Current Pharmaceutical Biotechnology - Volume 19, Issue 15, 2018

Chronic inflammation, synovial hyperplasia, and local hypoxia lead to synovial cell activation causing severe joint damage in chronic-inflammatory rheumatoid arthritis (RA). The proinflammatory and joint-destructive property of the increased release of extracellular nucleic acids has been demonstrated for extracellular mitochondrial DNA and oxidized DNA using an arthritis model. Microparticles derived from different cells are able to transport nucleic acids to distant cells and promote cellular activation in RA. In addition, extracellular RNA (eRNA) is present in the RA synovial lining layer, whereas eDNA could be detected in various areas of synovial tissue when compared to controls. The main source of eDNA is the formation of neutrophil extracellular traps (NETs) due to increased amounts of activated neutrophils in the synovial fluid in RA. A central cell type of joint destruction is the activated RA synovial fibroblast (RASF) characterized by increased production of proinflammatory factors, matrix-degrading enzymes, enhanced matrix adhesion, and cell migration. eRNA was shown to be released by RASF under hypoxia and RNase activity was increased in RA synovial fluid. In vitro, RNase-mediated reduction of eRNA decreased RASF adhesion to cartilage but not proliferation or adhesion to endothelial cells. In vivo, RNase1 treatment reduced RASF invasion into cartilage. Therefore, extracellular nucleic acids induced by (auto)immune responses in RA appear to promote inflammation and local joint destruction.

Extracellular RNA (eRNA), composed of mainly rRNA e.g. released upon cell injury, has previously been shown to have three main detrimental functions in the context of cardiovascular disease: (1) to promote tissue edema by activating the VEGF signal transduction cascade, disrupting endothelial tight junctions and increasing intercellular permeability; (2) to induce thrombus formation by activating the contact phase system of intrinsic blood coagulation; and (3) to increase inflammation by stimulating leukocyte adhesion and transmigration and the mobilization of pro-inflammatory cytokines. This review proposes eRNA to be a possible new drug target in cardiovascular disease. The effects of eRNA could potentially be limited by enhancing its degradation through the naturally occurring ribonuclease RNase. In acute settings such as transplantation or ischemia/reperfusion injury after myocardial infarction, this could be achieved by administering RNase intravenously; however, in chronic situations such as atherosclerosis, a new orally administrable chemical compound e.g. blocking the endogenous RNase inhibitor might be developed. In ischemia/reperfusion injury as well as in acute graft rejection, such an intervention would likely reduce edema, thrombosis, inflammation and cellular damage and hence improve survival. In atherosclerosis, antagonizing the RNase inhibitor would presumably reduce inflammation and slow plaque growth. Crucially, toxicological examinations of RNase administration did not find any adverse side effects, denoting it as potentially safe and well-tolerated. Therefore, eRNA appears to be a promising drug target in cardiovascular disease, and further investigations are required for the possible clinical use of an agent limiting its activity.

Neutrophil extracellular traps (NET), extruded decondensated chromatin entangled with neutrophil proteases, have been first identified in neutrophils stimulated with bacteria or phorbol myristate acetate (PMA) via activation of NADPH oxidase and the generation of reactive oxygen species. Although the first findings demonstrated the beneficial role of NET formation by trapping the bacteria and limiting their dissemination, numerous studies in the recent decade revealed the multifunctional aspects of NET formation which manifests itself not only in the context of anti-microbial effect but also as a pathological trigger. Uncontrolled and exaggerated NET formation or inability to digest and remove NET have been reported in thrombosis, auto-immune diseases, cancer or even in infertility. Studies are ongoing to disclose the role of NET in different pathological situations and most importantly, NET regulation via compounds that either interfere with NET formation or target NET components such as DNA or neutrophil proteases. Although the final product of NET formation seems to be quite common i.e. DNA entangled with proteases, stimuli that induce NET have a wide range of varieties and the involved pathways are diverse too. Therefore, in every pathological condition, it is necessary to consider carefully the type of stimulus and the signaling pathways in order to target the disease more specifically. Here we briefly summarize some (out of many) NET triggers/pathways and discuss the potential interventions in the pathological situations.

The innate immune system protects against infectious microbes by the recognition of pathogen- associated molecular patterns, which serve to detect pathogens on the host cell surface or in endosomes by pattern recognition receptors such as Toll-like receptors, nucleotide-binding oligomerization domain-containing protein-1-like receptors, mannose-receptor, or retinoic acid-inducible gene-1- like receptors that initiate proper host defense mechanisms. In addition to pathogen-associated molecular patterns, a series of endogenous danger-associated molecular patterns, such as nucleic acids, are recognized by pattern recognition receptors, which serve as body´s own alarm signals under sterile conditions, such as ischemic injuries, trauma, tumors, tissue transplants, or autoimmune diseases. Thus, exogenous as well as endogenous nucleic acids can function as “alarmins” to alert the body about danger or disease by triggering inflammation, dendritic cell maturation, and stimulate the immune response resulting in the release of cytokines, which in turn can augment the local inflammatory environment. Moreover, danger-associated molecular patterns such as nucleic acids can act as cofactor in the activation of pattern recognition receptors in situations of cellular stress or upon infection leading to a massive amplification of the inflammatory response. As a consequence, acute and also chronic inflammatory diseases such as rheumatoid arthritis, cancer, or atherosclerosis may depend on such perpetuated proinflammatory responses involving activities of nucleic acids. As antagonists, RNase1 or DNase administration or nucleic acid complexing agents may result in a significant blockade of the outcome of particular pathological situations and in considerable tissue protection.

Background: Glucocorticoids are powerful and effective anti-inflammatory and immunosuppressive drugs. They have extensive use in the treatment of different diseases, even though their side effects, such as hypertension, osteoporosis and, in particular, diabetes, are well known. They can exacerbate hyperglycemia in patients with diabetes mellitus or facilitate the development of metabolic disease in apparently healthy subjects, the so-called steroid-induced diabetes mellitus. The last one is an independent risk factor for other complications associated with the use of glucocorticosteroids. Objectives: We tried to clarify the physiopathology and the correct management of glucocorticoidsinduced diabetes, referring in particular to diabetic patients who have to take such drugs for long periods. Methods: A thorough literature search was performed using Pubmed database to cover all relative reports, using combinations of keywords: diabetes mellitus, insulin, glucocorticoids therapy, dipeptidyl peptidase- 4 inhibitors (DPP-4 inhibitor), glucagon-like peptide-1 receptor agonists (GLP-1 receptor agonists), sodium-glucose co-transporter-2 inhibitors (SGLT-2 inhibitor), hyperglycemia, glucose-lowering drugs. Results & Conclusion: Glucocorticoids-induced diabetes is a harmful and underestimated problem when the therapeutic program is not correct and well planned. Screening or more stringent monitoring must always be suggested before starting with corticosteroids; moreover, the hypoglycemic therapy should be personalized, based on the severity of hyperglycemia, type of steroid used and patients' comorbidities. All the oral glucose-lowering drugs can be used, but insulin is the most studied drug and the medication recommended for severe hyperglycemia, for patients with pre-existing diabetes or for those patients who have to undergo prolonged therapy over time.

Background: Plants extracts and their bioactive constituents can provide an alternative approach for new treatment. Pakistani flora reveals a huge, largely untapped source of potential antiviral constituents. Objective: High-cost concerns of direct-acting anti-HCV drugs limit their employment specifically in developing countries like Pakistan. Therefore, discovery of inexpensive and nontoxic agents is needed for HCV treatment. Methodology: In this study, we used plasmid constructs of pCR3.1/FLAGtag/HCV NS3/4A (genotype 1a & 3a) and Punica granatum extracts (PK AV 003) and semi-purified fractions (P1-P11) were evaluated for their anti-HCV activity. Acetone extract along with two fractions (P4 & P11) revealed a useful therapeutic index. Results: The fractions P4 (IC50=28.5±0.02 μg/ml; IC25=16±0.02 μg/ml) and P11 (IC50=72±0.02 μg/ml; IC25=41±0.03 μg/ml) dramatically suppressed HCV replication as measured by quantitative PCR (qPCR) and HCV NS3 protein expression level in transient transfection model. Consistent with suppression in genome replication, inhibition of HCV infectious particles by PK AV 003 extract was also judged in an infectious model system. This data is the first description of Pakistani indigenous cultivated fruit-producing plant, Punica granatum, possessing anti-HCV activity. Further analyses are being performed for investigating the mechanistic studies and structural characterization of purified fractions of PK AV 003. Conclusion: Our findings suggest that PK AV 003 fruit extract might be useful as an add-on therapeutic candidate for treating HCV infection.

Background: The aim of the study was to evaluate the changes in the expression of genes - TNF-α, IFN-γ, depending on the time and concentration of IL-12 used to expose the Normal Human Dermal Fibroblast (NHDF) cells. Methods: The material for the study included NHDF exposed to IL-12 in various IL-12 concentrations (1/10/100 ng/ml) and exposure time 0.5h, 1h, 2h, 4h, 8h, 24h, compared to the control. Changes in gene expression were evaluated with the use of the RTqPCR method. The statistical analysis was performed with the use of Statistica 12.5 PL The role of genes of the JAK-STAT signaling pathway in the induction of the inflammatory process was determined with the use of the PANTHER overrepresentation test. Results: Regardless of the time of NHDF exposure and the IL-12 concentrations used, we observed changes in the expressions of TNF-α and IFN-γ. Increased expression of one transcript involves the decreased expression of the other. We assessed that genes of the JAK-STAT signaling pathway are engaged in 6 biological processes, 8 molecular functions, 6 signaling pathways. Conclusion: The conducted studies indicate that conclusions about the intensity of the inflammatory process, and the efficacy of the anti-cytokine therapeutic strategy, may be made on the basis of the TNF-α, IFN-γ expression.

Background: The genus Lactobacillus has recently been the focal point of researchers due to their ability to produce secondary metabolites with antimicrobial effects. Objective: The aim of this study was to identify vaginal Lactobacillus sp. by analyzing 16S rRNA gene sequence, to investigate into antimicrobial activities of secondary metabolites produced by these isolates and to determine the quantities of lactic acid, acetic acid and hydrogen peroxide in secondary metabolites using High-Performance Liquid Chromatography. Methods: Antimicrobial activities of secondary metabolites were investigated against test microorganisms using Agar Well Diffusion and Agar Spot Methods. Results: According to the results, 18 L. crispatus, 17 L. gasseri, 5 L. jensenii, 4 L. vaginalis, 3 L. fermentum, 2 L. coleohominis, 1 L. saerimneri, 1 L. reuteri, 1 L. johnsonii and 1 L. helveticus were identified. Isolates were frequently found to be effective against Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae RSKK 578 and Bacillus subtilis ATCC 6633. None of the isolates showed activity against Staphylococcus aureus ATCC 43300, S. epidermidis ATCC 12228, S. epidermidis ATCC 35984 and Candida albicans ATCC 10231. Secondary metabolites of all tested isolates contain hydrogen peroxide between 7.306 and 0.33 μg/μL range. Conclusion: It was found that the secondary metabolites of some isolates contained both acetic and lactic acid, while some of them contained either acetic or lactic acid.