BNCT research activities at the Granada group and the project NeMeSis: Neutrons for medicine and sciences, towards an accelerator-based facility for new BNCT therapies, medical isotope production and other scientific neutron applications.

The Granada group in BNCT research is currently performing studies on: nuclear and radiobiological data for BNCT, new boron compounds and a new design for a neutron source for BNCT and other applications, including the production of medical radioisotopes. All these activities are described in this report.

Human decidual stromal cells secrete soluble pro-apoptotic factors during decidualization in a cAMP-dependent manner.

STUDY QUESTION: Is there a relationship between decidualization and apoptosis of decidual stromal cells (DSC)? SUMMARY ANSWER: Decidualization triggers the secretion of soluble factors that induce apoptosis in DSC. WHAT IS KNOWN ALREADY: The differentiation and apoptosis of DSC during decidualization of the receptive decidua are crucial processes for the controlled invasion of trophoblasts in normal pregnancy. Most DSC regress in a time-dependent manner, and their removal is important to provide space for the embryo to grow. However, the mechanism that controls DSC death is poorly understood. STUDY DESIGN, SIZE, DURATION: The apoptotic response of DSC was analyzed after exposure to different exogenous agents and during decidualization. The apoptotic potential of decidualized DSC supernatants and prolactin (PRL) was also evaluated. PARTICIPANTS/MATERIALS, SETTING, METHODS: DSC lines were established from samples of decidua from first trimester pregnancies. Apoptosis was assayed by flow cytometry. PRL production, as a marker of decidualization, was determined by enzyme-linked immunosorbent assay. MAIN RESULTS AND THE ROLE OF CHANCE: DSCs were resistant to a variety of apoptosis-inducing substances. Nevertheless, DSC underwent apoptosis during decidualization in culture, with cAMP being essential for both apoptosis and differentiation. In addition, culture supernatants from decidualized DSC induced apoptosis in undifferentiated DSC, although paradoxically these supernatants decreased the spontaneous apoptosis of decidual lymphocytes. Exogenously added PRL did not induce apoptosis in DSC and an antibody that neutralized the PRL receptor did not decrease the apoptosis induced by supernatants. LIMITATIONS, REASONS FOR CAUTIONS: Further studies are needed to examine the involvement of other soluble factors secreted by decidualized DSC in the induction of apoptosis. WIDER IMPLICATIONS OF THE FINDINGS: The present results indicate that apoptosis of DSC occurs in parallel to differentiation, in response to decidualization signals, with soluble factors secreted by decidualized DSC being responsible for triggering cell death. These studies are relevant in the understanding of how the regression of decidua, a crucial process for successful pregnancy, takes place. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the Consejería de Economía, Innovación y Ciencia, Junta de Andalucía (Grant CTS-6183, Proyectos de Investigación de Excelencia 2010 to C.R.-R.) and the Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Spain (Grants PS09/00339 and PI12/01085 to E.G.O.). E.L.-D. was supported by fellowships from the Ministerio de Educación y Ciencia, Spain and the University of Granada. The authors have no conflict of interest.

Fetal-placental hypoxia does not result from failure of spiral arterial modification in mice.

OBJECTIVES: To determine if fetal-placental hypoxia is a primary outcome of defective spiral artery remodeling. STUDY DESIGN: Pregnancies in Rag2(-/-)Il2rg(-/-) double knock-out mice, which fail to undergo normal physiological spiral arterial remodeling, were compared to syngeneic BALB/c control pregnancies. Mice at gestation day (gd)6, 8, 10, 12 and 18 were infused with Hypoxyprobe-1 before euthanasia to enable detection of cellular hypoxia by immunohistochemistry. RESULTS: In implantation sites of both phenotypes, trophoblast cells were reactive to Hypoxyprobe-1. No major differences were observed between the phenotypes in decidua or placenta at any gd or in gd18 fetal brain, lung, heart, liver or intestine or in maternal heart, brain, liver or spleen. Maternal kidneys from BALB/c were significantly hypoxic to Rag2(-/-)Il2rg(-/-) kidneys. CONCLUSIONS: In mice, lack of pregnancy-associated spiral artery remodeling does not impair oxygen delivery to the conceptus, challenging the concept that deficient spiral arterial remodeling leads to fetal hypoxia in human gestational complications such as preeclampsia and fetal growth restriction. The isolated hypoxic response of normal kidney has revealed that renal lymphocytes may have unique, tissue-specific regulatory actions on vasoconstriction that are pregnancy independent.

HDAC inhibitors with different gene regulation activities depend on the mitochondrial pathway for the sensitization of leukemic T cells to TRAIL-induced apoptosis.

Epigenetic modifications commonly associated with tumor development, such as histone deacetylation, may influence the resistance of some tumor cells to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) by regulating gene transcription of components of the TRAIL signalling pathway. In the present study we have analyzed the effect of six different histone deacetylase inhibitors (HDACi), belonging to the four classic structural families, on TRAIL-induced apoptosis in leukemic T cell lines. Non-toxic and functional doses of all HDACi but apicidin, similarly sensitized different leukemic T cell lines to TRAIL-induced apoptosis, while they showed no effect on the resistance of normal T lymphocytes. Sensitizing doses of vorinostat, valproic acid, sodium butyrate and MS-275 regulated the expression of TRAIL-R2, c-FLIP and Apaf-1 in leukemic cells while TSA modulated only the expression of Apaf-1. The synergistic effect of all HDACi and TRAIL was inhibited in Bcl-2-overexpressing leukemic T cells. Thus, different HDACi may affect the expression of different TRAIL-related genes, but regulation of the mitochondrial pathway seems to be essential for the TRAIL sensitizing effect of HDACi in leukemic T cells. Overall, HDACi represent a promising and safe strategy in combination with TRAIL for treatment of T-cell leukaemia.

Orphan receptor kinase ROR2 is expressed in the mouse uterus.

OBJECTIVE: Wingless-type mouse mammary tumor virus integration site family, member 5A (WNT5A), is expressed in mouse decidua and is thought to play an important role in decidualization. We examined expression of the receptor for WNT5A, receptor tyrosine kinase-like orphan receptor 2 (ROR2), in the uteri of cycling and pregnant mice. STUDY DESIGN: Reverse transcription (RT)-PCR and immunohistochemistry were performed. RESULTS: RT-PCR revealed that transcripts for Ror2, Wnt3a, Wnt5a and inhibitor of WNT signaling, Dickkopf homolog 1 (Dkk1), were present in the pregnant uterus. Immunohistochemistry revealed that in the virgin uterus, ROR2 is expressed in stromal cells and on the basal side of uterine gland and endometrial epithelial cells. During pregnancy, both the luminal and basal side of uterine gland epithelial cells expressed ROR2, stromal cell expression of ROR2 became more frequent and ROR2 expressing uterine Natural Killer (NK) cells and cells lining the maternal vascular space emerged. Immunofluorescence imaging and flow cytometry revealed that although uterine NK cells expressed ROR2, NK cells of the spleen were ROR2 negative. CONCLUSION: The expression of ROR2 by endometrial epithelial cells may suggest WNT signaling has roles in uterine epithelial cell polarity or implantation. Expression of ROR2 by uterine NK cells may suggest WNT signaling regulates uterine NK cell functions such angiogenesis and regulation of trophoblast migration. In summary, our results show that ROR2 expression by maternal uterine cells is influenced by pregnancy.

Human decidual stromal cells protect lymphocytes from apoptosis.

Human decidual stromal cells (DSC) have been shown to be involved in different immune functions that may be relevant for the relationship between the mother and fetus and hence for successful pregnancy. The expression of death ligands by fetal trophoblast and maternal decidual cells has been proposed as a mechanism for the establishment of materno-fetal immunotolerance. This study intended to elucidate the interrelations between DSC and lymphocytes. We analyzed the expression and function of death receptors and ligands in DSC maintained in culture. These DSC lines expressed CD95 and TNF-related apoptosis-inducing ligand receptor-2 (TRAIL-R2), although they were resistant to death receptor-mediated apoptosis. Regarding the expression of CD95L and TRAIL, it was variable among DSC lines although none of them induced apoptosis in death ligand-sensitive Jurkat T cells. Interestingly, most of the DSC lines, as well as fresh DSC, reduced apoptosis in Jurkat cells induced by anti-CD95 antibody and recombinant TRAIL. The protective effect of DSC was observed when they were co-cultured with Jurkat cells in Transwell plates, indicating that DSC may produce soluble factors of importance for lymphocyte survival. Moreover, the viability of peripheral blood lymphocytes and decidual lymphocytes was improved when co-cultured with DSC. Our results suggest that DSC, far from inducing apoptosis, may be relevant in the regulation of lymphocyte survival at the materno-fetal interface.

The differential sensitivity of Bc1-2-overexpressing human breast tumor cells to TRAIL or doxorubicin-induced apoptosis is dependent on Bc1-2 protein levels.

Bc1-2 protein is a potent anti-apoptotic protein that inhibits a mitochondria-operated pathway of apoptosis in many cells. DNA damaging agents and death receptor ligands can activate this mitochondrial apoptotic mechanism. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been suggested to escape from the inhibitory action of Bc1-2 protein. We show that in human breast tumor MCF-7 cells, TRAIL induced a mitochondrial pathway of apoptosis that involved cytochrome c release from mitochondria and activation of caspase 9. The DNA damaging drug doxorubicin also activated this mitochondria-regulated mechanism of apoptosis, which was inhibited in Bc1-2-overexpressing cells. We also demonstrate that in MCF-7 cells Bc1-2 might confer resistance to TRAIL-induced apoptosis, depending on the expression levels of the anti-apoptotic protein. These results indicate that enhanced expression of Bc1-2 in tumor cells can render these cells less sensitive not only to chemotherapeutic drugs but also to TRAIL.

Activation of protein kinase C inhibits TRAIL-induced caspases activation, mitochondrial events and apoptosis in a human leukemic T cell line.

TRAIL causes apoptosis in numerous types of tumor cells. However, the mechanisms regulating TRAIL-induced apoptosis remain to be elucidated. We have investigated the role of PKC in regulating TRAIL-induced mitochondrial events and apoptosis in the Jurkat T cell line. We found a caspase-dependent decline in mitochondrial membrane potential and translocation of cytochrome c from mitochondria into the cytosol in response to TRAIL. Both these events were prevented by PKC activation. Moreover, PKC activation considerably reduced the activation of caspases, PARP cleavage and apoptosis when induced upon TRAIL treatment. MAPK activation was involved in the mechanism of PKC-mediated inhibition of TRAIL-induced cytochrome c release from mitochondria. Furthermore, inhibition of the MAPK pathway partially reversed the PKC-mediated inhibition of TRAIL-induced apoptosis. Besides, PKC activation may also inhibit the TRAIL-induced apoptosis through a MAPK-independent mechanism. Altogether, these results indicate a negative role of PKC in the regulation of apoptotic signals generated upon TRAIL receptor activation.

Interferon-gamma sensitizes human myeloid leukemia cells to death receptor-mediated apoptosis by a pleiotropic mechanism.

The role of interferon (IFN)-gamma as a sensitizing agent in apoptosis induced by ligation of death receptors has been evaluated in human myeloid leukemia cells. Incubation of U937 cells with IFN-gamma sensitized these cells to apoptosis induced by tumor necrosis factor-alpha, agonistic CD95 antibody, and tumor necrosis factor-related apoptosis-inducing ligand. Other human myeloid leukemic cells were also sensitized by IFN-gamma to death receptor-mediated apoptosis. Treatment of U937 cells with IFN-gamma up-regulated the expression of caspase-8 and potently synergized with death receptor ligation in the processing of caspase-8 and BID cleavage. Concomitantly, a marked down-regulation of BCL-2 protein was also observed in cells incubated with IFN-gamma. Furthermore, the caspase-dependent generation of a 23-kDa fragment of BCL-2 protein, the release of cytochrome c from mitochondria and the activation of caspase-9 were also enhanced upon death receptor ligation in IFN-gamma-treated cells. Ectopically expressed Bcl-2 protein inhibited IFN-gamma-induced sensitization to apoptosis. In summary, these results indicate that IFN-gamma sensitizes human myeloid leukemic cells to a death receptor-induced, mitochondria-mediated pathway of apoptosis.

Interferon-gamma treatment elevates caspase-8 expression and sensitizes human breast tumor cells to a death receptor-induced mitochondria-operated apoptotic program.

In this report, we have assessed the role of IFN-gamma as a sensitizing agent in apoptosis mediated by activation of death receptor CD95 in breast tumor cells. Treatment of the tumor cell lines MCF-7 and MDA-MB231 with IFN-gamma significantly facilitated apoptosis induced by CD95 receptor ligation at the plasma membrane, independently of p53 status. In contrast, IFN-gamma treatment did not enhance the apoptotic effect of the DNA-damaging drug, doxorubicin. Analysis of apoptosis regulators indicated that caspase-8 mRNA and protein levels were up-regulated in both of the cell lines after treatment with IFN-gamma. Furthermore, IFN-gamma sensitized MCF-7 and MDA-MB231 cells to CD95-mediated activation of caspase-8, induction of cytochrome c release from mitochondria, and processing of caspase-9. Release of cytochrome c, caspases activation, and apoptosis were prevented in MCF-7 cells overexpressing Bcl-2. Altogether these results indicate that IFN-gamma, maybe through the elevation of caspase-8 levels, sensitizes human breast tumor cells to a death receptor-mediated, mitochondria-operated pathway of apoptosis.

Protein kinase C inhibits CD95 (Fas/APO-1)-mediated apoptosis by at least two different mechanisms in Jurkat T cells.

We have recently reported that activation of protein kinase C (PKC) plays a negative role in CD95-mediated apoptosis in human T cell lines. Here we present data indicating that although the PKC-induced mitogen-activated protein kinase pathway could be partially implicated in the abrogation of CD95-mediated apoptosis by phorbol esters in Jurkat T cells, the major inhibitory effect is exerted through a PKC-dependent, mitogen-activated protein kinase-independent signaling pathway. Furthermore, we demonstrate that activation of PKC diminishes CD95 receptor aggregation elicited by agonistic CD95 Abs. On the other hand, it has been reported that UV radiation-induced apoptosis is mediated at least in part by the induction of CD95 oligomerization at the cell surface. Here we show that activation of PKC also inhibits UVB light-induced CD95 aggregation and apoptosis in Jurkat T cells. These results reveal a novel mechanism by which T cells may restrain their sensitivity to CD95-induced cell death through PKC-mediated regulation of CD95 receptor oligomerization at the cell membrane.

[Assessment of 200 critically ill transferred children at a pediatric intensive care unit]. / Valoración de 200 traslados de niños críticos en una Unidad de Cuidados Intensivos Pediátricos.

BACKGROUND: Pediatric intensive care units have developed as treatment areas with a concentration of specialized equipment and personnel. Critically ill children often need to be moved to and from these critical care areas for diagnostic or therapeutic procedures. Such transport may pose additional risk to the critically ill patient. PATIENTS AND METHODS: In order to assess the problems encountered in our transport process, a prospective study was performed. A questionnaire was undertaken to evaluate the transport of critically ill children hospitalized in the Pediatric Intensive Care Unit of the Hospital Universitario Puerta del Mar from Cádiz over an eleven month period. RESULTS: Two hundred children transported were evaluated. Forty-seven (23.5%) were interhospital transported patients and one hundred fifty-three (76.5%) were intrahospital transported patients. The most common type of intrahospital transport involves transfers between the operating room and the intensive care unit (73 patients, 36.5%). Deterioration in respiratory, cardiovascular and other physiological systems was registered in twenty-two patients (11%). One hundred four equipment-related mishaps were noted in eighty-six patients (43%) during the transport process. Dislodgement of intravenous catheters, loss of oxygen supply, endotracheal tube problems and equipment malfunction were the most common mishaps noted. CONCLUSIONS: Our results would suggest that more training regarding the transport of the critically ill child are needed in our area.