Expression and localization of IGF family members in bovine antral follicles during final growth and in luteal tissue during different stages of estrous cycle and pregnancy.

The objectives of the study were to monitor the detailed pattern for mRNA expression (RT-PCR and RPA) of IGFs, IGFR-1, IGFBPs, GHR and localization of protein (immunohistochemistry) for IGF-1 and IGFR-1 in bovine follicle classes during final maturation and different corpus luteum (CL) stages during estrous cycle and during pregnancy. A relative high expression of IGF-1 in theca interna (TI) was observed before selection (E<0.5ng/mL). In GC, mRNA expression increased after selection. In contrast, IGF-2 was mainly expressed in the TI. The IGFR-1 mRNA was present in the TI and GC with increasing levels during final development. The expression results were confirmed by localization of IGF-1 and IGFR-1 proteins in GC and TI. There is clear evidence for the local expression of IGFBPs in TI and GC compartment with clear regulatory differences. In CL, the highest mRNA expression of IGF-1, IGF-2 and IGFR-1 was observed during early luteal phase, followed by a decrease, and then by a tendency of an increase during the mid and late luteal phases of the cyclic CL. This level remained low during pregnancy. Intense immunostaining for IGFR-1 in CL was observed mainly in large luteal cells. Evidence for a mRNA for all six IGFBPs were obtained with distinct differences for BP-3, -4 and -5. In conclusion, this comprehensive study gives clear evidence for an important role of the IGFs and IGFBPs in bovine follicular development and CL function. The relative amounts of IGFBPs may ultimately determine ovarian IGF action.

Stimulatory and synergistic effects of luteinising hormone and insulin like growth factor 1 on the secretion of vascular endothelial growth factor and progesterone of cultured bovine granulosa cells.

Vascular endothelial growth factor (VEGF) is the most important factor in the regulation of angiogenesis. Associated with luteinisation and formation of corpus luteum (CL) are alterations in luteal vascularity. The aim of the study was to test under in vitro conditions the stimulation of VEGF and progesterone (P) secretion of bovine granulosa cells by LH, IGF1 (insulin like growth factor) or by factors known to be produced by luteinised granulosa cells or in the early CL. Localisation of VEGF protein in preovulatory follicle and early CL were achieved by immunohistochemistry. LH and IGF1 stimulated dose dependently and significantly P and VEGF when tested alone. Both hormones added simultaneously had clear additive and even more interesting far greater (synergistic) effects on P with LH (0.1 ng/ml) plus 5 or 10 ng IGF1. In contrast, VEGF was stimulated only additively with 0.1 ng/ml of LH plus 5 or 10 ng IGF1. But with the higher dose of LH (1 ng/ml) additionally to the additive effect a tendency for a synergistic action (which was significant with 1 ng LH plus 5 ng IGF1/ml) was observed. Endothelin, oxytocin, progesterone, atrial natiuretic peptide, angiotensin II, prostaglandin F2 alpha alpha, prostaglandin E2, cortisol, fibroblast growth factor 1 and 2 and growth hormone showed no effect neither on P nor on VEGF. Tumour necrosis factor alpha (TNF alpha) stimulated (P < 0.05) VEGF with 10 or 100 ng/ml but not P. TPA (12-0 tetra decaenoyl-phorbol-13-acetate) or Ca2+ ionophore did not show a stimulatory effect in contrast to forskolin which increased P and VEGF secretion dose dependently. The VEGF protein was localised in follicle (granulosa cells, theca cells and some endothelial cells) and early (about 24 h after ovulation) CL (granulosa-lutein cells and endothelial cells). The same signalling pathway by stimulation of cAMP production and proteinkinase A activation for luteinisation and neo-vascularisation demonstrates a close temporal and spatial relationship of these normal physiological processes.

Expression and localisation of vascular endothelial growth factor and basic fibroblast growth factor during the final growth of bovine ovarian follicles.

Locally produced growth factors may have important modulatory roles in final ovarian follicular growth. The aim of this study was to investigate the possible participation of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF2) in bovine follicles during final growth. Ovaries were collected from a slaughterhouse within 10-20 min after exsanguination. A classification of follicles into five groups (<0.5; >0.5-5; >5-20; >20-180; >180 ng/ml) was performed according to the follicular fluid (FF) oestradiol-17 beta content. For a better characterisation of classes the mRNA expressions of FSH receptor, LH receptor and aromatase cytochrome P450 in theca interna (TI) and granulosa cells (GC) were determined. Analysis of VEGF transcript by RT-PCR showed that GC and theca cells express predominantly the smallest isoforms (VEGF(121) and VEGF(165)). VEGF mRNA expression in both tissues (TI and GC) and VEGF protein concentration in total follicle tissue increased significantly (and correlated) with developmental stages of follicle growth. The expression of mRNA for VEGF receptor (VEGFR)-1 and VEGFR-2 was very weak in GC, without any regulatory change during final follicle growth. In contrast, TI showed strong expression of mRNA for both receptors in all follicle classes examined. VEGF protein concentrations in FF increased significantly and continuously to maximum levels in preovulatory follicles. As shown by immunohistochemistry, VEGF protein was clearly localised in TI and GC of preovulatory follicles. FGF2 and FGF receptor (FGFR) mRNA expression in TI increased significantly during final growth of follicles. In contrast, the FGF2 and FGFR mRNA expression in GC was very weak and without any regulatory change during follicle growth. Histological observation revealed that FGF2 protein was localised in theca tissue (cytoplasm of endothelial cells and pericytes) but not in GC. Our results suggest that VEGF and FGF families are involved in the proliferation of capillaries that accompanies the selection of the preovulatory follicle resulting in an increased supply of nutrients and precursors, and therefore supporting the growth of the dominant follicle.

Expression and tissue concentration of vascular endothelial growth factor, its receptors, and localization in the bovine corpus luteum during estrous cycle and pregnancy.

The presence of vascular endothelial growth factor (VEGF) in the ovary has been reported in a number of species. The objective of the present study was to demonstrate the expression of VEGF, VEGF receptor (R)-1, and VEGFR-2 in detail by different methodological approaches in bovine corpora lutea (CL) obtained from different stages of the estrous cycle and during pregnancy. VEGF and VEGF receptor transcripts were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and ribonuclease protection assay. All components of the VEGF system were found in the bovine CL during the estrous cycle and pregnancy. Analysis of VEGF transcript by RT-PCR shows that CL tissues expressed predominantly the smallest isoforms (VEGF(121) and VEGF(165)). The highest mRNA expression for VEGF and VEGFR-2 mRNA was detected during the early luteal phase, followed by a significant decrease of expression during the mid and late luteal phase and a further decrease of VEGF mRNA after regression. During pregnancy, high levels of expression were always present. In contrast, no significant change in VEGFR-1 mRNA expression during the estrous cycle and pregnancy was found. The VEGF protein concentration in CL tissue was significantly higher (20.9-23.4 ng/g wet weight) during the early luteal phase (Days 1-7), followed by a decrease at the late luteal phase (14.3-18.7 ng/g wet weight) and, especially, after CL regression (2.8 ng/g wet weight). However, relatively high levels were found during pregnancy (10.1 ng/g wet weight). As achieved by immunohistochemistry, VEGF protein was localized predominantly in luteal cells. High VEGF protein and transcript concentrations and increased VEGFR-2 expression during the early luteal phase coincided with luteal vascularization. These results suggest an important role of VEGF in angiogenesis of the newly formed CL. The high VEGF mRNA expression and protein levels during matured vasculature in the mid-stage CL and pregnancy also suggest also a survival function for endothelial cells.

Regulation of angiotensin II production and angiotensin receptors in microvascular endothelial cells from bovine corpus luteum.

Recent findings suggest that the ovarian renin-angiotensin system regulates ovarian function through the paracrine/autocrine actions of angiotensin (Ang) II. The aims of this study were to investigate 1) the endothelial cell capacity to convert Ang I to Ang II, 2) the effects of endocrine and paracrine/autocrine factors on Ang II production in microvascular endothelial cells (MVE) derived from the developing corpora lutea (CL), and 3) the relationship between Ang II peptide concentration and expression of mRNA for angiotensin type 1 and 2 receptors (ATR1 and AT2R) in the bovine CL at different stages of the estrous cycle. When Ang I was added to the MVE at a concentration of 10(-9) M, it was converted to Ang II (21%). The production of Ang II from Ang I time-dependently rose for 24 h. Addition of captopril (an inhibitor of Ang-converting enzyme [ACE]) to the MVE cultures significantly inhibited Ang II production from 6 h to 24 h (P < 0.05). Addition of estradiol-17beta (E(2)) + vascular endothelial growth factor and E(2) + basic fibroblast growth factor to MVE cultures increased Ang II production, whereas E(2) or growth factors alone had no effect. Specific transcription for AT1R and AT2R was detected in bovine CL and MVE. There were no significant changes in Ang II tissue concentration or AT1R mRNA expression using reverse transcription-polymerase chain reaction during the estrous cycle. In contrast, AT2R mRNA expression decreased during the midluteal phase (P < 0.05) and increased to the highest level during the late luteal phase (P < 0.05). Results demonstrated that Ang II is generated from Ang I in MVE isolated from the developing bovine CL, indicating that MVE have ACE activity. In addition, mRNA expression for Ang II receptors was detected in the bovine CL and the luteal MVE. These results suggest that Ang II is produced by actions of the local renin-angiotensin system, at least in part, on MVE in the bovine CL, and that this peptide may be involved in the regulation of luteal function during early development and luteolysis.

Control of a prolonged outbreak of extended-spectrum beta-lactamase-producing enterobacteriaceae in a university hospital.

Extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBLPE) were isolated from clinical specimens from 130 to 140 patients/year in 1989-1991 in our hospital. In February 1992, a control program was initiated: screening tests in 3 intensive care units (ICUs) and contact-isolation precautions in all units. The septic surgical unit served as an isolation ward for surgical patients from whom ESBLPE was isolated. In 1992, the incidence of ESBLPE acquisition failed to decrease, and most acquisitions occurred in 3 ICUs. Critical evaluation of implementation of isolation procedures in these ICUs prompted corrective measures for barrier precautions. The incidence of acquired cases subsequently decreased, and a second evaluation determined that these measures had been correctly applied. The incidence of acquired cases in the septic surgical unit was lower than those in the other units. Decreases were also found in the incidence of acquisition of other hand-transmitted multidrug-resistant organisms. Barrier precautions, screening tests for ICU patients, and grouping of cohorts after ICU discharge are effective in controlling the spread of multidrug-resistant microorganisms by cross-contamination. The outbreak was effectively controlled without restricting antimicrobial use.

Possible role of growth hormone, IGFs, and IGF-binding proteins in the regulation of ovarian function in large farm animals.

The aim of the study and short review was to present evidence that growth hormone (GH), locally produced insulin-like growth factors (IGFs), and IGF-binding proteins (IGFBPs) may have an important role in the control of ovarian function. There is clear evidence for a distinct GH-receptor mRNA expression and protein production in follicles (oocytes and granulosa-cumulus cells) and corpus luteum (CL). In hypophysectomized ewes, GH and LH are necessary for normal CL development. IGF-1 mRNA in the follicles is expressed in theca interstitial cells (TIC) and granulosa cells (GC) with already higher levels in the TIC before follicle selection. In contrast, IGF-2 is mainly expressed in the TIC. The IGFR-1 mRNA is expressed in both the TIC and GC, with increasing levels in GC during the final development of dominant follicles. IGF-1 is a very potent stimulator of progesterone and oxytocin release in GC. IGFBP-1, -2, -3, -4, -5, and -6 have been isolated from follicular fluid or ovarian tissue. Studies indicate that IGFBP expression and production in the developing follicle is dependent on both cell type and follicle size and is regulated by IGF-1 and gonadotropins. The highest expression of IGF-1 and IGFR-1 mRNA was demonstrated during the early luteal phase. Distinct receptors for IGF-1 and IGF-2 were present in CL membrane preparations at all stages investigated. Intense immunostaining for IGF-1 was observed mainly in bovine large and small luteal cells and in a limited number of endothelial cells. In contrast, IGF-2 protein was localized in perivascular fibroblast and pericytes of the capillaries. With the use of a microdialysis system, we found that in vitro and in vivo IGF-1, IGF-2, and GH stimulated the release of progesterone in cultures of luteal cells or intact tissues. In conclusion, there is clear evidence for a central role of the IGFs, IGFBPs, and GH in follicular development and CL function.

Glutamate and taurine are increased in ventricular cerebrospinal fluid of severely brain-injured patients.

Glutamate contributes to secondary brain damage, resulting in cell swelling and brain edema. Under in vitro conditions, increased extracellular levels of the amino acid taurine reflect glutamate-induced osmotic cell swelling. In vivo, increases in cerebrospinal fluid (CSF) taurine could, therefore, unmask glutamate-mediated cytotoxic edema formation and possibly differentiate it from vasogenic edema. To test this hypothesis, ventricular CSF glutamate and taurine levels were measured in 28 severely brain-injured patients on days 1, 5, and 14 after trauma. Posttraumatic changes in CSF amino acids were investigated in regard to extent of tissue damage and alterations in brain edema as estimated by computerized tomography. On day 1, CSF glutamate and taurine levels were significantly increased in patients with subdural or epidural hematomas (8+/-0.8/71+/-12 microM), contusions (21+/-4.1/122+/-18 microM), and generalized brain edema (13+/-3.2/80+/-15 microM) compared to lumbar control CSF (1.3+/-0.1/12+/-1 microM; p < 0.001). CSF amino acids, however, did not reflect edema formation and resolution as estimated by computerized tomography. CSF taurine correlated positively with glutamate, eventually depicting glutamate-induced cell swelling. However, parallel neuronal release of taurine with its inhibitory function cannot be excluded. Thus, the sensitivity of taurine in unmasking cytotoxic edema formation is weakened by the inability in defining its origin and function under the conditions chosen in the present study. Overall, persisting pathologic ventricular CSF glutamate and taurine levels are highly suggestive of ongoing glial and neuronal impairment in humans following severe traumatic brain injury.

Risk factors for surgical wound infections in patients undergoing head and neck oncologic surgery.

Risk factors for surgical wound infection are difficult to establish in head and neck surgery. Flap reconstruction, which correlates with tumour size and surgical procedure, appears to be the main risk factor. Attempts should be made by the surgical staff to improve surgical procedures in terms of duration of surgery and choice of the procedure. The intraoperative choice between primary closure and flap reconstruction should be studied further. More subtle risk factors may appear in studies of large groups of patients and/or if a distinction is drawn between early and late SWI.

Growth factors and extracellular matrix proteins in interactions of cumulus-oocyte complex, spermatozoa and oviduct.

The expression and localization of selected growth factor systems and extracellular matrix (ECM) components that may influence oocyte maturation and fertilization within the mammalian oviduct are reported. Fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) systems could be detected by use of RT-PCR, RNase protection assay (RPA) and immunohistochemistry in bovine follicles, bovine cumulus-oocyte complexes (COC) and bovine and marmoset oviducts. Two different subtypes of the FGF receptor (FGFR-1 and -2) were identified in distinct cell types, indicating a functional difference. A complete epidermal growth factor (EGF) system was found in the porcine, but not in the bovine, oviduct. There were additional differences between bovine and primate oviducts: FGF-1/2 and FGFR were increased in the marmoset around ovulation, in contrast to an increase in FGF-1 in the cow. Immunohistochemistry revealed accumulation and storage of FGF and VEGF on the surface of the epithelium, possibly due to their binding property on heparanglycoproteins. Other ECM components, matrix metalloproteinase 1 (MMP-1) and tissue inhibitor of metalloproteinase 1 (TIMP-1), were found to be modulated in the ovarian follicle, COC and oviduct during the cycle. An oviduct-mediated depletion of sperm surface proteins (BSP1-3) was discovered as well as a sperm-induced novel oviductal mRNA related to an anti-oxidant protein family. Associated systems of growth factors and ECM components can be suggested as paracrine or autocrine mediators during fertilization in a species-, cycle- and tissue-dependent manner.

Notification of tuberculosis in a university hospital.

The aim of this study was to evaluate completeness of tuberculosis notification in Bichat Claude-Bernard University Hospital and to evaluate whether misclassification of atypical mycobacterial infection could have contributed to the inaccuracy of tuberculosis notification. Data from Microbiology Laboratory of the hospital and statutory notifications were compared. From 1 January 1994 to 31 December 1995, 299 tuberculosis cases were diagnosed in the Microbiology Laboratory and 316 cases were notified as tuberculosis. Notification rate for laboratory-documented tuberculosis was 57.5%, was significantly higher in cases with positive acid fast bacilli smear (75%) than without this feature (45%) and was similar in HIV-positive (59.4%) and HIV-negative (63.5%) patients. Among notified cases, diagnosis was established by laboratory proofs in only 54.4% and by clinical signs in 45.6%. Three cases with positive smear and culture growing atypical mycobacteria were wrongly notified. Notification of laboratory-documented tuberculosis was higher than that observed in a previous study in the same hospital, suggesting that the rise of tuberculosis incidence reported in our country could be partially artificial. Nevertheless, extent of notification remains insufficient and needs to be improved by combining microbiological data with current system of notification.