The molecular species responsible for α1 -antitrypsin deficiency are suppressed by a small molecule chaperone.

The formation of ordered Z (Glu342Lys) α1 -antitrypsin polymers in hepatocytes is central to liver disease in α1 -antitrypsin deficiency. In vitro experiments have identified an intermediate conformational state (M*) that precedes polymer formation, but this has yet to be identified in vivo. Moreover, the mechanism of polymer formation and their fate in cells have been incompletely characterised. We have used cell models of disease in conjunction with conformation-selective monoclonal antibodies and a small molecule inhibitor of polymerisation to define the dynamics of polymer formation, accumulation and secretion. Pulse-chase experiments demonstrate that Z α1 -antitrypsin accumulates as short-chain polymers that partition with soluble cellular components and are partially secreted by cells. These precede the formation of larger, insoluble polymers with a longer half-life (10.9 ± 1.7 h and 20.9 ± 7.4 h for soluble and insoluble polymers, respectively). The M* intermediate (or a by-product thereof) was identified in the cells by a conformation-specific monoclonal antibody. This was completely abrogated by treatment with the small molecule, which also blocked the formation of intracellular polymers. These data allow us to conclude that the M* conformation is central to polymerisation of Z α1 -antitrypsin in vivo; preventing its accumulation represents a tractable approach for pharmacological treatment of this condition; polymers are partially secreted; and polymers exist as two distinct populations in cells whose different dynamics have likely consequences for the aetiology of the disease.

An analog of glibenclamide selectively enhances autophagic degradation of misfolded α1-antitrypsin Z.

The classical form of α1-antitrypsin deficiency (ATD) is characterized by intracellular accumulation of the misfolded variant α1-antitrypsin Z (ATZ) and severe liver disease in some of the affected individuals. In this study, we investigated the possibility of discovering novel therapeutic agents that would reduce ATZ accumulation by interrogating a C. elegans model of ATD with high-content genome-wide RNAi screening and computational systems pharmacology strategies. The RNAi screening was utilized to identify genes that modify the intracellular accumulation of ATZ and a novel computational pipeline was developed to make high confidence predictions on repurposable drugs. This approach identified glibenclamide (GLB), a sulfonylurea drug that has been used broadly in clinical medicine as an oral hypoglycemic agent. Here we show that GLB promotes autophagic degradation of misfolded ATZ in mammalian cell line models of ATD. Furthermore, an analog of GLB reduces hepatic ATZ accumulation and hepatic fibrosis in a mouse model in vivo without affecting blood glucose or insulin levels. These results provide support for a drug discovery strategy using simple organisms as human disease models combined with genetic and computational screening methods. They also show that GLB and/or at least one of its analogs can be immediately tested to arrest the progression of human ATD liver disease.

Inactivation of α1-proteinase inhibitor by Candida albicans aspartic proteases favors the epithelial and endothelial cell colonization in the presence of neutrophil extracellular traps.

Candida albicans, a causative agent of opportunistic fungal infections in immunocompromised patients, uses ten secreted aspartic proteases (SAPs) to deregulate the homeostasis of the host organism on many levels. One of these deregulation mechanisms involves a SAP-dependent disturbance of the control over proteolytic enzymes of the host by a system of dedicated proteinase inhibitors, with one important example being the neutrophil elastase and alpha1-proteinase inhibitor (A1PI). In this study, we found that soluble SAPs 1-4 and the cell membrane-anchored SAP9 efficiently cleaved A1PI, with the major cleavage points located at the C-terminal part of A1PI in a close vicinity to the reactive-site loop that plays a critical role in the inhibition mechanism. Elastase is released by neutrophils to the environment during fungal infection through two major processes, a degranulation or formation of neutrophil extracellular traps (NET). Both, free and NET-embedded elastase forms, were found to be controlled by A1PI. A local acidosis, resulting from the neutrophil activity at the infection sites, favors A1PI degradation by SAPs. The deregulation of NET-connected elastase affected a NET-dependent damage of epithelial and endothelial cells, resulting in the increased susceptibility of these host cells to candidal colonization. Moreover, the SAP-catalyzed cleavage of A1PI was found to decrease its binding affinity to a proinflammatory cytokine, interleukin-8. The findings presented here suggest a novel strategy used by C. albicans for the colonization of host tissues and overcoming the host defense.

Small-molecule peptides inhibit Z alpha1-antitrypsin polymerization.

The Z variant of 1-antitrypsin (AT) polymerizes within the liver and gives rise to liver cirrhosis and the associated plasma deficiency leads to emphysema. In this work, a combinatorial approach based on the inhibitory mechanism of (alpha1)-AT was developed to arrest its pathogenic polymerization. One peptide, Ac-TTAI-NH(2), emerged as the most tight-binding ligand for Z (alpha1)-AT. Characterization of this tetrapeptide by gel electrophoresis and biosensor analysis revealed its markedly improved binding specificity and affinity compared with all previously reported peptide inhibitors. In addition, the peptide is not cytotoxic to lung cell lines. A model of the peptide-protein complex suggests that the peptide interacts with nearby residues by hydrogen bonds, hydrophobic interactions, and cavity-filling stabilization. The combinatorially selected peptide not only effectively blocks the polymerization but also promotes dissociation of the oligomerized (alpha1)-AT. These results are a significant step towards the potential treatment of Z (alpha1)-AT related diseases.

Effects of coronary prestenting platelet inhibition on coronary poststenting inflammation.

The aim of this study was to analyze the effect of 2 antiplatelet regimens on the inhibition of GP IIb/IIIa-dependent platelet activation and their association with the poststenting inflammatory response. Seventeen patients with acute myocardial infarction were divided into 2 groups: (A) clopidogrel plus tirofiban infusion administered together during inclusion (n = 10); (B) clopidogrel administered at inclusion and followed 2 hours after by tirofiban (n = 7). Blood samples were obtained at inclusion and at 24 and 48 hours after stenting. Before stenting, a greater reduction of GP IIb/IIIa-dependent platelet activation was found in both groups, although it was greater in group A than in group B. This statistical difference was not observed at 24 and 48 hours after the procedure. At 48 hours after stenting, interleukin-6, interleukin-10, soluble intracellular adhesion molecule-1, and soluble CD40 ligand plasma values were not different between experimental groups. By proteomics, different isoforms of the following proteins were identified: alpha 1-antitrypsin (ATT-1), fibrinogen gamma chain, apolipoprotein A-IV, apolipoprotein A-I, vitamin D binding protein, haptoglobin, and serotransferrin. At 48 hours after stenting, only the plasma expression of the ATT-1 isoform 5 was significantly increased in group A compared with group B. In conclusion, a greater inhibition of GP IIb/IIIa-dependent platelet activation before stenting was not correlated with a different inflammatory activity early after stenting.

L-asparaginase-induced antithrombin type I deficiency: implications for conformational diseases.

Serpinopathies, a group of diseases caused by mutations that disrupt the structurally sensitive serpins, have no known acquired cause. Interestingly, l-asparaginase treatment of acute lymphoblastic leukemia patients causes severe deficiency in the serpin antithrombin. We studied the consequences of this drug on antithrombin levels, activity, conformation, and immunohistological and ultrastructural features in plasma from acute lymphoblastic leukemia patients, HepG2 cells, and plasma and livers from mice treated with this drug. Additionally, we evaluated intracellular deposition of alpha1-antitrypsin. l-Asparaginase did not affect functional or conformational parameters of mature antithrombin; however, patients and mice displayed severe type I deficiency with no abnormal conformations of circulating antithrombin. Moreover, l-asparaginase impaired secretion of antithrombin by HepG2 cells. These effects were explained by the intracellular retention of antithrombin, forming aggregates within dilated endoplasmic reticulum cisterns. Similar effects were observed for alpha1-antitrypsin in plasma, cells, and livers, and intracellular aggregates of additional proteins were observed in frontal cortex and pancreas. This is the first report of a conformational drug-associated effect on serpins without genetic factors involved. l-Asparaginase treatment induces severe, acquired, and transient type I deficiency of antithrombin (and alpha1-antitrypsin) with intracellular accumulation of the nascent molecule, increasing the risk of thrombosis.

Sugar and alcohol molecules provide a therapeutic strategy for the serpinopathies that cause dementia and cirrhosis.

Mutations in neuroserpin and alpha1-antitrypsin cause these proteins to form ordered polymers that are retained within the endoplasmic reticulum of neurones and hepatocytes, respectively. The resulting inclusions underlie the dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB) and Z alpha1-antitrypsin-associated cirrhosis. Polymers form by a sequential linkage between the reactive centre loop of one molecule and beta-sheet A of another, and strategies that block polymer formation are likely to be successful in treating the associated disease. We show here that glycerol, the sugar alcohol erythritol, the disaccharide trehalose and its breakdown product glucose reduce the rate of polymerization of wild-type neuroserpin and the Ser49Pro mutant that causes dementia. They also attenuate the polymerization of the Z variant of alpha1-antitrypsin. The effect on polymerization was apparent even when these agents had been removed from the buffer. None of these agents had any detectable effect on the structure or inhibitory activity of neuroserpin or alpha1-antitrypsin. These data demonstrate that sugar and alcohol molecules can reduce the polymerization of serpin mutants that cause disease, possibly by binding to and stabilizing beta-sheet A.

Probiotic effects on faecal inflammatory markers and on faecal IgA in food allergic atopic eczema/dermatitis syndrome infants.

Probiotic bacteria are proposed to alleviate intestinal inflammation in infants with atopic eczema/dermatitis syndrome (AEDS) and food allergy. In such infants we investigated effects of probiotic bacteria on faecal IgA, and on the intestinal inflammation markers tumour necrosis factor-alpha (TNF-alpha), alpha1-antitrypsin (AT), and eosinophil cationic protein (ECP). A total of 230 infants with AEDS and suspected cow's milk allergy (CMA) received in a randomized double-blinded manner, concomitant with elimination diet, Lactobacillus GG (LGG), a mixture of four probiotic strains (MIX), or placebo for 4 wk. Four weeks after treatment, CMA was diagnosed with a double-blind placebo-controlled milk challenge. Faecal samples of 102 infants, randomly chosen for analysis, were collected before treatment, after 4-wk treatment, and on the first day of milk challenge. After treatment, IgA levels tended to be higher in probiotic groups than in the placebo group (LGG vs. placebo, p=0.064; MIX vs. placebo, p=0.064), and AT decreased in the LGG group, but not in other treatment groups. After challenge in IgE-associated CMA infants, faecal IgA was higher for LGG than for placebo (p=0.014), and TNF-alpha was lower for LGG than for placebo, but non-significantly (p=0.111). In conclusion, 4-wk treatment with LGG may alleviate intestinal inflammation in infants with AEDS and CMA.

Lack of effect of oral 4-phenylbutyrate on serum alpha-1-antitrypsin in patients with alpha-1-antitrypsin deficiency: a preliminary study.

OBJECTIVE: In homozygotes with ZZ genotype alpha-1-antitrypsin (alpha1AT) deficiency, mutant alpha1ATZ protein (alpha1ATZ) accumulates in hepatocytes, rather than being secreted into the blood. Homozygous individuals experience emphysema as a result of reduced levels of circulating alpha1AT in the lung with which to inhibit connective tissue breakdown. Homozygotes may also experience liver disease from the accumulation of alpha1ATZ within hepatocytes, which causes liver damage. A previous study indicated that the compound 4-phenylbutyrate (4-PBA) mediated a significant increase in release of alpha1ATZ from cells in tissue culture and in a mouse model of alpha1AT deficiency. The authors hypothesized that 4-PBA could be used to treat both the liver and lung disease of humans with alpha1AT deficiency. METHODS: In this preliminary, open label study the authors evaluated the effect of 14 days of oral 4-PBA therapy on alpha1AT blood levels in 10 patients with alpha1AT deficiency. RESULTS: There was no significant increase in alpha1AT blood level associated with 4-PBA administration. Symptomatic and metabolic side effects were significant. CONCLUSION: 4-PBA did not increase alpha1AT blood levels in humans with alpha1AT deficiency in this preliminary trial.

Antioxidant properties of sulfinates: protective effect of hypotaurine on peroxynitrite-dependent damage.

It has been proposed that hypotaurine may function as an antioxidant in vivo. We investigated whether this compound can act as protective agent able to prevent damage from peroxynitrite, a strong oxidizing and nitrating agent that reacts with several biomolecules. The results showed that the compound efficiently protects tyrosine against nitration, alpha1-antiproteinase against inactivation, and human low-density lipoprotein against modification by peroxynitrite. Hypotaurine is also highly effective in inhibiting peroxynitrite-mediated nitration of tyrosine in the presence of added bicarbonate. This result suggests that hypotaurine could play an important role as protective agent under physiological conditions. Moreover, it was found that cysteine sulfinic acid, but not taurine, possesses protective properties against peroxynitrite-dependent damage similar to hypotaurine. These findings indicate that the protective effects exerted by these compounds may be attributable to the presence of the sulfinic group oxidizable into sulfonate by scavenging peroxynitrite and/or its derived species.

Ambroxol inhibits peroxynitrite-induced damage of alpha1-antiproteinase and free radical production in activated phagocytic cells.

The present study examined the effect of ambroxol on toxic action of peroxynitrite and the respiratory burst in activated phagocytic cells. Ambroxol decreased the inactivation or destruction of alpha1-antiproteinase induced by peroxynitrite (ONOO-) or hypochlorous acid (HOCl), which was similar to penicillamine and glutathione and was greater than diclofenac sodium and naproxen sodium. Ambroxol significantly decreased ONOO--mediated tyrosine nitration and iron plus EDTA-mediated degradation of 2-deoxy-D-ribose. Ambroxol significantly attenuated the production of superoxide, hydrogen peroxide, HOCl, and nitric oxide in fMLP- or IL-1-activated phagocytic cells, while the inhibitory effects of antiinflammatory and thiol compounds were only observed in HOCl production. Ambroxol and antiinflammatory drugs did not show a cytotoxic effect on macrophages. The results suggest that ambroxol protects tissue components against oxidative damage by an action different from antiinflammatory drugs. Ambroxol may interfere with oxidative damage of alpha1-antiproteinase through a scavenging action on ONOO- and HOCl and inhibition of the respiratory burst of phagocytic cells.

Effect of beta propiolactone viral inactivation on alpha1 antitrypsin values.

AIMS: alpha1 Antitrypsin was undetectable in several patient samples treated with 0.5% beta propiolactone, which was used as a virucidal agent. This study was designed to confirm beta propiolactone as the cause and determine why it might have such an effect. METHODS: Volumes of 0, 5, 10, and 20 micro l of beta propiolactone were added to 2 ml aliquots of serum to make final concentrations of 0%, 0.25%, 0.5%, and 1% of beta propiolactone. alpha1 Antitrypsin concentrations and the pH were measured at different time intervals. The effects of adding buffer before the addition of beta propiolactone, NaOH after beta propiolactone, and 6M HCl instead of beta propiolactone were also measured. RESULTS: The addition of beta propiolactone to a volunteer's serum showed a fall in both alpha1 antitrypsin values and pH with increasing time and concentration of beta propiolactone. This effect was also seen when adding HCl, but was partially prevented by buffering the serum or adding NaOH. CONCLUSIONS: These results suggest that it is the acidity of the degradation products of beta propiolactone that is responsible for the fall in alpha1 antitrypsin values. This fall in alpha1 antitrypsin values was dependent on the concentration of beta propiolactone used and the length of time before the test was performed. The effect of beta propiolactone on laboratory tests should be re-evaluated, with attention being paid to sample pH, storage time, and storage temperature.

Prevention of polymerization of M and Z alpha1-Antitrypsin (alpha1-AT) with trimethylamine N-oxide. Implications for the treatment of alpha1-at deficiency.

alpha1-Antitrypsin (alpha1-AT) is the most abundant circulating proteinase inhibitor. The Z variant results in profound plasma deficiency as the mutant polymerizes within hepatocytes. The retained polymers are associated with cirrhosis, and the lack of circulating protein predisposes to early onset emphysema. We have investigated the role of the naturally occurring solute trimethylamine N-oxide (TMAO) in modulating the polymerization of normal M and disease-associated Z alpha1-AT. TMAO stabilized both M and Z alpha1-AT in an active conformation against heat-induced polymerization. Spectroscopic analysis demonstrated that this was due to inhibition of the conversion of the native state to a polymerogenic intermediate. However, TMAO did not aid the refolding of denatured alpha1-AT to a native conformation; instead, it enhanced polymerization. These data show that TMAO can be used to control the conformational transitions of folded alpha1-AT but that it is ineffective in promoting folding of the polypeptide chain within the secretory pathway.

Role of ubiquitin in proteasomal degradation of mutant alpha(1)-antitrypsin Z in the endoplasmic reticulum.

A delay in intracellular degradation of the mutant alpha(1)-antitrypsin (alpha(1)AT)Z molecule is associated with greater retention within the endoplasmic reticulum (ER) and susceptibility to liver disease in a subgroup of patients with alpha(1)AT deficiency. Recent studies have shown that alpha(1)ATZ is ordinarily degraded in the ER by a mechanism that involves the proteasome, as demonstrated in intact cells using human fibroblast cell lines engineered for expression of alpha(1)ATZ and in a cell-free microsomal translocation assay system programmed with purified alpha(1)ATZ mRNA. To determine whether the ubiquitin system is required for proteasomal degradation of alpha(1)ATZ and whether specific components of the ubiquitin system can be implicated, we have now used two approaches. First, we overexpressed a dominant-negative ubiquitin mutant (UbK48R-G76A) by transient transfection in the human fibroblast cell lines expressing alpha(1)ATZ. The results showed that there was marked, specific, and selective inhibition of alpha(1)ATZ degradation mediated by UbK48R-G76A, indicating that the ubiquitin system is at least in part involved in ER degradation of alpha(1)ATZ. Second, we subjected reticulocyte lysate to DE52 chromatography and tested the resulting well-characterized fractions in the cell-free system. The results showed that there were both ubiquitin-dependent and -independent proteasomal mechanisms for degradation of alpha(1)ATZ and that the ubiquitin-conjugating enzyme E2-F1 may play a role in the ubiquitin-dependent proteasomal mechanism.

Cefoperazone prevents the inactivation of alpha(1)-antitrypsin by activated neutrophils.

At sites of neutrophilic inflammation, tissue injury by neutrophil elastase is favored by phagocyte-induced hypochlorous acid-dependent inactivation of the natural elastase inhibitor alpha(1)-antitrypsin. In the present study, cefoperazone prevented alpha(1)-antitrypsin inactivation by neutrophils and reduced the recovery of hypochlorous acid from these cells. Moreover, the antibiotic reduced the free elastase activity in a neutrophil suspension supplemented with alpha(1)-antitrypsin without affecting the cells' ability to release elastase. These data suggest that the drug inactivates hypochlorous acid before its reaction with alpha(1)-antitrypsin, thereby permitting the antiprotease-mediated blockade of released elastase. In conclusion, cefoperazone appears to have the potential for limiting elastase-antielastase imbalances, attenuating the related tissue injury at sites of inflammation.

Modulation of peroxynitrite- and hypochlorous acid-induced inactivation of alpha1-antiproteinase by mercaptoethylguanidine.

1. Peroxynitrite is a cytotoxic species that can be formed, among other mechanisms, by the rapid reaction of superoxide with nitric oxide. Peroxynitrite formation has been implicated in a wide range of neurodegenerative and chronic inflammatory diseases, as has the formation of hypochlorous acid by myeloperoxidase. 2. There is considerable interest in the development of peroxynitrite scavengers as therapeutic agents. The thiol compound mercaptoethylguanidine has been suggested to fulfil this role since it has recently been shown to be not only a potent inhibitor of inducible nitric oxide synthase but also a scavenger of peroxynitrite. Indeed, it has been shown to be protective in some experimental models of circulatory shock and inflammation at plasma levels in the approximate range 100-300 microM. 3. One protein inactivated by peroxynitrite is the major inhibitor of serine proteinases in human body fluids, alpha1-antiproteinase. At high (250-1000 microM) concentrations, mercaptoethylguanidine was found to be effective in preventing peroxynitrite-mediated tyrosine nitration and alpha1-AP inactivation. 4. By contrast, lower concentrations of mercaptoethylguanidine (1-60 microM) enhanced the inactivation of alpha1-antiproteinase by peroxynitrite. 5. At all concentrations tested (1-1000 microM), mercaptoethylguanidine decreased the inactivation of alpha1-antiproteinase by hypochlorous acid. 6. We suggest that products of reaction of mercaptoethylguanidine with peroxynitrite or peroxynitrite-derived products could cause damage to alpha1-antiproteinase, and possibly other proteins in vivo, whereas scavenging of hypochlorous acid by mercaptoethylguanidine could contribute to its anti-inflammatory action in vivo.

Use of heparin in the treatment of protein-losing enteropathy after fontan operation for complex congenital heart disease.

Protein-losing enteropathy (PLE) is a serious complication of the Fontan operation and is associated with pronounced mortality. Medical management of PLE has been only partially successful. A recent report noted dramatic improvement in patients with PLE within 3 weeks of subcutaneous administration of heparin. We report a case of reversal of PLE with resolution of clinical symptoms and normalization of serum albumin, total protein, and fecal alpha1-antitrypsin values after several months of heparin treatment. Our findings substantiate those recently reported but suggest that reversal of PLE may necessitate more than a few weeks of heparin therapy.

Local control of alpha1-proteinase inhibitor levels: regulation of alpha1-proteinase inhibitor in the human cornea by growth factors and cytokines.

Alpha 1-proteinase inhibitor is a major serine proteinase inhibitor in the human cornea involved in the protection of the avascular corneal tissue against proteolytic damage. This inhibitor is upregulated systemically during infection, inflammation and injury. Cytokines that mediate the acute phase response such as IL-1beta and IL-2 increased alpha1-proteinase inhibitor present in corneal organ culture media. This released inhibitor represented mainly newly synthesized protein. However, IL-6, a general inducer of the acute phase response that upregulates alpha1-proteinase inhibitor in all other tissues and cells tested, failed to alter corneal alpha1-proteinase inhibitor levels over the tested period of 24 h. In addition to IL-1beta and IL-2, alpha1-proteinase inhibitor levels in the corneal organ culture medium increased following the addition of FGF-2 and IGF-I. The effect of the above growth factors and cytokines was relatively fast with maximal induction observed within the first 5 h. Among the tested growth factors and cytokines, IL-1beta was the most potent and increased total corneal alpha1-proteinase inhibitor levels approximately 2.4-fold in the cornea organ culture medium. Newly, synthesized alpha1-proteinase secreted into the medium increased 3.9-fold. In addition to the effect on corneal alpha1-proteinase inhibitor, IL-1beta also increased the amount of alpha1-proteinase inhibitor released by monocytes and macrophages but not by HepG2, CaCo2, and MCF-7 cells within 24 h. These results suggest that the cornea can locally control levels of alpha1-proteinase inhibitor in response to an inflammatory insult.

Oncostatin M is a potent stimulator of alpha1-antitrypsin secretion in lung epithelial cells: modulation by transforming growth factor-beta and interferon-gamma.

alpha1-Antitrypsin (alpha1-AT) plays a key role in lung homeostasis. Although the hepatocyte is considered as the primary source of alpha1-AT, we have previously demonstrated that rat alveolar epithelial type II cells as well as the human A549 cell line synthesize alpha1-AT, suggesting its local production within the lung. In the present study, we showed that oncostatin M, as opposed to interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), or IL-6, is a potent stimulator of alpha1-AT synthesis in the human A549 cell line. The oncostatin M-induced alpha1-AT secretion is modulated by interferon-gamma (IFN-gamma) and transforming growth factor-beta (TGF-beta) at both the protein and mRNA levels. IFN-gamma decreases oncostatin M-induced alpha1-AT secretion. By contrast, TGF-beta in combination with oncostatin M induces a dramatic and synergistic upregulation that is not observed in the HepG2 hepatocyte cell line. Our results suggest that during an inflammatory process, alveolar epithelial cells may contribute to the antiprotease defense within the lung.