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A number of epidemiological studies have evaluated the potential association between H. pylori and cardiovascular disease, but with contrasting results. We have previously shown that Helicobacter pylori infection is able to induce in mice and humans autoantibodies cross-reacting with histo-blood group Lewis antigens, expressed in different organs and in plasma glycoproteins and glycolipids. The aim of this study was to assess whether immunization of animals with H. pylori might induce myocardial histopathological changes. We have retrospectively examined, in detail, the histology of archived organs from mice and rabbits immunized with H. pylori in our previous studies. Human sera and cross-reacting monoclonal antibodies were also tested against bacterial preparations and tissue sections. Areas of myocardial necrosis, associated with coronary thrombotic occlusion, were found in 5 of 20 mice and 2 of 5 rabbits previously immunized with suspensions of H. pylori . No similar lesions were found in control animals, suggesting a causal link with H. pylori immunization. The animals bearing myocardial lesions had not been infected but only immunized months earlier with parenteral injections of dead H. pylori cells. This strongly suggests that immunization, by itself, might play a causative role. We propose that the cross-reactive autoimmune response induced by H. pylori could promote thrombotic occlusion through direct endothelial damage or by perturbing the coagulation process., (Copyright © 2020 Negrini, Villanacci, Poiesi and Savio.)

HIV-1 p17 contains C- and N-terminal sequences with positively charged residues and a consensus cluster for heparin binding. We have previously demonstrated by affinity chromatography that HIV-1 p17 binds strongly to heparin-agarose at physiological pH and to human activated CD4(+) T cells. In this study we demonstrated that the viral protein binds to heparan sulfate side chains of syndecan-2, syndecan-4, and CD44v3 purified from HeLa cells and that these heparan sulfate proteoglycans (HSPGs) co-localize with HIV-1 p17 on activated human CD4(+) T cells by confocal fluorescence analysis. Moreover, we observed a stimulatory or inhibitory activity when CD4(+) T cells were activated with mitogens together with nanomolar or micromolar concentrations of the matrix protein.

We have previously shown that HIV-1 p17 binds to activated peripheral blood mononuclear cells and enhances secretion of pro-inflammatory cytokines, but we were unable to define a ligand on activated cells. In this work we evaluate the hypothesis that HIV-1 p17 may be a heparin/heparan sulfate-binding protein. HIV-1 p17 contains C- and N-terminal sequences with positively charged residues and a consensus cluster for heparin binding. We demonstrated by affinity chromatography that HIV-1 p17 binds strongly to heparin-agarose at physiological pH. Soluble heparins and heparan sulfate but not chondroitin 4-sulfate and dextran sulfate inhibit binding of HIV-1 p17 to heparin solid phase and to activated CD4(+) T cells. Furthermore the inhibition of cell sulfatation by chlorate treatment completely counteracts HIV-1 p17 binding to activated cells. These results indicate for the first time that HIV-1 p17 can be ascribed to the heparin binding protein family and suggest that this interaction might play a key role in the ability of the protein to induce an inflammatory effect on activated cells.

Monocytes play a central role in the immune system by producing and reacting to different soluble factors. Cytokine dysregulation is an hallmark in HIV-infected individuals and it is one of the most significant factors leading to impaired immunity in HIV/AIDS disease. This study investigates the possibility of modulation in the secretion of some inflammatory cytokines and chemokines induced by HIV p17 in monocytes. The results show that p17, while ineffective on resting monocytes, exerts an inflammatory action on IL-4 mediated inhibition of TNF-alpha and IFN-gamma production induced by IL-15 stimulation. In addition, p17 is able to reduce MIP-1alpha secretion, but unable to influence IL-6 production. The ability of HIV p17 to contribute to an altered pattern of secreted soluble factors might imply a key role for this viral protein in the development of AIDS pathogenesis.

The allergenicity of seed storage proteins, the major components of edible legume seeds, may cause serious reactions in both children and adult population. Updated methodologies for evaluation of the activity of these proteins are needed. In this paper we used two-dimensional (2D) electrophoretic techniques to investigate the immuno-cross-reactivities of anti Ara h 3 basic subunit IgG to the seed proteomes of three legume species, namely, peanut, soybean, and lupin. The seed proteins, extracted with two different procedures, were separated by 2D electrophoresis, and the electrophoretic maps were analyzed by Western blot. In peanut proteome the antibodies strongly reacted with the 23 kDa polypeptides, corresponding to Ara h 3 basic isoforms, the antigen they were raised to, and three unidentified acidic polypeptides near 45 kDa. Remarkable cross-reactivities with lupin and soybean Ara h 3 homologous polypeptides and nonrelated proteins, namely, lupin conglutin gamma and soybean Bg7S, were detected. Therefore, these proteins may be regarded as new putative allergens. The present findings show the potentiality of 2D electrophoresis in the identification of food allergens and open the way to the traceability of the new cross-reacting proteins in the food chain.

Background: Several proteins have been identified as peanut allergens; among them, Ara h 1 (7S globulin) and Ara h 2 (2S globulin) are usually considered the major allergens., Objective: To identify the major allergens in a group of children selected for their specific pattern of immunoreactivity., Methods: We identified the dominant allergen by using (1) amino acid sequencing of the bands that show the strongest IgE immunoreactivity in 1-dimensional electrophoresis and immunoblotting and (2) specific animal IgGs raised against the dominant immunoreactive band to pinpoint the allergen(s) in peanut proteins separated by 2-dimensional electrophoresis and immunoblotting. To confirm these data, we further examined the peanut proteome using serum samples from the children with the unusual immunoreactivity., Results: We found a group of children with marked peanut allergy who are specifically sensitized to the basic subunit of Ara h 3 (11S globulin family)., Conclusion: That the dominant immunoreactivity in these patients is in a basic subunit of Ara h 3 was unexpected, because previous studies had indicated that Ara h 3 was only a minor peanut allergen and that the identified allergenic epitopes occurred mainly in the acidic Ara h 3 subunit.

High-grade gliomas are aggressive tumors of the central nervous system characterized by endothelial cell proliferation and a high degree of vascularity. Conventional antitumoral treatments (i.e., surgery, radiotherapy, and chemotherapy) do not achieve satisfactory results (median survival in glioblastoma 12-18 months). It has been suggested that immunotherapy with xenogenic endothelial cells could slow tumor growth rate in a number of tumors in a murine model, but the study did not include gliomas. In experiments performed in our laboratory, vaccination with proliferating bovine aortic endothelium increased survival time in Fischer rats inoculated intracerebrally with 9L. Immunotherapy was also able to reduce the growth of subcutaneously injected 9L gliosarcoma cells in Fischer rats and to decrease microvessel density within the tumors, in the absence of major organ toxicity. Immunoglobulins (Ig) in the sera from vaccinated rats stained bovine aortic endothelium as well as human umbilical vein endothelium in active proliferation. Moreover, immune sera from immunized rats stained microvessels of human malignant glioma specimens and vessels of intracerebrally implanted tumors. Two proteins of MW of 11 and 19 kDa were identified by Western blot as targets of Ig elicited by vaccination. A possible future development is to select peptides/proteins suitable for vaccination in humans, avoiding the biohazards connected with xenogenic whole-cell vaccination.

Sex steroids play an important role in modulating pulsatile growth hormone (GH) release, acting at both hypothalamic and pituitary level in both humans and experimental animals. Selective estrogen receptor modulators (SERMs) act as either estrogen receptor agonists or antagonists in a tissue-selective manner. In postmenopausal women, serum GH levels correlate positively with endogenous estradiol levels and insulin-like grwoth factor-I (IGF-I) is positively related to bone mineral density (BMD) at the spine and hip. The aim of the present study was to evaluate, for the first time, the direct effect of LY117018, an analog of raloxifene, on GH secretion from both human and rodent pituitary cells in vitro. Our results demonstrated that pharmacological concentrations of the raloxifene analog LY117018 can stimulate GH secretion through a direct action on the pituitary. LY117018 also showed an estrogen-like activity, inducing the proliferation of rat pituitary GH-secreting adenomatous cells (GH1).

Bovine beta-lactoglobulin was hydrolyzed with trypsin or chymotrypsin before, during and after treatment at 600 MPa and pH 6.8 for 10 min at 30, 37 and 44 degrees C. The extent of beta-lactoglobulin hydrolysis under pressure was noticeably higher than at atmospheric pressure, particularly when chymotrypsin was used. Addition of proteases at ambient pressure to previously pressure-treated beta-lactoglobulin gave only a modest increase in proteolysis with respect to the untreated protein. Products of enzyme hydrolysis under pressure were separated by reverse-phase HPLC, and were found to be different from those obtained at atmospheric pressure when chymotrypsin was used. The residual immunochemical reactivity of the products of combined pressure-enzyme treatment was assessed on the unresolved hydrolysates by ELISA tests using polyclonal and monoclonal antibodies, and on individual hydrolytic fractions by Western Blotting using sera of paediatric patients allergic to whey proteins in cow milk. The immunoreactivity of the whole hydrolysates was related to their content of residual intact beta-lactoglobulin, and no immunochemical reactivity was found for all the products of chymotrypsin hydrolysis under pressure. The results indicate that chymotrypsin effectively hydrolysed hydrophobic regions of beta-lactoglobulin that were transiently exposed during the pressure treatments and that were not accessible in the native protein or in the protein that had been previously pressure treated.

Purified recombinant HIV-1 p17 matrix protein significantly increased HIV-1 replication in preactivated peripheral blood mononuclear cell cultures obtained from healthy donors. Because HIV-1 infection and replication is related to cell activation and differentiation status, in the present study, we investigated the role played by p17 during the process of T cell stimulation. Using freshly isolated peripheral blood mononuclear cells, we demonstrate that p17 was able to enhance levels of tumor necrosis factor alpha and IFN-gamma released from cells stimulated by IL-2. IL-4 was found to down-regulate IFN-gamma and tumor necrosis factor alpha, and p17 restored the ability of cells to produce both cytokines. The property of p17 to increase production of proinflammatory cytokines could be a mechanism exploited by the virus to create a more suitable environment for HIV-1 infection and replication. Our data show that p17 exerts its biological activity after binding to a specific cellular receptor expressed on activated T lymphocytes. The functional p17 epitope involved in receptor binding was found to be located at the NH(2)-terminal region of viral protein. Immunization of BALB/c mice with a 14-aa synthetic peptide representative of the HIV-1 p17 functional region (SGGELDRWEKIRLR) resulted in the development of p17 neutralizing antibodies capable of blocking the interaction between p17 and its cellular receptor. Our results define a role for p17 in HIV-1 pathogenesis and contribute to our understanding of the molecular mechanism of HIV-1 infection and the development of additional antiviral therapeutic strategies.

Background: Products based on hydrolysed cow milk proteins or amino acid mixtures are recommended in children with cow's milk hypersensitivity. However, some children who are allergic to cow's milk and who clinically react to substitute milk formulas have been observed., Objective: To determine the tolerance and allergenicity of protein hydrolysate or amino acid-derived formulas in children with IgE-mediated cow's milk allergy., Methods: Twenty children with positive cow's milk challenges, positive skin prick tests and/or serum-specific IgE antibodies to cow's milk were selected. Oral challenges, skin prick tests and serum-specific IgE antibodies to extensively hydrolysed whey formula, partially hydrolysed whey formula, extensively hydrolysed casein formula and amino acid-derived formula were performed., Results: Five out of 17 (5/17) children reacted to partially hydrolysed whey formula, (3/16) to extensively hydrolysed whey formula, (2/10) to amino acid-derived formula, (1/16) to extensively hydrolysed casein formula. Only extensively hydrolysed casein formula was tolerated by at least 90% (with 95% confidence intervals) of children. Hydrolysates provoked early and delayed clinical reactions, amino acid mixtures only delayed reactions. Partially hydrolysed whey formula elicited a significantly higher number of positive skin prick test reactions than other formulas. Two children had specific IgE antibodies to extensively hydrolysed whey formula, one to partially hydrolysed whey formula, one to extensively hydrolysed casein formula and none to amino acid-derived formula., Conclusion: In this study, none of the cow's milk substitutes has been found to be non-allergenic. Our results suggest that in children with IgE-mediated cow's milk allergy, the first ingestions of extensively hydrolysed cow's milk protein formulas require strict medical supervision because of immediate reactions. This is not the case for amino acid-derived formula. Moreover, our data suggest that treatment of children allergic to cow's milk with cow's milk substitutes should be monitored for several days to document tolerance.

Somatostatin (SRIH), a cyclic tetradecapeptide hormone originally isolated from mammalian hypothalamus, is a potent suppressor of pituitary growth hormone (GH) secretion. SRIH acts through a family of G-protein-coupled membrane receptors containing seven transmembrane domains. Five genes encoding distinct SRIH receptor (SSTR) subtypes have so far been cloned in human and other species and termed SSTR1-5. In human somatotrophe pituitary adenomas GH secretion is controlled by both SSTR2 and SSTR5. However, in clinical practice only somatostatin analogs selective for SSTR2 (octreotide and lanreotide) are available. This may explain why clinical and in vitro responses to these analogs in acromegaly are only partial. In this study, we investigated the inhibitory effect of two new SRIH analogs with high selectivity for SSTR2 (NC-4-28B) and SSTR5 (BIM-23268) and compared it to that of native somatostatin (SRIH-14) on a large number of GH-secreting adenomas obtained by transphenoidal neurosurgery. Tissues from 16 adenomas were enzymatically dispersed and plated in 24-well dishes at 50,000 cells/well. After 3 days, groups of three wells were incubated for 4 h with medium alone, SRIH-14 or analogs NC-4-28B or BIM-23268, at the concentrations of 0.01, 0.1 and 1 microM. Our results show that 9 out of 16 adenomas were responsive (GH suppression: 20-40% vs. control, p < 0.05) to SRIH. In this group only 4 adenomas showed similar responses to both selective analogs, with 2 nonresponders (expression of other SRIH receptor subtypes) and 2 responders (concomitant expression of SSTR2 and SSTR5) to both analogs. GH release was selectively inhibited by NC-4-28B in 3 adenomas and by BIM-23268 in the remaining 2 adenomas, suggesting predominant expression of SSTR2 and SSTR5, respectively. SRIH failed to inhibit GH release in 7 adenomas (43%). Interestingly, in that group a better inhibitory effect was obtained with BIM-23268 (5 out of 7 adenomas) than with NC-4-28B, suggesting expression of a few SSTR5 receptors only, or of both SSTR2 and SSTR5, respectively. We conclude that the availability of somatostatin analogs selective for SSTR5 will enhance the treatment potency and spectrum in acromegaly., (Copyright 2001 S. Karger AG, Basel)

Proteolysis has a critical role in defining the typical organoleptic characteristics of Grana Padano, a well-known Italian cheese. During the ripening process, hydrolysis of beta-casein produces different fragments, the most abundant and widely studied of which are gamma-caseins, three polypeptides containing the HOOC-terminal portion of beta-casein. By sodium dodecyl sulfate-PAGE and a specific anti-beta-casein monoclonal antibody, two beta-casein-derived bands were identified in Grana Padano cheese: betaa and betab. Thanks to the identification of the amino acid sequences, it was shown that: a) betaa contains gamma1-casein [beta-casein (29-209)] and the correlated peptide [beta-casein (30-209)]; b) betab contains gamma2-casein [beta-casein (106-209)] and gamma3-casein [beta-casein (108-209)]. The production of betaa and betab by the three enzymes most involved in cheese proteolysis (pepsin, chymosin, and plasmin) was evaluated by performing in vitro digestions. A significant correlation between abundance of some polypeptides and ripening process was shown.

Since casein proteolysis has a critical role in defining the typical characteristics of Grana Padano cheese, we evaluated the hydrolysis of alphas-casein during the ripening process. Thanks to the high specificity of the anti-alphas((alphas1 + alphas2)-casein monoclonal antibody and amino acid sequence determination, it was possible to identify three main alphas-casein-derived polypeptides in cheese: alphaa, alphab, and alphac. Their production by the three enzymes most involved in cheese proteolysis (pepsin, chymosin, and plasmin) was evaluated by performing in vitro digestions. Data showed that alphaa was released in cheese mainly by the chymosin attack, while alphab and alphac were due to the action of plasmin. A significant correlation between the abundance of some polypeptides and ripening process was shown.

Background: It is generally believed that the elimination of certain foods from the diet of mothers during the lactation period produces a significant improvement in breast-fed children who develop allergic symptoms. Several studies have shown the presence of food proteins in human milk; on the other hand, no study has been able to correlate unequivocally the presence of these allergens in human milk with newborn sensitization., Objective: The aim of this study was to evaluate the presence of bovine proteins in breast milk., Methods: Milk samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). To detect bovine proteins in human milk, immunoblotting was performed by using monoclonal antibodies (MA) specific for beta-lactoglobulin and bovine caseins., Results: The results of this study do not confirm the presence of bovine proteins in breast milk suggested by other authors and shows unequivocally that the conflicting results reported in the literature about the presence of betalactoglobulin in human milk are due to cross-reactivity between bovine milk proteins and human proteins., Conclusions: Components other than bovine betalactoglobulin or caseins could be involved in the induction of allergic symptoms in exclusively breast-fed children.

Background: Cow's milk allergy is quite frequent in the first years of human life. When breast-feeding is not possible, a cow's milk substitute must be provided for allergic subjects. Different alternatives to cow's milk have been suggested as protein sources (soy, hydrolysed proteins, goat's milk, etc.), but all these dietetic solutions are not without risks for polyallergic or more sensitive subjects., Objective: To obtain new information on the suitability of other mammalian milks for allergic children, we evaluated the cross-reactivity between milk proteins from different animal species., Methods: Milk samples were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). To detect antibody-antigen complexes, immunoblotting was performed by using sera from children allergic to cow's and ewe's milk (RAST class >/= 4) and monoclonal antibodies (MoAb) specific for bovine proteins (caseins and beta-lactoglobulin)., Results: IgEs from children allergic to cow's milk are capable of recognizing most part of milk proteins from mammals bred in European countries (ewe, goat, buffalo), while no serum used in this study contains IgEs reacting with camel's milk proteins. Camel's milk was also not recognized from circulating IgEs from a child specifically allergic to ewe's milk. Specific antibovine monoclonal antibodies cross-reacted with proteins from other mammalian species, apart from those of camel., Conclusions: Homologies in amino acidic composition could justify the cross-reactivity observed between proteins from different animal species. On the other hand, the phylogenetic difference could be responsible for the failed recognition of camel's proteins by circulating IgEs and monoclonal antibodies.

Background: The aim of our study was to assess whether there could be any clinical and/or endocrine (spontaneous growth hormone [GH] secretion rate, baseline insulin-like growth factor-1 [IGF-1]) predictors and/or determinants of the acute effects of continuous intravenous infusion of recombinant human GH on hemodynamic parameters in 12 patients with dilated cardiomyopathy and congestive heart failure (CHF)., Methods and Results: The study involved 12 male patients with chronic CHF (ischemic in 8 patients and idiopathic in 4). Ten patients were in New York Heart Association functional class III or IV and 2 in class II. The first 24 hours were considered the control period; in fact, during the following 24 hours, all the patients underwent intravenous constant pump infusion of recombinant human GH. Blood samples for GH assay were taken every 20 minutes during the first night of the study (from 10 PM to 6 AM). Moreover, blood samples for GH assay were also taken during exogenous GH infusion. Blood samples for IGF-1 assays were taken at 8 AM of each of the 3 days of the study. Pulmonary artery pressure (PAP) and capillary wedge (PCWP) pressure, cardiac index, and arterial blood pressure were measured 30 minutes after right heart catheterization (baseline 1), at the end of the control period (baseline 2), and every 4 hours during GH infusion. A negative correlation has been found between mean nocturnal GH levels and baseline IGF-1 levels (r = -0.47, P =.124) and between mean nocturnal GH levels and both postinfusion absolute (r = -0.67, P <.05) and delta (postinfusion-preinfusion) (r = -0.58; P < 005) IGF-1 levels. No significant correlations have been found between several parameters of liver function (albumin, bilirubin, and pseudocholinesterase) and mean nocturnal GH. However, baseline IGF-1 levels showed a negative significant correlation (r = -0.76, P <.01) with total bilirubin and a positive correlation (r = 0.72, P <.01) with pseudocholinesterase. Baseline IGF-1 levels showed a significant negative correlation with baseline mean PAP (r = -0.68, P <.05) and PCWP (r = -0.70, P <.05) and a positive correlation with baseline cardiac index (r = 0.71, P <.05). Baseline IGF-1 levels also showed a significant negative correlation with absolute mean PAP (r = -0.63, P <.05) and mean PCWP (r = -0.67, P <.05) after GH infusion. After GH infusion, IGF-1 levels also negatively correlated with post-GH infusion mean PAP (r = -0.50, P =.09) and mean PCWP (r = -0.66, P <.05). The positive correlation between either baseline or postinfusion IGF-1 and the postinfusion cardiac index (r = 0.40 and 0.43, respectively) did not reach statistical significance., Conclusions: GH has acute functional effects on the heart in patients with CHF, including both an increase in myocardial contractility and a decrease in vascular resistances, and among patients with CHF, those with low baseline IGF-1 are likely to have fewer beneficial effects from GH infusion.

Objective: To analyse the role of recombinant HIV-1 protein p17 in the modulation of cell activity., Methods: Peripheral blood mononuclear cells (PBMC) obtained from healthy donors were cultured in the presence or absence of p17 with mitogens such as phytohaemagglutinin or interleukin-2 and their response assayed by cell proliferation. Cross-linking experiments were employed to investigate the presence of a binding between p17 and factor(s) present in human serum. An immunoenzymatic assay for p24 antigen detection was used to analyse the effect of the addition of exogenous p17 to cultures of PBMC infected with HIV-1 in vitro., Results: Purified recombinant p17 protein at a concentration of 0.25 microg/ml significantly increased the proliferation of preactivated PBMC obtained from healthy donors. This effect was obtained by binding p17 to factor(s) present in human serum and observed on both CD4+ and CD8+ T cells. Recombinant p17 also induced an increased rate of HIV-1 replication, probably due to enhanced T-cell proliferation. The activity of p17 protein was inhibited by anti-p17 antibodies generated by injecting recombinant p17 in rabbits, but not by human antibodies generated during the natural course of HIV infection., Conclusion: Characterization of the human factor(s) and identification of the interacting p17 epitope(s) will improve our understanding of the mechanisms used by HIV to efficiently replicate in our organisms.

Background: Hydrolysed casein and whey protein formulas have been developed with the aim of preventing sensitization in infants at high risk of cow milk allergy. Subsequently these products have also been used for treatment of children with cow milk allergy. However, severe reactions have occurred in some allergic infants fed with these formulas raising doubts about their absolute safety and suggest the need for developing in vitro techniques for detection of eventual residual allergenic activity in such preparations., Objectives: Our purpose was to evaluate the usefulness of monoclonal and polyclonal antibodies against casein components (alpha, beta and kappa casein) as reagents for the detection of the residual antigenic activity of casein components in several hydrolysed formulas., Methods: The monoclonal and polyclonal antibodies were produced according to standard procedures by immunizing female Balb/c mice with casein fraction (a mixture of alpha, beta and kappa casein). ELISA assays were developed to test the specificity of the antibodies and to detect and evaluate the amount of residual antigenic activity of the casein components in hydrolysed formulas., Results: Use of polyclonal antiserum specific for casein allowed detection of residual antigenic activity of casein components in all partial hydrolysates and in the two extensive whey protein hydrolysates in the amounts ranging from 0.05 to 0.67% of total protein. No such activity was detectable in either the two extensive casein hydrolysates tested or the aminoacid based formula. The polyclonal antiserum proved to be more suitable than monoclonals for detecting residual antigenic activity in the hydrolysates. The monoclonal antibodies were directed against epitopes expressed on different casein components., Conclusions: In this study the ELISA inhibition assay with polyclonal antibodies specific for casein components of cow milk proved to be a sensitive method for estimating residual antigenicity in the hydrolysed formulas commercially available for infants with cow milk allergy suggesting their potential application for the quality control of hypoallergenic infant formulas.

Insulin-dependent (type I) diabetic patients are known to have an exaggerated growth hormone (GH) response to GH-releasing hormone (GHRH), which is hypothesized to be due to a decrease in somatostatin tone. The aim of the study was to ascertain the influence of the presence and activity of the autoimmune process involving a key enzyme (glutamic acid decarboxylase [GAD]) in the synthetic pathway of a neurotransmitter regulating somatostatin secretion, ie, gamma-aminobutyric acid (GABA), on the GH response to GHRH alone or combined with an acetylcholinesterase inhibitor, pyridostigmine (PD), in patients with type I diabetes mellitus. Twenty non-obese type I diabetic patients and 17 normal subjects underwent an intravenous (IV) injection of 100 micrograms GHRH(1-29)NH2. Twelve of 20 diabetic subjects and all of the control subjects also underwent a second experimental procedure, administration of 120 mg oral PD 60 minutes before IV injection of 100 micrograms GHRH. Diabetic subjects with serum GAD antibody (GADA) levels more than 3 U (n = 10) showed significantly higher serum GH levels after GHRH injection as compared both with diabetic patients with GADA less than 3 U (n = 10) and with normal controls, whether expressed as absolute or peak values. GH peaks after GHRH were significantly (rs = .46, P < .05) correlated with the level of GADA in the whole population of type I diabetic subjects studied. PD significantly enhanced the GH response to GHRH, in terms of both absolute and peak values, in patients without GADA (n = 6) and in normal subjects. On the contrary, PD failed to enhance the GH response to GHRH in diabetic patients with GADA (n = 6). Our findings suggest that autoimmunity may play a key role in determining the exaggerated GH response to GHRH in type I diabetes mellitus. The mechanism underlying this effect is hypothesized to be the production of antibodies to GAD, a key enzyme in the synthesis of GABA, and in turn a reduced GABAergic stimulatory tone on somatostatin production at the hypothalamic level.

To study the onset of the action of gonadal sex steroids on the GH axis in spontaneous puberty, which is prolonged and sparingly predictable, we present a clinical investigative paradigm in which six previously untreated boys with isolated hypogonadotropic hypogonadism were exposed to progressively higher testosterone levels designed to mimic the androgen environment recognized during the early stages of puberty. We administered three incremental doses of testosterone (25-, 50-, and 100-mg im injections), each over a period of 4 weeks. Studies of overnight pulsatile GH secretion and GH responses to GHRH alone or combined with L-arginine (a functional somatostatin antagonist) were performed before testosterone administration and after each dose of testosterone. Serum testosterone, but not estrogen, levels increased progressively in all subjects during therapy. Deconvolution analysis of GH release profiles disclosed that GH secretory burst mass was stimulated significantly even by 25 mg testosterone. This parameter was not altered further by higher doses of testosterone. Spontaneous GH secretory burst number and amplitude increased significantly only after the 50- and 100-mg testosterone treatments, after which the serum GH response to GHRH and arginine also rose significantly. In contrast, the GH response to GHRH alone was not significantly affected by any dose of testosterone. Serum testosterone levels correlated significantly with the primary parameters of nocturnal GH secretion. In summary, our experimental model suggests that in males even very small increases in circulating testosterone occurring during the earliest stages of puberty are able to amplify pulsatile GH secretion. Our concomitant secretagogue data further suggest that testosterone exerts its action at different sites in the hypothalamo-somatotropic axis, i.e. directly at the pituitary level, and also at hypothalamic loci, possibly increasing both GHRH and somatostatin release.

The aim of the present study was to characterize in a large series (N = 12) of cultured somatotrope adenomas the in vitro effects of the neuropeptide galanin on growth hormone (GH) secretion. This was contrasted with two peptides known to be GH secretagogues (GH-releasing hormone [GHRH] and thyrotropin-releasing hormone [TRH]) and a peptide with a known GH-inhibitory effect (the somatostatin analog octreotide). Groups of three wells were incubated for 4 hours with growth medium alone (control incubation), galanin, GHRH(1-29)NH2, TRH, or octreotide. Galanin and octreotide were applied at concentrations of 0.1, 1, and 10 mumol/L, and GHRH and TRH at concentrations of 0.01, 0.1, and 1 mumol/L. Galanin was able to inhibit GH release in nine of 12 cultured somatotrope adenoma cells. This inhibitory effect was clearly dose-dependent in five adenomas. Overall, the mean GH nadir after galanin was -36.1% in nine responder adenoma cultures versus control wells. Octreotide inhibited GH release in five of eight cultured somatotrope adenoma cells. The mean GH nadir after octreotide was -32.7% in five responder adenoma cultures compared with control wells. GHRH and TRH were able to stimulate GH release, respectively, in seven of 11 and in six of seven cultured somatotrope adenoma cells. The mean GH peaks after either GHRH or TRH in responder adenoma cultures were, respectively, +71.5% and +143.7% compared with levels in the control wells. In conclusion, the consistency and potency of the in vitro GH-inhibitory effect of galanin in a large series of somatotrope adenomas are at least similar to those of the most effective available GH-lowering agent, the somatostatin analog octreotide.

Background: Partial and extensive hydrolysed protein formulae have been developed to lower or eliminate the antigenicity of milk proteins. Although normally well tolerated, extensive hydrolysates have been reported to induce serious allergic reactions in very sensitive children. Moreover, clinical practice has often raised concern about the role of partial hydrolysates in cow's milk allergy prevention., Objective: Starting from these considerations, we used anti-casein monoclonal antibodies to evaluate the presence of residual antigenic activity in both partially and extensively protein hydrolysates., Methods: Electrophoretic analyses associated with immunoblotting technique were performed using nine protein-enriched commercial formulae., Results: The presence of different amounts of residual intact cow's milk proteins and/or polypeptidic material with conserved antigenic activity (according to the extensive or partial hydrolysis) was verified in most milk-based samples considered., Conclusion: The use of monoclonal antibodies and immunoblotting could be useful for the quality of commercial 'hypoallergenic' formulae.

Background & Aims: The majority of patients with Helicobacter pylori infection have autoantibodies cross-reacting with gastric antigens. In this study, the relation between autoantibody status, histopathology of body mucosa, and antigenic profile of H. pylori was investigated., Methods: One hundred patients were examined for H. pylori infection, body gastritis, and gastric autoantibodies. Balb/c mice were analyzed for serum autoantibodies after immunization with H. pylori from patients with and without atrophic gastritis., Results: Immunoglobulin G autoantibodies were detected in 57 of the 87 infected patients (65.5%) but in none of the 13 patients without infection and gastritis. The autoreaction involved mainly the luminal surface of glandular cells and secretory canaliculi of parietal cells. The autoantibody status correlated with the presence and degree of inflammation and atrophy of the glands. H. pylori from patients with atrophic gastritis showed a higher capacity to induce autoantibodies than H. pylori from patients with a minimal superficial gastritis. Monoclonal antibodies showed differences in the bacterial expression of cross-reacting determinants., Conclusions: H. pylori-mediated autoimmunity is involved in the pathogenesis of chronic atrophic gastritis. The grade of antigenic mimicry of the infecting H. pylori strain plays a role in the progression of chronic gastritis to atrophy.

We describe the specific interaction between high-purity recombinant human immunodeficiency virus (HIV) type 1 p17 and human gamma interferon (hIFN-gamma) proteins. This interaction was found to be dose dependent and to involve conformational epitopes on both sides. Specificity was confirmed by competition ELISA, using monoclonal antibodies (MAbs) to hIFN-gamma as specific reagents. By competition experiments we also identified the epitope(s) on the hIFN-gamma molecule involved in p17 binding, very close to the receptor binding site. The kinetic constants were determined by surface plasmon resonance (SPR) analysis. The affinity constant (KA) of the complex was 2.78 x 10(8) M-1, that is, the ratio between a low dissociation rate constant (Koff)(1 x 10(-5)sec-1) and a high association rate constant (Kon) (3 x 10(3) M-1sec-1). However, p17 did not displace the binding of hIFN-gamma to its cellular receptor, nor did it interfere with the capability of the lymphokine to induce de novo expression of HLA-DR antigens on human monocytic cells or to inhibit the proliferation of tumor cells.

The aim of our study was to investigate the effect of hexarelin, a novel GH-releasing peptide-6 analog, and GH-releasing hormone (GHRH) (alone or in combination) on GH secretion in adult patients with increased somatostatin tone due to chronic glucocorticoid excess. We studied seven adult patients undergoing long-term (no less than 6 months) immunosuppressive glucocorticoid treatment for non-endocrine diseases (six females and one male, age range 42-68 years) and one subject (female, age 31 years) with endogenous hypercortisolism due to adrenal adenoma. Six normal subjects (four females and two males) matched for sex and age with the patients and not undergoing any therapy served as controls. All the subjects underwent the following three tests in random order: (1) human GHRH (1-29)NH2 (100 micrograms in 1 ml saline) injected as an i.v. bolus at 0 min, (2) hexarelin (100 micrograms in 1 ml saline) injected as an i.v. bolus at 0 min and (3) hexarelin (100 micrograms in 1 ml of saline) plus GHRH (100 micrograms in 1 ml saline) injected as an i.v. bolus at 0 min. After GHRH alone the patients with glucocorticoid excess showed a blunted GH response as compared with normal subjects (median delta GH: 0.9, range 0-5.6 micrograms/l vs 7:1, range 0.3-14.9 micrograms/l). No significant differences were observed in the steroid-treated group with respect to normal subjects after hexarelin alone (median delta GH: 15.5, range 1.9-45.2 micrograms/l vs 17.9, range 5.5-53.9 micrograms/l).(ABSTRACT TRUNCATED AT 250 WORDS)

Background: Extensively hydrolysed protein formulas are widely used as an alternative diet for children with cow's milk allergy. Partially hydrolysed protein formulas have been noted in some studies as useful in the prevention of allergy in infants at high risk of atopy. Although normally well tolerated, these 'hypoallergenic' products have been reported to cause serious immunological reactions in very sensitive subjects., Objective: Starting from these considerations, we studied some commercial hydrolysed formulas in search of biological data supporting the observed clinical reactions., Methods: We set up an electrophoretic method sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) which allowed us to study the molecular weight of peptides contained in hydrolysed products. Then, using the immunoblotting technique we evaluated the reactivity of circulating IgE (from serum of children allergic to cow's milk proteins) with the residual intact proteins and with the peptides present in these formulas., Results: Both group of milk proteins (caseins and whey proteins) were important allergens for children included in this study. The presence of high-molecular polypeptides was shown in partial hydrolysed formulas as such and in extensive hydrolysed products after protein enrichment by trichloroacetic acid (TCA) precipitation. Intact residual proteins were mainly responsible for the formation of IgE-antigen complexes observed in immunoblotting. More rarely, polypeptides of partial hydrolysed formulas were involved in immunological responses., Conclusions: Both partial and extensive hydrolysed formulas could induce clinical reactions in very sensitive subjects. These responses are mainly associated with allergy to the small amounts of residual intact proteins.

Renin-angiotensin-aldosterone system, plasma atrial natriuretic peptide (PANP), and blood volume (BV) have been investigated in 20 nephrotic patients with normal renal function and with (group 1; n = 12) or without (group 2; n = 8) sodium retention. Patients of group 1 had a plasma albumin (PALB) concentration < 1.7 g/dl, low BV and PANP levels, a reduced fractional excretion of lithium (FELi), and high plasma angiotensin II levels. Patients of group 2 had PALB > 1.7 g/dl, and the other parameters were normal. The spontaneous intake of dietary sodium was lower in group 1 than in group 2. In all patients the BV was directly correlated with PALB, and the plasma renin activity (PRA) was inversely correlated with both BV and PALB. A nonlinear inverse relationship was present between plasma aldosterone (PALD) levels and fractional excretion of sodium (FENa). The acute expansion of the BV in patients of group 1 normalized PRA, PALD, PAII, FENa, and FELi and increased PANP. The administration of spironolactone to the patients of both groups had variable effects on FENa, did not modify PRA and PALD, and reduced body weight, PANP, and FELi, thus suggesting that the reduction of BV induced by the drug increased the proximal reabsorption of sodium. Three additional patients who had sodium retention, PALB of 2.3-2.4 g/dl, normal PRA and PALD, elevated urinary excretion of aldosterone, and a slightly low PANP showed a spontaneous normalization of urinary aldosterone and PANP associated with natriuresis and weight loss, but thereafter urinary aldosterone increased, PANP decreased, and the sodium retention began again. Our data suggest that in nephrotic patients with severe hypoalbuminemia, contraction of BV plays a major role in promoting the sodium retention through the activation of compensatory hormonal mechanisms. On the other hand, when PALB is not severely reduced, the patients have normal BV, but they are very sensitive to small changes of BV which are better evidenced by modifications of the urinary excretion of aldosterone and PANP rather than by the profiles of PRA and PALD.

It has been previously reported that different quantitative results can be obtained when TNF alpha is measured in biological fluids by bioassay and immunoassay. This is thought to be related to the presence of antigenic forms of TNF alpha that cannot be detected by bioassay, such as complexes with soluble receptors (sTNF-R) or TNF alpha monomers. In this work we have observed discrepancies between antigenic and bioactive TNF alpha even when we used a sandwich-ELISA, unable to detect TNF alpha monomers, based on antibodies that bind epitopes overlapping with the soluble-receptor binding site of TNF alpha. Moreover, we found that antigenic TNF alpha levels in the presence of p55-sTNF-R (sTNF-R1) measured by different immunoassays were variable, depending on the immunoreagents and incubation time. To investigate whether TNF alpha-soluble receptor complex dissociation occurring during assay incubations contributes to the variability of results, we studied the kinetics of TNF alpha-soluble receptor interactions and examined the effect of complex dissociation using different analytical systems. TNF alpha association (k(on)) and dissociation (koff) rate constants with sTNF-R1, measured by real-time biospecific interaction analysis, were 5.01 x 10(5) s-1 M-1 and 2.8 x 10(-4) s-1, corresponding to an equilibrium constant (Kd) of 0.59 nM and to a half life for these complexes of 38 min. Complex dissociation and differential changes in the TNF alpha-sTNF-R1 bound:free ratio, in different analytical systems, markedly affects TNF alpha quantification.

Cow's milk protein is quite commune in infancy (2-3% in first year of age). Casein, beta-lactoglobulin and alpha-lactalbumin are the main allergens of cow's milk. The authors describe the immunological reaction involved in IgE synthesis and consequential inflammation after ingestion of cow's milk proteins and present soy and protein extensive hydrolysates as alternative diets for children with cow's milk allergy. Moreover, the authors present their studies on immunogenicity of hydrolysed formulae. At the end they suggest the therapeutic strategy in the cow's milk protein allergy.

The liver alpha-1-acid glycoprotein (AGP) gene promoter contains several positive cis-acting sequences that are involved in the hormone regulation of its expression. We have characterized a new functionally important sequence located at -155 to -143 upstream from the glucocorticoid regulatory element (GRE, -120 to -105). At least three nuclear proteins bind to this sequence (CTGTGGGAACAG), called the upstream regulatory element (URE). One of these proteins, AGP nuclear factor 4 (ANF-4), is the major component of the DNA-protein complex we detected in footprint and electrophoresis mobility shift assay (EMSA) experiments using rat liver, HTC(JZ-1) rat hepatoma cell extracts and affinity-purified proteins. Another is C/EBP beta, which also binds to three elements downstream from the GRE. The third protein is shown to have a molecular weight of 102 kD. Deletions and site-directed mutagenesis demonstrated that this complex of proteins is involved in the positive hormonal regulation of AGP gene transcription. Binding experiments revealed that ANF-4 and C/EBP beta binding sites are partially overlapping and require the palindromic structure of the URE for high-affinity binding. Southwestern (DNA-protein blot analysis) and cross-linking experiments with nuclear extracts from rat liver and HTC(JZ-1) rat hepatoma cells, revealed two identical constitutive binding activities with molecular masses of 66 and 102 kD. We concluded that this transcription complex is composed of three distinct proteins, ANF-4, C/EBP beta, and a 102-kD protein, and that they play an important role for the hormone regulation of AGP.

A biosensor system aimed at real-time measuring molecular interactions among label-free reactants has been used for a comparative analysis of the binding features (i.e., association-dissociation rates and affinity constants) as well as epitope mapping between bivalent monoclonal antibodies and the derived monovalent bispecific monoclonal antibody. The results show that observed different affinities between parental and derived bispecific antibodies concern the association rate constant, whereas the dissociation rate constants are unaltered. The apparent affinity-constant values determined by solid-phase radioimmunoassay yielded figures almost overlapping with those obtained with the biosensor instrument. The results of the present work indicate that the biosensor system has gained a key role not only as a tool for the study of antigen-antibody interactions, but also for setting up the reference parameters for the selection of the best candidates in the generation of bispecific monoclonal antibodies.

In this work we have studied the kinetic parameters of oligomeric tumour necrosis factor alpha (TNF-alpha) dissociation using biospecific interaction analysis (BIA), based on surface plasmon resonance (SPR) of TNF-alpha immobilized on a sensor chip, and by an ELISA technique able to detect TNF-alpha oligomers in solution. Validation studies, carried out with sensor chips bearing TNF-alpha oligomers or bovine albumin monomers, verified that: (a) TNF-alpha can be immobilized in the oligomeric form onto sensor chips; (b) the covalent linkage between TNF-alpha and sensor chips is stable under the experimental conditions; (c) TNF-alpha monomers are present on the sensor chips after dissociation; (d) immobilization and dissociation rate constant (kdiss) measurements are reproducible. The kdiss of recombinant TNF-alpha, measured under non denaturing conditions at pH 7.4 by BIA and ELISA were in good agreement, being 0.92 x 10(-5)/s and 1.1 x 10(-5)/s respectively (corresponding to a half life of about 20.9 h and 17.5 h, respectively). The dissociation rate was found to be significantly affected by the presence of detergents and by the pH of the solution, suggesting that TNF-alpha, at low concentrations, exists in solution with different molecular forms depending on the time of storage and buffer composition. Real-time BIA is rapid and does not require particular antibodies or reagents. Thus, the stability of the quaternary structure of natural or recombinant TNF-alpha from human or animal species as well as that of other oligomeric cytokines can probably be studied using this method.

Toxicological analysis of hair is becoming a popular method for investigating past, chronic use of illicit drugs. Several analytical methods using immunometry, chromatography and mass spectrometry have been reported. In this work, capillary electrophoresis was first used for the determination of illicit drugs, such as cocaine and morphine, in the hair of heroin and cocaine users. After rapid washing, hair samples were incubated overnight in 0.25 M HCl at 45 degrees C and the mixtures were extracted with ready-to-use Toxi-tubes A. The organic phase was evaporated and the residue dissolved in a suitable amount of electrophoresis buffer. Free zone capillary electrophoretic determinations of morphine, the main heroin metabolite, and cocaine were accomplished in 0.05 M borate buffer (pH 9.2) at a potential of 15,000 V, with UV detection at 214 and 238 nm, respectively. The use of the less selective wavelength of 200 nm allowed the simultaneous detection of both compounds. Efficient separations (up to 350,000 theoretical plates) and accurate and precise determinations (intra-day R.S.D.s in the range 3-5%) of cocaine and morphine in hair extracts were easily achieved. The analytical sensitivity was sufficient to determinate as little as 0.15 ng/mg of cocaine and morphine in hair using 100-mg samples. Interferences from more than 90 therapeutic drugs and drugs of abuse were excluded.

An automated biosensor system designed for measuring molecular interactions in real-time and without labelling of the reactants has been used to evaluate the association/dissociation rate and affinity constants of bivalent monoclonal antibodies and a monovalent bispecific monoclonal antibody. Observed differences in affinity between parental and bispecific antibody produced were related to the association rate constants, since the dissociation rate constants were in the same range. Values were also closely related to radioimmunochemical data. These results indicate that the biosensor system, besides presenting several advantages for characterizing antigen-antibody interaction, is valuable for selecting monoclonal antibodies with properties which might be useful in the development of bispecific monoclonal antibodies.

The aim of this study was to establish hybridomas capable of long-term production of human monoclonal antibodies (mAbs). Heterohybridization was performed between the mouse myeloma cell line P3X63Ag8.653 and activated human peripheral blood lymphocytes (PBL). In order to achieve better retention of human chromosomes, as well as to improve the stability of the heterohybrids, one HAT-sensitive immunoglobulin (Ig)-non-secreting human x mouse (h x m) heteromyeloma was fused for a second time with activated human PBL. In this way, a panel of HAT-sensitive Ig-non-secreting h x h x m heteromyelomas was obtained and tested for its ability to generate stable human Ig-secreting heterohybrids with activated human PBL. Six lines were selected on the basis of their enhanced characteristics of fusion efficiency and genetic stability. When fused with in vitro immunized human PBL, they generated several h x h x h x m hybridomas stably secreting high yields (10-23 micrograms/ml/24 h) of human mAbs reactive with recombinant HBV core antigen (rHBcAg). Moreover, a continuous production of human Ig was observed when two h x h x m heteromyelomas, previously made ouabaine-resistant, were hybridized with EBV-transformed lymphoblastoid cell lines. These h x h x m heteromyelomas are ideal fusion partners for the production of human mAbs.

Forty-nine monoclonal antibodies against Helicobacter pylori were screened to investigate their capacity to be used in enzyme-linked immunosorbent assay (ELISA) competitive systems for the serodiagnosis of Helicobacter pylori infection. On the basis of the inhibition pattern showed by the sera of five infected patients, the antibodies were subdivided into five groups. The immunoblotting analysis showed that the antibodies recognized a total of nine different antigenic determinants. In a study of the reaction of the antibodies with 12 isolates of H. pylori a total of 9 antigenic profiles were identified. Two monoclonal antibodies, HpN44 and HpN45, which recognized a 64-kD protein, were inhibited by all 5 positive sera. Antibody HpN45 was labeled with horseradish peroxidase, and the competitive ELISA was compared with an ordinary indirect ELISA in a study of 102 patients undergoing gastroscopy. Seventy-three patients proved to be infected by H. pylori according to urease or histologic tests. The sensitivity and specificity were 90.4% and 89.6%, respectively, for the indirect ELISA and 100% and 89.6% for the HpN45 competitive assay. The three patients who were 'false seropositive' with both serologic tests had atrophic gastritis. The high diagnostic performance and simplicity of the HpN45 monoclonal competitive ELISA make it suitable for routine serodiagnosis of H. pylori infection.

Aim of the study was to evaluate the effect of cardiopulmonary receptors activation and deactivation on antidiuretic hormone (ADH) and atrial natriuretic peptide (ANP) incretion in hypertensive and normotensive subjects. Twenty-one male subjects, 7 normotensives and 14 mild hypertensives, 7 without and 7 with left ventricular hypertrophy (LVH) were admitted to the study. Each subject underwent selective loading and unloading of cardiopulmonary receptors, by application of a positive (LBPP) or negative (LBNP) pressure to the lower body. Blood samples were taken for measurement of ANP, ADH, PRA, immunoreactive renin, aldosterone, noradrenaline and adrenaline. ADH plasma concentration increased during cardiopulmonary receptors inhibition, but this increase became statistically significant (p less than 0.05) at a step of LBNP (-40 mm Hg), in which an involvement of the sinoaortic receptors cannot be excluded. ANP plasma levels increased progressively during LBPP (p less than 0.05 at least). These changes were significantly reduced in hypertensive patients with LVH.

The authors' previous observation that many of the monoclonal antibodies against Helicobacter pylori cross-react with the cells of the human gastric mucosa prompted them to investigate the possibility that gastric self-antigens cross-reacting with H. pylori could be involved in the immune response against this organism. It was found that three antibodies against H. pylori, CB-4, CB-10, and CB-14, that cross-react with the human gastric mucosa also intensely cross-reacted with murine gastric epithelial cells. A strong reaction against autologous mucosa was also evident in the sera of mice immunized with H. pylori but not with other bacteria. A serological study performed in a group of 82 patients undergoing gastroscopy showed that the presence of seropositivity against H. pylori was strongly correlated with the presence of autoantibodies against human antral gastric mucosa. This activity was neutralized after absorption of the sera with H. pylori but not with other gram-negative bacteria. The antibodies in the mouse and in the human did not react with other segments of the gastrointestinal tract or with most of the other organs. Mice bearing hybridomas secreting a cross-reacting antibody (CB-4) had histopathologic abnormalities in their stomachs. These lesions were absent in the stomachs of mice bearing hybridomas secreting a non-cross-reacting antibody (CB-26). It was concluded that H. pylori infection can stimulate antibodies cross-reacting with gastric autoantigens and that this immunologic mechanism may represent a pathogenic link between H. pylori and gastritis.

The possible involvement of peripheral dopaminergic mechanisms in the action of atrial natriuretic peptides was investigated in 10 subjects by administering 200 micrograms h-ANP 99-126 intravenously for 30 min during treatment with 50 mg carbidopa, a peripheral inhibitor of dopamine synthesis, every 8 h, or during placebo. Atrial natriuretic peptide (ANP) infusion during placebo was associated with a significant increase of diuresis, natriuresis, kaliuresis, urinary noradrenaline, and dopamine excretion. Plasma aldosterone significantly decreased. Blood pressure was slightly reduced. The administration of carbidopa significantly reduced urinary dopamine excretion but did not modify natriuresis, diuresis, indexes of adrenergic and renin-aldosterone system activity, blood pressure, or heart rate, both in basal conditions and in response to ANP infusion. We conclude that the effects of exogenous ANP administration are independent from dopaminergic mechanisms that involve the synthesis of dopamine outside the central nervous system, particularly in the kidney.

The effects of angiotensin II (AII) on proximal tubular reabsorption have been evaluated in 6 healthy volunteers under normal salt and water balance. One-hour clearance periods were performed before, during and after the infusion of pressor doses of AII; in 3 of the 6 subjects, the study was repeated with lower doses of AII. Glomerular filtration rate (GFR) and renal plasma flow (RPF) were determined by the clearances of inulin and PAH, and the fractional excretion of lithium (FELi) was considered as an index of proximal sodium reabsorption. The effects of AII on the fractional excretion of beta 2 microglobulin (FE beta 2M) were also studied. Both doses of AII decreased GFR and RPF and increased the filtration fraction (FF); the modifications of these parameters, as well as the reduction of FELi and the fractional excretion of sodium (FENa) and the increase of plasma aldosterone and of plasma atrial natriuretic peptide (ANP), were more evident with pressor doses of AII, which increased the blood pressure from 129/83 to 142/95 mm Hg (p less than 0.01). AII did not modify FE beta 2M in either study. During AII, FELi decreased less than FENa and both were closely and inversely related to the variations of FF, whilst no relationship was present between FE beta 2M and FF. These results suggest that, in normal humans, the AII-induced rise of FF may be an important factor, even if not the only one, in enhancing the proximal reabsorption of lithium and thus of sodium, whilst it does not affect the absorption of beta 2M.

The involvement of cardiopulmonary and arterial sinoaortic receptors in the control of antidiuretic hormone (ADH) and atrial natriuretic peptide (ANP) release is still controversial in humans. Moreover, it is not clear if this control may be impaired in hypertensive patients with left ventricular hypertrophy (LVH). We studied 17 male subjects, age 18-58 (6 normotensives and 9 mild hypertensives, 5 without and 4 with LVH). Each subject underwent selective loading and unloading of cardiopulmonary receptors, in a randomized sequence, by application of a positive (LBPP) or negative (LENP) pressure to the lower body (steps: +10, +20, +40, -10, -40 mmHg, each for about 30 min), through a plexyglass-constructed tubular apparatus with a rubber adhesion round the patients' waist. Blood samples were taken at the end of every step for measurement of ADH, ANP, PRA, immunoreactive renin, aldosterone, noradrenaline and adrenaline. Cuff arterial pressure was measured every 5 min, while heart rate was evaluated by continuous ECG recording. Hypertensive subjects underwent right atrial pressure measurement by an iv catheter and forearm blood flow evaluation at rest and during the different steps (venous occlusion plethysmography). During LBNP, ADH plasma levels increased progressively, but the increase became statistically significant only at the step of -40 mmHg. ANP increased significantly during LBPP. Taking into account only hypertensive patients, a consistent reduction in the changes of ADH and ANP plasma levels, respectively during LBNP and LBPP in patients with LVH in respect to those without LVH was found.(ABSTRACT TRUNCATED AT 250 WORDS)

In this immunoenzymatic assay for human lutropin (hLH) we used bi-specific antibodies (BiAbs) obtained from the fusion of two hybridomas producing antibodies to beta-D-galactosidase and hLh. The BiAb complexed with the enzyme beta-D-galactosidase was used as tracer in a double-determinant assay. We compared the assay involving the BiAb (Bi-EIA) with an immunoenzymatic assay (EIA) in which the same capture antibody was used but the tracer was an enzyme-conjugated hLH-specific monoclonal antibody produced by the same parental cell line used to produce the BiAb. The coefficient of correlation (r) between the two assays was 0.979 but the Bi-EIA was more sensitive (detection limits: 0.8 int. units/L for the Bi-EIA, 2.0 int. units/L for the EIA) and more specific (less than 0.04% vs less than 1.2% cross-reactions with human choriogonadotropin). Mean intra- and interassay CVs for the Bi-EIA were 2.9% and 5.9%, respectively. Correlation (r) with an immunoradiometric assay (IRMA, Serono kit) was 0.960, with radioimmunoassay (RIA, Biodata kit) 0.909, and with an enzyme-linked immunosorbent assay (ELISA kit, Specialty Medical Industries Inc.) 0.888, (n = 25). Evidently, bi-specific antibodies can be used successfully in immunoenzymatic assays, and with potentially greater sensitivity and specificity than assay with a traditional antibody-enzyme conjugate.

Plasma immunoreactive atrial natriuretic factor (i-ANF) concentrations were measured in ten voluntary blood donors before and after acute blood loss induced after they had been supine for 20 min. Lying down caused i-ANF to increase significantly in all subjects. Bleeding (400 ml in 5 min) caused i-ANF to decrease to values significantly lower than either those observed before haemorrhage or those recorded in the upright position. These results provide evidence that in normal man acute changes in the intravascular volume, although limited within physiological limits, are able to induce opposite changes in i-ANF release.

We describe a radioimmunoassay (RIA) for measurement of atrial natriuretic peptide (ANP), based on one-step incubation and a simplified extraction procedure. The extraction was performed on a "Supelclean LC 18" column, with 2-mL plasma samples. Use of a diiodinated tracer improved the sensitivity of the RIA method. The minimal detectable value was 5 ng/L. Simplification of the extraction procedure and simultaneous incubation of the reagents provide a method more suitable for routine standard assay of ANP than those currently available. Intra- and interassay CVs were 6% (n = 12) and 11% (n = 10), respectively. The mean concentration of ANP in plasma of 32 healthy volunteers was 33 (SEM 4) ng/L. The ANP values for plasma after one-step incubation correlated well with those determined by a commercial RIA kit: r = 0.971, slope = 1.099, intercept = 1.949 ng/L (n = 25).

To verify if exogenous prostaglandin E2 (PGE2) is able to release antidiuretic hormone (ADH) and if endogenous angiotensin II plays a role in this eventual PGE2-induced stimulation of vasopressin, increasing doses of PGE2 were infused in 6 normal volunteers before (PGE2 study) and after the administration of 100 mg of captopril (captopril study). PGE2, even at an infusion rate of 40 and 60 ng/kg/min, did not modify blood pressure when it was infused alone; a significant fall of blood pressure was observed, in contrast, in the captopril study. PGE2 alone doubled the plasma levels of ADH. One hour after the subjects had been pre-treated with captopril, plasma levels of ADH fell by about 38%, then they increased by about 60% during the infusion of PGE2. These results suggest that in normal man endogenous angiotensin II is an important non-osmotic regulator of plasma ADH and that exogenous PGE2 can stimulate maximally the release of ADH only when the renin-angiotensin system is not impaired.