A monoclonal-based, two-site enzyme immunoassay of human insulin.

A procedure is described for the efficient production of insulin-specific monoclonal antibodies, which involves primary and secondary immunization of BALB/c mice in the hind footpads with bovine or porcine insulin and fusion of lymphocytes from popliteal lymph nodes with a P3x63 murine myeloma line. With this protocol, over 200 positive hybrids were obtained from four separate fusions. Dissociation constants of 31 purified monoclonals, cross-reacting with human insulin, were determined by two different methods and ranged between 4 X 10(-10) and 2 X 10(-6) mol/l. 24 monoclonals were biotinylated, paired in all possible combinations and tested by ELISA for their capacity to simultaneously bind to human insulin in a two-site assay. More than 40 monoclonal pairs were found which formed a sandwich with the hormone. The development of a simple and rapid one-step enzyme immunoassay is described, which involves a first monoclonal bound to the wells of a microtiter plate and a second monoclonal conjugated to alkaline phosphatase. With this assay, insulin can be determined in a range between 0.08 and 7.5 ng/ml in 3-4 h.

Synthesis and immunosuppressive activity of novel prodigiosin derivatives.

Prodigiosins (Ps) represent a family of naturally occurring red pigments characterized by a common pyrrolylpyrromethene skeleton. Some members of this family have been shown to possess interesting immunosuppressive properties exerted with a novel mechanism of action, different from that of currently used drugs. In fact, Ps inhibit phosphorylation and activation of JAK-3, a cytoplasmic tyrosine kinase associated with a cell surface receptor component called common gamma-chain, which is exclusive of all IL-2 cytokine family receptors. Blocking common gamma-chain transduction activity results in a potent and specific immunosuppressive activity. With respect to the interesting and unexploited immunomodulating properties of this family of compounds we initiated a medicinal chemistry program aimed at finding novel prodigiosin derivatives with improved immunosuppressive activity and lower toxicity. Utilizing an unprecedented and flexible way of assembling the prodigiosin frame, a number of new derivatives have been prepared and tested leading to the choice of 4-benzyloxy-5-[(5-undecyl-2H-pyrrol-2-ylidene)methyl]-2, 2'-bi-1H-pyrrole (PNU-156804, 16) as a lead immunosuppressant.

Overexpression of interleukin-6 in the central nervous system of transgenic mice increases central but not systemic proinflammatory cytokine production.

Production of inflammatory cytokines, including tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-6 (IL-6), in the brain is increased in various diseases. To investigate the relationships between the effect of overproduction of IL-6 in the brain on central and peripheral production of TNF, IL-1 beta and IL-6 itself, we used transgenic mice (NSE-hIL-6) where neuronal human IL-6 expression under the control of the neuronal specific enolase promoter results in astrocytosis and gliosis. These mice had higher cerebral endogenous IL-6 (12-fold), IL-1 beta (12-fold) and TNF (4-fold) production measured in brain homogenates after intracerebroventricular (i.c.v.) injection of 2.5 micrograms LPS, lipopolysaccharide (LPS) than wild-type mice (no TNF or IL-1 were detectable in saline-injected NSE or control mice). Cerebral cytokines production was also increased in NSE-hIL-6 mice treated i.p. with LPS doses that do not normally induce cytokines in the brain. The induction of peripheral (serum or spleen) TNF, IL-1 beta or IL-6 was the same in all these experiments in NSE-hIL-6 and wild-type mice. Furthermore, using microglial cell clone pretreated in vitro with IL-6, we noted an increase in LPS-induced TNF and IL-6 production and proliferation of pretreated cells than control. This study indicates that overproduction of IL-6 in the central nervous system (CNS) may ultimately result in increased central production of inflammatory cytokines, probably due to increased proliferation and activation of the cells which produce cytokine in the CNS.

Effects of methyl palmitate on cytokine release, liver injury and survival in mice with sepsis.

The effects of methyl palmitate (MP), a known inhibitor of Kupffer cells, were studied in a model of polymicrobial sepsis induced in CD-1 mice by cecal ligation and puncture (CLP). The inhibition of Kupffer cells by pretreatment with MP was shown by the reduced phagocytosis, the production of tumor necrosis factor (TNF) and interleukin-6 (IL-6) after lipopolysaccharide (LPS) challenge. The reduced activation of Kupffer cells resulted in lower levels of inflammatory products after CLP. TNF and IL-6 were significantly reduced in serum 2 h and 24 h respectively after CLP, interleukin-1 beta (IL-1 beta) was reduced in liver 4 h after CLP, nitric oxide (NO) and serum amyloid A (SAA) were significantly reduced 8 and 24 h respectively after CLP. Liver toxicity was significantly reduced in MP-treated mice and survival was significantly prolonged at all intervals, reaching 45% after six to ten days compared with 3% in control mice. These findings suggest that Kupffer cells play an important role in liver damage and survival in sepsis.

Protective effect of chlorpromazine on TNF-mediated hapten-induced irritant reaction.

Picryl chloride-induced irritant reaction (IR) was shown to be mediated by tumor necrosis factor (TNF). Anti-TNF monoclonal antibodies, but not interleukin 1 receptor antagonist (IL-1 Ra), had a protective effect. Chlorpromazine (CPZ), an inhibitor of TNF synthesis, protected against IR and inhibited the IR-associated TNF induction in ear homogenates. Investigation of the role of polymorphonuclear leukocyte (PMN) in neutropenic mice showed that neutropenia did not prevent the development of the IR.

Ultrasound-guided reduction of an incarcerated Spigelian hernia.

Spigelian hernia is a rare abdominal hernia. We report a case in which its diagnosis proved ultrasonography to be an effective tool, not only to diagnose an incarcerated Spigelian hernia but, moreover, to reduce it by echo-probe palpation. Ultrasound (US) is an aid for therapy of various diseases. In our experience, US-guidance prevented possible damage related to forced and wrongly applied compression during the hernia reduction, and allowed us to perform surgical repair on an elective basis. In conclusion, if an incarcerated Spigelian hernia is suspected, a US examination should be done on an emergency basis to confirm the diagnosis and to attempt US-guided reduction.

The usefulness of ultrasonography in the diagnosis of the Spigelian hernia.

Normally acquired, the Spigelian hernia is rare. Due to low incidence and frequent atypical presentations diagnosis is often difficult. Controversies exist about the diagnostic approach. We report our experience in two cases in which ultrasonography permitted a correct diagnosis of surgically confirmed Spigelian hernias. One of the two cases proved to be a carrier of bilateral Spigelian hernia at ultrasonography and at surgery. In our opinion ultrasonography represents the gold standard in the diagnosis of Spigelian hernias and we recommend its use as the first step in all cases of abdominal wall mass.

Ultrasonographic detection of portal branches and hepatic vein gas associated with mesenteric artery occlusion: a new finding correlated with patient prognosis?

The diagnosis of bowel infarction is still a challenge. In some cases, portal venous gas is an associated feature and in these patients, the prognosis is very poor. We report on our experience with two consecutive cases in which ultrasonography showed gas in the portal venous branches, and also in the hepatic veins in one of them. At laparotomy, advanced bowel necrosis was found, and both patients died within 24 hours. Other cases of portal venous gas associated with bowel infarction have been reported, but this is the first report of gas also being found in the hepatic veins. There may be a relationship between the amount of gas in the intrahepatic veins and the stage of bowel ischemia. Confirmation of this might improve the selection of patients and eliminate unnecessary procedures.

[Kinetics of hepatic enzymes in anorexia nervosa]. / Cinetica degli enzimi epatici nell'anoressia nervosa.

Nervous anorexia in advanced stage causes a generalized deterioration of organs' and apparatuses' functions. In particular, well-known are disorders of both CNS and PNS, changes of the haematic crasis and worsening of the metabolic-endocrine axis. Less well-known, however, is the compromission of function. Analysis of the data obtained in this trial points out a statistically significant correlation between the loss of weight and the variation of level of some liver enzymes (SGPT, SGOT, LDH). These indexes of hepatic necrosis in our opinion, can therefore, be regarded as sound markers not only for a more careful evaluation of the clinical evolution of the anorexic patient, but also for a better monitoring of the effectiveness of diet therapy.

Development and applications of a radioimmunoassay (RIA) for the in vitro and in vivo quantification of murine IL-1 beta.

A specific, precise, and accurate radioimmunoassay (RIA) for murine interleukin-1 beta (mIL-1 beta), with a sensitivity of 250 pg/ml, has been established. Although mIL-1 beta shares structural homology and multiple biological properties with mIL-1 alpha, this RIA did not detect mIL-1 alpha or other murine cytokines such as TNF and IL-6. Recombinant mIL-1 beta, freshly added in different concentrations to murine plasma, was recovered from 88 to 104%, and intra- and interassay coefficients of variation never exceeded the 10% value. Parallel analysis showed that murine plasma and cell or organ supernatants did not affect the test. This characteristic allowed mIL-1 beta analysis directly in the nonmanipulated biological specimens. In murine macrophage supernatants collected after 24 h of in vitro stimulation with LPS, nanogram fractions of IL-1 beta were detected by RIA. These values corresponded to approximately 50% of the total IL-1 detected by the LAF bioassay. In spleen, liver, and lung, IL-1 beta appeared at significant levels (110 ng/g of lung, 638 ng/g of spleen, and 78 ng/g of liver) as early as 1 h after LPS administration, reached the plateau 1-2 h later, and then slowly but progressively decreased. In plasma and brain, nanogram fractions of IL-1 beta were detectable by 4 h post-LPS. Thereafter, IL-1 beta levels progressively increased to reach the value of 44 ng/g in the brain and 2 ng/ml in plasma 8 h after LPS treatment.

Immunodepressive activity of FCE 23762 on humoral and cell-mediated immune responses in normal mice: comparison with doxorubicin.

FCE 23762 (3' desamino-3'[2(s)methoxyl-4-morpholinyl]doxorubicin) is a new doxorubicin (Dx) derivative that has been selected for clinical testing for its favourable antitumor characteristics, which include efficacy on Dx-resistant tumors. Immunosuppression is an undesirable side-effect of anti-cancer chemotherapy and the therapeutic efficacy of Dx is probably also related to its low immunotoxicity. It was, thus, of interest to compare the effects of FCE 23762 and its parental drug on the immune responses. Both compounds were injected i.v. into healthy mice at equitoxic doses and according to different treatment schedules. Single doses of FCE 23762 and Dx, given concomitant or after the antigen, suppressed at the same degree and dose-dependently the primary anti-SRBC antibody response. Following a multiple treatment schedule after the antigen, FCE 23762 was less suppressive than Dx on both primary and secondary antibody production. Differently from Dx, that was completely inactive, FCE 23762 moderately inhibited DTH reaction to SRBC, only at the highest single dose tested or for repeated administrations given simultaneously or after priming. Both drugs were totally ineffective in delaying skin allograft rejection. Since spleen cellularity and ex vivo lymphocyte proliferation to Con A and LPS were similarly impaired by the two drugs, the differentiated immunodepressive activity of FCE 23762 and Dx cannot be merely associated to their cytotoxic and antiproliferative action. The hypothesis of a selective effect on different regulatory cell subsets and/or immune mechanisms is discussed.

Inhibition of interleukin-2/p55 receptor subunit interaction by complementary peptides.

Complementary peptides to interleukin-2 (IL-2) sequences important for receptor binding were tested for their ability to mimic natural receptors and act as inhibitors of the IL-2/p55 receptor subunit interaction. Peptides hydropathically complementary to IL-2 sequences 15-27 and 40-54 were synthesized in a linear and in a multimeric form and then characterized first by solid-phase binding assays for their ability to interact with IL-2. Binding between the multimeric complementary peptides and biotinylated IL-2 was specific, saturable, and inhibited by linear as well as multimeric complementary peptides. Saturable interactions, characterized by dissociation constants in the micromolar range, occurred also between IL-2 immobilized on microtiter plates and biotinylated linear and multimeric complementary peptides. Peptides corresponding to the IL-2 target sequences were able to interfere with this interaction, as well as full-length IL-2. Peptide recognition was sequence dependent, since scrambling of complementary peptide sequences or IL-2 target peptide sequences abolished binding. Multimeric complementary peptides after immobilization on solid supports proved useful also for affinity purifications of recombinant IL-2 or IL-2 fragments corresponding to the target sites, directly from crude mixtures, in high yield and with high recovery. Complementary peptides to IL-2 sequence 15-27, but not to IL-2 sequence 40-54, in the linear or in the multimeric form, even if with different potency, interfered with the IL-2/p55 receptor subunit interaction in vitro, thus suggesting a possible role of this IL-2 site in receptor recognition.

Autocrine interleukin-1 beta regulates both proliferation and apoptosis in EL4-6.1 thymoma cells.

We demonstrate here that EL4-6.1 cells, a mouse thymoma that expresses high levels of membrane interleukin (IL)-1 receptors, produce IL-1 beta as an autocrine regulatory factor. Endogenous IL-1 beta sustains both proliferation and apoptosis: during the exponential phase, it mainly promotes proliferation, while during the plateau phase of cell growth, it induces death by apoptosis. Additionally, we show that exogenous IL-1 beta added to EL4-6.1 cells in lag phase induces apoptosis in a portion of the cells and proliferation in the remaining cells. Therefore, IL-1 beta can exert two completely opposite effects on a single cell type, depending on the state of the target cell.

Modulation of cytokine expression in mouse dendritic cell clones.

Dendritic cells (DC) play an essential role in the induction of primary immune responses; however, very little information is available on cytokine production by DC. Here we determined the cytokine gene expression profile of two immortalized DC clones, CB1 and D2SC/1, both generated from mouse spleen but differing in their activation requirements. Among the cytokines tested, only transforming growth factor-beta 1 was transcribed constitutively, but its production was detected only in D2SC/1 cells after treatment with granulocyte/macrophage colony-stimulating factor (GM-CSF). GM-CSF also promoted transcription and synthesis of interleukin (IL)-1 beta in CB1 cells that need pretreatment with GM-CSF to present major histocompatibility complex class II-restricted antigens efficiently in vitro. Lipopolysaccharide (LPS) up-regulated gene expression and induced release of tumor necrosis factor-alpha in both DC clones. In addition, LPS induced transcription of IL-1 alpha and both gene expression and synthesis of IL-1 beta in D2SC/1 cells. Interferon-gamma was ineffective in inducing cytokine gene expression, although it augmented the antigen-presentation capacity of DC, IL-4, IL-10 and IL-12 mRNA were not induced by any of the tested stimuli. The results suggest that DC have a limited cytokine gene expression pattern compared to macrophages and are heterogenous in some functional properties.

Role of xanthine oxidase and reactive oxygen intermediates in LPS- and TNF-induced pulmonary edema.

We studied the role of reactive oxygen intermediates (ROI) in lipopolysaccharide (LPS)-induced pulmonary edema. LPS treatment (600 micrograms/mouse, IP) was associated with a marked induction of the superoxide-generating enzyme xanthine oxidase (XO) in serum and lung. Pretreatment with the antioxidant N-acetylcysteine (NAC)--1 gm/kg orally, 45 minutes before LPS--or with the XO inhibitor allopurinol (AP)--50 mg/kg orally at -1 hour and +3 hours--was protective. On the other hand nonsteroidal antiinflammatory drugs (ibuprofen, indomethacin, and nordihydroguaiaretic acid) were ineffective. These data suggested that XO might be involved in the induction of pulmonary damage by LPS. However, treatment with the interferon inducer polyriboinosylic-polyribocytidylic acid, although inducing XO to the same extent as LPS, did not cause any pulmonary edema, indicating that XO is not sufficient for this toxicity of LPS. To define the possible role of cytokines, we studied the effect of direct administration of LPS (600 micrograms/mouse, IP), tumor necrosis factor (TNF, 2.5 or 50 micrograms/mouse, IV), interleukin-1 (IL-1 beta, 2.5 micrograms/mouse, IV), interferon-gamma (IFN-gamma, 2.5 micrograms/mouse, IV), or their combination at 2.5 micrograms each. In addition to LPS, only TNF at the highest dose induced pulmonary edema 24 hours later. LPS-induced pulmonary edema was partially inhibited by anti-IFN-gamma antibodies but not by anti-TNF antibodies, anti-IL-1 beta antibodies, or IL-1 receptor antagonist (IL-1Ra).

Kupffer cell depletion partially prevents hepatic heme oxygenase 1 messenger RNA accumulation in systemic inflammation in mice: role of interleukin 1beta.

The heme oxygenase 1 (HO-1) gene is rapidly activated in the liver after lipopolysaccharide (LPS) treatment. Ninety minutes after LPS treatment (0.1 mg/kg, intraperitoneally) hepatic HO-1 messenger RNA (mRNA) of mice was 40 times the control value. To investigate the hepatic cellular source of the increased HO-1 transcript, we treated mice with LPS and galactosamine (700 mg/kg, intraperitoneally), a selective transcriptional inhibitor of hepatocytes. Galactosamine prevented the LPS-mediated increase of HO-1 mRNA in the liver, indicating that hepatocytes are the main cell type in which HO-1 mRNA accumulates after LPS treatment. We then tested in vitro and in vivo the hypothesis that LPS-mediated hepatic accumulation of HO-1 mRNA is caused by intercellular communication between Kupffer cells and hepatocytes. Isolated rat hepatocytes showed an increase in HO-1 mRNA compared with controls after 90 minutes of exposure to a LPS stimulated Kupffer cell-conditioned medium. This suggests that soluble mediators from Kupffer cells were responsible for this effect. To study the role of Kupffer cells in vivo, we treated mice with Kupffer cell-inactivating or -depleting agents and LPS. Gadolinium chloride and liposome-encapsulated dichloromethylene diphosphonate lowered LPS-mediated HO-1 mRNA accumulation (by about 50%); in these groups hepatic levels of interleukin (IL)-1beta were decreased, by more than 75%. Methylpalmitate hardly affected hepatic HO-1 mRNA accumulation or IL-1beta content after LPS treatment. There was no relationship between HO-1 mRNA and serum TNF or IL-6 levels. These results suggest that LPS-mediated hepatic HO-1 mRNA accumulation is a hepatocyte response partly caused by soluble mediators, particularly IL-1beta, released from Kupffer cells.

Differential regulation of cytokine production in lipopolysaccharide tolerance in mice.

We investigated the pattern of down-regulation of cytokine production in endotoxin (lipopolysaccharide [LPS]) tolerance. A 4-day treatment with LPS (35 micrograms per mouse) was followed by a challenge on day 6 with one more injection of LPS. Circulating tumor necrosis factor (TNF) and interleukin-6 (IL-6) could not be induced (> 99% inhibition) by LPS in LPS-tolerant mice; colony-stimulating factor (CSF) was also down-regulated by more than 95%, whereas interferon (IFN) and IL-1 syntheses were only partially inhibited. To study the mechanism of cytokine down-regulation in tolerance, we attempted to reverse the tolerant state by pretreatment with phorbol 12-myristate 13-acetate (PMA) (4 micrograms per mouse) 10 min before the LPS challenge. PMA completely restored IL-6 production and partially that of CSF. PMA had no effect on IFN production and inhibited the induction of IL-1. TNF production was also not restored by PMA. To investigate the role of endogenously produced cytokines in the development of LPS tolerance, we administered IL-6, TNF, or IL-1 alpha, using the same treatment schedule as that for LPS. Whereas IL-6 had no effect, IL-1 alpha or TNF induced partial tolerance to LPS in terms of inhibition of LPS-stimulated TNF and IL-6 production. However, a full LPS-tolerant state could not be induced by administration of recombinant cytokines, suggesting the existence of additional mechanisms, such as a loss of LPS receptors or changes in release of soluble binding proteins.

Mechanisms of interleukin-2-induced hydrothoraxy in mice: protective effect of endotoxin tolerance and dexamethasone and possible role of reactive oxygen intermediates.

Interleukin (IL)-2 is known to induce vascular leak syndrome (VLS), which was suggested to be mediated by immune system-derived cytokines, including tumor necrosis factor (TNF). To characterize the role of TNF in IL-2 toxicity in C3H/HeN mice, we used two approaches to downregulate TNF production in vivo: treatment with dexamethasone (DEX) and induction of endotoxin (lipopolysaccharide) (LPS) tolerance by a 4-day pretreatment with LPS (35 micrograms/mouse/day). Mice were then treated with IL-2 for 5 days (1.8 x 10(5) IU/mouse, twice daily). Both DEX and LPS tolerance blocked development of hydrothorax in IL-2-treated mice and inhibited TNF induction. DEX and LPS tolerance also ameliorated IL-2 toxicity in terms of decrease in food intake and inhibited the increase of the acute-phase protein, serum amyloid A (SAA). The IL-2 activation of splenic natural killer (NK) cell activity was also diminished by DEX and, to a lesser extent, by LPS-tolerance. Treatment with IL-2 also caused induction of the superoxide-generating enzyme xanthine oxidase (XO) in tissues and serum and induced bacterial translocation in the mesenteric lymph nodes (MLN). These data suggest that multiple mechanisms, including NK cell activity, cytokines, and reactive oxygen intermediates, might be important in the vascular toxicity of IL-2.

Interleukin-10 inhibits lipopolysaccharide-induced tumor necrosis factor and interleukin-1 beta production in the brain without affecting the activation of the hypothalamus-pituitary-adrenal axis.

Interleukin (IL) 10 inhibits endotoxin (lipopolysaccharide; LPS) induced tumor necrosis factor (TNF) production in vivo and in vitro. In turn, IL-10 is induced by LPS and acts as a negative feedback to limit TNF production. We investigated the effects of IL-10 on brain TNF and IL-1 beta production induced by a central LPS administration in mice. Because central LPS also induces peripheral TNF, we also measured the serum TNF levels. A single intracerebroventricular injection of murine recombinant IL-10 (75 ng/mouse) simultaneously with LPS (2.5 micrograms/mouse) almost completely inhibited brain TNF production. The brain IL-1 beta production was also inhibited, as was the serum concentration of the acute-phase protein serum amyloid A. On the other hand, intracerebroventricular administration of an anti-IL-10 monoclonal antibody (JES5-2A5; 60 micrograms/mouse) potentiated brain TNF and IL-1 beta production. Identical results were obtained when the serum TNF levels were measured. IL-10 did not affect the LPS-induced increase of serum corticosterone, the main endogenous inhibitor of TNF production, or the induction of IL-6. These results indicate that LPS-induced IL-10 can act as an important endogenous inhibitor of brain TNF production and suggest an anti-inflammatory role for IL-10 in the central nervous system.