HIV p17 reverses the anti-inflammatory activity of IL-4 on IL-15 stimulated monocytes and modulates their ability to secrete MIP-1 alpha.

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.

Immunotherapy with bovine aortic endothelial cells in subcutaneous and intracerebral glioma models in rats: effects on survival time, tumor growth, and tumor neovascularization.

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.

Effects of the selective estrogen receptor modulator LY117018 on growth hormone secretion: In vitro studies.

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).

Differential inhibition of growth hormone secretion by analogs selective for somatostatin receptor subtypes 2 and 5 in human growth-hormone-secreting adenoma cells in vitro.

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.

Proteolysis of beta-casein as a marker of Grana Padano cheese ripening.

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.

Proteolysis of alphas-casein as a marker of Grana Padano cheese ripening.

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.

Endocrine predictors of acute hemodynamic effects of growth hormone in congestive heart failure.

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.

HIV p17 enhances lymphocyte proliferation and HIV-1 replication after binding to a human serum factor.

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.

Glutamate decarboxylase autoimmunity and growth hormone secretion in type I diabetes mellitus.

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.

Inhibitory effects of galanin on growth hormone (GH) release in cultured GH-secreting adenoma cells: comparative study with octreotide, GH-releasing hormone, and thyrotropin-releasing hormone.

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.

Use of immunoblotting and monoclonal antibodies to evaluate the residual antigenic activity of milk protein hydrolysed formulae.

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.

Aspects of molecular interaction between HIV p17 and human gamma interferon.

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.

Comparison of the effects of growth hormone-releasing hormone and hexarelin, a novel growth hormone-releasing peptide-6 analog, on growth hormone secretion in humans with or without glucocorticoid excess.

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)

Evaluation by SDS-PAGE and immunoblotting of residual antigenicity in hydrolysed protein formulas.

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.

Tumor necrosis factor (TNF) alpha quantification by ELISA and bioassay: effects of TNF alpha-soluble TNF receptor (p55) complex dissociation during assay incubations.

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.

Characterization of a novel transcription complex required for glucocorticoid regulation of the rat alpha-1-acid glycoprotein gene.

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.

Kinetic analysis of TNF-alpha oligomer-monomer transition by surface plasmon resonance and immunochemical methods.

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.

Real-time kinetic analysis applied to the production of bispecific monoclonal antibodies for radioimmunodetection of cancer.

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.

New heteromyeloma cell lines for the production of human 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.