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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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Mazzuca, Vitiello, Travaglini, Maurizi, Finamore, Santangelo, Rigon, Vadacca, Angeletti and Scarlata.)

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.

Background: Asthma is considered an incurable disease, although many advances have been made in asthma treatments in recent years. Therefore, elucidating the pathological mechanisms and seeking novel and effective therapeutic strategies for asthma are urgently needed., Methods: Airway resistance was measured by whole-body plethysmography. H&E staining was used to observe the morphological changes in the lung. Oxidative stress was assessed by measuring the levels of MDA, CAT and SOD. Gene expression was analysed by western blotting and RT-qPCR. ELISA was used to analyse the concentrations of IL-4, IL-5 and IFN-γ., Results: In the present study, we successfully established in vivo and in vitro asthma models. OVA administration led to elevated lung resistance, cell counts in BALF, and cytokine secretion, impaired airway structure and enhanced oxidative stress and autophagy in a mouse model of asthma, while IL-13 induced inflammation, oxidative stress and autophagy in BEAS-2B cells. A1AT reduced lung resistance and cell counts in BALF and suppressed inflammation, oxidative stress and autophagy in a mouse model of asthma and IL-13-induced BEAS-2B cells. Mechanistic investigations revealed that autophagy activation compromised the protective effect of A1AT on IL-13-induced BEAS-2B cells. Further mechanistic studies revealed that A1AT alleviated inflammation and oxidative stress by inhibiting autophagy in the context of asthma., Conclusion: We demonstrated that A1AT could alleviate inflammation and oxidative stress by suppressing autophagy in the context of asthma and thus ameliorate asthma. Our study revealed novel pathological mechanisms and provided novel potential therapeutic targets for asthma treatment., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Acute respiratory distress syndrome is characterized by hypoxemia, altered alveolar-capillary permeability, and neutrophil-dominated inflammatory pulmonary edema. Despite decades of research, an effective drug therapy for acute respiratory distress syndrome remains elusive. The ideal pharmacotherapy for acute respiratory distress syndrome should demonstrate antiprotease activity and target injurious inflammatory pathways while maintaining host defense against infection. Furthermore, a drug with a reputable safety profile, low possibility of off-target effects, and well-known pharmacokinetics would be desirable. The endogenous 52-kd serine protease α1-antitrypsin has the potential to be a novel treatment option for acute respiratory distress syndrome. The main function of α1-antitrypsin is as an antiprotease, targeting neutrophil elastase in particular. However, studies have also highlighted the role of α1-antitrypsin in the modulation of inflammation and bacterial clearance. In light of the current SARS-CoV-2 pandemic, the identification of a treatment for acute respiratory distress syndrome is even more pertinent, and α1-antitrypsin has been implicated in the inflammatory response to SARS-CoV-2 infection., (Copyright © 2021, the American Society of Anesthesiologists, Inc. All Rights Reserved.)

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359-394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1-0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.

Alpha-1 antitrypsin (AAT) is a major inhibitor of serine proteases in mammals. Therefore, its deficiency leads to protease-antiprotease imbalance and a risk for developing lung emphysema. Although therapy with human plasma-purified AAT attenuates AAT deficiency-related emphysema, its impact on lung antibacterial immunity is poorly defined. Here, we examined the effect of AAT therapy on lung protective immunity in AAT-deficient (KO) mice challenged with Streptococcus pneumoniae. AAT-KO mice were highly susceptible to S. pneumoniae, as determined by severe lobar pneumonia and early mortality. Mechanistically, we found that neutrophil-derived elastase (NE) degraded the opsonophagocytically important collectins, surfactant protein A (SP-A) and D (SP-D), which was accompanied by significantly impaired lung bacterial clearance in S. pneumoniae-infected AAT-KO mice. Treatment of S. pneumoniae-infected AAT-KO mice with human AAT protected SP-A and SP-D from NE-mediated degradation and corrected the pulmonary pathology observed in these mice. Likewise, treatment with Sivelestat, a specific inhibitor of NE, also protected collectins from degradation and significantly decreased bacterial loads in S. pneumoniae-infected AAT-KO mice. Our findings show that NE is responsible for the degradation of lung SP-A and SP-D in AAT-KO mice affecting lung protective immunity in AAT deficiency.

Based on previous results demonstrating that complexes of a mutant α1-antitrypsin with the heat shock proteins (HSP)70 and glucose-regulated protein94 (Grp94) circulate in the blood of patients with type 1 diabetes, we raised the hypothesis that these complexes could represent the primary antigen capable of triggering the autoimmune reactions leading to overt diabetes. As a first approach to this issue, we searched whether A1AT and HSPs had a sequence similarity to major islet antigen proteins so as to identify among the similar sequences those with potential relevance for the pathogenesis of diabetes. A thorough in silico analysis was performed to establish the score of similarity of the human proteins: A1AT, pro-insulin (INS), GAD65, IAPP, IA-2, ICA69, Grp94, HSP70 and HSP60. The sequences of A1AT and HSPs with the highest score of similarity to the islet peptides reported in the literature as the main autoantigens in human diabetes were recorded. At variance with other HSPs, also including HSP90 and Grp78, Grp94 contained the highest number and the longest sequences with structural similarity to A1AT and to well-known immunogenic peptides/epitopes of INS, GAD65, and IA-2. The similarity of A1AT with Grp94 and that of Grp94 with INS also suggested a functional relationship among the proteins. Specific sequences were identified in A1AT, Grp94 and HSP70, with the highest score of cross-similarity to a pattern of eight different islet protein epitopes. The similarity also involved recently discovered autoantigens in type 1 diabetes such as a hybrid peptides of insulin and the defective ribosomal insulin gene product. The significant similarity displayed by specific sequences of Grp94 and A1AT to the islet peptides considered main antigens in human diabetes, is a strong indication for testing these sequences as new peptides of immunogenic relevance in diabetes.

Alpha 1 antitrypsin deficiency (AATD) is an autosomal co-dominant disorder characterized by a low level of circulating AAT, which significantly reduces protection for the lower airways against proteolytic burden caused by neutrophils. Neutrophils, which are terminally differentiated innate immune cells and play a critical role to clear pathogens, accumulate excessively in the lung of AATD individuals. The neutrophil burden in AATD individuals increases the risk for early-onset destructive lung diseases by producing neutrophil products such as reactive oxygen radicals and various proteases. The level of AAT in AATD individuals is not sufficient to inhibit the activity of neutrophil chemotactic factors such as CXCL-8 and LTB4, which could lead to alveolar neutrophil accumulation in AATD individuals. However, as neutrophils have a short lifespan, and apoptotic neutrophils are rapidly cleared by alveolar macrophages that outnumber the apoptotic neutrophils in the pulmonary alveolus, the increased chemotaxis activity does not fully explain the persistent neutrophil accumulation and the resulting chronic inflammation in AATD individuals. Here, we propose that the ability of alveolar macrophages to clear apoptotic neutrophils is impaired in AATD individuals and it could be the main driver to cause neutrophil accumulation in their lung. This study demonstrates that Z-AAT variant significantly increases the expression of pro-inflammatory cytokines including CXCL-8, CXCL1, LTB4, and TNFα in LPS-treated macrophages. These cytokines play a central role in neutrophil recruitment to the lung and in clearance of apoptotic neutrophils by macrophages. Our result shows that LPS treatment significantly reduces the efferocytosis ability of macrophages with the Z-AAT allele by inducing TNFα expression. We incubated monocyte-derived macrophages (MDMs) with apoptotic neutrophils and found that after 3 h of co-incubation, the expression level of CXCL-8 is reduced in M-MDMs but increased in Z-MDMs. This result shows that the expression of inflammatory cytokines could be increased by impaired efferocytosis. It indicates that the efferocytosis ability of macrophages plays an important role in regulating cytokine expression and resolving inflammation. Findings from this study would help us better understand the multifaceted effect of AAT on regulating neutrophil balance in the lung and the underlying mechanisms., (Copyright © 2020 Lee, Lu, Oshins, West, Moneypenny, Han and Brantly.)

Rationale: Coronavirus disease (COVID-19) is a global threat to health. Its inflammatory characteristics are incompletely understood. Objectives: To define the cytokine profile of COVID-19 and to identify evidence of immunometabolic alterations in those with severe illness. Methods: Levels of IL-1β, IL-6, IL-8, IL-10, and sTNFR1 (soluble tumor necrosis factor receptor 1) were assessed in plasma from healthy volunteers, hospitalized but stable patients with COVID-19 (COVID stable patients), patients with COVID-19 requiring ICU admission (COVID ICU patients), and patients with severe community-acquired pneumonia requiring ICU support (CAP ICU patients). Immunometabolic markers were measured in circulating neutrophils from patients with severe COVID-19. The acute phase response of AAT (alpha-1 antitrypsin) to COVID-19 was also evaluated. Measurements and Main Results: IL-1β, IL-6, IL-8, and sTNFR1 were all increased in patients with COVID-19. COVID ICU patients could be clearly differentiated from COVID stable patients, and demonstrated higher levels of IL-1β, IL-6, and sTNFR1 but lower IL-10 than CAP ICU patients. COVID-19 neutrophils displayed altered immunometabolism, with increased cytosolic PKM2 (pyruvate kinase M2), phosphorylated PKM2, HIF-1α (hypoxia-inducible factor-1α), and lactate. The production and sialylation of AAT increased in COVID-19, but this antiinflammatory response was overwhelmed in severe illness, with the IL-6:AAT ratio markedly higher in patients requiring ICU admission ( P  < 0.0001). In critically unwell patients with COVID-19, increases in IL-6:AAT predicted prolonged ICU stay and mortality, whereas improvement in IL-6:AAT was associated with clinical resolution ( P  < 0.0001). Conclusions: The COVID-19 cytokinemia is distinct from that of other types of pneumonia, leading to organ failure and ICU need. Neutrophils undergo immunometabolic reprogramming in severe COVID-19 illness. Cytokine ratios may predict outcomes in this population.

Rheumatoid arthritis (RA) is a chronic autoimmune disease and rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA) are the most frequently detected autoantibodies (autoAbs). To date, more than 20% of RA cases are still defined as seronegative forms (seronegative RA, SN-RA). The aim of this study was to identify new antigenic targets of autoAbs in RA patients, which can also be recognized in SN-RA. Using a proteomic approach, we tested sera from SN-RA patients by analyzing synovial fluid (SF) proteins from these patients. Sera from SN-RA patients revealed a strong reactive spot, corresponding to alpha 1 antitrypsin (A1AT). Reverse-phase nanoliquid chromatography and tandem mass spectrometry (Matrix Assisted Laser Desorption/Ionization-Time Of Flight, MALDI-TOF/TOF) confirmed the presence of A1AT in SF and showed that homocysteinylation was one of the post-translational modifications of A1AT. Homocysteinylated (Hcy)-A1AT immunoprecipitated from SN-RA patients' SFs and in vitro modified Hcy-A1AT were used as antigens by Enzyme-Linked ImmunoSorbent Assay (ELISA) to test the presence of specific autoAbs in sera from 111 SN-RA patients, 132 seropositive (SP)-RA patients, and from 95 patients with psoriatic arthritis, 40 patients with osteoarthritis, and 41 healthy subjects as control populations. We observed that a large portion of SN-RA patients (75.7%), and also most of SP-RA patients' sera (87.1%) displayed anti-Hcy-A1AT autoAbs (anti-HATA). Native A1AT was targeted at a lower rate by SP-RA patients autoAbs, while virtually no SN-RA patients' sera showed the presence of anti-native A1AT autoAbs. In conclusion, anti-HATA can be considered potential biomarkers for RA, also in the SN forms. The discovery of novel autoAbs targeting specific autoantigens can represent higher clinic significance for all RA patients' population., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Multiple analytical and preclinical studies were performed to compare the biochemical characteristics, pharmacokinetics (PK), safety and neoantigenicity of a new 5% liquid formulation of Alpha-1 Proteinase Inhibitor (Liquid A1PI, Prolastin®-C Liquid) with the lyophilized version (Lyophilized A1PI, Prolastin®-C). Liquid A1PI and Lyophilized A1PI had similar average mass (~52 kDa), and both forms exhibited glycoform patterns consistent with the known banding pattern of A1PI (dominated by the M6 and M4 bands, including deconvoluted masses). Both Liquid A1PI and Lyophilized A1PI yielded average percent purity values ranging from 96% to 99% and had active content ranging from 53  mg/mL to 59  mg/mL. The PK profile of Liquid A1PI was similar to Lyophilized A1PI. Safety assessments in rabbits showed good tolerability and no test article-related changes in mortality, clinical signs, clinical pathology, body weight, food consumption, or urinalysis parameters. Following immunodepletion of antibodies that recognize Lyophilized A1PI, there were no significant differences in the anti-drug titers among animals immunized with Lyophilized A1PI and Liquid A1PI (p > 0.05), indicating that no antibodies to neoantigens were generated. Liquid A1PI and Lyophilized A1PI have similar profiles with respect to biochemical characteristics, PK, safety and neoantigenicity., (Copyright © 2019 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Although wasting marmoset syndrome (WMS) is one of the biggest problems facing captive marmoset colonies, the mechanisms underlying its pathogenesis remain unclear. In our clinical experience, it is difficult to cure WMS-affected marmosets with severe hypoalbuminemia. Thus, the mechanisms underlying hypoalbuminemia in WMS must be understood. In the present study, we investigated whether intestinal protein loss, a known reason for hypoalbuminemia, occurs in this disease. Fecal α1-proteinase inhibitor (α1-PI, also known as α1-antitrypsin) has been used to diagnose intestinal protein loss in other species. To develop an assay system for this protein, marmoset α1-PI was purified from plasma and antibodies against it were developed using the purified protein. Using the antibodies, a sandwich enzyme-linked immunosorbent assay (ELISA) to measure marmoset α1-PI was developed, and its detection sensitivity for fecal samples was ∼20-fold higher than that of a commercial kit for human α1-PI. From this ELISA, the reference intervals for serum and feces of healthy marmosets were 0.87-1.85 mg/ml and 0.53-395.58 μg/g, respectively. The average concentrations of α1-PI in serum and feces of seven WMS-affected marmosets were 1.17 mg/ml and 1357.58 μg/g, respectively. Although there were no significant differences in the serum concentrations between healthy and WMS-affected marmosets, the fecal concentrations were significantly higher in WMS-affected marmosets than in healthy individuals, suggesting that intestinal protein loss occurs in WMS. Intestinal protein loss of WMS-affected marmosets was significantly attenuated with treatment, suggesting that it is one of the mechanisms involved in the hypoalbuminemia observed in WMS., (© 2019 The Author(s).)

Rationale: The association between non-tuberculous mycobacterial lung disease and alpha-1-antitrypsin (AAT) deficiency is likely due, in part, to underlying emphysema or bronchiectasis. But there is increasing evidence that AAT itself enhances host immunity against microbial pathogens and thus deficiency could compromise host protection. Objectives: The goal of this project is to determine if AAT could augment macrophage activity against non-tuberculous mycobacteria. Methods: We compared the ability of monocyte-derived macrophages cultured in autologous plasma that were obtained immediately before and soon after AAT infusion-given to individuals with AAT deficiency-to control an ex vivo Mycobacterium intracellulare infection. Measurements and Main Results: We found that compared to pre-AAT infused monocyte-derived macrophages plus plasma, macrophages, and contemporaneous plasma obtained after a session of AAT infusion were significantly better able to control M. intracellulare infection; the reduced bacterial burden was linked with greater phagosome-lysosome fusion and increased autophagosome formation/maturation, the latter due to AAT inhibition of both M. intracellulare -induced nuclear factor-kappa B activation and A20 expression. While there was a modest increase in apoptosis in the M. intracellulare -infected post-AAT infused macrophages and plasma, inhibiting caspase-3 in THP-1 cells, monocyte-derived macrophages, and alveolar macrophages unexpectedly reduced the M. intracellulare burden, indicating that apoptosis impairs macrophage control of M. intracellulare and that the host protective effects of AAT occurred despite inducing apoptosis. Conclusion: AAT augments macrophage control of M. intracellulare infection through enhancing phagosome-lysosome fusion and autophagy.

Studies to identify novel immune-regulatory functions of active vitamin D (1,25(OH) 2 D3) in human CD4 + T cells revealed that 1,25(OH) 2 D3 potently induced expression of the gene SERPINA1, encoding the anti-protease α-1-antitrypsin. We confirmed α-1-antitrypsin protein expression by 1,25(OH) 2 D3-treated CD4 + T cells, but not in CD8 + T cells or monocytes. α-1-Antitrypsin promotes anti-inflammatory IL-10 synthesis in other immune cell populations. We therefore investigated its immune-regulatory effects in CD4 + T cells. Plasma-derived α-1-antitrypsin drove IL-10 synthesis by CD4 + T cells, which was not dependent on anti-protease activity, but appeared to require a serum-binding factor, since this could not be achieved with recombinant protein. α-1-Antitrypsin is reported to bind complement components, which regulate T cell function. A role for this interaction was therefore probed. Plasma-derived, but not recombinant α-1-antitrypsin contained C3a. Surface Plasmon Resonance and Microscale Thermophoresis demonstrated α-1-antitrypsin binding to C3a. Addition of C3a to CD4 + T cells cultured with recombinant α-1-antitrypsin restored induction of IL-10, whereas neutralisation of C3a abrogated IL-10 induced by plasma-derived α-1-antitrypsin. To interrogate an endogenous role for the α-1-antitrypsin-C3a axis in 1,25(OH) 2 D3-driven CD4 + T cell IL-10 synthesis, we treated cells from healthy or α-1-antitrypsin-deficient individuals (which transcribe SERPINA1 but do not secrete protein) with 1,25(OH) 2 D3. A significant correlation was identified between SERPINA1 and IL10 gene expression in healthy donor CD4 + T cells, which was absent in cells from α-1-antitrypsin-deficient individuals. Therefore, α-1-antitrypsin is required for 1,25(OH) 2 D3-induced IL-10 expression in CD4 + T cells, interacting with C3a to drive IL-10 expression., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Alpha-1 antitrypsin (AAT) is an acute phase protein that possesses immune-regulatory and anti-inflammatory functions independent of antiprotease activity. AAT deficiency (AATD) is associated with early-onset emphysema and chronic obstructive pulmonary disease. Of interest are the AATD nonsense mutations (termed null or Q0), the majority of which arise from premature termination codons in the mRNA coding region. We have recently demonstrated that plasma from an AATD patient homozygous for the Null Bolton allele ( Q0 bolton ) contains AAT protein of truncated size. Although the potential to alleviate the phenotypic consequences of AATD by increasing levels of truncated protein holds therapeutic promise, protein functionality is key. The goal of this study was to evaluate the structural features and anti-inflammatory capacity of Q0 bolton -AAT. A low-abundance, truncated AAT protein was confirmed in plasma of a Q0 bolton -AATD patient and was secreted by patient-derived induced pluripotent stem cell-hepatic cells. Functional assays confirmed the ability of purified Q0 bolton -AAT protein to bind neutrophil elastase and to inhibit protease activity. Q0 bolton -AAT bound IL-8 and leukotriene B 4 , comparable to healthy control M-AAT, and significantly decreased leukotriene B 4 -induced neutrophil adhesion ( p = 0.04). Through a mechanism involving increased mRNA stability ( p = 0.007), ataluren treatment of HEK-293 significantly increased Q0 bolton -AAT mRNA expression ( p = 0.03) and Q0 bolton -AAT truncated protein secretion ( p = 0.04). Results support the rationale for treatment with pharmacological agents that augment levels of functional Q0 bolton -AAT protein, thus offering a potential therapeutic option for AATD patients with rare mutations of similar theratype., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Background: Human α1-antitrypsin (hAAT) is a circulating anti-inflammatory serine-protease inhibitor that rises during acute phase responses. in vivo , hAAT reduces bacterial load, without directly inhibiting bacterial growth. In conditions of excess nitric-oxide (NO), hAAT undergoes S-nitrosylation (S-NO-hAAT) and gains antibacterial capacity. The impact of S-NO-hAAT on immune cells has yet to be explored. Aim: Study the effects of S-NO-hAAT on immune cells during bacterial infection. Methods: Clinical-grade hAAT was S-nitrosylated and then compared to unmodified hAAT, functionally, and structurally. Intracellular bacterial clearance by THP-1 macrophages was assessed using live Salmonella typhi . Murine peritoneal macrophages were examined, and signaling pathways were evaluated. S-NO-hAAT was also investigated after blocking free mambranal cysteine residues on cells. Results: S-NO-hAAT (27.5 uM) enhances intracellular bacteria elimination by immunocytes (up to 1-log reduction). S-NO-hAAT causes resting macrophages to exhibit a pro-inflammatory and antibacterial phenotype, including release of inflammatory cytokines and induction of inducible nitric oxide synthase (iNOS) and TLR2. These pro-inflammatory effects are dependent upon cell surface thiols and activation of MAPK pathways. Conclusions: hAAT duality appears to be context-specific, involving S-nitrosylation in a nitric oxide rich environment. Our results suggest that S-nitrosylation facilitates the antibacterial activity of hAAT by promoting its ability to activate innate immune cells. This pro-inflammatory effect may involve transferring of nitric oxide from S-NO-hAAT to a free cysteine residue on cellular targets.

Introduction: Human α1-antitrypsin (hAAT) is a 394-amino acid long anti-inflammatory, neutrophil elastase inhibitor, which binds elastase via a sequence-specific molecular protrusion (reactive center loop, RCL; positions 357-366). hAAT formulations that lack protease inhibition were shown to maintain their anti-inflammatory activities, suggesting that some attributes of the molecule may reside in extra-RCL segments. Here, we compare the protease-inhibitory and anti-inflammatory profiles of an extra-RCL mutation (cys232pro) and two intra-RCL mutations (pro357cys, pro357ala), to naïve [wild-type (WT)] recombinant hAAT, in vitro , and in vivo ., Methods: His-tag recombinant point-mutated hAAT constructs were expressed in HEK-293F cells. Purified proteins were evaluated for elastase inhibition, and their anti-inflammatory activities were assessed using several cell-types: RAW264.7 cells, mouse bone marrow-derived macrophages, and primary peritoneal macrophages. The pharmacokinetics of the recombinant variants and their effect on LPS-induced peritonitis were determined in vivo ., Results: Compared to WT and to RCL-mutated hAAT variants, cys232pro exhibited superior anti-inflammatory activities, as well as a longer circulating half-life, despite all three mutated forms of hAAT lacking anti-elastase activity. TNFα expression and its proteolytic membranal shedding were differently affected by the variants; specifically, cys232pro and pro357cys altered supernatant and serum TNFα dynamics without suppressing transcription or shedding., Conclusion: Our data suggest that the anti-inflammatory profile of hAAT extends beyond direct RCL regions. Such regions might be relevant for the elaboration of hAAT formulations, as well as hAAT-based drugs, with enhanced anti-inflammatory attributes.

While interleukin (IL)-1β is a potent pro-inflammatory cytokine involved in host defense, high levels can cause life-threatening sterile inflammation including systemic inflammatory response syndrome. Hence, the control of IL-1β secretion is of outstanding biomedical importance. In response to a first inflammatory stimulus such as lipopolysaccharide, pro-IL-1β is synthesized as a cytoplasmic inactive pro-form. Extracellular ATP originating from injured cells is a prototypical second signal for inflammasome-dependent maturation and release of IL-1β. The human anti-protease alpha-1 antitrypsin (AAT) and IL-1β regulate each other via mechanisms that are only partially understood. Here, we demonstrate that physiological concentrations of AAT efficiently inhibit ATP-induced release of IL-1β from primary human blood mononuclear cells, monocytic U937 cells, and rat lung tissue, whereas ATP-independent IL-1β release is not impaired. Both, native and oxidized AAT are active, suggesting that the inhibition of IL-1β release is independent of the anti-elastase activity of AAT. Signaling of AAT in monocytic cells involves the lipid scavenger receptor CD36, calcium-independent phospholipase A2β, and the release of a small soluble mediator. This mediator leads to the activation of nicotinic acetylcholine receptors, which efficiently inhibit ATP-induced P2X7 receptor activation and inflammasome assembly. We suggest that AAT controls ATP-induced IL-1β release from human mononuclear blood cells by a novel triple-membrane-passing signaling pathway. This pathway may have clinical implications for the prevention of sterile pulmonary and systemic inflammation.

Background: Human kallikrein-related peptidase 6 (KLK6) is a member of the kallikrein family of serine proteases. KLK6 is synthesized as a preproenzyme, mainly in tissues of the central nervous system (CNS), and secreted as an inactive precursor. Serum KLK6 is a biomarker of unfavorable prognosis for ovarian cancer, but its sensitivity for early detection is relatively low. Differential glycosylation of KLK6 has been identified in ascites fluid obtained from ovarian cancer patients, suggesting the presence of unique KLK6 isoforms in biological samples., Methods: In the present study, we applied a two-step enrichment approach for KLK6 in ovarian cancer ascites, followed by mice immunization and production of monoclonal antibodies. Immunoaffinity techniques coupled to mass spectrometric methods were employed for hybridoma screening and target antigen identification., Results: We found that the main target of the newly-generated monoclonal antibodies target was the serine protease inhibitor α1-antitrypsin (A1AT). Additional experiments confirmed that A1AT is the main inhibitor of KLK6 in biological fluids. One new antibody (24ED138) was chosen to build a hybrid assay for the accurate quantification of the A1AT-KLK6 complex in biological samples. The aforementioned assay was evaluated with serum samples collected from patients with ovarian cancer (n=24) and normal donors (n=16) and showed slight improvement in sensitivity (~12%) compared to the standard in-house KLK6 assay., Conclusions: We conclude that KLK6 is present in biological fluids either as free form, or bound to A1AT, and the bound form performs better than total KLK6 as a biomarker of ovarian carcinoma.

In 2011 a novel autoantibody system, anti-carbamylated protein (anti-CarP) antibodies, was described in rheumatoid arthritis (RA) patients. Anti-CarP antibody positivity associates with a more severe disease course, is observed years before disease onset, and may predict the development of RA in arthralgia patients. Although many clinical observations have been carried out, information on the antigenic targets of anti-CarP antibodies is limited. Most studies on anti-CarP antibodies utilize an ELISA-based assay with carbamylated fetal calf serum (Ca-FCS) as antigen, a complex mixture of proteins. Therefore, we analysed the molecular identity of proteins within Ca-FCS that are recognized by anti-CarP antibodies. Ca-FCS was fractionated using ion exchange chromatography, selecting one of the fractions for further investigation. Using mass-spectrometry, carbamylated alpha-1-antitrypsin (Ca-A1AT) was identified as a potential antigenic target of anti-CarP antibodies in RA patients. A1AT contains several lysines on the protein surface that can readily be carbamylated. A large proportion of the RA patients harbour antibodies that bind human Ca-A1AT in ELISA, indicating that Ca-A1AT is indeed an autoantigen for anti-CarP antibodies. Next to the Ca-A1AT protein, several homocitrulline-containing peptides of A1AT were recognized by RA sera. Moreover, we identified a carbamylated peptide of A1AT in the synovial fluid of an RA patient using mass spectrometry. We conclude that Ca-A1AT is not only a target of anti-CarP antibodies but is also present in the synovial compartment, suggesting that Ca-A1AT recognized by anti-CarP antibodies in the joint may contribute to synovial inflammation in anti-CarP-positive RA., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A novel sandwich-type biosensor was developed for the electrochemical detection of α-1 antitrypsin (AAT, a recognized biomarker for Alzheimer's disease). The biosensor was composed of 3, 4, 9, 10-perylene tetracarboxylic acid/carbon nanotubes (PTCA-CNTs) as a sensing platform and alkaline phosphatase-labeled AAT antibody functionalized silver nanoparticles (ALP-AAT Ab-Ag NPs) as a signal enhancer. CNTs offer high surface area and good electrical conductivity. Importantly, Ag NPs could increase the amount of ALP on the sensing surface and the ALP could dephosphorylate 4-amino phenyl phosphate (APP) enzymatically to produce electroactive species 4-aminophenol (AP). For detecting AAT based on the sandwich-type biosensor, the results show that the peak current value of AP using ALP-AAT Ab-Ag NPs as signal enhancer is much higher than that by using ALP-AAT Ab bioconjugate (without Ag NPs), the biosensor exhibited desirable performance for AAT determination with a wide linearity in the range from 0.05 to 20.0pM and a low detection limit of 0.01pM. Finally, the developed sensor was successfully applied to the analysis of AAT concentration in serum samples., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Adeno-associated virus (AAV)-mediated gene therapy is currently being pursued as a treatment for the monogenic disorder α-1-antitrypsin (AAT) deficiency. Results from phase I and II studies have shown relatively stable and dose-dependent increases in transgene-derived wild-type AAT after local intramuscular vector administration. In this report we describe the appearance of transgene-specific T-cell responses in two subjects that were part of the phase II trial. The patient with the more robust T-cell response, which was associated with a reduction in transgene expression, was characterized more thoroughly in this study. We learned that the AAT-specific T cells in this patient were cytolytic in phenotype, mapped to a peptide in the endogenous mutant AAT protein that contained a common polymorphism not incorporated into the transgene, and were restricted by a rare HLA class I C alleles present only in this patient. These human studies illustrate the genetic influence of the endogenous gene and HLA haplotype on the outcome of gene therapy.

Rice seed is a cost-effective bioreactor for the large-scale production of pharmaceuticals. However, convincing evidence of the immunogenicity of plant-specific glycans is still limited although plant-specific glycans are considered potential allergic antigens. In the present study, we found that the α-1,3-fucose content of the glycoprotein produced from rice seed was much lower than that in leaf, and conversely, a higher β-1,2-xylose content was detected in seed than that in leaf. We detected the α-1,6-fucose content in the glutelin and recombinant human α1-antitrypsin (OsrAAT). The further results in a line containing AAT and FUT8 genes indicated that the α-1,6-fucose content of modified glycosylated recombinant α1-antitrypsin (mgOsrAAT) was 38.4%, while glutelin was only 6.8%. Interestingly, the α-1,3-fucose content of mgOsrAAT was significantly reduced by 59.8% compared with that of OsrAAT. Furthermore, we assessed the immunogenicity of OsrAAT, mgOsrAAT and human α1-antitrypsin (hAAT) using an animal system. The PCA results indicated no significant differences in the IgG, IgM and IgE titers among OsrAAT, mgOsrAAT and hAAT. Further studies revealed that those antibodies were mainly from α-1,3-fucose, but not from β-1,2-xylose, indicating that α-1,3-fucose was the major immunogenic resource. Our results demonstrated that α-1,3-fucose contents in seed proteins was much less than that of leaf, and could not be a plant-specific glycan because it also exists in human proteins., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Alpha-1 antitrypsin deficiency (AATD) is characterized by low levels of circulating alpha-1 antitrypsin and an increased risk for emphysema, liver disease, and panniculitis. The reduced levels of alpha-1 antitrypsin in AATD predispose the lung to unopposed proteolytic activity, predominantly from neutrophil-derived proteases, chiefly neutrophil elastase. This leads to emphysema. The mechanisms subtending the liver disease are less well understood, but are probably due to a "gain-of function" inflammatory process in the liver, stoked by intracellular retention of aberrantly folded alpha-1 antitrypsin. The panniculitis associated with AATD is most likely due to unopposed proteolytic activity in the skin. Although AATD has been traditionally viewed as a condition arising from a protease-antiprotease imbalance in the lung, it is increasingly recognized that AATD is an inflammatory disorder, both in the lung and in the extrapulmonary manifestations associated with the condition. This inflammation is predominantly neutrophil driven, and there are several alpha-1 antitrypsin-related mechanisms involved in potentiating this neutrophilic response. The rationale for AAT augmentation therapy in AATD is classically based on restoring the antiprotease balance in the lung, but its beneficial effects may also be exerted systemically, further exposing the pathogenesis of AATD-related disease and indicating a potential usage for alpha-1 antitrypsin in other inflammatory conditions.

More than 50 years ago, the observation that absence of the α1 band from protein electrophoresis is associated with severe emphysema established the link between α1-antitrypsin deficiency (AATD) and lung damage. From this discovery, the classic paradigm of protease/antiprotease imbalance was derived, linking lung destruction in patients with AATD to the unopposed effect of proteases. By extension, this paradigm was also applied to patients with 'common' chronic obstructive pulmonary disease, in whom large increases in smoke-induced proteases could overwhelm the antiprotease capability of AAT. However, it has become increasingly evident that AAT has important anti-inflammatory and immunoregulatory activities which, beside its antiprotease function, may be critically involved in lung destruction. From this perspective, we will consider recent evidence, based on epidemiological, clinical and immunopathological studies, suggesting that it is time to move on from the original protease/antiprotease paradigm toward a more complex view of the condition, which embraces its immunomodulating functions. Of importance, the potent immunoregulatory, tolerogenic role of AAT may support its therapeutic use in a number of diseases other than AATD, particularly in immune-related disorders., (© 2016 S. Karger AG, Basel.)

In α1-antitrypsin-deficient HIV patients, an accelerated decline of CD4(+) T cell numbers is observed, suggesting that α1-antitrypsin is a potential endogenous HIV inhibitor. In infected T lymphocytes, α1-antitrypsin potently blocks NF-κB activation and HIV-1 replication by directly interacting with IκBα in the cytosol, thereby altering its ubiquitination pattern. However, the mechanism of α1-antitrypsin entry into the cytosol, where IκBα locates, remains unclear. In the present study, we investigated the mechanism of α1-antitrypsin internalization in CD4(+) T cells. Thus, primary CD4(+) T cells were infected with HIV-1 and then incubated with α1-antitrypsin to detect its internalization. We found that CD4(+) T cells internalized α1-antitrypsin through a clathrin-dependent endocytosis process. Next, intracellular α1-antitrypsin exerted the inhibitory effect on NF-κB activation and HIV-1 replication. On primary CD4(+) T cells, α1-antitrypsin interacted with low-density lipoprotein receptor-related protein 1 to initiate the internalization. Inside CD4(+) T lymphocytes, α1-antitrypsin was transported from the endosome to the lysosome and then released into the cytosol, where it is possible for α1-antitrypsin to directly interact with IκBα. These results together suggest that α1-antitrypsin internalization is a clathrin-dependent and low-density lipoprotein receptor-related protein 1-mediated endocytosis process. Internalized α1-antitrypsin is transported through the endosome-lysosome-cytosol routine to interact with cytosolic IκBα and block NF-κB activation and HIV-1 replication., (© Society for Leukocyte Biology.)

Leukotriene B4 (LTB4) contributes to many inflammatory diseases, including genetic and nongenetic forms of chronic obstructive pulmonary disease. α-1 Antitrypsin (AAT) deficiency (AATD) is characterized by destruction of lung parenchyma and development of emphysema, caused by low AAT levels and a high neutrophil burden in the airways of affected individuals. In this study we assessed whether AATD is an LTB4-related disease and investigated the ability of serum AAT to control LTB4 signaling in neutrophils. In vitro studies demonstrate that neutrophil elastase is a key player in the LTB4 inflammatory cycle in AATD, causing increased LTB4 production, and associated BLT1 membrane receptor expression. AATD patients homozygous for the Z allele were characterized by increased neutrophil adhesion and degranulation responses to LTB4. We demonstrate that AAT can bind LTB4 and that AAT/LTB4 complex formation modulates BLT1 engagement and downstream signaling events, including 1,4,5-triphosphate production and Ca(2+) flux. Additionally, treatment of ZZ-AATD individuals with AAT augmentation therapy decreased plasma LTB4 concentrations and reduced levels of membrane-bound neutrophil elastase. Collectively, these results provide a mechanism by which AAT augmentation therapy impacts on LTB4 signaling in vivo, and not only reinforces the utility of this therapy for resolving inflammation in AATD, but supports useful future clinical applications in treatment of other LTB4-related diseases., (Copyright © 2015 by The American Association of Immunologists, Inc.)

α1-Antitrypsin (A1AT) purified from human plasma upregulates expression and release of angiopoietin-like protein 4 (Angptl4) in adherent human blood monocytes and in human lung microvascular endothelial cells, providing a mechanism for the broad immune-regulatory properties of A1AT independent of its antiprotease activity. In this study, we demonstrate that A1AT (Prolastin), a potent inducer of Angptl4, contains significant quantities of the fatty acids (FA) linoleic acid (C18:2) and oleic acid (C18:1). However, only trace amounts of FAs were present in preparations that failed to increase Angplt4 expression, for example, A1AT (Zemaira) or M-type A1AT purified by affinity chromatography. FA pull-down assays with Western blot analysis revealed a FA-binding ability of A1AT. In human blood-adherent monocytes, A1AT-FA conjugates upregulated expression of Angptl4 (54.9-fold, p < 0.001), FA-binding protein 4 (FABP4) (11.4-fold, p < 0.001), and, to a lesser degree, FA translocase (CD36) (3.1-fold, p < 0.001) relative to A1AT devoid of FA (A1AT-0). These latter effects of A1AT-FA were blocked by inhibitors of peroxisome proliferator-activated receptor (PPAR) β/δ (ST247) and PPARγ (GW9662). When compared with controls, cell pretreatment with ST247 diminished the effect of A1AT-LA on Angptl4 mRNA (11.6- versus 4.1-fold, p < 0.001) and FABP4 mRNA (5.4- versus 2.8-fold, p < 0.001). Similarly, preincubation of cells with GW9662 inhibited inducing effect of A1AT-LA on Angptl4 mRNA (by 2-fold, p < 0.001) and FABP4 mRNA (by 3-fold, p < 0.001). Thus, A1AT binds to FA, and it is this form of A1AT that induces Angptl4 and FABP4 expression via a PPAR-dependent pathway. These findings provide a mechanism for the unexplored area of A1AT biology independent of its antiprotease properties., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Background: Several soluble factors have been reported to have the capacity of inhibiting HIV replication at different steps of the virus life cycle, without eliminating infected cells and through enhancement of specific cellular mechanisms. Yet, it is unclear if these antiviral factors play a role in the protection from HIV infection or in the control of viral replication. Here we evaluated two cohorts: i) one of 58 HIV-exposed seronegative individuals (HESNs) who were compared with 59 healthy controls (HCs), and ii) another of 13 HIV-controllers who were compared with 20 HIV-progressors. Peripheral blood, oral and genital mucosa and gut-associated lymphoid tissue (GALT) samples were obtained to analyze the mRNA expression of ELAFIN, APOBEC3G, SAMHD1, TRIM5α, RNase 7 and SerpinA1 using real-time PCR., Results: HESNs exhibited higher expression of all antiviral factors in peripheral blood mononuclear cells (PBMCs), oral or genital mucosa when compared with HCs. Furthermore, HIV-controllers exhibited higher levels of SerpinA1 in GALT., Conclusions: These findings suggest that the activity of these factors is compartmentalized and that these proteins have a predominant role depending on the tissue to avoid the infection, reduce the viral load and modulate the susceptibility to HIV infection.

Mutant Z α1-antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z α1-antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z α1-antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked α1-antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. A single-chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z α1-antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin., (© FASEB.)

A monoclonal antibody (mAb) that binds to a transient intermediate may act as a catalyst for the corresponding reaction; here we show this principle can extend on a macro molecular scale to the induction of mutant-like oligomerization in a wild-type protein. Using the common pathogenic E342K (Z) variant of α1-antitrypsin as antigen-whose native state is susceptible to the formation of a proto-oligomeric intermediate-we have produced a mAb (5E3) that increases the rate of oligomerization of the wild-type (M) variant. Employing ELISA, gel shift, thermal stability and FRET time-course experiments, we show that mAb5E3 does not bind to the native state of α1-antitrypsin, but recognizes a cryptic epitope in the vicinity of the post-helix A loop and strand 4C that is revealed upon transition to the polymerization intermediate, and which persists in the ensuing oligomer. This epitope is not shared by loop-inserted monomeric conformations. We show the increased amenity to polymerization by either the pathogenic E342K mutation or the binding of mAb5E3 occurs without affecting the energetic barrier to polymerization. As mAb5E3 also does not alter the relative stability of the monomer to intermediate, it acts in a manner similar to the E342K mutant, by facilitating the conformational interchange between these two states.

Type 1 diabetes (T1D) is a chronic autoimmune disease in which β cells are destroyed. Islet transplantation is the most promising therapeutic treatment for T1D patients. However, allograft rejection and autoimmune reaction have been recognized as primary causes of graft loss after transplantation. Alpha-1-antitrypsin (AAT) is an important serine protease inhibitor in serum. AAT is characterized by anti-inflammation, anti-apoptosis, and induction-specific immunological tolerance. In this study, we successfully established NIT-hAAT cell lines, which are murine islet β cell lines with stable expression of human AAT (hAAT) gene. These NIT-hAAT cells were transplanted under the left kidney capsule of BALB/c diabetic mice. Interestingly, the sustained expression of hAAT in vivo can block the inflammatory cell infiltration and reduce the production of proinflammatory cytokines to effectively prevent nonspecific inflammation. Results showed that hAAT can inhibit the proliferation of lymphocytes, shift the balance between Th17 and Treg, and suppress the maturation of dendritic cells. Therefore, hAAT can serve as a beneficial immunomodulator that limits immune rejection to prolong islet allograft survival and achieve long-term successful transplant outcomes.

Pre- and post-transplant infusions of donor splenocytes treated with 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide (ECDI-SPs) induce donor-specific tolerance and prolong rat renal allograft survival. However, proinflammatory cytokine production during peritransplantation negates the effects of ECDI-SPs. Therefore, we reasoned that blocking proinflammatory cytokines would promote long-term ECDI-SP-induced allograft survival. We therefore examined the effects of infusing ECDI-SPs alone or in combination with a short course of α1-Antitrypsin (AAT) on the long-term outcomes of a rat kidney allograft model. The data showed that ECDI-SPs+AAT promote renal allograft survival compared with ECDI-SPs alone. This effect was accompanied by expansion of Foxp3+ Tregs, enhanced alloantigen-specific Treg function, and modulation of expression levels of proinflammatory cytokines IL-1β, IL-6, TNF-α, and the anti-inflammatory cytokine IL-10. In conclusion, our strategy of combining ECDI-SPs and AAT provides a promising approach for inducing specific transplant tolerance., (Copyright © 2015 Elsevier B.V. All rights reserved.)

One would assume that the anti-inflammatory activity of α1-anti-trypsin (AAT) is the result of inhibiting neutrophil enzymes. However, AAT exhibits tolerogenic activities that are difficult to explain by serine-protease inhibition or by reduced inflammatory parameters. Targets outside the serine-protease family have been identified, supporting the notion that elastase inhibition, the only functional factory release criteria for clinical-grade AAT, is over-emphasized. Non-obvious developments in the understanding of AAT biology disqualify it from being a straightforward anti-inflammatory agent: AAT does not block dendritic cell activities, nor does it promote viral and tumour susceptibilities, stunt B lymphocyte responses or render treated patients susceptible to infections; accordingly, outcomes of elevated AAT do not overlap those attained by immunosuppression. Aside from the acute-phase response, AAT rises during the third trimester of pregnancy and also in advanced age. At the molecular level, AAT docks onto cholesterol-rich lipid-rafts and circulating lipid particles, directly binds interleukin (IL)-8, ADAM metallopeptidase domain 17 (ADAM17) and danger-associated molecular pattern (DAMP) molecules, and its activity is lost to smoke, high glucose levels and bacterial proteases, introducing a novel entity - 'relative AAT deficiency'. Unlike immunosuppression, AAT appears to help the immune system to distinguish between desired responses against authentic threats, and unwanted responses fuelled by a positive feedback loop perpetuated by, and at the expense of, inflamed injured innocent bystander cells. With a remarkable clinical safety record, AAT treatment is currently tested in clinical trials for its potential benefit in a variety of categorically distinct pathologies that share at least one common driving force: cell injury., (© 2014 British Society for Immunology.)

The traditional model of T helper differentiation describes the naïve T cell as choosing one of several subsets upon stimulation and an added reciprocal inhibition aimed at maintaining the chosen subset. However, to date, evidence is mounting to support the presence of subset plasticity. This is, presumably, aimed at fine-tuning adaptive immune responses according to local signals. Reprograming of cell phenotype is made possible by changes in activation of master transcription factors, employing epigenetic modifications that preserve a flexible mode, permitting a shift between activation and silencing of genes. The acute phase response represents an example of peripheral changes that are critical in modulating T cell responses. α1-antitrypsin (AAT) belongs to the acute phase responses and has recently surfaced as a tolerogenic agent in the context of adaptive immune responses. Nonetheless, AAT does not inhibit T cell responses, nor does it shutdown inflammation per se; rather, it appears that AAT targets non-T cell immunocytes towards changing the cytokine environment of T cells, thus promoting a regulatory T cell profile. The present review focuses on this intriguing two-way communication between innate and adaptive entities, a crosstalk that holds important implications on potential therapies for a multitude of immune disorders.

Alpha-1 antitrypsin (AAT) is a circulating serine protease inhibitor (serpin) that inhibits neutrophil elastase in the lung, and AAT deficiency is associated with early-onset emphysema. AAT is also a liver-derived acute-phase protein that, in vitro and in vivo, reduces production of pro-inflammatory cytokines, inhibits apoptosis, blocks leukocyte degranulation and migration, and modulates local and systemic inflammatory responses. In monocytes, AAT has been shown to increase intracellular cAMP, regulate expression of CD14, and suppress NFκB nuclear translocation. These effects may be mediated by AAT's serpin activity or by other protein-binding activities. In preclinical models of autoimmunity and transplantation, AAT therapy prevents or reverses autoimmune disease and graft loss, and these effects are accompanied by tolerogenic changes in cytokine and transcriptional profiles and T cell subsets. This review highlights advances in our understanding of the immune-modulating effects of AAT and their potential therapeutic utility.

The angiopoietin-like protein 4 (angptl4, also known as peroxisome proliferator-activated receptor [PPAR]γ-induced angiopoietin-related protein) is a multifunctional protein associated with acute-phase response. The mechanisms accounting for the increase in angptl4 expression are largely unknown. This study shows that human α1-antitrypsin (A1AT) upregulates expression and release of angplt4 in human blood adherent mononuclear cells and in primary human lung microvascular endothelial cells in a concentration- and time-dependent manner. Mononuclear cells treated for 1 h with A1AT (from 0.1 to 4 mg/ml) increased mRNA of angptl4 from 2- to 174-fold, respectively, relative to controls. In endothelial cells, the maximal effect on angptl4 expression was achieved at 8 h with 2 mg/ml A1AT (11-fold induction versus controls). In 10 emphysema patients receiving A1AT therapy (Prolastin), plasma angptl4 levels were higher relative to patients without therapy (nanograms per milliliter, mean [95% confidence interval] 127.1 [99.5-154.6] versus 76.8 [54.8-98.8], respectively, p = 0.045) and correlated with A1AT levels. The effect of A1AT on angptl4 expression was significantly diminished in cells pretreated with a specific inhibitor of ERK1/2 activation (UO126), irreversible and selective PPARγ antagonist (GW9662), or genistein, a ligand for PPARγ. GW9662 did not alter the ability of A1AT to induce ERK1/2 phosphorylation, suggesting that PPARγ is a critical mediator in the A1AT-driven angptl4 expression. In contrast, the forced accumulation of HIF-1α, an upregulator of angptl4 expression, enhanced the effect of A1AT. Thus, acute-phase protein A1AT is a physiological regulator of angptl4, another acute-phase protein., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Granulocyte-colony-stimulating factor (G-CSF)-induced hematopoietic stem and progenitor cell (HSPC) mobilization is associated with the release of neutrophil-derived proteases. Previously, we have shown that alpha-1-antitrypsin (AAT) inhibits these proteases in mice, resulting in inhibition of HSPC mobilization. Here, we studied the relationship between AAT and HSPC in steady state and cytokine-induced mobilization in humans. Patients with alpha-1-antitrypsin deficiency (AATD) have an 85-90 % decrease of AAT in the peripheral blood (PB). We hypothesized that this leads to increased proteolytic activity in the bone marrow and increased steady-state PB HSPC numbers. Using flow cytometry and semi-solid cell culture, we found no significant difference in PB HSPC in AATD patients (n = 18) as compared to controls (n = 22). Healthy stem cell donors (n = 43) were mobilized with G-CSF for 5 days and the number of CD45(+)/CD34(+) HSPC were determined in PB. We found that, during mobilization, PB AAT levels increased significantly, positively correlating with PB CD45(+)/CD34(+) cells (r = 0.31, p = 0.005). In conclusion, although serum AAT levels and HSPC mobilization in healthy stem cell donors are positively correlated, AAT is not an indispensable protease-inhibitor in the constitutive circulation of HSPC. These findings suggest a model in which both protease-dependent and -independent pathways contribute to HSPC mobilization.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides are a heterogeneous group of diseases corresponding to necrotising inflammation of small vessels with a wide range of clinical presentations. At least two of the diseases are believed to exhibit a common ground of pathophysiological mechanisms. These are granulomatosis with polyangiitis (GPA, formerly known as Wegener's granulomatosis) and microscopic polyangiitis (MPA). ANCA directed against proteinase 3 (PR3) are preferentially associated with GPA, and anti-myeloperoxidase (MPO) ANCA are associated mainly with MPA and eosinophilic GPA (formerly known as Churg-Strauss syndrome). Anti-MPO and anti-PR3 antibodies can activate neutrophils in vitro. In vivo data are available for humans and mice on the pathogenicity of anti-MPO but it is more controversial for PR3-ANCA. A recent genome-wide association study of patients with ANCA-associated vasculitides confirmed the genetic contribution to the pathogenesis of these conditions, with significant association of PR3-ANCA and human leukocyte antigen-DP and the genes encoding α1-antitrypsin and PR3. MPO-ANCA were significantly associated with human leukocyte antigen-DQ. Thus, recent results from epidemiological studies, genome-wide association study and therapeutic trials have suggested that these entities are, in fact, distinct. We have summarised these results and discuss the idea that these two entities should be studied separately as the nature of the two auto-antigens suggests at a molecular level despite shared ANCA involvement.

Islet transplantation has considerable potential as a cure for diabetes. However, the difficulties that arise from inflammation and the immunological rejection of transplants must be addressed for islet transplantation to be successful. Alpha 1-antitrypsin (AAT) inhibits the damage on β cells caused by inflammatory reactions and promotes β-cell survival and proliferation. This protein also induces specific immune tolerance to transplanted β cells. However, whether the expression of AAT in β cells themselves could eliminate or decrease immunological rejection of transplants is not clear. Therefore, we established a β cell line (NIT-hAAT) that stably expresses human AAT. Interestingly, in a cytotoxic T lymphocyte (CTL)-killing assay, we found that hAAT reduced apoptosis and inflammatory cytokine production in NIT-1 cells and regulated the Th1/Th2 cytokine balance in vitro. In vivo transplantation of NIT-hAAT cells into mice with diabetes showed hAAT inhibited immunological rejection for a short period of time and increased the survival of transplanted β cells. This study demonstrated that hAAT generated remarkable immunoprotective and immunoregulation effects in a model of β cell islet transplantation for diabetes model.

Aims: The ATZ11 antibody has been well established for the identification of α1-anti-trypsin (AAT) molecule type PiZ (Z-AAT) in blood samples and liver tissue. In this study, we systematically analyzed the antibody for additional binding sites in human tissue., Methods and Results: Ultrastructural ATZ11 binding was investigated immunoelectron microscopically in human umbilical vein endothelial cells (HUVECs) and in platelets of a healthy individual. Human embryonic kidney (HEK293) cells were transiently transfected with Von Willebrand factor (VWF) and analyzed immunocytochemically using confocal microscopy and SDS-PAGE electrophoresis followed by western blotting (WB). Platelets and serum samples of VWF-competent and VWF-deficient patients were investigated using native PAGE and SDS-PAGE electrophoresis followed by WB. The specificity of the ATZ11 reaction was tested immunohistochemically by extensive antibody-mediated blocking of AAT- and VWF-antigens. ATZ11-positive epitopes could be detected in Weibel-Palade bodies (WPBs) of HUVECs and α-granules of platelets. ATZ11 stains pseudo-WBP containing recombinant wild-type VWF (rVWF-WT) in HEK293 cells. In SDS-PAGE electrophoresis followed by WB, anti-VWF and ATZ11 both identified rVWF-WT. However, neither rVWF-WT-multimers, human VWF-multimers, nor serum proteins of VWF-deficient patients were detected using ATZ11 by WB, whereas anti-VWF antibody (anti-VWF) detected rVWF-WT-multimers as well as human VWF-multimers. In human tissue specimens, AAT-antigen blockade using anti-AAT antibody abolished ATZ11 staining of Z-AAT in a heterozygous AAT-deficient patient, whereas VWF-antigen blockade using anti-VWF abolished ATZ11 staining of endothelial cells and megakaryocytes., Conclusions: ATZ11 reacts with cellular bound and denatured rVWF-WT and human VWF as shown using immunocytochemistry and subsequent confocal imaging, immunoelectron microscopy, SDS-PAGE and WB, and immunohistology. These immunoreactions are independent of the binding of Z-AAT-molecules and non-Z-AAT complexes.

Background: Corneal inflammation has long been associated with contact lens wear and the use of extended-wear lenses enhances the risk of corneal injury. Elucidation of the molecular mediators of contact lens-associated inflammation has the potential to provide injury-identifying markers early in the inflammatory process, as well as determine potential therapeutic targets., Methods: This cross-over study investigated a potential correlation between overnight contact lens wear and the concentrations of two markers of inflammation, α1-antitrypsin and C-reactive protein, in tear fluid. To obtain baseline measurements, 17 subjects adapted to wearing silicone hydrogel contact lenses wore their prescribed eye glasses for one week, after which tears were collected and ocular health assessed by a licensed optometrist. Subjects then returned to wearing their prescribed silicone hydrogel lenses continuously for one week. A second tear sample was collected and ocular inflammation was again assessed. Enzyme-linked immunosorbent assays were performed on all tear samples for both α1-antitrypsin and C-reactive protein., Results: α1-antitrypsin was significantly (p = 0.01) elevated after continuous contact lens wear, with increases above baseline concentrations averaging 2.48-fold. Optometric assessment of inflammation loosely correlated with levels of this inflammatory marker. C-reactive protein was detected in the tears of subjects at both times and levels were also slightly elevated after extended lens wear, but not significantly (p > 0.5) and not consistently in all subjects., Conclusion: The results of this study suggest that α1-antitrypsin in tear fluid may be useful as an early marker of contact lens-associated ocular irritation and inflammation. The presence of C-reactive protein in the tears of contact lens wearers is a novel finding which, while not correlative with either α1-antitrypsin concentrations or clinically observable inflammation, may warrant further study., (© 2013 The Authors. Clinical and Experimental Optometry © 2013 Optometrists Association Australia.)

To analyze the possibility of using immunological parameters for the assessment of the activity of the process and prediction of the quality and completion of remission, we compared the dynamics of clinical and immunological parameters in 76 patients with endogenous attack-like psychoses during pharmacotherapy of a psychotic episode. Authors confirmed evidence for the activation of innate and adaptive immunity in the acute stage of psychosis as well as the correlation between immunological parameters (leukocyte elastase (LE) activity, alpha(1)-proteinase inhibitor (alpha(1)-PI), the level of autoantibodies to nerve growth factor (Aab-NGF)) and clinical symptoms assessed with the PANSS. The improvement of the clinical state assessed by the reduction in PANSS total score was noted in all patients though there were variations in the dynamics of immunological parameters. The increase of immunological parameters, along with the absence of changes at the discharge from the hospital, suggests that the remission was of low quality and the pathological process did not attenuate. Outpatient examination revealed the different dynamics of psychopathological disorders: stable state in 50% patients, moderate worsening of psychological state in 50% patients. Worsening of clinical symptoms after the discharge and in the outpatient stage was correlated with the elevation of the activity/level of immunological parameters. The changes in LE activity and Aab-NGF level precede the changes in mental state of patients in the following 1-2 months. These parameters may be used for monitoring of patients and prediction of quality and completion of remission.

Recombinant adeno-associated virus (rAAV) vectors have shown promise for the treatment of several diseases; however, immune-mediated elimination of transduced cells has been suggested to limit and account for a loss of efficacy. To determine whether rAAV vector expression can persist long term, we administered rAAV vectors expressing normal, M-type α-1 antitrypsin (M-AAT) to AAT-deficient subjects at various doses by multiple i.m. injections. M-specific AAT expression was observed in all subjects in a dose-dependent manner and was sustained for more than 1 year in the absence of immune suppression. Muscle biopsies at 1 year had sustained AAT expression and a reduction of inflammatory cells compared with 3 month biopsies. Deep sequencing of the TCR Vβ region from muscle biopsies demonstrated a limited number of T cell clones that emerged at 3 months after vector administration and persisted for 1 year. In situ immunophenotyping revealed a substantial Treg population in muscle biopsy samples containing AAT-expressing myofibers. Approximately 10% of all T cells in muscle were natural Tregs, which were activated in response to AAV capsid. These results suggest that i.m. delivery of rAAV type 1-AAT (rAAV1-AAT) induces a T regulatory response that allows ongoing transgene expression and indicates that immunomodulatory treatments may not be necessary for rAAV-mediated gene therapy.

B-lymphocyte activities are associated with allograft rejection. Interleukin-10 (IL-10) -expressing B cells, however, exhibit regulatory attributes. Human α1-antitrypsin (hAAT), a clinically available anti-inflammatory circulating glycoprotein that rises during acute-phase responses, promotes semi-mature dendritic cells and regulatory T (Treg) cells during alloimmune responses. Whether B lymphocytes are also targets of hAAT activity has yet to be determined. Here, we examine whether hAAT modulates B-cell responses. In culture, hAAT reduced the lipopolysaccharide-stimulated Ki-67(+) B-cell population, IgM release and surface CD40 levels, but elevated IL-10-producing cells 1.5-fold. In CD40 ligand-stimulated cultures, hAAT promoted a similar trend; reduction in the Ki-67(+) B-cell population and in surface expression of CD86, CD80 and MHCII. hAAT increased interferon-γ-stimulated macrophage B-cell activating factor (BAFF) secretion, and reduced BAFF-receptor levels. Draining lymph nodes of transgenic mice that express circulating hAAT (C57BL/6 background) and that received skin allografts exhibited reduced B-lymphocyte activation compared with wild-type recipients. BSA-vaccinated hAAT transgenic mice exhibited 2.9-fold lower BSA-specific IgG levels, but 2.3-fold greater IgM levels, compared with wild-type mice. Circulating Treg cells were 1.3-fold greater in transgenic hAAT mice, but lower in B-cell knockout (BKO) and chimeric hAAT-BKO mice, compared with wild-type mice. In conclusion, B cells are cellular targets of hAAT. hAAT-induced Treg cell expansion appears to be B-cell-dependent. These changes support the tolerogenic properties of hAAT during immune responses, and suggest that hAAT may be beneficial in pathologies that involve excessive B-cell responses., (© 2013 John Wiley & Sons Ltd.)

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides are a heterogeneous group of diseases corresponding to necrotising inflammation of small vessels with a wide range of clinical presentations. At least two of the diseases are believed to exhibit a common ground of pathophysiological mechanisms. These are granulomatosis with polyangiitis (GPA, formerly known as Wegener's granulomatosis) and microscopic polyangiitis (MPA). ANCA directed against proteinase 3 (PR3) are preferentially associated with GPA, and anti-myeloperoxidase (MPO) ANCA are associated mainly with MPA and eosinophilic GPA (formerly known as Churg-Strauss syndrome). Anti-MPO and anti-PR3 antibodies can activate neutrophils in vitro. In vivo data are available for humans and mice on the pathogenicity of anti-MPO but it is more controversial for PR3-ANCA. A recent genome-wide association study of patients with ANCA-associated vasculitides confirmed the genetic contribution to the pathogenesis of these conditions, with significant association of PR3-ANCA and human leukocyte antigen-DP and the genes encoding α1-antitrypsin and PR3. MPO-ANCA were significantly associated with human leukocyte antigen-DQ. Thus, recent results from epidemiological studies, genome-wide association study and therapeutic trials have suggested that these entities are, in fact, distinct. We have summarised these results and discuss the idea that these two entities should be studied separately as the nature of the two auto-antigens suggests at a molecular level despite shared ANCA involvement.

Clinical pancreatic islet transplantation is under evaluation for the treatment of autoimmune diabetes, yet several limitations preclude widespread use. For example, there is a critical shortage of human pancreas donors. Xenotransplantation may solve this problem, yet it evokes a rigorous immune response which can lead to graft rejection. Alpha-1-antitrypsin (AAT), a clinically available and safe circulating anti-inflammatory and tissue protective glycoprotein, facilitates islet alloimmune-tolerance and protects from inflammation in several models. Here, we examine whether human AAT (hAAT), alone or in combination with clinically relevant approaches, achieves long-term islet xenograft survival. Rat-to-mouse islet transplantation was examined in the following groups: untreated (n = 6), hAAT (n = 6, 60-240 mg/kg every 3 days from day -10), low-dose co-stimulation blockade (anti-CD154/LFA-1) and single-dose anti-CD4/CD8 (n = 5-7), either as mono- or combination therapies. Islet grafting was accompanied by blood glucose follow-up. In addition, skin xenografting was performed in order to depict responses that occur in draining lymph nodes. According to our results hAAT monotherapy and hAAT/anti-CD154/LFA-1 combined therapy, did not delay rejection day (11-24 days untreated vs. 10-22 day treated). However, host and donor intragraft inflammatory gene expression was diminished by hAAT therapy in both setups. Single dose T-cell depletion using anti-CD4/CD8 depleting antibodies, which provided 14-15 days of reduced circulating T-cells, significantly delayed rejection day (28-52 days) but did not achieve graft acceptance. In contrast, in combination with hAAT, the group displayed significantly extended rejection days and a high rate of graft acceptance (59, 61, >90, >90, >90). In examination of graft explants, marginal mononuclear-cell infiltration containing regulatory T-cells predominated surviving xenografts. We suggest that temporal T-cell depletion, as in the clinically practiced anti-thymocyte-globulin therapy, combined with hAAT, may promote islet xenograft acceptance. Further studies are required to elucidate the mechanism behind the observed synergy, as well as the applicability of the approach for pig-to-human islet xenotransplantation.

Evaluation of human antibody responses to alpha-1 antitrypsin (AAT) in clinical trials and clinical practice has been limited by the lack of a validated assay. Here we describe the development and validation of an ELISA method for quantification of human and nonhuman primate antibody responses to human AAT. A reference anti-human AAT serum standard was generated using sera from a cynomolgus macaque injected with a recombinant adeno-associated virus vector expressing human AAT. The ELISA was validated for use with human serum dilutions as low as 1:10 and was able to distinguish between specific responses in cynomolgus serum and non-specific increases in apparent antibody titer in serum from subjects in a clinical trial of an AAT gene therapy vector., (Copyright © 2012 Elsevier B.V. All rights reserved.)