A reappraisal of ICU and long-term outcome of allogeneic hematopoietic stem cell transplantation patients and reassessment of prognosis factors: results of a 5-year cohort study (2009-2013).

Epidemiology and prognosis of complications related to allogeneic hematopoietic stem cell transplant (HSCT) recipients requiring admission to intensive care unit (ICU) have not been reassessed precisely in the past few years. We performed a retrospective single-center study on 318 consecutive HSCT patients (2009-2013), analyzing outcome and factors prognostic of ICU admission. Among these patients, 73 were admitted to the ICU. In all, 32 patients (40.3%) died in ICU, 46 at hospital discharge (63%) and 61 (83.6%) 1 year later. Survivors had a significantly lower sequential organ failure assessment (SOFA) score, serum lactate and bilirubin upon ICU admission. Catecholamine support, mechanical ventilation (MV) and/or renal replacement therapy during ICU stay, a delayed organ support and an active graft versus host disease (GvHD) significantly worsen the outcome. By multivariate analysis, the worsening of SOFA score from days 1 to 3, the need for MV and the occurrence of an active GvHD were predictive of mortality. In conclusion, the incidence of HSCT-related complications requiring an admission to an ICU was at 22%, with an ICU mortality rate of 44%, and 84% 1 year later. A degradation of SOFA score at day 3 of ICU, need of MV and occurrence of an active GvHD are main predictive factors of mortality.

The H19 endodermal enhancer is required for Igf2 activation and tumor formation in experimental liver carcinogenesis.

The expression of the linked but reciprocally imprinted Igf2 and H19 genes is activated in adult liver in the course of tumor development. By in situ hybridization analysis we have shown that both the Igf2 and H19 RNAs are expressed in the majority of the neoplastic nodules, and that hepatocellular carcinomas are developed in an experimental model of liver carcinogenesis. H19 is also highly activated in smaller and less distinct hyperplastic regions. The few neoplastic areas showing Igf2 but no H19 RNA display loss of the maternally inherited allele at the Igf2/H19 locus. These data are compatible with the existence of a common activation mechanism of these two genes during liver carcinogenesis and with a stronger H19 induction in the pre-neoplastic lesions. By using mice carrying a deletion of the H19 endodermal enhancer, we show that this regulatory element is necessary for the activation of the Igf2 and H19 genes upon induction of liver carcinogenesis. Furthermore, multiple sites of the H19 endodermal enhancer region become hypersensitive to DNase I when the carcinogenesis process is induced. Lastly, liver tumors developed in mice paternally inheriting the H19 enhancer deletion are found to have marked growth delays, increased frequency of apoptotic nuclei, and lack of Igf2 mRNA expression, thus indicating that this regulatory element plays a major role in the progression of liver carcinogenesis, since it is required for the activation of the anti-apoptotic Igf2 gene.

Proteolytic activity is involved in changes in intrafollicular insulin-like growth factor-binding protein levels during growth and atresia of ovine ovarian follicles.

In the sheep, follicular growth is characterized by both an increase and a decrease in the level of intrafollicular insulin-like growth factor-binding protein-3 (IGFBP-3) and IGFBPs less than 40 kDa (IGFBP-2, -4, and -5), respectively. In contrast, follicular atresia is associated with a decrease and a large increase in levels of IGFBP-3 and IGFBPs less than 40 kDa, respectively. To assess whether intrafollicular proteases are involved in such changes, follicular fluid from follicles of different sizes and degrees of atresia was incubated alone or with pure human IGFBP-3, -4, or -5 or serum (as a source of exogenous IGFBP-2) for 20 h at 37 C. Samples were then analyzed by Western ligand blotting and by immunoblotting using specific antisera. Ovine follicular fluid from different classes of follicles contained proteolytic activity degrading IGFBP-2, -3, -4, and -5. Degradation of IGFBPs was accompanied by the generation of small proteolytic fragments visualized by immunoblotting or after autoradiography using radiolabeled IGFBP-4. Moreover, follicular growth and atresia were characterized by changes in IGFBP proteolytic activity. Indeed, follicular growth (between 2 and 6 mm in diameter) was characterized by 1) a decrease in IGFBP-3 proteolytic activity and 2) a dramatic increase in proteolytic activity degrading IGFBP-4 and, to a lesser extent, IGFBP-2 and -5. Atresia, in contrast, was associated with a strong increase in IGFBP-3 proteolytic activity in small ( < 3-mm diameter) follicles and a decrease in IGFBP-4 and -5 proteolytic activity in large ( > 5-mm diameter) follicles. Regardless of the follicle class, IGFBP proteolytic activity was strongly inhibited by EDTA and 1,10-phenanthroline, but very slightly or not at all inhibited by tissue inhibitor of matrix metalloprotease-1 and-2 and BB-2116 (natural and synthetic inhibitors of matrix metalloproteases, respectively) as well as cysteine and serine proteases inhibitors, with the exception of phenylmethylsulfonylfluoride (1 mM) in atretic follicles. In addition, IGFBP proteolytic activity was dependent on the presence of zinc and calcium chloride. Zymography experiments showed the presence of 72- and 92- to 96-kDa gelatinases in follicular fluid; their levels were dramatically increased during follicular atresia. These results suggest that 1) changes in intrafollicular IGFBP proteolytic activity could be at least partly responsible for the changes in intrafollicular IGFBP levels that occur during follicular growth and atresia in the sheep; and 2) metalloprotease(s) in healthy and atretic follicles as well as serine protease(s) in atretic follicles are involved in IGFBP degradation.

Unexpected high testis-specific transcriptional activity of the cyclin T1 promoter in transgenic mice.

The ubiquitously expressed cyclin T1 gene encodes for a protein involved in human immunodeficiency virus type 1 (HIV-1) transcription activation. The goat gene was recently shown to share an expression pattern similar to that of its endogenous counterpart when incorporated into mice using a BAC insert. To assess if its promoter could target ubiquitous expression of the bovine Prnp in transgenic mice, two constructs carrying either 1 or 30 kb of cyclin T1 5'-flanking sequences were built and microinjected. Both constructs resulted in the unexpected high male germ cell-specific expression of the prion protein. These data re-question the suspected location of the cyclin T1 gene regulatory elements.

Expression studies of the PIS-regulated genes suggest different mechanisms of sex determination within mammals.

In mammals, the Y-located SRY gene is known to induce testis formation from the indifferent gonad. A related gene, SOX9, also plays a critical role in testis differentiation in mammals, in birds and reptiles. It is now assumed that SRY acts upstream of SOX9 in the sex determination cascade, but the regulatory link which should exist between these two genes remains unknown. Studies on XX sex reversal in polled goats (PIS mutation: Polled Intersex Syndrome) have led to the discovery of a female-specific locus crucial for ovarian differentiation. This genomic region is composed of at least two genes, FOXL2 and PISRT1, which share a common transcriptional regulatory region, PIS. In this review, we present the expression pattern of these PIS-regulated genes in mice. The FOXL2 expression profile of mice is similar to that described in goats in accordance with a conserved role of this ovarian differentiating gene in mammals. On the contrary, the PISRT1 expression profile is different between mice and goats, suggesting different mechanisms of the primary switch in the testis determination process within mammals. A model based on two different modes of SOX9 regulation in mice and other mammals is proposed in order to integrate our results into the current scheme of gonad differentiation.

Automated quantitative determination of hepatitis C virus viremia by reverse transcription-PCR.

An automated reverse transcription-PCR was developed for the quantitative detection of hepatitis C virus. The quantitation is based on the coamplification and labelling with digoxigenin-dUTP during PCR of two similar templates, the viral genome and a modified RNA which acts as a mimic target. Known amounts of the mimic RNA sequence were introduced into the clinical samples. The automated quantitation of the two coamplified and labelled products depends on the use of two biotinylated caputre probes which are complementary, respectively, to a deleted sequence and to an inserted sequence introduced by site-directed mutagenesis in a wild viral cloned cDNA. This method proved to be simple, reproducible, and useful for quantitate hepatitis C virus viremia in chronically infected patients. This easy-to-perform, automated assay could also be used for the accurate determination of human immunodeficiency virus viremia or other RNA molecules.

Proteolytic activity degrading insulin-like growth factor-binding protein-2, -3, -4, and -5 in healthy growing and atretic follicles in the pig ovary.

In the pig, ovarian follicular growth is characterized by an increase in intrafollicular levels of insulin-like growth factor-binding protein (IGFBP)-3 and a decrease in the levels of IGFBPs < 40 kDa (IGFBP-2, -4 and, to a lesser extent, a 30-kDa IGFBP likely corresponding to IGFBP-5). In contrast, atresia is primarily associated with a strong increase in intrafollicular levels of IGFBP-2 and -4, with intrafollicular levels of IGFBP-3 and -5 varying slightly or not at all. The purpose of the present study was to determine whether intrafollicular proteases are involved in such changes. Porcine follicular development was synchronized with a progestin, and individual follicles were isolated 12 h and 96 h after progestin withdrawal. Follicular fluid from follicles of various sizes and qualities was collected and incubated alone or with a source of exogenous bovine IGFBP-2 or human IGFBP-3, -4, or -5 for 20 h at 37 degrees C. Samples were then analyzed by Western ligand blotting and by immunoblotting using specific antisera. Porcine follicular fluid from various classes of follicles contained proteolytic activity degrading IGFBP-2, -4, and -5. In contrast, intrafollicular IGFBP-3 proteolytic activity was very low or nondetectable. In preovulatory follicles, degradation of IGFBPs < 40 kDa was 1) accompanied by the generation of small proteolytic fragments visualized by immunoblotting, 2) strongly inhibited by EDTA and 1,10-phenanthroline, and 3) dependent on the presence of zinc and calcium chloride. PMSF (1 mM, serine protease inhibitor) inhibited degradation of IGFBP-2 and to a lesser extent IGFBP-4, but not IGFBP-5. Other serine and cysteine protease inhibitors as well as TIMP-2 and BB-2116 (natural tissue inhibitor-2 and synthetic inhibitor of matrix metalloproteinases [MMPs], respectively) were ineffective. Gelatin-substrate zymography revealed the presence of two major intrafollicular gelatinase MMPs at 60 kDa and 76-85 kDa (likely MMPs 2 and 9, respectively), the levels of which decreased (76-85 kDa) or strongly increased (60 kDa) during follicular atresia. Follicular growth at diameters between 2 and 6-7 mm was characterized by a dramatic increase in proteolytic activity degrading IGFBP-2, -5 and, to a lesser extent, IGFBP-4. Atresia, in contrast, was associated with a marked decrease in proteolytic activity degrading IGFBP-2, -4, and -5. These results suggest that 1) changes in proteolytic activity of intrafollicular IGFBPs < 40 kDa are at least partly responsible for the changes in intrafollicular IGFBP levels during follicular growth and atresia in the pig and 2) calcium- and zinc-dependent metalloprotease(s) as well as serine protease(s) are involved in degradation of intrafollicular IGFBPs < 40 kDa.

Prolactin and lipopolysaccharide treatment increased apoptosis and atresia in rat ovarian follicles.

Follicular atresia is associated with the presence of increased macrophages within the follicle. What is not known is whether, in the adult rat, macrophages are instrumental in inducing apoptosis and/or atresia or whether they are simply secondary to a hormonally mediated event. As prolactin is an immunoreactive hormone and stimulates the expression of monocyte chemoattractant, the present experiments compared the effects of prolactin treatment with that of an immune challenge with lipopolysaccharide (LPS) on the invasion of macrophages into the follicular and luteal compartments of the ovary and the occurrence of apoptosis/atresia in relation to macrophage invasion. Rats were treated for 3 days with either prolactin or LPS and ovaries obtained at pro-oestrus or oestrus. Prolactin and LPS increased the number of atretic vs. healthy follicles (P < 0.008, chi2) in pro-oestrus ovaries and increased the mean number of apoptotic cells and macrophages (P < 0.05 for some groups). Macrophages were typically observed in the thecal layer, apoptotic cells in the granulosa cell layer, although 84% follicles which had macrophages within the granulosa cell layer also contained relatively high numbers of apoptotic nuclei. Prolactin and LPS treatment in vivo reduced the progesterone response to follicle stimulating hormone (FSH) (P < 0.001) in cultures of ovarian dispersates but did not inhibit the response to forskolin. In contrast, prolactin or LPS added in vitro to the cultures inhibited the progesterone response to forskolin. Results show that both prolactin and LPS increase follicular apoptosis and atresia and reduce the progesterone response to FSH.

Insulin-like growth factor-binding proteins and ovarian folliculogenesis.

In the ovary, insulin-like growth factors (IGFs) enhance both proliferation and differentiation of follicular cells by potentiating gonadotropin's actions. The biological effects of IGFs are strikingly modulated by IGF-binding proteins (IGFBPs), whose levels in follicular fluid dramatically change during folliculogenesis. Indeed, in most mammalian species, follicular growth and atresia are characterized by an increase and a great decrease in the IGFBP-3/IGFBPs < 40 kD (IGFBP-2, -4 and -5) ratio in follicular fluid, respectively. These variations result from both changes in expression of these IGFBPs by follicular cells, and in local degradation by specific intrafollicular proteases. Such changes in IGFBP levels lead to great decrease and increase in IGF bioavailability in atretic and growing healthy follicles, respectively. Hence intrafollicular IGFBPs play a key role in the regulation of follicular development by modulating IGFs and therefore gonadotropin's actions.

Automated determination of amplified PCR products: application to HCV viremia detection and quantification.

A simple and reliable automated method was developed for the detection of amplified products after PCR, which provides an alternative to the time-consuming Southern blotting and hybridization procedure. For the determination and quantification of hepatitis C viremia, the digoxigenin labelling process was applied during the PCR of the amplicons, followed by an inverse hybridization assay performed with biotinylated probes. The detection of the PCR amplified products could be processed as simply as an ELISA, or by means of an automated analyser.

Expression of messenger ribonucleic acids of insulin-like growth factor binding protein-2, -4, and -5 in the ovine ovary: localization and changes during growth and atresia of antral follicles.

In the sheep as in many mammalian species, growth and atresia of antral follicles are characterized, respectively, by a decrease and a high increase in the intrafollicular levels of insulin-like growth factor binding proteins of less than 40 kDa (IGFBPs < 40 kDa), mainly IGFBP-2, -4, and -5. The objective of this study was to investigate whether such changes are associated with changes in follicular expression of the corresponding mRNA. For this purpose, ovaries were recovered from ewes slaughtered at the end of follicular phase (i.e., 30 h after progestagen sponge removal; control ewes) or at 24 h, 36 h or 72 h after hypophysectomy (hypox) performed 30 h after sponge removal. The expression of mRNA of IGFBPs of less than 40 kDa (IGFBPs < 40 kDa mRNA) was studied in ovine antral follicles from control and hypox ewes by in situ hybridization using [35S]-labeled human IGFBP-2, -4, and -5 cRNA as probes. In control ewes, IGFBP-2 mRNA was mainly expressed in granulosa as a gradient in healthy follicles, the expression being higher in granulosa cells close to the basal membrane than in granulosa cells bordering the antrum and within the cumulus. The level of IGFBP-2 mRNA was lower both in granulosa cells close to the basal membrane and in those bordering the antrum from small follicles than in the corresponding compartments of granulosa cells from large healthy follicles (p < 0.05). In healthy follicles, IGFBP-4 and -5 mRNA were mainly expressed in thecal cells. No change in level of IGFBP-4 mRNA was observed between small and large follicles, whereas the level of IGFBP-5 mRNA tended to be lower in thecal cells from large compared to small follicles (p = 0.055). In atretic follicles, expression of IGFBPs < 40 kDa mRNA strongly increased in granulosa (IGFBP-2 and -5, p < 0.01) and in thecal cells (IGFBP-2 and -4, p < 0.01). In hypox ewes, the chronology of changes in expression of follicular IGFBPs < 40 kDa mRNA and in intrafollicular levels of the corresponding proteins was studied during atresia of large antral follicles. Early atresia of large follicles was associated with a strong decrease in intrafollicular estradiol levels (p < 0.001); an increase in intrafollicular levels of IGFBP-2, -4, and -5 (p < 0.001) an increase in both IGFBP-2 (p < 0.001) and -5 (p < 0.01) mRNA expression in granulosa and thecal cells; but no changed in IGFBP-4 mRNA expression. Late atresia of large follicles was associated with a further decrease in intrafollicular estradiol levels (p < 0.01); a further increase in intrafollicular levels of IGFBP-2, -4, and -5 (p < 0.001); an increase in IGFBP-4 (p < 0.01) and -5 (p < 0.05) mRNA expression in theca and granulosa, respectively; a decrease in IGFBP-5 mRNA expression in theca (p < 0.05); but no further increase in IGFBP-2 mRNA expression. Overall, these data suggest that the decrease and the increase in expression of mRNA of follicular IGFBPs < 40 kDa during follicular growth and atresia, respectively, are involved in the decrease and the increase in intrafollicular levels of the corresponding proteins. Moreover, the increases in expression of follicular IGFBPs < 40 kDa during atresia of large follicles in hypophysectomized ewes followed a specific time course, the increase in IGFBP-2 and -5 mRNA expression being early than the increase in IGFBP-4 mRNA expression.

Follicular growth and ovarian dynamics in mammals.

General characteristics of female reproductive activity, such as seasonality, cyclicity and triggering of ovulation differ widely among mammals, but common mechanisms underlie ovarian function. In all mammals, follicles begin to grow from a pool of primordial follicles constituted early in life, continuously throughout the life of the female. Follicular development involves two phases. In a first phase (basal follicular growth), follicles grow slowly and follicular growth rate is tightly related to proliferation of granulosa cells. Basal follicular growth is mainly under the control of growth factors of paracrine origin. In these follicles, FSH may exert an indirect mitogenic effect on granulosa cells by enhancing expression of growth factors or growth factor receptors. In a second phase (terminal follicular growth), follicular growth is rapid and occurs by enlargement of the antrum. In addition, it is accompanied by important changes in differentiation of follicular cells. Terminal follicular development is strictly dependent on gonadotrophins. FSH plays determinant roles in enhancing granulosa cell differentiation and survival. These actions are mediated or modulated in an important way by paracrine factors, particularly steroids and growth factors. LH stimulates steroidogenesis in theca cells and sustains terminal maturation of granulosa cells in preovulatory follicles. Follicular growth, atresia and ovulation are accompanied by important tissue remodelling processes, which are under the fine control of proteinases and inhibitors of proteinases. In particular matrix metalloproteinases and their inhibitors are probably involved in the control of rapid terminal follicular growth and regression of atretic follicles as well as in follicular rupture at ovulation.

Distal element(s) is(are) required for position-independent expression of the goat alpha-lactalbumin gene in transgenic mice. Potential relationship with the location of the cyclin T1 locus.

We recently reported the site-independent and copy-number-related expression in mice of a goat alpha-lactalbumin gene with 150 kb and 10 kb of 5'- and 3'-flanking sequences, respectively. In the present study, we observed that the resection of the 5'-flanking region, leaving only 70 kb, resulted in a site-dependent expression of this milk protein-encoding transgene. This suggests that important cis-regulatory elements are located within the distal-deleted sequence. Within this region, we localised the promoter of the cyclin T1 gene, an ubiquitously expressed gene. So far, no other gene has been located between these two loci. Since these two genes are differentially expressed, our data suggest the potential location of an insulator within the deleted region that allows the two genes to be independently regulated.

Selectivity of the major histocompatibility complex class II presentation pathway of cortical thymic epithelial cell lines.

Major histocompatibility complex (MHC) restriction of the immune response is established during positive selection of T cells in the thymus. This occurs mainly through interactions of T cell receptor of developing thymocytes with MHC/peptide ligands on cortical thymic epithelial cells (TEC). An ongoing controversy concerns the origin and the role of peptides involved in the positive selection of thymocytes. Evidence provided here shows that processing of MHC class II complexes in cortical TEC differs from that of medullary TEC. Removal of the invariant chain associated with MHC class II complexes was rapid and complete in medullary TEC which present peptides from both exogenous and cytosolic origin. In cortical TEC, a large fraction of class II dimers remained associated with a 10-12-kDa fragment of invariant chain (Ii). Incomplete removal of Ii correlated with the inability of cortical TEC to present peptides from exogenous origin. However, presentation of peptides from cytosolic proteins by cortical TEC remained possible. Thus, most peptides from exogenous proteins may be excluded from participating in positive selection of CD4+ T cells by a mechanism limiting Ii breakdown.

Molecular cloning of a lipolysis-stimulated remnant receptor expressed in the liver.

The lipolysis-stimulated receptor (LSR) is a lipoprotein receptor primarily expressed in the liver and activated by free fatty acids. Antibodies inhibiting LSR functions showed that the receptor is a heterotrimer or tetramer consisting of 68-kDa (alpha) and 56-kDa (beta) subunits associated through disulfide bridges. Screening of expression libraries with these antibodies led to identification of mRNAs derived by alternate splicing from a single gene and coding for proteins with molecular masses matching that of LSR alpha and beta. Antibodies directed against a synthetic peptide of LSR alpha and beta putative ligand binding domains inhibited LSR activity. Western blotting identified two liver proteins with the same apparent molecular mass as that of LSR alpha and beta. Transient transfections of LSR alpha alone in Chinese hamster ovary cells increased oleate-induced binding and uptake of lipoproteins, while cotransfection of both LSR alpha and beta increased oleate-induced proteolytic degradation of the particles. The ligand specificity of LSR expressed in cotransfected Chinese hamster ovary cells closely matched that previously described using fibroblasts from subjects lacking the low density lipoprotein receptor. LSR affinity is highest for the triglyceride-rich lipoproteins, chylomicrons, and very low density lipoprotein. We speculate that LSR is a rate-limiting step for the clearance of dietary triglycerides and plays a role in determining their partitioning between the liver and peripheral tissues.

Markedly increased susceptibility to natural sheep scrapie of transgenic mice expressing ovine prp.

The susceptibility of sheep to scrapie is known to involve, as a major determinant, the nature of the prion protein (PrP) allele, with the VRQ allele conferring the highest susceptibility to the disease. Transgenic mice expressing in their brains three different ovine PrP(VRQ)-encoding transgenes under an endogenous PrP-deficient genetic background were established. Nine transgenic (tgOv) lines were selected and challenged with two scrapie field isolates derived from VRQ-homozygous affected sheep. All inoculated mice developed neurological signs associated with a transmissible spongiform encephalopathy (TSE) disease and accumulated a protease-resistant form of PrP (PrPres) in their brains. The incubation duration appeared to be inversely related to the PrP steady-state level in the brain, irrespective of the transgene construct. The survival time for animals from the line expressing the highest level of PrP was reduced by at least 1 year compared to those of two groups of conventional mice. With one isolate, the duration of incubation was as short as 2 months, which is comparable to that observed for the rodent TSE models with the briefest survival times. No survival time reduction was observed upon subpassaging of either isolate, suggesting no need for adaptation of the agent to its new host. Overexpression of the transgene was found not to be required for transmission to be accelerated compared to that observed with wild-type mice. Conversely, transgenic mice overexpressing murine PrP were found to be less susceptible than tgOv lines expressing ovine PrP at physiological levels. These data argue that ovine PrP(VRQ) provided a better substrate for sheep prion replication than did mouse PrP. Altogether, these tgOv mice could be an improved model for experimental studies on natural sheep scrapie.