CHECK - multilevel real-world pediatric asthma care coordination: results and lessons learned.

OBJECTIVE: Because asthma health disparities in children remain common, innovative approaches to obtain asthma health equity are essential. Comprehensive care coordination programs may address the social determinants of health that influence these disparities. This analysis aims to ascertain if receipt of Coordination of Healthcare for Complex Kids (CHECK) program services was associated with changes in school absence, cost, healthcare utilization, and controller prescription in children with asthma. METHODS: The CHECK program ran from December 1, 2014 through August 31, 2017. Engagement with community health workers was rolling and targeted based on risk level (low, medium, or high determined by healthcare utilization). This analysis included school-aged children with asthma (n = 2,629) and sufficient Chicago Public Schools attendance data (n = 430). RESULTS: Children engaged in CHECK were more likely to be female (p = .046) and to identify as Black and/or Hispanic/Latino than enrolled-only children. School absence was not different between the groups. Average total cost for engaged children was 21.3% more than enrolled-only children the first year (p = .027) but did not differ by the second year (p = .948). At baseline, 68.1% of the cohort had at least one ED visit 12 months prior to CHECK, this reduced to 49.5% post-1 and 41.9% post-2. Engaged children were 21% more likely to visit an ED (p = .010) and 40% more likely to have a controller. CONCLUSIONS: CHECK program receipt was associated with improved healthcare utilization and controller prescriptions. School attendance did not change. The CHECK model offers potential pathways to support low-income children with asthma.

Box and block test, hand grip strength and nine-hole peg test: correlations between three upper limb objective measures in multiple sclerosis.

BACKGROUND AND PURPOSE: Limited data are available in the literature for upper limb impairment in multiple sclerosis (MS). This study aimed to report the distribution of values of hand grip strength (HGS), of the box and block test (BBT) and of the nine-hole peg test (9HPT) correlated with demographic and clinical data in subjects with MS. METHODS: This study involved five Italian neurological centres. The inclusion criteria were age ≥ 18, MS diagnosis, stable disease phase, right-hand dominance. All subjects underwent HGS, BBT and 9-HPT evaluation. RESULTS: In all, 202 subjects with MS were enrolled: 137 females; mean age 48.4 years; mean Expanded Disability Status Scale (EDSS) 4.17; mean disease duration 14.12 years; disease course 129 relapsing-remitting, 21 primary progressive and 52 secondary progressive MS subjects; mean right HGS 25.3 kg, left 23.2 kg; mean right BBT 45.7 blocks, left 44.9 blocks; mean right 9-HPT 30.7 s, left 33.4 s. All results were statistically significantly different compared to healthy controls. HGS, BBT and 9-HPT were associated with age, EDSS and disease duration, whilst disease course correlated with BBT and 9-HPT. The BBT and 9-HPT scores significantly differed according to level of disability (EDSS ≤3.0, 3.5-5.5, ≥6.0). CONCLUSION: Hand grip strength and BBT value distribution in a large MS population is reported. Correlations between HGS, BBT and 9-HPT were generally low.

Comparative pattern of genetic structure in two Mediterranean killifishes Aphanius fasciatus and Aphanius iberus inferred from both mitochondrial and nuclear data.

In this study, genetic variation was assessed in Aphanius fasciatus and Aphanius iberus characterized by similar ecological traits but with very different distribution ranges in the Mediterranean area. Five populations of A. iberus and five of A. fasciatus were analysed using five polymorphic microsatellite loci and partial mitochondrial control region (D-loop) sequences. Congruent results were found with both nuclear and mitochondrial molecular markers. The results showed that similar levels of genetic divergence, based on mitochondrial control region sequences, are present among populations of A. iberus and among populations of A. fasciatus despite the very different geographic distance existing among the examined populations of the two species (low geographic distance in A. iberus and high in A. fasciatus). A possible explanation could be that the populations of A. iberus were isolated for a longer time than the populations of A. fasciatus supporting the hypothesis that the split in the lineage leading to A. iberus is older than the split in the lineage leading to A. fasciatus. The possibility that the wide circum-Mediterranean distribution of A. fasciatus ensures the high connectivity of its populations, preventing, in some cases, local differentiation, however, cannot be ruled out.

Influence of rotational speed on the cyclic fatigue of Mtwo instruments.

AIM: To evaluate the effect of rotational speed on cyclic fatigue of Mtwo nickel-titanium files. METHODOLOGY: A total of 120 new Mtwo rotary instruments sizes 10, 0.04 taper; 15, 0.05 taper; 20, 0.05 taper; and 25, 0.06 taper were randomly divided into three groups on the basis of the rotational speed used to shape nine standardized simulated canals: group A = 350 rpm; group B = 250 rpm; group C = 150 rpm. Each group consisted of 40 instruments, 10 for every size. The average preparation time (in seconds) and the average correlated numbers of cycles to instrument (NCI) the nine standardized canals were recorded for each file. The resistance to cyclic fatigue was determined by counting numbers of cycles to failure (NCF) with a rotational speed of 300 rpm in a 60° curve with a 5-mm radius. Data were analysed by two-way anova. RESULTS: Preparation time was significantly longer at 150 rpm than at 250 or 350 rpm. The average number of cycles needed for each file to instrument nine standardized canals was significantly higher at 350 and 250 rpm, than at 150 rpm. There were no significant differences in the NCF (P > 0.05) between A, B and C groups for instruments of the same size. CONCLUSIONS: Speed did not affect the cyclic fatigue of Mtwo instruments with the same size and taper. Preparation time was shorter at 350 or 250 rpm rather than at 150 rpm. However, there was no significant difference between 350 and 250 rpm rotational speed, neither in the preparation time of simulated canals nor in the resistance to fatigue fracture.

Activated γδ T cells inhibit osteoclast differentiation and resorptive activity in vitro.

Extensive evidence suggests that the immune system exerts powerful effects on bone cells, particularly in chronic disease pathologies such as rheumatoid arthritis (RA). The chronic inflammatory state in RA, particularly the excessive production of T cell-derived proinflammatory cytokines such as tumour necrosis factor (TNF)-α and interleukin (IL)-17, triggers bone erosions through the increased stimulation of osteoclast formation and activity. While evidence supports a role for IL-17 and TNF-α secreted by conventional CD4⁺ T cells in RA, recent evidence in animal models of RA have implicated γδ T cells as a major producer of pathogenic IL-17. However, the capacity of γδ T cells to influence osteoclast formation and activity in humans has not yet been investigated widely. To address this issue we investigated the effects of γδ T cells on osteoclast differentiation and resorptive activity. We have demonstrated that anti-CD3/CD28-stimulated γδ T cells or CD4⁺ T cells inhibit human osteoclast formation and resorptive activity in vitro. Furthermore, we assessed cytokine production by CD3/CD28-stimulated γδ T cells and observed a lack of IL-17 production, with activated γδ T cells producing abundant interferon (IFN)-γ. The neutralization of IFN-γ markedly restored the formation of osteoclasts from precursor cells and the resorptive activity of mature osteoclasts, suggesting that IFN-γ is the major factor responsible for the inhibitory role of activated γδ T cells on osteoclastogenesis and resorptive activity of mature osteoclasts. Our work therefore provides new insights on the interactions between γδ T cells and osteoclasts in humans.

Frequency and severity of headache is worsened by Interferon-ß therapy in patients with multiple sclerosis.

BACKGROUND: The relationship between multiple sclerosis (MS) and headache (HA) is not well known. It was reported that interferon-beta (IFNß) could induce or worsen HA. OBJECTIVE: To evaluate the impact of IFNß treatment on HA and the relationship between HA and the various commercial preparations of IFNß in mildly disabled patients with MS. METHODS: A specific questionnaire was administered to 357 relapsing-remitting MS patients. Characteristics of HAs were considered, including the temporal relationships with IFNß administration. RESULTS: One hundred and seventeen patients were treated with weekly intramuscular injections of interferon IFNß-1a (Avonex(®)), 84 with subcutaneous injections of IFNß-1b (Betaferon(®)) every other day, 48 and 108 with three times weekly subcutaneous injections of IFNß-1a (Rebif(®)) 22 mcg or IFNß-1a (Rebif(®)) 44 mcg, respectively. Three hundred and fourteen patients were affected by HA, and among them, 219 patients suffered of pre-existing HA. In this latter group, 121 subjects (55%) noted a worsening of their HA after starting IFNß therapy; this was more frequently reported by patients treated with Avonex(®) and Rebif(®) 44. Ninety-five patients experienced new HA. CONCLUSION: IFNß treatment could worsen HA in patients with pre-existing HA or cause the appearance of new HA. Among different IFNß preparations, Rebif(®) 44 and Avonex(®) seemed to be more cephalalgic than the other drugs.

Multiple sclerosis in Italy: cost-of-illness study.

This study estimates the direct costs of multiple sclerosis (MS) in Italy from the perspective of the National Health System. Patients diagnosed with MS for ≥1 year prior to study entry were included in the analysis; neurological disability was assessed using the Expanded Disability Status Scale (EDSS). Cost variables were analyzed according to: MS phenotype, disease course over the previous year and EDSS rating. A total of 510 patients were included in the analysis. Overall costs were significantly higher for relapsing-remitting MS and secondary progressive MS than for primary progressive MS (P < 0.05). Costs were higher for EDSS scores 0.0-3.5 and 4.0-6.0 than for scores > 6.0 (P < 0.05). The extrapolated data gave an estimated annual direct cost of MS per patient of 18,030. In conclusion, relapsing-remitting MS or secondary progressive MS phenotypes and lower estimated EDSS scores appear to be associated with higher costs.

Electromagnetic character of the competitive γγ/γ-decay from 137mBa.

Second-order processes in physics is a research topic focusing attention from several fields worldwide including, for example, non-linear quantum electrodynamics with high-power lasers, neutrinoless double-ß decay, and stimulated atomic two-photon transitions. For the electromagnetic nuclear interaction, the observation of the competitive double-γ decay from 137mBa has opened up the nuclear structure field for detailed investigation of second-order processes through the manifestation of off-diagonal nuclear polarisability. Here, we confirm this observation with an 8.7σ significance, and an improved value on the double-photon versus single-photon branching ratio as 2.62 × 10-6(30). Our results, however, contradict the conclusions from the original experiment, where the decay was interpreted to be dominated by a quadrupole-quadrupole component. Here, we find a substantial enhancement in the energy distribution consistent with a dominating octupole-dipole character and a rather small quadrupole-quadrupole component in the decay, hindered due to an evolution of the internal nuclear structure. The implied strongly hindered double-photon branching in 137mBa opens up the possibility of the double-photon branching as a feasible tool for nuclear-structure studies on off-diagonal polarisability in nuclei where this hindrance is not present.

Predictive models of surgical site infections after coronary surgery: insights from a validation study on 7090 consecutive patients.

BACKGROUND: The role of specific scoring systems in predicting risk of surgical site infections (SSIs) after coronary artery bypass grafting (CABG) has not been established. AIM: To validate the most relevant predictive systems for SSIs after CABG. METHODS: Five predictive systems (eight models) for SSIs after CABG were evaluated retrospectively in 7090 consecutive patients undergoing isolated (73.9%) or combined (26.1%) CABG. For each model, accuracy of prediction, calibration, and predictive power were assessed with area under receiver-operating characteristic curve (aROC), the Hosmer-Lemeshow test, and the Goodman-Kruskal γ-coefficient, respectively. Six predictive scoring systems for 30-day in-hospital mortality after cardiac operations were evaluated as to prediction of SSIs. The models were compared one-to-one using the Hanley-McNeil method. FINDINGS: There were 724 (10.2%) SSIs. Whereas all models showed satisfactory calibration (P = 0.176-0.656), accuracy of prediction was low (aROC: 0.609-0.650). Predictive power was moderate (γ: 0.315-0.386) for every model but one (γ: 0.272). When compared one-to-one, the Northern New England Cardiovascular Disease Study Group mediastinitis score had a higher discriminatory power both in overall series (aROC: 0.634) and combined CABG patients (aROC: 0.648); in isolated CABG patients, both models of the Fowler score showed a higher discriminatory power (aROC: 0.651 and 0.660). Accuracy of prediction for SSIs was low (aROC: 0.564-0.636) even for six scoring systems devised to predict mortality after cardiac surgery. CONCLUSION: In this validation study, current predictive models for SSIs after CABG showed low accuracy of prediction despite satisfactory calibration and moderate predictive power.

Static postural control disturbances among the different multiple sclerosis phenotypes: A Neurocom Balance Manager® evaluation study.

BACKGROUND: The computerized stabilometric platform can be used and privileged over clinical scales, as self-administered questionnaires to asses postural control and balance evaluation in Multiple sclerosis (MS). Aim of our study was to evaluate static postural control assessed by Neurocom Balance Manager® through the modified Clinical Test of Sensory Interaction on Balance (mCTSIB) in relapsing-remitting MS (RRMS), progressive MS (PMS) and CIS, compared to healthy controls (HC). METHODS: We screened MS patients consecutively referring to our MS Center at University of Catania, during July 2013-June 2014 diagnosed as CIS, RRMS and PMS. All MS patients underwent clinical and neurological evaluations and a complete postural exam by Neurocom Balance Manager® in order to evaluate Center of Pressure (COP), through mCTSIB. We evaluated the following parameters: Total Path Length-open eyes (TPL-OE), Total Path Length-closed eyes (TPL-CE), Sway Area-open eyes (SA-OE), Sway Area-closed eyes (SA-CE), Mean sway velocity-open eyes (MSV-OE), Mean sway velocity-closed eyes (MSV-CE). Additionally, patients were tested by Berg balance scale (BBS) for balance and Barthel Index (BI) for disability outcomes. RESULTS: Out of 170 MS patients assessed for eligibility, 163 met the inclusion/exclusion criteria and were finally enrolled. All balance parameters were found more impaired in MS group compared to controls and CIS. Moreover, no differences in terms of balance assessment were found between HC and CIS. The correlation analysis showed that BBS was strongly associated to SA-OE, SA-CE, TPL-OE and MSV-OE. We also found a correlation between BI and SA-CE. CONCLUSION: Our study revealed significant differences among HCs, CIS and MS. MS, especially PMS, exhibit the worst balance performances especially in EC trials. The higher correlation between balance parameters, especially sway area, and BBS score confirmed the reliability and sensibility of mCTSIB assessment in evaluating static postural control in MS patients.

Progesterone membrane receptor component 1 expression in the immature rat ovary and its role in mediating progesterone's antiapoptotic action.

Progesterone receptor membrane component-1 (PGRMC1) interacts with plasminogen activator inhibitor RNA binding protein-1 (PAIRBP1), a membrane-associated protein involved in the antiapoptotic action of progesterone (P4). In this paper, the first studies were designed to assess the ovarian expression pattern of PGRMC1 and PAIRBP1. Western blot analysis revealed that spontaneously immortalized granulosa cells (SIGCs) as well as granulosa and luteal cells express both proteins. Luteal cells were shown to express more PGRMC1 than granulosa cells. Immunohistochemical studies confirmed this and demonstrated that PGRMC1 was present in thecal/stromal cells, ovarian surface epithelial cells, and oocytes. PAIRBP1 was also expressed in thecal/stromal cells and ovarian surface epithelial cells but not oocytes. Furthermore, PAIRBP1 and PGRMC1 were detected among the biotinylated surface proteins that were isolated by avidin affinity purification, indicating that they localized to the extracellular surface of the plasma membrane. Confocal microscopy revealed that both of these proteins colocalize to the plasma membrane as well as the cytoplasm. The second studies were designed to assess PGRMC1's role in P4's antiapoptotic actions. These studies showed that overexpression of PGRMC1 increased 3H-P4 binding and P4 responsiveness. Conversely, treatment with a PGRMC1 antibody blocked P4's antiapoptotic action. Taken together, the present findings indicate that both PAIRBP1 and PGRMC1 show a similar expression pattern within the ovary and colocalize to the extracellular surface of the plasma membrane. At the plasma membrane, these two proteins interact to form a complex that is required for P4 to transduce its antiapoptotic action.

Morphostructural investigation of the female reproductive system and molecular evidence for Wolbachia in Balclutha brevis Lindberg 1954 (Hemiptera, Cicadellidae).

Balclutha brevis Lindberg 1954 (Homoptera, Cicadellidae) is an allochthonous species that is rapidly spreading in Sicily and in mainland Europe due to the wide spread of its host plant and therefore could also compete with populations of native species. Considering these ecological implications, based on the lacking ultrastructural data about the reproductive systems of the Auchenorrhyncha and since previous investigations on the male reproductive system of B. brevis have shown some interesting features, we carried out morphostructural investigations on the female reproductive system of this alien leafhopper. Moreover, given the high interest in literature on Wolbachia entomoparasite and based on our previous studies, we provided a contribution to further investigations in applied sciences. For this aim we performed a molecular analysis on males and females of B. brevis to detect the possible presence of strains of the bacterium known to alter host reproductive biology. The female reproductive system has a morphological organization comparable to the general anatomical features of most of the Auchenorrhyncha species; however, comparing our data with the literature, some considerations are discussed. As for the histological and ultrastructural investigations, our results show a secretory activity of the various examined structures. In the spermatheca of B. brevis, in particular, the secretory activity is more marked in the sac-shaped tract, where histochemical investigations showed a lipid component of the secretion; possible origin of this component is discussed. Moreover, mainly free spermatozoa are found in the sac-shaped tract of the spermatheca and in the common oviduct. As for the latter, an interesting findings is the lack of cuticular intima on the epithelial surface of the common oviduct; furthermore, the observed features and the literature in this regards led us to review the significance of the structure called as spermatheca. The molecular screening study (PCR) with three Wolbachia specific genes (16SrRNA, ftsZ,wsp) strongly supports Wolbachia infections in both males and females of the host species. This is the first study demonstrating the presence of Wolbachia in Balclutha genus, though no morphostructural alteration commonly related to the presence of the bacterium has been found in all the examined specimens.

Role of water content in dielectric properties and delayed luminescence of bovine Achilles' tendon.

In order to investigate the role of water network in collagen structure, measurement of dielectric permittivity was performed on bovine Achilles' tendon as a function of water content. The data show a sudden decrease of the permittivity at each measured frequency value when the tendon humidity decreases. A similar behaviour is shown by the total number of photons emitted in delayed luminescence (DL) experiments. The comparison of the two results is in agreement with the hypothesis that DL is connected to the excitation and subsequent decay of collective electronic states, whose properties depend on the organized structure of the system.

Silicon detectors for monitoring neutron beams in n-TOF beamlines.

During 2014, the second experimental area (EAR2) was completed at the n-TOF neutron beam facility at CERN (n-TOF indicates neutron beam measurements by means of time of flight technique). The neutrons are produced via spallation, by means of a high-intensity 20 GeV pulsed proton beam impinging on a thick target. The resulting neutron beam covers the energy range from thermal to several GeV. In this paper, we describe two beam diagnostic devices, both exploiting silicon detectors coupled with neutron converter foils containing (6)Li. The first one is based on four silicon pads and allows monitoring of the neutron beam flux as a function of the neutron energy. The second one, in beam and based on position sensitive silicon detectors, is intended for the reconstruction of the beam profile, again as a function of the neutron energy. Several electronic setups have been explored in order to overcome the issues related to the gamma flash, namely, a huge pulse present at the start of each neutron bunch which may blind the detectors for some time. The two devices were characterized with radioactive sources and also tested at the n-TOF facility at CERN. The wide energy and intensity range they proved capable of sustaining made them attractive and suitable to be used in both EAR1 and EAR2 n-TOF experimental areas, where they became immediately operational.

Morphostructural analysis of the male reproductive system and DNA barcoding in Balclutha brevis Lindberg 1954 (Homoptera, Cicadellidae).

Balclutha brevis Lindberg 1954 is an allochthonous leafhopper infesting an invasive grass, Pennisetum setaceum, in Sicily and in mainland Europe; therefore, this species could compete with populations of native species, thus contributing to the loss of biodiversity. Considering the ecological implications of B. brevis, investigations on all its biological aspects represent, therefore, a premise for further studies in applied sciences. Based on the lacking ultrastructural data about the reproductive systems of the Auchenorrhyncha, we carried out morphostructural investigations on the male reproductive system of B. brevis. Further, a first report of DNA barcoding analysis (amplification and sequencing of Cytochrome Oxidase I gene) has also been performed to characterize B. brevis compared to other congeneric species. From a morphological point of view, the male reproductive system of B. brevis has an organization comparable to the general anatomical features of most of the Auchenorrhyncha species; however, comparing our data with those concerning the different groups of Cicadomorpha, some considerations are discussed. As for the histological and ultrastructural investigations, our results show a secretory activity of the various examined structures, mainly in the lateral ejaculatory ducts and in the accessory glands. The latter, in particular, show morphostructural differences comparing the distal tract to the proximal one; moreover, the histochemical techniques showed the possible presence of a lipid component in the peculiar cytoplasmic granules found in the gland cells. The significance of these findings in the accessory glands is discussed. Finally, the ultrastructural features found in the seminal vesicles are different from those of the lateral ejaculatory ducts and are indicative of the different roles played by these structures in the organization of the spermatozoa bundles.

N-cadherin-mediated cell contact inhibits granulosa cell apoptosis in a progesterone-independent manner.

Previous studies have shown that both progesterone and cell contact inhibit granulosa cell (GC) apoptosis in vitro. Since the progesterone concentration associated with aggregated GCs may be higher than that of single GCs, experiments were conducted to differentiate progesterone's action from that of cell contact. For these studies, GCs were isolated from immature rats. Large GCs were collected after Percoll gradient centrifugation and placed in serum-free culture for 24 h. These studies confirmed that the rate of apoptosis was 2-3 times higher for single GCs than for aggregated GCs. This relationship was observed in the presence of aminoglutethimide, where progesterone concentrations were 3 ng/ml or less. A dose-response studied revealed that a minimum of 100 ng/ml progesterone were required to suppress apoptosis of single GCs. In addition, a single cell contact was shown to be sufficient to suppress apoptosis, with a small nonsteroidogenic GC being as effective as a large steroidogenic GC. Taken together, these data support the concept that cell contact blocks apoptosis in a progesterone-independent manner. GC contact is due to the presence of gap and adhesion-type junctions. To assess which, if either, of these junctions is involved in mediating the antiapoptotic action of cell contact, cocultures were set-up between GCs and R2C cells. Contact with R2C cells inhibits GC apoptosis, but does not result in the formation of functional gap junctions. This demonstrates that gap junctions are not essential to maintain GC viability. Adhesion-type junctions result from a homophilic binding of N-cadherin, which is expressed by both GCs and R2C cells. When this binding is inhibited by treatment with either an antibody to N-cadherin or a synthetic N-cadherin peptide, cell aggregation is attenuated. For those cells that form cell contacts in the presence of these N-cadherin-binding inhibitors, the percentage of apoptotic cells is increased compared to that in controls. These observations suggest that homophilic binding of N-cadherin molecules on the surface membranes of adjacent GCs initiates a signal transduction cascade that ultimately inhibits apoptosis.

Basic fibroblast growth factor and N-cadherin maintain rat granulosa cell and ovarian surface epithelial cell viability by stimulating the tyrosine phosphorylation of the fibroblast growth factor receptors.

Both granulosa cells (GCs) and ovarian surface epithelial cells undergo apoptosis in vivo. Although basic fibroblast growth factor (bFGF) and N-cadherin-mediated cell contact inhibit GC apoptosis, little is known about the factors that influence rat ovarian surface epithelial (ROSE) cell apoptosis. The present studies were designed to determine whether bFGF and N-cadherin maintain the viability of both GC and ROSE cells by stimulating separate signaling pathways. For the GC studies, large GCs were collected from immature rat ovaries after Percoll gradient centrifugation and placed in serum-free culture for 24 h. These studies confirmed that about 10% of the aggregated GCs and more than 50% of single GCs were apoptotic after culture. bFGF reduced the percentage of apoptotic single GCs, but did not influence aggregated GCs. A neutralizing antibody to bFGF blocked bFGF's antiapoptotic action, but did not alter the percentage of apoptotic aggregated GCs. The antibody to N-cadherin not only increased the percentage of aggregated apoptotic GCs, but also blocked bFGF's ability to maintain the viability of single GCs. The effect of the FGF receptor antibody was similar to that of the N-cadherin antibody. Like GCs, ROSE cells also undergo apoptosis in serum-free medium. Exposure to either the N-cadherin or FGF receptor antibody, even in the presence of serum, increased the percentage of apoptotic aggregated ROSE cells. As tyrosine kinase activity is involved in maintaining cell viability, the pattern of tyrosine-phosphorylated proteins was examined after culture in control (ascites) or N-cadherin antibody-supplemented medium. Exposure to the N-cadherin antibody altered the pattern of tyrosine-phosphorylated proteins, decreasing the tyrosine phosphorylation of proteins in the 130- to 180-kDa range and increasing the tyrosine phosphorylation of one or more proteins of about 50 kDa. The identity of the 50-kDa protein is unknown. However, immunoprecipitation studies demonstrated that the N-cadherin antibody reduced the amount of tyrosine-phosphorylated FGF receptor in both GCs and ROSE cells by 50%. This decrease corresponds to an increase in apoptosis among aggregated cells. Taken together, these data suggest that homophilic N-cadherin binding and bFGF-FGF receptor binding activate signal transduction pathways that converge at the level of the FGF receptor and subsequently promote the viability of both GC and ROSE cells.

Basic fibroblast growth factor maintains calcium homeostasis and granulosa cell viability by stimulating calcium efflux via a PKC delta-dependent pathway.

Previous studies have demonstrated that basic fibroblast growth factor prevents granulosa cell apoptosis. The following six observations provide insight into the mechanism by which basic fibroblast growth factor mediates its antiapoptotic action. First, loading granulosa cells with 1,2 bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, an intracellular calcium chelator, prevented apoptosis when granulosa cells were deprived of basic fibroblast growth factor. Second, treatment with thapsigargin, an agent known to increase intracellular free calcium, induced granulosa cell apoptosis even in the presence of basic fibroblast growth factor. Third, an activator of PKC mimicked, whereas PKC inhibitors blocked, basic fibroblast growth factor's antiapoptotic action. Fourth, continuous basic fibroblast growth factor exposure maintained relatively constant levels of intracellular free calcium, and a PKC inhibitor induced a sustained 2- to 3-fold increase in intracellular free calcium. Fifth, granulosa cells, as well as spontaneously immortalized granulosa cells, were shown to express PKC delta, -lambda, and -zeta. Finally, the PKC delta-specific inhibitor, rottlerin, blocked basic fibroblast growth factor's antiapoptotic action in granulosa cells and spontaneously immortalized granulosa cells. These studies suggest that basic fibroblast growth factor regulates intracellular free calcium through a PKC delta-dependent mechanism and that a sustained increase in intracellular free calcium is sufficient to induce and is required for granulosa cell apoptosis. Additional studies demonstrated that in spontaneously immortalized granulosa cells, basic fibroblast growth factor increased PKC delta activity by 60% within 2.5 min compared with serum-free control levels. Rottlerin attenuated basic fibroblast growth factor's ability to stimulate PKC delta activity and to maintain intracellular free calcium. Further, intracellular free calcium levels in spontaneously immortalized granulosa cells transfected with a PKC delta antibody in the presence of basic fibroblast growth factor were 2-fold higher than those spontaneously immortalized granulosa cells transfected with IgG. Similarly, transfecting spontaneously immortalized granulosa cells with a specific PKC delta-substrate increased intracellular free calcium compared with spontaneously immortalized granulosa cells transfected with a specific substrate for PKC epsilon. Moreover, basic fibroblast growth factor increased and rottlerin attenuated (45)Ca efflux by 50% compared with that in basic fibroblast growth factor-treated cells. Finally, an inhibitor of the plasma membrane calciumadenosine triphosphatase pump suppressed (45)Ca efflux, elevated intracellular free calcium, and induced apoptosis. Collectively, these studies demonstrate that basic fibroblast growth factor activates PKC delta, which, in turn, stimulates calcium efflux, accounting in part for basic fibroblast growth factor's ability to maintain calcium homeostasis and, ultimately, granulosa cell viability.

Steroidogenic factor-1 as a positive regulator of rat granulosa cell differentiation and a negative regulator of mitosis.

Ovarian follicles contain small nonaromatase-expressing and large aromatase-expressing granulosa cells (GCs). The present studies were designed to determine whether small GCs can differentiate into large GCs and/or express aromatase. Additional studies were conducted to assess the role of steroidogenic factor-1 (SF-1), an orphan nuclear receptor, in regulating GC differentiation and proliferation. For these studies, small GCs were isolated from immature rats by Percoll gradient centrifugation and cultured for up to 48 h with FSH and/or 8-bromo-cAMP (8-br-cAMP). FSH/8-br-cAMP induced a 2-fold increase in SF-1 messenger RNA levels within 4 h. This increase was maintained throughout the culture period. By 24 h culture, FSH/8-br-cAMP increased the percentage of large GCs. It was not until 48 h of culture with FSH and 8-br-cAMP that aromatase expression increased. This increase was detected by both Western blot and quantitative immunocytochemistry. 8-br-cAMP alone did not promote GC differentiation. Small GCs were then cultured with FSH/8-br-cAMP in the presence or absence of an antisense oligonucleotide complementary to the putative SF-1 ligand-binding site (SF-1 AS). As a control, small GCs were cultured with FSH/8-br-cAMP and an 18-mer nonsense oligonucleotide (SF-1 NS). The SF-1 AS, but not the SF-1 NS, prevented FSH/8-br-cAMP from increasing 1) SF-1 messenger RNA levels, 2) transcription of a SF-1(x2) promoter/luciferase construct, 3) GC size, and 4) aromatase expression. In a third series of experiments, small GCs were cultured for 24 h in 1) control media supplemented with 2) a mitogen, phorbol ester [12-O-tetraphorbol acetate (TPA)], 3) FSH/8-br-cAMP, or 4) both. TPA increased the number of GCs by 51 +/- 9%. FSH/8-br-cAMP completely blocked TPA-induced mitosis. When small GCs were cultured with FSH/cAMP, TPA, and SF-1 AS, the number of GCs increased by 50 +/- 7%. This increase was not observed with SF-1 NS. Taken together, these data demonstrate that SF-1 is expressed in both small and large GCs, and enhanced SF-1 expression is part of the molecular mechanism associated with GC differentiation. Interestingly, SF-1 not only regulates differentiation, but also inhibits TPA-induced GC mitosis.