NPY regulates catecholamine secretion from human adrenal chromaffin cells.

The aim of the present work was to find out whether NPY synthesized in human adrenal chromaffin cells controls in an autocrine/paracrine fashion the release of catecholamines by these cells. Accordingly, the constitutive and regulated release of both NPY and catecholamines was measured simultaneously in cultured human chromaffin cells. In addition, by using both RT-PCR and a combination of specific agonists and antagonists, we characterized the expression of NPY receptors on these cells as well as their pharmacology. Our results were as follows. 1) Human chromaffin cells constitutively secrete NPY. 2) Nicotine elicits a rapid increase in the release of both catecholamines and NPY; this release of NPY is more sustained than that of catecholamines. 3) RT-PCR shows expression of Y1, Y2, Y4, and Y5 receptor mRNA by chromaffin cells; these receptors are functional, as various receptor specific agonists elicit an increase in intracellular calcium. 4) Peptide YY, in contrast to NPY, is not able to stimulate the release of catecholamines. This finding was corroborated by the observation that no receptor-specific antagonists were able to reduce constitutive catecholamine release, whereas an NPY-immunoneutralizing antibody markedly attenuated the secretion. Taken together, these data suggest that NPY originating from the adrenal medulla locally enhances the secretion of catecholamines, presumably by acting via the putative y3 receptor.

Discovery of amino acid variants in the human glucose-dependent insulinotropic polypeptide (GIP) receptor: the impact on the pancreatic beta cell responses and functional expression studies in Chinese hamster fibroblast cells.

The two incretins, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), are insulinotropic factors released from the small intestine to the blood stream in response to oral glucose ingestion. The insulinotropic effect of GLP-1 is maintained in patients with Type II (non-insulin-dependent) diabetes mellitus, whereas, for unknown reasons, the effect of GIP is diminished or lacking. We defined the exon-intron boundaries of the human GIP receptor, made a mutational analysis of the gene and identified two amino acid substitutions, A207 V and E354Q. In an association study of 227 Caucasian Type II diabetic patients and 224 matched glucose tolerant control subjects, the allelic frequency of the A207 V polymorphism was 1.1% in Type II diabetic patients and 0.7% in control subjects (p = 0.48), whereas the allelic frequency of the codon 354 polymorphism was 24.9% in Type II diabetic patients versus 23.2% in control subjects. Interestingly, the glucose tolerant subjects (6% of the population) who were homozygous for the codon 354 variant had on average a 14% decrease in fasting serum C-peptide concentration (p = 0.01) and an 11% decrease in the same variable 30 min after an oral glucose load (p = 0.03) compared with subjects with the wild-type receptor. Investigation of the function of the two GIP receptor variants in Chinese hamster fibroblasts showed, however, that the GIP-induced cAMP formation and the binding of GIP to cells expressing the variant receptors were not different from the findings in cells expressing the wildtype GIP receptor. In conclusion, amino acid variants in the GIP receptor are not associated with random Type II diabetes in patients of Danish Caucasian origin or with altered GIP binding and GIP-induced cAMP production when stably transfected in Chinese hamster fibroblasts. The finding of an association between homozygosity for the codon 354 variant and reduced fasting and post oral glucose tolerance test (OGTT) serum C-peptide concentrations, however, calls for further investigations and could suggest that GIP even in the fasting state regulates the beta-cell secretory response.

Glucose and leptin induce apoptosis in human beta-cells and impair glucose-stimulated insulin secretion through activation of c-Jun N-terminal kinases.

c-Jun N-terminal kinases (SAPK/JNKs) are activated by inflammatory cytokines, and JNK signaling is involved in insulin resistance and beta-cell secretory function and survival. Chronic high glucose concentrations and leptin induce interleukin-1beta (IL-1beta) secretion from pancreatic islets, an event that is possibly causal in promoting beta-cell dysfunction and death. The present study provides evidence that chronically elevated concentrations of leptin and glucose induce beta-cell apoptosis through activation of the JNK pathway in human islets and in insulinoma (INS 832/13) cells. JNK inhibition by the dominant inhibitor JNK-binding domain of IB1/JIP-1 (JNKi) reduced JNK activity and apoptosis induced by leptin and glucose. Exposure of human islets to leptin and high glucose concentrations leads to a decrease of glucose-induced insulin secretion, which was partly restored by JNKi. We detected an interplay between the JNK cascade and the caspase 1/IL-1beta-converting enzyme in human islets. The caspase 1 gene, which contains a potential activating protein-1 binding site, was up-regulated in pancreatic sections and in isolated islets from type 2 diabetic patients. Similarly, cultured human islets exposed to high glucose- and leptin-induced caspase 1 and JNK inhibition prevented this up-regulation. Therefore, JNK inhibition may protect beta-cells from the deleterious effects of high glucose and leptin in diabetes.

Exercise aggravates cardiovascular risks and mortality in rats with disrupted nitric oxide pathway and treated with recombinant human erythropoietin.

Chronic administration of recombinant human erythropoietin (rHuEPO) can generate serious cardiovascular side effects such as arterial hypertension (HTA) in clinical and sport fields. It is hypothesized that nitric oxide (NO) can protect from noxious cardiovascular effects induced by chronic administration of rHuEPO. On this base, we studied the cardiovascular effects of chronic administration of rHuEPO in exercise-trained rats treated with an inhibitor of NO synthesis (L-NAME). Rats were treated or not with rHuEPO and/or L-NAME during 6 weeks. During the same period, rats were subjected to treadmill exercise. The blood pressure was measured weekly. Endothelial function of isolated aorta and small mesenteric arteries were studied and the morphology of the latter was investigated. L-NAME induced hypertension (197 ± 6 mmHg, at the end of the protocol). Exercise prevented the rise in blood pressure induced by L-NAME (170 ± 5 mmHg). However, exercise-trained rats treated with both rHuEPO and L-NAME developed severe hypertension (228 ± 9 mmHg). Furthermore, in these exercise-trained rats treated with rHuEPO/L-NAME, the acetylcholine-induced relaxation was markedly impaired in isolated aorta (60% of maximal relaxation) and small mesenteric arteries (53%). L-NAME hypertension induced an internal remodeling of small mesenteric arteries that was not modified by exercise, rHuEPO or both. Vascular ET-1 production was not increased in rHuEPO/L-NAME/training hypertensive rats. Furthermore, we observed that rHuEPO/L-NAME/training hypertensive rats died during the exercise or the recovery period (mortality 51%). Our findings suggest that the use of rHuEPO in sport, in order to improve physical performance, represents a high and fatal risk factor, especially with pre-existing cardiovascular risk.

Identification of estrogen-responsive DNA sequences by transient expression experiments in a human breast cancer cell line.

The expression of a hybrid gene formed by the promoter region of the Xenopus laevis vitellogenin gene B1 and the CAT coding region is regulated by estrogen when the gene is transfected into hormone-responsive MCF-7 cells. Furthermore, the 5' flanking region of the gene B1 alone can confer inducibility to heterologous promoters, although to a varying extent depending on the promoter used. Deletion mapping of he vitellogenin hormone-responsive sequences revealed that a 13 bp element 5'-AGTCACTGTGACC-3' at position -334 is essential for estrogen inducibility. We have shown previously that this 13 bp element is present upstream of several liver-specific estrogen-inducible genes.

IL-12 controls cytotoxicity of a novel subset of self-antigen-specific human CD28+ cytolytic T cells.

Activated CD8 T cells develop cytotoxicity against autologous cells bearing foreign Ags and self/tumor Ags. However, self-specific cytolysis needs to be kept under control to avoid overwhelming immunopathology. After peptide vaccination of melanoma patients, we studied molecular and functional properties of T cell subsets specific for the self/tumor Ag Melan-A/MART-1. Ex vivo analysis revealed three Ag-specific effector memory (EM) populations, as follows: CD28-negative EM (EM28(-)) T cells strongly expressing granzyme/perforin, and two EM28(+) subsets, one with high and the other with low level expression of these cytotoxic proteins. For further functional characterization, we generated 117 stable CD8 T cell clones by ex vivo flow cytometry-based sorting of these subsets. All EM28(-)-derived clones lysed target cells with high efficacy. In contrast, EM28(+)-derived clones were heterogenous, and could be classified in two groups, one with high and the other with low killing capacity, correlating with granzyme/perforin expression. High and low killer phenotypes remained surprisingly stable for several months. However, strongly increased granzyme expression and cytotoxicity were observed after exposure to IL-12. Thus, the data reveal a newly identified subset of CD28(+) conditional killer T cells. Because CD28 can mediate strong costimulatory signals, tight cytotoxicity control, as shown in this study through IL-12, may be particularly important for subsets of T cells expressing CD28.

A soluble hexameric form of CD40 ligand activates human dendritic cells and augments memory T cell response.

A strategy to improve the immunogenicity of candidate vaccines is to trigger the innate immune system. Triggering of CD40 at the surface of dendritic cells (DC) is essential in the induction of an efficient immune response. Although CD40 agonist antibodies have been shown to be potent inducers of immune responses in experimental models, serious safety concerns have been raised for their use in humans. In addition, the production of soluble functional CD40 ligand has been challenging and the soluble form existing so far is not developed anymore. Here, we have evaluated the potency of a new soluble form of hexameric CD40 ligand (sCD40L) to serve as an adjuvant for anti-viral T cell responses. sCD40L was able to activate human DC and to enhance virus-specific memory T cell responses. These results demonstrate that this soluble form of CD40 ligand may serve as an adjuvant for T cell response and thus provide the rationale for its potential use in T cell based vaccine strategies.

Analysis of interaction of cloned human and/or sheep fetal hemoglobin gamma-chain and LPS in augmenting induction of inflammatory cytokine production in vivo and in vitro.

We have reported earlier that purified preparations of sheep fetal hemoglobin, but not adult hemoglobin, in concert with non-stimulatory doses of lipopolysaccharide (LPS) (lipid A), act cooperatively to regulate in vitro production of a number of cytokines, including TNFalpha, TGFbeta and IL-6 from murine and human leukocytes. Following in vivo treatment of mice with the same combination of hemoglobin and LPS, harvested spleen or peritoneal cells showed a similar augmented capacity to release these cytokines into culture supernatants. We report below that genetically cloned gamma-chain of human or sheep fetal hemoglobin, but not cloned alpha- or beta-chains, can produce this cooperative effect, as indeed can HPLC purified, heme-free, gamma-chains derived from cord blood fetal hemoglobin, and that purified haptoglobin completely abolishes the cooperative interaction.

Population pharmacokinetics and effects of efavirenz in patients with human immunodeficiency virus infection.

OBJECTIVE: The reverse transcriptase inhibitor efavirenz is currently used at a fixed dose of 600 mg/d. However, dosage individualization based on plasma concentration monitoring might be indicated. This study aimed to assess the efavirenz pharmacokinetic profile and interpatient versus intrapatient variability in patients who are positive for human immunodeficiency virus, to explore the relationship between drug exposure, efficacy, and central nervous system toxicity and to build up a Bayesian approach for dosage adaptation. METHODS: The population pharmacokinetic analysis was performed by use of NONMEM based on plasma samples from a cohort of unselected patients receiving efavirenz. With the use of a 1-compartment model with first-order absorption, the influence of demographic and clinical characteristics on oral clearance and oral volume of distribution was examined. The average drug exposure during 1 dosing interval was estimated for each patient and correlated with markers of efficacy and toxicity. The population kinetic parameters and the variabilities were integrated into a Bayesian equation for dosage adaptation based on a single plasma sample. RESULTS: Data from 235 patients with a total of 719 efavirenz concentrations were collected. Oral clearance was 9.4 L/h, oral volume of distribution was 252 L, and the absorption rate constant was 0.3 h(-1). Neither the demographic covariates evaluated nor the comedications showed a clinically significant influence on efavirenz pharmacokinetics. A large interpatient variability was found to affect efavirenz relative bioavailability (coefficient of variation, 54.6%), whereas the intrapatient variability was small (coefficient of variation, 26%). An inverse correlation between average drug exposure and viral load and a trend with central nervous system toxicity were detected. This enabled the derivation of a dosing adaptation strategy suitable to bring the average concentration into a therapeutic target from 1000 to 4000 microg/L to optimize viral load suppression and to minimize central nervous system toxicity. CONCLUSIONS: The high interpatient and low intrapatient variability values, as well as the potential relationship with markers of efficacy and toxicity, support the therapeutic drug monitoring of efavirenz. However, further evaluation is needed before individualization of an efavirenz dosage regimen based on routine drug level monitoring should be recommended for optimal patient management.

Cyclooxygenase-2 in human and experimental ischemic proliferative retinopathy.

BACKGROUND: Intravitreal neovascular diseases, as in ischemic retinopathies, are a major cause of blindness. Because inflammatory mechanisms influence vitreal neovascularization and cyclooxygenase (COX)-2 promotes tumor angiogenesis, we investigated the role of COX-2 in ischemic proliferative retinopathy. METHODS AND RESULTS: We describe here that COX-2 is induced in retinal astrocytes in human diabetic retinopathy, in the murine and rat model of ischemic proliferative retinopathy in vivo, and in hypoxic astrocytes in vitro. Specific COX-2 but not COX-1 inhibitors prevented intravitreal neovascularization, whereas prostaglandin E2, mainly via its prostaglandin E receptor 3 (EP3), exacerbated neovascularization. COX-2 inhibition induced an upregulation of thrombospondin-1 and its CD36 receptor, consistent with the observed antiangiogenic effects of COX-2 inhibition; EP3 stimulation reversed effects of COX-2 inhibitors on thrombospondin-1 and CD36. CONCLUSIONS: These findings point to an important role for COX-2 in ischemic proliferative retinopathy, as in diabetes.

Human endometrial carcinomas serially transplanted in nude mice and established in continuous cell lines.

Three out of five human endometrial carcinomas were successfully grafted into nude mice (BALB/c/nu/nu). Two of these tumors could be maintained by serial transplantation. The morphological characteristics displayed by the grafted tumors were comparable to those of the original carcinomas. Permanent cell lines were established from these two tumors. Reinjection of cells grown in vitro into nude mice produced nodules of identical histology as compared to original solid transplants. The influence of medroxyprogesterone acetate on tumor growth in vivo and cell proliferation in vitro was studied. This hormonal treatment did not produce any significant effect on tumor cells, either in vitro or in vivo, for the two endometrial carcinomas. After medroxyprogesterone administration, a slight but non-significant growth inhibition of the tumor cells in vitro was observed and the tumor transplants in vivo did not appear to be influenced. The experiments illustrate the possible use of this model for testing potential anti-cancer agents.

Antitumour effects of single or combined monoclonal antibodies directed against membrane antigens expressed by human B cells leukaemia.

Background: The increasing availability of different monoclonal antibodies (mAbs) opens the way to more specific biologic therapy of cancer patients. However, despite the significant success of therapy in breast and ovarian carcinomas with anti-HER2 mAbs as well as in non-Hodkin B cell lymphomas with anti-CD20 mAbs, certain B cell malignancies such as B chronic lymphocytic leukaemia (B-CLL) respond poorly to anti-CD20 mAb, due to the low surface expression of this molecule. Thus, new mAbs adapted to each types of tumour will help to develop personalised mAb treatment. To this aim, we analyse the biological and therapeutic properties of three mAbs directed against the CD5, CD71 or HLA-DR molecules highly expressed on B-CLL cells. Results: The three mAbs, after purification and radiolabelling demonstrated high and specific binding capacity to various human leukaemia target cells. Further in vitro analysis showed that mAb anti-CD5 induced neither growth inhibition nor apoptosis, mAb anti-CD71 induced proliferation inhibition with no early sign of cell death and mAb anti-HLA-DR induced specific cell aggregation, but without evidence of apoptosis. All three mAbs induced various degrees of ADCC by NK cells, as well as phagocytosis by macrophages. Only the anti-HLA-DR mAb induced complement mediated lysis. Coincubation of different pairs of mAbs did not significantly modify the in vitro results. In contrast with these discrete and heterogeneous in vitro effects, in vivo the three mAbs demonstrated marked anti-tumour efficacy and prolongation of mice survival in two models of SCID mice, grafted either intraperitoneally or intravenously with the CD5 transfected JOK1-5.3 cells. This cell line was derived from a human hairy cell leukaemia, a type of malignancy known to have very similar biological properties as the B-CLL, whose cells constitutively express CD5. Interestingly, the combined injection of anti-CD5 with anti-HLA-DR or with anti-CD71 led to longer mouse survival, as compared to single mAb injection, up to complete inhibition of tumour growth in 100% mice treated with both anti-HLA-DR and anti-CD5. Conclusions: Altogether these data suggest that the combined use of two mAbs, such as anti-HLA-DR and anti-CD5, may significantly enhance their therapeutic potential.

No implication of thromboxane prostanoid receptors in reactive hyperemia of skin and skeletal muscle in human forearm.

There is evidence that reactive hyperemia (ie, the transient increase of blood flow above resting level after a short period of ischemia) could be negatively modulated by vasoconstrictor prostanoids. The present study tested whether pharmacological blockade of the thromboxane prostanoid receptors with the specific antagonist S18886 (terutroban) would amplify reactive hyperemia in human skin and skeletal muscle. Twenty healthy young male volunteers were enrolled in a randomized, blinded, crossover trial of oral S18886 30 mg/d for 5 days versus placebo. Reactive hyperemia was evaluated in forearm skin and skeletal muscle, after occlusion of the brachial artery with a pneumatic cuff inflated at suprasystolic pressure. Blood flow was measured with laser Doppler imaging (skin) and strain gauge venous occlusion plethysmography (muscle). On the first and last day of each treatment period, recordings of reactive hyperemia were obtained immediately before and 2 hours after drug intake. Whether in forearm muscle or skin, S18886 had no discernible effect on peak postocclusion blood flow, nor on the global hyperemic response as quantified by the area under curve. These results do not support that thromboxane prostanoid receptor activation could exert a moderating influence on reactive hyperemia in human skin and skeletal muscle, at least in young subjects.

The T cell-specific CXC chemokines IP-10, Mig, and I-TAC are expressed by activated human bronchial epithelial cells.

Recruitment of activated T cells to mucosal surfaces, such as the airway epithelium, is important in host defense and for the development of inflammatory diseases at these sites. We therefore asked whether the CXC chemokines IFN-induced protein of 10 kDa (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha-chemoattractant (I-TAC), which specifically chemoattract activated T cells by signaling through the chemokine receptor CXCR3, were inducible in respiratory epithelial cells. The effects of proinflammatory cytokines, including IFN-gamma (Th1-type cytokine), Th2-type cytokines (IL-4, IL-10, and IL-13), and dexamethasone were studied in normal human bronchial epithelial cells (NHBEC) and in two human respiratory epithelial cell lines, A549 and BEAS-2B. We found that IFN-gamma, but not TNF-alpha or IL-1 beta, strongly induced IP-10, Mig, and I-TAC mRNA accumulation mainly in NHBEC and that TNF-alpha and IL-1 beta synergized with IFN-gamma induction in all three cell types. High levels of IP-10 protein (> 800 ng/ml) were detected in supernatants of IFN-gamma/TNF-alpha-stimulated NHBEC. Neither dexamethasone nor Th2 cytokines modulated IP-10, Mig, or I-TAC expression. Since IFN-gamma is up-regulated in tuberculosis (TB), using in situ hybridization we studied the expression of IP-10 in the airways of TB patients and found that IP-10 mRNA was expressed in the bronchial epithelium. In addition, IP-10-positive cells obtained by bronchoalveolar lavage were significantly increased in TB patients compared with normal controls. These results show that activated bronchial epithelium is an important source of IP-10, Mig, and I-TAC, which may, in pulmonary diseases such as TB (in which IFN-gamma is highly expressed) play an important role in the recruitment of activated T cells.

Hemodynamic, renal, and endocrine effects of 4-h infusions of human atrial natriuretic peptide in normal volunteers.

A synthetic human atrial natriuretic peptide of 26 aminoacids [human (3-28)ANP or hANP] was infused into normal male volunteers. Six subjects were infused for 4 h at 1-wk intervals with either hANP at the rate of 0.5 or 1.0 microgram/min or its vehicle in a single-blind randomized order. Human (3-28)ANP at the dose of 0.5 microgram/min raised immunoreactive plasma ANP levels from 104 +/- 17 to 221 +/- 24 pg/ml (mean +/- SEM), but it induced no significant change in blood pressure, heart rate, effective renal plasma flow, glomerular filtration rate, or renal electrolyte excretion. At the rate of 1.0 microgram/min, human (3-28)ANP increased immunoreactive plasma ANP levels from 89 +/- 12 to 454 +/- 30 pg/ml. It reduced effective renal plasma flow from 523 +/- 40 to 453 +/- 38 ml/min (P less than 0.05 vs. vehicle), but left glomerular filtration rate unchanged. Natriuresis rose from 207 +/- 52 to 501 +/- 69 mumol/min (P less than 0.05 vs. vehicle) and urinary magnesium excretion from 3.6 +/- 0.5 to 5.6 +/- 0.5 mumol/min (P less than 0.01 vs. vehicle). The excretion rate of the other electrolytes, blood pressure, and heart rate were not significantly modified. At both doses, human (3-28)ANP tended to suppress the activity of the renin-angiotensin-aldosterone system. In 3 additional volunteers, the skin blood flow response to human (3-28)ANP, infused for 4 h at the rate of 1.0 microgram/min, was studied by means of a laser-doppler flowmeter. The skin blood flow rose during the first 2 h of peptide administration, then fell progressively to values below baseline. After the infusion was discontinued, it remained depressed for more than 2 h. Thus, in normal volunteers, human (3-28)ANP at the dose of 1.0 microgram/min produced results similar to those obtained previously with rat (3-28)ANP. It enhanced natriuresis without changing the glomerular filtration rate while effective renal plasma flow fell. It also induced a transient vasodilation of the skin vascular bed.