Alleviation of radiation combined skin injury in rat model by topical application of ascorbate formulation.

PURPOSE: This research endeavor was undertaken to elucidate the impact of an innovative ascorbate formulation on the regeneration process of full-thickness excision wounds in a rat model exposed to whole-body gamma irradiation, replicating conditions akin to combat or radiation emergency scenarios. MATERIALS AND METHODS: We established a comprehensive rat model by optimizing whole body γ-radiation doses (5-9 Gy) and full-thickness excision wound sizes (1-3 cm2) to mimic radiation combined injury (RCI). The developed RCI model was used to explore the healing potential of ascorbate formulation. The study includes various treatment groups (i.e., sham control, radiation alone, wound alone, radiation + wound, and radiation + wound + formulation). The ascorbate formulation was applied twice daily, with a 12-hour gap between each application, starting 1 hour after the initiation of the wound. The healing potential of the formulation in the RCI context was evaluated over 14 days through hematological, molecular, and histological parameters. RESULTS: The combination of a 5 Gy radiation dose and a 1 cm2 wound was identified as the optimal setting to develop the RCI model for subsequent studies. The formulation was used topically immediately following RCI, and then twice daily until complete healing. Treatment with the ascorbate formulation yielded noteworthy outcomes and led to a substantial reduction (p < .05) in the wound area, accelerated epithelialization periods, and an increased wound contraction rate. The formulation's localized healing response improved organ weights, normalized blood parameters, and enhanced hematopoietic and immune systems. A gene expression study revealed the treatment up-regulated TGF-ß and FGF, and down-regulated PDGF-α, TNF-α, IL-1ß, IL-6, MIP-1α, and MCP-1 (p < .05). Histopathological assessments supported the formulation's effectiveness in restoring cellular architecture and promoting tissue regeneration. CONCLUSION: Topical application of the ascorbate formulation in RCI resulted in a significant improvement in delayed wound healing, leading to accelerated wound closure by mitigating the expression of inflammatory responses.

Exploration of Antileishmanial Compounds Derived from Natural Sources.

AIMS: Leishmaniasis is a deadly tropical disease that is neglected in many countries. World Health Organization, along with a few other countries, has been working together to protect against these parasites. Many novel drugs from the past few years have been discovered and subjected against leishmaniasis, which have been effective but they are quite expensive for lower-class people. Some drugs showed no effect on the patients, and the longer use of these medicines has made resistance against these deadly parasites. Researchers have been working for better medication by using natural products from medicinal plants (oils, secondary metabolites, plant extracts) and other alternatives to find active compounds as an alternative to the current synthetic drugs. MATERIALS AND METHODS: To find more potential natural products to treat Leishmania spp, a study has been conducted and reported many plant metabolites and other natural alternatives from plants and their extracts. Selected research papers with few term words such as natural products, plant metabolites, Leishmaniasis, in vivo, in vitro, and treatment against leishmaniasis; in the Google Scholar, PubMed, and Science Direct databases with selected research papers published between 2015 and 2021 have been chosen for further analysis has been included in this report which has examined either in vivo or in vitro analysis. RESULTS: This paper reported more than 20 novel natural compounds in 20 research papers that have been identified which report a leishmanicidal activity and shows an action against promastigote, axenic, and intracellular amastigote forms. CONCLUSION: Medicinal plants, along with a few plant parts and extracts, have been reported as a possible novel anti-leishmanial medication. These medicinal plants are considered nontoxic to Host cells. Leishmaniasis treatments will draw on the isolated compounds as a source further and these compounds compete with those already offered in clinics.

Therapeutic applications of nanobiotechnology.

Nanobiotechnology, as a novel and more specialized branch of science, has provided a number of nanostructures such as nanoparticles, by utilizing the methods, techniques, and protocols of other branches of science. Due to the unique features and physiobiological characteristics, these nanostructures or nanocarriers have provided vast methods and therapeutic techniques, against microbial infections and cancers and for tissue regeneration, tissue engineering, and immunotherapies, and for gene therapies, through drug delivery systems. However, reduced carrying capacity, abrupt and non-targeted delivery, and solubility of therapeutic agents, can affect the therapeutic applications of these biotechnological products. In this article, we explored and discussed the prominent nanobiotechnological methods and products such as nanocarriers, highlighted the features and challenges associated with these products, and attempted to conclude if available nanostructures offer any scope of improvement or enhancement. We aimed to identify and emphasize the nanobiotechnological methods and products, with greater prospect and capacity for therapeutic improvements and enhancements. We found that novel nanocarriers and nanostructures, such as nanocomposites, micelles, hydrogels, microneedles, and artificial cells, can address the associated challenges and inherited drawbacks, with help of conjugations, sustained and stimuli-responsive release, ligand binding, and targeted delivery. We recommend that nanobiotechnology, despite having few challenges and drawbacks, offers immense opportunities that can be harnessed in delivering quality therapeutics with precision and prediction. We also recommend that, by exploring the branched domains more rigorously, bottlenecks and obstacles can also be addressed and resolved in return.

Silver Nanoparticles Phytofabricated through Azadirachta indica: Anticancer, Apoptotic, and Wound-Healing Properties.

Silver nanoparticles (AgNPs) have unlocked numerous novel disciplines in nanobiotechnological protocols due to their larger surface area-to-volume ratios, which are attributed to the marked reactivity of nanosilver, and due to their extremely small size, which enables AgNPs to enter cells, interact with organelles, and yield distinct biological effects. AgNPs are capable of bypassing immune cells, staying in the system for longer periods and with a higher distribution, reaching target tissues at higher concentrations, avoiding diffusion to adjacent tissues, releasing therapeutic agents or drugs for specific stimuli to achieve a longer duration at a specific rate, and yielding desired effects. The phytofabrication of AgNPs is a cost-effective, one-step, environmentally friendly, and easy method that harnesses sustainable resources and naturally available components of plant extracts (PEs). In addition, it processes various catalytic activities for the degradation of various organic pollutants. For the phytofabrication of AgNPs, plant products can be used in a multifunctional manner as a reducing agent, a stabilizing agent, and a functionalizing agent. In addition, they can be used to curtail the requirements for any additional stabilizing agents and to help the reaction stages subside. Azadirachta indica, a very common and prominent medicinal plant grown throughout the Indian subcontinent, possesses free radical scavenging and other pharmaceutical properties via the regulation of proinflammatory enzymes, such as COX and TOX. It also demonstrates anticancer activities through cell-signaling pathways, modulating tumor-suppressing genes such as p53 and pTEN, transcriptional factors, angiogenesis, and apoptosis via bcl2 and bax. In addition, it possesses antibacterial activities. Phytofabricated AgNPs have been applied in the areas of drug delivery, bioimaging, biosensing, cancer treatment, cosmetics, and cell biology. Such pharmaceutical and biological activities of phytofabricated AgNPs are attributed to more than 300 phytochemicals found in Azadirachta indica, and are especially abundant in flavonoids, polyphenols, diterpenoids, triterpenoids, limonoids, tannins, coumarin, nimbolide, azadirachtin, azadirone, azadiradione, and gedunin. Parts of Azadirachta indica, including the leaves in various forms, have been used for wound healing or as a repellent. This study was aimed at examining previously biosynthesized (from Azadirachta indica) AgNPs for anticancer, wound-healing, and antimicrobial actions (through MTT reduction assay, scratch assay, and microbroth dilution methods, respectively). Additionally, apoptosis in cancer cells and the antibiofilm capabilities of AgNPs were examined through caspase-3 expression, dentine block, and crystal violet methods. We found that biogenic silver nanoparticles are capable of inducing cytotoxicity in HCT-116 colon carcinoma cells (IC50 of 744.23 µg/mL, R2: 0.94), but are ineffective against MCF-7 breast cancer cells (IC50 >> 1000 µg/mL, R2: 0.86). AgNPs (IC50 value) induced a significant increase in caspase-3 expression (a 1.5-fold increase) in HCT-116, as compared with control cells. FITC-MFI was 1936 in HCT-116-treated cells, as compared to being 4551 in cisplatin and 1297 in untreated cells. AgNPs (6.26 µg/mL and 62.5 µg/mL) induced the cellular migration (40.2% and 33.23%, respectively) of V79 Chinese hamster lung fibroblasts; however, the improvement in wound healing was not significant as it was for the controls. AgNPs (MIC of 10 µg/mL) were very effective against MDR Enterococcus faecalis in the planktonic mode as well as in the biofilm mode. AgNPs (10 µg/mL and 320 µg/mL) reduced the E. faecalis biofilm by >50% and >80%, respectively. Natural products, such as Syzygium aromaticum (clove) oil (MIC of 312.5 µg/mL) and eugenol (MIC of 625 µg/mL), showed significant antimicrobial effects against A. indica. Our findings indicate that A. indica-functionalized AgNPs are effective against cancer cells and can induce apoptosis in HCT-116 colon carcinoma cells; however, the anticancer properties of AgNPs can also be upgraded through active targeting (functionalized with enzymes, antibiotics, photosensitizers, or antibodies) in immunotherapy, photothermal therapy, and photodynamic therapy. Our findings also suggest that functionalized AgNPs could be pivotal in the development of a novel, non-cytotoxic, biocompatible therapeutic agent for infected chronic wounds, ulcers, and skin lesions involving MDR pathogens via their incorporation into scaffolds, composites, patches, microgels, or formulations for microneedles, dressings, bandages, gels, or other drug-delivery systems.

Chitosan: Applications in Drug Delivery System.

Chitin and chitosan have unique structures with significant functional groups carrying useful chemical capabilities. Chitin and chitosan are acknowledged as novel biomaterials with advantageous biocompatibility and biodegradability. Chitosan is a polysaccharide that is made from chitin. There have been several attempts to employ this biopolymer in the biomedical area. This material's application in the production of artificial skin, drug targeting, and other areas is explored. The most prevalent strategies for recovering chitin from sea organisms are described and various pharmacological and biological uses are discussed. This review article targets drug delivery with the help of chitosan derived nanomaterial. The drug delivery system applications through nonmaterial have encountered a considerable role in the pharmaceutical, medical, biological, and other sectors in recent years. Nanomaterials have advanced applications as novel drug delivery systems in many fields, especially in industry, biology, and medicine. In the biomedical and pharmaceutical arena, the natural polymer-based nanoparticulate method has now been widely studied as particulate vehicles. By mixing alginate with other biopolymers, by immobilizing specific molecules such as sugar molecules and peptides by chemical or physical cross-linking, different properties and structures such as biodegradability, gelling properties, mechanical strength, and cell affinity can be obtained. Owing to their inherent ability to deliver both hydrophilic and hydrophobic drug molecules, increase stability, decrease toxicity, and enhance commonly formulated medications, these particles are now widely used in imaging and molecular diagnostics, cosmetics, household chemicals, sunscreens, radiation safety, and novel drug delivery.

The Association between Biofilm Formation and Antimicrobial Resistance with Possible Ingenious Bio-Remedial Approaches.

Biofilm has garnered a lot of interest due to concerns in various sectors such as public health, medicine, and the pharmaceutical industry. Biofilm-producing bacteria show a remarkable drug resistance capability, leading to an increase in morbidity and mortality. This results in enormous economic pressure on the healthcare sector. The development of biofilms is a complex phenomenon governed by multiple factors. Several attempts have been made to unravel the events of biofilm formation; and, such efforts have provided insights into the mechanisms to target for the therapy. Owing to the fact that the biofilm-state makes the bacterial pathogens significantly resistant to antibiotics, targeting pathogens within biofilm is indeed a lucrative prospect. The available drugs can be repurposed to eradicate the pathogen, and as a result, ease the antimicrobial treatment burden. Biofilm formers and their infections have also been found in plants, livestock, and humans. The advent of novel strategies such as bioinformatics tools in treating, as well as preventing, biofilm formation has gained a great deal of attention. Development of newfangled anti-biofilm agents, such as silver nanoparticles, may be accomplished through omics approaches such as transcriptomics, metabolomics, and proteomics. Nanoparticles' anti-biofilm properties could help to reduce antimicrobial resistance (AMR). This approach may also be integrated for a better understanding of biofilm biology, guided by mechanistic understanding, virtual screening, and machine learning in silico techniques for discovering small molecules in order to inhibit key biofilm regulators. This stimulated research is a rapidly growing field for applicable control measures to prevent biofilm formation. Therefore, the current article discusses the current understanding of biofilm formation, antibiotic resistance mechanisms in bacterial biofilm, and the novel therapeutic strategies to combat biofilm-mediated infections.

Determination of Antioxidants by DPPH Radical Scavenging Activity and Quantitative Phytochemical Analysis of Ficus religiosa.

The use of F. religiosa might be beneficial in inflammatory illnesses and can be used for a variety of health conditions. In this article, we studied the identification of antioxidants using (DPPH) 2,2-Diphenyl-1-picrylhydrazylradical scavenging activity in Ficus religiosa, as F. religiosa is an important herbal plant, and every part of it has various medicinal properties such as antibacterial properties that can be used by the researchers in the development and design of various new drugs. The 2,2-Diphenyl-1-picrylhydrazyl (DPPH) is a popular, quick, easy, and affordable approach for the measurement of antioxidant properties that includes the use of the free radicals used for assessing the potential of substances to serve as hydrogen providers or free-radical scavengers (FRS). The technique of DPPH testing is associated with the elimination of DPPH, which would be a stabilized free radical. The free-radical DPPH interacts with an odd electron to yield a strong absorbance at 517 nm, i.e., a purple hue. An FRS antioxidant, for example, reacts to DPPH to form DPPHH, which has a lower absorbance than DPPH because of the lower amount of hydrogen. It is radical in comparison to the DPPH-H form, because it causes decolorization, or a yellow hue, as the number of electrons absorbed increases. Decolorization affects the lowering capacity significantly. As soon as the DPPH solutions are combined with the hydrogen atom source, the lower state of diphenylpicrylhydrazine is formed, shedding its violet color. To explain the processes behind the DPPH tests, as well as their applicability to Ficus religiosa (F. religiosa) in the manufacture of metal oxide nanoparticles, in particular MgO, and their influence on antioxidants, a specimen from the test was chosen for further study. According to our findings, F. religiosa has antioxidant qualities and may be useful in the treatment of disorders caused by free radicals.

Potential of nanoparticles encapsulated drugs for possible inhibition of the antimicrobial resistance development.

The immune system is a dynamic network of cells and cytokines are the major mediators of immune responses which combat pathogens. Based on the cytokine production, effector T cells differentiate into subsets known as Th1, Th2, Th17, or Treg. This system serves as a barrier to intracellular pathogens, bacterial infections and stimulates the production of reactive oxygen species (ROS), reactive nitrogen intermediates, and nitric oxide, which diffuses across membranes and engulfs intracellular pathogens. Oxidative stress occurs when ROS, reactive nitrogen species (RNS) production, and antioxidant defences become imbalanced. Oxidative stress generated by infected cells produces a substantial amount of free radicals which enables the killing of intracellular pathogens. Intracellular pathogens are exposed to endogenous ROS as part of normal aerobic respiration, also exogenous ROS and RNS are generated by the host immune system in response to infection. Nanoparticles which are designed for drug delivery are capable of trapping the desired drug in the particles which protect the drug from enzymatic degradation in a biological system. The subcellular size of nanoparticles enables higher intracellular uptake of the drug which results in the reduction of the concentration of free drugs reducing their toxic effect. Research on the modulation of immune response and oxidative stress using nanoparticles used to encapsulate drugs has yet to be explored fully. In this review, we illustrate the immune activation and generation of oxidative stress properties which are mediated by nanoparticle encapsulated drug delivery systems which can make the therapy more effective in case of diseases caused by intracellular pathogens.

Seeding drug discovery: Telomeric tankyrase as a pharmacological target for the pathophysiology of high-altitude hypoxia.

Cellular exposure to extreme environments leads to the expression of multiple proteins that participate in pathophysiological manifestations. Hypobaric hypoxia at high altitude (HA) generates reactive oxygen species (ROS) that can damage telomeres. Tankyrase (TNKS) belongs to multiple telomeric protein complexes and is actively involved in DNA damage repair. Although published research on TNKS indicates its possible role in cancer and other hypoxic diseases, its role in HA sicknesses remains elusive. Understanding the roles of telomeres, telomerase, and TNKS could ameliorate physiological issues experienced at HA. In addition, telomeric TNKS could be a potential biomarker in hypoxia-induced sicknesses or acclimatization. Thus, a new research avenue on TNKS linked to HA sickness might lead to the discovery of drugs for hypobaric hypoxia.

Shorter telomere length, higher telomerase activity in association with tankyrase gene polymorphism contribute to high-altitude pulmonary edema.

High-altitude pulmonary edema (HAPE) is a noncardiogenic form of pulmonary edema, which is induced upon exposure to hypobaric hypoxia at high altitude (HA). Hypobaric hypoxia generates reactive oxygen species that may damage telomeres and disturb normal physiological processes. Telomere complex comprises of multiple proteins, of which, tankyrase (TNKS) is actively involved in DNA damage repairs. We hence investigated the association of TNKS and telomeres with HAPE to delineate their potential role at HA. The study was performed in three groups, High-altitude pulmonary edema patients (HAPE-p, n = 200), HAPE-resistant sojourners (HAPE-r, n = 200) and highland permanent healthy residents (HLs, n = 200). Variants of TNKS were genotyped using polymerase chain reaction-restriction fragment length polymorphism. Plasma TNKS level was estimated using enzyme-linked immunosorbent assay, expression of TNKS and relative telomere length were assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and telomerase activity was assessed by the telomere repeat amplification protocol assay. TNKS poly-ADP ribosylates the telomere-repeat factor (TRF), which is a negative regulator of telomere length. Consequently, TRF expression was also measured by RT-qPCR. The TNKS heterozygotes rs7015700GA were prevalent in HLs compared to the HAPE-p and HAPE-r. The plasma TNKS was significantly decreased in HAPE-p than HAPE-r (P = 0.006). TNKS was upregulated 9.27 folds in HAPE-p (P = 1.01E-06) and downregulated in HLs by 3.3 folds (P = 0.02). The telomere length was shorter in HAPE-p compared to HAPE-r (P = 0.03) and HLs (P = 4.25E-4). The telomerase activity was significantly higher in HAPE-p compared to both HAPE-r (P = 0.01) and HLs (P = 0.001). HAPE-p had the lowest TNKS levels (0.186 ± 0.031 ng/µl) and the highest telomerase activity (0.0268 amoles/µl). The findings of the study indicate the association of TNKS and telomeres with HA adaptation/maladaptation.

Use Chou's 5-steps rule to evaluate protective efficacy induced by antigenic proteins of Mycobacterium tuberculosis encapsulated in chitosan nanoparticles.

The study focuses on whether antigenic proteins encapsulated in biopolymeric nanoparticles can augment protective efficacy. Chitosan nanoparticles (ChN) were prepared by ionic gelation method and Culture Filtrate Proteins (CFP) - CFP-10 and CFP-21 of Mycobacterium tuberculosis (Mtb) were encapsulated in ChN. The binding efficiency of nanoparticles with CFP-10 and CFP-21 proteins was confirmed by UV-Spectrophotometer. The efficacy of nanoparticles-encapsulated antigenic proteins administered intraperitoneal against Mtb aerosol infection was evaluated in Balb/c mice. Protection study was done by bacterial counts [CFU]. CFP-10 and CFP-21 proteins primed cells demonstrated a Th1 bias T cell response in an ex vivo assay. ChN-CFP10 and ChN-CFP21 nanoparticles have both protective and therapeutic potential against Mtb. In the group of mice immunized with CHN-CFP-10 the number of colonies reduced significantly from day 15 to day 60. ChN-CFP-21 showed maximum protection in ChN-CFP-21 immunized mice. ChN-CFP-10 and ChN-CFP-21 clearly showed enhanced protection against Mtb.

Dengue Symptoms Significance in Anti-Dengue Drug Development: Road Less Travelled.

Dengue outbreak has affected rural areas of Delhi-NCR, Haryana widely but it lacks in surveillance. High cases of dengue symptoms were reported in these regions whereas dengue symptoms have been a neglected issue in the anti-dengue drug development. Therefore, this study aims to analyze the status of the dengue infection, a rural issue of Delhi-NCR, Haryana and to identify the significance of dengue symptoms in anti-dengue drug development. The study was conducted when there is high chance of dengue infection i.e. from August 2015 to October 2015 at OPD Unit of PR Institute of Medical Science & Research, Delhi-NCR, Sonepat. It includes 158 patients from 24 rural areas of Haryana comprising both males and females from different age groups. Out of 20% cases, 6% were IgG-Positive, 9% were IgMPositive and 88% were NS1-Positive and rest 80% was normal. It includes 44% cases of thrombocytopenia. Badkhalsa village (28%), age group 18-24 (34%) and males (63%) reported cases of high infection. It was found that people with fewer platelet counts (Rai village) were not suffering from dengue whereas people with more platelet count reported dengue infection (Badkhalsa village). INTERPRETATION & CONCLUSION: This study focuses on new research directions by highlighting the dengue symptoms importance in anti-dengue drug development also it is a first attempt to investigate the status of dengue, a rural issue of Delhi-NCR, Haryana and suggests that health authorities and people living in these regions should take initiatives for better health.

Molecular docking NS4B of DENV 1-4 with known bioactive phyto-chemicals.

Dengue disease is a global disease that has no effective treatment. The dengue virus (DENV) NS4B is a target for designing specific antivirals due to its importance in viral replication. Medicinal plants have been a savior for dengue virus as they consist of a class of phytochemicals having anti-viral activity and can pose a new approach ofstrong drug against viruses. The present study analyzes the activity of compounds against NS4B of DENV (1-4) serotypes. In this study Catechin, Cianidanol, Epicatechin, Eupatoretin, Glabranin, Laurifolin, DL-Catechin, astherapeutic agents were filtered by using Lipinski rule's five and the drug-likeness property of these agents were used for assessment of pharmacological properties. The molecular docking results presented the 2-D structures of bioactive complex, which interacted with especially conserved residues of target domains. Interestingly, we find the Catechin, Laurifolin, Cianidanol have highest binding energy against NS4B in DENV-1,2,4 which is evident by the formation of more hydrogen bonds with the amino acid residues at the binding site of the receptor. Our results revealed that the bioactive compound, especially Catechin has significant anti-dengue activities. In addition, this study may be helpful in further experimental investigations.

CYBA and GSTP1 variants associate with oxidative stress under hypobaric hypoxia as observed in high-altitude pulmonary oedema.

HAPE (high-altitude pulmonary oedema) is characterized by pulmonary hypertension, vasoconstriction and an imbalance in oxygen-sensing redox switches. Excess ROS (reactive oxygen species) contribute to endothelial damage under hypobaric hypoxia, hence the oxidative-stress-related genes CYBA (cytochrome b-245 α polypeptide) and GSTP1 (glutathione transferase Pi 1) are potential candidate genes for HAPE. In the present study, we investigated the polymorphisms -930A/G and H72Y (C/T) of CYBA and I105V (A/G) and A114V (C/T) of GSTP1, individually and in combination, in 150 HAPE-p (HAPE patients), 180 HAPE-r (HAPE-resistant lowland natives) and 180 HLs (healthy highland natives). 8-Iso-PGF2α (8-iso-prostaglandin F2α) levels were determined in plasma and were correlated with individual alleles, genotype, haplotype and gene-gene interactions. The relative expression of CYBA and GSTP1 were determined in peripheral blood leucocytes. The genotype distribution of -930A/G, H72Y (C/T) and I105V (A/G) differed significantly in HAPE-p compared with HAPE-r and HLs (P≤0.01). The haplotypes G-C of -930A/G and H72Y (C/T) in CYBA and G-C and G-T of I105V (A/G) and A114V (C/T) in GSTP1 were over-represented in HAPE-p; in contrast, haplotypes A-T of -930A/G and H72Y (C/T) in CYBA and A-C of I105V (A/G) and A114V (C/T) in GSTP1 were over-represented in HAPE-r and HLs. 8-Iso-PGF2α levels were significantly higher in HAPE-p and in HLs than in HAPE-r (P=2.2×10(-16) and 1.2×10(-14) respectively) and the expression of CYBA and GSTP1 varied differentially (P<0.05). Regression analysis showed that the risk alleles G, C, G and T of -930A/G, H72Y (C/T), I105V (A/G) and A114V (C/T) were associated with increased 8-iso-PGF2α levels (P<0.05). Interaction between the two genes revealed over-representation of most of the risk-allele-associated genotype combinations in HAPE-p and protective-allele-associated genotype combinations in HLs. In conclusion, the risk alleles of CYBA and GSTP1, their haplotypes and gene-gene interactions are associated with imbalanced oxidative stress and, thereby, with high-altitude adaptation and mal-adaptation.

Distinct and shared roles of ß-arrestin-1 and ß-arrestin-2 on the regulation of C3a receptor signaling in human mast cells.

BACKGROUND: The complement component C3a induces degranulation in human mast cells via the activation of cell surface G protein coupled receptors (GPCR; C3aR). For most GPCRs, agonist-induced receptor phosphorylation leads to the recruitment of ß-arrestin-1/ß-arrestin-2; resulting in receptor desensitization and internalization. Activation of GPCRs also leads to ERK1/2 phosphorylation via two temporally distinct pathways; an early response that reflects G protein activation and a delayed response that is G protein independent but requires ß-arrestins. The role of ß-arrestins on C3aR activation/regulation in human mast cells, however, remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: We utilized lentivirus short hairpin (sh)RNA to stably knockdown the expression of ß-arrestin-1 and ß-arrrestin-2 in human mast cell lines, HMC-1 and LAD2 that endogenously expresses C3aR. Silencing ß-arrestin-2 attenuated C3aR desensitization, blocked agonist-induced receptor internalization and rendered the cells responsive to C3a for enhanced NF-κB activity as well as chemokine generation. By contrast, silencing ß-arrestin-1 had no effect on these responses but resulted in a significant decrease in C3a-induced mast cell degranulation. In shRNA control cells, C3a caused a transient ERK1/2 phosphorylation, which peaked at 5 min but disappeared by 10 min. Knockdown of ß-arrestin-1, ß-arrestin-2 or both enhanced the early response to C3a and rendered the cells responsive for ERK1/2 phosphorylation at later time points (10-30 min). Treatment of cells with pertussis toxin almost completely blocked both early and delayed C3a-induced ERK1/2 phosphorylation in ß-arrestin1/2 knockdown cells. CONCLUSION/SIGNIFICANCE: This study demonstrates distinct roles for ß-arrestins-1 and ß-arrestins-2 on C3aR desensitization, internalization, degranulation, NF-κB activation and chemokine generation in human mast cells. It also shows that both ß-arrestin-1 and ß-arrestin-2 play a novel and shared role in inhibiting G protein-dependent ERK1/2 phosphorylation. These findings reveal a new level of complexity for C3aR regulation by ß-arrestins in human mast cells.

CYP1A1, CYP1A2 and CYBA gene polymorphisms associated with oxidative stress in COPD.

BACKGROUND: The genetic susceptibility to chronic obstructive pulmonary disease (COPD) depends on detoxification and antioxidant enzymes, which detoxify cigarette smoke reactive components that, otherwise, generate oxidative stress. METHODS: In a case-control study of 346 subjects with and without COPD, we examined the polymorphisms 462Ile/Val, 3801T/C of CYP1A1, -3860G/A of CYP1A2 and -930A/G, 242C/T of CYBA individually or in combination and their contribution to oxidative stress markers by measuring malondialdehyde (MDA), catalase (CAT), glutathione (GSH) and glutathione peroxidase (GPx). RESULTS: COPD patients had significantly increased MDA concentration (p<0.001) and decreased CAT activity, GSH concentration, GPx activity (p< or =0.01). The patients were over-represented by the alleles 462Val, 3801C of CYP1A1 and -930G, 242C of CYBA (p<0.001, p=0.003, p=0.030 and p=0.031, respectively) and consequently the haplotypes of same alleles i.e. 462Val:3801C, 462Val:3801T and -930G:242C (p=0.048, p=0.016 and p=0.039, respectively). Similarly, CYP1A1 and CYP1A2 haplotypes, 462Val:3860G and 462Val:3801T:3860G were significantly over-represented (p=0.001 and p=0.003), respectively in patients. The same alleles-associated genotype-combinations between genes were more prevalent in patients. Of note, the genotypes, 462Ile/Val+Val/Val, 3801TC+CC of CYP1A1 and -930AG+GG of CYBA associated with increased MDA concentration (p=0.018, p=0.045 and p=0.017, respectively), decreased CAT activity (p<0.0001, p=0.080 and p<0.0001, respectively) and GSH concentration (p<0.0001, p=0.0002 and p=0.011, respectively) in patients. CONCLUSION: The identified alleles, its haplotypes and the genotype-combination along with increased oxidative stress, signify the importance in susceptibility to COPD.

COX2 and p53 risk-alleles coexist in COPD.

BACKGROUND: Cigarette smoke stimulates airway epithelial cells to release pro-inflammatory cytokines which influence various inflammation-related genes, including COX2, whereas p53 expression is known to alter in such a condition. Since both the genes share several common physiological functions including inflammation and oxidative stress, we investigated within gene and gene-gene interactions towards susceptibility to the disease. METHOD: In a prospective gene-association study we conducted PCR-RFLP for genotyping the COX2 -765G/C and 8473T/C and p53 72Pro/Arg polymorphisms in 229 COPD patients and 147 healthy controls. RESULTS: The -765GC+CC genotypes of COX2 and Pro/Pro+Pro/Arg genotypes of p53 were prevalent in patients with significant odds ratio, 2.05 and 2.30, respectively (p=0.001; p=0.009, respectively), as a consequence, the -765C and 72Pro alleles were prevalent (p

Correlation of oxidative status with BMI and lung function in COPD.

OBJECTIVES: The imbalance in oxidative status together with nutrition depletion and low body weight play a vital role in the pathogenesis and severity of chronic obstructive pulmonary disease (COPD). The study was undertaken to ascertain if a relationship existed between oxidative status and BMI in COPD. In addition, association of oxidative status and BMI with lung function of the disease was also examined. MATERIALS AND METHODS: In 202 COPD patients and 136 healthy controls plasma lipid peroxidation (LPO), reduced glutathione (GSH), glutathione peroxidase (GPx), catalase (CAT) activities, BMI and FEV(1)% predicted were looked for interactions. RESULTS: The patients had increased LPO (p=0.006) and decreased antioxidants (GSH, p=0.005; GPx, p=0.035 and CAT, p=0.008, respectively). Of note are the correlations of oxidative stress markers with BMI and FEV(1)% predicted in the patients. LPO inversely and GSH, GPx, and CAT positively correlated with both BMI (p=0.007, p<0.001, p=0.045 and p=0.009, respectively), and FEV(1)% of predicted (LPO, p=0.001; GSH, p<0.001; GPx, p=0.043 and CAT, p<0.001) in the patients. Further, a positive correlation existed between BMI and FEV(1)% predicted (p=0.016) in COPD. CONCLUSION: The intimate relationship of oxidative status with BMI and lung function, and the direct correlation between BMI and FEV(1) may potentiate severity of the disease.

Endothelial nitric oxide synthase gene variants contribute to oxidative stress in COPD.

Nitric oxide (NO) plays critical role in endothelial dysfunction and oxidative stress in COPD, pointing to the significance of endothelial nitric oxide synthase gene (eNOS) variants. We investigated the association of -786T/C, -922A/G, 4B/4A, and 894G/T polymorphisms of eNOS with the disease and its impact on nitrite and malonaldehyde levels in 190 COPD patients and 134 healthy controls, all smokers. The -786C, -922G and 4A alleles were significantly over-represented in patients (p=0.02, p=0.02, and p=0.03, respectively). The haplotypes, -786C:4A, 4A:894G, -786C:894G, and -786C:4A:894G were significantly over-represented in patients (p<0.0001, p =0.02, p=0.02, and p <0.0001, respectively), whereas, haplotypes, -786T:4B, 4B:894G, -786T:894G, and -786T:4B:894G were significantly under-represented in the patients (p<0.0001). The patients had significantly increased levels of nitrite (p=0.003) and malonaldehyde (p<0.0001). Combination of genotypes containing -786C and 4A alleles were greater in patients (p 0.05), and these combinations associated with decreased FEV1 value and nitrite level (p=0.03 and p=0.04, respectively) and with increased malonaldehyde levels (p=0.02). The eNOS -786C, -922G, and 4A alleles, these alleles associated haplotypes and genotype combinations were over-represented in patients. The variants and their combinations of four polymorphisms of eNOS contribute to disturbed pulmonary function and oxidative stress in COPD.

Genetic polymorphisms of GSTP1 and mEPHX correlate with oxidative stress markers and lung function in COPD.

The genetic susceptibility to COPD might depend on variations in detoxification enzymes that activate and detoxify cigarette smoke products, which otherwise generate oxidative stress causing pathogenesis. In a case-control study of 202 COPD patients and 136 normals, we examined the association of polymorphisms I105V, A114V of GSTP1 and Y113H, H139R of mEPHX individually or in combination with disease and their contribution to oxidative stress markers such as MDA, GSH, GPx and airflow obstruction. Patients were over-represented by the alleles 105V, 114V of GSTP1 and 113H, 139H of mEPHX (chi(2)=10.63, p=0.001, chi(2)=13.92, p<0.001, chi(2)=13.02, p<0.001 and chi(2)=4.48, p=0.034, respectively) and the haplotypes of same alleles i.e. 105V-114V and 113H-139H (chi(2)=14.58, p<0.001 and chi(2)=23.14, p<0.001). Moreover, there was marked over-representation of combination of genotypes, I105I+A114A of GSTP1 (53% vs. 36%) in controls; whereas, the combinations with 105V/114V alleles (64% vs. 47%) of GSTP1 (OR=1.99; 95% CI=1.28-3.09; p=0.002) and the homozygotes H113H+H139H (27% vs.10%) of mEPHX (OR=3.26; 95% CI=1.73-6.15; p=0.0001) in patients. Patients had significantly elevated MDA level (p<0.001) and decreased GSH level (p<0.001) and GPx activity (p=0.035), respectively. Of note, the genotypes, I105V/V105V, A114V/V114V of GSTP1 and Y113H/H113H of mEPHX associated with increased MDA level (p=0.04, p=0.03 and p=0.003), decreased GSH level (p=0.019, p=0.007 and p=0.0006) and lower FEV1 (p=0.23, p=0.037 and p=0.029), respectively, in patients; so was the correlation of these biomarkers and lung function with the combinations of the genotypes. In conclusion, 105V/114V alleles of GSTP1 and 113H/139H alleles of mEPHX and the combination of genotypes with same alleles associated with imbalanced oxidative stress and lung function in patients, signifying the importance in the disease.