Parallel oxygenators in the same circuit for refractory hypoxemia on veno-venous extracorporeal membrane oxygenation. A 3-patient series.

Extracorporeal membrane oxygenator (ECMO) is a well-established therapy for respiratory failure. Refractory hypoxemia, despite the use of ECMO, remains a challenging problem. The ECMO circuit may not provide enough oxygenation support in the presence of high cardiac output, increased physiologic demand, and impaired gas exchange. Adding a second ECMO oxygenator using the same pump (sometimes needing a second drainage cannula) can improve oxygenation and facilitate lung-protective ventilation in selected patients. We describe a 3-patient series with severe ARDS secondary to SARS-CoV-2 infection and refractory hypoxemia during ECMO support successfully treated with this approach.

Prostate cancer cell type-specific involvement of the VDR and RXR in regulation of the human PTHrP gene via a negative VDRE.

Parathyroid hormone-related protein (PTHrP) increases the growth and osteolytic potential of prostate cancer cells, making it important to control PTHrP expression in these cells. We show that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its non-hypercalcemic analog, EB1089, decrease PTHrP mRNA and cellular protein levels in the androgen-dependent human prostate cancer cell line LNCaP and its androgen-independent derivative, the C4-2 cell line. This effect is mediated via a negative Vitamin D response element (nVDREhPTHrP) within the human PTHrP gene and involves an interaction between nVDREhPTHrP and the Vitamin D receptor (VDR). The retinoid X receptor (RXR) is a frequent heterodimeric partner of the VDR. We show that RXRalpha forms part of the nuclear protein complex that interacts with nVDREhPTHrP along with the VDR in LNCaP and C4-2 cells. We also show that the RXR ligand, 9-cis-retinoic acid, downregulates PTHrP mRNA levels; this decrease is more pronounced in LNCaP than in C4-2 cells. In addition, 9-cis-retinoic acid enhances the 1,25(OH)2D3-mediated downregulation of PTHrP expression in both cell lines; this effect also is more pronounced in LNCaP cells. Proliferation of LNCaP, but not C4-2, cells is decreased by 9-cis-retinoic acid. Promoter activity driven by nVDREhPTHrP cloned upstream of the SV40 promoter and transiently transfected into LNCaP and C4-2 cells is downregulated in response to 1,25(OH)2D3 and EB1089 in both cell lines. Co-treatment with these compounds and 9-cis-retinoic acid further decreases CAT activity in LNCaP, but not C4-2, cells. These results indicate that PTHrP gene expression is regulated by 1,25(OH)2D3 in a cell type-specific manner in prostate cancer cells.

Reemplazo de aorta ascendente con paro circulatorio con hipotermia sistémica moderada y perfusión cerebral retrógrada / Total replacement of the ascending aorta with moderate systemic hypothermic circulatory arrest and retrograde cerebral perfusion

Resumen: Antecedentes: El daño neurológico es una complicación devastadora de la cirugía con paro circulatorio del cayado aórtico y aorta ascendente. La perfusión anterógrada del encéfalo permite disminuir la incidencia de esta complicación, pero es un procedimiento engorroso que interfiere el campo quirúrgico. Para procedimientos más simples que requieran de paro circulatorio de menor duración, la Perfusión Cerebral Retrógrada (PCR) es una alternativa válida. Objetivo: Evaluar nuestros resultados en la cirugía del reemplazo total de la aorta ascendente tubular con paro circulatorio con hipotermia sistémica moderada y PCR. Material y Método: Entre enero de 2015 y enero de 2020 se identificaron los pacientes en la Base de Datos del Servicio de Cirugía Cardiaca de nuestra institución, se revisaron los protocolos operatorios, registros de perfusión y epicrisis, para obtener datos demográficos, clínicos y quirúrgicos pertinentes. La supervivencia alejada se certificó a través del "Servicio Registro Civil e Identificación de Chile". Resultados: En el periodo en estudio, 27 pacientes (21 hombres) tuvieron un reemplazo total de la aorta ascendente tubular con paro circulatorio con hipotermia moderada y PCR. Ocho pacientes tenían una cirugía previa; 7 de estos un reemplazo valvular aórtico. El 75% de los otros 20 pacientes tenía una válvula aórtica bicúspide. El diámetro máximo de la aorta ascendente fue en promedio 53,7 mm (45 a 67), y fue reemplazada en el 52% de los casos con un tubo protésico de 34 mm (promedio:32,4 mm; margen:30 a 34 mm). En 20 pacientes se efectuó un reemplazo valvular aórtico (15 con prótesis biológica). El tiempo promedio de circulación extracorpórea fue 174,6 min (97 a 243) y la temperatura sistémica mínima promedio fue 21ºC (18 a 25). El tiempo promedio de paro circulatorio fue 22,3 min (12 a 40) y de PCR 13 min (6 a 27). No hubo mortalidad operatoria. La morbilidad más frecuente fue la fibrilación auricular (33%). Una paciente presentó un episodio convulsivo aislado y otro fue reoperado por hemorragia postoperatoria. Una paciente falleció a los 48 meses de su operación. Conclusión: El paro circulatorio con hipotermia sistémica moderada y PCR para la cirugía de reemplazo total de la aorta ascendente facilitó la operación, con baja mortalidad y morbilidad en este grupo de pacientes.

Abstract: Background. Neurological damage is a devastating complication of aortic arch and ascending aorta surgery with deep hypothermic circulatory arrest. Antegrade cerebral perfusion significantly decreases the incidence of this complication, but it is a cumbersome procedure that interfere the surgical field. For more simple procedures, requiring a shorter period of circulatory arrest, retrograde cerebral perfusion (RCP) would be a valid alternative. Objective. To evaluate the results of total surgical replacement of the tubular ascending aorta with moderate hypothermic circulatory arrest and retrograde cerebral perfusion (RCP). Methods. Patients operated between January 2015 and January 2020 were included.Demographic, clinical and surgical information was obtained from the operatives notes, perfusion registry and discharge reports. Long-term survival was certified by the "Chilean Civil and Identification Registry". Results. 27 patients (21 men) underwent a total replacement of the tubular ascending aorta with circulatory arrest with moderate hypothermia and RCP. Eight patients had been previously operated on;7 of them had a previous aortic valve replacement. Of the remaining 20 patients, 75% had a bicuspid aortic valve. Average maximum diameter of the ascending aorta was 53.7 mm (45 - 67). Average size of the ascending aorta replacement graft was 32.4 mm (30 -34). In 20 patients a concomitant aortic valve replacement was performed (15 with a biological valve). Mean extracorporeal circulation time was 174.6 min (97 - 243) and mean minimal systemic temperature was 21ºC (18 - 25). Mean circulatory arrest time was 22.3 min (12 - 40) and mean RCP time was 13 min (6 - 27), There was no operative mortality. Atrial fibrillation was the most frequent post-operative morbidity (33%). One patient presented an isolated convulsive episode and another was re-operated due to postoperative hemorrhage. One patient died, 48 months after her operation. Conclusion. Moderate hypothermic circulatory arrest with RCP simplifies total tubular ascending aorta replacement, with low mortality and morbidity.

Intracrine PTHrP protects against serum starvation-induced apoptosis and regulates the cell cycle in MCF-7 breast cancer cells.

PTHrP is secreted by breast cancer cells in vivo and in vitro. In the breast cancer cell line MCF-7, PTHrP overexpression is associated with increased mitogenesis. We used this cell line to study the mechanism for the proliferative effects of PTHrP. Clonal MCF-7 lines were established overexpressing wild-type PTHrP or PTHrP mutated in the nuclear localization signal (NLS). Mutation of the NLS negated the proliferative effects and nuclear trafficking of PTHrP, indicating that increased mitogenesis is mediated via an intracrine pathway. Cells overexpressing wild-type PTHrP were enriched in G2 + M stage of the cell cycle compared with cells overexpressing NLS-mutated PTHrP, indicating an intracrine role for PTHrP in cell cycle regulation. Wild-type PTHrP also protected MCF-7 cells from serum starvation-induced apoptosis. Cells overexpressing wild-type PTHrP showed significantly greater cell survival than cells overexpressing NLS-mutated PTHrP. The ratios of the apoptosis-regulating proteins Bcl-2 to Bax and Bcl-x(L) to Bax were higher in cells overexpressing wild-type, but not NLS-mutated, PTHrP compared with control cells. These findings suggest that the proliferative effects of PTHrP in breast cancer cells are mediated through regulation of the cell cycle and apoptosis, and that controlling PTHrP production in breast cancer may be therapeutically useful.

Regulation of PTH-related protein gene expression by vitamin D in PC-3 prostate cancer cells.

Parathyroid hormone-related protein (PTHrP) is expressed by prostate cancer cells. Since PTHrP increases prostate cancer cell growth and enhances the osteolytic effects of prostate cancer cells, it is important to control PTHrP expression in prostate cancer. Vitamin D exerts a protective effect against prostate cancer through its antiproliferative actions. We investigated whether this steroid also downregulates PTHrP gene transcription, using the human prostate cancer cell line PC-3 as a model system. We report that PTHrP mRNA and secreted protein levels are downregulated by 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) via a transcriptional mechanism. We also show that PTHrP gene expression is upregulated, also via a transcriptional mechanism, by epidermal growth factor (EGF), which is normally secreted by prostate cancer cells. 1,25(OH)(2)D(3) reversed the EGF-induced PTHrP upregulation at both the mRNA and protein levels. Since PTHrP enhances prostate cancer cell growth, this study demonstrates the importance of maintaining adequate levels of 1,25(OH)(2)D(3).

Prostate cancer cell type-specific regulation of the human PTHrP gene via a negative VDRE.

Parathyroid hormone-related protein (PTHrP) is expressed by prostate cancer cells. Since PTHrP increases the growth and enhances the osteolytic effects of prostate cancer cells, it is important to control the level of PTHrP expression in these cells. We show that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its non-calcemic analogue, EB1089, suppress PTHrP mRNA and protein levels in the human prostate cancer cell lines PC-3 and LNCaP. The human PTHrP gene contains a sequence element homologous to the negative vitamin D response element within the parathyroid hormone gene. This DNA sequence (nVDRE(hPTHrP)) bound the vitamin D receptor (VDR) present in nuclear extracts from both PC-3 and LNCaP cells. However, when cloned upstream of the SV40 promoter and transiently transfected into PC-3 and LNCaP cells, nVDRE(hPTHrP) downregulated promoter activity in response to 1,25(OH)2D3 or EB1089 treatment in LNCaP, but not in PC-3, cells. These results may help to explain why some prostate cancers appear to be refractory to treatment with vitamin D analogues.

Parathyroid hormone-related protein enhances PC-3 prostate cancer cell growth via both autocrine/paracrine and intracrine pathways.

Parathyroid hormone-related protein (PTHrP) is expressed by human prostatic tissue and prostate cancer cell lines, and positively influences primary prostate tumor growth in vivo. The human prostate cancer cell line PC-3, which expresses functional PTH/PTHrP receptors, was used as a model to study the effects of PTHrP on prostate cancer cell growth. Addition of PTHrP (1-34), (1-86), and (1-139) increased cell number and [3H]thymidine incorporation; these effects were reversed by anti-PTHrP antiserum. This antiserum also decreased endogenous PC-3 cell growth. Clonal PTHrP-overexpressing PC-3 cell lines also showed enhanced cell growth and [3H]thymidine incorporation and were enriched in the G2+M phase of the cell cycle, suggesting an effect of PTHrP on mitosis. Overexpression of PTHrP with the nuclear localization sequence (NLS) deletion partially reversed the growth-stimulatory effects. The growth rate of these cells was midway between that of wild-type PTHrP-overexpressing and control cells, presumably because NLS-mutated PTHrP is still secreted and acts through the cell surface PTH/PTHrP receptor. In contrast to NLS-mutated PTHrP, wild-type protein showed preferential nuclear localization. These results suggest that the proliferative effects of PTHrP in PC-3 cells are mediated via both autocrine/paracrine and intracrine pathways, and that controlling PTHrP production in prostate cancer may be therapeutically beneficial.

Chemoprevention of BBN-Induced Bladder Carcinogenesis by the Selective Estrogen Receptor Modulator Tamoxifen.

Bladder cancer is the fifth most frequent tumor in men and ninth in women in the United States. Due to a high likelihood of recurrence, effective chemoprevention is a significant unmet need. Estrogen receptors (ERs), primarily ERß, are expressed in normal urothelium and urothelial carcinoma, and blocking ER function with selective ER modulators such as tamoxifen inhibits bladder cancer cell proliferation in vitro. Herein, the chemoprotective potential of tamoxifen was evaluated in female mice exposed to the bladder-specific carcinogen, N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Carcinogen treatment resulted in a 76% tumor incidence and increased mean bladder weights in comparison to controls. In contrast, mice receiving tamoxifen concurrent (8-20 weeks) or concurrent and subsequent (8-32 weeks) to BBN administration had no change in bladder weight and only 10% to 14% incidence of tumors. Non-muscle-invasive disease was present in animals treated with tamoxifen before (5-8 weeks) or after (20-32 weeks) BBN exposure, while incidence of muscle-invasive bladder carcinoma was reduced. ERß was present in all mice and thus is a potential mediator of the tamoxifen chemoprotective effect. Surprisingly, ERα expression, which was detected in 74% of the mice exposed to BBN alone but not in any controlmice, was correlated with tumor incidence, indicating a possible role for this receptor in carcinogen-induced urothelial tumorigenesis. Thus, these data argue that both ERα and ERß play a role in modulating carcinogen-induced bladder tumorigenesis. Administration of tamoxifen should be tested as a chemopreventive strategy for patients at high risk for bladder cancer recurrence.

The functional consequences of cross-talk between the vitamin D receptor and ERK signaling pathways are cell-specific.

The actions of the active metabolite of 1,25-(OH)2D3 (1,25-D) are mediated primarily by the vitamin D receptor (VDR), a member of the nuclear receptor family of ligand-activated transcription factors. Although their ligands cause transcriptional activation, many of the ligands also rapidly activate cellular signaling pathways through mechanisms that have not been fully elucidated. We find that 1,25-D causes a rapid, but sustained activation of ERK (extracellular signal-regulated kinase) in bone cell lines. However, the effect of ERK activation on VDR transcriptional activity was cell line-specific. Inhibition of ERK activation by the MEK inhibitor, U0126, stimulated VDR activity in MC3T3-E1 cells, but inhibited the activity in MG-63 cells as well as in HeLa cells. VDR is not a known target of ERK. We found that the ERK target responsible for reduced VDR activity in MC3T3-E1 cells is RXRalpha. MC3T3-E1 cells express lower levels of RXRbeta and RXRgamma than either HeLa or MG-63 cells. Although overexpression of RXRalpha in MC3T3-E1 cells increased VDR activity, U0126 further enhanced the activity. In contrast, overexpression of RXRgamma stimulated VDR activity but abrogated the stimulation by U0126. Thus, although 1,25-D treatment activates ERK in many cell types, subsequently inducing changes independent of VDR, the effects of treatment with 1,25-D on the transcriptional activity of VDR are RXR isoform-specific. In cells in which RXRalpha is the VDR partner, the transcriptional activation of VDR by 1,25-D is attenuated by the concomitant activation of ERK. In cells utilizing RXRgamma, ERK activation enhances VDR transcriptional activity.

Sales biliares (colilglicina) en líquido amniótico y en sangre de cordón umbilical en pacientes con colestasia intrahepática del embarazo (CIE) / Biliary salts (cholylglicine) in amniotic fluid and blood of umbilical cord in patients with intrahepatic cholestasia of pregnancy

Se diseña un estudio para conocer el comportamiento de la colilglicina, CG, en sangre materna, sangre total de cordón umbilical y en líquido amniótico, en embarazadas sanas y con colestasia intrahepática del embarazo, CIE, en muestras tomadas simultáneamente. A las pacientes seleccionadas y sin otra patología materna o fetal se las dividió en dos grupos. El grupo control está formado por 14 embarazadas sanas, y el grupo con CIE, por ocho pacientes. Se hizo un diagnóstico de CIE por el cuadro clínico, niveles de CG en ayunas superiores a 0,7 ug/ml., y desaparición del prurito después del parto. Durante la cesárea se obtuvo muestra simultánea de líquido amniótico, sangre total del cordón y sangre venosa materna, y se determino CG mediante enzimo-inmunoensayo ENDAB-cholylglicine. Se encontraron valores promedios de CG algo más elevados en sangre de cordón que en sangre materna en ambos grupos, pero sin diferencias significativas: grupo control, X=0,6 más menos 0,7 ug/ml vs. X=0,14 más menos 0,1 ug/ml; grupo con CIE= 2,3 más menos 1,5 ug/ml vs. X=1,6 más menos 0,6 ug/ml. Al comparar por separado cada conjunto madre-hijo se encontró que los valores CG fetal pueden ser diferentes a los de su madre (3 casos), o presentar una correlación estadísticamente significativa: después de eliminar valares extremos, tres muestras donde Z > 1,65 al aplicar prueba de covarianza. Grupo control, R=0,621, p < 0,02; grupo CIE, R=0,958 p < 0,01. Los niveles CG en sangre de cordón en los hijos de madres CIE son tres veces mayores que en los hijos del grupo control. Los valores de CG en líquido amniótico presentaron amplias variaciones en el grupo con CIE.