, biosynthetic, and contractile capacity, but that an adequate source of nutrients is necessary to demonstrate this advantage in wound healing.


Assuntos
Envelhecimento/fisiologia , Cicatrização/fisiologia , Animais , Divisão Celular/fisiologia , Colágeno/genética , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , RNA Mensageiro/biossíntese
16.
Oncogene ; 9(10): 2935-42, 1994 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-7916148

RESUMO

Reduced expression of the tissue transglutaminase in both murine and human tumours has been consistently associated with tumour growth and progression. To investigate the functional effects of transglutaminase expression we have transfected a constitutive human tissue transglutaminase expression construct into a highly malignant hamster fibrosarcoma cell line Met B. Met B clones expressing the exogenous tissue transglutaminase exhibited a reduced incidence of primary tumour formation and an increased adherence to tissue culture plastic and fibronectin coated surfaces when compared to transfected and non transfected control cells. Transglutaminase transfected clones exhibited no significant differences in their growth rates measured in vitro, cell morphology or levels of spontaneous apoptosis measured by the determination of detergent insoluble apoptotic envelopes. The data demonstrates a suppressive effect of tissue transglutaminase on tumour growth and confirms its importance in the phenotypic changes associated with the cancer process.


Assuntos
Fibrossarcoma/enzimologia , Transglutaminases/genética , Animais , Apoptose , Adesão Celular , Divisão Celular/genética , Clonagem Molecular , Cricetinae , Dipeptídeos/metabolismo , Fibrossarcoma/patologia , Humanos , Camundongos , Transfecção
17.
Anal Quant Cytol Histol ; 10(6): 423-58, 1988 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-3064764

RESUMO

Flow cytometric (FCM) methodology represents a powerful analytical tool for screening and detecting abnormal and malignant cells, for subclassifying malignancies beyond conventional morphologic type and grade and for transcending light microscopic features by providing more biologically meaningful information. This paper reviews the historical background leading to the development of the FCM methodology and instrumentation that is presently being used for cytopathologic (and histopathologic) diagnosis, tumor subclassification and identification of aggressive cancers. Emphasis is placed on human carcinomas, with reference to hematologic malignancies when appropriate. Examples of FCM DNA content and cytokinetic techniques are described for the various cytologic sampling methods. The advantages, limitations and future prospects for the application of flow cytometric techniques in cytopathology are discussed.


Assuntos
Citometria de Fluxo/métodos , Patologia/métodos , Biópsia/métodos , Técnicas Citológicas/tendências , Humanos , Patologia/tendências
18.
Arch Otolaryngol Head Neck Surg ; 114(10): 1109-13, 1988 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-3415817

RESUMO

Surgical revision of the upper airway for obstructive sleep apnea has repeatedly improved subjective more than objective laboratory outcome measures. To examine this disparity, we obtained subjective sleepiness questionnaire scores, Continuous Performance test, and polysomnography (PSG) in 40 patients with mild to moderate obstructive sleep apnea (mean apnea index, 33.9; mean minimum oxygen saturation during sleep, 75.4%). Continuous Performance test confirmed abnormal daytime sleepiness and correlated with minimum oxygen saturation and number of transitions between stages. Postoperatively, questionnaire scores fell a mean of 62%, indicating a marked improvement in subjective sleepiness. Changes in questionnaire score correlated with changes in minimum oxygen saturation. Mean PSG indexes showed no change. Individual patients without PSG improvement reported long-term improvement in daytime functioning, as confirmed by family members. These results suggest that measures in addition to PSG, including patient subjective response, would more fully characterize the outcome of revision of the upper airway for sleep apnea.


Assuntos
Sistema Respiratório/cirurgia , Síndromes da Apneia do Sono/cirurgia , Adulto , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Síndromes da Apneia do Sono/fisiopatologia
19.
Gynecol Oncol ; 26(1): 41-56, 1987 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-3792935

RESUMO

The flow cytometric measured DNA content (i.e., DNA index), S-fractions, and histopathologic malignancy grades were studied for ninety uterine cervical squamous cell carcinomas using tissue biopsies taken prior to radiotherapy. The DNA aneuploidy frequency for low S-phase tumors (%S less than 14) was only 9/29 (31%) compared to 22/30 (73%) for intermediate (%S 15-23) and 30/31 (97%) for high (%S greater than 24) tumors. An overall mean %S of 20 +/- 7 (range 2-45%) was observed for these cervical cancers, with the S-fraction significantly increasing (P = less than 0.01) from 12 +/- 5, to 18 +/- 8, and 26 +/- 7 for diploid/near diploid, low-degree DNA aneuploidy, and high-degree DNA aneuploidy tumors, respectively. Broad heterogeneity was observed for the microscopic scored malignancy grades within DNA index and the cell-cycle S-fraction subgroups. Generally, multifactorial histopathology scoring was not significantly correlated with either the tumor DNA index or %S variables. Based on statistical analysis, the malignancy grades more closely reflected the tumor proliferative activity than the DNA index, with nuclear polymorphism, mitotic frequency, and the invasion pattern showing the lowest P values (which were not significant at P = 0.05). High tumor S-fraction was associated with high malignancy grade, as evidenced by 19/25 (75%) of high S-fraction, high degree DNA aneuploidy tumors having greater than average malignancy grade compared to only 2/14 (14%) low to moderate S-fraction tumors having similar DNA index. The results indicate that more reliable identification of biologically different cervical cancers can be achieved by evaluating the tumor DNA index in relationship to the cell-cycle %S and malignancy grading.


Assuntos
Carcinoma de Células Escamosas/patologia , DNA de Neoplasias/análise , Interfase , Neoplasias do Colo do Útero/patologia , Biópsia , Carcinoma de Células Escamosas/análise , DNA de Neoplasias/genética , Feminino , Humanos , Mitose , Invasividade Neoplásica , Ploidias , Neoplasias do Colo do Útero/análise
20.
Gynecol Oncol ; 26(1): 57-70, 1987 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-3792936

RESUMO

In an attempt to achieve more biologically meaningful subclassification of squamous cell cervical cancers and ultimately more reliable prediction of tumor behavior, we have studied the tumor pretreatment DNA content (i.e., DNA index), cell-cycle S-fraction, and histomorphologic malignancy grading properties for tumors having similar and different clinical staging. The tumor DNA index, %S-phase cells, and average malignancy grades were statistically tested against known clinical predictive variables for 90 primary squamous cell carcinomas of the uterine cervix studied prior to radiotherapy. We observed that the biological and morphological tumor properties of cervical cancers broadly overlapped between different stages of disease and for other clinical criteria including the gross morphology, lymph node status, and intravenous positivity. No significant differences were detected in the DNA indices, %S, or malignancy grades between the stages of disease or the other clinical criteria. It was concluded that: the gross clinical staging criteria inadequately reflected the tumor biological properties; and that the possibility exists for refining prognosis by use of the biological and morphological noncodependent tumor parameters to supplement staging criteria; and the use of cell-cycle cytokinetic and malignancy grading criteria in addition to tumor DNA index is superior to DNA index (or DNA content) alone for identifying biologically different cervical cancers.


Assuntos
Carcinoma de Células Escamosas/patologia , DNA de Neoplasias/análise , Interfase , Neoplasias do Colo do Útero/patologia , DNA de Neoplasias/genética , Feminino , Humanos , Metástase Linfática , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Ploidias , Prognóstico
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