FLOTAC can detect parasitic and pseudoparasitic elements in reptiles.

Reptiles have increased in popularity worldwide; snakes and lizards are frequently used as pets. As a consequence of their popularity, the interest of the scientific community in these animals has increased. In order to acquire epidemiological data on the parasitic infections affecting reptiles in Italy a survey was carried out in 125 snakes and 25 lizards bred in the Campania region of southern Italy. Individual fecal samples were collected and FLOTAC was used for copromicroscopic diagnosis. Eimeriidae, oxyurids, strongylids, other gastro-intestinal nematodes and pulmonary nematodes were the most representative parasites found. Eggs of pseudoparasites (mites, oxyurids and trichurids affecting rodents) were also found. The use of FLOTAC for diagnosis of parasitic infections in reptiles has demonstrated to be a rapid and sensitive test to improve diagnosis and acquire new information on the parasitological fauna of reptiles.

Prevalence and molecular identification of Cryptosporidium isolates from pet lizards and snakes in Italy.

In order to acquire prevalence and genetic data on Cryptosporidium infections in captive lizards and snakes kept as pets, a survey was conducted on 150 individual reptiles from southern Italy. Fecal samples were preserved in 5% formalin and analyzed using a commercial immunofluorescence assay (IFA) for the detection of Cryptosporidium oocysts and Giardia cysts. IFA revealed the presence of Cryptosporidium oocysts in nine of the 150 samples examined (6.0%), precisely in 6/125 snakes (4.8%) and in 3/25 lizards (12.0%); all fecal samples tested negative for the presence of Giardia cysts. Molecular characterization based on nested PCR amplification and sequencing of the SSU-rRNA gene, revealed the presence of Cryptosporidium serpentis in three samples from snakes (Boa constrictor constrictor, Elapheguttata guttata guttata and Python molurus).

Therapeutic effect of neutralizing endogenous IL-18 activity in the collagen-induced model of arthritis.

Two distinct IL-18 neutralizing strategies, i.e. a rabbit polyclonal anti-mouse IL-18 IgG and a recombinant human IL-18 binding protein (rhIL-18BP), were used to treat collagen-induced-arthritic DBA/1 mice after clinical onset of disease. The therapeutic efficacy of neutralizing endogenous IL-18 was assessed using different pathological parameters of disease progression. The clinical severity in mice undergoing collagen-induced arthritis was significantly reduced after treatment with both IL-18 neutralizing agents compared to placebo treated mice. Attenuation of the disease was associated with reduced cartilage erosion evident on histology. The decreased cartilage degradation was further documented by a significant reduction in the levels of circulating cartilage oligomeric matrix protein (an indicator of cartilage turnover). Both strategies efficiently slowed disease progression, but only anti-IL-18 IgG treatment significantly decreased an established synovitis. Serum levels of IL-6 were significantly reduced with both neutralizing strategies. In vitro, neutralizing IL-18 resulted in a significant inhibition of TNF-alpha, IL-6, and IFN-gamma secretion by macrophages. These results demonstrate that neutralizing endogenous IL-18 is therapeutically efficacious in the murine model of collagen-induced arthritis. IL-18 neutralizing antibody or rhIL-18BP could therefore represent new disease-modifying anti-rheumatic drugs that warrant testing in clinical trials in patients with rheumatoid arthritis.

Anti-inflammatory effect of FK-506 on human skin mast cells.

FK-506 and the structurally related macrolide rapamycin are high-affinity ligands for a specific binding protein (FK-506 binding protein). We examined the effects of FK-506 and rapamycin on the release of pre-formed (histamine) and de novo synthesized inflammatory mediators (prostaglandin D2) from mast cells isolated from human skin tissue. FK-506 (0.1 to 100 nM) concentration-dependently inhibited (5 to 65%) histamine release from skin mast cells activated by anti-IgE. FK-506 was more potent in skin mast cells than in basophils (IC40 = 2.15 +/- 0.78 nM versus 5.12 +/- 1.34 nM; p < 0.001), whereas the maximal inhibitory effect was higher in basophils than in skin mast cells (88.77 +/- 2.44% versus 67.30 +/- 3.98%; p < 0.01). FK-506 had little or no inhibitory effect on histamine release from skin mast cells challenged with compound A23187 and substance P, respectively, whereas it completely suppressed A23187-induced histamine release from basophils. FK-506 (0.1 to 100 nM) also inhibited (up to 65%) the de novo synthesis of prostaglandin D2 from skin mast cells challenged with anti-IgE. Despite its structural similarity to FK-506, rapamycin (10 to 300 nM) had little or no effect on the release of histamine from skin mast cells induced by anti-IgE, A23187, and substance P. However, rapamycin competitively antagonized the inhibitory effect of FK-506 on anti-IgE-induced histamine release from skin mast cells with a dissociation constant of about 14 nM. These data indicate that FK-506, but not rapamycin, is a potent anti-inflammatory agent acting on skin mast cells presumably by binding to the FK-506 binding protein. It thus appears that binding to the FK-506 binding protein is necessary, but not sufficient, to deliver an inhibitory signal to skin mast cells.

Anti-inflammatory effect of cyclosporin A on human skin mast cells.

We have examined the effects of cyclosporin A (CsA) and cyclosporin H (CsH), which bind with different affinity to cyclophilin, to evaluate the role of this protein in the release of preformed (histamine) and de novo synthesized (prostaglandin D2[PGD2]) mediators of inflammatory reactions from human skin mast cells (HSMC). CsA (2.4-800 nM)-inhibited (5-60%) histamine release from HSMC challenged with anti-IgE. CsA exerted little, if any, inhibitory effect on histamine release from HSMC challenged with compound A23187 and substance P, whereas it completely suppressed A23187-induced histamine release from human basophils. Inhibition of histamine release from HSMC challenged with anti-IgE was extremely rapid and was not abolished by washing (three times) the cells before anti-IgE challenge. CsA (2.4-800 nM) markedly inhibited (25-70%) the de novo synthesis of PGD2 from HSMC challenged with anti-IgE. CsH, which has an extremely low affinity for cyclophilin, had no effect on skin mast-cell mediator release. These data suggest that CsA is a potent anti-inflammatory agent acting on HSMC, presumably by interacting with cyclophilin.

Relationship between nocturnal serum thyrotropin peak and metabolic control in diabetic patients.

Circadian variations in serum TSH, especially its nocturnal rise, are often blunted in nonthyroidal illness. We analyzed TSH secretion in 15 diabetic patients (7 with type I and 8 with type II diabetes mellitus). Patients were evaluated when diabetes was poorly controlled (fasting blood glucose ranging from 13.7-19.2 mmol/L with absence of ketoacidosis) and after achieving glycemic control. Before correction of hyperglycemia, the nocturnal serum TSH peak (2230-0200 h) was abolished in 11 of 15 patients (73%); the mean (+/- SE) night TSH/morning TSH x 100 was 109.0 +/- 9.5 (range, 66.7-166.7) vs. a mean of 216.5 +/- 27.0 (range, 139.8-462.5) in normal controls. The mean morning TSH value in diabetics (1.9 +/- 0.4 mU/L) did not differ from that in normal age- and sex-matched controls. The mean TSH increase after iv administration of TRH was only slightly reduced (8.4 +/- 1.2 mU/L pretreatment vs. 10.8 +/- 1.6 mU/L posttreatment), with the TRH test blunted in 3 cases. No differences were found between type I and type II patients. Correction of hyperglycemia was associated with the reappearance of a nocturnal TSH peak in all but 1 patient (mean TSH peak, 198.2 +/- 13.0; P = NS vs. controls). This change paralleled the normalization of serum total T3 and rT3, which were reduced and increased, respectively, when diabetes was poorly controlled. An inverse relationship was found between serum fructosamine levels and the nocturnal TSH peak, suggesting that metabolic decompensation accounts for the abolishment of the latter.

Effect of tibolone on glucose and lipid metabolism in postmenopausal women.

The effect of tibolone, a new therapeutic agent for menopause, on glucose and lipid metabolism was investigated in 11 healthy postmenopausal women. At baseline and after 3 months of tibolone administration (2.5 mg/day), glucose metabolism was evaluated in each subject using both an oral glucose tolerance test (75 g) and the minimal model method of a frequently sampled intravenous glucose tolerance test. Frequently sampled intravenous glucose tolerance test allows the calculation of insulin sensitivity and peripheral glucose use independent of insulin. High-density lipoprotein-cholesterol, total cholesterol, apoprotein-A, and apoprotein-B measured in fasting conditions were not modified by tibolone, whereas triglycerides were reduced significantly (P < 0.01). Fasting levels of glucose were reduced significantly (P < 0.025), whereas those of insulin, C-peptide, and the C-peptide/insulin ratio were not modified. Glucose, insulin, C-peptide, and the C-peptide/insulin ratio responses to oral or iv glucose were not modified. Insulin sensitivity was inversely correlated to body mass index, and independent on that body mass index was significantly enhanced (P < 0.01). Glucose utilization independent of insulin was not modified. The present data indicate that tibolone does not negatively influence glucose metabolism and may indeed improve both the peripheral tissue sensitivity to insulin and the lipid profile.

Functional responses of hindlimb circulation in aged normal and WHHL rabbits.

Normal New Zealand and Watanabe heritable hyperlipidemic (WHHL) rabbits, about 24 months old, were prepared, under anaesthesia, for recording blood pressure and hindlimb blood flow. Changes in hindlimb vascular resistance were measured after local intra-arterial bolus injection of increasing doses of acetylcholine, bradykinin, serotonin, sodium nitroprusside and phenylephrine. In WHHL rabbits basal hindlimb blood flow was reduced (from 22.6 +/- 3.0 to 12.5 +/- 1.8 ml/min; P less than 0.05) and hindlimb vascular resistance was increased (from 4.6 +/- 0.5 to 8.2 +/- 1.5 mmHg/ml per min; P less than 0.05). No difference was observed in response to acetylcholine, serotonin, sodium nitroprusside and phenylephrine. The only marked alteration found in WHHL rabbits was a clear deficit to bradykinin stimulation. Morphological analysis, using scanning and transmission electron microscopy, indicated a clear damage of the femoral artery, like the presence of atherosclerotic plaques, and an abnormal distribution of patent microvessels in the WHHL muscles of the leg. Peripheral circulation in WHHL rabbits shows some peculiar features, like increased basal vascular resistance and a selective impairment of bradykinin responses. Together with these abnormalities, it seems that responses to various other dilating or contracting agents are normal, suggesting that in this interesting animal model of atherosclerosis the alterations are more specific than in other models.

Evidence for the presence of early vascular lesions in newborn Watanabe heritable hyperlipidemic (WHHL) rabbits.

We have investigated the morphology of the aortic wall of newborn New Zealand White (NZW) (n = 10) and newborn Watanabe heritable hyperlipidemic (WHHL) (n = 10) rabbits. In both strains, lipid levels (cholesterol and triglycerides) were elevated above the concentrations expected. This was particularly evident in WHHL. The morphology of the aortas of NZW rabbits suggested an intensive biosynthetic and bioenergetic activity of endothelium. This was most evident in areas where blood flow underwent division. No major abnormalities were noted in the endothelium or subendothelium. In newborn WHHL rabbits, leucocyte adhesion (usually monocytes) to endothelium and migration into the subendothelium was apparent, particularly on the aortic arch and around areas of dividing blood flow in the thoracic aorta. Tuberous raised structures were present in low numbers and distributed randomly on the aortic wall. Endothelial cells had elevated nuclear zones projecting into the vessel lumen. At regions of blood flow division, endothelium was polygonal in shape and silver staining of cell borders was more intense. Fatty streaks were present at blood flow divisions and micro-plaque was seen. Transmission electron microscopy of fatty streak-like areas showed the presence of up to two layers of smooth muscle cells and in some areas, lipid-laden macrophages were seen. The presence of atherosclerotic lesions in newborn WHHL rabbits suggests that the process may commence in utero.

4-Diazinyl- and 4-pyridinylimidazoles: potent angiotensin II antagonists. A study of their activity and computational characterization.

A series of N-[biphenylyl(tetrazolyl)methyl]-2-butylimidazoles containing variously substituted diazine or pyridine moieties either as their free bases or N-oxide derivatives attached to the 4-position of the imidazole ring was synthesized and tested for interaction with the AT1 receptors of rat adrenal cortex membranes (receptor binding assay). Some compounds were then chosen for further evaluation in vivo in the A II-induced pressor response in conscious normotensive rats. The most potent in the AT1 binding assay were found to be compounds in which the diazine or pyridine ring nitrogen is adjacent to the point of attachment between the two heteroaromatic rings such as 2-butyl-4-(3,6-dimethylpyrazin-2-yl)-1-[[2'-(1H-tetrazol-5-y l)-biphenyl-4- yl]methyl]-1H-imidazole (3b) or 2-butyl-4-[5-(methoxycarbonyl)pyrid-2-yl]-1-[[2'-(1H-tetrazol++ +-5- yl)biphenyl-4-yl]methyl]-1H-imidazole (6c). The binding affinities and oral activities of the pyridine N-oxide imidazoles in which a stabilizing group ortho to the pyridine ring nitrogen is present were markedly improved as in 2-butyl-4-[(3-methoxycarbonyl)-6-methyl-N-oxopyridin-2-yl]-1-[[2'- (1H- tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-imidazole 31b. Molecular modeling studies were carried out to determine the molecular electrostatic potential values of related model systems and to correlate their receptor interaction energies with the observed activities of our compounds.

N-3-substituted pyrimidinones as potent, orally active, AT1 selective angiotensin II receptor antagonists.

A novel series of nonpeptide angiotensin II (A II) antagonists containing a pyrimidinone ring which carries a C-linked biphenyltetrazole moiety and a carboxyheteroaryl group on the 3-position have been prepared. Their affinity for the AT1 receptor was determined in a binding assay on rat adrenal cortical membranes. The in vivo antihypertensive properties were tested by evaluating the inhibition of the pressor response to A II followed by iv and id administration. Extensive molecular modeling studies, including comparison of molecular electrostatic potential distributions, conformational analysis, and overlays on a computational pharmacophore model of A II, were used to evaluate structural parameters of the new compounds, in comparison to other known A II antagonists (e.g., DUP-753 and SK&F 108566). According to the modeling studies, the introduction of a (carboxyheteroaryl)methyl moiety at the 3-position of the pyrimidinone ring led to derivatives with increased potency. Methyl 2-[[4-butyl-2-methyl-6-oxo-5-[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl ]- 4-yl]methyl]-1-(6H)-pyrimidinyl]methyl]-3-thiophenecarboxylate (3k, LR-B/081), one of the most potent compounds in the series (Ki = 1.4 nM), exhibited a marked antihypertensive activity on oral administration to conscious renal hypertensive rats, with long duration of action. It was selected for clinical evaluation in the treatment of hypertension in man.

Experimental pharmacology of hirunorm: a novel synthetic peptide thrombin inhibitor.

Enhanced thrombin activity has been associated with coronary thrombosis and with acute and long-term complications following coronary balloon angioplasty. Blocking thrombin activity with specific inhibitors is proposed as a promising antithrombotic therapy. We describe the anticoagulant and antithrombotic properties of hirunorm, a novel synthetic 26-aminoacid peptide thrombin inhibitor, in comparison with r-hirudin and hirulog-1. Hirunorm was equipotent to hirulog-1 and 1/30 as potent as r-hirudin in blocking alpha-thrombin amidolytic activity (IC50 = 10 +/- 2, 15 +/- 1 and 0.3 +/- 0.1 nM, respectively), but it did not affect trypsin, plasmin and t-PA activities at 10 microM. All the compounds inhibited clot-bound thrombin to clots prepared by thrombin hydrolysis of purified fibrinogen in buffer. Hirunorm and hirulog-1 showed similar species-dependent potency in doubling basal in vitro clotting times of human, rat and rabbit plasma (EC200 varied 70 to 200 nM for TT, 0.7 to 16 microM for aPTT and 0.8 to 17 microM for PT), while r-hirudin was always at least three times more active. When assayed by HPLC or by bioassay of the intact peptide, hirunorm was stable against alpha-thrombin and plasma hydrolases, but it was catabolized by rat liver and kidney enzymes. Venous thrombosis was produced in anaesthetized rats by vena cava ligation following a procoagulant serum injection. Intravenous and subcutaneous hirunorm inhibited venous thrombosis at doses (< or = 0.3 mg/kg) two-three times higher than those of r-hirudin. Hirulog-1 was as active as hirunorm only after i.v. infusion. Arterial thrombosis was obtained in the anaesthetized rat by chemical (FeCl2) stimulation of a common carotid and i.v. infused hirunorm (1-3 mg/kg/30 min) inhibited it dose-dependently; r-hirudin was partly active only at 3 mg/kg, but hirulog-1 was inactive at either dose. Full antithrombotic doses of hirunorm did not affect the bleeding time as measured from punctured mesenteric vessels, in anaesthetized rats. In conclusion, hirunorm is a potent peptide thrombin inhibitor endowed with antithrombotic activity in models of venous and arterial thrombosis.

Dissociation of the anti-ischaemic effects of cloricromene from its anti-platelet activity.

1. Cloricromene is a non-anticoagulant coumarin derivative with anti-platelet and anti-leukocyte properties, which has beneficial effects in various models of ischaemia and shock. 2. We have assessed the effects of cloricromene on (a) ex vivo platelet aggregation, and (b) infarct size using a model of myocardial ischaemia in the anaesthetized rabbit. 3. Cloricromene (1-1000 micrograms kg-1 min-1 for 15 min) induced a dose-dependent inhibition of ex vivo platelet aggregation, causing only a minimal increase in heart rate and no change in mean arterial blood pressure. The inhibitory activity was considerably stronger when platelet aggregation was induced by collagen than by ADP. 4. Cloricromene inhibited ex vivo platelet aggregation in rabbits pretreated with indomethacin (5 mg kg-1) and this inhibition persisted for 30-60 min. 5. The model of myocardial ischaemia involved 1 h occlusion of the first antero-lateral branch of the left coronary artery followed by 2 h of reperfusion. Infusion of cloricromene (30 or 300 micrograms kg-1 min-1), ibuprofen (80 micrograms kg-1 min-1) or vehicle began 15 min prior to occlusion, and continued throughout the experiment. 6. While area at risk was similar for all groups studied, cloricromene (30 or 300 micrograms kg-1 min-1) or ibuprofen caused a reduction in infarct size, and decreased myeloperoxidase activity in the tissue of the infarcted myocardium. 7. Cloricromene at 300 micrograms kg-1 min-1 also reduced the occlusion-induced elevation of the ST-segment of the rabbit electrocardiogram, and inhibited platelet aggregation ex vivo. Ibuprofen or cloricromene at 30 fg kg-1 min-1 had no effect on either the ST-elevation or platelet reactivity.8. Thus, cloricromene exhibits a cardioprotective activity via an inhibition of leukocyte infiltration, in the presence (300 microg kg-l min-1) or absence (30 microg kg-1 min-1) of inhibition of platelet activity ex vivo.The anti-aggregatory activity of cloricromene acts via a mechanism that is either different from, or in addition to, inhibition of cyclo-oxygenase, and is of long duration.

Pharmacology of LR-B/081, a new highly potent, selective and orally active, nonpeptide angiotensin II AT1 receptor antagonist.

1. The pharmacological profile of LR-B/081, (methyl 2-[[4-butyl-2-methyl- 6-oxo-5-[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl]-1(6H)- pyrimidinyl]methyl]-3-thiophenecarboxylate), a novel antagonist at the angiotensin II (AII) AT1-receptor, was studied in vitro and in vivo. 2. In rabbit aortic strips incubated with LR-B/081 (1-1,000 nM), the concentration-response curve to AII was displaced to the right in a nonparallel fashion and the maximal contraction was progressively reduced, indicating that the compound is an insurmountable antagonist in this preparation (apparent pKB = 9.50 +/- 0.23). However, the interaction of LR-B/081 with AII receptors was found to be reversible, since the maximal response to AII was restored by coincubation with losartan, a surmountable AII AT1-antagonist. Contractions elicited by KCl or phenylephrine were not affected by 10 microM LR-B/081. 3. In rat isolated perfused kidney, LR-B/081 and losartan antagonized the AII-induced vasoconstriction [IC50 (95% confidence limits) = 17(13-24) and 39(32-54) nM, respectively]. The LR-B/081 antagonism was incompletely reversed by excess AII, while losartan was fully displaced. The IC50 values of LR-B/081 and losartan obtained against vasoconstriction induced by endothelin-1 and noradrenaline were two orders of magnitude higher. 4. In pithed rats, the intravenous administration of LR-B/081 (0.2-2 mumol kg-1) dose-dependently shifted to the right in a nonparallel fashion the dose-pressor response curve to AII. The maximal pressor response to AII was reduced by LR-B/081 in a dose-dependent fashion. The coadministration of losartan induced a progressive recovery of the maximal pressor response to All, indicating that in vivo the interaction of LR-B/081 with All receptors is reversible. LR-B/081 at 6 micromol kg-1, i.v. also did not affect the vasopressor response induced by noradrenaline in the pithed rat.5. In conscious normotensive rats, single oral administration of LR-B/081 at 6 micromol kg-1 markedly inhibited the All-induced pressor response; the inhibition lasted more than 24 h.6. In conscious renal hypertensive rats, intravenous LR-B/081 appeared as potent as losartan (ED40mmHg(95% confidence limits) = 0.50(0.36-0.70) and 0.86(0.57-1.3) micromol kg-1, respectively). A single intravenous(2 micromol kg-1) or oral (6 micromol kg-1) administration of LR-B/081 induced a marked fall in blood pressure which lasted for at least 12 h.7. In conscious spontaneously hypertensive rats, LR-B/081 at 20 micromol kg-1 , p.o., induced a marked and sustained fall in blood pressure. The duration of the antihypertensive effect was longer than 12 h.Heart rate was not modified by LR-B/081 treatment. Repeated oral administration of 17 micromol kg-1LR-B/081 for 16 days did not result in the development of tolerance.8 These results demonstrate that LR-B/081 is a potent, selective and orally active antagonist of All at the AT1-receptor subtype, which markedly lowers the blood-pressure in conscious renal and spontaneously hypertensive rats.

Inhibition of IgE-mediated release of histamine and peptide leukotriene from human basophils and mast cells by forskolin.

Forskolin, a diterpene compound isolated from the roots of Coleus forskohlii, activates adenylate cyclase in membranes from a variety of mammalian tissues. We found that forskolin (10(-7) to 3 X 10(-5) M) caused a concentration-related inhibition of IgE-mediated release of histamine and peptide leukotriene C4 (LTC4) from human basophils and lung mast cells. There was a significant linear correlation between the per cent inhibition of histamine and LTC4 release from both cell types. However, in both systems forskolin exerted a significantly greater inhibitory effect on LTC4 release than on histamine release. The concentration-response inhibition curve was paralleled by a forskolin-induced rise in cAMP levels in human leukocyte and mast cell preparations. The relationship between the effect of forskolin and the cAMP concentration was supported by the finding that forskolin inhibited the "first stage" of antigen-induced histamine release, but not the release caused by the Ca2+ ionophore, A23187. Propranolol, a competitive beta-receptor antagonist, did not block the inhibition of mediator release or the cAMP accumulation caused by forskolin. These data suggest that forskolin modulates the release of mediators of immediate hypersensitivity reactions via the activation of adenylate cyclase in human basophils and mast cells.

Serological and molecular studies of HLA in insulin-dependent diabetes mellitus in Sardinia.

This study was carried out in Sardinia, an Italian region with a very high IDDM incidence. HLA class I and class II antigens were studied in 97 unrelated IDDM patients, 33 complete families with at least one affected member each, and 559 healthy controls. Molecular typing of the DQB1 alleles was carried out in 31 patients and 61 controls. The haplotypes were determined by family studies. The HLA-DR3, DQw2, and DR4 antigens were positively associated with IDDM. The DR3 antigen was nearly always associated to B18 and frequently carried by the extended haplotype A30 Cw5 B18 3F130 DR3 DQw2. The genotype analysis of the patients showed a strong increase of the DR3/DR4 heterozygotes with a relative risk higher than that of the DR3 and DR4 homozygotes. The DR2 antigen was negatively associated with IDDM in the central island districts but not in the southern districts. The DQB1 molecular analysis showed only three alleles in the patients: DQB1*0201 (75.8 per cent), DQB1*0302 (16.1 per cent), and DQB1*0502 (8.1 per cent). These alleles are non Asp 57, so it would seem that nearly if not all Sardinian IDDM patients are NA/NA homozygotes. The DQB1*0502 allele, extremely rare in other Caucasian populations, represents in Sardinia about 70 per cent of the HLA-DR2 haplotypes, contributing to the increase of the pool of IDDM susceptible genes. Moreover it is carried in 27 per cent of the DR2 positive individuals with the extended haplotype A2 Cw7 Bw58 3F31 DR2 DQw1.AZH.

Pharmacological profile of MEN 11066, a novel potent and selective aromatase inhibitor.

MEN 11066 is a new non-steroidal compound which potently inhibits human placenta (K(i)=0.5 nM) and rat ovarian (K(i)=0.2 nM) aromatase in vitro. In vivo, a single oral dose of 0.3 mgkg(-1) significantly decreased uterus weight in immature rats after stimulation of uterus growth by androstenedione. MEN 11066 reduced in a dose-dependent manner plasma estradiol levels in adult female rats treated with pregnant mare serum gonadotropin (PMSG). After 2 weeks of repeated daily treatment in adult rats, a significant decrease in uterine weight was observed together with a 65% decrease in plasma estradiol, whereas plasma levels of testosterone, progesterone, aldosterone, corticosterone, cholesterol, LH and FSH were not affected. The lack of any effect by MEN 11066 on adrenal steroids was confirmed by the unchanged plasma corticosterone and aldosterone levels in immature rats and also in adult rats when the repeated treatment with MEN 11066 (15 days) was followed by the administration of a synthetic ACTH analogue. No change in 11beta-hydroxylase or 21-hydroxylase activities was produced in vitro by the addition of 10 microM MEN 11066. Fifteen-day treatment with MEN 11066 did not produce changes in several rat hepatic enzymatic activities involved in the metabolism of xenobiotics. These results demonstrated that MEN 11066 is a potent inhibitor of aromatase which does not interfere with the cytochrome P450 involved in the synthesis of other steroids or in the metabolism of xenobiotics.

Protective effect of the tachykinin NK2 receptor antagonist nepadutant in acute rectocolitis induced by diluted acetic acid in guinea-pigs.

We have evaluated the potential protective activity of nepadutant, a selective tachykinin NK2 receptor antagonist, in a model of acute rectocolitis induced by an enema with 7.5% acetic acid in guinea-pigs. The injury was quantified visually by using a macroscopic injury score, and histologically by using a necrosis score. In addition, changes in myeloperoxidase activity, a marker for neutrophil infiltration, and plasma protein extravasation were evaluated. The injury caused by 7.5% acetic acid was mild, affecting the superficial layers and producing a strong edema of the submucosa. A single administration of nepadutant (0.3-10 mg/kg s.c., 1 h before acetic acid) markedly reduced the macroscopic damage and necrosis score and the increase in plasma protein extravasation induced by 7.5% acetic acid in the early phase of the injury. Single administration of nepadutant (3 mg/kg s.c.) reduced the macroscopic score and myeloperoxidase activity at the top (24 h) of inflammation. Repeated administration (3 mg/kg s.c. three times during 24 h) or co-administration of the tachykinin NK1 receptor antagonist MEN 11467 (3 mg/kg s.c.) did not enhance the antiulcer effect obtained with the single treatment with nepadutant. These data suggest the involvement of tachykinin NK2 receptors in the first phases of inflammation induced by acetic acid.

Pharmacology of MEN 11467: a potent new selective and orally- effective peptidomimetic tachykinin NK(1) receptor antagonist.

We have investigated the pharmacological properties of MEN 11467, a novel partially retro-inverse peptidomimetic antagonist of tachykinin NK(1) receptors. MEN 11467 potently inhibits the binding of [(3)H] substance P (SP) to tachykinin NK(1) receptors in the IM9 limphoblastoid cell line (pK(i) = 9.4 +/- 0.1). MEN 11467 is highly specific for the human tachykinin NK(1) receptors, since it has negligible effects (pK(i) <6) on the binding of specific ligands to tachykinin NK(2) or NK(3) receptors and to a panel of 30 receptors ion channels unrelated to tachykinin receptors. The antagonism exerted by MEN 11467 at tachykinin NK(1) receptors is insurmountable in saturation binding experiments, both K(D) and B(max) of SP were significantly reduced by MEN 11467 (0.3-10 nM). In the guinea-pig isolated ileum, MEN 11467 (0.03-1 nM) produced a nonparallel rightward shift of the concentration-response curve to SP methylester with a concomitant reduction of the Emax to the agonist (pK(B) = 10.7 +/- 0.1). Moreover the antagonist activity of MEN 11467 was hardly reversible despite prolonged washout. In vivo, MEN 11467 produced a long lasting (> 2-3h) dose-dependent antagonism of bronchoconstriction induced by the selective tachykinin NK(1) receptor agonist, [Sar(9), Met(O(2))(11)]SP in anaesthetized guinea-pigs (ID(50)s' = 29+/-5, 31+/-12 and 670+/-270 microg/kg, after intravenous, intranasal and intraduodenal administration, respectively), without affecting bronchoconstriction induced by methacholine. After oral administration MEN 11467 produced a dose-dependent inhibition of plasma protein extravasation induced in guinea-pig bronchi by [Sar(9), Met(O(2))(11)] (ID(50) = 6.7 +/- 2 mg/kg) or by antigen challenge in sensitized animals (ID(50) = 1.3 mg/kg). After i.v. administration MEN 11467 weakly inhibited the GR 73632-induced foot tapping behaviour in gerbil (ED(50) = 2.96 +/- 2 mg/kg), indicating a poor ability to block central tachykinin NK(1) receptors. These results demonstrate that MEN 11467 is a potent, highly selective and orally effective insurmountable pseudopeptide antagonist of peripheral tachykinin NK(1) receptors with a long duration of action.

Lesions of the anterior temporal stem and the performance of delayed match-to-sample and visual discriminations in monkeys.

Resection of the medial temporal lobes in humans produces an anterograde amnesia in which past memories are seemingly intact, but the ability to form new memories is compromised. Efforts to reproduce these symptoms in animals have relied extensively on the delayed non-match-to-sample (DNMS) and the delayed match-to-sample (DMS) tasks. DNMS deficits have been found with combined damage to the amygdala and hippocampus, but not to the adjacent white matter (the temporal stem) that connects the temporal cortex to other brain areas. DMS deficits are, however, produced by lesions to either the anteroventral temporal cortex or the orbital frontal cortex. These two areas are interconnected through the anterior temporal stem. The present study examined the hypothesis that an anterior temporal stem lesion would impair DMS in monkeys. The anterior extreme of the temporal stem was transected in 4 Macaca fascicularis and resulted in a powerful deficit on DMS at all delays. Postoperative retention of preoperatively learned visual discriminations and postoperative learning of new visual discriminations were not reliably impaired.