- Hybrid poly-l-lactic acid/poly(ε-caprolactone) nanofibrous scaffold can improve biochemical and molecular markers of human induced pluripotent stem cell-derived hepatocyte-like cells. [Journal Article]
- JCJ Cell Physiol 2018 Dec 04
- A suitable alternative strategy for liver transplantation is the use of nanofibrous scaffolds together with stem cells. In this study, a random hybrid of poly-l-lactic acid (PLLA) and poly(ε-caprolac...
A suitable alternative strategy for liver transplantation is the use of nanofibrous scaffolds together with stem cells. In this study, a random hybrid of poly-l-lactic acid (PLLA) and poly(ε-caprolactone) (PCL) was used as a three-dimensional (3D) culture for differentiation of hepatocyte-like cells and compared with routine culture (two-dimensional [2D]). The expression of the endodermal marker, forkhead box A2 (FOXA2), was assessed on Day 3 and the hepatic markers; albumin (ALB), α-1 antitrypsin (AAT), and cytokeratin-18 (CK-18) were evaluated on Day 18 using quantitative polymerase chain reaction qPCR. As well as, ALB, α-fetoprotein (AFP), and low-density lipoprotein (LDL) uptake were evaluated using immunocytochemistry; moreover, periodic acid-Schiff and Oil Red were done by cell staining. In addition, AFP and urea production were evaluated by chemiluminescence and colorimetric assays. Light and scanning electron microscopy (SEM) showed changes in the cells in 2D and 3D models. The gene expression of hepatic markers was significantly higher in the 3D cultures. In addition, immunocytochemistry and cell staining showed that ALB, AFP, LDL-uptake, periodic acid-Schiff, and Oil Red were expressed in both cells derived on 2D and 3D. Furthermore, the evaluation of AFP and urea secretion was significantly different between 2D and 3D strategies. These findings suggest that functionally cells cultured on a PLLA/PCL scaffold may be suitable for cell therapy and regenerative medicine.
- Glyco-engineered CHO cell lines producing alpha-1-antitrypsin and C1 esterase inhibitor with fully humanized N-glycosylation profiles. [Journal Article]
- MEMetab Eng 2018 Dec 01
- Recombinant Chinese hamster ovary (CHO) cells are able to provide biopharmaceuticals that are essentially free of human viruses and have N-glycosylation profiles similar, but not identical, to humans...
Recombinant Chinese hamster ovary (CHO) cells are able to provide biopharmaceuticals that are essentially free of human viruses and have N-glycosylation profiles similar, but not identical, to humans. Due to differences in N-glycan moieties, two members of the serpin superfamily, alpha-1-antitrypsin (A1AT) and plasma protease C1 inhibitor (C1INH), are currently derived from human plasma for treating A1AT and C1INH deficiency. Deriving therapeutic proteins from human plasma is generally a cost-intensive process and also harbors a risk of transmitting infectious particles. Recombinantly produced A1AT and C1INH (rhA1AT, rhC1INH) decorated with humanized N-glycans are therefore of clinical and commercial interest. Here, we present engineered CHO cell lines producing rhA1AT or rhC1INH with fully humanized N-glycosylation profiles. This was achieved by combining CRISPR/Cas9-mediated disruption of 10 gene targets with overexpression of human ST6GAL1. We were able to show that the N-linked glyco-structures of rhA1AT and rhC1INH are homogeneous and similar to the structures obtained from plasma-derived A1AT and C1INH, marketed as Prolastin®-C and Cinryze®, respectively. rhA1AT and rhC1INH produced in our glyco-engineered cell line showed no detectable differences to their plasma-purified counterparts on SDS-PAGE and had similar enzymatic in vitro activity. The work presented here shows the potential of expanding the glyco-engineering toolbox for CHO cells to produce a wider variety of glycoproteins with fully humanized N-glycan profiles. We envision replacing plasma-derived A1AT and C1INH with recombinant versions and thereby decreasing our dependence on human donor blood, a limited and possibly unsafe protein source for patients.
- Protein-losing enteropathy secondary to nonocclusive mesenteric ischemia: A case report. [Journal Article]
- MMedicine (Baltimore) 2018; 97(48):e13403
- CONCLUSIONS: NOMI has a high mortality rate, often requiring intestinal resection immediately after onset. To our knowledge, there is no report of PLE developing after conservative treatment, as in our case. Further study of cases is necessary to determine the reversibility of the condition, which will influence the therapeutic plan. We herein present an extremely rare case of PLE after conservative treatment for NOMI. The possibility of PLE also needs to be considered when hypoalbuminemia occurs after conservative treatment of NOMI.
- HSP27, ALDH6A1 and Prohibitin Act as a Trio-biomarker to Predict Survival in Late Metastatic Prostate Cancer. [Journal Article]
- ARAnticancer Res 2018; 38(11):6551-6560
- CONCLUSIONS: Trio-biomarker composed of HSP27, ALDH6A1 and prohibitin may predict survival of metastatic prostate cancer patients.
- In-depth quantitative proteome analysis of seminal plasma from men with oligoasthenozoospermia and normozoospermia. [Journal Article]
- RBReprod Biomed Online 2018; 37(4):467-479
- CONCLUSIONS: Our seminal plasma proteome research provides new complementary high-confidence data, and also enhances understanding of the pathogenic mechanisms in oligoasthenozoospermia.
- Oxidation-resistant and thermostable forms of alpha-1 antitrypsin from Escherichia coli inclusion bodies. [Journal Article]
- FOFEBS Open Bio 2018; 8(10):1711-1721
- Native α1-antitrypsin (AAT) is a 52-kDa glycoprotein that acts as an antiprotease and is the physiological inhibitor of neutrophil serine proteases. The main function of AAT is to protect the lung fr...
Native α1-antitrypsin (AAT) is a 52-kDa glycoprotein that acts as an antiprotease and is the physiological inhibitor of neutrophil serine proteases. The main function of AAT is to protect the lung from proteolytic damage induced by inflammation. AAT deficiency (AATD) is a codominant autosomal disorder caused by pathogenic mutations in SERPINA1 gene, leading to reduced levels of serum AAT. The deficiency is known to increase the risk of pulmonary emphysema and chronic obstructive pulmonary disease as a consequence of proteolytic imbalance induced by inflammation, associated in many instances with cigarette smoking and other environmental hazards. Currently, the available therapy for lung disease associated with AATD is serum purified human AAT injected into patients on a weekly basis. It would be advantageous to replace serum-derived AAT with a recombinant version which is stable and resistant to oxidation. We have expressed AAT in Escherichia coli as inclusion bodies and developed a highly efficient refolding and purification process. We engineered a series of mutant forms of AAT to achieve enhance thermostability and oxidation resistance. Moreover, we synthesized an active form of AAT via cysteine-pegylation to achieve a markedly extended half-life in vivo. The resulting molecule, which retains comparable activity to the wild-type form, is expected to be an improved therapeutic agent for treating hereditary emphysema. In addition, the molecule may also be used to treat other types of emphysema caused by smoking, cystic fibrosis, pulmonary hypertension, pulmonary fibrosis, and chronic obstructive pulmonary disease.
- Expression, Purification, and Characterization of Recombinant Human α1-Antitrypsin Produced Using Silkworm-Baculovirus Expression System. [Journal Article]
- MBMol Biotechnol 2018; 60(12):924-934
- Human α1-antitrypsin (AAT) is the most abundant serine proteinase inhibitor (serpin) in the human plasma. Commercially available AAT for the medications of deficiency of α1-antitrypsin is mainly puri...
Human α1-antitrypsin (AAT) is the most abundant serine proteinase inhibitor (serpin) in the human plasma. Commercially available AAT for the medications of deficiency of α1-antitrypsin is mainly purified from human plasma. There is a high demand for a stable and low-cost supply of recombinant AAT (rAAT). In this study, the baculovirus expression vector system using silkworm larvae as host was employed and a large amount of highly active AAT was recovered from the silkworm serum (~ 15 mg/10 ml) with high purity. Both the enzymatic activity and stability of purified rAAT were comparable with those of commercial product. Our results provide an alternative method for mass production of the active rAAT in pharmaceutical use.
- Therapeutic potential of alpha-1 antitrypsin in human disease. [Journal Article]
- APAnn Pediatr Endocrinol Metab 2018; 23(3):131-135
- Alpha-1 antitrypsin (AAT), an alpha globulin glycoprotein, is a member of the serine protease inhibitor (serpin) superfamily. The clinical significance of AAT is highlighted by AAT deficiency. Geneti...
Alpha-1 antitrypsin (AAT), an alpha globulin glycoprotein, is a member of the serine protease inhibitor (serpin) superfamily. The clinical significance of AAT is highlighted by AAT deficiency. Genetic deficiency of AAT can present as several neutrophilic diseases associated with emphysema, liver cirrhosis, panniculitis, and systemic vasculitis. Recently, animal and human studies have shown that AAT can control inflammatory, immunological, and tissue-protective responses. In addition, AAT treatment can prevent overt hyperglycemia, increase insulin secretion, and reduce cytokine-mediated apoptosis of pancreatic β-cells in diabetes. These multifunctional roles of AAT draw attention to the glycoprotein's therapeutic potential for many inflammatory and autoimmune diseases beyond AAT deficiency. As underlying mechanisms, recent studies have suggested the importance of serine protease inhibitory activity of AAT in obesity-associated insulin resistance, chronic obstructive pulmonary disease, and cystic fibrosis. In this review, we explore the multiple functions of AAT, in particular, the anti-inflammatory and serine protease inhibitory functions, and AAT's therapeutic potential in a variety of human diseases through published literature.
- α1-antitrypsin mitigates NLRP3-inflammasome activation in amyloid β1-42-stimulated murine astrocytes. [Journal Article]
- JNJ Neuroinflammation 2018 Sep 27; 15(1):282
- CONCLUSIONS: We conclude that Aβ1-42-stimulation results in an upregulation of NLRP3, caspase 1, and its cleavage products in astrocytes. A1AT time-dependently hampers neuroinflammation by downregulation of Aβ1-42-mediated NLRP3-inflammasome expression and thus may serve as a pharmaceutical opportunity for the treatment of Alzheimer's disease.
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- SERPINA1 methylation and lung function in tobacco-smoke exposed European children and adults: a meta-analysis of ALEC population-based cohorts. [Journal Article]
- RRRespir Res 2018 Aug 22; 19(1):156
- CONCLUSIONS: The results do not support a role of SERPINA1 gene methylation as determinant of lung function across the life course in the tobacco smoke exposed general population exposed.