Depletion of macrophages in mice results in higher dengue virus titers and highlights the role of macrophages for virus control

Monocytes and macrophages are target cells for dengue infection. Besides their potential role for virus replication, activated monocytes/macrophages produce cytokines that may be critical for dengue pathology. To study the in vivo role of monocytes and macrophages for virus replication, we depleted monocytes and macrophages in IFN‐αβγR knockout mice with clodronate liposomes before dengue infection. Although less virus was first recovered in the draining LN in the absence of macrophages, monocyte/macrophage depletion eventually resulted in a ten‐fold higher systemic viral titer. A massive infiltration of CD11b⁺CD11c^lowLy6C^low monocytes into infected organs was observed in parallel with increasing virus titers before viremia was controlled. Depletion of monocytes in the blood before or after local infection had no impact on virus titers, suggesting that monocytes are not required as “virus‐shuttles”. Our data provide evidence that systemic viremia is established independently of tissue macrophages present at the site of infection and blood monocytes. Instead, we demonstrate the importance of monocytes/macrophages for the control of dengue virus.     

Preclinical in vitro and in vivo evaluation of the potent and specific cyclin-dependent kinase 2 inhibitor NU6102 and a water soluble prodrug NU6301

To facilitate the evaluation of CDK2 (cyclin-dependent kinase 2) as a cancer target, the in vitro and in vivo properties of NU6102 (O⁶-cyclohexylmethyl-2-(4′-sulphamoylanilino)purine) and a water soluble prodrug (NU6301) were investigated. NU6102 selectively inhibited the growth of CDK2 WT (wild type) versus KO MEFs (knockout mouse embryo fibroblasts) (GI₅₀ (concentration required to inhibit cell growth by 50%) 14 μM versus >30 μM), and was more growth-inhibitory in p53 mutant or null versus p53 WT cells (p = 0.02), and in Rb (retinoblastoma protein) WT SKUT-1B versus SKUT 1 Rb deficient cells (p = 0.01). In SKUT-1B cells NU6102 induced a G2 arrest, inhibition of Rb phosphorylation and cytotoxicity (LC₅₀ 2.6 μM for a 24 h exposure). The prodrug NU6301 rapidly generated NU6102 in vitro in mouse plasma, and tumour NU6102 levels in vivo consistent with activity in vitro. Eight or 12 hourly dosing of 120 mg/kg NU6301 for 10 days was well tolerated in SKUT-1B tumour-bearing mice and inhibited Rb phosphorylation in tumour tissue. Two (8 hourly dosing) and 3 (12 hourly dosing) day tumour growth delay was observed (p = 0.04 and p = 0.007, respectively) following NU6301 administration. NU6102 and its prodrug NU6301 have pharmacological properties consistent with CDK2 inhibition, and represent useful tool molecules for the evaluation of CDK2 as a target in cancer.     

Decreased FGF8 signaling causes deficiency of gonadotropin-releasing hormone in humans and mice

Idiopathic hypogonadotropic hypogonadism (IHH) with anosmia (Kallmann syndrome; KS) or with a normal sense of smell (normosmic IHH; nIHH) are heterogeneous genetic disorders associated with deficiency of gonadotropin-releasing hormone (GnRH). While loss-of-function mutations in FGF receptor 1 (FGFR1) cause human GnRH deficiency, to date no specific ligand for FGFR1 has been identified in GnRH neuron ontogeny. Using a candidate gene approach, we identified 6 missense mutations in FGF8 in IHH probands with variable olfactory phenotypes. These patients exhibited varied degrees of GnRH deficiency, including the rare adult-onset form of hypogonadotropic hypogonadism. Four mutations affected all 4 FGF8 splice isoforms (FGF8a, FGF8b, FGF8e, and FGF8f), while 2 mutations affected FGF8e and FGF8f isoforms only. The mutant FGF8b and FGF8f ligands exhibited decreased biological activity in vitro. Furthermore, mice homozygous for a hypomorphic Fgf8 allele lacked GnRH neurons in the hypothalamus, while heterozygous mice showed substantial decreases in the number of GnRH neurons and hypothalamic GnRH peptide concentration. In conclusion, we identified FGF8 as a gene implicated in GnRH deficiency in both humans and mice and demonstrated an exquisite sensitivity of GnRH neuron development to reductions in FGF8 signaling.     

Brain glucagon-like peptide-1 regulates arterial blood flow, heart rate, and insulin sensitivity

OBJECTIVE— To ascertain the importance and mechanisms underlying the role of brain glucagon-like peptide (GLP)-1 in the control of metabolic and cardiovascular function. GLP-1 is a gut hormone secreted in response to oral glucose absorption that regulates glucose metabolism and cardiovascular function. GLP-1 is also produced in the brain, where its contribution to central regulation of metabolic and cardiovascular homeostasis remains incompletely understood.RESEARCH DESIGN AND METHODS— Awake free-moving mice were infused with the GLP-1 receptor agonist exendin-4 (Ex4) into the lateral ventricle of the brain in the basal state or during hyperinsulinemic eu-/hyperglycemic clamps. Arterial femoral blood flow, whole-body insulin-stimulated glucose utilization, and heart rates were continuously recorded.RESULTS— A continuous 3-h brain infusion of Ex4 decreased femoral arterial blood flow and whole-body glucose utilization in the awake free-moving mouse clamped in a hyperinsulinemic-hyperglycemic condition, only demonstrating that this effect was strictly glucose dependent. However, the heart rate remained unchanged. The metabolic and vascular effects of Ex4 were markedly attenuated by central infusion of the GLP-1 receptor (GLP-1R) antagonist exendin-9 (Ex9) and totally abolished in GLP-1 receptor knockout mice. A correlation was observed between the metabolic rate and the vascular flow in control and Ex4-infused mice, which disappeared in Ex9 and GLP-1R knockout mice. Moreover, hypothalamic nitric oxide synthase activity and the concentration of reactive oxygen species (ROS) were also reduced in a GLP-1R–dependent manner, whereas the glutathione antioxidant capacity was increased. Central GLP-1 activated vagus nerve activity, and complementation with ROS donor dose-dependently reversed the effect of brain GLP-1 signaling on peripheral blood flow.CONCLUSIONS— Our data demonstrate that central GLP-1 signaling is an essential component of circuits integrating cardiovascular and metabolic responses to hyperglycemia.There is now compelling evidence supporting the interplay between metabolic and vascular diseases (1,2) in which neuronal circuits in the central nervous system seem to play a critical role in orchestrating the control of glucose homeostasis (3). We recently demonstrated that the central infusion of insulin decreased blood pressure and increased arterial blood flow and heart rate through a molecular mechanism depending on the synthesis of nitric oxide in the hypothalamus (4). Importantly, the central regulation of nitric oxide (NO) metabolism affected whole-body glucose utilization (5). This mechanism was impaired during high-fat diet–induced insulin resistance and diabetes and reverted upon central NO supplementation (4). These findings raise the possibility that signals from peripheral tissues, which act on the brain to control glucose metabolism, could also regulate vascular function.Enteroendocrine cells have important roles in regulating energy intake and glucose homeostasis through their actions on peripheral target organs, including the endocrine pancreas. Enteroendocrine cells secrete multiple hormones, including glucagon-like peptide (GLP)-1, which controls pancreatic endocrine secretion (6). GLP-1 is also a neuropeptide synthesized by neurons in the caudal regions of the nucleus of the solitary tract (NTS) (7,8). GLP-1 is released into the hypothalamus and controls food intake, blood pressure, and heart rate (9,10). Whereas most of the glucose-lowering actions of GLP-1 have been attributed to the direct effect of the hormone on the endocrine pancreas, i.e., to stimulation of insulin and inhibition of glucagon secretion, we demonstrated the importance of extra-pancreatic GLP-1 receptor–dependent control of insulin secretion (11) and whole-body glucose distribution (12). The infusion into the brain of the GLP-1 receptor antagonist exendin-9 (Ex9) inhibited insulin secretion induced by gut glucose (11). Conversely, central administration of the GLP-1 receptor agonist exendin-4 (Ex4) augmented intravenous glucose-stimulated insulin secretion to a level similar to that obtained during an intragastric glucose infusion (11). Our data suggested that the absorptive state was associated with the stimulation of the gut-to-brain axis leading to the activation of brain GLP-1 signaling and, consequently, to hyperinsulinemia. During the absorptive state, blood flow redistribution toward mesenteric organs is also observed, which has been proposed to favor nutrient redistribution into the liver (13). Importantly, stimulation of the central GLP-1 receptor increases blood pressure and heart rate and activates autonomic regulatory neurons (8,14,15). However, recently it has been shown that GLP-1 reduced islet blood flow after glucose administration (16). Therefore, the role of brain GLP-1 signaling also in the control of cardiovascular homeostasis remains incompletely understood.We have now pursued the importance of GLP-1 action in the central nervous system for control of cardiovascular function using studies in conscious free-moving mice. After central GLP-1 infusion, we simultaneously recorded femoral arterial blood flow, heart rate, and insulin and glucose sensitivity during hyperinsulinemic-euglycemic or hyperglycemic clamps. We now demonstrate that hypothalamic reactive oxygen and nitrogen species are controlled by brain GLP-1 and are essential for the coordinated regulation of metabolic and cardiovascular function.     

Discovery of potent and selective adamantane-based small-molecule P2X(7) receptor antagonists/interleukin-1beta inhibitors

A novel series of antagonists of the human P2X7 receptor is described. Modification of substituents enabled identification of compounds selective for the rat P2X7 receptor and provides useful pharmacological tools for evaluation of the role of P2X7 in disease.     

Laparoscopic management of adnexal masses: a gold standard?

PURPOSE OF REVIEW: To review recent literature on the laparoscopic management of adnexal masses, when this approach may be considered as a gold standard. RECENT FINDINGS: Cyst rupture was recently demonstrated to be a significant prognostic factor in stage I invasive epithelial carcinoma, and it was recommended to restrict the laparoscopic approach to patients with preoperative evidence that the cyst was benign. The laparoscopic approach is still highly controversial in masses suspicious at ultrasound. The limits of the laparoscopic approach are discussed reviewing recent literature and our experience. The laparoscopic management of adnexal masses appears to be safe in most hospitals even in developing countries. This approach is being used with increasing frequency in unusual indications such as newborns, children, adolescents and pregnant women. The learning curve for endoscopic surgery appears to be longer than expected. Many patients with benign adnexal masses, such as ovarian endometrioma, are still treated by laparotomy or with an inadequate endoscopic technique. Several studies have suggested that the stripping technique is a tissue-sparing procedure. SUMMARY: The laparoscopic puncture of malignant ovarian tumours confined to the ovaries is uncommon, and should be avoided whenever possible. The teaching of endoscopy is essential to promote adequate procedures performed according to the principles of microsurgery and to preserve postoperative ovarian physiology.