Alpha1-antitrypsin inhibits the activity of the matriptase catalytic domain in vitro

Matriptase is a type II transmembrane protease that is characterized by an N-terminal transmembrane and multiple extracellular domains, in addition to the conserved extracellular serine protease catalytic domain. The expression pattern of matriptase suggests that this protease may play broad roles in the biology of surface lining epithelial cells. In this study we report that alpha1-antitrypsin (AAT), an endogenous inhibitor of serine proteases, inhibits the catalytic domain of human recombinant matriptase in vitro. Co-incubation of AAT with matriptase (at a molar ratio 1:2) resulted in the formation of heat stable complexes, clearly seen in sodium dodecyl sulfate electrophoresis and Western blots. AAT was found to be a slow, tight-binding inhibitor of the catalytic domain of matriptase with a second order reaction rate constant of 0.31 x 10(3) M(-1)s(-1). Notably, the oxidized form of AAT, which lacks serine protease inhibitor activity, failed to generate matriptase complexes and to inhibit matriptase activity. Since matriptase is involved in a number of physiologic processes, including activation of epithelial sodium channels, our findings offer considerable new insights into new regulatory function of AAT in vivo.     

Alpha1-antitrypsin deficiency and toxic shock: a Japanese autopsy case

A 74-year-old Japanese female presented with the sudden appearance of hemorrhagic purpuric ecchymoses on her lower extremities and with fever and chills, and died on the fifth day of hospitalization. A diagnosis of alpha1-antitrypsin (AT) deficiency was made postmortem. The liver weighed 1260 g. Histological sections from the liver revealed rather severe fatty changes of the hepatocytic parenchyma and partial loss of the normal hepatic architecture with fibrosis. The hepatocytes contained periodic acid-Schiff (PAS)-positive, diastase-resistant and alpha1-AT-positive intracytoplasmic globules. There was markedly increased inflammatory infiltration with severe edema and congestion, accompanied by fibrous, thickened pulmonary alveolar walls with fibrin deposition in the lungs (right, 410 g; left, 280 g), which suggest findings similar to those seen in multiple organ failure. Mild pulmonary emphysema was also present in the upper lobes of the lungs. Histological sections from the hemorrhagic necrotic ecchymoses of the skin showed marked neutrophil infiltration over the subcutaneous tissue with bleeding and blistering. A finding of thrombophlebitis was also found in the subcutaneous tissue. No bacteria were detected in the ecchymoses, the urine or the blood. Plasma protein analysis revealed a lower level (9.5 micromol/L) of alpha1-AT and a higher level (330 U) of anti-streptolysin O (ASO). These findings suggest that the patient died of toxic shock-like syndrome and that alpha1-AT deficiency might have facilitated the development of the toxic shock. To our knowledge, this is the first case of toxic shock associated with alpha1-AT deficiency.     

Alpha1-antitrypsin suppresses TNF-alpha and MMP-12 production by cigarette smoke-stimulated macrophages

We have previously observed that mice exposed to cigarette smoke and treated with exogenous alpha(1)-antitrypsin (A1AT) were protected against the development of emphysema and against smoke-induced increases in serum TNF-alpha. To investigate possible mechanisms behind this latter observation, we cultured alveolar macrophages lavaged from C57 mice. Smoke-conditioned medium caused alveolar macrophages to increase secretion of macrophage metalloelastase (MMP-12) and TNF-alpha, and this effect was suppressed in a dose-response fashion by addition of A1AT. Macrophages from animals exposed to smoke in vivo and then lavaged also failed to increase MMP-12 and TNF-alpha secretion when the animals were pretreated with A1AT. Because proteinase activated receptor-1 (PAR-1) is known to control MMP-12 release, macrophages were treated with the G protein-coupled receptor inhibitor, pertussis toxin; this suppressed both TNF-alpha and MMP-12 release, while a PAR-1 agonist (TRAP) increased TNF-alpha and MMP-12 release. Smoke-conditioned medium caused increased release of the prothrombin activator, tissue factor, from macrophages. Hirudin, a thrombin inhibitor, and aprotinin, an inhibitor of plasmin, reduced smoke-mediated TNF-alpha and MMP-12 release, and A1AT inhibited both plasmin and thrombin activity in a cell-free functional assay. These findings extend our previous suggestion that TNF-alpha production by alveolar macrophages is related to MMP-12 secretion. They also suggest that A1AT can inhibit thrombin and plasmin in blood constituents that leak into the lung after smoke exposure, thereby preventing PAR-1 activation and MMP-12/TNF-alpha release, and decreasing smoke-mediated inflammatory cell influx.     

Effects of the protease inhibitor (Pi) polymorphism on alpha-1-antitrypsin concentration and elastase inhibitory capacity in human serum

The concentration of alpha-1-antitrypsin (AAT) and its elastase inhibitory capacity (EIC) have been investigated in vitro in sera from 1688 healthy Canberra blood donors typed for electrophoretic variants of the protease inhibitor (Pi) locus. Nine Pi alleles were recorded in the sample, of which M1 was found at a frequency of nearly 70% and the other eight were each at frequencies below 15%. As a class, heterozygotes among the three Pi M subtype alleles, M1, M2 and M3, have higher means and lower variances for AAT and EiC than do the three M subtype homozygotes. Among the three homozygotes M1M1 has highest AAT and EIC and among the heterozygotes dominance in M1M2 and M1M3 is towards or beyond the high M1M1 values. Of the six other Pi alleles recorded, two (F and G) have similar values to the M subtypes but the other four (I, N, S and Z) have lower values. The patterns of means and variances in AAT and EIC for the different M subtype genotypes do not support the precise threshold function postulated by Martin and Oakeshott (1983) to relate activity to Darwinian fitness. Nevertheless, several aspects of the results are consistent with a general positive relationship between activity and fitness.     

ICAM-1 isoforms: specific activity and sensitivity to cleavage by leukocyte elastase and cathepsin G

The extracellular moiety of ICAM-1 consists of five Ig-like domains, the first and third domains mediating adhesion to integrin ligands. The ICAM-1 gene, however, gives rise to the expression of five alternative splice variants containing two, three, or four Ig-like domains. In this work, we have investigated whether the rearrangement of the architecture of ICAM-1 affects its structural properties and function. We showed that, in contrast to the common form, all alternative isoforms of ICAM-1 were susceptible to cleavage by leukocyte elastase and cathepsin G. We found that the length of an isoform did not influence the susceptibility to proteolysis. The molecular diversity provided by the skipping of entire Ig domains and the level of expression on the APC, however, significantly influenced their ability to potentiate the proliferation of T cells. Finally, we found that the expression of minor ICAM-1 isoforms encoding the third Ig-like domains was sufficient to sustain neutrophil infiltration in the liver and confer exon-5-targeted ICAM-1-deficient mice susceptibility to LPS-induced septic shock. These findings not only demonstrate that ICAM-1 isoforms are fully functional, but support the concept that alternative RNA splicing in the Ig superfamily may fulfill distinct roles during the development of the immune response.     

Inhibition of human leukocyte elastase bound to elastin: relative ineffectiveness and two mechanisms of inhibitory activity

Human leukocyte elastase (HLE) has been demonstrated on lung elastic fibers in areas of pulmonary emphysema. In vitro studies in our laboratory have shown that HLE-elastin complexes may be remarkably stable. We tested the possibility that elastin-bound HLE may retain catalytic activity in the presence of inhibitors that are effective against free HLE and found: (1) alpha-1-proteinase inhibitor (alpha 1PI), antileukoprotease (ALP), and eglin C inhibited free HLE on an approximately 1:1 molar basis, measured with either 3H-elastin or a synthetic peptide substrate; (2) the ability of each inhibitor to control catalytic activity of HLE when complexed with elastin was impaired (e.g., in a 24-h assay, a 70-fold molar excess of alpha 1PI gave only 93% inhibition of HLE); and (3) a chloromethyl ketone inhibitor of HLE gave qualitatively similar results, although at the low enzyme concentrations used it was a less effective inhibitor of free and elastin-bound enzyme than were the polypeptide inhibitors. Further, we found evidence for two distinct mechanisms of inhibition of elastin-bound HLE. alpha 1PI and eglin C prevented elastin solubilization largely by enhancing net dissociation of HLE from the complexes; enzyme remaining bound to the substrate retained essentially full activity. In contrast, ALP and the chloromethyl ketone prevented elastin solubilization by binding to the complexes and inhibiting the enzyme in situ. These results may have implications regarding progressive elastin solubilization in vivo and should stimulate further investigation of enzyme activity in heterogeneous systems in which one or more reactants are insoluble.     

Human follicle-stimulating hormone produced by recombinant DNA technology: a review for clinicians

Human follicle-stimulating hormone (FSH) is now produced in vitro by recombinant DNA technology. FSH being a complex heterodimeric protein, a eukaryotic cell line has been selected for expression work (Chinese hamster ovary cells). The pharmaceutical preparation of recombinant human FSH (r-FSH) differs from that of human menopausal gonadotrophin (HMG) and the first generation of urinary human FSH (u-FSH) in terms of (i) source of bulk materials, (ii) purity and specific activity, (iii) batch to batch consistency, and (iv) complete absence of luteinizing hormone (LH) activity. Pharmacokinetic characterization of r-FSH has shown an absolute bioavailability of approximately 75% after both s.c. and i.m. administration and an apparent terminal half-life of 37 +/- 25 h. These characteristics are very similar to those of u-FSH. Clinical efficacy and safety are currently demonstrated through several randomized, well controlled studies, comparing r-FSH administered s.c. with u-FSH administered i.m. for stimulating follicular development prior to assisted reproduction treatment and in World Health Organization (WHO) group II anovulation. To date, approximately 1000 patients have been treated with r-FSH. Moreover, r-FSH has recently been used successfully in association with recombinant human LH for inducing ovulation and pregnancy in WHO group I anovulatory patients. At this stage of r-FSH preparation assessment, it is likely that r-FSH will replace all urinary-derived FSH preparations for stimulating ovarian follicular development. For clinicians, current experience with r-FSH indicates that it should be used with the regimes and doses applied to u-FSH.     

Characterization of natural Dac g 1 variants: an alternative to recombinant group 1 allergens

BACKGROUND: Production of soluble correctly folded recombinant group 1 allergens has proven to be difficult. Purified natural group 1 allergens could be an alternative for application in immunotherapy. OBJECTIVE: Cloning and expression of recombinant Dac g 1; purification of natural Dac g 1 variants and immunochemical characterization of these molecules. METHODS: Dac g 1 was cloned and expressed in the yeast Pichia pastoris. Hydrophobic interaction (HIC), size exclusion, and/or affinity chromatography were used to purify Dac g 1 from Dactylis glomerata pollen extract. Dac g 1 variants were analyzed by N-terminal sequencing. Immune reactivity was assessed by sandwich ELISA, competitive RIA, RAST (inhibition), and in vitro basophil histamine release tests. RESULTS: Dac g 1 was cloned, revealing up to 98% amino acid sequence homology to other group 1 allergens. Purification of natural Dac g 1 revealed at least 3 variants, with an apparent molecular mass (Mr) on SDS-PAGE of 33 kd (HMr), 30 kd (IMr) and 28 kd (LMr). Extraction of IMr Dac g 1 required 0.9% saline, whereas the other 2 variants were also extractable in water. The N-terminus of HMr and IMr Dac g 1 differs at 2 positions, and LMr Dac g 1 was shown to be N-terminally truncated, lacking the first 30 amino acids. The nonretarded fraction of HIC commonly used in group 1 purification protocols does not contain this LMr molecule. IMr Dac g 1 was poorly recognized in 2 of 3 sandwich ELISAs and competitive RIA but demonstrated similar biological activity compared with HMr Dac g 1. CONCLUSIONS: Natural Dac g 1 variants can be separated by extraction of pollen in the presence or absence of saline followed by HIC and size exclusion chromatography. Thus, purified Dac g 1 is an alternative to recombinant group 1 allergens.     

Alpha 1-antitrypsin, alpha 1-antichymotrypsin, and alpha 2-macroglobulin in human gastric carcinomas: a retrospective immunohistochemical study

One hundred twenty-six gastric carcinomas (68 advanced cancers and 58 early cancers) were examined immunohistochemically for alpha 1-antitrypsin (AAT), alpha 1-antichymotrypsin (ACT), and alpha 2-macroglobulin (AMG) within tumor cells. The incidence of these three protease inhibitors was markedly higher in advanced than in early cancers, regardless of the histologic type of gastric carcinoma. In advanced cancers the incidence of both AAT and AMG was significantly higher in well-differentiated adenocarcinomas than in poorly differentiated adenocarcinomas, but no difference was observed in the expression of ACT between these two types of advanced carcinomas. Eighty per cent of the AAT-positive advanced carcinomas had ACT, and 40 per cent of these tumors also contained AMG. The two-year survival rates clearly indicated that well-differentiated adenocarcinomas with AAT have worse prognoses than well-differentiated adenocarcinomas without AAT, but there was no relation between the expression of ACT or AMG and prognosis. These results strongly suggest that the presence of protease inhibitors in gastric carcinomas is related to the invasive growth of the tumors and that AAT is a tissue tumor marker of well-differentiated adenocarcinomas of the stomach. It may also serve as a biologic marker of high malignancy in patients with these gastric cancers.     

Susceptibility of HIV-1 non-B subtypes and recombinant variants to Enfuvirtide.

BACKGROUND: Data on susceptibility of HIV-1 non-B subtypes to Enfuvirtide (ENF) is rather limited. OBJECTIVE: To determine if ENF could be active in vitro against HIV-1 non-B subtypes and how the gp41 genetic variability across variants may influence ENF susceptibility. METHODS: Using PHENOSCRIPT Env, a recombinant envelope virus assay, ENF susceptibility was investigated in isolates from 19 drug-naive HIV-1-infected individuals harboring non-B subtypes. RESULTS: Using phylogenetic analyses of the gp41 gene, distinct HIV-1 subtypes were recognized: A (2), C (5), D (1), F (2), G (1), J (1), CRF02_AG (6) and CRF06 (1). Susceptibility to ENF and IC(50) values in vitro could be obtained in only 13 (68.4%) specimens, most likely due to the high genetic variability in HR1 and HR2 regions in the remaining cases. A wide range of IC(50) values with a median of 0.013 microg/ml was observed (range, 0.005-0.180 microg/ml). Natural polymorphisms, but not classical ENF resistance associated mutations within HR1 (residues 36-45) were identified in most non-B viruses. CONCLUSION: This study provides information about the baseline susceptibility to ENF in antiretroviral-naive subjects infected with different HIV-1 non-B subtypes. Susceptibility to ENF seems to be preserved despite high genetic variability in HR1 and HR2 regions.     

Alpha 1-antitrypsin binds to and interferes with functionality of EspB from atypical and typical enteropathogenic Escherichia coli strains

Enteropathogenic Escherichia coli (EPEC), including diffusely adhering atypical E. coli, strains use a type III secretion system to deliver effector proteins into the membrane and cytoplasm of infected cells. The E. coli secreted proteins A, B, and D (EspA, EspB, and EspD) are required for the formation of the characteristic attaching and effacing (A/E) lesions. EspB and EspD are thought to form a translocation pore in the host cell membrane through which effector proteins are injected into the host cytosol. Besides its function in pore formation, EspB has been found in the cytosol and implicated to function as an effector protein. We screened for putative host cell proteins interacting with EspB of atypical EPEC strains and identified alpha(1)-antitrypsin (AAT) as a binding partner for EspB. AAT binds to EspB in pull-down and overlay experiments and also to EspD in overlay experiments. In agreement with the role of EspB and EspD in pore formation, EPEC-mediated hemolysis of red blood cells is strongly reduced by AAT in a concentration-dependent manner, indicating that AAT interferes with type III secretion by inhibiting the formation of the translocation pore. This is further supported by a decreased actin polymerization after infection of HeLa or CaCo-2 cells with EPEC in the presence of physiologically relevant concentrations of AAT. In this study, we identify AAT as a new binding partner for EspB and EspD, suggesting a previously unappreciated role for AAT in host cell defense against EPEC infections and potentially also against other bacterial pathogens.     

Relationship of DNA strand breakage produced by bromodeoxyuridine to topoisomerase II activity in Bloom-syndrome fibroblasts

Cells from patients with Bloom syndrome, a cancer-prone disorder with cutaneous photosensitivity and spontaneous chromosome breakage, exhibit an abnormally increased number of sister-chromatid exchanges following treatment with 5-bromodeoxyuridine (BrdU). This effect has been postulated to be mediated by abnormal topoisomerase II activity. We used alkaline elution to measure DNA single-strand breakage following prolonged exposure to BrdU. Five-day exposure to BrdU produced equal numbers of alkali-labile sites in normal and Bloom-syndrome fibroblasts. These breaks were not protein-associated but were produced by alkali. Treatment with topoisomerase II inhibitors induced similar frequencies of DNA single-strand breaks in normal and Bloom-syndrome fibroblasts. These findings imply that BrdU incorporation into cellular DNA induces alkali-labile DNA lesions that are independent of topoisomerase II activity in Bloom and normal cells.     

Novel identification of differences in the kinetoplast DNA of Leishmania isolates by recombinant DNA techniques and in situ hybridisation

Kinetoplast DNA (kDNA) was extracted from marker isolates of three 'Old World' cutaneous leishmania, L. tropica, L. aethiopica and L. major. Restriction endonuclease digestion followed by electrophoresis showed that each isolate produced a unique pattern of DNA fragments. The kDNA of each isolate was hybridised to Southern blots of digests of all three kDNAs. This showed that the kDNA of each isolate contained sequences unique to that isolate as well as sequences common to all three isolates. The kDNA sequences of L. tropica and L. aethiopica were more closely related than either were to those of L. major. kDNA of each isolate was cloned and plasmids selected which contained a fragment of DNA unique to the isolate from which the kDNA originated. Whole kDNA was hybridised to organisms fixed on a microscope slide. In each case the kDNA hybridised strongly to the kinetoplast of the organism from which the DNA had been extracted. There was a small amount of cross-hybridisation between L. tropica and L. aethiopica confirming the result of the Southern blot hybridisation. The feasibility of a method of isolate identification using recombinant DNA probes containing sequences unique to a particular isolate or group of isolates is discussed.     

Alpha1-antitrypsin as a risk for infant and adult respiratory outcomes in a national birth cohort

Reduced alpha1-antitrypsin (AAT) encoded by the gene SERPINA1 is a potential risk for pulmonary disease. We investigated SERPINA1 polymorphism as a risk for infant and adult pulmonary morbidity, and adult respiratory function and its change between 43 and 53 yr. We used data on a British national representative sample (n = 5,362) studied since birth in 1946 to age 53 yr (when n = 3,035), when DNA was first obtained. SERPINA1 Z and, to a lesser extent, S carriers had an increased risk of infant lower respiratory infection compared with those who were neither S nor Z carriers (Z carriers: odds ratio = 2.32, 95% confidence interval = 1.37-3.92; S but not Z carriers odds ratio = 1.58, 95% confidence interval = 1.10-2.28) after adjustment for environmental, socioeconomic, and developmental factors, and breast-feeding. There was no difference in the adult outcomes at 53 yr according to genotype, nor was there any association of genotype with change in forced expiratory volume at 1 s between 43 and 53 yr. Lower alpha1-antitrypsin, as indicated by carrier status for the Z and S alleles, was a risk for infant lower respiratory infection, but not for adult respiratory outcomes.     

Alpha1-antitrypsin and alpha-fetoprotein. Protein markers in endodermal sinus (yolk sac) tumors.

A combined immunocytochemical and quantitative serum and tissue study was performed on a group of endodermal sinus (yolk sac) tumors, localizing and measuring both alpha1-antitrypsin (AAT) and alpha-fetoprotein (AFP) in tumor tissue and patient sera. Utilizing indirect immunofluorescent and triple-sandwich immunoperoxidase methods, both proteins were demonstrated within intra- and extracellular periodic acid-Schiff-positive hyaline globules characteristic of the tumor, as well as within the cytoplasm of tumor epithelial cells lining endodermal sinuses, where AAT deposition predominated. Tumor tissue extracts confirmed the presence of significant quantities of both proteins, and pretreatment serum elevations of both showed a parallel decline during therapy. In this study, AAT is characterized as a tumor protein marker for the first time, and a parallelism between AAT and AFP is demonstrated in both serum and tumor tissue. These findings represent additional supportive evidence for the yolk sac origin of endodermal sinus tumors in man.     

Alpha 1-antitrypsin deficiency and liver cirrhosis in adults. An analysis of 35 Swedish autopsied cases

alpha 1-Antitrypsin (AAT) deficiency in adults predisposes to lung and liver disease, but its natural history is incompletely known. To better characterize the liver disease, all known deceased adult Swedish patients known to us with homozygous (PiZZ) AAT-deficiency, who had undergone autopsy during the 20-year period 1963-82 were reviewed. Of 94 such patients, 35 had cirrhosis (27 males and eight females) with a mean age at death of 65.5 +/- 10.5 (SD) years compared to 53.6 +/- 12.8 years (p less than 0.01) for the 59 non-cirrhotic patients. The longer survival suggests less severe lung disease in the cirrhotic group. Clinically these patients most frequently presented with ascites or other signs of portal hypertension. Evidence of alcohol overconsumption, chronic viral hepatitis, or autoimmune disease was rare. Aside from low plasma AAT levels, laboratory and other clinical features were indistinguishable from those of decompensated cirrhosis of any etiology. The prognosis was generally grave with a mean survival of two years after diagnosis. Fourteen of the 35 cirrhotics (10 males and four females) had primary liver cancer (PLC) at autopsy. We conclude that cirrhosis and PLC are more frequent complications in elderly patients with AAT-deficiency than was previously known. These complications had a marked male predominance.