Search

Aims α1-Antitrypsin (AAT) is a serine protease inhibitor which recently has been shown to prevent Type 1 diabetes development, to prolong islet allograft survival and to inhibit pancreatic B-cell apoptosis in vivo. It has also been reported that Type 1 diabetic patients have significantly lower plasma concentrations of AAT, suggesting the potential role of AAT in the pathogenesis of Type 1 diabetes. We have investigated whether plasma AAT levels are altered in Type 2 diabetes. Methods The study included patients with Type 2 diabetes ( n = 163) and non-diabetic control subjects matched for age, sex and smoking habits ( n = 158) derived from the population-based Malmö Diet and Cancer study. Plasma samples were analysed for AAT concentration and phenotype and serum glucose, insulin, C-reactive protein and lipid levels were measured. Glycated haemoglobin was also measured. Results In the diabetic group, the women had higher mean plasma AAT levels than men ( P < 0.05). The mean plasma AAT levels did not differ between diabetic and control subjects. However, the number of individuals with low AAT levels (< 1.0 mg/ml) was 50% higher in the diabetic group ( P < 0.05) and the frequency of AAT deficiency genotypes was 50% higher (NS) in diabetic compared with control subjects. In the group of diabetic patients with AAT < 1 mg/ml, AAT directly correlated with systolic blood pressure ( P = 0.048) and inversely correlated with waist–hip ratio ( P = 0.031). Conclusions Our results provide evidence that deficiency of AAT may be associated with an increased risk of developing Type 2 diabetes. [ABSTRACT FROM AUTHOR]

The article focuses on the study which examines the increased 8-isoprostane, a marker of oxidative stress in exhaled breath condensate (EBC) in subjects with asbestos exposure. It is found that asbestosis and pleural plaques exhibit unpredictable but progressive development. Oxidative stress due to asbestos is the primary cause of increased 8-isoprostane in EBC. The measurement of 8-isoprostane in EBC is a promising non-invasive means for assessing the activity of asbestos-induced diseases.

Abstract: Members of the serine proteinase inhibitor or serpin superfamily inhibit their target proteinases by a conformational transition that involves the enzyme being translocated from the upper to the lower pole of the protein. This sophisticated mechanism is subverted by point mutations to form ordered polymers that are retained within the endoplasmic reticulum of the cell of synthesis. These polymers activate NF-κB and cause cytotoxicity by a pathway that is independent of the unfolded protein response. As diverse conditions can be explained the same mechanism of polymerisation we have grouped them together as a new class of disease, the serpinopathies. We review here the structural basis of the serpinopathies and discuss how the ordered accumulation of polymers causes cell death. [Copyright &y& Elsevier]

Abstract: Insulin-like growth factors (IGFs) belong to a family of growth factors with structural homology to proinsulin. Up till now, no specific details regarding the transcriptional regulation by autocrine, paracrine or endocrine effector molecules in vivo have been described for the IGF-II gene. This is in big contrast to IGF-I gene transcription which has been studied more extensively. To better understand how the IGF-II gene is controlled at the gene transcription level, we have isolated the common carp IGF-II gene together with the 5′-flanking region by genomic library screening. The mature IGF-II protein was encoded by exon 2 and exon 3. Transient transfection of the 5′-flanking region containing a TATA box-like sequence into cultured eukaryotic cells revealed that it is a strong promoter with definitive tissue specificity. Nucleotides between −301 and −62 in the promoter are essential to drive the basal IGF-II gene expression; whereas nucleotides between −891 and −416 in the promoter are responsible for the growth hormone activation. Using electrophoretic mobility shift assay and yeast one-hybrid screening, it was demonstrated that α1-antitrypsin could bind specifically to the nucleotide position −301 to −262 of the gene promoter. Co-transfection studies revealed that the over-expression of α1-antitrypsin increased the IGF-II promoter activity by 3.4-fold, further confirming that α1-antitrypsin acts as a trans-acting factor on the IGF-II promoter. [Copyright &y& Elsevier]

Abstract: Endothelial cells are among the main physiological targets of the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α). In endothelial cells TNF-α elicits a broad spectrum of biological effects including differentiation, proliferation and apoptosis. α1-antitrypsin (AAT), an endogenous inhibitor of serine proteases plays a vital role in protecting host tissue from proteolytic injury at sites of inflammation. Recently, it has been shown that AAT can be internalized by pulmonary endothelial cells, raising speculation that it may modulate endothelial cell function in addition to suppressing protease activity. Using Affymetrix microarray technology, real time PCR and ELISA methods we have investigated the effects of AAT on un-stimulated and TNF-α stimulated human primary lung microvascular endothelial cell gene expression and protein secretion. We find that AAT and TNF-α generally induced expression of distinct gene families with AAT exhibiting little activity in terms of inflammatory gene expression. Approximately 25% of genes up regulated by TNF-α were inhibited by co-administration of AAT including TNF-α-induced self expression. Surprisingly, the effects of AAT on TNF-α-induced self expression was inhibited equally well by oxidized AAT, a modified form of AAT, which lacks serine protease inhibitor activity. Overall, the pattern of gene expression regulated by native and oxidized AAT was similar with neither inducing pro-inflammatory gene expression. These findings suggest that inhibitory effects of native and oxidized forms of AAT on TNF-α stimulated gene expression may play an important role in limiting the uncontrolled endothelial cell activation and vascular injury in inflammatory disease. [Copyright &y& Elsevier]

Abstract: Terminally misfolded or unassembled proteins are degraded by the cytoplasmic ubiquitin-proteasome pathway in a process known as ERAD (endoplasmic reticulum-associated protein degradation). Overexpression of ER α1,2-mannosidase I and EDEMs target misfolded glycoproteins for ERAD, most likely due to trimming of N-glycans. Here we demonstrate that overexpression of Golgi α1,2-mannosidase IA, IB, and IC also accelerates ERAD of terminally misfolded human α1-antitrypsin variant null (Hong Kong) (NHK), and mannose trimming from the N-glycans on NHK in 293 cells. Although transfected NHK is primarily localized in the ER, some NHK also co-localizes with Golgi markers, suggesting that mannose trimming by Golgi α1,2-mannosidases can also contribute to NHK degradation. [Copyright &y& Elsevier]

The article presents a study that evaluates 8-isoprostane and leukotrienes in exhaled breath condensate in patients with silicosis. It shows several impaired lung functions parameters in silicotics. It states that 39% of silicotics have antineutrophilic cytoplasmic antibodies. It also reveals the positive association between the silica exposure category and the level of 8-isoprostane.

The native form of serpins (serine protease inhibitors) is a metastable conformation, which converts into a more stable form upon complex formation with a target protease. It has been suggested that movement of helix-F (hF) and the following loop connecting to strand 3 of β-sheet A (thFs3A) is critical for such conformational change. Despite many speculations inferred from analysis of the serpin structure itself, direct experimental evidence for the mobilization of hF/thFs3A during the inhibition process is lacking. To probe the mechanistic role of hF and thFs3A during protease inhibition, a disulfide bond was engineered in α1-antitrypsin, which would lock the displacement of thFs3A from β-sheet A. We measured the inhibitory activity of each disulfide-locked mutant and its heat stability against loop-sheet polymerization. Presence of a disulfide between thFs3A and s5A but not between thFs3A and s3A caused loss of the inhibitory activity, suggesting that displacement of hF/thFs3A from strand 5A but not from strand 3A is required during the inhibition process. While showing little influence on the inhibitory activity, the disulfide between thFs3A and s3A retarded loop-sheet polymerization significantly. This successful protein engineering of α1-antitrypsin is expected to be of value in clinical applications. Based on our current studies, we propose that the reactive-site loop of a serpin glides through between s5A and thFs3A for the full insertion into β-sheet A while a substantial portion of the interactions between hF and s3A is kept intact. [ABSTRACT FROM AUTHOR]

Golgi-endomannosidase provides an alternate glucosidase-independent pathway of glucose trimming. Activity for endomannosidase is detectable in various tissues and cell lines but not in CHO cells. Cloning of CHO cell endomannosidase revealed that the highly conserved Trp188 and Arg177 of vertebrate endomannosidase were both substituted by Cys. The Trp188Cys substitution was functionally important since it alone resulted in endoplasmic reticulum (ER) mislocalization of endomannosidase and caused the greatly reduced in vivo activity. These effects could be reversed in cells with a back-engineered Cys188Trp CHO cell endomannosidase, in particular N-glycans of α1-antitrypsin became fully processed. The intramolecular disulfide bridge of CHO cell endomannosidase formed with the additional Cys188 was not solely responsible for the reduced enzyme activity since endomannosidase with engineered Cys188Ala or Ser substitutions did not restore enzyme activity and was ER mislocalized. Thus, the conserved Trp188 residue in endomannosidases is of critical importance for correct subcellular localization and in vivo activity of the enzyme. [ABSTRACT FROM AUTHOR]

Abstract: Members of the serine proteinase inhibitor (serpin) family play important roles in the inflammatory and coagulation cascades. Interaction of a serpin with its target proteinase induces a large conformational change, resulting in insertion of its reactive center loop (RCL) into the main body of the protein as a new strand within β-sheet A. Intermolecular insertion of the RCL of one serpin molecule into the β-sheet A of another leads to polymerization, a widespread phenomenon associated with a general class of diseases known as serpinopathies. Small peptides are known to modulate the polymerization process by binding within β-sheet A. Here, we use fluorescence correlation spectroscopy (FCS) to probe the mechanism of peptide modulation of α1-antitrypsin (α1-AT) polymerization and depolymerization, and employ a statistical computationally-assisted design strategy (SCADS) to identify new tetrapeptides that modulate polymerization. Our results demonstrate that peptide-induced depolymerization takes place via a heterogeneous, multi-step process that begins with internal fragmentation of the polymer chain. One of the designed tetrapeptides is the most potent antitrypsin depolymerizer yet found. [Copyright &y& Elsevier]

Abstract: The recognition of bacterial lipopolysaccharide (LPS) is principally mediated by either membrane-bound or soluble form of the glycoprotein CD14 and CD14-associated signal transducer, toll-like receptor 4 (TLR4). Recent findings indicate that the serine protease inhibitor, α1-antitrypsin (AAT), may not only afford protection against proteolytic injury, but may also neutralize microbial activities and affect regulation of innate immunity. We postulated that AAT affects monocyte responses to LPS by regulating CD14 expression and soluble CD14 release. Here we show that a short-term (up to 2h) monocyte exposure to AAT alone or in combination with LPS leads to a remarkable induction of CD14 levels. In parallel, a short-term (2h) cell exposure to AAT/LPS significantly enhances LPS-induced NFκB (p50 and p65) activation in conjunction with increased TNFα, IL-1β and IL-8 release. In contrast, longer term incubation (18h) of monocytes with combined AAT/LPS results in a significant reduction in expression of both CD14 and TLR4, inhibition of LPS-induced TNFα, IL-1β and IL-8 mRNA and protein expression. These findings provide evidence that AAT is an important regulator of CD14 expression and release in monocytes and suggest that AAT may be involved in LPS neutralization and prevention of over-activation of monocytes in vivo. [Copyright &y& Elsevier]

Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor (serpin) protein family, is unique among the serpins in its conformational lability. This lability allows spontaneous conversion of the active form to a more stable, latent conformation under physiological conditions. In other serpins, polymerization, rather than latency transition, is induced under pathological conditions or upon heat treatment. To identify specific factors promoting latency conversion in PAI-1, we mutated PAI-1 at various positions and compared the effects with those of equivalent mutations in α1-antitrypsin, the archetypal serpin. Mutations that improved interactions with the turn between helix F and the third strand of β-sheet A (thFs3A) or the fifth strand of β-sheet A (s5A), which are near the site of latency transition-associated insertion of the reactive center loop, retarded latency conversion but did not greatly increase structural stability. Mutations that decreased interactions with s2C facilitated conformational conversion, possibly by releasing the reactive center loop from β-sheet C. Mutations of Thr93 that filled a hydrophobic surface pocket on s2A dramatically increased structural stability but had a negligible effect on the conformational transition. Our results suggest that the structural features controlling latency transition in PAI-1 are highly localized, whereas the conformational strain of the native forms of other inhibitory serpins is distributed throughout the molecule and induces polymerization. [ABSTRACT FROM AUTHOR]

Matrix metalloproteinase (MMP)-9, secreted as pro-MMP-9, is cleaved by serine proteases at the N-terminus to generate active MMP-9. Pro-MMP-9 has been found in the bronchoalveolar lavage fluid of patients with asthma. Because many inhaled aeroallergens contain active proteases, the authors sought to determine whether German cockroach (GC) fecal remnants (frass) and house dust mite (HDM) were able to cleave pro-MMP-9. Treatment of recombinant human (rh) pro-MMP-9 with GC frass resulted in a dose- and time-dependent cleavage. This was abrogated by pretreating frass with an inhibitor of serine, but not cysteine protease activity. GC frass also induced cleavage of pro-MMP-9 from primary human neutrophils dependent on the active serine proteases in GC frass. HDM was less potent at cleaving pro-MMP-9. α1-Antitrypsin (A1AT), a naturally occurring protease inhibitor, attenuated GC frass-induced cleavage of pro-MMP-9. A1AT partially inactivated the serine protease activity in GC frass, while GC frass cleaved A1AT in a dose- and time-dependent manner. These data suggest that GC frass-derived serine proteases could regulate the activity of MMP-9 and that A1AT may play an important role in modulating GC frass activity in vivo. These data suggest a mechanism by which inhalation of GC frass could regulate airway remodeling through the activation of pro-MMP-9. [ABSTRACT FROM AUTHOR]

Abstract: α1-Antitrypsin is well known for its ability to inhibit human neutrophil elastase. Pretreatment of α1-antitrypsin with hypohalous acids HOCl and HOBr as well as with the myeloperoxidase-hydrogen peroxide-chloride (or bromide) system inactivated this proteinase. The flavonols rutin, quercetin, myricetin, and kaempferol inhibited the inactivation of α1-antitrypsin by HOCl and HOBr with rutin having the most pronounced effect. In contrast, these flavonols did not remove the proteinase inactivation by the myeloperoxidase–hydrogen peroxide–halide system. Taurine did not protect against the inactivation of α1-antitrypsin by HOCl, HOBr, or the myeloperoxidase–hydrogen peroxide–halide system, while methionine was efficient in all systems. A close association between myeloperoxidase and α1-antitrypsin was revealed by native gel electrophoresis and in-gel peroxidase staining. In addition, α1-antitrypsin binds to the myeloperoxidase components transferred after SDS–PAGE on a blotting membrane. With this complex formation, myeloperoxidase overcomes the natural antioxidative protective system of plasma and prevents the inactivation of α1-antitrypsin. [Copyright &y& Elsevier]

The therapeutic use of autologous cells with the capacity for extensive in vitro expansion and manipulation prior to host administration has been an area of significant investigation over the last decade. Blood outgrowth endothelial cells (BOECs) are derived from the circulation and exhibit proliferative growth, in vivo engraftment, and survival characteristics for long-term expression of endogenously secreted proteins, such as factor VIII (FVIII). The authors describe a modified method for the isolation, culture, and expansion of these cells that is readily accomplished using standard laboratory methods. Using a commercially available transfection reagent, ∼30% of these primary cells can be routinely transfected with the nonviral Sleeping Beauty transposon for long-term, stable transgene expression. Moreover, the results indicate that these cells have the ability to secrete functionally active proteins that are synthesized endogenously by hepatocytes and require post-translational modification including α1-antitrypsin and clotting factors VII and IX. This, coupled with their notably long half-life of years, suggests that these cells may provide an appropriate vehicle for secretion of a variety of proteins produced by different cell types in vivo. Thus, BOECs have the potential to provide clinically relevant secreted proteins for diseases other than those of endothelial origin. [ABSTRACT FROM AUTHOR]

Two-dimensional electrophoresis was used to investigate the effects of a QTL for muscle hypertrophy on sarcoplasmic protein expression in ovine muscles. In the Belgian Texel breed, the QTL for muscle hypertrophy is localized in the myostatin-encoding gene. Based on microsatellite markers flanking the myostatin gene, we compared the hypertrophied genotype with the normal genotype. The average age of the sheep was 3 mo. Among the 4 muscles studied, in the hypertrophied genotype only the vastus medialis was normal, whereas the semimembranosus, tensor fasciae latae, and LM were hypertrophied. In the hypertrophied genotype, these muscles showed upregulation of enzymes involved in glycolytic metabolism together with oxidative metabolism in LM. Certain chaperone proteins, including glutathione S-transferase-Pi, heat shock protein-27, and heat shock cognate-70, were also more highly expressed, probably due to increased use of energetic pathways. Expression of the iron transport protein transferrin was increased. Alpha-1-antitrypsin was the only protein showing a similar pattern of expression (i.e., less expressed) in all 4 muscles of the hypertrophied genotype. It is suggested that transferrin and alpha-1-antitrypsin may interact to reinforce myogenic proliferative signaling. [ABSTRACT FROM AUTHOR]

Background and Objectives The aim of this study was to increase the yield of active α1-proteinase inhibitor (α1-PI) from Cohn fraction IV-1 paste during the manufacture of this therapeutic protein and to investigate the molecular mechanism for this yield increase. Materials and Methods Dissolution experiments with IV-1 paste investigated the impact of different variables on the yield of α1-PI activity. Solutions of IV-1 paste prepared under different conditions were assayed for evidence of protease activity by Western blots of α1-PI following SDS-PAGE, by azocaseinolytic and amidolytic (S-2288) assays, and by zymography, and for the extent of α1-PI oligomerization by Western blots following nondenaturing PAGE. Results Minor modification of the manufacturing process by combining dissolution of IV-1 paste with the subsequent pH adjustment (to 9·25–9·50 with NaOH), achieved by addition of a standard concentration of NaOH to the 10-mm Tris base dissolvent for IV-1 paste, was found to give a highly reproducible 9·4 ± 0·9% increase in yield of active α1-PI. Solutions of IV-1 paste prepared with this combined dissolvent contained reduced amounts of low molecular weight fragments of α1-PI, reduced protease activity, and reduced amounts of oligomers of α1-PI. Addition of the protease inhibitor leupeptin to the 10-mm Tris base dissolvent for IV-1 paste also caused an increase in the yield of α1-PI activity. Conclusions Dissolution of IV-1 paste in a more alkaline medium gave a significant increase in the yield of active α1-PI. This yield increase was attributed to a reduction both in protease activity and in the extent of oligomerization of α1-PI. [ABSTRACT FROM AUTHOR]

Copyright of Archivos de Bronconeumología (English Edition) is the property of Sociedad Espanola de Neumologia y Cirugia Toracica (SEPAR) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

A novel cucumber mosaic virus inducible viral amplicon (CMViva) expression system has been developed that allows for tightly regulated chemically inducible expression of heterologous genes in plant hosts. Transient production of recombinant α1-antitrypsin (rAAT), a human blood protein, was demonstrated in Nicotiana benthamiana leaves. The highest production levels were obtained by co-infiltrating leaves with Agrobacterium tumefaciens cells containing CMViva carrying the AAT gene and A. tumefaciens cells carrying a binary vector constitutively expressing the gene silencing suppressor p19. Accumulation of up to thirty-fold more rAAT was observed in leaves (24 mg per 100 g leaf tissue) when compared with the expression levels observed using the cauliflower mosaic virus (CaMV) 35S promoter. Significantly, 70% of the rAAT produced using the CMViva expression system was found to be biologically active, a 170-fold increase in functional protein compared with the CaMV 35S expression system. [ABSTRACT FROM AUTHOR]

Copyright of Der Internist is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Mesotrypsin, an inhibitor-resistant human trypsin isoform, does not activate or degrade pancreatic protease zymogens at a significant rate. These observations led to the proposal that mesotrypsin is a defective digestive protease on protein substrates. Surprisingly, the studies reported here with α1-antitrypsin (α1AT) revealed that, even though mesotrypsin was completely resistant to this serpin-type inhibitor, it selectively cleaved the Lys10–Thr11 peptide bond at the N-terminus. Analyzing a library of α1AT mutants in which Thr11 was mutated to various amino acids, we found that mesotrypsin hydrolyzed lysyl peptide bonds containing Thr or Ser at the P1′ position with relatively high specificity ( kcat/ KM∼105 m−1·s−1). Compared with Thr or Ser, P1′ Gly or Met inhibited cleavage 13- and 25-fold, respectively, whereas P1′ Asn, Asp, Ile, Phe or Tyr resulted in 100–200-fold diminished rates of proteolysis, and Pro abolished cleavage completely. Consistent with the Ser/Thr P1′ preference, mesotrypsin cleaved the Arg358–Ser359 reactive-site peptide bond of α1AT Pittsburgh and was rapidly inactivated by the serpin mechanism ( ka∼106 m−1 s−1). Taken together, the results indicate that mesotrypsin is not a defective protease on polypeptide substrates in general, but exhibits a relatively high specificity for Lys/Arg–Ser/Thr peptide bonds. This restricted, thrombin-like subsite specificity explains why mesotrypsin cannot activate pancreatic zymogens, but might activate certain proteinase-activated receptors. The observations also identify α1AT Pittsburgh as an effective mesotrypsin inhibitor and the serpin mechanism as a viable stratagem to overcome the inhibitor-resistance of mesotrypsin. [ABSTRACT FROM AUTHOR]

The native form of serine protease inhibitors (serpins) is kinetically trapped in a metastable state, which is thought to play a central role in the inhibitory mechanism. The initial binding complex between a serpin and a target protease undergoes a conformational change that forces the protease to translocate toward the opposite pole. Although structural determination of the final stable complex revealed a detailed mechanism of keeping the bound protease in an inactive conformation, it has remained unknown how the serpin exquisitely translocates a target protease with an acyl-linkage unhydrolyzed. We previously suggested that the acyl-linkage hydrolysis is strongly suppressed by active site perturbation during the protease translocation. Here, we address what induces the transient perturbation and how the serpin metastability contributes to the perturbation. Inhibitory activity of α1-antitrypsin (α1AT) toward elastase showed negative correlations with medium viscosity and Stokes radius of elastase moiety, indicating that viscous drag directly affects the protease translocation. Stopped-flow measurements revealed that the change in the inhibitory activity is primarily caused by the change in the translocation rate. The native stability of α1AT cavity mutants showed a negative correlation with the translocation rate but a positive correlation with the acyl-linkage hydrolysis rate, suggesting that the two kinetic steps are not independent but closely related. The degree of active site perturbation was probed by amino acid nucleophiles, supporting the view that the changes in the acyl-linkage hydrolysis rate are due to different perturbation states. These results suggest that the active site perturbation is caused by local imbalance between a pulling force driving protease translocation and a counteracting viscous drag force. The structural architecture of serpin metastability seems to be designed to ensure the active site perturbation by providing a sufficient pulling force, so the undesirable hydrolytic activity of protease is strongly suppressed during the translocation. [Copyright &y& Elsevier]

The article examines the effects of α1-Antitrypsin (AAT)-Z on lipopolysaccharide-stimulated human monocyte activation. The proinflammatory effects of native and polymeric AAT may be dependent on the presence of other cell activators, bacterial or otherwise while pure preparations of AAT appear to exert predominantly anti-inflammatory activity.

Mutations in neuroserpin and α1-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 α1-antitrypsin-associated cirrhosis. Polymers form by a sequential linkage between the reactive centre loop of one molecule and β-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 α1-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 α1-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 β-sheet A. [ABSTRACT FROM AUTHOR]

Abstract: Antiretroviral chemotherapy penetrates the CNS poorly. CNS HIV, thus sheltered, may injure the brain and complicate control of systemic HIV infection. Microglial cells play a major role in HIV persistence in the CNS but are rarely targeted for gene delivery. Because recombinant SV40 vectors (rSV40s) transduce other phagocytic cells efficiently, we tested rSV40 delivery of anti-HIV genetic therapy to microglial cells. Microglia prepared as enriched cultures from human fetal brain, were transduced with marker vectors, SV(RFP) and SV(Nef/FLAG), respectively, carrying DsRed and HIV-1 Nef bearing a FLAG epitope. By immunostaining and FACS, 95% of unselected cells expressed the transgenes, without detectable toxicity. Microglia were transduced with SV(AT), carrying human α1-antitrypsin (α1AT), which blocks Env and Gag processing. SV(AT)-treated microglia strongly resisted challenge with HIV-1BaL, even when microglia were transduced with SV(AT) following HIV challenge. Thus, rSV40s effectively transduce microglia and protect them from HIV. [Copyright &y& Elsevier]

Abstract: Serpin A1 (α1-proteinase inhibitor) inhibits human immunodeficiency virus 1 (HIV-1) production by mechanisms which remain to be elucidated. The complex formation of serpin A1 with proteinases eliminates the proteolytic activity and generates a fragment corresponding to the serpin C-terminal 36-residue peptide. Here, we show that the C-terminal 26-residue peptide of serpin A1 (A1-C26) inhibits HIV-long terminal repeat (LTR)-driven transcription in epithelial cells transfected with HIV-1 LTR promoter-driven genes. A1-C26 increased STAT1 phosphorylation and strongly reduced viral expression in a monocytic cell line infected with HIV-1 NL4-3. This reduction of expression was also observed in HIV-1 infected, PHA-activated peripheral blood mononuclear cells. In HIV-1 infected cells, the inhibitory activity of HIV-1 caused by B9-C23 and C1-C26, the A1-C26 homologues corresponding to the C-terminal sections of serpin B9 and serpin C1, was much lower than that obtained with A1-C26. These serpin peptides represent a novel class of antiviral agents. [Copyright &y& Elsevier]

Abstract: Human α1-antitrypsin-deficient variants may aggregate in the liver, with subsequent deficiency in the plasma, which can lead to emphysema. The structural and functional characteristics of 10 dysfunctional α1-antitrypsin variants (R39C, S53F, V55P, I92N, G115S, N158K, E264V, A336T, P369S, and P369L) were analyzed in detail. Most of them were unstable, as compared to the wild-type molecule, and many of the variants folded into an intermediate form. When five thermostable mutations (T68A, A70G, M374I, S381A, and K387R) were introduced into dysfunctional α1-antitrypsin variants, the stabilities and inhibitory activities of most of the variants were restored to levels comparable to those of the wild-type molecule. However, the extremely unstable S53F variant was not stabilized sufficiently by these mutations so as to exhibit function. N158K variant, which carries a mutation in the region critical for the reactive site loop insertion into β-sheet A, exhibited a reduced level of inhibitory activity, despite conformational stabilization. These results show that aberrant folding caused by conformational destabilization due to mutations can be compensated for by increasing the overall stability of the α1-antitrypsin molecule, with exception of a mutation in the highly localized region critical for functional execution. [Copyright &y& Elsevier]

α1-antitrypsin (α1-AT) is a physiological inhibitor of activated protein C (APC) and therefore decreased APC activity. APC itself causes an anticoagulant effect by inactivating factors Va and VIIIa. The present case-control study was performed to evaluate the role of the elevated α1-AT concentration in pediatric patients with ischemic stroke (IS). α1-AT concentrations were measured along with established prothrombotic risk factors 6–12 months after the acute thrombotic onset in 81 Caucasian children with IS aged 1 month to 18 years. The cutoff values defined as age-dependent 90th percentiles were obtained from 229 healthy controls. Median (range) values of α1-AT were significantly higher in patients compared with control subjects [122.0 mg/dl (61.4–224.0) vs. 114.0 mg/dl (66.8–156.0); p = 0.016]. In addition, 14 of the 81 patients (17.3%) compared with 10 of the 162 controls (6.2%) had α1-AT concentrations above the 90th age-dependent percentiles (p = 0.012). Multivariate analysis performed in a 1:2 matched case-control setting adjusted for the presence of established prothrombotic risk factors showed a significantly increased odds ratio (OR) and 95% confidence interval (CI) for patients with elevated α1-AT >90th percentiles and IS (OR/CI: 4.0/1.64–9.92; p = 0.0024). Data shown here give evidence that total α1-AT concentrations above the 90th age-dependent percentiles independently increase the risk of IS 4.0-fold in Caucasian children. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Microscopic poly(styrene-divinylbenzene) beads coated with a monomolecular film of fibrinogen agglutinate when stirred in the presence of thrombin, a consequence of interbead fibrin formation. Trypsin, by digesting bead-bound fibrin, dissociates bead aggregates at a rate proportional to the amount of enzyme activity. The agglutination of beads and the dissociation of bead aggregates can be monitored turbidimetrically using a platelet aggregometer or other photometric device equipped with a stirred cell. We have exploited the behavior of aggregates of fibrin-coated beads to develop a rapid, sensitive, and accurate method for measuring the activity of trypsin and its inhibition, in aqueous media, including serum. The new method yields serum antitrypsin activity levels that correlate well with immunological levels of α1-antitrypsin and, thus, may prove useful for assessing antitrypsin activity in clinical specimens. [Copyright &y& Elsevier]

Abstract: α1-Antitrypsin (AAT), a major endogenous inhibitor of serine proteases, plays an important role in minimizing proteolytic injury to host tissue at sites of infection and inflammation. There is now increasing evidence that AAT undergoes post-translational modifications to yield by-products with novel biological activity. One such molecule, the C-terminal fragment of AAT, corresponding to residues 359–394 (C-36 peptide) has been reported to stimulate significant pro-inflammatory activity in monocytes and neutrophils in vitro. In this study we showed that C-36 peptide is present in human lung tissue and mimics the effects of lipopolysaccharide (LPS), albeit with lower magnitude, by inducing monocyte cytokine (TNFα, IL-1β) and chemokine (IL-8) release in conjunction with the activation of nuclear factor-κB (NF-κB). Using receptor blocking antibodies and protein kinase inhibitors, we further demonstrated that C-36, like LPS, utilizes CD14 and Toll-like receptor 4 (TLR4) receptors and enzymes of the mitogen-activated protein kinase (MAPK) signaling pathways to stimulate monocyte TNFα release. The specificity of C-36 effects were demonstrated by failure of a shorter peptide (C-20) to elicit biological activity and the failure of C-36 to inhibit CD3/CD28-stimulated IL-2 receptor expression or proliferation in T-cells which lack TLR4 and CD14. We suggest that C-36 mediates its effects though the activation of LPS signaling pathways. [Copyright &y& Elsevier]

Abstract: α1-Antitrypsin (AAT) is a major circulating and tissues inhibitor of serine proteinases implicated in the regulation of inflammation and host defence. There is now increasing evidence that AAT may also exhibit anti-inflammatory activities independent of its protease inhibitor function. This study was undertaken to investigate the effects of native (inhibitory) and polymerized (non-inhibitory) forms of AAT on MID (Moraxella IgD binding protein)-induced human tonsillar B cell activation in vitro. We found that 0.5μg/ml MID induces B cell proliferation and stimulates IL-6 release (p <0.001) relative to non-stimulated controls. Both native and polymerized AAT (0.5mg/ml) inhibited MID-stimulated B cell proliferation in a similar manner (by 70%, p <0.001), whereas MID-induced IL-6 release was more strongly suppressed by polymerized (9.9-fold, p <0.001) as compared to native AAT (2.8-fold, p <0.01). Electrophoretic analysis of cell culture media did not indicate any interaction between AAT and MID, and flow cytometry data showed no competition for the same receptor. The effects of AATs were observed whether added together with MID or 2h after MID-addition to cell cultures. Thus, our data demonstrate that AAT inhibits MID-induced B cell activation in vitro that is unrelated to its protease inhibitory activity and is not dependent on MID binding to the cell surface. [Copyright &y& Elsevier]

α1-Antitrypsin (α1AT) deficiency is a genetic disorder causing emphysema if serum α1AT levels are <570 μg/ml. We have shown that intrapleural administration of an AAV5α1AT vector yielded persistent therapeutic α1AT serum levels. Since anti-AAV2 and -AAV5 antibodies prevalent in humans may limit the use of these common serotypes in gene therapy, we screened 25 AAV vectors derived from humans and nonhuman primates for α1AT expression following intrapleural administration to mice. The rhesus AAVrh.10 serotype yielded the highest levels and was chosen for further study. Following intrapleural administration, 77% of total body transgene expression was in the chest wall, diaphragm, lung, and heart. Intrapleural administration of AAVrh.10α1AT provided long-term, therapeutic α1AT expression in mice, although higher doses were required to achieve therapeutic levels in female mice than in male mice. Intrapleural administration of AAVrh.10α1AT produced the same levels in AAV2/AAV5-preimmune and naive mice. In mice administered with AAV5α1AT and subsequently “boosted” with the AAVrh.10α1AT vector, serum levels were increased by 300%. These data indicate that AAVrh.10 is the most effective known AAV vector for intrapleural gene delivery and has the advantage of circumventing human immunity to AAV.Molecular Therapy (2006) 13, 67–76; doi: 10.1016/j.ymthe.2005.09.003 [ABSTRACT FROM AUTHOR]

Abstract: Haptoglobin (Hp), a hemoglobin-binding protein, is known as an acute phase protein and increases during the acute phase of inflammation in most mammals. We reported previously in brown bears that the mean Hp concentrations were higher in blood samples obtained in winter than those in spring. To examine a possible relation of the seasonal variations of Hp to hibernation, in the present study, we measured the plasma concentrations of Hp as well as some other acute phase proteins (α2-macroglobulin, α1-antitrypsin, C-reactive protein) in 6 European brown bears (Ursus arctos), from which blood samples were obtained at 5–6 different months of year including February, the time of hibernation. The Hp concentrations showed clear seasonal variations, being highest in February. The α2-macroglobulin concentrations also showed a similar but much smaller rise in February, but those of α1-antitrypsin and C-reactive protein did not show any seasonal variations. Our results suggest that the seasonal variation of plasma Hp concentration in brown bears is associated with a hibernation-specific mechanism more than that of acute phase response. [Copyright &y& Elsevier]

Abstract: In degradative articular diseases such as rheumatoid arthritis and osteoarthritis, loss of the extracellular matrix occurs, resulting in the destruction of joint cartilage. Proteolysis of aggrecan is one of the early events that leads to breakdown of the extracellular matrix. Aggrecanase-1 (ADAMTS-4) is considered to play a pivotal role in the abrasion of cartilage aggrecan in rheumatoid arthritis and osteoarthritis. To identify an endogenous inhibitor of aggrecanase-1, we performed a yeast two-hybrid screen using the catalytic domain of human aggrecanase-1 as a bait and transformed an EGY48 yeast strain carrying the bait plasmid with a human liver cDNA library plasmid. This screen identified α1-antitrypsin, a member of the family of plasma serine proteinase inhibitors, as a prey. Recombinant aggrecanase-1 and α1-antitrypsin were expressed in mammalian cells and used in coimmunoprecipitation experiments, which showed that full-length aggrecanase-1 and α1-antitrypsin are also associated in vivo. However, aggrecanase-1 did not interfere with the inhibitory activity of α1-antitrypsin against elastase, and α1-antitrypsin had no effect on the proteolytic activity of aggrecanase-1. Taken together, these data suggest that aggrecanase-1 and α1-antitrypsin bind in vivo, although the physiological significance of the interaction between aggrecanase-1 and α1-antitrypsin remains unclear. [Copyright &y& Elsevier]

Abstract: Gene transfer into pancreatic cells in vivo could be of immense therapeutic benefit in cases of type 1 diabetes (T1D) through the production of molecules capable of interrupting the progression of autoimmunity or promoting regeneration of insulin-secreting β cells. We adapted a clinically relevant surgical technique (endoscopic retrograde cholangiopancreatography) to deliver rAAV encoding human α1-antitrypsin (approved gene symbol SERPINA1) to the pancreas of 3-week-old Fisher 344 rats and C57BL/6 mice. We compared natural as well as bioengineered serotypes of rAAV (rAAV1, rAAV2/Apo (B. R. Burkhardt et al., 2003, Ann. N. Y. Acad. Sci. 1005, 237–241) [1], rAAV8) as well as different promoters (chicken β-actin, human insulin) for their expression in vivo. Rats injected with rAAV1 showed the highest hAAT expression (week 2, rAAV1/CB-AT, 579 ± 457 ng/ml). In mice, rAAV8 vector delivered the highest serum concentration of hAAT (week 2, rAAV8/CB-AT, 19 ± 6 μg/ml). The chicken β-actin promoter provided the highest expression in both rodent experiments. Immunohistochemical staining indicated transduction primarily of pancreatic acinar cells with either the rAAV1/CB-AT vector in the rat or the rAAV8/CB-AT vector in the mouse. This study demonstrates that rAAV vectors can be designed to deliver therapeutic genes efficiently to the pancreas and achieve high levels of gene expression and may be useful in treating pancreatic disorders, including T1D. [Copyright &y& Elsevier]

Background: The major house dust mite group 1 allergens Der p 1 and Der f 1 are the most potent indoor allergens. Der p 1 cleaves human cell surface molecules, the low-affinity IgE receptor (CD23/FcεRII), the α-subunit of the IL-2 receptor (CD25), and a protease inhibitor α1-antitrypsin, and in vitro and in vivo studies suggested the importance of its proteolytic activity in the pathogenesis of allergy. Recently, we established an efficient system to prepare correctly folded active recombinant Der p 1 and Der f 1 (Der p 1-N52Q and Der f 1-N53Q) with similar molecular sizes, secondary structures and allergenicities as their natural types. To evaluate whether Der p 1-N52Q and Der f 1-N53Q are suitable for use in future in vitro and in vivo studies as alternatives to the natural types, we investigate their proteolytic activity to cleave the human proteins and IgE-eliciting activity in mice. Methods: Proteolytic activities of Der p 1-N52Q and Der f 1-N53Q against a short peptide substrate, a collagen substrate Azocoll, human CD23 and CD25 expressed on the cells and human α1-antitrypsin were analyzed by kinetic assays for proteolysis of the fluorogenic or colorimetric substrates, flow cytometry and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively. Mice were intraperitoneally immunized with Der p 1-N52Q and Der f 1-N53Q adsorbed on Alum, and the serum IgE levels were measured by sandwich ELISA. Results: Der p 1-N52Q and Der f 1-N53Q showed proteolytic specificities against the short peptide substrate, Azocoll, human cell surface CD23 and CD25 and human α1-antitrypsin, and elicited significant serum IgE levels in immunized mice. Conclusion: The recombinant forms, Der p 1-N52Q and Der f 1-N53Q, will be useful tools as alternatives to the natural Der p 1 and Der f 1 for various in vitro and in vivo analyses. Copyright © 2005 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

To investigate the presence and concentration ofα1-antitrypsin in aqueous humour at the time of corneal rejection and to compare results obtained from patients with reversible and irreversible rejection.Samples of aqueous humour were obtained from 17 patients with acute corneal endothelial allograft rejection. The presence ofα1-antitrypsin in aqueous humour was confirmed by immunoblotting and measured employing a sandwich ELISA. Total protein concentrations in aqueous humour were measured using Bradford's method. The outcome of corneal rejection episodes was determined 1 month after diagnosing corneal rejection and described as reversible or irreversible rejection.α1-antitrypsin was detected in aqueous humour. Patients with reversible rejection had significantly higherα1-antitrypsin concentration than patients with irreversible rejection (p = 0.044). There was no significant difference in total protein concentrations (p = 0.745), and no correlation was found betweenα1-antitrypsin and total protein concentrations (p = 0.368).α1-antitrypsin in aqueous humour seems to signal a favourable outcome of corneal rejection. The possible mechanism is discussed. [ABSTRACT FROM AUTHOR]

Furin proteolytically cleaves a wide variety of proprotein substrates mainly within the trans-Golgi network (TGN) but also at the cell membrane and in endosomal compartments where pH is more acidic. Incorporation of furin recognition sequences within the reactive site loop (RSL) of α1-antitrypsin (AT) leads to the production of furin inhibitors. In an attempt to design more stable, potent, and specific serpin-based inhibitors, we constructed a series of AT and α1-antichymotrypsin (ACT) mutants by modifying the P7-P1 region of their RSLs. The biochemical properties of these variants were assessed by evaluating their propensity to establish SDS-resistant complexes with furin in a variety of conditions (pH 6.0-9.0) and by measuring their association rate constants. The effect of pH during the initial steps of complex formation was minimal, suggesting that the acylation step is not rate-limiting. The decrease in stoichiometry of inhibition (SI) values observed in AT variants at high pHs was a result of the reduced pH-dependent deacylation rate, which is rate-limiting in this mechanism and which suggests increased complex stability. Conversely, the SI values for ACT mutants had a tendency to be lower at acidic pH. Transiently transfecting HEK293 cells with these mutants abolished processing of the pro-von Willebrand factor precursor but, interestingly, only the ACT variants were secreted in the media as uncleaved forms. Our results suggest that reengineering the reactive site loops of serpins to accommodate and target furin or other serine proteases must take into account the intrinsic physicochemical properties of the serpin. [ABSTRACT FROM AUTHOR]

Aims: The damage to lung tissue in chronic obstructive pulmonary disease (COPD) may involve the progressive loss of pulmonary vascular endothelial cells. Endothelial binding of α1-antitrypsin (α1-AT) derived from plasma has been identified, and α1-AT deficiency is a known genetic risk factor associated with α1-AT polymerization and COPD development. Therefore, in the present study we aimed to investigate if α1-AT is present on the lung vascular endothelium, and if it is in a polymeric form. Methods and results: Postmortem paraffin-embedded tissue specimens from 15 COPD (chronic bronchitis and emphysema) cases with and without Z α1-AT (GIu342Lys) deficiency and from 10 cases without signs of COPD were studied. Immunohistochemistry was performed using the streptavidin-biotin method with a monoclonal ATZ11 antibody specific for polymeric α1-AT, and polyclonal antibodies against human α1-AT and neutrophil elastase. Vascular endothelium showed intense staining for α1-AT with the ATZ11 antibody in all cases; however, intensity of staining in patients with α1-AT deficiency was greater. No endothelial staining was observed with the anti-elastase antibody. Conclusions: This is the first demonstration that α1-AT bound to the vascular endothelium of lungs is in a polymeric form, which also suggests a possible previously unknown role for polymeric α1-AT in vivo. [ABSTRACT FROM AUTHOR]

Background: Non-invasive diagnostic tools to evaluate the severity of acute, radiation-induced proctitis are not readily available. The faecal excretion of eight markers of gut inflammation was therefore examined. Five proteins and three lipid derivates were analysed in sequential stool samples taken before and during radiation therapy. Methods: Stool samples from 15 patients with prostate cancer scheduled for radiation therapy were examined. Pretreatment and in-treatment samples (2nd and 6th weeks) were measured by enzyme-linked immunosorbent assay (ELISA) (calprotectin, lactoferrin, transferrin, leukotriene B4, prostaglandin E2, thromboxane B2 and TNFα) or nephelometry (α1-antitrypsin). Results: Calprotectin and lactoferrin concentrations increased significantly during radiation treatment ( P = 0.0005 and P = 0.019). Transferrin was detected in only 9 out of 45 samples. There were no changes in tumour necrosis factor α (TNFα), leukotriene B4, prostaglandin E2 and thromboxane B2 during treatment. α1-antitrypsin could not be detected in any sample. Conclusions: This study indicates that faecal calprotectin and lactoferrin concentrations could be markers of acute, radiation-induced proctitis. Patient compliance and stability of the markers make this a promising method for clinical research. Eicosanoids could be measured in stool samples, but the concentrations did not increase with increasing radiation dose. [ABSTRACT FROM AUTHOR]