Production of recombinant streptokinase from Streptococcus pyogenes isolate and its potential for thrombolytic therapy

Objectives:

To produce an effective recombinant streptokinase (rSK) from pathogenic Streptococcus pyogenes isolate in yeast, and evaluate its potential for thrombolytic therapy.

Methods:

This study was conducted from November 2012 to December 2013 at King Khalid University, Abha, Kingdom of Saudi Arabia (KSA). Throat swabs collected from 45 pharyngitis patients in Asser Central Hospital, Abha, KSA were used to isolate Streptococcus pyogenes. The bacterial DNA was used for amplification of the streptokinase gene (1200 bp). The gene was cloned and in vitro transcribed in an eukaryotic expression vector that was transformed into yeast Pichia pastoris SMD1168, and the rSK protein was purified and tested for its thrombolytic activity.

Results:

The Streptococcus pyogenes strain was isolated and its DNA nucleotide sequence revealed similarity to other Streptococcus pyogenes in the Gene bank. Sequencing of the amplified gene based on DNA nucleotide sequence revealed a SK gene closely related to other SK genes in the Gene bank. However, based on deduced amino acids sequence, the gene formed a separate cluster different from clusters formed by other examined genes, suggesting a new bacterial isolate and accordingly a new gene. The purified protein showed 82% clot lysis compared to a commercial SK (81%) at an enzyme concentration of 2000 U/ml.

Conclusion:

The present yeast rSK showed similar thrombolytic activity in vitro as that of a commercial SK, suggesting its potential for thrombolytic therapy and large scale production.There has been increasing interest in the use of thrombolytic therapy with fibrinolytic (thrombolytic) agents for the treatment of various circulating disorders, for example, pulmonary embolism, deep venous thrombosis, and myocardial infarction.1,2 These disorders are increasingly becoming the leading causes of mortality wide world.3 Among the commonly used fibrinolytic agents in thrombolytic therapy are urokinase (UK), tissue type plasminogen activators (TPA), and streptokinase (SK). These agents are collectively named as plasminogen activators, as they convert the enzymatically inactive plasminogen (PG) to an active protease, plasmin, which dissolves the fibrin clots and solubilizes the degradation products that can be removed by phagocytosis, and thus help the restoration of blood flow through the occluded vessels.3 Unlike UK and TPA, SK has no enzymatic activity of its own, but it has an indirect PG activity property, by first forming a high affinity 1:1 stoichiometric complex with PG or plasmin. The resultant activator complex is a highly specific protease that changes PG molecules to proteolytically active plasmin.4 Streptokinase was the first drug introduced as a therapy for acute myocardial infarction more than 40 years ago.5 It is now one of the leading fibrinolytic agents in the treatment of thrombotic conditions,6 and is included in the WHO Model List of Essential Medicines.7 Currently, despite the wide use of TPA in developed nations, SK remains essential to the management of MI and other thrombotic events in developing countries.8 Streptokinase (E.C.3.4.99.22) is an extracellular single chain, non-enzymatic, monomeric protein, consisting of 440 amino acids, including a 26-amino acid N-terminal single peptide, which is cleaved during secretion to give the mature 414 amino acids production residues of 47 KDs molecular weight.9,10 Streptokinase has been produced by many strains of β-hemolytic streptococci isolated from the upper respiratory tract. Streptokinase produced by different groups of streptococci differs greatly in structure.11 However, due to its bacterial origin, the natural (native) SK might have a potential antigenic capacity.12 This can be avoided by the use of rSK that has been produced by recombinant DNA technology,13-15 and found to have similar thrombolytic activity as that of the native SK.16 Indeed, rSK has been extensively used in clinical trials in myocardial infarction and proved to be an effective and safe thrombolytic agent.17 The aim of the present study is the production of a purified recombinant streptokinase from pathogenic Streptococcus pyogenes isolate by its expression in yeast, using recombinant DNA technology, and evaluation of its thrombolytic activity in vitro.     

Targeted complement inhibition and microvasculature in transplants: a therapeutic perspective

Active complement mediators play a key role in graft‐versus‐host diseases, but little attention has been given to the angiogenic balance and complement modulation during allograft acceptance. The complement cascade releases the powerful proinflammatory mediators C3a and C5a anaphylatoxins, C3b, C5b opsonins and terminal membrane attack complex into tissues, which are deleterious if unchecked. Blocking complement mediators has been considered to be a promising approach in the modern drug discovery plan, and a significant number of therapeutic alternatives have been developed to dampen complement activation and protect host cells. Numerous immune cells, especially macrophages, develop both anaphylatoxin and opsonin receptors on their cell surface and their binding affects the macrophage phenotype and their angiogenic properties. This review discusses the mechanism that complement contributes to angiogenic injury, and the development of future therapeutic targets by antagonizing activated complement mediators to preserve microvasculature in rejecting the transplanted organ.     

Outcome of posterior chamber phakic intraocular lens procedure to correct myopia

Purpose

To assess the safety and efficacy of the implantable contact lens (ICL™) to treat myopia.

Design

Clinical, retrospective, single center, non-randomized case series.

Participants

Sixty-nine eyes of 46 patients with myopia ranging from −3.00 to 25.00 D were included in this study.

Intervention

Implantation of the ICL™.

Main outcome measures

Uncorrected Visual Acuity (UCVA), refraction, best spectacle corrected visual acuity (BSCVA), adverse events, operative and postoperative complications, subjective assessment and symptoms.

Results

The mean follow-up was 12.35 ± 6.13 (SD) months (range, 6 months–32 months). At the last visit, 49.20% of eyes had 20/20 or better UCVA compared to preoperative 20/20 or better BSCVA of 31.9% of eyes; 69.23% of eyes had postoperative UCVA better than or equal to preoperative BSCVA. The mean manifest refractive cylinder was 1.93 ± 1.21 D at baseline and 1.00 ± 0.92 D postoperatively. The mean manifest refraction spherical equivalent (MRSE) was −11.70 ± 4.24 D preoperatively and −0.69 ± 1.13 D postoperatively. A total of 69.8% of eyes were within ±0.5 D of the predicted MRSE; 84.1% were within ±1.0 D, and 88.90% were within ±2.0 D. BSCVA of 20/20 or better was achieved in 64.6% of eyes postoperatively, compared to 31.9% preoperatively. Mean improvement in BSCVA was 1line. One eye (1.5%) lost ⩾2 lines of BSCVA at the last visit, whereas 20% of eyes improved by ⩾2 lines. A total of 56.92% of cases gained ⩾1 line of BSCVA and 4.62% of cases lost ⩾1 line. Four ICL lenses were removed without significant loss of BSCVA, and 2 eyes with clinically significant lens opacities were observed. Four eyes (5.8%) developed a pupillary block the first day postoperatively. One eye (1.4%) developed a hypotony and AC shallowing.

Conclusion

Implantation of ICL for the correction of myopia was a safe procedure with good visual and refractive results from the early postoperative period to 1 year. Long-term follow-up is required to confirm the long-term safety of this implant.     

Immunochemical Studies on Catechol-Estrogen Modified Plasmid: Possible Role in Rheumatoid Arthritis

Introduction  

Increased concentrations of estrogen metabolites (catecholestrogens) have been found in rheumatoid arthritis (RA) but the exact patho-etiology remains elusive.     

Corticosteroid Administration and Outcome of Adolescents and Adults With Acute Bacterial Meningitis: A Meta-analysis

OBJECTIVE: To systematically assess the effect of the adjunctive administration of corticosteroids in the treatment of acute bacterial meningitis.METHODS: We performed a systematic review and meta-analysis by searching several databases for reports (published from January 1966 through February 2008) of placebo-controlled randomized trials of corticosteroid use in the treatment of adolescents and adults with acute bacterial meningitis. We used random-effects models. Sources of heterogeneity were explored by preplanned subgroup analyses.RESULTS: The 4 eligible trials (published between 1999 and 2007) were of high methodological quality and included 1261 adult patients. Overall, the short-term mortality rate associated with corticosteroid administration was not significantly lower than that associated with placebo (relative risk [RR], 0.81; 95% confidence interval [CI], 0.54-1.20; I²=54%). A significant interaction was found between the effect of corticosteroids and the income status of the country (P=.02) and the prevalence of infection with human immunodeficiency virus (HIV) among study populations (P=.03). The administration of corticosteroids resulted in a lower short-term mortality rate than did the administration of placebo in high-income countries (pooled RR, 0.5; 95% CI, 0.27-0.92; I²=0%) and in the studies with a low prevalence of infection with HIV (RR, 0.66; 95% CI, 0.44-0.99; I²=0%). In studies from high-income countries, the number needed to treat with corticosteriods to prevent 1 death and 1 neurologic sequela was 12.5 (95% CI, 7.1-100.0) and 11.0 (95% CI, 5.6-100.0), respectively.CONCLUSION: Our meta-analysis suggests that the adjunctive administration of corticosteroids is beneficial in the treatment of adolescents and adults with bacterial meningitis in patient populations similar to those seen in high-income countries and in areas with a low prevalence of HIV infection.CI = confidence interval; CSF = cerebrospinal fluid; HIV = human immunodeficiency virus; NNT = number needed to treat; RR = relative riskAcute bacterial meningitis can lead to morbidity and mortality, even when highly effective antibiotic therapy is administered.¹ Because neurologic damage is at least partially caused by the host immune response,² anti-inflammatory drug therapy should, in theory, improve outcome. Indeed, cumulative evidence, including the results of a Cochrane review³ and a systematic review and meta-analysis⁴ of adjunctive corticosteroid therapy for adults with acute bacterial meningitis, indicates that early treatment with dexamethasone improves outcome. In these studies, the beneficial effect was most apparent for patients with meningitis caused by Streptococcus pneumoniae, the leading causative pathogen of adult meningitis. Accordingly, practice guidelines currently recommend the adjunctive administration of dexamethasone to all adult patients with pneumococcal meningitis.⁵ However, 2 subsequent large randomized trials^6,7 that included 900 patients from developing countries did not show similar benefits in terms of mortality and neurologic sequelae. Because the previous meta-analysis⁴ included quasi-randomized and open-label trials,^8,9 concerns exist about the validity of their pooled estimates. The findings of meta-epidemiological studies^10,11 suggest that the introduction of cointerventions during the conduct of such trials, even for well-defined end points such as mortality, cannot always be excluded and thus could weaken the inferences based on the pooled results of a meta-analysis.To further examine the ongoing controversy concerning corticosteroid therapy for bacterial meningitis, we performed a meta-analysis of published reports of randomized, placebo-controlled trials of adjunctive corticosteroid therapy for adolescents and adults with bacterial meningitis.     

Whole liver engineering: A promising approach to develop functional liver surrogates

Liver donor shortage remains the biggest challenge for patients with end‐stage liver failures. While bioartificial liver devices have been developed as temporary supports for patients waiting for transplantation, their applications have been limited clinically. Whole liver engineering is a biological scaffold based regenerative medicine approach that holds promise for developing functional liver surrogates. Significant advancements have been made since the first report in 2010. This review focuses on the recent achievements of whole liver engineering studies.