Human Alpha 1-antitrypsin Research Articles

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

Intestinal anastomosis is a routine procedure in pediatric surgery, with leakage being a significantcomplication. 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. Wehypothesized that AAT might enhance anastomotic healing. In a monolayer of non-tumorigenic HIEC-6 epithelial cells derived from fetal intestine a scratch wascreated. Standard medium without (control) or with AAT (0.5 and 1 mg/ml) was added. Cells were observed using aLife-Cell Imaging System. Cell proliferation was assessed, and the expression of proliferation-related genes wasmeasured by qRT-PCR. 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-expressionof CDKN1A, CDKN2A, ANGPTL4, WNT3 and COL3A1 genes. AAT did not change the mRNA-expression of CXCL8 butdecreased levels of IL-8 as compared to controls. At physiological concentrations AAT accelerates the confluence of intestinal cells and increases cellproliferation. The local administration of AAT may bear therapeutic potential to improve anastomotic healing.

Alpha-1 Antitrypsin Augmentation Therapy in Chronic Pancreatitis Patients Undergoing Total Pancreatectomy and Islet Autotransplantation: A Randomized, Controlled Study.

Stress-induced islet graft loss during the peri-transplantation period reduces the efficacy of islet transplantation. In this prospective, randomized, double-blind clinical trial, we evaluated the safety and efficacy of 60 mg/kg human alpha-1 antitrypsin (AAT) or placebo infusion weekly for four doses beginning before surgery in chronic pancreatitis (CP) patients undergoing total pancreatectomy and islet autotransplantation (TP-IAT). Subjects were followed for 12 months post-TP-IAT. The dose of AAT was safe, as there was no difference in the types and severity of adverse events in participants from both groups. There were some biochemical signals of treatment effect with a higher oxygen consumption rate in AAT islets before transplantation and a lower serum C-peptide (an indicator of islet death) in the AAT group at 15 min after islet infusion. Findings per the statistical analysis plan using a modified intention to treat analysis showed no difference in the C-peptide area under the curve (AUC) following a mixed meal tolerance test at 12 months post-TP-IAT. There was no difference in the secondary and exploratory outcomes. Although AAT therapy did not show improvement in C-peptide AUC in this study, AAT therapy is safe in CP patients and there are experiences gained on optimal clinical trial design in this challenging disease.

ER chaperones use a protein folding and quality control glyco-code

N-glycans act as quality control tags by recruiting lectin chaperones to assist protein maturation in the endoplasmic reticulum. The location and composition of N-glycans (glyco-code) are key to the chaperone-selection process. Serpins, a class of serine protease inhibitors, fold non-sequentially to achieve metastable active states. Here, the role of the glyco-code in assuring successful maturation and quality control of two human serpins, alpha-1 antitrypsin (AAT) and antithrombin III (ATIII), is described. We find that AAT, which has glycans near its N terminus, is assisted by early lectin chaperone binding. In contrast, ATIII, which has more C-terminal glycans, is initially helped by BiP and then later by lectin chaperones mediated by UGGT reglucosylation. UGGT action is increased for misfolding-prone disease variants, and these clients are preferentially glucosylated on their most C-terminal glycan. Our study illustrates how serpins utilize N-glycan presence, position, and composition to direct their proper folding, quality control, and trafficking.

Beneficial effects of alpha-1 antitrypsin therapy in a mouse model of colitis-associated colon cancer

BackgroundIt is widely accepted that chronic inflammatory bowel diseases significantly higher a risk for colorectal cancer development. Among different types of treatments for patients with colon cancer, novel protein-based therapeutic strategies are considered.AIMTo explore the effect of human plasma alpha-1 antitrypsin (AAT) protein in the chemically induced mouse model of colorectal cancer.MethodsBALB/c mice with azoxymethane/dextran sodium sulfate (AOM/DSS)-induced colitis-associated colorectal cancer (CAC), we intraperitoneally treated with commercial preparation of human plasma AAT (4 mg per mouse). Effects of this therapy were evaluated histologically, and by immunohistochemical and gene expression assays.ResultsWhen compared with non-treated controls, AOM/DSS mice receiving AAT therapy exhibited significantly longer colons, and less anal bleeding. Concurrently, AAT-treated mice had significantly fewer polyps, and lower numbers of large colon tumors. Immunohistochemical examinations of colon tissues showed significantly lower neutrophil counts, more granzyme B-positive but fewer MMP9 (gelatinase B)-positive cancer cells and lower numbers of apoptotic cells in mice receiving AAT therapy. The expression levels of IL4 were significantly higher while TNFA was slightly reduced in tumor tissues of AOM/DSS mice treated with AAT than in AOM/DSS mice.ConclusionHuman AAT is an acute phase protein with a broad-protease inhibitory and immunomodulatory activities used as a therapeutic for emphysema patients with inherited AAT deficiency. Our results are consistent with previous findings and support an idea that AAT alone and/or in combination with available anti-cancer therapies may represent a new personalized approach for patients with colitis-induced colon cancer.

839-P: Alpha-1 Antitrypsin Augmentation Therapy in Chronic Pancreatitis Patients Undergoing Total Pancreatectomy and Islet Autotransplantation

Stress-induced islet graft loss reduces the efficacy of islet transplants. In this single-center, randomized, double-blind, and controlled clinical trial, we evaluated the safety and efficacy of human alpha-1 antitrypsin (AAT) infusion in chronic pancreatitis (CP) patients undergoing total pancreatectomy and islet autotransplantation (TP-IAT) at the Medical University of South Carolina. Among 48 qualified subjects randomized in a 1:2 (placebo vs AAT) ratio, 43 received TP-IAT. Subjects were given saline or AAT infusion at 60mg/kg weekly for 4 weeks and were followed for 12 months. The primary efficacy endpoint was the islet function measured by the C-peptide area under the curve during a mixed meal tolerance test (MMTT) at Month 12 (M12). Adverse events (AEs) were measured throughout the study. Safety, glycemic control, insulin use, and quality of life (QoL) were compared between groups. Safety evaluation showed no significant differences in the types and severity of participant AEs. The primary efficacy outcome was not significantly different between groups. Baseline characteristics including age, body weight, BMI, HbA1C, etc, were comparable in the randomized populations. The oxygen consumption rate before the transplant was higher in the AAT group. Serum C-peptide (an indicator of islet death) was lower in the AAT group, suggesting less islet injury. AAT subjects trended toward better SF-12 physical QoL and lower insulin use at both 75 and 365 days post-IAT. Despite intensive efforts to get participants to return for the Day 365 MMTT, only 10/14 (71%) in the placebo group and 18/29 (62%) in the AAT group returned for this test. Although we did not observe a statistical difference in islet function using AAT therapy, we gained a better understanding of this patient population. Minimizing numbers of follow-up visits, use of fewer doses of medication with higher doses, and providing travel are all important for future clinical trials in TP-IAT. Disclosure H. Wang: None. W. Gou: None. D. Adams: None. K. Morgan: Speaker's Bureau; Johnson &amp; Johnson Medical Devices Companies. P.J. Nietert: None. J. Hirsch: None. C. Strange: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (1R01DK105183, DK120394, DK118529); U.S. Department of Veterans Affairs (I01BX004536 to H.W.)

Correction of F8 intron 1 inversion in hemophilia A patient-specific iPSCs by CRISPR/Cas9 mediated gene editing.

Introduction: Hemophilia A (HA) is the most common genetic bleeding disorder caused by mutations in the F8 gene encoding coagulation factor VIII (FVIII). As the second predominant pathogenic mutation in hemophilia A severe patients, F8 Intron one inversion (Inv1) completely splits the F8 gene into two parts and disrupts the F8 transcription, resulting in no FVIII protein production. The part which contains exon 2-exon 26 covers 98% of F8 coding region. Methods: We hypothesized that in situ genetic manipulation of F8 to add a promoter and exon one before the exon two could restore the F8 expression. The donor plasmid included human alpha 1-antitrypsin (hAAT) promoter, exon one and splicing donor site (SD) based on homology-mediated end joining (HMEJ) strategy was targeted addition in hemophilia A patient-derived induced pluripotent stem cell (HA-iPSCs) using CRISPR/Cas9. The iPSCs were differentiated into hepatocyte-like cells (HPLCs). Results: The hAAT promoter and exon one were targeted addition in HA-iPSCs with a high efficiency of 10.19% via HMEJ. The FVIII expression, secretion, and activity were detected in HPLCs derived from gene-targeted iPSCs. Discussion: Thus, we firstly rescued the 140kb reversion mutation by gene addition of a 975bp fragment in the HA-iPSCs with Inv1 mutation, providing a promising gene correction strategy for genetic disease with large sequence variants.

Mice inflammatory responses to inhaled aerosolized LPS: effects of various forms of human alpha1-antitrypsin.

Rodent models of lipopolysaccharide (LPS)-induced pulmonary inflammation are used for anti-inflammatory drug testing. We aimed to characterize mice responses to aerosolized LPS alone or with intraperitoneal (i.p.) delivery of alpha1-antitrypsin (AAT). Balb/c mice were exposed to clean air or aerosolized LPS (0.21 mg/mL) for 10 min per day, for 3 d. One hour after each challenge, animals were treated i.p. with saline or with (4 mg/kg body weight) one of the AAT preparations: native (AAT), oxidized (oxAAT), recombinant (recAAT), or peptide of AAT (C-36). Experiments were terminated 6 h after the last dose of AATs. Transcriptome data of mice lungs exposed to clean air versus LPS revealed 656 differentially expressed genes and 155 significant gene ontology terms, including neutrophil migration and toll-like receptor signaling pathways. Concordantly, mice inhaling LPS showed higher bronchoalveolar lavage fluid neutrophil counts and levels of myeloperoxidase, inducible nitric oxide synthase, IL-1β, TNFα, KC, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF). Plasma inflammatory markers did not increase. After i.p. application of AATs, about 1% to 2% of proteins reached the lungs but, except for GM-CSF, none of the proteins significantly influenced inflammatory markers. All AATs and C-36 significantly inhibited LPS-induced GM-CSF release. Surprisingly, only oxAAT decreased the expression of several LPS-induced inflammatory genes, such as Cxcl3, Cd14, Il1b, Nfkb1, and Nfkb2, in lung tissues. According to lung transcriptome data, oxAAT mostly affected genes related to transcriptional regulation while native AAT or recAAT affected genes of inflammatory pathways. Hence, we present a feasible mice model of local lung inflammation induced via aerosolized LPS that can be useful for systemic drug testing.

Human Alpha-1 Antitrypsin Attenuates ENaC and MARCKS and Lowers Blood Pressure in Hypertensive Diabetic db/db Mice.

Hypertension may develop before or after the onset of diabetes and it is known to increase the risk of developing diabetic nephropathy. Alpha-1 antitrypsin (AAT) is a multi-functional protein with beneficial effects in various diseases but its role in reducing blood pressure in the diabetic kidney has not been thoroughly studied. Like blood pressure, epithelial sodium channels (ENaC) and its adaptor protein myristoylated alanine-rich C-kinase substrate (MARCKS) are regulated by circadian rhythms. Our hypothesis is that administration of human AAT (hAAT) reduces blood pressure in hypertensive diabetic mice by attenuating membrane expression of ENaC and its association with the actin cytoskeleton. First, we show hAAT administration results in reduced blood pressure in diabetic db/db mice compared to vehicle treatment in both the inactive and active cycles. Western blotting and immunohistochemistry analyses showed a reduction of ENaC and the actin cytoskeleton protein, MARCKS in the kidneys of diabetic db/db mice treated with hAAT compared to vehicle. hAAT treatment resulted in elevated amounts of extracellular vesicles present in the urine of diabetic db/db mice compared to vehicle treatment both in the inactive and active cycles. Multiple hexosylceramides, among other lipid classes increased in urinary EVs released from hAAT treated hypertensive diabetic mice compared to vehicle treated mice. Taken together, these data suggest hAAT treatment could normalize blood pressure in the diabetic kidney in a mechanism involving attenuation of renal ENaC and MARCKS protein expression and possibly ceramide metabolism to hexosylceramide in kidney cells.

Recombinant Alpha-1 Antitrypsin as Dry Powder for Pulmonary Administration: A Formulative Proof of Concept.

Alpha-1 antitrypsin (AAT) deficiency is a genetic disorder associated with pulmonary emphysema and bronchiectasis. Its management currently consists of weekly infusions of plasma-purified human AAT, which poses several issues regarding plasma supplies, possible pathogen transmission, purification costs, and parenteral administration. Here, we investigated an alternative administration strategy for augmentation therapy by combining recombinant expression of AAT in bacteria and the production of a respirable powder by spray drying. The same formulation approach was then applied to plasma-derived AAT for comparison. Purified, active, and endotoxin-free recombinant AAT was produced at high yields and formulated using L-leucine and mannitol as excipients after identifying compromise conditions for protein activity and good aerodynamic performances. An oxygen-free atmosphere, both during formulation and powder storage, slowed down methionine-specific oxidation and AAT inactivation. This work is the first peer-reviewed report of AAT formulated as a dry powder, which could represent an alternative to current treatments.

Characterization of hepatic inflammatory changes in a C57BL/6J mouse model of alpha1-antitrypsin deficiency.

Alpha-1 antitrypsin deficiency (AATD) is a genetic disease caused by a hepatic accumulation of mutant alpha-1 antitrypsin (ZAAT). Individuals with AATD are prone to develop a chronic liver disease that remains undiagnosed until late stage of the disease. Here, we sought to characterize the liver pathophysiology of a human transgenic mouse model for AATD with a manifestation of liver disease compared with normal transgenic mice model. Male and female transgenic mice for normal (Pi*M) and mutant variant (Pi*Z) human alpha-1 antitrypsin at 3 and 6 mo of age were subjected to this study. The progression of hepatic ZAAT accumulation, hepatocyte injury, steatosis, liver inflammation, and fibrotic features were monitored by performing an in vivo study. We have also performed a Next-Gene transcriptomic analysis of the transgenic mice liver tissue 16 h after lipopolysaccharide (LPS) administration to delineate liver inflammatory response in Pi*Z mice as compared with Pi*M. Our results show hepatic ZAAT accumulation, followed by hepatocyte ballooning and liver steatosis developed at 3 mo in Pi*Z mice compared with the mice carrying normal variant of human alpha-1 antitrypsin. We observed higher levels of hepatic immune cell infiltrations in both 3- and 6-mo-old Pi*Z mice compared with Pi*M as an indication of liver inflammation. Liver fibrosis was observed as accumulation of collagen in 6-mo-old Pi*Z liver tissues compared with Pi*M control mice. Furthermore, the transcriptomic analysis revealed a dysregulated liver immune response to LPS in Pi*Z mice compared with Pi*M. Of particular interest for translational work, this study aims to establish a mouse model of AATD with a strong manifestation of liver disease that will be a valuable in vivo tool to study the pathophysiology of AATD-mediated liver disease. Our data suggest that the human transgenic mouse model of AATD could provide a suitable model for the evaluation of therapeutic approaches and preventive reagents against AATD-mediated liver disease.NEW & NOTEWORTHY We have characterized a mouse model of human alpha-1 antitrypsin deficiency with a strong manifestation of liver disease that can be used as an in vivo tool to test preventive and therapeutic reagents. Our data explores the altered immunophenotype of alpha-1 antitrypsin-deficient liver macrophages and suggests a relationship between acute inflammation, immune response, and fibrosis.

Alpha-1 Antitrypsin Reduces Disease Progression in a Mouse Model of Charcot-Marie-Tooth Type 1A: A Role for Decreased Inflammation and ADAM-17 Inhibition

Charcot-Marie-Tooth disease type 1 (CMT1A) is a hereditary peripheral neuropathy for which there is no available therapy. Alpha-1 antitrypsin (AAT) is an abundant serine protease inhibitor with anti-inflammatory and immunomodulating properties. Here, we tested whether treatment with human AAT (hAAT) would have a therapeutic effect on CMT1A in a PMP22 transgenic mouse model. Our results show that hAAT significantly improved compound muscle action potential and histopathological features and decreased circulating IL-6 in CMT1A mice. We also investigated some of the possible underlying mechanisms in vitro. We confirmed that hAAT inhibits ADAM-17, a protease that has been implicated in blocking myelination. Furthermore, both hAAT and recombinant human AAT (rhAAT) were able to attenuate the activation of a macrophage/microglia cell line, markedly decreasing the activation of the MHC class II promoter and the expression of pro-inflammatory genes such as IL-1β and the endoplasmic reticulum (ER) stress marker ATF3. Taken together, our results demonstrate for the first time that hAAT is able to reduce the progression of CMT1A, possibly by dampening inflammation and by regulating ADAM-17. Given the already well-established safety profile of hAAT, specifically in AAT deficiency disease (AATD), we suggest that the findings of our study should be promptly investigated in CMT1A patients.

The unfolded protein response to PI*Z alpha-1 antitrypsin in human hepatocellular and murine models.

Alpha‐1 antitrypsin (AAT) deficiency (AATD) is an inherited disease caused by mutations in the serpin family A member 1 (SERPINA1, also known as AAT) gene. The most common variant, PI*Z (Glu342Lys), causes accumulation of aberrantly folded AAT in the endoplasmic reticulum (ER) of hepatocytes that is associated with a toxic gain of function, hepatocellular injury, liver fibrosis, and hepatocellular carcinoma. The unfolded protein response (UPR) is a cellular response to improperly folded proteins meant to alleviate ER stress. It has been unclear whether PI*Z AAT elicits liver cell UPR, due in part to limitations of current cellular and animal models. This study investigates whether UPR is activated in a novel human PI*Z AAT cell line and a new PI*Z human AAT (hAAT) mouse model. A PI*Z AAT hepatocyte cell line (Huh7.5Z) was established using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing of the normal ATT (PI*MM) gene in the Huh7.5 cell line. Additionally, novel full‐length genomic DNA PI*Z hAAT and PI*M hAAT transgenic mouse models were established. Using these new models, UPR in Huh7.5Z cells and PI*Z mice were comprehensively determined. Robust activation of UPR was observed in Huh7.5Z cells compared to Huh7.5 cells. Activated caspase cascade and apoptosis markers, increased chaperones, and autophagy markers were also detected in Z hepatocytes. Selective attenuation of UPR signaling branches was observed in PI*Z hAAT mice in which the protein kinase R‐like ER kinase and inositol‐requiring enzyme1α branches were suppressed while the activating transcription factor 6α branch remained active. This study provides direct evidence that PI*Z AAT triggers canonical UPR and that hepatocytes survive pro‐apoptotic UPR by selective suppression of UPR branches. Our data improve understanding of underlying pathological molecular mechanisms of PI*Z AATD liver disease.

Combined multivariate statistical and flux balance analyses uncover media bottlenecks to the growth and productivity of Chinese hamster ovary cell cultures.

Chinese hamster ovary (CHO) cells are widely used for producing recombinant proteins. To enhance their productivity and product quality, media reformulation has been a key strategy, albeit with several technical challenges, due to the myriad of complex molecular mechanisms underlying media effects on culture performance. Thus, it is imperative to characterize metabolic bottlenecks under various media conditions systematically. To do so, we combined partial least square regression (PLS-R) with the flux balance analysis of a genome-scale metabolic model to elucidate the physiological states and metabolic behaviors of human alpha-1 antitrypsin producing CHO-DG44 cells grown in one commercial and another two in-house media under development. At the onset, PLS-R was used to identify metabolite exchanges that were correlated to specific growth and productivity. Then, by comparing metabolic states described by resultant flux distributions under two of the media conditions, we found suboptimal level of four nutrients and two metabolic wastes, which plausibly hindered cellular growth and productivity; mechanistically, lactate and ammonia recycling were modulated by glutamine and asparagine metabolisms in the media conditions, and also by hitherto unsuspected folate and choline supplements. Our work demonstrated how multivariate statistical analysis can be synergistically combined with metabolic modeling to uncover the mechanistic elements underlying differing media performance. It thus paved the way for the systematic identification of nutrient targets for medium reformulation to enhance recombinant protein production in CHO cells.

Alpha-1 antitrypsin deficiency

Alpha-1 antitrypsin deficiency (DAAT) is a rare autosomal recessive genetic disorder caused by mutations in the Serpina1 gene. The role of alpha-1 antitrypsin (A1AT) is to maintain homeostasis in the acute phase of inflammation. DAAT manifests itself primarily in carriers of the Z allele, especially in the homozygous state, as emphysema and chronic liver disease. Although the diagnostic strategy is well defined and screening is fully reimbursed, DAAT is still largely underdiagnosed. In addition to simple lifestyle advice, which is essential once the diagnosis has been made, the specific treatment for severe deficiency and lung involvement is based on substitution with purified human A1AT, which slows the development of pulmonary emphysema.

Proteomic Analysis of Exosomes Secreted from Human Alpha-1 Antitrypsin Overexpressing Mesenchymal Stromal Cells.

Simple SummaryThe overexpression of human alpha-1 antitrypsin (hAAT) in mesenchymal stromal cells (MSCs) improved their intrinsic properties with enhanced protective effects in previous murine models of type 1 diabetes. This study compared the protein profiles of extracellular vesicles (EVs) from bone marrow-derived control or hAAT-MSCs. EVs from both cell types share common features in size and exosome marker expression. By comparing common proteins in EVs from three donor cell lines using Gene Ontology (GO) analysis, we found that common EV proteins from all donors are important to cell adhesion and extracellular matrix organization. Differentially expressed proteins from hAAT-MSCs and MSCs are involved in cytokine signaling of the immune system, stem cell differentiation, and carbohydrate metabolism. This study shows that hAAT-MSCs have different profiles of paracrine effector exosomal proteins compared to control MSCs.Extracellular vesicles (EVs) mediate many therapeutic effects of stem cells during cellular therapies. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) were manufactured to overexpress the human antiprotease alpha-1 antitrypsin (hAAT) and studied to compare the EV production compared to lentivirus treated control MSCs. The goal of this study was to compare protein profiles in the EVs/exosomes of control and hAAT-MSCs using unbiased, high resolution liquid chromatography and mass spectrometry to explore differences. Nanoparticle tracking analysis (NTA) showed that the particle size of the EVs from control MSCs or hAAT-MSCs ranged from 30 to 200 nm. Both MSCs and hAAT-MSCs expressed exosome-associated proteins, including CD63, CD81, and CD9. hAAT-MSCs also expressed high levels of hAAT. We next performed proteomic analysis of EVs from three healthy donor cell lines. Exosomes collected from cell supernatant were classified by GO analysis which showed proteins important to cell adhesion and extracellular matrix organization. However, there were differences between exosomes from control MSCs and hAAT-MSCs in cytokine signaling of the immune system, stem cell differentiation, and carbohydrate metabolism (p < 0.05). These results show that hAAT-MSC exosomes contain a different profile of paracrine effectors with altered immune function, impacts on MSC stemness, differentiation, and prevention of cell apoptosis and survival that could contribute to improved therapeutic functions.

Production and characterization of mono-PEGylated alpha-1 antitrypsin for augmentation therapy

Alpha-1 antitrypsin (AAT) is an endogenous inhibitor of serine proteases which, in physiological conditions, neutralizes the excess of neutrophil elastase and other serine proteases in tissues and especially the lungs. Weekly intravenous infusion of plasma-purified human AAT is used to treat AAT deficiency-associated lung disease. However, only 2 % of the AAT dose reach the lungs after intravenous infusion. Inhalation of AAT might offer an alternative route of administration. Yet, the rapid clearance of AAT from the respiratory tract results in high and frequent dosing by inhalation and limited efficacy. In the present study, we produced and characterized in vitro a PEGylated version of AAT which could offer a prolonged body residence time and thereby be useful for augmentation therapy by the intravenous and inhalation routes. Two PEGylation reactions – N-terminal and thiol PEGylation – and three polyethylene glycol (PEG) chains – linear 30 kDa, linear 40 kDa and 2-armed 40 kDa – were used. The yields of mono-PEGylated AAT following purification by anion exchange chromatography were 40–50 % for N-terminal PEGylation and 60–70% for thiol PEGylation. The PEG-AAT conjugates preserved the ability to form a protease-inhibitor complex with neutrophil elastase and proteinase 3 as well as the full inhibitory capacity to neutralize neutrophil elastase activity. These results open up interesting prospects for PEGylated AAT to achieve a prolonged half-life and an improved therapeutic efficacy in vivo.

A Novel Cellular Therapy to Treat Pancreatic Pain in Experimental Chronic Pancreatitis Using Human Alpha-1 Antitrypsin Overexpressing Mesenchymal Stromal Cells.

Chronic pancreatitis (CP) is characterized by pancreatic inflammation, fibrosis, and abdominal pain that is challenging to treat. Mesenchymal stromal cells (MSCs) overexpressing human alpha-1 antitrypsin (hAAT-MSCs) showed improved mobility and protective functions over native MSCs in nonobese diabetic mice. We investigated whether hAAT-MSCs could mitigate CP and its associated pain using trinitrobenzene sulfonic acid (TNBS)-induced CP mouse models. CP mice were given native human MSCs or hAAT-MSCs (0.5 × 106 cells/mouse, i.v., n = 6–8/group). The index of visceral pain was measured by graduated von Frey filaments. Pancreatic morphology and pancreatic mast cell count were analyzed by morphological stains. Nociceptor transient receptor potential vanilloid 1 (TRPV1) expression in dorsal root ganglia (DRG) was determined by immunohistochemistry. hAAT-MSC-treated CP mice best preserved pancreatic morphology and histology. MSC or hAAT-MSC infusion reduced abdominal pain sensitivities. hAAT-MSC therapy also suppressed TRPV1 expression in DRG and reduced pancreatic mast cell density induced by TNBS. Overall, hAAT-MSCs reduced pain and mitigated pancreatic inflammation in CP equal to MSCs with a trend toward a higher pancreatic weight and better pain relief in the hAAT-MSC group compared to the MSC group. Both MSCs and hAAT-MSCs might be used as a novel therapeutic tool for CP-related pain.

Development of anti-inflammatory peptidomimetics based on the structure of human alpha1-antitrypsin

Human α1-antitrypsin (hAAT) has two distinguishing functions: anti-protease activity and regulation of the immune system. In the present study we hypothesized that those two protein functions are mediated by different structural domains on the hAAT surface. Indeed, such biologically active immunoregulatory sites (not associated with canonical anti-protease activity) on the surface of hAAT were identified by in silico methods. Several peptides were derived from those immunoregulatory sites. Four peptides exhibited impressive biological effects in pharmacological concentration ranges. Peptidomimetic (14) was developed, based on the structure of the most druggable and active peptide. The compound exhibited a potent anti-inflammatory activity in vitro and in vivo. Such a compound could be used as a basis for developing novel anti-inflammatory drug candidates and as a research tool for better understanding hAAT functions.

Neutrophil-Derived Proteases Contribute to the Pathogenesis of Early Diabetic Retinopathy.

PurposePrevious studies indicate that leukocytes, notably neutrophils, play a causal role in the capillary degeneration observed in diabetic retinopathy (DR), however, the mechanism by which they cause such degeneration is unknown. Neutrophil elastase (NE) is a protease released by neutrophils which participates in a variety of inflammatory diseases. In the present work, we investigated the potential involvement of NE in the development of early DR.MethodsExperimental diabetes was induced in NE-deficient mice (Elane−/−), in mice treated daily with the NE inhibitor, sivelestat, and in mice overexpressing human alpha-1 antitrypsin (hAAT+). Mice were assessed for diabetes-induced retinal superoxide generation, inflammation, leukostasis, and capillary degeneration.ResultsIn mice diabetic for 2 months, deletion of NE or selective inhibition of NE inhibited diabetes-induced retinal superoxide levels and inflammation, and inhibited leukocyte-mediated cytotoxicity of retinal endothelial cells. In mice diabetic for 8 months, genetic deletion of NE significantly inhibited diabetes-induced retinal capillary degeneration.ConclusionsThese results suggest that a protease released from neutrophils contributes to the development of DR, and that blocking NE activity could be a novel therapy to inhibit DR.

Transgenic Mice Overexpressing Human Alpha-1 Antitrypsin Exhibit Low Blood Pressure and Altered Epithelial Transport Mechanisms in the Inactive and Active Cycles.

Human alpha-1 antitrypsin (hAAT) is a versatile protease inhibitor, but little is known about its targets in the aldosterone-sensitive distal nephron and its role in electrolyte balance and blood pressure control. We analyzed urinary electrolytes, osmolality, and blood pressure from hAAT transgenic (hAAT-Tg) mice and C57B/6 wild-type control mice maintained on either a normal salt or high salt diet. Urinary sodium, potassium, and chloride concentrations as well as urinary osmolality were lower in hAAT-Tg mice maintained on a high salt diet during both the active and inactive cycles. hAAT-Tg mice showed a lower systolic blood pressure compared to C57B6 mice when maintained on a normal salt diet but this was not observed when they were maintained on a high salt diet. Cathepsin B protein activity was less in hAAT-Tg mice compared to wild-type controls. Protein expression of the alpha subunit of the sodium epithelial channel (ENaC) alpha was also reduced in the hAAT-Tg mice. Natriuretic peptide receptor C (NPRC) protein expression in membrane fractions of the kidney cortex was reduced while circulating levels of atrial natriuretic peptide (ANP) were greater in hAAT-Tg mice compared to wild-type controls. This study characterizes the electrolyte and blood pressure phenotype of hAAT-Tg mice during the inactive and active cycles and investigates the mechanism by which ENaC activation is inhibited in part by a mechanism involving decreased cathepsin B activity and increased ANP levels in the systemic circulation.