Association of increase in Serping1 level with dopaminergic cell reduction in an MPTP-induced Parkinson's disease mouse model.

Parkinson's disease (PD) is a progressive neurodegenerative disease, which shows distinct manifestations such as significant loss of dopaminergic neurons in the substantia nigra (SN). Gene expression was analyzed in the SN of mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), wherein downregulation of dopaminergic neurons occurred to examine the possible causes leading to the loss of dopaminergic neurons. In addition, a serine/cysteine protease inhibitor (Serping1) was studied as one of the genes that were prominently upregulated in mice chronically intoxicated with MPTP. Western blot analysis showed that, concomitant to the downregulation of dopaminergic cells, there was a substantial increase in Serping1 expression within the SN of the MPTP-induced PD mouse model. The SH-SY5Y cells were transfected with Serping1 short interfering RNA (siRNA) to evaluate the correlation between the expression of Serping1 and the loss of dopaminergic cells. Serping1 depletion elicited the upregulation of dopaminergic cells. Moreover, neuroprotective effect against dopaminergic cell loss was demonstrated upon the inhibition of Serping1 expression by siRNA in the MPP+ (1-methyl-4-phenylpyridinium)- treated SH-SY5Y cells. These results show that increased expression of Serping1 may play a critical role in dopaminergic cell death in the SN of chronic MPTP-induced PD mouse model and in SH-SY5Y cells.

Expression and production of the SERPING1-encoded endogenous complement regulator C1-inhibitor in multiple cohorts of tuberculosis patients.

BACKGROUND: To facilitate better discrimination between patients with active tuberculosis (TB) and latent TB infection (LTBI), whole blood transcriptomic studies have been performed to identify novel candidate host biomarkers. SERPING1, which encodes C1-inhibitor (C1-INH), the natural inhibitor of the C1-complex has emerged as candidate biomarker. Here we collated and analysed SERPING1 expression data and subsequently determined C1-INH protein levels in four cohorts of patients with TB. METHODS: SERPING1 expression data were extracted from online deposited datasets. C1-INH protein levels were determined by ELISA in sera from individuals with active TB, LTBI as well as other disease controls in geographically diverse cohorts. FINDINGS: SERPING1 expression was increased in patients with active TB compared to healthy controls (8/11 cohorts), LTBI (13/14 cohorts) and patients with other (non-TB) lung-diseases (7/7 cohorts). Serum levels of C1-INH were significantly increased in The Gambia and Italy in patients with active TB relative to the endemic controls but not in South Africa or Korea. In the largest cohort (n = 50), with samples collected longitudinally, normalization of C1-INH levels following successful TB treatment was observed. This cohort, also showed the most abundant increase in C1-INH, and a positive correlation between C1q and C1-INH levels. Combined presence of increased levels of both C1q and C1-INH had high specificity for active TB (96 %) but only very modest sensitivity 38 % compared to the endemic controls. INTERPRETATION: SERPING1 transcript expression is increased in TB patients, while serum protein levels of C1-INH were increased in half of the cohorts analysed.

Glyco-engineered CHO cell lines producing alpha-1-antitrypsin and C1 esterase inhibitor with fully humanized N-glycosylation profiles.

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.

Therapeutic targeting of classical and lectin pathways of complement protects from ischemia-reperfusion-induced renal damage.

Ischemia-reperfusion injury is the major cause of delayed graft function in transplanted kidneys, an early event significantly affecting long-term graft function and survival. Several studies in rodents suggest that the alternative pathway of the complement system plays a pivotal role in renal ischemia-reperfusion injury. However, limited information is currently available from humans and larger animals. Here we demonstrated that 30 minutes of ischemia resulted in the induction of C4d/C1q, C4d/MLB, and MBL/MASP-2 deposits in a swine model of ischemia-reperfusion injury. The infusion of C1-inhibitor led to a significant reduction in peritubular capillary and glomerular C4d and C5b-9 deposition. Moreover, complement-inhibiting treatment significantly reduced the numbers of infiltrating CD163(+), SWC3a(+), CD4a(+), and CD8a(+) cells. C1-inhibitor administration led to significant inhibition of tubular damage and tubular epithelial cells apoptosis. Interestingly, we report that focal C4d-deposition colocalizes with C1q and MBL at the peritubular and glomerular capillary levels also in patients with delayed graft function. In conclusion, we demonstrated the activation and a pathogenic role of classical and lectin pathways of complement in a swine model of ischemia-reperfusion-induced renal damage. Therefore, inhibition of these two pathways might represent a novel therapeutic approach in the prevention of delayed graft function in kidney transplant recipients.

Potential biomarkers of muscle injury after eccentric exercise.

Proteomics was utilized to identify novel potential plasma biomarkers of exercise-induced muscle injury. Muscle injury was induced in nine human volunteers by eccentric upper extremity exercise. Liquid chromatography-mass spectrometry identified 30 peptides derived from nine proteins which showed significant change in abundance post-exercise. Four of these proteins, haemoglobin alpha chain, haemoglobin beta chain, alpha1-antichymotrypsin (ACT) and plasma C-1 protease inhibitor (C1 Inh), met the criterion for inclusion based on changes in at least two distinct peptides. ACT and C1 Inh peptides peaked earlier post-exercise than creatine kinase, and thus appear to provide new information on muscle response to injury.

Appraisal of danazol prophylaxis for hereditary angioedema.

Hereditary angioedema (HAE) is an autosomal dominant disease characterized by painful, recurrent attacks of inflammation affecting the hands, feet, face, abdomen, urogenital tract, and the larynx. The inflammation can be disfiguring, debilitating, quite painful, and, in the case of laryngeal attacks, life-threatening. Attacks are frequently the source of unnecessary exploratory abdominal procedures, extended hospital stays affecting a patient's ability to retain employment, and severe compromise of the patient's quality of life. HAE is estimated to affect 10,000 people in the US and is caused by deficient or dysfunctional C1-inhibitor, a naturally occurring molecule that is known to inhibit kallikrein, bradykinin, and other serine proteases in plasma. The treatment and management of HAE have been hampered by the dearth of safe and effective therapies. In the United States, there are currently no approved therapies for the treatment of acute HAE attacks. Although prophylactic HAE therapies do exist, they are often viewed as suboptimal due to moderate degrees of efficacy and the existence of adverse effects associated with therapy. Danazol, an attenuated androgen, is the most commonly prescribed prophylaxis treatment for HAE in the United States. Although it has demonstrated moderate efficacy in the prevention of HAE attacks, danazol's side-effect profile can be problematic because there is a correlation between frequency and severity of adverse events and dosage and duration of therapy.