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The universal proteinase inhibitor α2-macroglobulin (α₂-MG) exhibiting antiviral and immunomodulatory activities, is considered as an important participant in the infectious process. The activity of α₂-MG in the new coronavirus infection and post-covid syndrome (long COVID) has not been studied yet. We examined 85 patients diagnosed with community-acquired bilateral polysegmental pneumonia developed under conditions of a new coronavirus infection SARS-CoV-2. For assessment of the post-COVID period, 60 patients were examined 5.0±3.6 months after the coronavirus infection. Among these patients, 40 people had complications, manifested in the form of neurological, cardiological, gastroenterological, dermatological, bronchopulmonary symptoms. The control group included 30 conditionally healthy individuals with a negative PCR result for SARS-CoV-2 RNA and lack of antibodies to the SARS-CoV-2 virus. The α₂-MG activity in serum samples of patients with coronavirus infection dramatically decreased, up to 2.5% of the physiological level. This was accompanied by an increase in the activity of the α₁-proteinase inhibitor, elastase- and trypsin-like proteinases by 2.0-, 4.4- and 2.6-fold respectively as compared with these parameters in conditionally healthy individuals of the control. In the post-COVID period, despite the trend towards normalization of the activity of inhibitors, the activity of elastase-like and especially trypsin-like proteinases in serum remained elevated. In overweight individuals, the increase in the activity of trypsin-like proteinases was most pronounced and correlated with an increase in the antibody titer to the SARS-CoV-2 virus. In the post-COVID period, the α₂-MG activity not only normalized, but also exceeded the control level, especially in patients with dermatological and neurological symptoms. In patients with neurological symptoms or with dermatological symptoms, the α₂-MG activity was 1.3 times and 2.1 times higher than in asymptomatic persons. Low α₂-MG activity in the post-COVID period persisted in overweight individuals. The results obtained can be used to monitor the course of the post-COVID period and identify risk groups for complications.

Alpha-1 antitrypsin (A1AT), a circulating acute-phase reactant antiprotease, is produced and secreted by cells of endodermal epithelial origin, primarily hepatocytes, and by immune cells. Deficiency of A1AT is associated with increased risk of excessive lung inflammation and injury, especially following chronic cigarette smoke (CS) exposure. Exogenous administration of mesenchymal progenitor cells, including adipose tissue-derived stromal/stem cells (ASC), alleviates CS-induced lung injury through paracrine effectors such as growth factors. It is unknown, however, if mesodermal ASC can secrete functional A1AT and if CS exposure affects their A1AT production. Human ASC collected via liposuction from nonsmoking or smoking donors were stimulated by inflammatory cytokines tumor necrosis alpha (TNFα), oncostatin M (OSM), and/or dexamethasone (DEX) or were exposed to sublethal concentrations of ambient air control or CS extract (0.5%-2%). We detected minimal expression and secretion of A1AT by cultured ASC during unstimulated conditions, which significantly increased following stimulation with TNFα or OSM. Furthermore, TNFα and OSM synergistically enhanced A1AT expression and secretion, which were further increased by DEX. The A1AT transcript variant produced by stimulated ASC resembled that produced by bronchial epithelial cells rather than the variant produced by monocytes/macrophages. While the cigarette smoking status of the ASC donor had no measurable effect on the ability of ASC to induce A1AT expression, active exposure to CS extract markedly reduced A1AT expression and secretion by cultured ASC, as well as human tracheobronchial epithelial cells. ASC-secreted A1AT covalently complexed with neutrophil elastase in control ASC, but not in cells transfected with A1AT siRNA. Undifferentiated ASC may require priming to secrete functional A1AT, a potent antiprotease that may be relevant to stem cell therapeutic effects. [ABSTRACT FROM AUTHOR]

Serpins are a widely distributed family of serine proteases. A key determinant of their specificity is the reactive centre loop (RCL), a surface motif of ∼20 amino acids in length. Expression libraries of variant serpins could be rapidly probed with proteases to develop novel inhibitors if optimal systems were available. The serpin variant alpha-1 proteinase inhibitor M358R (API M358R) inhibits the coagulation protease thrombin, but at sub-maximal rates compared to other serpins. Here we compared two approaches to isolate functional API variants from serpin expression libraries, using the same small library of API randomized at residue 358 (M358X): flow cytometry of transfected HEK 293 cells expressing membrane-displayed API; and a thrombin capture assay (TCA) performed on pools of bacterial lysates expressing soluble API. No enrichment for specific P1 residues was observed when the RCL codons of the 1% of sorted transfected 293 cells with the highest fluorescent thrombin-binding signals were subcloned and sequenced. In contrast, screening of 16 pools of bacterial API-expressing transformants led to the facile identification of API M358R and M358K as functional variants. Kinetic characterization showed that API M358R inhibited thrombin 17-fold more rapidly than API M358K. Reducing the incubation time with immobilized thrombin improved the sensitivity of TCA to detect supra-active API M358R variants and was used to screen a hypervariable library of API variants expressing 16 different amino acids at residues 352–357. The most active variant isolated, with TLSATP substituted for FLEAI, inhibited thrombin 2.9-fold more rapidly than API M358R. Our results indicate that flow cytometric approaches used in protein engineering of antibodies are not appropriate for serpins, and highlight the utility of the optimized TCA for serpin protein engineering. [ABSTRACT FROM AUTHOR]

Highlights: [•] Structure/function understanding of its reactive centre loop (RCL) is incomplete. [•] We describe a method of screening pooled bacterial lysates for rapid screening of RCL variants. [•] The approach successfully detected functional API M358R present in a 1:99 mixture of inactive API clones. [•] The method identified novel functional variants of API M358R altered at residue 347 (P12) and at multiple residues 352–356 (P7–P3) validated by purification and kinetic analysis. [ABSTRACT FROM AUTHOR]

The aim was to establish the 1st International Standard (IS) for alpha-1-antitrypsin (AAT) to standardise potency assignment of therapeutic products, calibrated in moles and mg active AAT in line with product labelling practice. Assigning total protein and antigen values to the IS was also investigated. The active concentration of four candidate AAT preparations was determined in an international collaborative study by inhibition of trypsin (calibrated by active-site titration). Total protein and antigen content were determined for each candidate using local methods and in-house standards, and a common AAT preparation. The total protein content of the IS was also determined by amino acid analysis. Potency determination of recombinant and transgenic materials against the IS was investigated in a follow-up study. Data analysis for potency determination indicated no statistical difference between any of the candidates, or between the results for recombinant and plasma-derived products. Total protein content of the IS determined by amino acid analysis was consistent with the potency value. The variability in the total protein and antigen results for the other candidates was reduced when the data were recalculated relative to the IS. Candidate C (05/162) was established by the WHO Expert Committee on Biological Standardization (ECBS) in 2006 as the WHO 1st IS for AAT with a potency of 243 nmoles (12·4 mg) active inhibitor per ampoule. In 2008, ECBS approved the IS for potency determination of recombinant material and assigned a total protein and antigen value of 12·4 mg. [ABSTRACT FROM AUTHOR]

Abstract: Human alpha-1-proteinase inhibitor [1] Also known as antitrypsin. 1 (α1-PI) is the most abundant serine protease inhibitor in plasma. Its major function is inhibition of neutrophil elastase in lungs. α1-PI deficiency may result in severe, ultimately fatal emphysema. Three plasma-derived (pd-) α1-PI products are licensed in the US for replacement therapy of deficient patients. The recombinant versions (r-α1-PI), proposed as alternatives to pd-α1-PI products, have been under intensive investigation. For accurate determination of α1-PI from different sources and in various forms, there is an obvious need for reliable standardized assays for α1-PI quantification and potency measurements. As a part of our multi-step research focused on α1-PI structure-function investigation, we have established a simple and reproducible double-sandwich ELISA based on commercially available polyclonal antibodies. The developed ELISA allows the quantification of both pd-α1-PI and r-α1-PI in various complex matrices. A validation of the ELISA was performed with the working range of the assay (3.1–50ng/ml) established on the bases of the following parameters: linearity (3–100ng/ml, r 2 =0.995); accuracy (87.3–114.6% recovery); intra-assay precision (%CV, 2.8%); inter-assay plate-to-plate precision (3.9% per day and 4.1% day-to-day); detection limit (1.10ng/ml); and quantification limit (3.34ng/ml). The analytical performance of the α1-PI ELISA indicates that this assay can be used for monitoring concentration levels of α1-PI in multi-component biological matrices, based on the following: (a) quantification of r-α1-PI in various fermentation mixtures (E. coli and A. niger); (b) investigation of α1-PI enzymatically digested in the conditions of harsh fungal proteolysis; (c) evaluation of thermally polymerized α1-PI; (d) quantification of α1-PI in human serum; and (e) comparative quantification of α1-PI in commercially available products. [Copyright &y& Elsevier]

Abstract: The objective of this study was to determine whether the viral reduction processes of nanofiltration and solvent/detergent treatment used in the manufacture of alpha-1 proteinase inhibitor (API) cause neoantigenic changes. Polyclonal antibodies were raised in rabbits against the treated API and quantitatively absorbed with an affinity column containing API that had not undergone viral reduction treatment. Antibodies before and after absorption were measured in a validated ELISA using the immunogen for antibody capture. Antibodies against novel API epitopes were not found after antiserum from rabbits inoculated with treated API was absorbed with untreated API. A positive control, consisting of serum obtained from rabbits inoculated with trinitrophenylated API, showed substantial amounts of measurable antibody following absorption with untreated API. The results suggest that the viral reduction process used does not result in the creation of API neoantigens. [Copyright &y& Elsevier]

Alpha-1-proteinase inhibitor activity was studied in presence of resting and activated polymorphonuclear leucocytes. Four different agonists; phorbol myristic acetate, N-formyl-methionyl-leucyl-phenylalanine, opsonised zymosan and arachidonic acid decreased the inhibitor activity by 23.3%, 20%, 12% and 16.6^ respectively. The inhibitor activity was protected by using various free radical scavengers. Catalase and superoxide dismutase both restored activity by about 18%, mannitol by 13% and sodium azide by 17.3%. The inhibitor activity was also protected significantly by pretreatment of polymorphs with L-Arg, a precursor of nitric oxide, before activation. L-Arg was also observed to suppress the generation of superoxide and hydroxyl radical appreciably. The nitric oxide synthase inhibitor, aminoguanidine drastically inhibited the nitrite release and reversed the protection offered by L-Arg to the inhibitor activity. Our results indicate a multifactorial nature of the inactivation process, the culprit species being superoxide, hydrogen peroxide, hydroxyl radical and hypohalides. Nitric oxide seems to scavenge the superoxide radical directly after its formation rather than inhibiting its generation by NADPH oxidase as was believed earlier. [ABSTRACT FROM AUTHOR]

In an infection, polymorphonuclear neutrophils (PMN) become activated and they produce oxidizing compounds and elastase in the extracellular medium. Alpha-1-proteinase inhibitor (α1PI), a protease inhibitor which is inactivated by oxidants, is the main endogenous inhibitor of elastase helping to limit excessive elastase activity.This study evaluates the ability of a plant extract, Cola nitida nuts, to protect α1PI from inactivation by oxidizing compounds as reactive oxygen species.On the one hand, we have evaluated the direct effect of cola nut extract on neutrophil elastase, and on the H2O2 and myeloperoxidase (MPO)–H2O2 system via cell-free systems. Results showed that cola nut extract scavenges H2O2 and therefore protects α1PI from HOCl which is produced from the MPO–H2O2 system. Experiments also showed that cola extract has the capacity to limit elastase activity.On the other hand, we have worked on cellular systems including isolated PMN with the aim to study the effect of cola extract on PMN metabolism. PMN were stimulated with PMA, calcium ionophore or fMLP. Each stimulant possesses its own stimulation pathway. According to the inhibitory concentration obtained at 50%, the results on cellular systems led to the conclusion that cola extract can reduce elastase liberation from PMN.It can then be concluded that cola nut extract can protect α1PI from inactivation, and has an effect both on elastase liberation and elastase activity. The cola nut extract effect is rather biased towards a reduction in elastase release, thus limiting the injurious effects caused by this enzyme. [Copyright &y& Elsevier]

The organization and sequence of genes encoding the α-proteinase inhibitor (αPI), a major serine proteinase inhibitor of the mammalian bloodstream, have been compared in several species, including murine rodents (genus Mus). Analysis of gene copy number indicates that amplification of αPI genes occurred at some time during evolution of the Mus genus, leading to fixation of a family of about three to five genes in several existing species (e.g., M. domesticus and M. saxicola), and only a single gene in others (e.g., M. caroli). A phylogeny for the various mammalian αPI mRNAs was constructed based upon synonymous substitutions within coding regions. The mRNAs in different murine species diverged from a common ancestor before the formation of the first species lineages of the Mus genus, i.e., about 10-13 million years ago. Thus, αPI gene amplification must have occurred prior to Mus speciation; gene families were retained in some, but not all, murine species. The reactive center region of the αPI polypeptide, which determines target protease specificity, has diverged rapidly during evolution of the Mus species, but not during evolution of other mammalian species included in the analysis. It is likely that this accelerated evolution of the reactive center, which has been noted previously for serine proteinase inhibitors, was driven by some sort of a positive Darwinian selection that was exerted in a taxon-specific manner. We suggest that evolution of αPI genes of murine rodents has been characterized by both modification of gene copy number and rapid reactive center divergence. These processes may have resulted in a broadened repertoire of proteinase inhibitors that was evolutionarily advantageous during Mus speciation. [ABSTRACT FROM AUTHOR]

To unravel the complexities of the glycosylation of a protein is a substantial task, which requires considerable effort and resources. However, in many situations this is unnecessary, because only a limited amount of information is required. A new lectin-binding assay is described which is rapid, cheap and versatile. A purified glycoprotein is absorbed on to the plastic surface of a microtitre plate. After removing unbound protein by washing, uncoated sites on the plate are blocked and digoxigenin or biotin-labelled lectin is added. The degree of lectin binding is measured using either an anti-DIG antibody or streptavidin conjugated enzyme, which is subsequently used to develop a colour reaction. Using this method it is possible to screen multiple specimens with high sensitivity and excellent precision. In addition, very small amounts of lectin are used, background absorbances are low, and the procedure does not require a high degree of technical skill. Because very small amounts of glycoprotein are needed, a glycoprotein can often be rapidly purified by batch affinity chromatography. The method has been successfully applied to several purified proteins using the lectins, Con A, LCA, LTA, MAA, and SNA, and the information obtained agrees with that produced by more sophisticated approaches, eg Dionex Carbohydrate Analyser. Using a panel of lectins, a carbohydrate structural profile is quickly built-up, and subtle differences in glycosylation identified. This method should be particularly useful for screening glycosylation in multiple clinical specimens; in specimens where very small amounts of material are available, such as membrane molecules; and in the screening of recombinant proteins produced commercially. [ABSTRACT FROM AUTHOR]

By a regular immunoperoxidase method, alpha-1-proteinase inhibitor (Api) was demonstrated in pancreatic islets in individuals with and without genetic deficiency of Api. Subsequently a double immunoperoxidase method, with two different chromogens (diaminobenzidine-brown and 4-chloro-1-naphthol-blue), was applied on the same tissue section in order to identify cells containing Api and cells secreting polypeptide hormones. Api-positive cells and hormone-secreting cells were found to be mutually exclusive indicating that Api is synthesized by previously unrecognized islet cell. The population of Api cells was significantly higher in persons with genetic deficiency than in other individuals, implying a possible compensatory hyperplasia of those cells triggered by a low level of circulating Api. [ABSTRACT FROM AUTHOR]

Biochemically, there is usually much less elastase activity in gingival tissue than in crevicular fluid. The tissue distributions of active and inactive elastase and the endogenous inhibitors alpha-l-proteinase inhibitor (α1PI) and alpha-2-macroglobulin (α2M) were therefore compared. Inflamed tissue was obtained from chronic periodontitis patients, and cryostat sections were incubated with the histochemical elastase substrate MeOSuc-Ala-Ala-Pro-Val- MNA. Adjacent sections were examined immunocyto- chemically with antibodies to neutrophil elastase, α1PI, α2 2M, and leukocyte differentiation antigens. Antigenic elastase was widely distributed in CD15-positive granulocytes in both the epithelium and lamina propria as well as in granulomatous tissue from infrabony defects. However, there was very limited histochemical staining of these cells, and biochemical activity against the equivalent substrate MeOSuc-Ala-Ala-Pro-Val-AFC could be extracted only from sections with such staining. The pH optimum and effector response of the activity in the extracts were, nevertheless, consistent with those of leukocyte elastase. The large difference between the total elastase content of the tissue, as determined immunocytochemically, and the limited amount of active enzyme, as demonstrated histochemically, indicated that the majority was in an inactive form. The involvement of tissue inhibitors was suggested by the fact that extracts from sections with no histochemical staining reduced biochemical elastase activity in crevicular fluid. α2M was found in many fibroblasts and also some CD68-positive macrophages, which additionally contained α1PI. Both inhibitors appeared to be present extracellularly, since there was significant background staining. Thus, α1PI and α2M could contribute to the regulation of elastase activity in the gingiva. [ABSTRACT FROM AUTHOR]