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Immunoinhibition assays are hypothesized to work by antibodies blocking substrate access to enzyme active sites. To test this hypothesis, the inhibition of amylase isoenzymes by monoclonal and polyclonal antisera was assessed using substrates of varying sizes: chromogenic sustrates 3, 5, or 7 glucose units in length, novel synthetic macromolecular substrates, and starch. The synthetic macromolecular substrates consisted of small oligosaccharide substrates linked to an inert polymer that conferred a large size to substrate molecules as determined by gel filtration chromatography. When substrate size increased, amylase activity could be inhibited equivalently by antibody concentrations that are 10-fold lower. Progressively less polyclonal serum was required to inhibit amylase activity as substrate length increased from 3 to 5 to 7 glucose units and as size was increased by linkage to a polymer. Different effects of substrate size were observed with two monoclonal antibodies. One monoclonal antibody blocked amylase activity independent of substrate size, while another monoclonal antibody had little inhibitory effect except using starch as substrate. We conclude that use of larger substrates can expand the repertoire of inhibitory epitopes on enzymes and convert a noninhibitory antibody into an inhibitory one.

Histone deacetylase inhibitors (HDACIs) have been used to focus on the effects of inducing gene expression through the acetylation of histones which results in chromatin remodeling. The study explored whether HDACIs could induce the expression of costimulatory/adhesion molecules on acute myeloid leukemia (AML) cells, thereby effectively inducing tumor immunity. The expression of CD80, CD86, human leukocyte antigen (HLA)-DR, HLA-ABC, and intracellular adhesion molecule-1 (ICAM-1) was tested in human AML cell lines after the addition of HDACI, sodium butyrate (SB). Generally, increased expression of CD86 was observed by SB treatment in a majority of cell lines, and ICAM-1 was expressed in fewer cell lines. Essentially the same results were obtained using other HDACIs such as FR901228, trichostatin A, and trapoxin A. Quantitation of transcripts of CD86 accompanied with RNA synthesis inhibition assay and nuclear run-on assay revealed that SB up-regulates the CD86 expression transcriptionally. Furthermore, chromatin immunoprecipitation experiments showed that HDACI treatment caused remarkable acetylation on histone H3 and H4 at CD86 promoter chromatin in vivo. In 30 clinical AML samples, CD86 expression was significantly increased (P <.001) by SB treatment, and the expression of HLA-DR and ICAM-1 was moderately increased (P <.05) by SB treatment. Finally, the allogeneic mixed leukocyte reaction (allo-MLR) against HL60 cells pretreated with SB was enhanced 4-fold compared with allo-MLR obtained with non-treated HL60 cells. These results suggest that the immunotherapeutic use of HDACIs may become a novel tool for treatment of AML. (Blood. 2000;96:3847-3856)

Neurturin (NTN) a structural and functional relative of glial cell line-derived neurotrophic factor, was originally identified based on its ability to support the survival of sympathetic neurons in culture. Similar to glial cell line-derived neurotrophic factor (GDNF), Neurturin has been shown to bind to a high affinity glycosylphosphatidylinositol (GPI)-linked receptor (GFRalpha2) and induce phosphorylation of the tyrosine kinase receptor Ret, resulting in the activation of the mitogen activated protein kinase (MAPK) signalling pathway. A panel of six novel murine monoclonal antibodies (MAbs) specific to human Neurturin has been developed and characterized. Four of the MAbs tested inhibit, to varying degrees, binding of NTN to the GPI-linked GFRalpha2 receptor. Three MAbs cross-react with the murine homolog. These antibodies have been shown to be useful reagents for Western blotting, immunohistochemistry, and also for the development of a sensitive, quantitative enzyme-linked immunosorbent assay (ELISA) for human NTN. Novel, specific MAbs with varying epitope specificities and blocking activity will be valuable tools for both the in vitro and in vivo characterization of NTN and its relationship to the GFRalpha2 and Ret receptors.

Various antiphospholipid and/or antiprotein antibodies have been suspected to be associated with recurrent early foetal loss in absence of any habitual aetiology. We conducted a hospital-based case control study on women with no antecedent of thromboembolic or autoimmune disease. We studied 3 groups of 518 women: patients with unexplained primary recurrent early foetal loss, patients with explained episodes and mothers with no previous obstetrical accident. Matching the 3 groups was carried out on the basis of age, number or pregnancies and time elapsed since the end of the last pregnancy. Significant biological markers were then prospectively tested. The various antibodies were shown to be dependent on parity and on the presence of previous foetal loss: cut-off values were thus calculated using data obtained from the group of explained accidents, and adjusted for parity. Only anti-phosphatidylethanolamine IgM [odds ratio: 6.0, 95% confidence interval (2.3-15.7), p = 0.0003], anti-beta2-glycoprotein I IgG [4.4, (1.6-11.7), p = 0.0035] anti-annexin V IgG antibodies [3.2 (1.2-8.1), p = 0.015] and lupus anticoagulant [3.0, (1.3-6.8), p = 0.009], were found to be independent retrospective risk factors for unexplained early foetal loss. These four markers were subsequently found to be, during the following pregnancy, associated with a significant risk of foetal loss despite a low-dose aspirin treatment. In non-thrombotic, non-auto-immune women with unexplained primary recurrent early foetal loss, subgroups of patients with positive anti-phosphatidylethanolamine IgM antibodies, or positive anti-beta2-glycoprotein-I IgG antibodies, or positive anti-annexin V IgG antibodies or lupus anticoagulant must be particularised. This should allow therapeutic trials to be carried in well-defined patients.

Previous studies documenting the existence of cross-reactivity between the lipoated (but not unlipoated) forms of the inner lipoyl domain (E2L2) of PDC-E2 [the major autoantigen in Primary biliary cirrhosis (PBC)] and trifluoroacetylated (TFA) proteins, led us to hypothesize that PBC may be due to an initial insult with an environmental agent that cross-reacts with TFA. Therefore, we performed a comparative study of the reactivity of rabbit anti-TFA antibody and anti-lipoic acid (LA) antibody against the mitochondrial autoantigens of human PBC and various TFA and LA conjugated proteins. Whereas both anti-TFA and anti-LA reacted with PDC-E2, the wild-type lipoated form of E2L2, OGDC-E2, E3-BP and LA-KLH, neither reacted with BCOADC-E2 or the non-lipoated form of E2L2. Of interest was that while anti-TFA reacted with PDC-E2, TFA-RSA and LA-KLH, it failed to inhibit PDC-E2 enzyme function. In contrast, anti-LA demonstrated cytoplasmic and mitochondrial staining, and inhibited PDC enzyme activity. Hence, although considerable cross reactivity exists between anti-TFA and anti-LA, the molecular nature of the interaction is clearly different. One of 14 PBC sera reacted weakly with TFA-albumin, whereas four of 14 PBC sera reacted with LA-KLH. Immunohistochemically, both anti-TFA and anti-LA antibodies reacted focally with periportal hepatocytes and bile ducts in both PBC and controls. However, anti-LA produced much stronger focalized staining of the bile ducts of diseased liver. This study suggests that while anti-TFA antibody recognizes lipoic acid-linked enzymes and proteins, the epitope recognized differs from that of anti-LA antibody and PBC autoantibodies. It is unlikely that a response to TFA is the triggering event in PBC. Anti-LA antibodies share a higher degree of similarity to PBC sera providing suggestive evidence that anti-LA antibodies or anti-LA like antibodies (mimotopes) may help define the initiator of the autoimmune response., (Copyright 2000 Academic Press.)

Previous studies focused on indels in the complement C345 protein family identified a number of potential protein-protein interaction sites in components C3 and C5. Here, one of these sites in C5, near the alpha-chain C terminus, was examined by alanine-scanning mutagenesis at 16 of the 18 non-alanine residues in the sequence KEALQIKYNFSF RYIYPLD. Alanine substitutions affected activities in the highly variable manner characteristic of binding sites. Substitutions at the lysine or either phenylalanine residue in the central KYNFSF sequence had the greatest effects, yielding mutants with <20% of the normal activity. These three mutants were also resistant to the classical pathway (CP) C5 convertase, with sensitivities roughly proportional to their hemolytic activities, but had normal susceptibilities to the cobra venom factor (CVF)-dependent convertase. Synthetic peptide MGKEALQIKYNFS-NH2 was found similarly to inhibit CP but not CVF convertase activation, and the effects of alanine substitutions in this peptide largely reflected those of the equivalent mutations in C5. These results indicate that residues KYNFSF form a novel, distal binding site for the CP, but not CVF convertase. This site lies approximately 880 residues downstream of the convertase cleavage site within a module that has been independently named C345C and NTR; this module is found in diverse proteins including netrins and tissue inhibitors of metalloproteinases.

Adult respiratory distress syndrome (ARDS) is characterized by acute lung injury with a high mortality rate and yet its mechanism is poorly understood. Sepsis syndrome and acid aspiration are the most frequent causes of ARDS, leading to increased lung permeability, enhanced polymorphonuclear neutrophil (PMN) sequestration and respiratory failure. Using a murine model of acute lung injury induced by septic syndrome or acid aspiration, we investigated the role of cytosolic phospholipase A2 (cPLA2) in ARDS. We found that disruption of the gene encoding cPLA2 significantly reduced pulmonary edema, PMN sequestration and deterioration of gas exchange caused by lipopolysaccharide and zymosan administration. Acute lung injury induced by acid aspiration was similarly reduced in mice with a disrupted cpla2 gene. Our observations suggest that cPLA2 is a mediator of acute lung injury induced by sepsis syndrome or acid aspiration. Thus, the inhibition of cPLA2-initiated pathways may provide a therapeutic approach to acute lung injury, for which no pharmaceutical agents are currently effective.

During Gram-negative sepsis bacterial LPS induces endothelial cell contraction, actin reorganization, and loss of endothelial integrity by an unknown signal mechanism. In this study, we provide evidence that LPS-stimulation of endothelial cells (HUVEC) decreases myosin light chain (MLC) phosphatase, resulting in an increase in MLC phosphorylation followed by cell contraction. All of these LPS effects could be blocked by the Rho-GTPase inhibitor C3 transferase from Clostridium botulinum or the Rho kinase inhibitor Y-27632. These data suggest that LPS induces MLC phosphorylation via Rho/Rho kinase-mediated inhibition of MLC phosphatase in HUVEC. Furthermore, we observed that cAMP-elevating drugs, known to exert a vasoprotective function, mimicked the effects of C3 transferase and Y-27632, i.e., inhibited LPS-induced MLC phosphatase inactivation and MLC phosphorylation. cAMP elevation did not inhibit myosin phosphorylation induced by constitutively active V14Rho or the MLC phosphatase inhibitor calyculin and did not induce phosphorylation of RhoA in HUVEC, indicating inhibition of an upstream regulator of Rho/Rho kinase. Taken together, Rho/Rho kinase appears to be a central target for inflammatory mediators causing endothelial cell contraction such as bacterial toxins, but also for vasoprotective molecules elevating intracellular cAMP.

Our previous reports indicated that C57BL/6 mice infected with a lethal variant of Plasmodium yoelii 17X (P. yoelii 17XL) produced high levels of interleukin 10 (IL-10) and interferon-gamma (IFN-gamma) while mice infected with the nonlethal variant of the parasite did not produce detectable levels of IL-10. In the present study, the involvement of IL-10 and IFN-gamma in exacerbation or regulation of blood-stage malaria was investigated by using the lethal variant of P. yoelii 17XL and monoclonal antibodies (mAb) against the cytokines. C57BL/6 mice were injected intraperitoneally with a neutralizing anti-IL-10 mAb or anti-IFN-gamma mAb after inoculation with P. yoelii 17XL. Treatment of mice with anti-IL-10 mAb resulted in substantial prolongation of survival and 60% of treated mice survived while 100% of control mice died by day 11. On the contrary, treatment of mice with anti-IFN-gamma mAb exacerbated infection and all mice died after an earlier period than those treated with normal rat Ig. No differences in parasitemias were found between treated and untreated mice. To elucidate the involvement of nitric oxide in the host protection or exacerbation, mice were treated with aminoguanidine, an inhibitor of nitric oxide synthetase, after inoculation of P. yoelii 17XL. Neither mortality nor parasitemia was influenced by the treatment. These results indicate that an IFN-gamma response is associated with protective immunity in mice infected with P. yoelii 17XL, while an IL-10 response is associated with disease exacerbation during the infection.

Using anti-microcystin-LR monoclonal antibodies, an immunoblotting procedure was developed to monitor the formation of microcystin-protein phosphatase adducts in vitro and in vivo. The detection limits for the covalent binding of MCYST-LR with the recombinant protein phosphatase 1 (PP1) and rabbit liver cytosol proteins were found to be 0.1 ng and 0.3 ng per assay, respectively. MCYST-PP1 adducts were detected 30 s after the addition of MCYST-LR into the reaction mixture. Reduction of the methyldehydroalanine (Mdha) residue of MCYST-LR with ethanethiol totally abolished the covalent binding of the toxin to PP1, but retained its inhibitory toxicity on PP1. Immunoblotting analyses and enzyme-linked immunosorbant assay showed that between 5 min to 16 h after i.p. injection of single dose (35 microg/kg) of MCYST-LR into mice, approximately 0-27% of the injected toxin was found covalently bound while 0.2-9.2% existed as free form in liver cytosol.

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by autoantibodies reactive with the pyruvate dehydrogenase complex. A conformational epitope has been mapped to aa 91-227 within the inner lipoyl domain of the E2 subunit (pyruvate dehydrogenase complex E2 (PDC-E2)). We have used phage display to further localize this epitope. A random heptapeptide library was screened using IgG from two patients with PBC, with negative selection using pooled normal IgG. Phage that contained peptide inserts (phagotopes) selected using PBC sera differed from those selected using IgG from patients with RA or polychondritis. Two motifs occurred only among the PBC-selected phagotopes; these were MH (13 sequences, 16 phagotopes) and FV (FVEHTRW, FVEIYSP, FVLPWRI). The phagotopes selected were tested for reactivity with anti-PDC-E2 affinity purified from four patients with PBC. Phagotopes that contained 1 of 15 different peptide sequences were reactive with one or more of these four anti-PDC-E2 preparations, whereas phagotopes that contained 1of the remaining 28 sequences were negative. The peptides (FVLPWRI, MHLNTPP, MHLTQSP) encoded by three phagotopes that were strongly reactive with all four preparations of anti-PDC-E2 were synthesized. Each of the selected peptides, but not an irrelevant peptide, inhibited the reactivity by ELISA of PBC serum with recombinant PDC-E2 and reduced the inhibition of the enzyme activity of PDC by a PBC serum. The peptide sequences, along with the known NMR structure of the inner lipoyl domain of PDC-E2, allow the prediction of nonsequential residues 131HM132 and 178FEV180 that contribute to a conformational epitope.

A solid phase method for direct radioimmunoassay in plasma of the new renin inhibitor CGP 60536 has been developed which does not require the extraction of the parent drug with organic solvents. The assay showed a good reproducibility down to plasma concentrations of 0.15 ng/ml (LOQ) with intra- and inter-assay coefficients of variation < or = 20%. The procedure, which requires only small volumes of plasma (25 microl), is simple to use and well suited for routine analysis. The method allows the investigation of the pharmacokinetics of CGP 60536 in animals and man given low oral doses of the drug.

Inhibition of cyclin dependent kinases (cdk) by proteins of two families of cdk inhibitors (CKIs) represents one of the key modes of cell-cycle control. Although not fully understood at present, the functions of the individual members of the Cip/Kip and INK4 families of CKIs have been implicated in fundamental biological processes as diverse as cellular proliferation, responses to genotoxic stress, regulation of cellular differentiation, and senescence. In addition, the seven currently known CKIs qualify as either established or candidate tumor suppressors whose loss or inactivation contribute to molecular pathogenesis of a wide range of tumor types. In this study, we report the isolation and characterization of a panel of 10 mouse monoclonal antibodies (MAbs) that specifically recognize p21WAF1/CIP1 (p21) or the individual members of the INK4 family of CKIs: p15INK4b (p15), p16INK4a (p16), p18INK4c (p18), or p19INK4d (p19). These antibodies are proving to be invaluable molecular probes for analyses of protein abundance, subcellular localization, interacting cellular proteins, and ultimately the function(s) of these cell cycle regulators. Epitopes targeted by the antibodies were mapped by peptide enzyme-linked immunoadsorbent assay (ELISA), and performance of the MAbs assessed in a range of immunochemical techniques. Individual MAbs of our series recognize distinct pools of the respective CKIs, a feature reflected by their differential applicability in immunoblotting, immunoprecipitation, and immunostaining including immunohistochemistry on archival paraffin-embedded tissue sections. Together, these antibodies represent useful reagents to study CKIs in cells and tissues, a set of tools that should help elucidate the physiological roles played by the individual CKIs, and better understand the molecular mechanisms of loss or inactivation of these (candidate) tumor suppressors in human malignancies.

Concerning schistosomiasis, little is known about the intracellular signaling response of human peripheral blood mononuclear cells (PBMC) to Schistosoma mansoni antigens. To understand the critical role of protein tyrosine kinases (PTKs) in PBMC activation by S. mansoni antigens, we investigated how inhibition of PTKs by genistein, a tyrosine kinase inhibitor, affects proliferation, cytokine production and activation of mitogen-activated protein kinases (MAPKs). Our studies showed that PTKs have an important role in proliferation of PBMC from chronic schistosomiasis patients as cells stimulated with S. mansoni soluble antigens in the presence of genistein had an impaired proliferation. In contrast, PTK inhibition failed to cause any effect on MAPKs activity. We also evaluated the cytokine production for interleukin (IL)-2, interferon gamma (IFN-gamma), and IL-10 in culture supernatants of PBMC treated with or without PTKs inhibitor. Our results show that PBMC from chronic patients produced a high amount of IL-10 when stimulated with soluble egg antigen preparation (SEA), however, the amount produced of IL-2 and IFN-gamma was not significant. In the presence of PTKs inhibitor only the production of IL-10 was decreased. The findings suggest that PTKs are involved on signal transduction pathway for PBMC activation, but may not be an absolute requirement for all signaling responses to S. mansoni antigens.

In primary biliary cirrhosis, autoantibodies are produced to the family of 2-oxoacid dehydrogenase complexes. These 'anti-mitochondrial' antibodies are traditionally detected by immunofluorescence but this method of detection is subjective and labour-intensive. We assessed an enzymatic mitochondrial antibody (EMA) assay based on antibody inhibition of enzymatic activity of pyruvate dehydrogenase complex in wells of microtitre plates with a colorimetric read-out. We tested 48 Australian and 1947 Japanese patients with primary biliary cirrhosis, 306 normal subjects and 691 patients with various hepatic and non-hepatic diseases. The overall sensitivity of the EMA for the diagnosis of primary biliary cirrhosis, 82%, was slightly lower than that of immunofluorescence, 90% The advantages of the EMA test include high specificity, >99%, and semi-automated features facilitating objectivity, rapidity, simplicity and economy. The EMA test could be particularly applicable to population screening for early primary biliary cirrhosis.

Peptides derived from the HLA class I heavy chain (a.a. 75-84) have been shown to modulate immune responses in vitro and in vivo in a non-allele-restricted fashion. In vivo studies in rodents have demonstrated prolonged allograft survival following peptide therapy. The immunomodulatory effect of these peptides has been correlated with peptide-mediated modulation of heme oxygenase 1 activity (HO-1). Recently, we used a rational approach for designing novel peptides with enhanced immunosuppressant activity. These peptides were also more potent inhibitors of HO-1 activity in vitro. Here we evaluated one of these peptides, RDP1258, for its ability to prolong heterotopic heart graft survival in rats. The peptide mediated effect on HO-1 was analyzed in vitro and in vivo. Peptide RDP1258 was shown to inhibit rat HO-1 in vitro in a dose-dependent fashion. However, RDP1258, like other HO-inhibitors, when administered to rats, secondarily resulted in an up-regulation of splenic HO-1 activity. Up-regulation of HO-1 was associated with prolonged heart allograft survival (6.6 +/- 0.6 vs. 2/14 > 100 days and 12/14 16.2 +/- 1.7 days; p < 0.001). The analysis of graft infiltrating cells on day 5 after transplantation showed a significant decrease in the number of graft infiltrating cells in RDP1258-treated recipients compared to untreated ones (14.8 vs. 32.7%; p < 0.01). In addition, grafts from peptide-treated animals showed significantly decreased expression of TNF-alpha mRNA and increased levels of iNOS mRNA. Our results are consistent with the recent observation that up-regulation of HO-1 results in the inhibition of several immune effector functions. Modulation of HO-1 activity may enable the development of novel immunomodulatory strategies in humans.

We have been interested for some time in establishing a strategy for deriving lead compounds from macromolecule ligands such as minibody variants. A minibody is a minimized antibody variable domain whose two loops are amenable to combinatorial mutagenesis. This approach can be especially useful when dealing with 'difficult' targets. One such target is the NS3 protease of hepatitis C virus (HCV), a human pathogen that is believed to infect about 100 million individuals worldwide and for which an effective therapy is not yet available. Based on known inhibitor specificity (residues P6-P1) of NS3 protease, we screened a number of minibodies from our collection and we were able to identify a competitive inhibitor of this enzyme. We thus validated an aspect of recognition by HCV NS3 protease, namely that an acid anchor is necessary for inhibitor activity. In addition, the characterization of the minibody inhibitor led to the synthesis of a constrained hexapeptide mimicking the bioactive loop of the parent macromolecule. The cyclic peptide is a lead compound prone to rapid optimization through solid phase combinatorial chemistry. We therefore confirmed that the potential of turning a protein ligand into a low molecular weight active compound for lead discovery is achievable and can complement more traditional drug discovery approaches.

Leukocyte infiltration in the pancreas is involved in the aggravation of acute pancreatitis from edematous phase into necrotic change, and mild disease into severe disease; however, the mechanism responsible for leukocyte accumulation is not fully understood. This study was designed to clarify the mechanism underlying leukocyte accumulation into the pancreas and to elucidate the therapeutic efficacy of a novel diamino-pyridine derivative, IS-741 on leukocyte-endothelial cell interaction using rat necrotizing pancreatitis model. The number of adherent leukocytes to pancreatic collecting venules assessed by in vivo fluorescence microscopy increased significantly in necrotizing pancreatitis animals in a time-dependent manner. The expression of CD11b on circulating neutrophils determined by flow cytometric analysis was enhanced to approximately 500% after 2 h. IS-741 attenuated the leukocyte adherence significantly, accompanied by a lower up-regulation of CD11b. These findings were further supported by the histological examination that the accumulation of leukocytes in the pancreas was remarkably inhibited by IS-741. These results suggest that the leukocyte accumulation in the early phase of acute necrotizing pancreatitis may be mediated by leukocyte-endothelial cell interaction via leukocyte integrin CD11b/18. IS-741 attenuated the leukocyte endothelial cell interaction as a consequence of its inhibitory effect on CD11b upregulation.

A sixty-five year old white male presented with an acquired Factor V inhibitor after an episode of cholecystitis and cefotaxime therapy. Plasma Factor V activity was less than 1%. He developed lower gastrointestinal bleeding a week after onset of coagulopathy, and was treated with plasmapheresis, fresh frozen plasma, oral cyclophosphamide, and prednisone. The coagulopathy resolved within four days of treatment, and within two weeks of presentation. Laboratory studies revealed an IgG inhibitor to Factor V that closely mimicked the more commonly encountered lupus anticoagulant. We would like to alert clinicians to this entity because, in contrast to a lupus anticoagulant, the acquired Factor V inhibitor can be associated with clinical bleeding as in our patient, and requires therapy prior to any surgical procedures.

We have investigated the mechanism whereby nuclear DNA fragmentation activity emerging during early apoptosis is inhibited during normal cell life. In a cell-free system, cytosol fractions from diverse nonapoptotic human cell lines (Jurkat T-cell leukemia, HeLa carcinoma, SK-N-MC neuroblastoma, and WI-38 embryonic lung fibroblast) potently neutralized the nuclear DNA fragmentation activity of cytosol from apoptotic anti-Fas treated Jurkat cells. Recombinant human DNA fragmentation factor 45 kDa subunit (DFF45/ICAD), an inhibitor of the caspase-activated DNase DFF40/CAD, substituted for healthy cytosol in inhibiting DNA fragmentation. An antiserum against human DFF45 detected 44 and 34 kDa proteins (major and minor, respectively) in the cytosols but not in the nuclear or membrane fractions of various cultured human cells. Cytosols depleted of DFF45/ICAD by immunoadsorption had little or no inhibitor of nuclear DNA fragmentation activity and no caspase-activated DNA fragmentation activity. We conclude that immunoreactive DFF45/ICAD is the principal inhibitor of apoptotic DNase activity in the cytosol of healthy cells.

The influence of nitric oxide (NO) on eosinophil infiltration into the airways was investigated in rats actively sensitized with ovalbumin. The animals were treated chronically with the NO synthase inhibitor, N omega-Nitro-L-arginine methyl ester (L-NAME; 75 mumol rat-1 day-1), for 4 weeks. Bronchoalveolar lavage was performed at 6, 24, 48 and 72 h after intratracheal injection of ovalbumin. Intratracheal challenge of the sensitized rats with ovalbumin caused a significant increase in total leucocyte infiltration in bronchoalveolar lavage fluid both 24 and 48 h post-ovalbumin injection. Neutrophils and eosinophils peaked, respectively, at 24 h (29%) and 48 h (30%) in bronchoalveolar lavage fluid whereas the mononuclear cell did not differ significantly from the counts in non-sensitized rats at any time. At both 6 and 24 h post-ovalbumin injection, the chronic treatment of the animals with L-NAME affected neither the total nor the differential leucocyte content. However, at 48 h post-ovalbumin challenge, the total cell count was reduced by approximately 48% in the L-NAME-treated animals and this was associated with a marked inhibition (81%) of the eosinophil influx. Histological examination of the lungs from these animals (48 h post-ovalbumin challenge) also showed a prominent reduction (69.5%; P < 0.05) of the eosinophil infiltration in the respiratory segments. Our results demonstrate that NO plays a pivotal role in the eosinophil infiltration in airways of actively sensitized rats.

Background: Cereal flours are used in the wood industry to improve the quality of the glues necessary to produce veneer panels. However, up to now, no cases of sensitization to cereal flour in this kind of industry have been reported. Cereal alpha-amylase inhibitors have been previously described as important occupational allergens responsible for baker's asthma., Objective: To determine whether cereal allergens were responsible for occupational sensitization in three wood industry workers., Methods: The diagnosis was made by clinical questionnaire, physical examination, skin-prick tests to cereals, CAP and immunoblotting., Results: The three patients had positive skin prick tests and CAP to cereal flours. An IgE-immunoblotting revealed that only low molecular weight proteins (under 20 kDa) were detected by the three sera. These main IgE-binding proteins were members of the alpha-amylase inhibitor family which have been described as one of the group of main allergenic proteins in rye, barley and wheat. The three patients changed their workplace and remain asymptomatic in spite of the fact that they are still in contact with different woods and exposed to high concentrations of wood dust and other chemicals such as formaldehyde., Conclusion: Proteins from cereal flours are important occupational allergens in some wood industries.

To clarify the roles of increased apoptosis and cell proliferation in chronic autoimmune lymphocytic thyroiditis and thyroid tumorigenesis, expression of p53 and p21(WAF1) proteins was immunohistochemically investigated in a series of 158 cases. Positive epithelial cells were quantified to give numbers per unit square and to score for distribution. They were found scattered in nontumorous thyroid tissue, their numbers increasing with the severity of thyroiditis and the correlation between expression of the two proteins, regardless of the presence or absence of thyroid neoplasms. Simultaneous expression of both proteins was occasionally found in the same cells by analysis of serial histologic sections. In thyroid tumors, increased expression was found to be diffuse, focal, or scattered for the distribution of p53- or p21(WAF1)-immunopositive cells in accordance with tumor cell dedifferentiation, showing significant correlation between expression of the two proteins. Correlated with these findings, enhanced apoptosis along with decreased Bcl-2 expression and increased Ki-67 labeling in lymphocytic thyroiditis and thyroid tumors was also confirmed in the same series, using in situ DNA nick-end labeling and immunohistochemical methods. Increased expression of p53 and/or p21(WAF1) proteins was thus suggestive of possible DNA damage and increased apoptosis in autoimmune thyroiditis. In addition, a significant correlation between protein overexpression and dedifferentiation of thyroid tumor cells was apparent., (Copyright 1998 Academic Press.)

Cyclic ADP-ribose (cADPR) has been shown to be a mediator for intracellular Ca2+ mobilization for insulin secretion by glucose in pancreatic beta cells, and CD38 shows both ADP-ribosyl cyclase to synthesize cADPR from NAD+ and cADPR hydrolase to hydrolyze cADPR to ADP-ribose. We show here that 13.8% of Japanese non-insulin-dependent diabetes (NIDDM) patients examined have autoantibodies against CD38 and that the sera containing anti-CD38 autoantibodies inhibit the ADP-ribosyl cyclase activity of CD38 (P

Evidence is provided that dromedary heavy-chain antibodies, in vivo-matured in the absence of light chains, are a unique source of inhibitory antibodies. After immunization of a dromedary with bovine erythrocyte carbonic anhydrase and porcine pancreatic alpha-amylase, it was demonstrated that a considerable amount of heavy-chain antibodies, acting as true competitive inhibitors, circulate in the bloodstream. In contrast, the conventional antibodies apparently do not interact with the enzyme's active site. Next we illustrated that peripheral blood lymphocytes are suitable for one-step cloning of the variable domain fragments in a phage-display vector. By bio-panning, several antigen-specific single-domain fragments are readily isolated for both enzymes. In addition we show that among those isolated fragments active site binders are well represented. When produced as recombinant protein in Escherichia coli, these active site binders appear to be potent enzyme inhibitors when tested in chromogenic assays. The low complexity of the antigen-binding site of these single-domain antibodies composed of only three loops could be valuable for designing smaller synthetic inhibitors.

A material crossreacting with antibodies against the bufadienolide proscillaridin A and inhibiting the sodium pump was found in human blood plasma. The concentration of the material with a retention time similar to ouabain in a reversed phase HPLC correlated to systolic blood pressure and pulse pressure. Affinity purification of this compound from bovine adrenals resulted in the isolation of a compound with molecular mass of 600 Da that was not identical with ouabain. Consistent with the postulate that endogenous ouabain and proscillaridin A immunoreactivities may belong to a new class of cardiotonic steroid hormones, a protein of Mr = 60 kDa has been found in bovine serum by affinity-labeling with N-hydroxysuccimidyl digoxigenin-3-O-methylcarbonyl-epsilon-aminocaproate.

Coagulation factor XIIIa, plasma transglutaminase (endo-gamma-glutamine:epsilon-lysine transferase EC 2.3.2.13) catalyzes isopeptide bond formation between glutamine and lysine residues and rapidly cross-links fibrin clots. A monoclonal antibody (5A2) directed to a fibrinogen Aalpha-chain segment 529-539 was previously observed from analysis of end-stage plasma clots to block fibrin alpha-chain cross-linking. This prompted the study of its effect on nonfibrinogen substrates, with the prospect that 5A2 was inhibiting XIIIa directly. It inhibited XIIIa-catalyzed incorporation of the amine donor substrate dansylcadaverine into the glutamine acceptor dimethylcasein in an uncompetitive manner with respect to dimethylcasein utilization and competitively with respect to dansylcadaverine. Uncompetitive inhibition was also observed with the synthetic glutamine substrate, LGPGQSKVIG. Theoretically, uncompetitive inhibition arises from preferential interaction of the inhibitor with the enzyme-substrate complex but is also found to inhibit gamma-chain cross-linking. The conjunction of the uncompetitive and competitive modes of inhibition indicates in theory that this bireactant system involves an ordered reaction in which docking of the glutamine substrate precedes the amine exchange. The presence of substrate enhanced binding of 5A2 to XIIIa, an interaction deemed to occur through a C-terminal segment of the XIIIa A-chain (643-658, GSDMTVTVQFTNPLKE), 55% of which comprises sequences occurring in the fibrinogen epitope Aalpha-(529-540) (GSESGIFTNTKE). Removal of the C-terminal domain from XIIIa abolishes the inhibitory effect of 5A2 on activity. Crystallographic studies on recombinant XIIIa place the segment 643-658 in the region of the groove through which glutamine substrates access the active site and have predicted that for catalysis, a conformational change may accompany glutamine-substrate binding. The uncompetitive inhibition and the substrate-dependent binding of 5A2 provide evidence for the conformational change.

VH (heavy-chain variable region) and VL (light-chain variable region) genes were amplified by PCR from hybridomas producing MAb-11 and MAb-18 which inhibited Japanese radish acid phosphatase. Nucleotide sequencing of the V genes demonstrates that the MAbs contained similar VH and identical VL domains. Initially, the VH and VL genes were expressed in Escherichia coli as single-chain Fv (ScFv) fragments. Fragments ScFv-11 and ScFv-18, named for MAb-11 and MAb-18, respectively, inhibited the enzyme activity to the same extent as the intact MAbs. Both of the antibody fragments widely cross-reacted with other phosphatases, including some phosphomonoesterases and phosphodiesterases from different sources. ScFv-18 also inhibited acid phosphatase from a different origin, but stimulated the activity of alkaline phosphatase from calf intestine. The PCR-amplified VH and VL genes were subsequently expressed separately in Escherichia coli as fusion products with glutathione S-transferase. The fusion proteins had little effect on Japanese radish acid phosphatase. Furthermore, a large number of recombinant ScFv fragments specific to the acid phosphatase were generated by using a bacteriophage expression system and a mouse ScFv gene library. These ScFv fragments had a range of effects on the enzyme activity, including inhibition, stimulation, and none. Among them, an ScFv fragment, designated ScFv-G7, inhibited more strongly than ScFv-11 and ScFv-18.

Antibodies were generated against the positively charged chair-like glycosidase inhibitor nojirimycin by in vitro immunization. A number of catalytic antibodies were isolated, one of which catalyzes the hydrolysis of p-nitrophenyl beta-D-glucopyranoside 3 with a rate enhancement (kcat/kuncat) of 10(5) M over the HOAC-catalyzed reaction. The antibody discriminates modifications in the pyranoside ring of substrate 3 at the C2, C4, and the anomeric positions. The pH dependence of the reaction and chemical modification studies suggest the presence of an active-site Asp or Glu residue that may function as a general acid. This study further defines those requirements necessary to generate antibodies that efficiently cleave glycosidic bonds.

Histidase (histidine ammonia-lyase) is a cytosolic enzyme responsible for catalyzing the non-oxidative deamination of histidine to urocanic acid. Full-length cDNAs encoding rat histidase have been isolated from a lambdaZAP liver cDNA library using a partial cDNA fragment obtained by PCR. Whereas the initial description of the rat histidase 3' untranslated sequence contained a rare polyadenylation signal sequence, the data presented encompass a more distant 28-bp region, possessing a nucleotide stretch (AATATAAA), identical to that in the mouse histidase cDNA. Dideoxynucleotide chain-termination sequencing of two clones obtained by in vivo excision yielded an additional 376 bp and 105 bp of 5' and 3' untranslated sequences, respectively. A selected rat histidase cDNA clone was introduced into the pET-16b prokaryotic vector and expressed in BL21(DE3)pLysS Escherichia coli. After purification by nickel-chelation chromatography, recombinant histidine-tagged protein was employed to raise anti-(rat histidase) immunoglobulin in a Japanese white rabbit. The polyclonal rabbit antibody recognized and formed immune complexes with rat and recombinant human histidase proteins. Immunoblots of crude rat organ extracts detected a spectrum of histidase expression extending beyond that observed in liver and skin. Among other histidase-positive cells were those of the renal cortex tubular epithelium, fundic mucosal glands of stomach, gastric intramuscular (Auerbach's) plexus, and adrenal cortex. Immunohistochemical studies of histidase in rat liver produced discrete staining of hepatocytes in association with portal triads (Rappaport zone I). Furthermore, in contrast with previous reports of activity confined to epidermal stratum corneum, our findings demonstrate immunoreactive protein within and limited to the adjacent stratum granulosum.

Background: The development of simple and standardised methods to measure airborne levels of workplace biological allergens is an important step in reducing the incidence of occupational asthma. Such a method would be useful for measuring wheat flour allergens which cause asthma in bakers. Measurement of allergen per se rather than total dust enables exposure to be better defined., Methods: Monoclonal antibodies were produced, their specificity analysed by immunoblotting and then used to affinity-purify a putative flour allergen. The importance of this protein as an allergen was tested by RAST using sera from allergic bakers and it was identified by N-terminal sequencing. Suitable monoclonal antibodies were chosen to develop an enzyme-linked immunosorbent assay. Commercial baking flours and personal airborne dust samples were analysed using the immunoassay., Results: A sensitive and specific monoclonal antibody-based enzyme-linked immunosorbent assay was developed to measure a wheat alpha-amylase inhibitor. The wheat alpha-amylase inhibitor content of bulk wheat flours was 0.124% (95% confidence limits 0.083-0.164%) and airborne levels in bakeries had a geometric mean of 744 ng/m3 (95% confidence limits 371-1,496 ng/m3)., Conclusion: This assay is suitable for widespread use as the monoclonal antibodies and standards are well defined and potentially infinitely available. The assay therefore offers distinct advantages over those exposure assessment methods currently in use. Comparable results would be obtained by different investigators over a prolonged time period. The assessment of flour allergen exposure and the relationship with clinical response could then be investigated using a multi-centered approach.

Pyrazole, acetone, and ethanol are known to induce cytochrome P450 2E1 (CYP2E1) and fatty acid (omega-1)-hydroxylation in rat liver microsomes. However, the nature of the P450 enzyme involved in this (omega-1)-hydroxylation has not been clearly established in extrahepatic tissues such as kidney. Four enzymatic activities (hydroxylations of chlorzoxazone, 4-nitrophenol, and two fatty acids) were assayed in kidney microsomal preparations of rats treated with CYP2E1 inducers. Per os treatment resulted in large increases (threefold to fivefold) in the chlorzoxazone and 4-nitrophenol hydroxylations, and up to a ninefold increase when ethanol was administered by inhalation. However, neither the omega-hydroxylation nor the (omega-1)-hydroxylation of fatty acids was modified. Immunoinhibition specific to CYP2E1 did not significantly decrease the omega and (omega-1)-lauric acid hydroxylations, while the polyclonal anti-CYP4A1 antibody inhibited in part both the omega- and (omega-1)-hydroxylations. Chemical inhibitions using either CYP2E1 competitive inhibitors (such as chlorzoxazone, DMSO, and ethanol) or P450 mechanism-based inhibitors (such as diethyldithiocarbamate and 17-octadecynoic acid) led to a partial inhibition of the hydroxylations. All these results suggest that fatty acid (omega-1)-hydroxylation, a highly specific probe for CYP2E1 in rat and human liver microsomes, is not mediated by CYP2E1 in rat kidney microsomes. In contrast to liver, where two different P450 enzymes are involved in fatty acid omega- and (omega-1)-hydroxylations, the same P450 enzyme, mainly a member of the CYP4A family, was involved in both hydroxylations in rat renal microsomes.

BALB/c mice are genetically susceptible to infection with Leishmania major (L major). When such mice infected with L. major were treated with specific inhibitors of cathepsin B, a lysosomal cysteine protease that digests exogenous antigenic proteins, the mice acquired resistance against L. major infection. T cells from these mice produced large amounts of IFN-gamma and low amounts of IL-4 as compared with those of untreated BALB/c mice. In addition, the mice treated with cathepsin B inhibitor produced a high titer of IgG2a specific antibodies and only low titers of IgG1 and IgE antibodies. This type of response is in contrast with the high specific IgG1 or IgE antibody responses which are the usual antibody responses in BALB/c mice infected with L. major. These findings indicate that cathepsin B may be critically involved in processing antigens of L. major to promote exclusively the development of Th2 type CD 4+T cell responses.

Levels of free anti-(xanthine oxidoreductase) (XOR) antibodies in the serum of normal healthy human subjects were determined, using both human and bovine enzyme as antigen, in an enzyme-linked immunosorbent assay (ELISA). Levels of IgM class anti-(human XOR) antibodies were found to be particularly high (mean values representing approximately 3% of total IgM) and to be significantly higher than levels of IgM anti-(bovine XOR) antibodies, indicating that endogenous XOR, rather than ingested bovine milk XOR, is the immunogen. IgM anti-XOR antibody levels were significantly higher in women under 50 years than in age-matched men, or in older women. Levels of IgG class anti-XOR antibodies were much lower and showed no correlation with gender or age. Affinity-purified anti-(human XOR) antibodies only partially inhibited enzymic activities of XOR. The majority of both IgM and IgG anti-(human XOR) antibodies in serum occurred as immune complexes, suggesting that the specific antibodies have a protective role in removing potentially damaging XOR from the circulation.

Objective: Platelet factor 4 (PF-4) is a critical alpha chemokine in inflammation and injury responses, with multiple effects upon cellular activities. Discrete peptide sequences of the PF-4 molecule have been shown to retain biological activity. Our aim was to examine the influence of the PF-4 derived octapeptide (CT-112; TTSQVRPR) on type II collagen induced arthritis in mice, to determine if this peptide exhibited antiinflammatory properties., Methods: DBA/1 mice were treated with CT-112 from either the time of immunization with type II collagen or from the initial onset of arthritis., Results: CT-112 both prevented the development of arthritis in mice treated prophylactically and reduced progression of disease in animals treated therapeutically, and was active when delivered by either subcutaneous injection or oral gavage. No marked immunosuppressive effects were observed during CT-112 treatment, with only moderate decrease in antibody levels and mitogen responses. A significant reduction of the circulating levels of IL-1 was a consistent finding in mice treated therapeutically with CT-112., Conclusion: These data suggest PF-4 derived octapeptide exerts antiinflammatory effects of experimental arthritis in mice.

To generate conventional or monoclonal antibodies for the serological detection of drugs, antibiotics, toxins and other low molecular mass substances, a suitable and effective adjuvant is needed. Lipopeptides derived from a major component of the bacterial cell wall constitute potent nontoxic and nonpyrogenic immunoadjuvants when mixed with conventional antigens. Here we demonstrate that the synthetic lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteinyl- serine (P3CS) coupled to a Th-cell epitope (P3CS-Th) can efficiently enhance the specific immune response against low molecular weight compounds in different species. In the presence of the synthetic lipopeptide P3CS-Th, the peptides which are per se non-immunogenic stimulated a specific humoral immune response in mice after intraperitoneal application. Mixtures containing adjuvants without the Th sequence showed no significant antibody induction. A marked enhancement of the humoral immune response was obtained with the low molecular mass antigens Iturin AL, Herbicolin A and Microcystin (MLR) coupled to poly-l-lysin (MLR-PLL), in rabbits and in chickens. Lipopeptide-Th cell epitope conjugates also constituted adjuvants for the in vitro immunization of either human mononuclear cells or mouse B-cells with MLR-PLL; after fusion of the immunized cultures with the heteromyeloma cell lines CB-F7 or the mouse myeloma cell line SP 2/0, respectively, we observed a significantly increased yield of antibody secreting hybridomas.

Background: Low molecular weight allergens may be responsible for hypersensitivity reactions after the ingestion of wheat., Objective: The purpose of this investigation was to identify relevant, low molecular weight allergens after the ingestion of wheat protein., Methods: Serum samples were collected from seven children with wheat allergy and one adult with baker's asthma. Control serum samples were collected from wheat-tolerant patients. Wheat extracts were prepared and separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 12.5% gels revealing numerous protein bands. IgE immunoblot analysis of crude wheat extracts identified multiple IgE-binding proteins. Wheat proteins were separated further with two-dimensional gel electrophoresis, which was followed by IgE immunoblotting investigations., Results: Immunoblot analysis identified a 15 kd wheat protein that bound IgE from all five children with wheat allergy who were evaluated. No IgE binding to this wheat protein was demonstrated in any of the control subjects. Samples representing the 15 kd wheat protein (isoelective point, 5.85) were selected. The N-terminal peptide sequence of this protein (residues 1 to 20) matched to a wheat alpha-amylase inhibitor., Conclusion: These data demonstrate that wheat alpha-amylase inhibitor is a relevant allergen in patients experiencing hypersensitivity reactions after the ingestion of wheat protein. This wheat protein, which has been implicated as an important allergen in patients with baker's asthma, represents a sensitizing allergen after both ingestion and inhalation.

Vasoconstrictor and Na/K pump inhibitory properties of a bufodienolide Na/K-ATPase inhibitor, marinobufagenin, were studied in isolated rings of 2 to 3 order branches of human pulmonary arteries respectively. Marinobufagenin displayed concentration-dependent vasoconstrictor activity (0.01 to 10 mmol/L). In sarcolemma membranes prepared from pulmonary artery marinobufagenin inhibited Na/K-ATPase (IC50 = 50 nmol/L). In eight healthy male Caucasians, concentrations of marinobufagenin-like immunoreactive material in C-18 extracted plasma were 1.38 +/- 0.60 nmol/L. Twenty-four-hour urinary release of marinobufagenin-like immunoreactive material in eight healthy males was 1.20 +/- 0.95 nmol/day. Chloroform extract of human urine was fractionated using reverse-phase high-performance liquid chromatography (32% acetonitrile, Deltapak). The HPLC fraction coeluting with marinobufagenin in 7 min, cross reacted with antimarinobufagenin and antidigoxin, but not antiouabain antibody. These results demonstrate that human plasma and urine contains a bufodienolide vasoconstrictor EDLF, marinobufagenin-like immunoreactive Na,K pump inhibitor.

In the first part of our work (1) it was found that the cultivation of blood of sensitized people and animals with specific allergen (AI) caused the phenomenon of specific inhibition stimulated luminol-dependent chemiluminescence of leukocytes (PhSISCL). In the current study the mechanism of this inhibition was investigated. It was revealed that PhSISCL resulted from the direct influence of AI on polymorphonuclear leukocytes (PMNL). The activity of NADPH-dependent oxidase was established by the Wymann M. et al. method (2). It was determined that the values of chemiluminescence (CL) for the cultivation of sensitized rabbit blood with specific AI did not differ from the control. This demonstrated that the activity of NADPH-oxidase was not inhibited. On these grounds it is possible to assume that PhSISCL is connected to either the inhibition of myeloperoxidase (MPO) activity or to a release of this ferment from PMNL during cultivation. The latter was specifically investigated and was not confirmed.

Human gelatinase B was produced from peripheral blood neutrophils and purified by affinity chromatography on gelatin sepharose. This material was used as an antigen to prepare mouse monoclonal antibodies (mAb). The resulting hybridomas were selected on the basis of binding to biotinylated antigen and by a sandwich ELISA using gelatinase-B-specific polyclonal rabbit antiserum and pure natural antigen. Five of these mAb were selected for further characterization. They all displayed variable epitope specificity, binding capacity and inhibitory activity. Whereas mAb REGA-2D9 and REGA-3G12 showed the strongest binding to biotinylated gelatinase B and natural gelatinase B, respectively, mAb REGA-2F9 did not bind biotinylated antigen. None of the mAb displayed cross-reactivity to gelatinase A in a direct ELISA. The mAb REGA-1G8 was found to cross-react with human serum albumin. The binding capacity of the other four mAb with leukocyte gelatinase B was compared and a sensitive sandwich ELISA was developed with the antibodies REGA-3G12 and REGA-2D9 (detection limit 0.5 ng/ml). The mAb REGA-3G12 was unique in that it inhibited catalysis by gelatinase B. This was shown by assaying the degradation of nasal septum type II gelatin in the presence and absence of each of the five mAb. Furthermore, mAb REGA-3G12 inhibited the degradation of biotinylated gelatin in a microtiterplate solution assay. In addition to the potential use of the inhibitory mAb REGA-3G12 in the treatment of diseases with excessive gelatinase B production, several of the described mAb are useful as diagnostic probes to detect gelatinase B in body fluids and tissue samples of patients with multiple sclerosis, rheumatoid arthritis and cancer.

It is confirmed by several studies that in normal subjects a substance recognized by antibodies anti digoxin exists. Such a substance can be found at increased concentration in pregnant women, neonates, in liver or kidney diseases. A limited increase in concentration has been also registered in patients with essential hypertension and in normotensive patients with a family history of hypertension. Serum or urines rich in such a substance show an increased capacity of inhibiting in vitro the sodium-potassium pump and therefore in reducing also in vivo the capacity of reabsorption of sodium and with it, of water. The investigators interest for this substance has two main reasons: 1) the interference that such a substance has in dosages of digitalis in therapeutic monitorizing; 2) the possibility that such a substance has an important physiological role in hydroelectrolytic metabolism.

Seven cDNA clones encoding rice allergenic proteins were newly isolated. Comparison of the sequences of ten cDNA clones, including the previously isolated three clones results in their classification into four subfamilies. Homologies in the nucleotide sequences among and within subfamilies are 70-85% and above 95%, respectively. A sequence of twenty five amino-acid residues at the C-terminal proximal region is highly conserved among all clones and resembles that of plant lipid transfer proteins.

Two peptides that correspond to sequences within the major 33-amino acid sequence recognized by human liver-kidney microsomal-1 autoantibodies were used to elicit antibodies in rabbits (four per peptide) against CYP2D6. Peptide 1(DPAQPPRDLTEAFLA) corresponded to amino acids 263-277, and peptide 2 (LLTEHRMTWDPAQPPRDLTE) corresponded to amino acids 254-273 of CYP2D6. The peptide-keyhole limpet hemocyanin conjugates elicited good immune responses against their respective peptides as judged by enzyme-linked immunosorbent assay (titers of 1/10,000 to 1/30,000). The antisera recognized CYP2D6 on Western blots and, to varying extents, inhibited recombinant CYP2D6 and liver microsomal CYP2D6 activity. Immunization with peptide 2 produced antisera with the greatest inhibitory potency. Antiserum from a rabbit (#236) immunized with peptide 2 inhibited up to 95% of dextromethorphan O-demethylase activity in human liver microsomes at the highest concentration tested (40% v/v) but did not significantly inhibit CYP1A2, CYP2C9, CYP2E1, or CYP3A4 marker activities. On Western blot, only a single immunoreactive protein comigrating with recombinant CYP2D6 was recognized. In liver microsomes from a CYP2D6-deficient individual, no proteins were recognized, and the antisera did not cross-react with recombinant CYP1A2, CYP2C9, CYP2E1, or CYP3A4. There was a significant correlation between the quantity of immunoreactive CYP2D6 as determined by immunoblotting with anti-peptide 2 antiserum and dextromethorphan O-demethylation in a panel of 10 human liver microsomes (r = 0.95). These data identify a peptide sequence (peptide 2) that can be used to raise antisera that specifically recognize and inhibit CYP2D6.

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor with three tandem inhibitory domains, which inhibits the initial reactions of the extrinsic blood coagulation pathway through the first and second Kunitz domains. We prepared a monoclonal antibody against recombinant human TFPI (rTFPI) and determined the epitope as the third Kunitz domain, using fragments derived from rTFPI (K1-K2 fragment and K3 fragment) and synthetic peptides. We then developed an enzyme immunoassay (EIA) method using a combination of the monoclonal antibody and a polyclonal antibody. Although TFPI activity is distributed among LDL/VLDL-associated, HDL-associated, and free forms of TFPI after gel-filtration of human plasma, only the free form was detected by the EIA method. After incubation with LDL, the antigenicity of rTFPI was reduced, but that of K3 fragment was not. Gel-filtration analysis of the mixture of radiolabeled rTFPI or K3 with LDL demonstrated that rTFPI, but not K3, bound LDL. From these results, we concluded that the monoclonal antibody against TFPI recognized only a free form of TFPI in plasma, since the epitope of lipoprotein-associated TFPI had been masked by the interaction with lipoproteins.

The distribution of inhibitor-1 was analysed in the neocortex of cat, ferret and rat by immunocytochemistry (at the light and electron microscope levels) and by immunoblotting using an affinity purified antibody which recognises both the phosphorylated and dephosphorylated forms of the protein. In each mammalian cortex immunocytochemical techniques identified inhibitor-1 predominantly in infragranular pyramidal neurons and, at a lower concentration, in supragranular pyramidal neurons of cortical layers II-III, and V-VI. Within the cortical layers, neuronal cell bodies and apical dendrites were stained strongly but no immunoreactivity was associated with dendritic spines. Regional differences in intensity of staining were revealed when appropriate antibody concentrations were used; the concentration of inhibitor-1 appeared to follow a gradient with the highest levels in layer VI and the lowest in layer I. The results were confirmed by immunoblotting of microdissected cortical regions which identified the inhibitor-1 protein unambiguously. The distribution of inhibitor-1 is different from that reported by other investigators.

The positive inotropic effect of the beta-adrenoceptor agonist isoproterenol is accompanied by inhibition of phosphatase type 1 activity in myocardium. Indirect assays suggest that this effect is due to activation of protein phosphatase inhibitor-1, which inhibits phosphatase activity only when phosphorylated. To test this hypothesis directly, electrically stimulated (3 Hz) guinea pig ventricular preparations were perfused according to the Langendorff method with physiological buffers with or without 5 mCi 32P/heart, and then various concentrations of isoproterenol were applied. Contractility was recorded. Hearts were freeze-clamped and cAMP and inhibitor-1 activities were measured. In 32P-labeled hearts a protein at about 26 kd on autoradiograms of 12% sodium dodecyl sulfate gels was detected. Isoproterenol (1 microM) increased rate of tension development to 238% of the predrug value, cAMP concentrations 1.5-fold, and inhibitor-1 activity threefold. Concomitantly, there was an increase in a 32P-labeled band at about 26 kd from 380 to 540 pmol 32P/mg protein. This protein at about 26 kd, after transfer to nitrocellulose, was recognized by an antiserum prepared against rabbit skeletal muscle inhibitor-1. More radioactive protein of about 26 kd could be immunoprecipitated by the antiserum from isoproterenol-treated than from untreated hearts. It is concluded that a protein, probably identical to phosphatase inhibitor-1, is phosphorylated in vivo in the heart in the presence of isoproterenol. Phosphorylation of inhibitor-1 with consequent modification of type 1 phosphatase activity may contribute to the effects of isoproterenol in the heart.

Since the early recognition that leukotrienes are generated in response to allergen exposure, a role for these multipurpose mediators has been sought. The pharmacologic actions of the leukotrienes and their cell sources were strong evidence that they should contribute to allergic airway disease. That promise is now being fulfilled. Potent leukotriene receptor antagonists and enzyme inhibitors of leukotriene generation are now being investigated. With the availability of these new compounds, not only will greater insight to leukotrienes in asthma become apparent, but possibly newer, more effective therapeutics. Of additional interest and relevance is the potential role of leukotrienes in other nonrespiratory inflammatory diseases such as inflammatory bowel disease, rheumatoid arthritis, and psoriasis. Therefore, the role of leukotrienes in inflammation is not limited to the respiratory system but are more universal in their ability to cause tissue injury. Consequently, studies that have shown benefit from inhibition of leukotriene synthesis and antagonism of the LTD4 receptor in respiratory diseases are suggestive that such an approach will also be beneficial in other inflammatory diseases. For example, a study of 72 patients with A-64077 (zileuton) has demonstrated that 5-lipoxygenase inhibitors are efficacious in the treatment of inflammatory bowel disease. Therefore, the contribution of leukotrienes to inflammation is likely to be a global phenomenon, and the introduction of leukotriene antagonists and 5-lipoxygenase inhibitors may represent the beginning of a new era for the treatment of many inflammatory diseases.

An anticoagulant factor with phospholipase A2 activity has been isolated from Vipera berus venom. Phospholipase activity was studied on platelet phospholipid and on brain cephalin. The venom factor showed a potent anticoagulant activity: 1 mug impaired the clotting of 1 ml of citrated recalcified platelet-poor plasma. The anticoagulant inhibited clotting by antagonism to phospholipid. The antagonism constant (Kan = 6.8-10(-9) M) demonstrated the high affinity of the inhibitor for phospholipid. As with other phospholipases A2, the venom factor was thermoresistant but very sensitive to photo-oxidation. Both activities (anticoagulant activity and phospholipase activity) were not markedly dissociated by either denaturation or neutralization processes. Slightly different curves of photo-oxidative inactivation of both activities suggested the presence, on the molecule, of two very close sites responsible for phospholipase and anticoagulant activities. The inhibitor effect on coagulation was independent of the hydrolysis process. In fact, lysoderivatives and fatty acids, resulting from complete hydrolysis with the venom factor, were as active as the native phospholipids. Moreover phospholipase A2 from other viperidae venom, which did not have anticoagulant activity, produced similarly active lysoderivatives. This showed that the cleavage of the beta-acyl bond does not interfere with the activity of phospholipid. A possible mechanism of clotting inhibition by the venom factor was proposed. Owing to its high affinity for phospholipid, the inhibitor would complex phospholipid at its protein binding site impairing the normal arrangement of coagulation protein factors and, consequently, their activation. The positive charges of the inhibitor (pI = 9.2) could bind with phosphoryl or carboxyl groups of phospholipid, making them unavailable for protein binding. The complex formation involves a loss of dissociating capacity of the enzyme towards its substrate. This required an additional interaction of the inhibitor with a coagulation protein factor. The inhibitor could be removed from the complex by specific antibodies, permitting recovery of normal phospholipid-protein interaction. The role of calcium in the complex has not yet been elucidated. This venom factor affords a useful tool for investigating the phospholipid-clotting protein interaction.