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Context: Mutations in the GHRH receptor (GHRHR) have been detected in the familial type-IB isolated GH deficiency (IGHD-IB) inherited as an autosomal recessive disorder and characterized by a low but detectable serum GH level and good response to substitutive GH therapy., Objective: The aim of our study was the identification of mutations in sporadic patients with a IGHD-IB phenotype., Subjects and Methods: The GHRHR gene was systematically screened by DHPLC in 134 IGHD patients with no family history of the disorder or declared parental consanguinity., Results: We identified a novel variation, Val10Gly, within the signal peptide at the heterozygous state in three patients and in one of 1084 controls (P = 0.004), suggesting that it might contribute to IGHD. The functional analysis showed that the signal peptide is not cleaved from the mutant GHRHR, which in turn is not translocated to the cellular surface, demonstrating that 10Gly drastically affects the receptor correct processing. Because 10Gly was also present in normal-stature relatives of the patients as well as in a control, it is likely that it exerts its effects in the context of other genetic and environmental susceptibility factors., Conclusion: At difference from previous papers reporting GHRHR mutations in familial cases with a clear recessive mode of inheritance, our study was conducted on a large sample of sporadic patients and allowed to discover a novel mechanism of the disease caused by a recurrent dominant mutation in the GHRHR signal peptide associated with incomplete penetrance.

Background and Purpose: Substance P (SP) and peroxisome proliferator-activated receptor-gamma (PPAR-gamma) play important roles in different inflammatory conditions and are both expressed in human monocytes and macrophages. However, it is not known whether or not they interact. This study was undertaken to evaluate the effects of SP on PPAR-gamma protein expression in monocytes and macrophages (MDMs: monocyte-derived macrophages) from healthy smokers and non-smokers., Experimental Approach: PPAR-gamma protein was detected by western blot and quantified by calculating the ratio between PPAR-gamma and beta-actin protein expression. Constitutive tachykinin NK(1) receptor expression in monocytes and MDMs from healthy smokers and non-smokers was evaluated by western blot. Cytokine release was evaluated by ELISA., Key Results: In the concentration range 10(-10)-10(-6) M, SP stimulated PPAR-gamma protein expression in monocytes and MDMs, being more effective in cells from healthy smokers. Moreover, in these cells there was a constitutively increased expression of NK(1) receptors. SP-induced expression of the PPAR-gamma protein was receptor-mediated, as it was reproduced by the NK(1) selective agonist [Sar(9)Met(O(2))(11)]SP and reversed by the competitive NK(1) antagonist GR71251. SP-induced maximal effects were similar to those evoked by 15-deoxy-Delta(12,14)-prostaglandin J(2); an endogenous PPAR-gamma agonist, and were significantly reduced by a PPAR-gamma antagonist. NK(1) and PPAR-gamma agonists exerted opposite effects on TNF-alpha release from monocytes and MDMs., Conclusions and Implications: Enhancement of PPAR-gamma protein expression represents a novel activity for SP, which could contribute to a range of chronic inflammatory disorders.

Epidemiological studies demonstrate that the Mediterranean diet, in which olive oil is the major source of fat, reduces the risk of coronary heart disease and cancer. It has been proposed that the beneficial effects of olive oil not only depend on oleic acid, but are also associated with minor polar compounds (MPC). A positive correlation between inflammation and cardiovascular diseases has long been described, monocyte/macrophages and NF-kappaB playing a pivotal role. The aim of this work was to investigate the effects of an extra-virgin olive oil extract (MPC-OOE), particularly rich in MPC and prepared by some of us, on NF-kappaB translocation in monocytes and monocyte-derived macrophages (MDM) isolated from healthy volunteers. In a concentration-dependent manner, MPC-OOE inhibited p50 and p65 NF-kappaB translocation in both un-stimulated and phorbol-myristate acetate (PMA)-challenged cells, being particularly effective on the p50 subunit. Interestingly, this effect occurred at concentrations found in human plasma after nutritional ingestion of virgin olive oil and was quantitatively similar to the effect exerted by ciglitazone, a PPAR-gamma ligand. However, MPC-OOE did not affect PPAR-gamma expression in monocytes and MDM. These data provide further evidence of the beneficial effects of extra-virgin olive oil by indicating its ability to inhibit NF-kappaB activation in human monocyte/macrophages.

Previous observations demonstrated that Peroxisome Proliferator-Activated Receptor-gamma (PPAR-gamma), a key regulator of adipocyte differentiation, is expressed in a large variety of cells, including cells of the monocyte/macrophage lineage. This study was aimed to quantify both the constitutive and ligand-induced PPAR-gamma expression in monocytes and monocyte-derived macrophages (MDM) isolated from healthy smokers and non-smokers, and to evaluate the possible direct effect of nicotine. PPAR-gamma protein was detected by Western blot and quantification was performed by calculating the ratio between PPAR-gamma and beta-actin protein expression. Cytokine release was measured with enzyme-linked immunoassay kits. Constitutive PPAR-gamma protein was detected in human monocytes and its expression was up-regulated along with differentiation to MDM. The endogenous ligand 15-deoxy-delta(12,14)-prostaglandin J(2) and the synthetic agonist ciglitazone enhanced PPAR-gamma expression, the former being effective also at low micromolar concentrations. Both agonists significantly inhibited the basal secretion of pro-inflammatory cytokines (e.g., TNF-alpha, IL-6), ciglitazone being more potent. Monocytes and MDM from healthy smokers presented a significantly enhanced (4-fold and 2.5-fold, respectively) constitutive PPAR-gamma expression, as compared to those from healthy non-smokers. However, ligand-induced PPAR-gamma expression and inhibition of cytokine secretion were similar in healthy smokers and non-smokers. Nicotine dose-dependently enhanced PPAR-gamma expression with a maximum at 10 muM, and inhibited release of pro-inflammatory cytokines; these effects were reversed by alpha-bungarotoxin. Nicotine and PPAR-gamma agonists did not exert synergistic effects. In conclusion, monocytes and MDM from healthy smokers present a constitutively enhanced PPAR-gamma expression; this effect is reproduced, to some extent, by nicotine in vitro.

Background: Diabetes and obesity, components of the metabolic syndrome, are common longterm complications in women with previous gestational diabetes (pGDM). Long-term follow-up of women with mild gestational hyperglycemia is lacking., Methods: Fifty women with previous positive oral glucose challenge test and negative oral glucose tolerance test (pOGCT+OGTT-), 161 with previous normal glucose tolerance (pNGT), and 182 pGDM were studied after 6.5 years from the index pregnancy., Results: Patients with pGDM showed a worse metabolic pattern than pNGT. Women with pOGCT+OGTT- had significantly higher levels of fasting glucose, homeostasis model assessment (HOMA), percentage of impaired fasting glucose, and low age and high-density lipoprotein (HDL)-cholesterol than pNGT subjects. Prevalence of the metabolic syndrome (MS) was, respectively, sixfold and twofold higher in pGDM and pOGCT+OGTT- than in pNGT. In a Cox proportional hazard model, after multiple adjustments, pGDM was significantly associated with subsequent hyperglycemia (hazard ratio [HR] = 4.2; 95% CI 1.6-11.1), low HDL-cholesterol (HR = 1.7, 1.1-2.8), hypertriglyceridemia (HR = 4.2, 1.2-14.9), hypertension (HR = 2.2, 1.3-3.6), MS (HR = 3.7, 1.3-10.8), while pOGCT+OGTT- was associated with subsequent hyperglycemia (HR = 4.3, 1.3-14.7), and low HDL-cholesterol (HR = 2.0, 1.0-3.8). The metabolic syndrome was present in 52.6% of obese pGDM, 50% of obese pOGCT+OGTT-, and 28.6% of obese pNGT women; the corresponding HRs were, respectively, HR = 2.20, 0.74-6.57 (pGDM), and HR = 3.56, 1.10-11.5 (pOGCT+OGTT-)., Conclusions: Women who failed the OGCT, but not the OGTT, showed a subsequent worse metabolic pattern than pNGT subjects, independently of confounding factors. In the presence of obesity, the prevalence of the metabolic syndrome was similar to that of obese pGDM women, and almost twofold higher than in obese pNGT controls.

Macrophage activation is a key feature of inflammatory reactions occurring during bacterial infections, immune responses and tissue injury. We previously demonstrated that human macrophages of different origin express the tyrosine kinase receptor recepteur d'origine nantaise, the human receptor for MSP (RON) and produce superoxide anion (O(2)(-)) when challenged with macrophage-stimulating protein (MSP), the endogenous ligand for RON. This study was aimed to evaluate the role of MSP in alveolar macrophages (AM) isolated from healthy volunteers and patients with interstitial lung diseases (sarcoidosis, idiopathic pulmonary fibrosis), either smokers or non-smokers, by evaluating the respiratory burst, cytokine release and nuclear factor-kappa B (NF-kappaB) activation. MSP effects were compared with those induced by known AM stimuli, for example, phorbol myristate acetate, N-formyl-methionyl-leucyl-phenylalanine, lipopolysaccharide.MSP evokes O(2)(-) production, cytokine release and NF-kappaB activation in a concentration-dependent manner. By evaluating the respiratory burst, we demonstrate a significantly increased O(2)(-) production in AM from healthy smokers or smokers with pulmonary fibrosis, as compared to non-smokers, thus suggesting MSP as an enhancer of cigarette smoke toxicity. Besides inducing interleukin-1 beta (IL-1beta) and interleukin-10 (IL-10) production, MSP triggers an enhanced tumor necrosis factor-alpha release, especially in healthy and pulmonary fibrosis smokers. On the contrary, MSP-induced IL-10 release is higher in AM from healthy non-smokers. MSP activates the transcription factor NF-kappaB; this effect is more potent in healthy and fibrosis smokers (2.5-fold increase in p50 subunit translocation). This effect is receptor-mediated, as it is prevented by a monoclonal anti-human MSP antibody. The higher effectiveness of MSP in AM from healthy smokers and patients with pulmonary fibrosis is suggestive of its role in these clinical conditions.

We previously showed that the Kaposi Sarcoma line KS-IMM express a functional Met tyrosine kinase receptor, which, upon HGF stimulation, activates motogenic, proliferative, and invasive responses. In this study, we investigated the signalling pathways activated by HGF, as well as by Met monoclonal antibodies (Mabs), acting as full or partial agonists. The full agonist Mab mimics HGF in all biological and biochemical aspects. It elicits the whole spectrum of responses, while the partial agonist Mab induces only wound healing. These differences correlated with a more prolonged and sustained tyrosine phosphorylation of the receptor and MAPK evoked by HGF and by the full agonist Mab, relative to the partial agonist Mab. Since Gab1, JNK and PI 3-kinase are activated with same intensity and kinetics by HGF and by the two agonist antibodies, it is concluded that level and duration of MAPK activation by Met receptor are crucial for the induction of a full HGF-dependent mitogenic and invasive program in KS cells.

1 Substance P (SP) is deeply involved in lung pathophysiology and plays a key role in the modulation of inflammatory-immune processes. We previously demonstrated that SP activates guinea-pig alveolar macrophages (AMs) and human monocytes, but a careful examination of its effects on human AMs is still scarce. 2 This study was undertaken to establish the role of SP in human AM isolated from healthy smokers and non-smokers, by evaluating the presence of tachykinin NK(1) receptors (NK-1R) and SP's ability to induce superoxide anion (O(2)(-)) production and cytokine release, as well as activation of the nuclear factor-kappaB (NF-kappaB) pathway. 3 By Western blot analysis and immunofluorescence, we demonstrate that authentic NK-1R are present on human AMs, a three-fold enhanced expression being observed in healthy smokers. These NK-1R are functional, as SP and NK(1) agonists dose-dependently induce O(2)(-) production and cytokine release. In AMs from healthy smokers, SP evokes an enhanced respiratory burst and a significantly increased release of tumor necrosis factor-alpha as compared to healthy non-smokers, but has inconsistent effects on IL-10 release. The NK(1) selective antagonist CP 96,345 ((2S,3S)-cis-2-diphenylmethyl-N[(2-methoxyphenyl)-methyl]-1-azabicyclo-octan-3-amine)) competitively antagonized SP-induced effects. 4 SP activates the transcription factor NF-kappaB, a three-fold increased nuclear translocation being observed in AMs from healthy smokers. This effect is receptor-mediated, as it is reproduced by the NK(1) selective agonist [Sar(9)Met(O(2))(11)]SP and reverted by CP 96,345. 5 These results clearly indicate that human AMs possess functional NK-1R on their surface, which are upregulated in healthy smokers, providing new insights on the mechanisms involved in tobacco smoke toxicity.

Inhibition of tumor necrosis factor-alpha (TNF-alpha) represents a relevant target in rheumatoid arthritis therapy. Besides inhibiting cyclooxygenase, anti-inflammatory drugs can affect the activation of transcription factors. We investigated the ability of dexamethasone, indomethacin, and rofecoxib to modulate nuclear factor-kappaB (NF-kappaB) activation and TNF-alpha release from human monocytes challenged with lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Both stimuli induced NF-kappaB nuclear translocation and TNF-alpha secretion. Dexamethasone potently inhibited TNF-alpha release, indomethacin inhibited only PMA-evoked release, while rofecoxib had no effect. In the electrophoretic mobility shift assay, dexamethasone and rofecoxib dose-dependently inhibited the DNA binding activity of NF-kappaB in stimulated monocytes, whereas indomethacin failed to inhibit the LPS-evoked one. These results were further confirmed by evaluating the drugs' ability to reduce nuclear NF-kappaB subunits, as well as the amount of phosphorylated IkappaBalpha in cytosolic fractions. In conclusion, these results indicate that anti-inflammatory drugs differ largely in their ability to inhibit NF-kappaB activity and/or TNF-alpha release from human monocytes. These effects can be relevant to rheumatoid arthritis therapy.

Aims: To evaluate the prevalence of beta-cell autoantibodies in women with gestational diabetes and impaired glucose tolerance, and identify clinical characteristics differentiating hyperglycaemic patients with and without autoantibodies., Methods: One hundred and twenty-three pregnant patients with gestational diabetes, 84 with impaired glucose tolerance and 290 with normoglycaemia were evaluated for anti-islet cell antibodies, glutamic acid decarboxylase (GAD) autoantibodies, and the components of the metabolic syndrome., Results: Autoantibody positivity was 8.9%, 17.9% and 0.3% in patients with diabetes, impaired tolerance and normoglycaemia, respectively. Hyperglycaemic patients with autoantibodies had lower body mass index, waist, weight gain at the time of the screening test and a lower percentage of previous pregnancies than those without autoantibodies. In addition, their fasting insulin values were significantly lower and inversely related to the presence of autoantibodies (odds ratio (OR) = 0.64; 95% confidence interval (CI) 0.42-0.96), the lowest values being found in anti-GAD+ patients. Autoantibody-positive women with diabetes were more frequently treated with insulin than negative patients (OR = 7.21; 95% CI 1.85-28.08)., Conclusions: Autoantibody-positive women with gestational hyperglycaemia displayed fewer features of insulin resistance and required more frequent insulin therapy than negative women and presumably had presymptomatic Type 1 diabetes. If this conclusion is corroborated by the follow-up of larger series, clinical and immunological distinction of types of gestational hyperglycaemia would be useful.