The effect of age on aqueous humor of humans with high myopia.

Background: High myopia is a common cause of vision loss. Age is an important factor in the development of high myopia. However, the effect of age on aqueous humor proteins in the context of high myopia is unknown. This study explored the effect of age on the aqueous humor protein of humans with high myopia. Methods: The aqueous humor of high myopia patients of different ages with implantable collamer lens implantation (ICL) was collected. Data-independent acquisition proteomic analysis was employed to explore differentially expressed proteins (DEPs). Two different bioinformatics analysis methods were used to interpret the proteomic results. Furthermore, three proteins were confirmed by enzyme-linked immunosorbent assay (ELISA). Results: The study showed 18 upregulated and 20 downregulated proteins. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the upregulated DEPs were highly enriched in coagulation and complement cascades. Weighted gene coexpression network analysis showed that the blue module was identified as a key module for high myopia and that the plasminogen (PLG) protein is a hub protein. ELISA confirmed that the expression levels of Alpha-1-antitrypsin were significantly upregulated in the aqueous humor of older patients presenting with high myopia. Conclusions: This is the first study to investigate the effect of age on the level of aqueous humor protein in high myopia. Our study provided a comprehensive data set on the overall protein changes of different ages of human high myopia, shedding light on its potential molecular mechanism in high myopia damage to the eyeball.

Alpha-1 antitrypsin inhibits Clostridium botulinum C2 toxin, Corynebacterium diphtheriae diphtheria toxin and B. anthracis fusion toxin.

The bacterium Clostridium botulinum, well-known for producing botulinum neurotoxins, which cause the severe paralytic illness known as botulism, produces C2 toxin, a binary AB-toxin with ADP-ribosyltranferase activity. C2 toxin possesses two separate protein components, an enzymatically active A-component C2I and the binding and translocation B-component C2II. After proteolytic activation of C2II to C2IIa, the heptameric structure binds C2I and is taken up via receptor-mediated endocytosis into the target cells. Due to acidification of endosomes, the C2IIa/C2I complex undergoes conformational changes and consequently C2IIa forms a pore into the endosomal membrane and C2I can translocate into the cytoplasm, where it ADP-ribosylates G-actin, a key component of the cytoskeleton. This modification disrupts the actin cytoskeleton, resulting in the collapse of cytoskeleton and ultimately cell death. Here, we show that the serine-protease inhibitor α1-antitrypsin (α1AT) which we identified previously from a hemofiltrate library screen for PT from Bordetella pertussis is a multitoxin inhibitor. α1AT inhibits intoxication of cells with C2 toxin via inhibition of binding to cells and inhibition of enzyme activity of C2I. Moreover, diphtheria toxin and an anthrax fusion toxin are inhibited by α1AT. Since α1AT is commercially available as a drug for treatment of the α1AT deficiency, it could be repurposed for treatment of toxin-mediated diseases.

Immunological and homeostatic pathways of alpha -1 antitrypsin: a new therapeutic potential.

α -1 antitrypsin (A1AT) is a 52 kDa acute-phase glycoprotein belonging to the serine protease inhibitor superfamily (SERPIN). It is primarily synthesized by hepatocytes and to a lesser extent by monocytes, macrophages, intestinal epithelial cells, and bronchial epithelial cells. A1AT is encoded by SERPINA1 locus, also known as PI locus, highly polymorphic with at least 100 allelic variants described and responsible for different A1AT serum levels and function. A1AT inhibits a variety of serine proteinases, but its main target is represented by Neutrophil Elastase (NE). However, recent attention has been directed towards its immune-regulatory and homeostatic activities. A1AT exerts immune-regulatory effects on different cell types involved in innate and adaptive immunity. Additionally, it plays a role in metal and lipid metabolism, contributing to homeostasis. An adequate comprehension of these mechanisms could support the use of A1AT augmentation therapy in many disorders characterized by a chronic immune response. The aim of this review is to provide an up-to-date understanding of the molecular mechanisms and regulatory pathways responsible for immune-regulatory and homeostatic activities of A1AT. This knowledge aims to support the use of A1AT in therapeutic applications. Furthermore, the review summarizes the current state of knowledge regarding the application of A1AT in clinical and laboratory settings human and animal models.

Limb Perfusion Delivery of a rAAV1 Alpha-1 Antitrypsin Vector in Non-Human Primates Is Safe but Insufficient for Therapy.

BACKGROUND/OBJECTIVES: α-1 antitrypsin (AAT) deficiency is an inherited, genetic condition characterized by reduced serum levels of AAT and increased risk of developing emphysema and liver disease. AAT is normally synthesized primarily in the liver, but muscle-targeting with a recombinant adeno-associated virus (rAAV) vector for α-1 antitrypsin (AAT) gene therapy has been used to minimize liver exposure to the virus and hepatotoxicity. Clinical trials of direct intramuscular (IM) administration of rAAV1-hAAT have demonstrated its overall safety and transgene expression for 5 years. However, the failure to reach the therapeutic target level after 100 large-volume (1.5 mL) IM injections of maximally concentrated vector led us to pursue a muscle-targeting approach using isolated limb perfusion. This targets the rAAV to a greater muscle mass and allows for a higher total volume (and thereby a higher dose) than is tolerable by multiple direct IM injections. Limb perfusion has been shown to be feasible in non-human primates using the rAAV1 serotype and a ubiquitous promoter expressing an epitope-tagged AAT matched to the host species. METHODS: In this study, we performed a biodistribution and preclinical safety study in non-human primates with a clinical candidate rAAV1-human AAT (hAAT) vector at doses ranging from 3.0 × 1012 to 1.3 × 1013 vg/kg, bracketing those used in our clinical trials. RESULTS: We found that limb perfusion delivery of rAAV1-hAAT was safe and showed a biodistribution pattern similar to previous studies. However, serum levels of AAT obtained with high-dose limb perfusion still reached only ~50% of the target serum levels. CONCLUSIONS: Our results suggest that clinically effective AAT gene therapy may ultimately require delivery at doses between 3.5 × 1013-1 × 1014 vg/kg, which is within the dose range used for approved rAAV gene therapies. Muscle-targeting strategies could be incorporated when delivering systemic administration of high-dose rAAV gene therapies to increase transduction of muscle tissues and reduce the burden on the liver, especially in diseases that can present with hepatotoxicity such as AAT deficiency.

Alpha-1-antitrypsin improves anastomotic healing in intestinal epithelial cells model.

PURPOSE: Intestinal anastomosis is a routine procedure in pediatric surgery, with leakage being a significant complication. Human alpha1-antitrypsin (AAT), whose physiological serum concentrations range from 0.9-2.0 mg/ml, is known to accelerate wound healing and stimulate the expression of cell proliferation-related genes. We hypothesized that AAT might enhance anastomotic healing. METHODS: In a monolayer of non-tumorigenic HIEC-6 epithelial cells derived from fetal intestine a scratch was created. Standard medium without (control) or with AAT (0.5 and 1 mg/ml) was added. Cells were observed using a Life-Cell Imaging System. Cell proliferation was assessed, and the expression of proliferation-related genes was measured by qRT-PCR. RESULTS: In the presence of AAT, the scratch closed significantly faster. Cells treated with 1 mg/ml AAT showed 53% repopulation after 8 h and 97% after 18 h, while control cells showed 24% and 60% repopulation, respectively (p < 0.02). The treatment with AAT induced HIEC-6-cell proliferation and significantly increased the mRNA-expression of CDKN1A, CDKN2A, ANGPTL4, WNT3 and COL3A1 genes. AAT did not change the mRNA-expression of CXCL8 but decreased levels of IL-8 as compared to controls. CONCLUSION: At physiological concentrations AAT accelerates the confluence of intestinal cells and increases cell proliferation. The local administration of AAT may bear therapeutic potential to improve anastomotic healing.

Berberine potentiates liver inflammation and fibrosis in the PI*Z hAAT transgenic murine model.

BACKGROUND: Alpha-1 antitrypsin deficiency (AATD) is an inherited disease, the common variant caused by a Pi*Z mutation in the SERPINA1 gene. Pi*Z AAT increases the risk of pulmonary emphysema and liver disease. Berberine (BBR) is a nature dietary supplement and herbal remedy. Emerging evidence revealed that BBR has remarkable liver-protective properties against various liver diseases. In the present study, we investigated the therapeutic effects and toxicities of BBR in Pi*Z hepatocytes and Pi*Z transgenic mice. METHODS: Huh7.5 and Huh7.5Z (which carries the Pi*Z mutation) cells were treated with different concentrations of BBR for 48 hours. MTT was performed for cell viability assay. Intracellular AAT levels were evaluated by western blot. In vivo studies were carried out in wild type, native phenotype AAT (Pi*M), and Pi*Z AAT transgenic mice. Mice were treated with 50 mg/kg/day of BBR or solvent only by oral administration for 30 days. Western blot and liver histopathological examinations were performed to evaluate therapeutic benefits and liver toxicity of BBR. RESULTS: BBR reduced intracellular AAT levels in Huh7.5Z cells, meanwhile, no Pi*Z-specific toxicity was observed. However, BBR did not reduce liver AAT load but significantly potentiated liver inflammation and fibrosis accompanying the activation of unfolded protein response and mTOR in Pi*Z mice, but not in wild type and Pi*M mice. CONCLUSIONS: BBR exacerbated liver inflammation and fibrosis specifically in Pi*Z mice. This adverse effect may be associated with the activation of unfolded protein response and mTOR. This study implicates that BBR should be avoided by AATD patients.

Prevalence of Alpha-1 Antitrypsin Deficiency Alleles in a Lithuanian Cohort of Wheezing Small Children.

Severe inherited alpha-1 antitrypsin deficiency (AATD) is an autosomal genetic condition linked to chronic obstructive pulmonary disease (COPD). The significance of heterozygous, milder deficiency variants (PiSZ, PiMZ, PiMS) is less clear. We studied AATD genotypes in 145 children (up to 72 months old) with assessed wheezing severity using the Pediatric Respiratory Assessment Measure (BCCH PRAM score). A control group of 74 children without airway obstruction was included. AAT concentration and Pi phenotype were determined from dry blood spot samples using nephelometry and real-time PCR; PiS and PiZ alleles were identified by isoelectrofocusing. Among the wheezers, the Pi*S allele incidence was 2.07% (3 cases) and the Pi*Z allele was 6.9% (10 cases). The Pi*Z allele frequency was higher in wheezers compared to controls (44.8% vs. 20.27%) and the general Lithuanian population (44.8% vs. 13.6%) and was similar to adult COPD patients in Lithuania: Pi*S 10.3% vs. 15.8% and Pi*Z 44.8% vs. 46.1%. No association was found between AAT genotypes and wheezing severity. Finding that wheezer children exhibit a frequency of Z* and S* alleles like that found in adults with COPD suggests a potential genetic predisposition that links early wheezing in children to the development of COPD in adulthood. Larger cohort studies are needed to confirm this finding.

[Validation of a method for measuring the antielastolytic activity of human circulating alpha1-antitrypsin]. / Validation d'une méthode de mesure de l'activité antiélastasique de l'alpha1-antitrypsine circulante humaine.

The existence of alpha-1 antitrypsin variants with apparently unremarkable phenotypes and serum concentrations, contrasting with a clinical picture suggestive of a severe deficiency, led us to investigate whether in these cases there was a reduction or even suppression of the capacity of alpha-1 antitrypsin to inhibit elastase. To this end, in two different laboratories, we adapted and validated a method for measuring the functional activity of alpha-1 antitrypsin, based on spectrophotometric kinetic analysis of the inhibition by serum alpha-1 antitrypsin of the hydrolytic activity of porcine pancreatic elastase on a chromogenic substrate. This method has proved to be robust, reproducible and transferable and made possible to define, on the basis of an analysis of a hospital population, a functionality index with a confidence interval comprised between 0.87 and 1.2, allowing to identify subjects likely to have a functional deficiency of alpha-1 antitrypsin, whether this deficiency being of a genetic origin without any quantitative or phenotypic translation, or whether being acquired under the effect of external agents (cigarette smoke or viruses).

α-1 antitrypsin is promising for the identification of glaucoma severity and is associated with glaucomatous neural damage.

Aim: To investigate the association between plasma AAT level and glaucoma.Methods: 163 glaucoma patients and 111 healthy controls were recruited. The plasma AAT levels were measured by ELISA.Results: Plasma AAT level was significantly higher in glaucoma patients than those in healthy controls (p < 0.001). Patients with higher plasma AAT level exhibited severer disease stage (early vs. severe: p < 0.05; H-P-A; early vs. severe: p < 0.05; early vs. end-stage: p < 0.01; AGIS). ROC curves yielded that AAT can distinguish patients with early glaucoma from those with advanced glaucoma (early vs. severe: AUC: 0.616; H-P-A; early vs. severe: AUC: 0.763; early vs. end-stage: AUC: 0.660; AGIS).Conclusion: Plasma AAT is a useful biomarker for the identification of glaucoma severity.

[Box: see text].

Personalised indication of augmentation therapy for emphysema associated with severe alpha-1 antitrypsin deficiency: a case series.

Severe alpha-1 antitrypsin deficiency (AATD) is associated with an increased risk of emphysema. However, the clinical manifestations are very heterogeneous, and an individual prognosis is very difficult to establish. Intravenous augmentation therapy with alpha-1 antitrypsin (AAT) from pooled blood donors is the only specific treatment available, but it requires weekly or biweekly administration for life. Several guidelines provide the indication criteria for the initiation of AAT augmentation therapy. However, in clinical practice, there are situations in which the decision as to when to start treatment becomes uncertain and some studies have shown great variability in the indication of this treatment even among specialists. The usual dilemma is between initiating augmentation therapy in individuals who may not develop significant lung disease or in whom disease will not progress or delaying it in patients who may otherwise rapidly and irreversibly progress. We illustrate this dilemma with five clinical cases: from the case of a patient with normal lung function who requests initiation of therapy to a moderately stable patient without augmentation or a mild patient who, after several years of remaining stable without treatment, deterioration in lung function initiated and, consequently, augmentation therapy was begun. All the nuances associated with the indication of augmentation justify a personalised approach and the decision about initiating augmentation therapy must be made after careful consideration of the pros and cons with the patient in reference centres with experience in treatment. These reference centres can work in collaboration with local hospitals where patients can be closely followed and augmentation therapy can be administered to avoid unnecessary travelling, making periodical administrations more comfortable for the patient.

Citrullination, a novel posttranslational modification of elastin, is involved in COPD pathogenesis.

Elastin is an extracellular matrix protein (ECM) that supports elasticity of the lung, and in patients with chronic obstructive pulmonary disease (COPD) and emphysema, the structural changes that reduce the amount of elastic recoil, lead to loss of pulmonary function. We recently demonstrated that elastin is a target of peptidyl arginine deiminase (PAD) enzyme-induced citrullination, thereby leading to enhanced susceptibility of this ECM protein to proteolysis. This study aimed to investigate the impact of PAD activity in vivo and furthermore assessed whether pharmacological inhibition of PAD activity protects against pulmonary emphysema. Using a Serpina1a-e knockout mouse model, previously shown to develop inflammation-mediated emphysema, we validated the involvement of PADs in airway disease. In line with emphysema development, intratracheal administration of lipopolysaccharide in combination with PADs provoked significant airspace enlargement (P < 0.001) and diminished lung function, including loss of lung tissue elastance (P = 0.0217) and increases in lung volumes (P = 0.0463). Intraperitoneal treatment of mice with the PAD inhibitor, BB-Cl-amidine, prevented PAD/LPS-mediated lung function decline and emphysema and reduced levels of citrullinated airway elastin (P = 0.0199). These results provide evidence for the impact of PADs on lung function decline, indicating promising potential for the future development of PAD-based therapeutics for preserving lung function in patients with COPD.NEW & NOTEWORTHY This study provides evidence for the impact of peptidyl arginine deiminase (PAD) enzymes on lung function decline, indicating promising potential for the future development of PAD-based therapeutics for preserving lung function in patients with COPD.

Enhancement of high-density lipoprotein-associated protease inhibitor activity prevents atherosclerosis progression.

BACKGROUND AND AIMS: Inflammatory cells within atherosclerotic lesions secrete proteolytic enzymes that contribute to lesion progression and destabilization, increasing the risk for an acute cardiovascular event. Elastase is a serine protease, secreted by macrophages and neutrophils, that may contribute to the development of unstable plaque. We previously reported interaction of endogenous protease-inhibitor proteins with high-density lipoprotein (HDL), including alpha-1-antitrypsin, an inhibitor of elastase. These findings support a potential role for HDL as a modulator of protease activity. In this study, we test the hypothesis that enhancement of HDL-associated elastase inhibitor activity is protective against atherosclerotic lesion progression. METHODS: We designed an HDL-targeting protease inhibitor (HTPI) that binds to HDL and confers elastase inhibitor activity. Lipoprotein binding and the impact of HTPI on atherosclerosis were examined using mouse models. Histology and immunofluorescence staining of aortic root sections were used to examine the impact of HTPI on lesion morphology and inflammatory features. RESULTS: HTPI is a small (1.6 kDa) peptide with an elastase inhibitor domain, a soluble linker, and an HDL-targeting domain. When incubated with human plasma ex vivo, HTPI predominantly binds to HDL. Intravenous administration of HTPI to mice resulted in its binding to plasma HDL and increased elastase inhibitor activity on isolated HDL. Accumulation of HTPI within plaque was observed after administration to Apoe-/- mice. To examine the effect of HTPI treatment on atherosclerosis, prevention and progression studies were performed using Ldlr-/- mice fed Western diet. In both study designs, HTPI-treated mice had reduced lipid deposition in plaque. CONCLUSIONS: These data support the hypothesis that HDL-associated anti-elastase activity can improve the atheroprotective potential of HDL and highlight the potential utility of HDL enrichment with anti-protease activity as an approach for stabilization of atherosclerotic lesions.

Allelic frequency of pathogenic α1-antitrypsin variants in the Japanese population: Results from a survey of open Japanese genetic variation databases.

α1-antitrypsin deficiency (AATD) is a hereditary disorder with a global prevalence that differs across regions. AATD is highly prevalent in Europe and North America but rarely found in Asian countries, including Japan, possibly because of the founder effect of the pathogenic SERPINA1 variants PI*Z and PI*S. However, AATD remains underdiagnosed even in high-prevalence and low-prevalence regions, possibly because of lack of awareness. In this study, we surveyed open Japanese genetic variation databases to estimate AATD prevalence in Japan. We identified allelic frequencies (AFs) of 5 among the 14 major pathogenic SERPINA1 variants from three datasets, collectively derived from 63,119 Japanese participants. The mean AF was determined to be 8.56 × 10-4 (95% confidence interval [CI]: 6.43 × 10-4 to 1.12 × 10-3). Given that this represents the entire Japanese population, one AATD patient was speculated to be born per 1.37 × 106 births (95% CI: 7.97 × 105 to 2.42 × 106) in Japan. Our results support the prevailing notion that AATD is extremely rare in Japan.

Analysis of alpha-1-antitrypsin (AAT)-regulated, glucocorticoid receptor-dependent genes in macrophages reveals a novel host defense function of AAT.

Alpha-1-antitrypsin (AAT) plays a homeostatic role in attenuating excessive inflammation and augmenting host defense against microbes. We demonstrated previously that AAT binds to the glucocorticoid receptor (GR) resulting in significant anti-inflammatory and antimycobacterial consequences in macrophages. Our current investigation aims to uncover AAT-regulated genes that rely on GR in macrophages. We incubated control THP-1 cells (THP-1control) and THP-1 cells knocked down for GR (THP-1GR-KD) with AAT, performed bulk RNA sequencing, and analyzed the findings. In THP-1control cells, AAT significantly upregulated 408 genes and downregulated 376 genes. Comparing THP-1control and THP-1GR-KD cells, 125 (30.6%) of the AAT-upregulated genes and 154 (41.0%) of the AAT-downregulated genes were significantly dependent on GR. Among the AAT-upregulated, GR-dependent genes, CSF-2 that encodes for granulocyte-monocyte colony-stimulating factor (GM-CSF), known to be host-protective against nontuberculous mycobacteria, was strongly upregulated by AAT and dependent on GR. We further quantified the mRNA and protein of several AAT-upregulated, GR-dependent genes in macrophages and the mRNA of several AAT-downregulated, GR-dependent genes. We also discussed the function(s) of selected AAT-regulated, GR-dependent gene products largely in the context of mycobacterial infections. In conclusion, AAT regulated several genes that are dependent on GR and play roles in host immunity against mycobacteria.

Alpha-1 antitrypsin targeted neutrophil elastase protects against sepsis-induced inflammation and coagulation in mice via inhibiting neutrophil extracellular trap formation.

AIMS: Sepsis pathophysiology is complex and identifying effective treatments for sepsis remains challenging. The study aims to identify effective drugs and targets for sepsis through transcriptomic analysis of sepsis patients, repositioning analysis of compounds, and validation by animal models. MAIN METHODS: GSE185263 obtained from the GEO database that includes gene expression profiles of 44 healthy controls and 348 sepsis patients categorized by severity. Bioinformatic algorithms revealed the molecular, function, and immune characteristics of the sepsis, and constructed sepsis-related protein-protein interaction networks. Subsequently, Random Walk with Restart analysis was applied to identify candidate drugs for sepsis, which were tested in animal models for survival, inflammation, coagulation, and multi-organ damage. KEY FINDINGS: Our analysis found 1862 genes linked to sepsis development, enriched in functions like neutrophil extracellular trap formation (NETs) and complement/coagulation cascades. With disease progression, immune activation-associated cells were inhibited, while immune suppression-associated cells were activated. Next, the drug repositioning method identified candidate drugs, such as alpha-1 antitrypsin, that may play a therapeutic role by targeting neutrophil elastase (NE) to inhibit NETs. Animal experiments proved that alpha-1 antitrypsin treatment can improve the survival rate, reduce sepsis score, reduce the levels of inflammation markers in serum, and alleviate muti-organ morphological damage in mice with sepsis. The further results showed that α-1 antitrypsin can inhibit the NETs by suppressing the NE for the treatment of sepsis. SIGNIFICANCE: Alpha-1 antitrypsin acted on the NE to inhibit NETs thereby protecting mice from sepsis-induced inflammation and coagulation.