Putative correlates of protection against shigellosis assessing immunomarkers across responses to S. sonnei investigational vaccine.

Shigella spp. are a leading bacterial cause of diarrhea. No widely licensed vaccines are available and there is no generally accepted correlate of protection. We tested a S. sonnei Generalized Modules for Membrane Antigen (GMMA)-based vaccine (1790GAHB) in a phase 2b, placebo-controlled, randomized, controlled human infection model study (NCT03527173) enrolling healthy United States adults aged 18-50 years. We report analyses evaluating immune responses to vaccination, with the aim to identify correlates of risk for shigellosis among assessed immunomarkers. We found that 1790GAHB elicited S. sonnei lipopolysaccharide specific α4ß7+ immunoglobulin (Ig) G and IgA secreting B cells which are likely homing to the gut, indicating the ability to induce a mucosal in addition to a systemic response, despite parenteral delivery. We were unable to establish or confirm threshold levels that predict vaccine efficacy facilitating the evaluation of vaccine candidates. However, serum anti-lipopolysaccharide IgG and bactericidal activity were identified as potential correlates of risk for shigellosis.

Age-dependent acquisition of IgG antibodies to Shigella serotypes-a retrospective analysis of seroprevalence in Kenyan children with implications for infant vaccination.

Background: Shigellosis mainly affects children under 5 years of age living in low- and middle-income countries, who are the target population for vaccination. There are, however, limited data available to define the appropriate timing for vaccine administration in this age group. Information on antibody responses following natural infection, proxy for exposure, could help guide vaccination strategies. Methods: We undertook a retrospective analysis of antibodies to five of the most prevalent Shigella serotypes among children aged <5 years in Kenya. Serum samples from a cross-sectional serosurvey in three Kenyan sites (Nairobi, Siaya, and Kilifi) were analyzed by standardized ELISA to measure IgG against Shigella sonnei and Shigella flexneri 1b, 2a, 3a, and 6. We identified factors associated with seropositivity to each Shigella serotype, including seropositivity to other Shigella serotypes. Results: A total of 474 samples, one for each participant, were analyzed: Nairobi (n = 169), Siaya (n = 185), and Kilifi (n = 120). The median age of the participants was 13.4 months (IQR 7.0-35.6), and the male:female ratio was 1:1. Geometric mean concentrations (GMCs) for each serotype increased with age, mostly in the second year of life. The overall seroprevalence of IgG antibodies increased with age except for S. flexneri 6 which was high across all age subgroups. In the second year of life, there was a statistically significant increase of antibody GMCs against all five serotypes (p = 0.01-0.0001) and a significant increase of seroprevalence for S. flexneri 2a (p = 0.006), S. flexneri 3a (p = 0.006), and S. sonnei (p = 0.05) compared with the second part of the first year of life. Among all possible pairwise comparisons of antibody seropositivity, there was a significant association between S. flexneri 1b and 2a (OR = 6.75, 95% CI 3-14, p < 0.001) and between S. flexneri 1b and 3a (OR = 23.85, 95% CI 11-54, p < 0.001). Conclusion: Children living in low- and middle-income settings such as Kenya are exposed to Shigella infection starting from the first year of life and acquire serotype-specific antibodies against multiple serotypes. The data from this study suggest that Shigella vaccination should be targeted to infants, ideally at 6 or at least 9 months of age, to ensure children are protected in the second year of life when exposure significantly increases.

Salmonella Vaccine Study in Oxford (SALVO) trial: protocol for an observer-participant blind randomised placebo-controlled trial of the iNTS-GMMA vaccine within a European cohort.

INTRODUCTION: Invasive non-typhoidal Salmonellosis (iNTS) is mainly caused by Salmonella enterica serovars Typhimurium and Enteritidis and is estimated to result in 77 500 deaths per year, disproportionately affecting children under 5 years of age in sub-Saharan Africa. Invasive non-typhoidal Salmonellae serovars are increasingly acquiring resistance to first-line antibiotics, thus an effective vaccine would be a valuable tool in reducing morbidity and mortality from infection. While NTS livestock vaccines are in wide use, no licensed vaccines exist for use in humans. Here, a first-in-human study of a novel vaccine (iNTS-GMMA) containing S. Typhimurium and S. Enteritidis Generalised Modules for Membrane Antigens (GMMA) outer membrane vesicles is presented. METHOD AND ANALYSIS: The Salmonella Vaccine Study in Oxford is a randomised placebo-controlled participant-observer blind phase I study of the iNTS-GMMA vaccine. Healthy adult volunteers will be randomised to receive three intramuscular injections of the iNTS-GMMA vaccine, containing equal quantities of S. Typhimurium and S. Enteritidis GMMA particles adsorbed on Alhydrogel, or an Alhydrogel placebo at 0, 2 and 6 months. Participants will be sequentially enrolled into three groups: group 1, 1:1 randomisation to low dose iNTS-GMMA vaccine or placebo; group 2, 1:1 randomisation to full dose iNTS-GMMA vaccine or placebo; group 3, 2:1 randomisation to full dose or lower dose (dependant on DSMC reviews of groups 1 and 2) iNTS-GMMA vaccine or placebo.The primary objective is safety and tolerability of the vaccine. The secondary objective is immunogenicity as measured by O-antigen based ELISA. Further exploratory objectives will characterise the expanded human immune profile. ETHICS AND DISSEMINATION: Ethical approval for this study has been obtained from the South Central-Oxford A Research Ethics Committee (Ethics REF:22/SC/0059). Appropriate documentation and regulatory approvals have been acquired. Results will be disseminated via peer-reviewed articles and conferences. TRIAL REGISTRATION NUMBER: EudraCT Number: 2020-000510-14.

Efficacy, safety, and immunogenicity of the Shigella sonnei 1790GAHB GMMA candidate vaccine: Results from a phase 2b randomized, placebo-controlled challenge study in adults.

BACKGROUND: Shigellosis accounts for substantial morbidity and mortality worldwide and is the second most common cause of moderate and severe diarrhoea in children. METHODS: This phase 2b study (NCT03527173), conducted between August 2018 and November 2019, evaluated vaccine efficacy (VE), safety, and immunogenicity of a Shigella sonnei GMMA candidate vaccine (1790GAHB) in adults, using a S. sonnei 53 G controlled human infection model. Participants (randomized 1:1) received two doses of 1790GAHB or placebo (GAHB-Placebo), at day (D) 1 and D29, and an oral challenge of S. sonnei 53 G at D57. VE was evaluated using several endpoints, reflecting different case definitions of shigellosis. For the primary endpoint, the success criterion was a lower limit of the 90% confidence interval >0. FINDINGS: Thirty-six and 35 participants received 1790GAHB or placebo, respectively; 33 and 29 were challenged, 15 and 12 developed shigellosis. VE was not demonstrated for any endpoint. Adverse events were more frequent in 1790GAHB versus placebo recipients post-vaccination. Anti-S. sonnei lipopolysaccharide (LPS) IgG responses increased at D29 and remained stable through D57 in group 1790GAHB; no increase was shown in placebo recipients. INTERPRETATION: 1790GAHB had an acceptable safety profile and induced anti-LPS IgG responses but did not demonstrate clinical efficacy against shigellosis. Baseline/pre-challenge antibody levels were higher in participants who did not develop shigellosis post-challenge, suggesting a role of anti-LPS IgG antibodies in clinical protection, although not fully elucidated in this study. For further vaccine development an increased S. sonnei O-antigen content is likely needed to enhance anti-LPS immune responses. FUNDING: GlaxoSmithKline Biologicals SA, Bill and Melinda Gates Foundation.

Booster Vaccination With GVGH Shigella sonnei 1790GAHB GMMA Vaccine Compared to Single Vaccination in Unvaccinated Healthy European Adults: Results From a Phase 1 Clinical Trial.

The investigational Shigella sonnei vaccine (1790GAHB) based on GMMA (generalized modules for membrane antigens) is immunogenic, with an acceptable safety profile in adults. However, pre-vaccination anti-S. sonnei lipopolysaccharide (LPS) antibody levels seemed to impact vaccine-related immune responses. This phase 1, open-label, non-randomized extension study (ClinicalTrials.gov: NCT03089879) evaluated immunogenicity of a 1790GAHB booster dose in seven adults with undetectable antibodies prior to priming with three 1790GAHB vaccinations 2-3 years earlier (boosted group), compared to one dose in 28 vaccine-naïve individuals (vaccine-naïve group). Anti-S. sonnei LPS serum IgG geometric mean concentrations and seroresponse (increase of ≥25 EU or ≥50% from baseline antibody ≤ 50 EU and ≥50 EU, respectively) rates were calculated at vaccination (day [D]1), D8, D15, D29, D85. Safety was assessed. Geometric mean concentrations at D8 were 168 EU (boosted group) and 32 EU (vaccine-naïve group). Response peaked at D15 (883 EU) and D29 (100 EU) for the boosted and vaccine-naïve groups. Seroresponse rates at D8 were 86% (boosted group) and 24% (vaccine-naïve group) and increased at subsequent time points. Across both groups, pain (local) and fatigue (systemic) were the most frequent solicited adverse events (AEs). Unsolicited AEs were reported by 57% of boosted and 25% of vaccine-naïve participants. No deaths, serious AEs, or AEs of special interest (except one mild neutropenia case, possibly vaccination-related) were reported. One 1790GAHB dose induced a significant booster response in previously-primed adults, regardless of priming dose, and strong immune response in vaccine-naïve individuals. Vaccination was well tolerated.

Extracellular vesicles as shuttles of tumor biomarkers and anti-tumor drugs.

Extracellular vesicles (EV) include vesicles released by either normal or tumor cells. EV may exceed the nanometric scale (microvesicles), or to be within the nanoscale, also called exosomes. Thus, it appears that only exosomes and larger vesicles may have the size for potential applications in nanomedicine, in either disease diagnosis or therapy. This is of particular interest for research in cancer, also because the vast majority of existing data on EV are coming from pre-clinical and clinical oncology. We know that the microenvironmental features of cancer may favor cell-to-cell paracrine communication through EV, but EV have been purified, characterized, and quantified from plasma of tumor patients as well, thus suggesting that EV may have a role in promoting and maintaining cancer dissemination and progression. These observations are prompting research efforts to evaluate the use of nanovesicles as tumor biomarkers. Moreover, EVs are emerging as natural delivery systems and in particular, exosomes may represent the ideal natural nanoshuttles for new and old anti-tumor drugs. However, much is yet to be understood about the role of EV in oncology and this article aims to discuss the future of EV in cancer on the basis of current knowledge.

In vitro and in vivo characterization of a novel Hedgehog signaling antagonist in human glioblastoma cell lines.

Glioblastoma multiforme (GBM) is composed of heterogeneous and genetically different cells, which are highly invasive and motile. The standard chemotherapeutic agent, temozolomide, affects GBM cell proliferation but is generally unable to prevent tumor recurrence. Hedgehog pathway activation has been reported to be relevant in GBM and different pharmacological pathway modulators have been identified. We report that by growing a commercially available recurrent GBM cell line (DBTRG-05MG) without serum and in the presence of defined growth factors; we obtained a less differentiated cell population, growing in suspension as neurospheres, in which the Hedgehog pathway is activated. Furthermore, the expression profile of Hedgehog pathway components found in DBTRG-05MG neurospheres is similar to primary stem-like cells derived from recurrent GBM patients. We report the effect of our novel specific Smoothened receptor antagonist (SEN450) on neurosphere growing cells and compared its effect to that of well known benchmark compounds. Finally, we showed that SEN450 is both antiproliferative on its own and further reduces tumor volume after temozolomide pretreatment in a mouse xenograft model using DBTRG-05MG neurosphere cells. Altogether our data indicate that the Hedgehog pathway is not irreversibly switched off in adherent cells but can be reactivated when exposed to well-defined culture conditions, thus restoring the condition observed in primary tumor-derived material, and that pharmacological modulation of this pathway can have profound influences on tumor proliferation. Therefore, pharmacological inhibition of the Hedgehog pathway is a potentially useful therapeutic approach in GBM.

KIF11 inhibition for glioblastoma treatment: reason to hope or a struggle with the brain?

BACKGROUND: Glioblastomas (GBM) are typically comprised of morphologically diverse cells. Despite current advances in therapy, including surgical resection followed by radiation and chemotherapy, the prognosis for patients with GBM remains poor. Unfortunately, most patients die within 2 years of diagnosis of their disease. Molecular abnormalities vary among individual patients and also within each tumor. Indeed, one of the distinguishing features of GBM is its marked genetic heterogeneity. Due to the brain location of the tumor, the potential target inhibition for anticancer therapy must exhibit a manageable neurotoxicity profile in the concentration range in which the compounds show anti-proliferative activity.Kinesin KIF11 inhibition by small molecules such as Monastrol or Ispinesib is currently under investigation in the field of malignant tumors. In the current study we have assessed the relevance of the anti-mitotic Kinesin-like protein KIF11 in human GBM cell-lines. RESULTS: In this study the target was validated using a set of well characterised and potentially specific small molecule inhibitors of KIF11: an ispinesib analog, Monastrol, a Merck compound and 3 simplified derivatives of the Merck compound. Following an in silico selection, those compounds predicted to bear a favorable BBB permeation profile were assessed for their phenotypic effect on cell lines derived both from primary (U87MG) as well as treated (DBTRG-05-MG) glioblastomas. For some compounds, these data could be compared to their effect on normal human astrocytes, as well as their neurotoxicity on primary rat cortical neurons. The ispinesib analogue 1 showed an anti-proliferative effect on GBM cell lines by blocking them in the G2/M phase in a concentration range which was shown to be harmless to primary rat cortical neurons. Furthermore, ispinesib analog increased caspase 3/7-induced apoptosis in U87MG cells. CONCLUSION: In the area of cell cycle inhibition, KIF11 is critical for proper spindle assembly and represents an attractive anticancer target. Our results suggest that KIF11 inhibitors, when able to permeate the blood-brain-barrier, could represent an interesting class of anticancer drugs with low neurotoxic effects in the treatment of brain tumors.

The vitamin D analogue BXL-628 inhibits growth factor-stimulated proliferation and invasion of DU145 prostate cancer cells.

PURPOSE: Suppression of the invasive phenotype is essential in developing new therapeutic tools to treat advanced prostate cancer (PC) indicating that androgen-independent prostate cancer (AI-PC) is characterized by increased metastatic potential. In the present study, we have investigated the effect of the nonhypercalcemic vitamin D analogue BXL-628 on proliferation and invasive properties of the human PC cell line DU145. In particular, the effect of the analogue was tested following stimulation with a potent growth factor, keratinocyte growth factor (KGF), which stimulates both proliferation and invasion of these cells. We have also evaluated the effect of the analogue on KGF stimulation of PI3K/AKT signaling pathway. METHODS: Cell proliferation was determined by cell counting. Invasion through Matrigel was evaluated using Boyden chambers. PI3K activity was measured by immunokinase assay and AKT phosphorylation was evaluated by western blot analysis. Keratinocyte growth factor receptor (KGFR) autotransphosphorylation was evaluated by western blot after immunoprecipitation of the receptor. RESULTS: BXL-628 is able to inhibit both proliferation and invasion of DU145 cells in basal conditions and in response to KGF. Following stimulation with KGF, the inhibition is due to suppression of KGFR autotransphosphorylation and downstream PI3K/AKT activation, both achieved following a brief (5 min) incubation with the analogue. This effect on KGFR autophosphorylation was still present when cells were treated with the alpha-amanitin, an inhibitor of RNA transcription, indicating a rapid, nongenomic effect. CONCLUSIONS: Our results demonstrate that the vitamin D analogue BXL-628 is able to suppress KGF-induced proliferation and invasion of AI-PC cells in vitro, prospecting a possible use of the drug, which is currently in phase II clinical studies for benign prostatic hyperplasia, in the treatment of advanced prostate cancer.

Non-genomic effects of the androgen receptor and vitamin D agonist are involved in suppressing invasive phenotype of prostate cancer cells.

Suppression of invasive phenotype is essential in developing new therapeutic tools to treat prostate cancer (PC). Evidence indicates that androgen-dependent (AD) prostate cancer cells are characterized by a lower malignant phenotype. We have demonstrated that transfection with an androgen receptor (AR) expression vector of the androgen-independent (AI) prostate cancer cell line PC3 decreases invasion of these cells through modulation of alpha6beta4 integrin expression, indicating a genotropic effect of androgens in inhibiting invasion ability of AD PC cells. Later on, we have shown that also a non-genotropic mechanism is involved in such an effect. By using immunoconfocal fluorescent microscopy, we demonstrated that AR in PC3-AR cells co-localizes with the EGFR receptors (EGFR) in PC3-AR cells. Co-immunoprecipitation studies both in PC3-AR cells and in the AD cell line LNCaP that physiologically express both receptors, confirm the occurrence of an interaction between of the two proteins. In PC3-AR cells, we demonstrated a disruption of EGFR signalling properties (reduced EGF-induced EGFR autotransphosphorylation, reduced EGF-stimulated PI3K activity as well as EGFR-PI3K interaction) contributing to the lower invasive phenotype of these cells. In another study, we investigated the effects of a new Vitamin D analogue, BXL628, on invasion in response to KGF in the androgen-independent PC cell line DU145. We found that the compound was able to reduce proliferation and invasion of the cells in response to the growth factor. In addition, we found that KGF-induced autotransphosphorylation of KGF receptor (KGFR) and PI3K activation were suppressed after short-term (5min) pre-treatment with the analogue before addition of KGF. Collectively, these studies demonstrate that a non-genotropic effect due to a direct interaction of the androgen receptor with EGFR and to a rapid effect of a Vitamin D agonist on KGFR may disrupt signalling of GF leading to decreased tumorigenicity and a less malignant phenotype of PC cells in vitro.

Oxytocin mediates the estrogen-dependent contractile activity of endothelin-1 in human and rabbit epididymis.

Epididymis is a sex steroid (androgen + estrogen)-sensitive duct provided with spontaneous motility, allowing sperm transport. We previously reported that the oxytocin (OT) receptor (OTR) mediates an estrogen-dependent increase in epididymal contractility. Because endothelin (ET)-1 also regulates epididymal motility, we tested its sex steroid dependence in a rabbit model. We demonstrated that estrogens up-regulate responsiveness to ET-1, which is reduced by blocking aromatase activity (letrozole, 2.5 mg/kg) or by triptorelin (2.9 mg/kg)-induced hypogonadism, whereas it is fully restored by estradiol valerate (3.3 mg/kg weekly) but not by testosterone enanthate (30 mg/kg weekly). However, changing sex steroid milieu did not affect either ET-1, its receptor gene, or protein expression. Two structurally distinct OTR-antagonists [(d(CH2)5(1), Tyr(Me)(2), Orn(8))-OT and atosiban] almost completely abolished ET-1 contractility, without competing for [125I]ET-1 binding, suggesting that OT/OTR partially mediates ET-1 action. Immunohistochemical studies in human and rabbit epididymis demonstrated that both OT and its synthesis-associated protein, neurophysin I, are expressed in the epithelial cells facing the muscular layer, suggesting local OT production. Quantitative RT-PCR demonstrated a high abundance of OT transcripts in human epididymis. OT transcript was also originally detected and partially sequenced in rabbit epididymis. To verify whether ET-1 regulates OT release, we used rabbit epididymal epithelial cell cultures. These cells expressed a high density of [125I]ET-1 binding sites and responded to ET-1 with a dose-dependent OT release. Hence, we propose that an ET-1-induced OT/OTR system activation underlies the estrogen-dependent hyperresponsiveness to ET-1. These local sources might promote the spontaneous motility necessary for sperm transport.

Elevated serum interferon-gamma-inducible chemokine-10/CXC chemokine ligand-10 in autoimmune primary adrenal insufficiency and in vitro expression in human adrenal cells primary cultures after stimulation with proinflammatory cytokines.

Chemokines are a large family of cytokines involved in the pathogenesis of inflammatory and autoimmune diseases. Among CXC chemokines, CXC chemokine ligand 10 (CXCL10) has been identified to play an important role in several endocrinological autoimmune diseases, such as Hashimoto's thyroiditis, Graves' disease, and type 1 diabetes mellitus. Although the mechanisms leading to glandular autoimmune process may be at least in part shared by different endocrine organs, the role of CXCL10 in autoimmune adrenal insufficiency is unknown. The aim of this study was to evaluate the role of CXCL10 in Addison's disease (AD). Serum CXCL10 levels were assayed in 64 patients with clinically evident autoimmune AD, 20 patients with autoimmune subclinical AD, nine patients with nonautoimmune AD, and 48 healthy volunteers. Clinically evident and subclinical AD, but not nonautoimmune AD patients, showed a significant increase in serum CXCL10 levels compared with healthy subjects: 119.9 pg/ml (range, 39.8-427.6) and 124.0 pg/ml (range, 37.0-384.7) vs. 75.6 pg/ml (range, 22.4-164.0; P < 0.001 for both groups). Comparable serum CXCL10 levels were found between patients with an isolated form of AD and patients with other autoimmune conditions associated with AD, suggesting a specific influence of the adrenal autoimmune process in determining elevated CXCL10 concentrations in such patients. No relationship was found between serum CXCL10 levels and anti-21-hydroxylase or adrenal cortex autoantibody titers or between CXCL10 levels and duration of disease. The role of CXCL10 in the adrenal gland was also evaluated in vitro in human zona fasciculata cells (hZFC). CXCL10, although not basally detected in cultured hZFC, was strongly induced by interferon-gamma and synergistically increased by TNF-alpha addition. Hydrocortisone or ACTH alone had no effect on CXCL10 secretion in hZFC, but they both significantly inhibited cytokine-induced CXCL10 secretion. Taken together, these data suggest a potential role of hZFC, through the production of CXCL10, in regulating the recruitment of specific subsets of activated lymphocytes in autoimmune AD.

Identification, characterization and biological activity of oxytocin receptor in the developing human penis.

Although abnormalities of the male external genitalia (MEG) are a relatively common problem, little is known concerning the molecular mechanisms that finely regulate penile development. We report here the expression of the oxytocin receptor (OTR) gene by real-time RT-PCR in human fetal tissues (11th-12th week of gestation), including the MEG. The developing penis expressed a very high level of OTR mRNA, only a half log(10) unit lower than fetal central nervous system, used as a positive control. The OTR protein is also highly expressed (western, immunohistochemistry and binding studies) and immunolocalized both in the mesenchymal body and in the surrounding blood capillaries, which will later constitute penile trabeculae and sinusoids. Binding studies using [125I]oxytocin antagonist ([125I]OTA) in cultured human fetal penile smooth muscle cells (hfPSMC) revealed the presence of specific OTR with a high capacity and affinity for oxytocin (OT) and for OTA. Increasing concentrations of OT dose-dependently induced intracellular Ca2+ mobilization. Furthermore, OTR mediated an increase in the proliferation and the migration of hfPSMC. In conclusion, we demonstrate that in the developing human MEG, OTR is highly expressed and might be involved in coordinating timely and appropriate proliferation and migration of the penile cells. Thus, OTR might represent an additional target for investigating human fetal MEG organogenesis.

Human bladder as a novel target for vitamin D receptor ligands.

Human prostate is now considered a target for vitamin D receptor (VDR) ligands, such as BXL-628. Because BXL-628 inhibited prostate growth without interfering with androgen signaling, it represents a new option for benign prostate hyperplasia (BPH) therapy. However, BPH symptoms are related not only to prostate size, but also to compensatory bladder hypertrophy and eventual overactivity. We now report that human bladder expresses VDR (determined by real-time PCR immunohistochemistry and Western blot) and responds to VDR agonists, such as the natural ligand, calcitriol, and its synthetic and less hypercalcemic derivative, BXL-628. Experiments were conducted with stromal cells derived from human bladder neck obtained at surgery from BPH patients. BXL-628 counteracted keratinocyte growth factor (KGF) and androgen-induced cell proliferation and stimulated apoptosis with a parallel reduced expression of the survival oncoprotein Bcl-2. Prolonged serum starvation time-dependently pushed bladder stromal cells to express activated myofibroblast markers, such as desmin and smoothelin, without changing other contractile-related proteins and intermediate filaments, such as vimentin. Chronic exposure to BXL-628 prevented starvation-induced cell phenotype modification. Because hypertrophy and starvation-induced bladder remodeling are supposed to underlie bladder overactivity, it is possible that BXL-628 might be helpful in reducing not only cumbersome symptoms related to prostate overgrowth, but also those related to bladder irritation.

Thiazolidinediones inhibit growth and invasiveness of the human adrenocortical cancer cell line H295R.

Thiazolidinediones (TZDs) are a new class of antidiabetic drugs that have also been shown to possess antitumoral properties in different human cancers. TZDs bind and activate the peroxisome proliferator-activated receptor (PPAR)-gamma, which is a nuclear receptor acting as a transcription factor in several tissues. In the present study, we evaluated PPARgamma mRNA and protein expression in tissue samples of human adrenocortical carcinomas (ACCs), normal adrenal glands, and the human ACC cell line H295R. PPARgamma mRNA was expressed in six of eight ACC, two of three normal adrenal glands and the H295R cells. These results were confirmed by immunohistochemistry. PPARgamma transcriptional activity in H295R cells, monitored by a reporter gene assay, was induced 2- to 3-fold by TZDs, such as rosiglitazone (RGZ) and pioglitazone, whereas in PPARgamma-transfected cells RGZ alone or RGZ plus 9-cis retinoic acid further increased reporter activity. TZDs inhibited both the proliferation and invasiveness of H295R cells in a dose-dependent manner. Thymidine incorporation was reduced by about 60% by 20 mum of both TZDs. Cotreatment with the retinoic X receptor ligand 9-cis retinoic acid had an additive effect. TZDs increased the number of cells in the G(0)/G(1) phase and decreased them in the S phase. Western blot analysis showed that TZDs increased the expression of the cell cycle inhibitors p21 and p27 and reduced the expression of cyclin D1. Twenty micromoles of RGZ and pioglitazone reduced H295R invasiveness through Matrigel by about 85%. Zymography and ELISA tests showed that TZD inhibited metalloproteinase-2 secretion by H295R cells in a dose-dependent manner. These data suggest that TZDs reduce the malignant potential of the H295R ACC cell line and, therefore, might potentially constitute a novel tool in the medical treatment of human ACCs.

Expression of the novel adrenocorticotropin-responsive gene selective Alzheimer's disease indicator-1 in the normal adrenal cortex and in adrenocortical adenomas and carcinomas.

Selective Alzheimer's disease indicator-1 (seladin-1) is a novel gene with antiapoptotic activity that is down-regulated in vulnerable brain regions in Alzheimer's disease. This gene encodes 3-beta-hydroxysterol Delta-24-reductase (DHCR24), which converts desmosterol into cholesterol. In the adrenal cortex, increased expression of seladin-1/DHCR24, which appears to be modulated by ACTH, has been recently reported in cortisol-secreting adenomas, compared with the adjacent atrophic tissue. In our study, we measured the expression level of seladin-1/DHCR24 in cortisol- (n = 18) and aldosterone-secreting (n = 16) adrenocortical adenomas, in carcinomas (n = 17), and in normal adrenal glands (n = 8) by quantitative real-time RT-PCR. The amount of seladin-1/DHCR24 mRNA was significantly reduced in carcinomas (total RNA, 2.5 +/- 0.8 pg/ micro g) compared with the other groups (P < 0.01). Western blot analysis confirmed the mRNA results. Similarly, in adrenal malignancies, significantly reduced levels of expression of the ACTH receptor gene were found. In the adrenal cancer cell line H295R and in primary cultures from adrenocortical cells, ACTH (1 nM) and forskolin (10 micro M) effectively increased seladin-1/DHCR24 expression, confirming that seladin-1/DHCR24 is modulated by the ACTH/cAMP-driven pathway. In summary, this is the first demonstration that seladin-1/DHCR24 expression is reduced in adrenal cancer, suggesting that it might be viewed as a new potential marker of adrenal malignancies.

Increased phosphorylation of AKAP by inhibition of phosphatidylinositol 3-kinase enhances human sperm motility through tail recruitment of protein kinase A.

Sperm motility is regulated by a complex balance between kinases and phosphatases. Among them, phosphatidylinositol 3-kinase (PI 3-kinase) has been recently suggested to negatively regulate sperm motility (Luconi, M., Marra, F., Gandini, L., Lenzi, A., Filimberti, E., Forti, G. and Baldi, E. (2001). Hum. Reprod. 16, 1931-1937). We demonstrate the presence and activity of PI 3-kinase in human spermatozoa and have investigated the molecular mechanism(s) by which the PI 3-kinase inhibitor, LY294002, triggers an increase in sperm motility. PI 3-kinase inhibition results in an increase in intracellular cAMP levels and in tyrosine phosphorylation of the protein kinase A-anchoring protein AKAP3. These effects finally result in a stimulation of protein kinase A (PKA) binding to AKAP3 in sperm tails through the regulatory subunit RIIbeta. The increased binding of RIIbeta to AKAP3 induced by LY294002 is mainly due to tyrosine phosphorylation of AKAP3, since it is completely blocked by the tyrosine kinase inhibitor erbstatin, which also reverses the effects of LY294002 on motility and suppresses PKA-AKAP3 interaction. The requirement of PKA binding to AKAP3 for sperm motility is confirmed by the reduction of motility induced by an inhibitor of RIIbeta-AKAP3 binding, Ht31, whose effects on sperm motility and PKA binding to AKAP3 are reversed by LY294002. These results demonstrate that PI 3-kinase negatively regulates sperm motility by interfering with AKAP3-PKA binding, providing the first evidence of a molecular mechanism by which PKA can be targeted to sperm tails by interaction with tyrosine phosphorylated form of AKAP3.

Expression and function of gonadotropin-releasing hormone (GnRH) receptor in human olfactory GnRH-secreting neurons: an autocrine GnRH loop underlies neuronal migration.

Olfactory neurons and gonadotropin-releasing hormone (GnRH) neurons share a common origin during organogenesis. Kallmann's syndrome, clinically characterized by anosmia and hypogonadotropic hypogonadism, is due to an abnormality in the migration of olfactory and GnRH neurons. We recently characterized the human FNC-B4 cell line, which retains properties present in vivo in both olfactory and GnRH neurons. In this study, we found that FNC-B4 neurons expressed GnRH receptor and responded to GnRH with time- and dose-dependent increases in GnRH gene expression and protein release (up to 5-fold). In addition, GnRH and its analogs stimulated cAMP production and calcium mobilization, although at different biological thresholds (nanomolar for cAMP and micromolar concentrations for calcium). We also observed that GnRH triggered axon growth, actin cytoskeleton remodeling, and a dose-dependent increase in migration (up to 3-4-fold), whereas it down-regulated nestin expression. All these effects were blocked by a specific GnRH receptor antagonist, cetrorelix. We suggest that GnRH, secreted by olfactory neuroblasts, acts in an autocrine pattern to promote differentiation and migration of those cells that diverge from the olfactory sensory lineage and are committed to becoming GnRH neurons.

Cushing's syndrome in a patient with bilateral macronodular adrenal hyperplasia responding to cisapride: an in vivo and in vitro study.

Cortisol secretion in adrenal Cushing's syndrome can be regulated by aberrant hormone receptors, such as gastric inhibitory polypeptide, V1 vasopressin, catecholamines, LH/human chorionic gonadotropin, and serotonin receptors. We report the case of a patient with Cushing's syndrome due to bilateral adrenal macronodular hyperplasia. Extensive in vivo testing for the presence of aberrant receptors revealed a 5-fold increase of plasma cortisol after the administration of cisapride, an agonist of the serotonin 4 (5-HT(4)) receptor. Primary cell cultures were established from adrenocortical specimens obtained at surgery, and in vitro studies also showed that cisapride determined an increase [133.7 +/- 5.5% (mean +/- SE) of baseline, considered 100%) of cortisol secretion from cultured cells. The presence of 5-HT(4) receptor transcript, and in particular of isoforms c, g, and n, was confirmed by RT-PCR, and the determination of the mRNA levels by real-time RT-PCR revealed a higher expression than in normal adrenal glands. To our knowledge, this is one of the first reports of Cushing's syndrome in which cortisol secretion is regulated mainly by the 5-HT(4) receptor, among known aberrant receptors. In addition, it is noteworthy that hypocortisolism ensued after the removal of the most enlarged adrenal gland, but the in vivo response to cisapride persisted.

Vitamin D3 analogue inhibits keratinocyte growth factor signaling and induces apoptosis in human prostate cancer cells.

BACKGROUND: Prostate cancer is a worldwide significant health care problem, due to its high incidence and mortality. In particular, androgen-independent tumors have the worst prognosis, because they are refractory to almost all kinds of available therapy. Hence, there is the need of new treatment opportunities targeting androgen-independent, growth factor-mediated, tumor signaling. One of these new promising opportunities is vitamin D3 and its related analogues. METHODS: We investigated the effect of a vitamin D3 analogue, analogue (V), on proliferation of several human prostate cancer cells in basal condition and after treatment with KGF, one of the intraprostatic growth factors that might participate in the progression of prostate cancer. In addition, in the androgen-independent cell line DU 145, we also studied the effect of analogue (V), KGF, and their mutual interaction on protein tyrosine phosphorylation, bcl-2 expression and apoptosis. RESULTS: Overall, we found that analogue (V) dose-dependently decreased basal and KGF-induced prostate cancer cell growth, although to a different extent. Maximal effect was obtained in DU 145 cells. In these cells, KGF stimulated tyrosine phosphorylation of a protein corresponding to its receptor, induced bcl-2 expression, and prolonged cell survival. Analogue (V) not only counteracted all these KGF-mediated events, but also decreased basal bcl-2 expression, therefore, allowing DU 145 cells to undergo an apoptotic program. CONCLUSIONS: Our results indicated that in prostate cancer cells analogue (V) decreased basal and KGF-induced cell proliferation. This effect, at least in DU 145 cells, is in part mediated by negative interactions with cell survival and KGF signaling.