A psychometric analysis of the Early Trauma Inventory-Short Form in Colombia: CTT and Rasch model.

BACKGROUND: Potential childhood traumatic experiences increase risk for mental and physical health disorders and their precise assessment can help to promote health prevention and promotion strategies for countries with limited data and measurement strategies like Colombia. OBJECTIVE: The goal of the present study is to strengthen evidence for the validity of scores from an adapted version of the Early Trauma Inventory self report-short form (ETI-SF) using Item Response Theory and by assessing factorial invariance across gender and education level. PARTICIPANTS AND SETTING: The study assessed a total of 1909 Colombian participants (66.16 % women, 32.16 % men, 1.68 % other gender; age range 18-72 years old). METHODS: Participants answered the ETI-SF via a web-based sampling strategy. RESULTS: The total scores of the scale showed good reliability coefficients (α = 0.81 and ω = 0.60). A specific analysis for the subscales showed good reliability for the emotional, physical, and sexual trauma subscales (αs and ωs >0.64), while general trauma showed lower than accepted reliability values (α =0.56 and ω = 0.37). Most of the individual items of the scale showed good calibration. The factorial invariance analysis suggests the possibility of some gender and educational differences. CONCLUSIONS: The study confirms particularly high rates of potential childhood traumatic experiences in Colombia and complement data for specific trauma types. Overall, the ETI-SF is confirmed as useful for Colombia, which highlights this scale as a good tool to use for public health assessment. Future research can continue the integration of diverse methods for estimating the quality of the scale.

Aproximaciones evolutivas y psicopatología: Teoría de historia de vida y sistemas psicobiológicos en el trastorno de personalidad límite / Evolutionary approaches and psychopathology: Life history theory and psychobiological systems for borderline personality disorder

La psicología clínica requiere de constantes desarrollos científicos que lleven a una explicación de la complejidad de los trastornos mentales y sus bases causales. Las aproximaciones evolutivas han mostrado ser de particular poder heurístico para esta tarea. Entre ellas, la Teoría de Historia de Vida (THV) incorpora avances teóricos y empíricos novedosos y significativos. No obstante, existe la necesidad de incorporar investigación y aproximaciones evolutivas adicionales de interés. Por lo tanto, en este artículo se propondrá el potencial de integración al ampliar la causalidad evolutiva en conjunción con aproximaciones de sistemas psicobiológicos de conducta. Para esto se utilizará como ejemplo el Trastorno Límite de Personalidad, ampliando su comprensión como una interacción de causas próximas entre los sistemas psicobiológicos de estrés y apego, dentro del marco de causas últimas de THV. Finalmente, se demarcarán aspectos que nutren el campo clínico con implicaciones para la evaluación y los dominios de intervención.(AU)

Clinical psychology requires continuous research to encourage integrative explanations for the complexity of mental disorders and their underlying causes. Biological evolutionary approaches have shown particular heuristic power for this endeavor. Life history theory (LHT) is an evolutionary model that incorporates novel and significant theoretical and empirical advances. However, there is a growing need for the incorporation of other successful evolutionary approaches. Thus, the goal of the present paper is to propose potential integrative connections between evolutionary causal modes, behavior systems, and LHT. For this, borderline personality disorder is used as an example of a condition that can be understood as an interaction between stress and attachment psychobiological systems (proximate causes), within the framework of ultimate causes clarified by LHT. To conclude, we will outline several aspects that could enhance the clinical field with implications for assessment and intervention.(AU)

Prevalencia de anticuerpos anti-Leptospira spp. en personas con exposición laboral en el departamento del Tolima / Prevalence of anti-Leptospira spp. antibodies among people with occupational exposure in Tolima department / Prevalência de anticorpos anti-Leptospira spp. em pessoas com exposição laboral no Departamento do Tolima

Objetivo: estimar la prevalencia de anticuerpos IgM contra Leptospira spp., mediante el Ensayo de Inmunoabsorción Ligado a Enzimas (ELISA), en la población de riesgo laboral de 8 municipios del Tolima. Metodología: se obtuvieron muestras de sangre de 261 empleados, las cuales fueron analizadas mediante la técnica de elisa para la detección de anticuerpos IgM anti-Leptospira spp., seguido de MAT y serotipificación. Resultado : se estimó una seroprevalencia del 25,29%, con una seroreactividad mayor en trabajadores de plantas de beneficio animal (34,2%), recolección de residuos sólidos (27,1%) y trabajadores de acueducto y alcantarillado (14,8%). La actividad en plantas de beneficio animal se identificó como factor de riesgo de Leptospira spp. (OR=1,86). Los serovares identificados fueron L. Bratislava (16), Ballum (5), Tarassovi (3), Hebdomadis (2), Sejroe (2) y Icterhemorragiae (1). El municipio de Libano presentó el mayor porcentaje de positividad (36,96%), seguido de Espinal y Guamo con 28,57% cada uno. Discusión: La evaluación del sistema de vigilancia indicó deficiencia en recursos y debilidades de los profesionales de la salud al desconocer los procedimientos, investigación, diagnóstico y notificación de la enfermedad. Conclusión: la leptospirosis está presente en poblaciones de riesgo laboral en el Tolima y se hace necesario abordar esta problemática en la población de otros municipios y los animales transmisores de la enfermedad.

Objective: to estimate the prevalence of IgM antibodies against Leptospira spp. Using the enzyme-linked immunosorbent assay (elisa) in a population at occupational risk from 8 municipalities of the Tolima department, Colombia. Methodology: blood samples were collected from 261 employees and analyzed with the elisa technique to detect IgM and anti-Leptospira spp. antibodies. This was followed by mat and serotyping. Result: a seroprevalence of 25.29% was estimated, with higher seroreactivity for individuals working at slaughter plants (34.2%), collecting solid waste (27.1%) and those in contact with water and sewage waste (14.8%). Activity in slaughter plants was identified as a risk factor for Leptospira spp. (OR = 1.86). The serovars identified were L. Bratislava (16), Ballum (5), Tarassovi (3), Hebdomadis (2), Sejroe (2), and Icterhemorragiae (1). The municipality of Libano had the highest percentage of positivity (36.96%), followed by Espinal and Guamo with 28.57% each. Discussion: assessment of the current surveillance system for leptospirosis indicated deficient resources and health professionals who are lacking in terms of knowledge regarding appropriate procedures, research on, diagnosis and reporting mechanisms for the disease. Conclusions: leptospirosis is present in public workers with occupational hazard in Tolima. In addition, this issue should be approached while taking into account the population from other municipalities as well as the animals associated with its transmission

Objetivo: estimam a prevalência de anticorpos IgM contra Leptospira spp., por ensaio de ensaio de imunossorvente ligado a enzima (ELISA) na população de risco ocupacional de 8 municípios de Tolima. Metodologia: Coletaram-se amostras de sangue de 261 empregados, e analisaram-se com a técnica de ELISA para detectar anticorpos IgM anti-Leptospira spp., seguido de MAT e de serotipificação. Resultados : estimou-se uma seroprevalência de 25,29%, com seroatividade superior nos trabalhadores dos matadouros (34,2%), da recolecção de lixo sólido (27,1%), e nos trabalhadores dos esgotos (14,8%). A atividade nos matadouros foi identificada como fator de risco de Leptospira spp. (OR=1,86). Os serovares identificados foram L. Bratislava (16), Ballum K(5), Tarassovi K(3), Hebdomadis (2), Sejroe (2) e Icterhemorragiae (1). O município de Libano apresentou a percentagem mais alta de positividade (36,96%), seguido por Espinal e por Guamo, com 28,57 cada um. Discussão: a avaliação do sistema de vigilância revelou deficiência de recursos e fraquezas dos profissionais da saúde, porque desconhecem os procedimentos, a investigação, o diagnóstico e a notificação da doença. Conclusão: a leptospirosis está nas populações de risco laboral no Departamento do Tolima, o que faz necessário acometer este problema na população de outros municípios e nos animais transmissores da doença.

Genotype-environment interaction in multibreedbovine populations in the Colombian low tropic / Interacción genotipo-ambiente en una población bovina multirracialen el trópico bajo colombiano / Interação genótipo ambiente em populações bovinas multiraciaisno trópico baixo colombiano

With the aim o f quantifying the genotype-environment interaction (GEI) and the phenotypic stabilityin multibreed bovine population of the Colombian Northwest, registries from 16 herds located in threeagroecological regions (E1, E2, E3) from low tropic systems (humid subtropic forest, humid tropic forest anddry tropic forest, were collected from 1995 to 2007. Weight at 12-mo (W12), weight at 18-mo (W18), and weightat 24-mo (W24), were evaluated with 1806, 1455, and 1197 data, 14, 11, and 10 genetic groups respectively;animals of the breeds and crossbred between Angus (A), Blanco Orejinegro (B), Zebu (Z), Holstein (H),Romosinuano (R), and Senepoll (S) were used. In a mixed model, the fixed effects of contemporary group(year-season-sex) and the age covariate were used, which showed a significant effect (p<0.001) on the threetraits. Random effects were region, genetic group (breed or crosses), and GEI, but the last one (GEI) showedsignificant effect (p<0.05) for this last one. The Shukla’s variance in Bayesian methodology was used forthe phenotypic stability analysis. The results indicated that the groups with high proportion of Zebu wereassociated with E2 and groups with greater levels of Romosinuano were associated with E3. Holstein andBlanco Orejinegro tended to give greater phenotypic stability than the groups that used these breeds.

Con el objetivo de cuantificar la interacción genotipo-ambiente (IGA) y la estabilidad fenotípica en unapoblación bovina multirracial del noreste colombiano, se usaron registros de 16 rebaños localizados en tresregiones agroecológicas del trópico bajo: bosque húmedo subtropical (R1), bosque húmedo tropical (R2)y bosque seco tropical (R3), entre los años 1995 y 2007. Los pesos fueron evaluados a los 12 (P12), 18(P18) y 24 meses (P24), con 1806, 1455 y 1197 datos, y 14, 11 y 10 grupos genéticos, respectivamente.Fueron usados animales puros y cruzados entre las razas Angus (A), Blanco Orejinegro (B), Cebú (C),Holstein (H), Romosinuano (R) y Senepol (S). Se utilizó un modelo mixto, en el que los efectos fijos de grupocontemporáneo y la edad presentaron efecto significativo (p<0.001) sobre las tres características. Los efectosaleatorios fueron región, grupo genético (raza o cruce) e IGA, la que presentó efecto significativo (p<0.05).Para el análisis de estabilidad fenotípica se utilizó la varianza de Shukla mediante metodología bayesiana.Los resultados indican que los grupos genéticos con altas proporciones de Cebú fueron asociados con R2 ylos grupos genéticos con altos niveles de Romosinuano fueron asociados con R3. Las razas Holstein y BlancoOrejinegro tendieron a dar mayor estabilidad fenotípica a los grupos donde estas razas fueron usadas.

Com o propósito de quantificar a interação genótipo ambiente (IGA) e estabilidade fenotípica empopulações bovinas multiraciais no trópico baixo colombiano foram utilizados registros desde 1995 até2007 de 16 fazendas localizadas em três regiões agroecológicas: bosque subtropical úmido (E1) bosquetropical úmido (E2) e bosque tropical seco (E3). Foram avaliadas o peso aos 12, 18 e 24 meses, com1806, 1455 e 1197 registros, respectivamente de 10 grupos genéticos das raças Angus, Blanco Orejinegro,Zebu, Holandês, Romosinuano e Senepol. O Modelo mixto utilizado incluiu os efeitos fixos de grupocontemporâneo (ano, época e sexo) e a idade como covariavel, os quais foram significativos (p<0.001)nas três características. Foram considerados os efeitos aleatórios de regiao, grupo genético e a interação(GEI), onde este último foi significativo. A analise de estabilidade fenotípica foi realizada utilizandoa variância de Shukla por metodologia Bayesiana. Os resultados indicaram que os grupos com maiorproporção de Zebu foram associados com E2 e os grupos com maior proporção de Romosinuano foramassociados com E3. Os animais que tinham composição racial de Holandês e Blanco Orejinegro tiverammaior estabilidade fenotípica que os outros grupos raciais.

Crecimiento de hembras cruzadas en el trópico colombiano / Crescimento de fêmeas cruzadas no tropico colombiano / Growth of crossbred females in the Colombian tropic

El presente trabajo tuvo como objetivo describir el crecimiento de hembras cruzadas de seis gruposgenéticos por medio del modelo de Brody. Los grupos genéticos evaluados fueron 50% Angus 50% Cebú(AC), 50% BON 50% Cebú (BC), 100% Cebú (C), 50% Romosinuano 50% Cebú (RC), 50% Senepol 25%Angus 25% Cebú (SAC), 50% Senepol 50%Cebú (SC). Se estimó el Porcentaje de madurez a los 12, 18 y24 meses y las edades al 75% y al 95% de madurez. La madurez a los 12 meses varió entre 42% y 48.5%, alos 18 entre 53% y 60% y a los 24 meses entre 61% y 69%; el grupo genético más precoz fue AC y el menosprecoz BC. La edad al 75% de madurez varió entre 1067 y 1468 días; y la edad al 95% de madurez estuvoentre 2396 y 3322 días.

The aim of this study was to describe the growth of crossbred females of six genetic groups using themodel of Brody. Genetic groups evaluated were: 50% Angus 50% Zebu (AZ), 50% BON 50% Zebu (BZ),100% Zebu (Z), 50% Romosinuano 50% Zebu (RZ), 50%Senepol 25% Angus 25% Zebu (SAZ), and 50%Senepol 50%Zebu (SZ). The percent of maturity at 12, 18, and 24 months and ages at 75% and 95% ofmaturity was estimated. The maturity at 12 months varied between 42% and 48.5%, at 18 months between 53% and 60%, and at 24 months between 61% and 69%. The genetic group more precocious was AZ, andless precocious was BZ. The age at 75% of maturity varied between 1067 and 1468 days; and age at 95%of maturity varied between 2396 and 3322 days.

Este estudo teve como objetivo descrever o crescimento de fêmeas cruzadas de seis grupos genéticos,utilizando o modelo de Brody. Os grupos genéticos foram: 50% Angus 50% Zebu (AZ), 50% BON 50% Zebu(BC), 100% Zebu (Z) 50% Romosinuano 50% Zebu (RZ), 50%Angus 25% Senepol e 25 % Zebu (SAZ), 50%Zebu e 50% Senepol (ZC). Foi estimada a percentagem de maturidade aos 12, 18 e 24 meses e a idade ao75% e 95% de maturidade. A madures aos 12 meses oscilou entre 42% e 48.5%, para 18 entre 53% e 60%, e24 meses entre 61% e 69%, o mais antigo grupo genético e da AC foi menos precoce BC. A idade variou de75% vencimento entre 1067 e 1468 dias, e idade de maturidade foi de 95% entre 2396 e 3322 dias.

Association between increased CCL2 (MCP-1) expression in lesions and persistence of disease activity in giant-cell arteritis.

OBJECTIVE: Patients with giant-cell arteritis (GCA) usually respond dramatically to corticosteroid treatment. However, recurrences are frequent and corticosteroid requirements are highly variable among patients. The aim of our study was to identify genes potentially involved in disease persistence. METHODS: Gene expression was explored with cDNA arrays in temporal artery biopsies from six GCA patients with relapsing disease and six patients who easily achieved sustained remission. Differentially expressed genes of interest were subsequently analysed by quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry in temporal artery biopsies from 35 patients with biopsy-proven GCA and nine controls. RESULTS: CCL2 (MCP-1) was up-regulated in temporal artery samples from relapsing individuals. In the extended series of patients, CCL2 mRNA concentration in lesions was significantly higher than in controls (31 +/- 15.6 vs 0.44 +/- 0.10, P = 0.0001). In addition, CCL2 was more abundant in patients who experienced two or more relapses during the first year compared with those who endured sustained remission (127 +/- 82 vs 11 +/- 5.5, P = 0.0233) and correlated with the cumulated prednisolone dose (R = 0.533, P = 0.0024). CCL2 mRNA concentration correlated with IL-1beta (R = 0.45, P = 0.02), tumour necrosis factor-alpha (TNF-alpha) (R = 0.47, P = 0.013) and IL-6 (R = 0.52, P = 0.0053) mRNA. However, circulating CCL2 determined by ELISA was decreased in patients with strong systemic inflammatory response, suggesting that reduction in circulating CCL2 may reinforce the local gradient in lesions. CONCLUSION: Increased CCL2 (MCP-1) expression in lesions is associated with persistence of disease activity in GCA.

Tumor suppressor p53 regulates heparanase gene expression.

Mammalian heparanase degrades heparan sulfate, the most prominent polysaccharide of the extracellular matrix. Causal involvement of heparanase in tumor progression is well documented. Little is known, however, about mechanisms that regulate heparanase gene expression. Mutational inactivation of tumor suppressor p53 is the most frequent genetic alteration in human tumors. p53 is a transcription factor that regulates a wide variety of cellular promoters. In this study, we demonstrate that wild-type (wt) p53 binds to heparanase promoter and inhibits its activity, whereas mutant p53 variants failed to exert an inhibitory effect. Moreover, p53-H175R mutant even activated heparanase promoter activity. Elimination or inhibition of p53 in several cell types resulted in a significant increase in heparanase gene expression and enzymatic activity. Trichostatin A abolished the inhibitory effect of wt p53, suggesting the involvement of histone deacetylation in negative regulation of the heparanase promoter. Altogether, our results indicate that the heparanase gene is regulated by p53 under normal conditions, while mutational inactivation of p53 during cancer development leads to induction of heparanase expression, providing a possible explanation for the frequent increase of heparanase levels observed in the course of tumorigenesis.

Molecular properties and involvement of heparanase in cancer progression and mammary gland morphogenesis.

Tumor spread involves degradation of various components of the extracellular matrix and blood vessel wall. Among these is heparan sulfate proteoglycan, which plays a key role in the self-assembly, insolubility and barrier properties of basement membranes and extracellular matrices. Expression of an endoglycosidase (heparanase) which degrades heparan sulfate correlates with the metastatic potential of tumor cells, and treatment with heparanase inhibitors markedly reduces the incidence of metastasis in experimental animals. Heparin-binding angiogenic proteins are stored as a complex with heparan sulfate in the microenvironment of tumors. These proteins are released and can induce new capillary growth when heparan sulfate is degraded by heparanase. Here, we describe the molecular properties, expression and involvement in tumor progression of a human heparanase. The enzyme is synthesized as a latent approximately 65 kDa protein that is processed at the N-terminus into a highly active approximately 50 kDa form. The heparanase mRNA and protein are preferentially expressed in metastatic human cell lines and in tumor biopsy specimens, including breast carcinoma. Overexpression of the heparanase cDNA in low-metastatic tumor cells conferred a high metastatic potential in experimental animals, resulting in an increased rate of mortality. The heparanase enzyme also released ECM-resident bFGF in vitro, and its overexpression elicited an angiogenic response in vivo. Heparanase may thus facilitate both tumor cell invasion and neovascularization, two critical steps in tumor progression. Mammary glands of transgenic mice overexpressing the heparanase enzyme exhibit precocious branching of ducts and alveolar development, suggesting that the enzyme promotes normal morphogenesis and possibly pre-malignant changes in the mammary gland.

Stimulation of tumor growth by human soluble intercellular adhesion molecule-1.

Because serum levels of soluble intercellular adhesion molecule-1 (sICAM-1) are elevated in cancer and sICAM-1 is angiogenic, we tested the ability of sICAM-1 to promote tumor growth. Our preliminary experiments showed that exogenous sICAM-1 significantly stimulated the growth of human tumors in vivo. Human fibrosarcoma transfectants, which express ICAM-1, produce ICAM-1 on the cell surface and release sICAM-1 into the medium without any apparent effect on cell growth in vitro. We found that conditioned medium from sense ICAM-1 transfectants compared with mock or antisense ICAM-1 transfectants stimulates endothelial cell migration in vitro and neovascularization in the chick chorioallantoic membrane assay. Tumor cells transfected with sense constructs form faster growing tumors than mock- and antisense-transfected cells in both chick embryos and nude mice models. Serum levels of human sICAM-1 from nude mice bearing sense ICAM-1 transfectants correlate positively with tumor weight. Sense ICAM-1 transfectants are more proliferative and induce more blood vessel formation than mock and antisense transfectants in nude mice. Because expression of ICAM-1 does not affect tumor cell growth in vitro, the angiogenic activity of sICAM-1 produced by sense ICAM-1 transfectants may be involved in the stimulation of tumor growth. Therefore, sICAM-1 may perform dual functions that are essential for tumor growth: angiogenesis and escape from immune surveillance.

Halofuginone: a potent inhibitor of critical steps in angiogenesis progression.

We have previously demonstrated that halofuginone, a low molecular weight quinazolinone alkaloid, is a potent inhibitor of collagen alpha1(I) and matrix metalloproteinase 2 (MMP-2) gene expression. Halofuginone also effectively suppresses tumor progression and metastasis in mice. These results together with the well-documented role of extracellular matrix (ECM) components and matrix degrading enzymes in formation of new blood vessels led us to investigate the effect of halofuginone on the angiogenic process. In a variety of experimental system, representing sequential events in the angiogenic cascade, halofuginone treatment resulted in profound inhibitory effect. Among these are the abrogation of endothelial cell MMP-2 expression and basement membrane invasion, capillary tube formation, and vascular sprouting, as well as deposition of subendothelial ECM. The most conclusive anti-angiogenic activity of halofuginone was demonstrated in vivo (mouse corneal micropocket assay) by showing a marked inhibition of basic fibroblast growth factor (bFGF) -induced neovascularization in response to systemic administration of halofuginone, either i.p. or in the diet. The ability of halofuginone to interfere with key events in neovascularization, together with its oral bioavailability and safe use as an anti-parasitic agent, make it a promising drug for further evaluation in the treatment of a wide range of diseases associated with pathological angiogenesis.

Perfil sociodemográfico y laboral de los odontólogos en la ciudad de Medellín / Socialdemographic and labor profile of the odontologists in Medellin, Colombia

Como parte de una investigación sobre el mercado de trabajo de los odontólogos en Medellín, realizada por los mismos autores de 1999, en este artículo se analizan las características de la demanda de dichos profesionales con base en una muestra de 303 odontólogos ocupados en esta ciudad. De conjunto, estos profesionales se caracterizan por ser adultos jóvenes con edad promedio de 40 años formados en un 65 por ciento en la Universidad de Antioquia y en un 22 por ciento en el CES. El 47 por ciento procede de hogares paternos localizados en los dos estratos socioeconómicos más altos. Comparados con estos, los profesionales han logrado cierta movilidad social, en tanto que los hogares formados por fuera de la esfera paterna alcanzan en un 62 por ciento los dos estratos más altos. Cerca del 39 por ciento de los odontólogos labora en dos o más instituciones. Sin embargo, este pluriempleo no deriva en excesos de la jornada laboral. De hecho, el 77 por ciento trabaja menos de 45 horas a la semana, lo cual se corresponde con la alta proporción de profesionales o subempleados (45 por ciento ). La antiguedad promedio en el trabajo asciende a 8,3 años con diferencias estadísticamente significativas entre los que laboran en el sector privado (7,4 años) y los que lo hacen el el sector público (10,9 años). Con todo, la proporción actual de empleo temporal no es despreciable (7,3 por ciento). El ingreso promedio de un odontólogo en la ciudad de Medellín (todos lo empleos), ascienden a $ 1.813.862 mensuales, con diferencia estadísticamete significativas entre quienes solo tienen pregrado ($ 1.695.689) y los que han cursado al menos un posgrado ( $2.166.382). El empleo principal, el promedio de ingresos alcanza $1.839.806 mensuales, con diferencias estadísticamente significativas entre los del sector público y los del sector privado o entre quienes laboran bajo contrato permanente y los de contratación temporal

Mammalian heparanase as mediator of tumor metastasis and angiogenesis.

Expression of heparan sulfate-degrading endoglycosidases, commonly referred to as heparanases, correlates with the metastatic potential of tumor cell lines, and treatment with heparanase inhibitors markedly reduces the incidence of metastasis in experimental animals. We purified a 50 kDa heparanase from human hepatoma and placenta and cloned a cDNA and gene encoding a protein of 543 amino acids. Only one heparanase sequence was identified, suggesting that this enzyme is the dominant endoglucuronidase in mammalian tissues. Expression of the cloned cDNA in insect and mammalian cells yielded 65 kDa and 50 kDa recombinant proteins. The 50 kDa enzyme represents an N-terminal processed enzyme that is at least 200-fold more active than the full-length 65 kDa form. Processing was demonstrated following incubation of the full-length recombinant enzyme with intact tumor cells. The heparanase mRNA and protein are preferentially expressed in metastatic cell lines and in specimens of human melanomas and carcinomas. In the colon, both the heparanase mRNA and protein are expressed already at the stage of tubulovillous adenoma, but not in the adjacent 'normal-looking' colon epithelium. Non-metastatic murine T lymphoma and melanoma cells transfected with the heparanase gene acquired a highly metastatic phenotype in vivo. Apart from its involvement in the egress of cells from the vasculature, heparanase is tightly involved in angiogenesis, both directly--by promoting invasion of endothelial cells (vascular sprouting), and indirectly--by releasing heparan sulfate-bound basic fibroblast growth factor, and generating HS degradation fragments that promote bFGF activity. The angiogenic potential of heparanase was demonstrated in vivo (Matrigel plug assay) by showing a three to fourfold increase in neovascularization induced by Eb T lymphoma cells following their transfection with the heparanase gene. The ability of heparanase to promote both tumor angiogenesis and metastasis makes it a promising target for cancer therapy.

Inhibition of matrix metalloproteinase-2 expression and bladder carcinoma metastasis by halofuginone.

Matrix metalloproteinase-2 (MMP-2) plays a critical role in tumor cell invasion and metastasis. Inhibitors of this enzyme effectively suppress tumor metastasis in experimental animals and are currently being tested in clinical trials. MMP-2 transcriptional regulation is a part of a delicate balance between the expression of various extracellular matrix (ECM) constituents and ECM degrading enzymes. Halofuginone, a low-molecular-weight quinazolinone alkaloid, is a potent inhibitor of collagen type alpha1 (I) gene expression and ECM deposition. We now report that expression of the MMP-2 gene by murine (MBT2-t50) and human (5637) bladder carcinoma cells is highly susceptible to inhibition by halofuginone. Fifty percent inhibition was obtained in the presence of as little as 50 ng/ml halofuginone. This inhibition is due to an effect of halofuginone on the activity of the MMP-2 promoter, as indicated by a pronounced suppression of chloramphenicol acetyltransferase activity driven by the MMP-2 promoter in transfected MBT2 cells. There was no effect on chloramphenicol acetyltransferase activity driven by SV40 promoter in these cells. Halofuginone-treated cells failed to invade through reconstituted basement-membrane (Matrigel) coated filters, in accordance with the inhibition of MMP-2 gene expression. A marked reduction (80-90%) in the lung colonization of MBT2 bladder carcinoma cells was obtained after the i.v. inoculation of halofuginone-treated cells as compared with the high metastatic activity exhibited by control untreated cells. Under the same conditions, there was almost no effect of halofuginone on the rate of MBT2 cell proliferation. These results indicate that the potent antimetastatic activity of halofuginone is due primarily to a transcriptional suppression of the MMP-2 gene, which results in a decreased enzymatic activity, matrix degradation, and tumor cell extravasation. This is the first description, to our knowledge, of a drug that inhibits experimental metastasis through the inhibition of MMP-2 at the transcriptional level. Combined with its known inhibitory effect on collagen synthesis and ECM deposition, halofuginone is expected to exert a profound anticancerous effect by inhibiting both the primary tumor stromal support and metastatic spread.

Inhibition of heparanase activity and tumor metastasis by laminarin sulfate and synthetic phosphorothioate oligodeoxynucleotides.

Heparanase activity correlates with the metastatic potential of tumor cells. Moreover, the anti-metastatic effect of non-anti-coagulant species of heparin and certain sulfated polysaccharides was attributed to their heparanase-inhibiting activity. We investigated the effect of a chemically sulfated polysaccharide (laminarin), consisting primarily of beta-1,3 glucan (sodium laminarin), and of synthetic phosphorothioate oligodeoxynucleotides, primarily phosphorothioate homopolymer of cytidine (SdC28), on heparanase activity and tumor metastasis. Investigation of the ability of tumor cells to degrade heparan sulfate in intact extracellular matrix revealed that heparanase activity expressed by B16-BL6 mouse melanoma cells and 13762 MAT rat mammary adenocarcinoma cells was effectively inhibited by LS (50% inhibition at 0.2-1 microgram/ml), but there was no inhibition by sodium laminarin up to a concentration of 50 microgram/ml. Complete inhibition of the melanoma heparanase was obtained in the presence of 0.1 microM SdC28. A single i.p. injection of laminarin sulfate, but not of sodium laminarin, before i.v. inoculation of the melanoma or breast-carcinoma cells inhibited the extent of lung colonization by the tumor cells by 80 to 90%. Similar inhibition was exerted by 0.1 microM SdC28. At the effective concentrations, both compounds had a small effect on proliferation of the tumor cells and on growth of the primary tumors in vivo. These results further emphasize the involvement of heparanase in tumor metastasis and the potential clinical application of diverse heparanase-inhibiting molecules such as sulfated polysaccharides and synthetic polyanionic molecules.

Inhibition of bladder carcinoma angiogenesis, stromal support, and tumor growth by halofuginone.

We have previously demonstrated that halofuginone, a widely used alkaloid coccidiostat, is a potent inhibitor of collagen alpha1(I) and matrix metalloproteinase 2 gene expression. Halofuginone also suppresses extracellular matrix deposition and cell proliferation. We investigated the effect of halofuginone on transplantable and chemically induced mouse bladder carcinoma. In both systems, oral administration of halofuginone resulted in a profound anticancerous effect, even when the treatment was initiated at advanced stages of tumor development. Although halofuginone failed to prevent proliferative preneoplastic alterations in the bladder epithelium, it inhibited further progression of the chemically induced tumor into a malignant invasive stage. Histological examination and in situ analysis of the tumor tissue revealed a marked decrease in blood vessel density and in both collagen alpha1(I) and H19 gene expression. H19 is regarded as an early marker of bladder carcinoma. The antiangiogenic effect of halofuginone was also demonstrated by inhibition of microvessel formation in vitro. We attribute the profound antitumoral effect of halofuginone to its combined inhibition of the tumor stromal support, vascularization, invasiveness, and cell proliferation.

Mammalian heparanase: gene cloning, expression and function in tumor progression and metastasis.

Heparan sulfate proteoglycans interact with many extracellular matrix constituents, growth factors and enzymes. Degradation of heparan sulfate by endoglycosidic heparanase cleavage affects a variety of biological processes. We have purified a 50-kDa heparanase from human hepatoma and placenta, and now report cloning of the cDNA and gene encoding this enzyme. Expression of the cloned cDNA in insect and mammalian cells yielded 65-kDa and 50-kDa recombinant heparanase proteins. The 50-kDa enzyme represents an N-terminally processed enzyme, at least 100-fold more active than the 65-kDa form. The heparanase mRNA and protein are preferentially expressed in metastatic cell lines and specimens of human breast, colon and liver carcinomas. Low metastatic murine T-lymphoma and melanoma cells transfected with the heparanase cDNA acquired a highly metastatic phenotype in vivo, reflected by a massive liver and lung colonization. This represents the first cloned mammalian heparanase, to our knowledge, and provides direct evidence for its role in tumor metastasis. Cloning of the heparanase gene enables the development of specific molecular probes for early detection and treatment of cancer metastasis and autoimmune disorders.

The dynamics of the imprinted H19 gene expression in the mouse model of bladder carcinoma induced by N-butyl-N-(4-hydroxybutyl)nitrosamine.

The imprinted H19 gene product is an oncofetal RNA molecule in humans. It is expressed in fetal bladder, down-regulated postnatally and is re-expressed in human bladder carcinoma. This study was designed to investigate the dynamics of the expression of H19 in the mouse bladder carcinoma induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) and its relation to stages of neoplastic transformation. BBN was administered to mice in the drinking water for 26-28 weeks. The bladders were removed at 5-10 week intervals for histopathological examination and for in situ hybridization for H19 RNA, using a 35S-labeled probe. Following BBN administration expression of H19 first appeared after 5 weeks in the lamina propria adjacent to the basement membrane, concomitant with mucosal hyperplasia. At 11 weeks focal expression was noted in epithelial cells. Invasive carcinomas, of the transitional and squamous sub-types, were seen after 20 weeks and more of BBN administration. At this stage H19 expression was observed in scattered tumor cells, in the connective tissue stroma of the tumor and in the lamina propria underlying the remaining hyperplastic/dysplastic mucosa. Abundant expression of H19 was evident in fetal bladder but was absent in normal adult bladder. We conclude that, similar to humans, the H19 gene product is an oncofetal RNA molecule in the experimental mouse model of bladder carcinoma. In this model H19 is expressed in the connective tissue of the lamina propria prior to its expression in epithelial cells, concurrent with preneoplastic changes in the transitional epithelium of the bladder.

Characterization of the imprinted IPW gene: allelic expression in normal and tumorigenic human tissues.

IPW (Imprinted gene in the Prader-Willi syndrome region) is a recently identified paternally expressed gene. Previous work has demonstrated IPW expression in the human fetus and adult, with monoallelic expression in adult lymphoblasts and fibroblasts, and in fetal tissues. To further examine the expression of IPW, a series of experiments were carried out using RT-PCR to measure IPW expression in placentae and various fetal and tumor tissues. Biallelic expression of IPW was found in testicular germ cell tumor and bladder cancer cells, suggesting loss of imprinting in the latter case. Both H19 and Insulin-like growth Factor 2 (IGF2), two additional imprinted genes, also showed biallelic expression in those same tumors that demonstrated IPW biallelic expression. Of note, the naturally occurring parthenogenetic-derived mature teratoma unexpectedly expressed large amounts of IPW. Lastly, the pluripotent embryonal cancer cell line Tera-2 expressed IPW at the same level before and after differentiation induced by retinoic acid, suggesting that this gene functions in a 'housekeeping' capacity throughout cell growth. This was in contradistinction to H19 and IGF2, both of which showed significant transcriptional upregulation after Tera-2 cell differentiation.

Purification and characterization of placental heparanase and its expression by cultured cytotrophoblasts.

The role of different extracellular matrix (ECM)-degrading enzymes in the normal functioning of the placenta is well documented. Heparan sulphate proteoglycan (HSPG) is an integral constituent of the placental and decidual ECM. Because this proteoglycan specifically interacts with various macromolecules in the ECM, its degradation may disassemble the matrix. Hence, in the case of the placenta, this may facilitate normal placentation and trophoblast invasion. Crude placental specimens were collected from first and third trimester placentas. Heparanase (endo-beta-glucuronidase) was isolated and purified by ammonium sulphate precipitation followed by sequential chromatographies on carboxymethyl-, heparin- and ConA-Sepharose columns. The placental enzyme was further characterized for its molecular weight and specific inhibition by heparin, and was shown to resemble heparanase expressed by highly metastatic tumor cells and activated cells of the immune system. In order to locate the source of heparanase activity in the placenta, primary cytotrophoblast cultures were established. Intact cells, as well as conditioned medium and cell lysates, were analysed for heparanase activity using metabolically sulphate-labelled ECM as a natural substrate. Heparanase was highly active in lysates of cytotrophoblasts. This activity was also expressed by intact cytotrophoblasts seeded on ECM, but no activity could be detected in the culture medium. Incubation of the cytotrophoblasts in contact with ECM resulted in release of ECM-bound basic fibroblast growth factor (bFGF). We propose that the cytotrophoblastic heparanase facilitates placentation, through cytotrophoblast extravasation and localized neovascularization.