Platelets enhance tissue factor protein and metastasis initiating cell markers, and act as chemoattractants increasing the migration of ovarian cancer cells.

BACKGROUND: An increase in circulating platelets, or thrombocytosis, is recognized as an independent risk factor of bad prognosis and metastasis in patients with ovarian cancer; however the complex role of platelets in tumor progression has not been fully elucidated. Platelet activation has been associated with an epithelial to mesenchymal transition (EMT), while Tissue Factor (TF) protein expression by cancer cells has been shown to correlate with hypercoagulable state and metastasis. The aim of this work was to determine the effect of platelet-cancer cell interaction on TF and "Metastasis Initiating Cell (MIC)" marker levels and migration in ovarian cancer cell lines and cancer cells isolated from the ascetic fluid of ovarian cancer patients. METHODS: With informed patient consent, ascitic fluid isolated ovarian cancer cells, cell lines and ovarian cancer spheres were co-cultivated with human platelets. TF, EMT and stem cell marker levels were determined by Western blotting, flow cytometry and RT-PCR. Cancer cell migration was determined by Boyden chambers and the scratch assay. RESULTS: The co-culture of patient-derived ovarian cancer cells with platelets causes: 1) a phenotypic change in cancer cells, 2) chemoattraction and cancer cell migration, 3) induced MIC markers (EMT/stemness), 3) increased sphere formation and 4) increased TF protein levels and activity. CONCLUSIONS: We present the first evidence that platelets act as chemoattractants to cancer cells. Furthermore, platelets promote the formation of ovarian cancer spheres that express MIC markers and the metastatic protein TF. Our results suggest that platelet-cancer cell interaction plays a role in the formation of metastatic foci.

Independent anti-angiogenic capacities of coagulation factors X and Xa.

Knockout models have shown that the coagulation system has a role in vascular development and angiogenesis. Herein, we report for the first time that zymogen FX and its active form (FXa) possess anti-angiogenic properties. Both the recombinant FX and FXa inhibit angiogenesis in vitro using endothelial EA.hy926 and human umbilical cord vascular endothelial cells (HUVEC). This effect is dependent on the Gla domain of FX. We demonstrate that FX and FXa use different mechanisms: the use of Rivaroxaban (RX) a specific inhibitor of FXa attenuated its anti-angiogenic properties but did not modify the anti-angiogenic effect of FX. Furthermore, only the anti-angiogenic activity of FXa is PAR-1dependent. Using in vivo models, we show that FX and FXa are anti-angiogenic in the zebrafish intersegmental vasculature (ISV) formation and in the chick embryo chorioallantoic membrane (CAM) assays. Our results provide further evidence for the non-hemostatic functions of FX and FXa and demonstrate for the first time a biological role for the zymogen FX.

[In vitro cell response to chemotherapeutic agents, to personalize ovarian cancer treatment: report of two cases]. / Potencial herramienta en la personalización del tratamiento oncológico: casos clínicos.

Our laboratory has implemented an in vitro assay to estimate the response to chemotherapy in ovarian cancer cells pertaining to individual patients. In two selected patients, we determined the correlation between an in vitro assay of cells from suspected ovarian cancer ascites, with the clinical chemotherapy response. Cancer cells isolated from peritoneal fluid with suspected ovarian cancer were tested for cytotoxicity with corresponding chemotherapy regimens. Circulating Cal25 levels and attending physician consultation determined clinical course and response to chemotherapy. The in vitro assay result correlated with Cal25 levels, progression free survival and attending physician evaluation. The assay predicted correctly the failure of two successive chemotherapy regimes in the first patient, while predicting a favorable clinical response in the second subject.

Progesterone utilizes distinct membrane pools of tissue factor to increase coagulation and invasion and these effects are inhibited by TFPI.

Tissue factor (TF) serving as the receptor for coagulation factor VII (FVII) initiates the extrinsic coagulation pathway. We previously demonstrated that progesterone increases TF, coagulation and invasion in breast cancer cell lines. Herein, we investigated if tissue factor pathway inhibitor (TFPI) could down-regulate progesterone-increased TF activity in these cells. Classically, TFPI redistributes TF-FVII-FX-TFPI in an inactive quaternary complex to membrane associated lipid raft regions. Herein, we demonstrate that TF increased by progesterone is localized to the heavy membrane fraction, despite progesterone-increased coagulation originating almost exclusively from lipid raft domains, where TF levels are extremely low. The progesterone increase in coagulation is not a rapid effect, but is progesterone receptor (PR) dependent and requires protein synthesis. Although a partial relocalization of TF occurs, TFPI does not require the redistribution to lipid rafts to inhibit coagulation or invasion. Inhibition by TFPI and anti-TF antibodies in lipid raft membrane fractions confirmed the dependence on TF for progesterone-mediated coagulation. Through the use of pathway inhibitors, we further demonstrate that the TF up-regulated by progesterone is not coupled to the progesterone increase in TF-mediated coagulation. However, the progesterone up-regulated TF protein may be involved in progesterone-mediated breast cancer cell invasion, which TFPI also inhibits.

Encefalitis amebiana granulomatosa por Balamuthia mandrillaris en Chile confirmada con secuenciación de ADN

Las enfermedades causadas por amebas de vida libre son infecciones oportunistas que pueden tener un curso fatal. Pueden producir afecciones diseminadas graves con compromiso del sistema nervioso central, como la encefalitis amebiana granulomatosa. Esta infección es cada vez más frecuente en América Latina, aunque se reconocen tardíamente debido a la similitud con otras patologías o porque es inusual incluirla en el diagnóstico diferencial. Comunicamos un caso fatal de una encefalitis amebiana granulomatosa por Balamuthia mandrillaris en una niña de 10 años. Destacamos la gravedad de la afectación cerebral y la falta de esquemas antimicrobianos validados para su tratamiento. Hoy en el mundo esta infección es considerada una enfermedad emergente, influenciada por el cambio climático, lo que llama a estar atentos a su presencia.

Diseases caused by free-living amoebae are opportunistic infections that can have a fatal course. They can cause very serious disseminated conditions with involvement of the central nervous system such as granulomatous amoebic encephalitis. This infection has become more common in Latin America, although its recognition is late due to the similarity with other pathological conditions or because it is unusual to include it in the differential diagnosis. We report a fatal case of granulomatous amoebic encephalitis due to Balamuthia mandrillaris in a 10-year-old girl. We highlight the severity of the brain involvement and the lack of validated schemes for its treatment. Today in the world this infection is considered an emerging disease, influenced by climate change, which calls for being attentive to its presence.

Diabetic concentrations of metformin inhibit platelet-mediated ovarian cancer cell progression.

Clinical studies have suggested a survival benefit in ovarian cancer patients with type 2 diabetes mellitus taking metformin, however the mechanism by which diabetic concentrations of metformin could deliver this effect is still poorly understood. Platelets not only represent an important reservoir of growth factors and angiogenic regulators, they are also known to participate in the tumor microenvironment implicated in tumor growth and dissemination. Herein, we investigated if diabetic concentrations of metformin could impinge upon the previously reported observation that platelet induces an increase in the tube forming capacity of endothelial cells (angiogenesis) and upon ovarian cancer cell aggressiveness. We demonstrate that metformin inhibits the increase in angiogenesis brought about by platelets in a mechanism that did not alter endothelial cell migration. In ovarian cancer cell lines and primary cultured cancer cells isolated from the ascitic fluid of ovarian cancer patients, we assessed the effect of combinations of platelets and metformin upon angiogenesis, migration, invasion and cancer sphere formation. The enhancement of each of these parameters by platelets was abrogated by the present of metformin in the vast majority of cancer cell cultures tested. Neither metformin nor platelets altered proliferation; however, metformin inhibited the increase in phosphorylation of focal adhesion kinase induced by platelets. We present the first evidence suggesting that concentrations of metformin present in diabetic patients may reduce the actions of platelets upon both endothelial cells and cancer cell survival and dissemination.

Recomendaciones para el diagnóstico y manejo de la infección por citomegalovirus en la mujer embarazada y el recién nacido / Recommendations for the diagnosis and management of cytomegalovirus infection in pregnant woman and newborn infant

Resumen La Sociedad Chilena de Infectología, a través de su Comité de Infecciones Neonatales, en conjunto con la Sociedad Chilena de Obstetricia y Ginecología, proponen un documento de diagnóstico y manejo de la Infección por Citomegalovirus en la Mujer Embarazada y el Recién Nacido. Esta guía aborda el manejo de la infección en el binomio, su enfrentamiento diagnóstico y terapéutico, orientado al equipo de salud que atiende a mujeres embarazadas y recién nacidos con infección por citomegalovirus (CMV) en Chile. Considera la situación epidemiológica global y latinoamericana, con recomendaciones para la evaluación clínica y de laboratorio; establece criterios de diagnóstico, propone enfoques terapéuticos de acuerdo a la situación clínica, analiza las medidas de prevención y establece una propuesta nacional para el seguimiento de esta enfermedad. Se ha puesto especial énfasis en entregar, de forma práctica, y con la mayor evidencia posible, las recomendaciones para el manejo del binomio con infección por CMV.

Abstract The Chilean Society of Infectology, through its Neonatal Infections Committee in conjunction with the Chilean Society of Obstetrics and Gynecology, propose a document for the Diagnosis and Management of Cytomegalovirus Infection in Pregnant Woman and Newborn Infant. This guideline suggests the management of mother and child infection, its diagnostic and therapeutic options. Considers the global and Latin American epidemiology, with recommendations for clinical and laboratory evaluation; diagnostic criteria, therapeutic approaches according to the clinical situation, analyzes prevention measures and establishes a national proposal for monitoring this disease.

Recomendaciones para el diagnóstico y el manejo de la infección por citomegalovirus en la mujer embarazada y el recién nacido / Recommendations for the diagnosis and management of cytomegalovirus infection in pregnant women and newborns

Resumen La Sociedad Chilena de Infectología, a través de su Comité de Infecciones Neonatales, en conjunto con la Sociedad Chilena de Obstetricia y Ginecología, proponen un documento de diagnóstico y manejo de la infección por citomegalovirus (CMV) en la mujer embarazada y el recién nacido. Esta guía aborda el manejo de la infección en el binomio, su enfrentamiento diagnóstico y terapéutico, orientado al equipo de salud que atiende a mujeres embarazadas y recién nacidos con infección por CMV en Chile. Considera la situación epidemiológica global y latinoamericana, con recomendaciones para la evaluación clínica y de laboratorio; establece criterios de diagnóstico, propone enfoques terapéuticos de acuerdo a la situación clínica, analiza las medidas de prevención y establece una propuesta nacional para el seguimiento de esta enfermedad. Se ha puesto especial énfasis en entregar, de forma práctica, y con la mayor evidencia posible, las recomendaciones para el manejo del binomio con infección por CMV.

Abstract The Chilean Society of Infectology, through its Neonatal Infections Committee in conjunction with the Chilean Society of Obstetrics and Gynecology, propose a document for the Diagnosis and Management of Cytomegalovirus Infection in Pregnancy and Newborn. This guideline suggests the management of mother and child infection, its diagnostic and therapeutic options. Considers the global and Latin American epidemiology, with recommendations for clinical and laboratory evaluation, diagnostic criteria, therapeutic approaches according to the clinical situation, analyzes prevention measures and establishes a national proposal for monitoring this disease.

Progesterone regulation of tissue factor depends on MEK1/2 activation and requires the proline-rich site on progesterone receptor.

To characterize the molecular mechanism and map the response element used by progesterone (P) to upregulate tissue factor (TF) in breast cancer cells. TF expression and mRNA levels were analyzed in breast cancer ZR-75 and T47D cells, using Western blot and real-time PCR, respectively. Mapping of the TF promoter was performed using luciferase vectors. Progesterone receptor (PR) and specificity protein 1 (Sp1) binding to the TF promoter were analyzed by chromatin immuno precipitation assay. Specific or selective inhibitors were used for the MEK1/2 and the c-Src pathways (UO126 and PP2, respectively). TF mRNA increase peaks at 18 h following P treatment in ZR-75 and T47D cells. P upregulation occurs via a transcriptional mechanism that depends on PR and MEK1/2 activation, PR and Sp1 transcription factors bind to a region in the TF promoter that contains three Sp1 sites. TF mRNA upregulation requires an intact PR proline-rich site (mPRO), but it is independent from c-Src. TF upregulation by P is mediated by Sp1 sites in the TF promoter region. Transcriptional upregulation in breast cancer cells occurs via a new mechanism that requires MEK1/2 activation and the mPRO site but independent of c-Src activity. PR Phosphorylation at serine 294 and 345 is not essential.