AAT Activity Research Articles

Nitric oxide fumigation can affect the metabolism of volatile compounds derived from analyses of fatty acids and amino acids in post-harvest flat peach during cold storage

The loss of aroma volatiles can significantly reduce the economic value of postharvest flat peach during cold storage. In this study, the mechanism of NO fumigation regulating the aroma volatiles during cold storage at 1 ± 0.5 °C was investigated. The possible effects of NO on storage quality, VOCs, enzyme activity related to the fatty acid metabolism pathway, and the level of fatty acids and amino acids in fruit were analyzed. The results indicated that treatment with 10 μL L−1 NO maintained higher VC, firmness, SSC, TA, and restrained increase of MDA as well as the rotting rate. 10 μL L−1 NO treatment significantly supported a higher abundance of aldehydes before day 18 and encouraged the production of esters after day 18 of fruit under cold storage, which was associated with higher activity of ADH, AAT, HPL and LOX after day 18.10 μL L−1 NO treatment led to higher content of unsaturated acids, including OA, LA before day 18 and maintained higher content of LeA during the whole cold storage. According to these findings, 10 μL L−1 NO treatment may be a viable strategy to preserve the main aromatic volatiles and extend the shelf life of flat peaches.

COVID-19 Pathology Sheds Further Light on Balance between Neutrophil Proteases and Their Inhibitors

Excessive neutrophil influx and activation in lungs during infections, such as manifest during the ongoing SARS CoV-2 pandemic, have brought neutrophil extracellular traps (NETs) and the concomitant release of granule contents that damage surrounding tissues into sharp focus. Neutrophil proteases, which are known to participate in NET release, also enable the binding of the viral spike protein to cellular receptors and assist in the spread of infection. Blood and tissue fluids normally also contain liver-derived protease inhibitors that balance the activity of proteases. Interestingly, neutrophils themselves also express the protease inhibitor alpha-1-antitrypsin (AAT), the product of the SERPINA-1 gene, and store it in neutrophil cytoplasmic granules. The absence of AAT or mutations in the SERPINA-1 gene promotes lung remodeling and fibrosis in diseases such as chronic obstructive pulmonary disease (COPD), and acute respiratory distress syndrome (ARDS) and increases the risk of allergic responses. Recent observations point to the fact that reduced activity of AAT presents a major susceptibility factor for severe COVID-19. Here, we focus attention on the mechanism of neutrophil elastase (NE) in NET release and its inhibition by AAT as an additional factor that may determine the severity of COVID-19.

Secretion of functional α1-antitrypsin is cell type dependent: Implications for intramuscular delivery for gene therapy

Heterologous expression of proteins is used widely for the biosynthesis of biologics, many of which are secreted from cells. In addition, gene therapy and messenger RNA (mRNA) vaccines frequently direct the expression of secretory proteins to nonnative host cells. Consequently, it is crucial to understand the maturation and trafficking of proteins in a range of host cells including muscle cells, a popular therapeutic target due to the ease of accessibility by intramuscular injection. Here, we analyzed the production efficiency for α1-antitrypsin (AAT) in Chinese hamster ovary cells, commonly used for biotherapeutic production, and myoblasts (embryonic progenitor cells of muscle cells) and compared it to the production in the major natural cells, liver hepatocytes. AAT is a target protein for gene therapy to address pathologies associated with insufficiencies in native AAT activity or production. AAT secretion and maturation were most efficient in hepatocytes. Myoblasts were the poorest of the cell types tested; however, secretion of active AAT was significantly augmented in myoblasts by treatment with the proteostasis regulator suberoylanilide hydroxamic acid, a histone deacetylase inhibitor. These findings were extended and validated in myotubes (mature muscle cells) where AAT was transduced using an adeno-associated viral capsid transduction method used in gene therapy clinical trials. Overall, our study sheds light on a possible mechanism to enhance the efficacy of gene therapy approaches for AAT and, moreover, may have implications for the production of proteins from mRNA vaccines, which rely on the expression of viral glycoproteins in nonnative host cells upon intramuscular injection.

Effects of Fluoride on Metabolic enzymes of brain and Gastrocnemius muscle of mice: protective role of selenium and alpha-tocopherol

The present study aimed to investigate the ameliorative effects of Selenium and Alpha-tocopherol on activities of enzymes involved in metabolic functions, energy production and transfer in the brain and gastrocnemius muscle of mice treated with sodium fluoride. Animals were separated into five groups and treated with Selenium (05 µg/kg BW), Alpha-tocopherol (2mg/kg BW) alone, and in combination for 15 days to evaluate their ameliorative effects against fluoride (20mg/kg BW) toxicity. The obtained results showed a statistically significant (p<0.05) decrease in the body weight, organo-somatic index, protein content, ALAT, AAT, CPK, acid phosphatase and alkaline phosphatase enzymes activities in the fluoride-treated group in comparison with control group indicates oxidative stress induced by fluoride. The effect of fluoride on enzymes of muscle was comparatively higher, which suggests the greater accumulation of fluoride in muscle than brain. These changes were reverted moderately in Selenium and alpha-tocopherol alone treated groups, and significantly (p<0.05) in Selenium + alpha-tocopherol treated group suggests the combinatorial effect of Selenium and alpha-tocopherol in the amelioration of fluoride-induced toxicity. In conclusion, the findings all pointed to the fact that Selenium and alpha-tocopherol showed combinatorial effects in the amelioration of oxidative stress induced by fluoride.

Enoxaparin augments alpha-1-antitrypsin inhibition of TMPRSS2, a promising drug combination against COVID-19

The cell surface serine protease Transmembrane Protease 2 (TMPRSS2) is required to cleave the spike protein of SARS-CoV-2 for viral entry into cells. We determined whether negatively-charged heparin enhanced TMPRSS2 inhibition by alpha-1-antitrypsin (AAT). TMPRSS2 activity was determined in HEK293T cells overexpressing TMPRSS2. We quantified infection of primary human airway epithelial cells (hAEc) with human coronavirus 229E (HCoV-229E) by immunostaining for the nucleocapsid protein and by the plaque assay. Detailed molecular modeling was undertaken with the heparin–TMPRSS2–AAT ternary complex. Enoxaparin enhanced AAT inhibition of both TMPRSS2 activity and infection of hAEc with HCoV-229E. Underlying these findings, detailed molecular modeling revealed that: (i) the reactive center loop of AAT adopts an inhibitory-competent conformation compared with the crystal structure of TMPRSS2 bound to an exogenous (nafamostat) or endogenous (HAI-2) TMPRSS2 inhibitor and (ii) negatively-charged heparin bridges adjacent electropositive patches at the TMPRSS2–AAT interface, neutralizing otherwise repulsive forces. In conclusion, enoxaparin enhances AAT inhibition of both TMPRSS2 and coronavirus infection. Such host-directed therapy is less likely to be affected by SARS-CoV-2 mutations. Furthermore, given the known anti-inflammatory activities of both AAT and heparin, this form of treatment may target both the virus and the excessive inflammatory consequences of severe COVID-19.

α1-Antitrypsin attenuates acute rejection of orthotopic murine lung allografts

Backgroundα1-Antitrypsin (AAT) is an acute phase glycoprotein, a multifunctional protein with proteinase inhibitory, anti-inflammatory and cytoprotective properties. Both preclinical and clinical experiences show that the therapy with plasma purified AAT is beneficial for a broad spectrum of inflammatory conditions. The potential effects of AAT therapy have recently been highlighted in lung transplantation (LuTx) as well.MethodsWe used a murine fully mismatched orthotopic single LuTx model (BALB/CJ as donors and C57BL/6 as recipients). Human AAT preparations (5 mg, n = 10) or vehicle (n = 5) were injected to the recipients subcutaneously prior to and intraperitoneally immediately after the LuTx. No immune suppressive drugs were administered. Three days after the transplantation, the mice were sacrificed, and biological samples were assessed.ResultsHistological analysis revealed significantly more severe acute rejection in the transplanted lungs of controls than in AAT treated mice (p < 0.05). The proportion of neutrophil granulocytes, B cells and the total T helper cell populations did not differ between two groups. There was no significant difference in serum CXCL1 (KC) levels. However, when compared to controls, human AAT was detectable in the serum of mice treated with AAT and these mice had a higher serum anti-elastase activity, and significantly lower proportion of Th1 and Th17 among all Th cells. Cleaved caspase-3-positive cells were scarce but significantly less abundant in allografts from recipients treated with AAT as compared to those treated with vehicle.ConclusionTherapy with AAT suppresses the acute rejection after LuTx in a mouse model. The beneficial effects seem to involve anti-protease and immunomodulatory activities of AAT.

Fingerprints and changes analysis of volatile compounds in fresh-cut yam during yellowing process by using HS-GC-IMS

The purpose of this study was to investigate the dynamic change in volatile components during the yellowing process. The volatile components were analyzed by headspace gas chromatography–ion mobility spectrometry (HS-GC-IMS) and the critical enzyme activities were determined by commercial kits. The results revealed that 29 signal peaks for 20 compounds were identified, which were quantified in all samples: 1 furan, 1 ester, 15 aldehydes, 3 ketones, and 9 alcohols. The contents of most of these compounds increased first and then decreased at 36 h, which were basically consistent with the enzyme activities of LOX, HPL, ADH and AAT. Subsequently, principal component analysis (PCA) results clearly showed that the fresh-cut yams for different yellowing processes were well distinguished by the volatile compounds. These results showed that the potential of HS-GC-IMS-based approaches to evaluate the volatile compound profiles of fresh-cut yam at different stages in the yellowing period.

Boosted Pro-Inflammatory Activity in Human PBMCs by Lipopolysaccharide and SARS-CoV-2 Spike Protein Is Regulated by α-1 Antitrypsin

For the treatment of severe COVID-19, supplementation with human plasma-purified α-1 antitrypsin (AAT) to patients is currently considered. AAT inhibits host proteases that facilitate viral entry and possesses broad anti-inflammatory and immunomodulatory activities. Researchers have demonstrated that an interaction between SARS-CoV-2 spike protein (S) and lipopolysaccharides (LPS) enhances pro-inflammatory responses in vitro and in vivo. Hence, we wanted to understand the potential anti-inflammatory activities of plasma-derived and recombinant AAT (recAAT) in a model of human total peripheral blood mononuclear cells (PBMCs) exposed to a combination of CHO expressed trimeric spike protein and LPS, ex vivo. We confirmed that cytokine production was enhanced in PBMCs within six hours when low levels of LPS were combined with purified spike proteins (“spike”). In the presence of 0.5 mg/mL recAAT, however, LPS/spike-induced TNF-α and IL-1β mRNA expression and protein release were significantly inhibited (by about 46–50%) relative to LPS/spike alone. Although without statistical significance, recAAT also reduced production of IL-6 and IL-8. Notably, under the same experimental conditions, the plasma-derived AAT preparation Respreeza (used in native and oxidized forms) did not show significant effects. Our findings imply that an early pro-inflammatory activation of human PBMCs is better controlled by the recombinant version of AAT than the human plasma-derived AAT used here. Considering the increasing clinical interest in AAT therapy as useful to ameliorate the hyper-inflammation seen during COVID-19 infection, different AAT preparations require careful evaluation.

Conversion of the death inhibitor ARC to a killer activates pancreatic β cell death in diabetes.

Loss of insulin-secreting pancreatic β cells through apoptosis contributes to the progression of type 2 diabetes, but underlying mechanisms remain elusive. Here, we identify a pathway in which the cell death inhibitor ARC paradoxically becomes a killer during diabetes. While cytoplasmic ARC maintains β cell viability and pancreatic architecture, a pool of ARC relocates to the nucleus to induce β cell apoptosis in humans with diabetes and several pathophysiologically distinct mouse models. β cell death results through the coordinate downregulation of serpins (serine protease inhibitors) not previously known to be synthesized and secreted by β cells. Loss of the serpin α1-antitrypsin from the extracellular space unleashes elastase, triggering the disruption of β cell anchorage and subsequent cell death. Administration of α1-antitrypsin to mice with diabetes prevents β cell death and metabolic abnormalities. These data uncover a pathway for β cell loss in type 2 diabetes and identify an FDA-approved drug that may impede progression of this syndrome.

Взаимосвязь протеинурии, регистрируемой перед родоразрешением, c исходом беременности и родов у пациенток с преэклампсией

Study Objective: To assess the association between pre-delivery proteinuria (PU) in patients with preeclampsia and pregnancy/ delivery outcomes for mother and child. Study Design: retrospective study. Materials and Methods. We have conducted a retrospective study of 149 labour and delivery medical records of patients with PU diagnosed before delivery, who had preeclampsia during their pregnancy. Patients were divided into two groups depending on PU severity: moderate and significant PU. We studied the rate of complications for mother and foetus, pregnancy/delivery outcomes for foetus, and anthropometric and demographic parameters of mothers, their somatic conditions, and obstetric history. Study Results. Women with significant PU diagnosed before delivery demonstrated higher statistically and clinically significant risk of HELLP syndrome, as well as increased AAT and ALT activity vs. patients with moderate PU. As for outcomes for newborns, there was a clear statistically significant association between respiratory disorders in child and rate of pre-delivery PU in mother. This complication was recorded more frequently in newborns born to mothers with significant PU vs. moderate PU. Conclusion. An absolute PU quantification before delivery is a method to forecast unfavourable outcomes for mother and child. Keywords: preeclampsia, moderate proteinuria, severe proteinuria, pregnancy and delivery outcomes

Endogenous sulfur dioxide is a novel inhibitor of hypoxia-induced mast cell degranulation

IntroductionMast cell (MC) degranulation is an important step in the pathogenesis of inflammatory reactions and allergies; however, the mechanism of stabilizing MC membranes to reduce their degranulation is unclear. MethodsSO2 content in MC culture supernatant was measured by HPLC-FD. The protein and mRNA expressions of the key enzymes aspartate aminotransferase 1 (AAT1) and AAT2 and intracellular AAT activity were detected. The cAMP level in MCs was detected by immunofluorescence and ELISA. The release rate of MC degranulation marker β-hexosaminidase was measured. The expression of AAT1 and cAMP, the MC accumulation and degranulation in lung tissues were detected. ObjectivesTo exam whether an endogenous sulfur dioxide (SO2) pathway exists in MCs and if it serves as a novel endogenous MC stabilizer. ResultsWe firstly show the existence of the endogenous SO2/AAT pathway in MCs. Moreover, when AAT1 was knocked down in MCs, MC degranulation was significantly increased, and could be rescued by a SO2 donor. Mechanistically, AAT1 knockdown decreased the cyclic adenosine monophosphate (cAMP) content in MCs, while SO2 prevented this reduction in a dose-independent manner. Pretreatment with the cAMP-synthesizing agonist forskolin or the cAMP degradation inhibitor IBMX significantly blocked the increase in AAT1 knockdown-induced MC degranulation. Furthermore, in hypoxia-stimulated MCs, AAT1 protein expression and SO2 production were markedly down regulated, and MC degranulation was activated, which were blunted by AAT1 overexpression. The cAMP synthesis inhibitor SQ22536 disrupted the suppressive effect of AAT1 overexpression on hypoxia-induced MC degranulation. In a hypoxic environment, mRNA and protein expression of AAT1 was significantly reduced in lung tissues of rats. Supplementation of SO2 elevated the cAMP level and reduced perivascular MC accumulation and degranulation in lung tissues of rats exposed to a hypoxic environment in vivo. ConclusionSO2 serves as an endogenous MC stabilizer via upregulating the cAMP pathway under hypoxic circumstance.

Distinct anti-inflammatory properties of alpha1-antitrypsin and corticosteroids reveal unique underlying mechanisms of action

Alpha1-antitrypsin (AAT) is a serum protease inhibitor that rises during inflammation and healthy pregnancies. Plasma-derived AAT, indicated for genetic AAT deficiency, is presently being explored for additional medical indications. Unlike corticosteroids, some anti-inflammatory activities of AAT involve NF-κB–dependent outcomes, e.g., induction of IL-1R antagonist. AAT activities were compared to dexamethasone (DEX), using various in-vitro cells assays, animal studies, and NF-κB–p65 localization and activity studies. Results demonstrate a cytokine shift towards resolution in AAT-treated cells, as opposed to pan-suppression in DEX-treated cells. AAT enhanced, while DEX suppressed LPS-induced IL-1Ra production and re-epithelialization. When drugs were combined, AAT allowed the immunosuppressive DEX activities, while DEX at medium to high levels antagonized beneficial AAT effects. Interestingly, lower levels of DEX maintained the immunosuppressive effect, while allowing upregulation of IL-1Ra. Therefore, AAT may represent a distinct endogenous anti-inflammatory, resolution-promoting agent that may improve tissue well-being while preventing undesired corticostroids side effects.

Negative auto-regulation of sulfur dioxide generation in vascular endothelial cells: AAT1 S-sulfenylation

Recently, endogenous sulfur dioxide (SO2) has been found to exert an important function in the cardiovascular system. However, the regulatory mechanism for SO2 generation has not been entirely clarified. Hence, we aimed to explore the possible auto-regulation of endogenous SO2 generation and its mechanisms in vascular endothelial cells. We showed that SO2 did not affect the protein expression of aspartate aminotransferase 1 (AAT1), a major SO2 synthesis enzyme, but significantly inhibited AAT activity in primary human umbilical vein endothelial cells (HUVECs) and porcine purified AAT1 protein. An AAT1 enzymatic kinetic study showed that SO2 reduced the Vmax (1.89 ± 0.10 vs 2.55 ± 0.12, μmol/mg/min, P < 0.05) and increased the Km (35.97 ± 9.54 vs 19.33 ± 1.76 μmol/L, P < 0.05) values. Furthermore, SO2 induced S-sulfenylation of AAT1 in primary HUVECs and purified AAT1 protein. LC-MS/MS analysis indicated that SO2 sulfenylated AAT1 at Cys192. Mechanistically, thiol reductant DTT treatment or C192S mutation prevented SO2-induced AAT1 sulfenylation and the subsequent inhibition of AAT activity in purified AAT1 protein and primary HUVECs. Our findings reveal, for the first time, a mechanism of auto-regulation of SO2 generation through sulfenylation of AAT1 at Cys192 to suppress AAT activity in vascular endothelial cells. These findings will greatly deepen the understanding of regulatory mechanisms in the cardiovascular homeostasis.

The influence of different amounts of zinc citrate on blood biochemical indices and productivity of rabbit organism

Deficiency of micro- and macronutrients in the organism of young rabbits causes disorders of metabolism and decreased productivity. Inclusion of essential micronutrients made by nanotechnology to the rabbit diet can eliminate this problem. Therefore, the purpose of our research was to investigate the influence of different amounts of zinc citrate in combination with cobalt and chromium citrates on the biochemical processes in the organism and the productivity of rabbits from 62 to 86 days of age. Researches were performed on rabbits 40 days of age, weighing 1.2–1.4 kg. The animals of the experimental groups were fed with fodder ration of the control group and during the day were fed with cobalt citrate in the amount of 40 μg Co/kg of body weight, chromium citrate – 40 mcg Cr/kg of body weight and zinc citrate, calculated respectively in I, II and III of experimental groups 0.25; 0.50 and 0.75 mg Zn/kg of body weight. In the preparatory period – on the 10th day and in the experimental on the 12th, 24th and 36th days of drinking the supplements in 4 animals from the group blood samples were taken for biochemical researches. Body weight gain was also determined by weighing. It was found that in the blood of animals of the first experimental group on the 12th day of drinking mineral additives increased the cholesterol content on 28.8%, on 24th day – decrease by 39.1% and increase – creatinine by 11.6%, and on 36th day – increase in blood of total protein on 12.3%, increase of alkaline phosphatase activity by 23.0%, creatinine content by 15.5% and average daily growth by 9.4%. A similar additive containing zinc citrate of 0.50 mg Zn/kg of body weight in the blood of rabbits of the second experimental group increased the activity of ACAT by 12 and 24 days, respectively, by 37.3 and 22.1%, respectively, and reduced their cholesterol content by 33.9 and 63.1% compared to the control. Whereas in the same group the growth of ALT activity was noted by 23.7%, and the average daily increase was higher than the control indicator by 7.9% 36 days of the research in comparison with the control group. The high content of zinc citrate in the mineral supplement (0.75 mg Zn/kg) in the blood of rabbits of the third experimental group contributed to the increase of the total protein concentration in all periods of the research, AST activity increased by 26.1% on the 24th day, AAT by 34.9% and the content of triacylglycerols and cholesterol was decreased, while on the 36th day the AAT activity increased by 20.3%, under these conditions, the average daily weight gain of rabbits was the highest and amounted to 38.4 g, which was 12.9% higher than the control. Therefore, the additive in the content of citrates of Co and Cr in the amount of 40 μg/kg of body weight and Zn citrate in the amount of 0.75 mg Zn/kg of body weight contributed to the improvement of metabolic processes and increase of growth indices, which may indicate the dose-dependent influence of the additives used in the rabbit young body.

Effects of grafting on free fatty acid contents and related synthetic enzyme activities in peel and flesh tissues of oriental sweet melon during the different development period

In this experiment, white seed pumpkin ‘Shengzhen No. 1’ was used as rootstock for the oriental sweet melon (Cucumis melo L.) ‘Yumeiren’. The fruits of own-rooted melons were used as control. The objective of this experiment was to study the effects of grafting on the following parameters: content of free fatty acids and activities of synthetic enzymes (LOX, ADH, and AAT). These parameters were measured in the peel and flesh tissues of oriental sweet melon at different time-points. Due to grafting, the content of oleic acid and linolenic acid decreased in peel tissues of melons at early stages of maturity. Therefore, total contents of free fatty acids were lower than those of control. However, there was no significant difference in the content of three free fatty acids in flesh tissue; this content was lower than that in peel tissue. Due to grafting, enzymatic activities of ADH and AAT decreased in peel tissue of melons; however, these enzymes showed different activities in flesh tissues of melons subjected to grafting. Nevertheless, there was no significant difference in enzymatic activities of flesh tissues; however, these activities were lower than that of peel tissue. These results indicate that free fatty acid content and related synthetic enzyme activities decreased when grafting was included in the cultivation of oriental sweet melons. Consequently, the aroma of these melons decreased due to grafting.

Effect of endogenous sulfur dioxide on the apoptosis induced by cobalt chloride in the human pulmonary arterial endothelial cells

Objective To explore the effect of endogenous sulfur dioxide (SO2) on the apoptosis induced by cobalt chloride (CoCl2) in the human pulmonary arterial endothelial cells (HPAECs). Methods CoCl2 was used in the primary HPAECs to mimize hypoxia-induced cell apoptosis.The aspartate aminotransferase 1(AAT1), and the key enzyme generating endogenous SO2 were over-expressed by transfecting HPAECs with lentivirus containing AAT1 cDNA.HPAECs were divided into 4 groups: vehicle group, vehicle+ CoCl2 group, AAT1 group and AAT1+ CoCl2 group.The expressions of AAT1, B-cell lymphoma-2 (bcl-2), bcl-associated X protein (bax), Caspase-3 and activated Caspase-3 (cleaved Caspase-3) in the HPAECs were measured by Western blot.The AAT activity was assessed with colorimetry method.The SO2 content in the HPAECs was in situ observed by SO2-specific fluorescent probe.The HPAECs apoptosis was investigated by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. Results There were significant differences in the endogenous SO2 content, the expre-ssions of AAT1 and bcl-2, and the ratio of cleaved Caspase-3/Caspase-3 among 4 groups of HPAECs were (F=147.364, 23.738, 6.521, 64.884, all P 0.05). Compared with vehicle group, AAT activity [(0.96±0.24) Carmen′s unit/μg vs.(2.21±0.60) Carmen′s unit/μg], endogenous SO2 content (40.71±7.72 vs.105.60±16.20) and bcl-2 expression (0.59±0.19 vs.1.02±0.20) in the HPAECs of vehicle+ CoCl2 group were significantly decreased, while the cell apoptosis assessed by TUNEL and the ratio of cleaved Caspase-3/Caspase-3 (1.56±0.25 vs.0.95±0.13) were significantly increased (all P 0.05). The SO2 content (351.50±42.43 vs.105.60±16.20) and AAT1 expression (1.22±0.33 vs.0.53±0.11) in the HPAECs of AAT1 group were higher than those of vehicle group (all P 0.05). Moreover, no difference was observed in the HPAECs apoptosis assessed by TUNEL and the ratio of cleaved Caspase-3/Caspase-3 (0.51±0.17 vs.0.50±0.11) between the two AAT1-overexpressed groups (all P>0.05). Conclusion Endo-genous SO2 inhibited the hypoxic HPAECs apoptosis stimulated by the treatment of CoCl2. Key words: Sulfur dioxide; Aspartate aminotransferase 1; Cobalt chloride; Human pulmonary arterial endothelial cells; Apoptosis

SERPINA1 mRNA as a Treatment for Alpha-1 Antitrypsin Deficiency.

Alpha-1-antitrypsin (AAT) deficiency is a genetic disorder that produces inactive/defective AAT due to mutations in the SERPINA1 gene encoding AAT. This disease is associated with decreased activity of AAT in the lungs and deposition of excessive defective AAT protein in the liver. Currently there is no specific treatment for liver disease associated with AAT deficiency. AAT lung disease is often treated with one of several serum protein replacement products; however, long-term studies of the effectiveness of SerpinA1 replacement therapy are not available, and it does not reduce liver damage in AAT deficiency. mRNA therapy could potentially target both the liver and lungs of AAT deficient patients. AAT patient fibroblasts and AAT patient fibroblast-derived hepatocytes were transfected with SERPINA1-encoding mRNA and cell culture media were tested for SerpinA1 expression. Our data demonstrates increased SerpinA1 protein in culture media from treated AAT patient fibroblasts and AAT patient fibroblast-derived hepatocytes. In vivo studies in wild type mice demonstrate SERPINA1 mRNA biodistribution in liver and lungs, as well as SerpinA1 protein expression in these two target organs which are critically affected in AAT deficiency. Taken together, our data suggests that SerpinA1 mRNA therapy has the potential to benefit patients suffering from AAT deficiency.

Large-scale purification of high purity α1-antitrypsin from Cohn Fraction IV with virus inactivation by solvent/detergent and dry-heat treatment.

α1-Antitrypsin (AAT) is widely used to treat patients with congenital AAT deficiency. Cohn Fraction IV (Cohn F IV) is normally discarded during the manufacturing process of albumin but contains approximately 33% of plasma AAT. We established a new process for large-scale purification of AAT from it. liquid chromatography-electrospray ionization-tandem mass spectrometry and high-performance liquid chromatography were applied for qualitative identification and composition analysis, respectively. Stabilizers were optimized for AAT activity protection during lyophilization and dry-heat. Virus inactivation by dry-heat and solvent/detergent (S/D) was validated on a range of viruses. AAT with purity of 95.54%, specific activity of 3,938.5IU/mg, and yield of 26.79%, was achieved. More than 95% activity was reserved after S/D. More than 96% activity was obtained after lyophilization or dry-heat. After S/D, pseudorabies virus (PRV) and vesicular stomatitis virus (VSV) were inactivated below detectable level within 1H. Virus titer reductions of more than 5.50 log10 and 5.38 log10 were achieved for PRV and VSV, respectively. Porcine parvovirus and encephalomyocarditis virus were inactivated by 3.17 log10 and 5.88 log10 reduction after dry-heat. The advantages of this process, including suitability for large-scale production, high purity, better utilization of human plasma, viral safety, commercial and inexpensive chromatography medium, may facilitate its further application.

Changes in endogenous sulfur dioxide pathway in angiotensin II- induced myocardial hypertrophy in mice

Objective To explore the changes in the endogenous sulfur dioxide (SO2) pathway in the myocardial hypertrophy induced by the angiotensin Ⅱ(Ang Ⅱ) in mice. Methods Fourteen healthy C57BL mice, 9 weeks old, were randomly divided into control group(n=7) and Ang Ⅱ group(n=7), and capsule osmotic pump with pre-loaded 9 g/L saline and Ang Ⅱ was implanted into the back of each mouse subcutaneously.After 2 weeks, the mice were executed.The heart weight/body weight (HW/BW) and the left heart weight/full heart weight (LVW/HW) of the mice were measured.The microstructure of the cardiac myocyte was observed by hematoxylin-eosin (HE) staining under the microscope.The expression of myocardial alpha myosin heavy chain (α-MHC) was detected by immunohistochemistry and Western blot methods.SO2 enzymes aspartate aminotransferase 1 (AAT1) and AAT2 protein expression were detected by Western blot method.Myocardial SO2 content and AAT activity were measured by high performance liquid chromatography with fluorimetric detection and colometric method. Results Compared with control group, the HW/BW and LVW/HW in mice of Ang Ⅱ group were significantly increased(all P<0.01), the cardiac myocytes were hypertrophy, and α-MHC positive staining in the cytoplasm of myocardium was weakened.Moreover, Western blot data showed that α-MHC protein expression in heart tissue of Ang Ⅱ-treated mice was decreased significantly (all P<0.05). Simultaneously, the data showed that AAT2 protein expression, SO2 content and AAT activity in heart tissue of Ang Ⅱ-treated mice were also decreased markedly[(1.093±0.131) μmol/g protein vs.(0.737±0.233) μmol/g protein, P<0.05; (7.979±1.317) U/mg protein vs.(6.470±0.516) U/mg protein, P<0.01]. Furthermore, there was a negative correlation between LVW/HW and cardiac SO2 content in heart tissue (r=-0.56, P<0.05). Conclusions Myocardial endogenous SO2/AAT2 pathway is down-regulated in the development of myocardial hypertrophy induced by Ang Ⅱ in mice. Key words: Myocardial hypertrophy; Sulfur dioxide; Angiotensin Ⅱ

IL-32-induced Inflammatory Cytokines Are Selectively Suppressed by α1-antitrypsin in Mouse Bone Marrow Cells

The induction of interleukin (IL)-32 in bone marrow (BM) inflammation is crucial in graft versus host disease (GvHD) that is a common side effect of allogeneic BM transplantation. Clinical trials on α-1 antitrypsin (AAT) in patients with GvHD are based on the preliminary human and mouse studies on AAT reducing the severity of GvHD. Proteinase 3 (PR3) is an IL-32-binding protein that was isolated from human urine. IL-32 primarily induces inflammatory cytokines in myeloid cells, probably due to PR3 expression on the membrane of the myeloid lineage cells. The inhibitory activity of AAT on serine proteinases may explain the anti-inflammatory effect of AAT on GvHD. However, the anti-inflammatory activity of AAT on BM cells remains unclear. Mouse BM cells were treated with IL-32γ and different inflammatory stimuli to investigate the anti-inflammatory activity of AAT. Recombinant AAT-Fc fusion protein inhibited IL-32γ-induced IL-6 expression in BM cells, but failed to suppress that induced by other stimuli. In addition, the binding of IL-32γ to PR3 was abrogated by AAT-Fc. The data suggest that the specific anti-inflammatory effect of AAT in mouse BM cells is due to the blocking of IL-32 binding to membrane PR3.