Search Results (22)

Somatic embryogenesis (SE) is a morphogenetic pathway widely employed in the commercial micropropagation of plants. This route enables the generation of somatic embryos from somatic tissues, which give rise to complete (bipolar) plants that develop like zygotic embryos. SE can proceed via direct or indirect pathways, and both approaches have been adapted not only for large-scale clonal propagation but also for the regeneration of genetically modified plants. In this context, SE can be harnessed as a versatile platform for recombinant protein production, including vaccine antigens and therapeutic proteins, by combining plant tissue culture with genetic transformation strategies. Successful examples include non-model plants, as Daucus carota and Eleutherococcus senticosus expressing the cholera and heat-labile enterotoxin B subunits, respectively; Oryza sativa, Nicotiana tabacum, and Medicago sativa producing complex proteins such as human serum albumin (HSA), α₁-antitrypsin (AAT), and monoclonal antibodies. However, challenges remain in optimizing transformation efficiency, scaling up bioreactor-based suspension cultures, and ensuring proper post-translational modifications under Good Manufacturing Practice (GMP) standards. Recent advances in synthetic biology, modular vector design, and glycoengineering have begun to address these limitations, improving control over transcriptional regulation and protein quality. This review highlights the application of SE as a biotechnological route for recombinant protein production, discusses current challenges, and presents innovative strategies and perspectives for the development of sustainable plant-derived biopharmaceutical systems. Full article

Objective: The aim of this study was to examine the safety and tolerance of Bifidobacterium longum subsp. infantis YLGB-1496 (B. infantis YLGB-1496) in toddlers with respiratory illness. Methods: In this randomized controlled trial, 120 toddlers with respiratory illness were randomly assigned to the probiotic (YLGB-1496) or control group for a 12-week intervention. Follow-up examinations were conducted at baseline (week 0) and at weeks 6 and 12 of the intervention. Toddlers’ height and weight were measured by trained personnel, and defecation characteristics and gastrointestinal symptoms were recorded by parents or guardians. Stool samples were collected to determine the fecal pH, fecal calprotectin (FC) concentration, and fecal α1-antitrypsin (AAT) concentration. Results: A total of 115 toddlers completed the 12-week intervention (58 in the YLGB-1496 group and 57 in the control group). The height-for-age Z score (HAZ) in the YLGB-1496 group was significantly greater than that in the control group (p = 0.006). The weight-for-age Z score (WAZ) in the YLGB-1496 group increased between weeks 6 and 12, whereas the WAZ in the control group continuously decreased during the intervention. No differences in the frequency or consistency of defecation between the groups were observed. Toddlers in the YLGB-1496 group had lower incidences of poor appetite, nausea, vomiting, stomachache, lower abdominal pain, diarrhea, and dehydration (p < 0.05) but higher fecal AAT concentrations (p = 0.008) than did those in the control group. No differences in the fecal pH or FC concentration were observed between the groups. Conclusions: B. infantis YLGB-1496 demonstrated excellent safety and tolerability in toddlers and effectively reduced the gastrointestinal discomfort associated with respiratory illnesses. Full article

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

α1-Antitrypsin (AAT), an acute-phase reactant not unsimilar to C-reactive protein (CRP), is a serine protease inhibitor that harbors tissue-protective and immunomodulatory attributes. Its concentrations appropriately increase during conditions of extensive tissue injury, and it induces immune tolerance, in part, by inhibiting the enzymatic activity of the inflammatory serine protease, proteinase 3 (PR3). Typically administered to patients with genetic AAT deficiency, AAT treatment was recently shown to improve outcomes in patients with steroid-refractory graft-versus-host disease (GVHD). GVHD represents a grave outcome of allogeneic hematopoietic stem cell transplantation (HSCT), a potentially curative intervention for hematological diseases. The procedure requires radio/chemotherapy conditioning of the prospective marrow recipient, a cytotoxic process that causes vast tissue injury and, in some formats, interferes with liver production of AAT. To date, changes in the functional profile of AAT during allogeneic HSCT, and during the cytotoxic intervention that precedes HSCT, are unknown. The present study followed 53 patients scheduled for allogeneic HSCT (trial registration NCT03188601). Serum samples were tested before and after HSCT for AAT and CRP levels and for intrinsic anti-proteolytic activity. The ex vivo response to clinical-grade AAT was tested on circulating patient leukocytes and on a human epithelial cell line treated with patient sera in a gap closure assay. According to the ex vivo experiments, circulating leukocytes responded to AAT with a favorable immune-regulated profile, and epithelial gap closure was enhanced by AAT in sera from GVHD-free patients but not in sera from patients who developed GVHD. According to serum collected prior to HSCT, non-relapse mortality was reliably predicted by combining three components: AAT and CRP levels and serum anti-proteolytic activity. Taken together, HSCT outcomes are significantly affected by the anti-proteolytic function of circulating AAT, supporting early AAT augmentation therapy for allogeneic HSCT patients. Full article

Human neutrophil elastase (HNE) is involved in SARS-CoV-2 virulence and plays a pivotal role in lung infection of patients infected by COVID-19. In healthy individuals, HNE activity is balanced by α1-antitrypsin (AAT). This is a 52 kDa glycoprotein, mainly produced and secreted by hepatocytes, that specifically inhibits HNE by blocking its activity through the formation of a stable complex (HNE–AAT) in which the two proteins are covalently bound. The lack of this complex, together with the detection of HNE activity in BALf/plasma samples of COVID-19 patients, leads us to hypothesize that potential functional deficiencies should necessarily be attributed to possible structural modifications of AAT. These could greatly diminish its ability to inhibit neutrophil elastase, thus reducing lung protection. The aim of this work was to explore the oxidation state of AAT in BALf/plasma samples from these patients so as to understand whether the deficient inhibitory activity of AAT was somehow related to possible conformational changes caused by the presence of abnormally oxidized residues. Full article

PiZZ (Glu342Lys) α1-antitrypsin deficiency (AATD) is characterized by intrahepatic AAT polymerization and is a risk factor for liver disease development in children. The majority of PiZZ children are disease free, hence this mutation alone is not sufficient to cause the disease. We investigated Z-AAT polymers and the expression of fibrosis-related genes in liver tissues of PiZZ children with different clinical courses. Liver biopsies obtained during 1979–2010 at the Department of Paediatrics, Karolinska University Hospital, Sweden, were subjected to histological re-evaluation, immunohistochemistry and NanoString-based transcriptome profiling using a panel of 760 fibrosis plus 8 bile acid-related genes. Subjects were divided into three groups based on clinical outcomes: NCH (neonatal cholestasis, favourable outcome, n = 5), NCC (neonatal cholestasis, early cirrhosis and liver transplantation, n = 4), and NNCH (no neonatal cholestasis, favourable outcome, n = 5, six biopsies). Hepatocytes containing Z-AAT polymers were abundant in all groups whereas NCC showed higher expression of genes related to liver fibrosis/cirrhosis and lower expression of genes related to lipid, aldehyde/ketone, and bile acid metabolism. Z-AAT accumulation per se cannot explain the clinical outcomes of PiZZ children; however, changes in the expression of specific genes and pathways involved in lipid, fatty acid, and steroid metabolism appear to reflect the degree of liver injury. Full article

Physical activity has a positive effect on human health and well-being, but intense exercise can cause adverse changes in the organism, leading to the development of oxidative stress and inflammation. The aim of the study was to determine the effect of short-term cold water immersion (CWI) and a sauna bath as methods of postexercise regeneration on the indicators of inflammation and oxidative damage in the blood of healthy recreational athletes. Forty-five male volunteers divided into two groups: ‘winter swimmers’ who regularly use winter baths (n = 22, average age 43.2 ± 5.9 years) and ‘novices’ who had not used winter baths regularly before (n = 23, mean age 25 ± 4.8 years) participated in the study. The research was divided into two experiments, differing in the method of postexercise regeneration used, CWI (Experiment I) and a sauna bath (Experiment II). During Experiment I, the volunteers were subjected to a 30-min aerobic exercise, combined with a 20-min rest at room temperature (RT-REST) or a 20-min rest at room temperature with an initial 3-min 8 °C water bath (CWI-REST). During the Experiment II, the volunteers were subjected to the same aerobic exercise, followed by a RT-REST or a sauna bath (SAUNA-REST). The blood samples were taken before physical exercise (control), immediately after exercise and 20 min after completion of regeneration. The concentrations of selected indicators of inflammation, including interleukin 1β (IL-1β), interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 8 (IL-8), interleukin 10 (IL-10), transforming growth factor β1 (TGF-β1) and tumor necrosis factor α (TNF-α), as well as the activity of indicators of oxidative damage: α1-antitrypsin (AAT) and lysosomal enzymes, including arylsulfatase A (ASA), acid phosphatase (AcP) and cathepsin D (CTS D), were determined. CWI seems to be a more effective post-exercise regeneration method to reduce the inflammatory response compared to a sauna bath. A single sauna bath is associated with the risk of proteolytic tissue damage, but disturbances of cellular homeostasis are less pronounced in people who regularly use cold water baths than in those who are not adapted to thermal stress. Full article

Colitis-associated colon cancer (CAC) accompanies the massive infiltration of neutrophils during tumorigenesis and progression of CAC. Depletion of neutrophils in circulation results in significant inhibition of tumor incidence in CAC. However, the underlying mechanisms are largely unclear. In this study, we provide evidence for the crucial involvement of inflammatory neutrophil-activated serine proteases (NSPs) on the dysregulation of the anti-inflammatory and antitumor IGFBP-3/IGFBP-3R signaling axis in CAC using a chronic AOM/DSS mouse model. We also provide preclinical evidence for α1-antitrypsin (AAT) as a preventive and as a therapeutic for CAC. AAT administration not only prevented colitis-associated tumorigenesis but also inhibited established CAC. AOM/DSS treatment results in the significant activation of NSPs, leading to CAC through increased pro-inflammatory cytokines and decreased anti-inflammatory and antitumor IGFBP-3. Collectively, these data suggest that the NSPs proteolyze IGFBP-3, whereas AAT inhibits chronic colonic inflammation-induced NSP activity and subsequently suppresses IGFBP-3 proteolysis. Therefore, the anti-inflammatory and antitumor functions of the IGFBP-3/IGFBP-3R axis are restored. AAT mimicking small peptides also showed their inhibitory effects on NSP-induced IGFBP-3 proteolysis. These results suggest that targeting the NSP-IGFBP-3/IGFBP-3R axis using NSP inhibitors such as AAT and the AAT mimics and IGFBP-3R agonists could lead to novel approaches for the prevention and treatment of CAC. Full article

Protecting tissues from excessive inflammation by glucocorticoids results in an effective blockade of inflammation; however, it does not instigate processes of inflammatory resolution or tissue repair. Moreover, glucocorticoids have side effects such as a susceptibility to infections. In otolaryngology—specifically, within the inner ear—surgical and non-surgical pathologies include cochlear implantation, stapes surgery, perilymph fistulas and Meniere’s disease. For these, steroids are indicated in order to prevent excessive inflammation that might lead to hearing and vestibular failure. Unless tissue homeostasis is restored, the compromised tissue is at risk of a functional loss. α1-Antitrypsin (AAT) is a circulating inflammation-modulating molecule that rises during the molecular signs of a tissue injury; it manipulates inflammation towards an inflammatory resolution and advances tissue repair. Lifelong infusions of AAT are currently indicated for genetic AAT deficiencies and are safe. In the present review, we discuss the advantages and downfalls of glucocorticoid treatments across several surgical inner ear injuries alongside evidence of the beneficial attributes of treatments with AAT. Collectively, the present knowledge places AAT treatments, wither independent or in combination with glucocorticoids, as adding focus on tissue repair in the context of unmet medical needs in otolaryngology. Full article

Wound healing requires a non-compromising combination of inflammatory and anti-inflammatory processes. Human α1-antitrypsin (hAAT), a circulating glycoprotein that rises during acute-phase responses and during healthy pregnancies, is tissue-protective and tolerance-inducing; although anti-inflammatory, hAAT enhances revascularization. hAAT blocks tissue-degrading enzymes, including neutrophil elastase; it is, therefore, unclear how wound healing might improve under hAAT-rich conditions. Here, wound healing was examined in the presence of recombinant hAAT (hAAT^WT) and protease-inhibition-lacking hAAT (hAAT^CP). The impact of both hAAT forms was determined by an epithelial cell gap closure assay, and by excisional skin injuries via a microemulsion optimized for open wounds. Neutrophilic infiltration was examined after 8 h. According to results, both hAAT forms accelerated epithelial gap closure and excisional wound closure, particularly at early time points. Unlike dexamethasone-treated wounds, both resulted in closed borders at the 8-h time point. In untreated and hAAT^CP-treated wounds, leukocytic infiltrates were widespread, in hAAT^WT-treated wounds compartmentalized and in dexamethasone-treated wounds, scarce. Both hAAT forms decreased interleukin-1β and increased VEGF gene expression. In conclusion hAAT improves epithelial cell migration and outcomes of in vivo wounds irrespective of protease inhibition. While both forms of hAAT allow neutrophils to infiltrate, only native hAAT created discrete neutrophilic tissue clusters. Full article

Neutrophils play a pathogenic role in COVID-19 by releasing Neutrophils Extracellular Traps (NETs) or human neutrophil elastase (HNE). Given that HNE is inhibited by α1-antitrypsin (AAT), we aimed to assess the content of HNE, α1-antitrypsin (AAT) and HNE–AAT complexes (the AAT/HNE balance) in 33 bronchoalveolar lavage fluid (BALf) samples from COVID-19 patients. These samples were submitted for Gel-Electrophoresis, Western Blot and ELISA, and proteins (bound to AAT or HNE) were identified by Liquid Chromatography-Mass Spectrometry. NETs’ release was analyzed by confocal microscopy. Both HNE and AAT were clearly detectable in BALf at high levels. Contrary to what was previously observed in other settings, the formation of HNE–AAT complex was not detected in COVID-19. Rather, HNE was found to be bound to acute phase proteins, histones and C3. Due to the relevant role of NETs, we assessed the ability of free AAT to bind to histones. While confirming this binding, AAT was not able to inhibit NET formation. In conclusion, despite the finding of a high burden of free and bound HNE, the lack of the HNE–AAT inhibitory complex in COVID-19 BALf demonstrates that AAT is not able to block HNE activity. Furthermore, while binding to histones, AAT does not prevent NET formation nor their noxious activity. Full article

The most common hereditary disorder in adults, α1-antitrypsin deficiency (AATD), is characterized by reduced plasma levels or the abnormal functioning of α1-antitrypsin (AAT), a major human blood serine protease inhibitor, which is encoded by the SERine Protein INhibitor-A1 (SERPINA1) gene and produced in the liver. Recently, it has been hypothesized that the geographic differences in COVID-19 infection and fatality rates may be partially explained by ethnic differences in SERPINA1 allele frequencies. In our review, we examined epidemiological data on the correlation between the distribution of AATD, SARS-CoV-2 infection, and COVID-19 mortality rates. Moreover, we described shared pathogenetic pathways that may provide a theoretical basis for our epidemiological findings. We also considered the potential use of AAT augmentation therapy in patients with COVID-19. Full article

Cold-water immersion (CWI) after exercise is a method used by sportsmen to improve recovery. The aim of the study was to assess the effect of a 3 min CWI on the inflammatory state by measuring levels of interleukin 6 (IL-6), interleukin 10 (IL-10), tumor necrosis factor α (TNF-α), and transforming growth factor β1 (TGF-β1), and activities of α1-antitrypsin (AAT) and lysosomal enzymes, including arylsulfatase (ASA), acid phosphatase (AcP), and cathepsin D (CTS D), in the blood of healthy recreational athletes. Male volunteers (n = 22, age 25 ± 4.8 yr) performed a 30 min submaximal aerobic exercise, followed by a 20 min rest at room temperature (RT-REST) or a 20 min rest at room temperature with an initial 3 min 8 °C water bath (CWI-REST). Blood samples were taken at baseline, immediately after exercise, and after 20 min of recovery. The IL-6, IL-10, and TNF-α levels and the AAT activity increased significantly immediately after exercise. The IL-6 level was significantly higher after CWI-REST than after RT-REST. No changes in the activities of the lysosomal enzymes were observed. The effect of a 3 min CWI on the level of inflammatory markers during post-exercise recovery was limited. Thus, it might be considered as a widely available method of regeneration for recreational athletes. Full article

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. Full article

Airway inflammation plays a central role in bronchiectasis. Protease–antiprotease balance is crucial in bronchiectasis pathophysiology and increased presence of unopposed proteases activity may contribute to bronchiectasis onset and progression. Proteases’ over-reactivity and antiprotease deficiency may have a role in increasing inflammation in bronchiectasis airways and may lead to extracellular matrix degradation and tissue damage. Imbalances in serine proteases and matrix-metallo proteinases (MMPs) have been associated to bronchiectasis. Active neutrophil elastase has been associated with disease severity and poor long-term outcomes in this disease. Moreover, high levels of MMPs have been associated with radiological and disease severity. Finally, severe deficiency of α1-antitrypsin (AAT), as PiSZ and PiZZ (proteinase inhibitor SZ and ZZ) phenotype, have been associated with bronchiectasis development. Several treatments are under study to reduce protease activity in lungs. Molecules to inhibit neutrophil elastase activity have been developed in both oral or inhaled form, along with compounds inhibiting dipeptydil-peptidase 1, enzyme responsible for the activation of serine proteases. Finally, supplementation with AAT is in use for patients with severe deficiency. The identification of different targets of therapy within the protease–antiprotease balance contributes to a precision medicine approach in bronchiectasis and eventually interrupts and disrupts the vicious vortex which characterizes the disease. Full article