The mitochondrial carrier homolog 2 is involved in down-regulation of influenza A virus replication.

BACKGROUND: The mitochondrial carrier homolog 2 (MTCH2) is a mitochondrial outer membrane protein regulating mitochondrial metabolism and functions in lipid homeostasis and apoptosis. Experimental data on the interaction of MTCH2 with viral proteins in virus-infected cells are very limited. Here, the interaction of MTCH2 with PA subunit of influenza A virus RdRp and its effects on viral replication was investigated. METHODS: The human MTCH2 protein was identified as the influenza A virus PA-related cellular factor with the Y2H assay. The interaction between GST.MTCH2 and PA protein co-expressed in transfected HEK293 cells was evaluated by GST-pull down. The effect of MTCH2 on virus replication was determined by quantification of viral transcript and/or viral proteins in the cells transfected with MTCH2-encoding plasmid or MTCH2-siRNA. An interaction model of MTCH2 and PA was predicted with protein modeling/docking algorithms. RESULTS: It was observed that PA and GST.MTCH2 proteins expressed in HEK293 cells were co-precipitated by glutathione-agarose beads. The influenza A virus replication was stimulated in HeLa cells whose MTCH2 expression was suppressed with specific siRNA, whereas the increase of MTCH2 in transiently transfected HEK293 cells inhibited viral RdRp activity. The results of a Y2H assay and protein-protein docking analysis suggested that the amino terminal part of the viral PA (nPA) can bind to the cytoplasmic domain comprising amino acid residues 253 to 282 of the MTCH2. CONCLUSION: It is suggested that the host mitochondrial MTCH2 protein is probably involved in the interaction with the viral polymerase protein PA to cause negative regulatory effect on influenza A virus replication in infected cells.

Human sorting nexin 2 protein interacts with Influenza A virus PA protein and has a negative regulatory effect on the virus replication.

BACKGROUND: Replication of the influenza A viruses occurs in the cells through the viral RdRP consisting of PB1, PB2, and PA. Several cellular proteins are involved in these processes. This study aims to reveal the interaction between human SNX2 protein and the PA protein and the effects of the SNX2 on the virus replication. RESULTS: To identify potential host interacting proteins to the PA, yeast two-hybrid assay was carried out with HEK293 cell cDNA library and the PA as a bait. We focused on SNX2 protein, which interacts with the PA in the yeast cells. By using the co-immunoprecipitation assays, it has been demonstrated that the amino-terminal part of the PA was important for binding to the SNX2. Immunolocalization of the proteins in HeLa cells supported this interaction. Knockdown of the SNX2 with siRNA in the cells resulted in a significant increase in both viral transcripts and virus growth. However, the increase of SNX2 in transfected cells didn't cause a significant change in the viral RdRP activity in minireplicon assay. This may suggest that the negative effect of SNX2 on the virus replication could be saturated with its authentic intra-cellular amount. CONCLUSIONS: This study revealed that the SNX2 and PA protein interact with each other in both yeast and HEK293 cells, and the SNX2 has a negative regulatory function on the virus replication. However, more knowledge is required to elucidate the action mechanism of the SNX2 on the influenza A virus replication at the molecular level.

Dual-drug delivery of Ag-chitosan nanoparticles and phenytoin via core-shell PVA/PCL electrospun nanofibers.

Dual-drug delivery systems were constructed through coaxial techniques, which were convenient for the model drugs used the present work. This study aimed to fabricate core-shell electrospun nanofibrous membranes displaying simultaneous cell proliferation and antibacterial activity. For that purpose, phenytoin (Ph), a well-known proliferative agent, was loaded into a polycaprolactone (PCL) shell membrane, and as-prepared silver-chitosan nanoparticles (Ag-CS NPs), as biocidal agents, were embedded in a polyvinyl alcohol (PVA) core layer. The morphology, chemical composition, mechanical and thermal properties of the nanofibrous membranes were characterized by FESEM/STEM, FTIR and DSC. The coaxial PVA-Ag CS NPs/PCL-Ph nanofibers (NFs) showed more controlled Ph release than PVA/PCL-Ph NFs. There was notable improvement in the morphology, thermal, mechanical, antibacterial properties and cytobiocompatibility of the fibers upon incorporation of Ph and Ag-CS NPs. The proposed core-shell PVA/PCL NFs represent promising scaffolds for tissue regeneration and wound healing by the effective dual delivery of phenytoin and Ag-CS NPs.

Development and characterization of vancomycin-loaded levan-based microparticular system for drug delivery.

Encapsulation of vancomycin (VANCO) into biodegradable levan microparticles was achieved using a simple preparation technique. Microparticles were prepared by using levan polysaccharide produced by a halophilic bacterium Halomonas smyrnensis AAD6T. To optimize efficiency of encapsulation process by precipitation method, three parameters were studied: drug and polymer concentrations and preparation rotating speed. The particles were characterized in vitro. The size of levan microparticles was changed between 0.404 µm and 1.276 µm. The surface charge was detected between +4.1 mV and +6.5 mV. The highest drug encapsulation capacity of the system was 74.7% and was depending on the polymer concentration. In dissolution studies, initial burst effect around 10-20% from all the formulations was observed and then the release was slowed down and continued at a constant level. In vitro antibiotic release from the microparticles was controlled with the drug carrier system and release fit to Higuchi kinetic model. All the released samples collected at different time intervals during dissolution studies have exhibited intrinsic bactericidal activity against Bacillus subtilis ATCC 6633. WST-1 cell proliferation and viability studies showed that VANCO-loaded levan microparticles at concentrations between 100 µg/mL and 1000 µg/mL were nontoxic to L929 cells. As conclusion, levan microparticulate system could be a potential carrier of antibiotic drugs such as VANCO.

Effect of Antibiotics on Polymorphonuclear Leukocyte Functions and Myeloperoxidase Activity, Glutathione and Malondialdehyde Levels in Allergic Asthma.

We investigated the effect of ciprofloxacin, rifampicine and doxycycline on myeloperoxidase (MPO) activity, glutathione (GSH) and malondialdehyde (MDA) levels in allergic asthma patients and healthy volunteers. Polymorphonuclear leukocytes (PMNs) were isolated with ficoll-hypaque gradient centrifugation method. MPO activity was assayed with modified o-dianisidine, GSH by Ellman's and MDA levels by Beuge's method. PMN functions and MDA levels of patients significantly decreased when compared with healthy volunteers. Ciprofloxacin significantly increased PMN functions, MPO activity and MDA levels of both groups. We have demonstrated that ciprofloxacin has beneficial effects on MPO activity and PMN functions in allergic asthma patients and healthy volunteers.

Optimization and evaluation of clarithromycin floating tablets using experimental mixture design.

The purpose of the study was to prepare and evaluate clarithromycin (CLA) floating tablets using experimental mixture design for treatment of Helicobacter pylori provided by prolonged gastric residence time and controlled plasma level. Ten different formulations were generated based on different molecular weight of hypromellose (HPMC K100, K4M, K15M) by using simplex lattice design (a sub-class of mixture design) with Minitab 16 software. Sodium bicarbonate and anhydrous citric acid were used as gas generating agents. Tablets were prepared by wet granulation technique. All of the process variables were fixed. Results of cumulative drug release at 8th h (CDR 8th) were statistically analyzed to get optimized formulation (OF). Optimized formulation, which gave floating lag time lower than 15 s and total floating time more than 10 h, was analyzed and compared with target for CDR 8th (80%). A good agreement was shown between predicted and actual values of CDR 8th with a variation lower than 1%. The activity of clarithromycin contained optimizedformula against H. pylori were quantified using well diffusion agar assay. Diameters of inhibition zones vs. log10 clarithromycin concentrations were plotted in order to obtain a standard curve and clarithromycin activity.

Potential adjuvant effects of Nigella sativa seeds to improve specific immunotherapy in allergic rhinitis patients.

OBJECTIVE: To investigate the effects of Nigella sativa seed supplementation on symptom levels, polymorphonuclear leukocyte (PMN) functions, lymphocyte subsets and hematological parameters of allergic rhinitis. SUBJECTS AND METHODS: Twenty-four patients randomly selected from an experimental group of 31 (mean age 34 years) sensitive to house dust mites with allergic rhinitis and a control group of 8 healthy volunteers (mean age 23 years) were treated with allergen-specific immunotherapy in conventional doses for 30 days. After a month of immunotherapy, 12 of the 24 patients and the 8 healthy volunteers were given N. sativa seed supplementation (2 g/day orally) for 30 days. The remaining 12 patients continued only on immunotherapy during the same period. The other 7 patients were given 0.1 ml saline solution subcutaneously once a week as a placebo. The symptom scores, PMN functions, lymphocyte subsets and other hematological parameters were evaluated before and after all treatment periods. RESULTS: There was a statistically significant increase in the phagocytic and intracellular killing activities of PMNs of patients receiving specific immunotherapy, especially after the addition of N. sativa seed. The CD8 counts of patients receiving specific immunotherapy plus N. sativa seed supplementation significantly increased compared to patients receiving only specific immunotherapy. PMN functions of healthy volunteers significantly increased after N. sativa seed supplementation compared to baseline. CONCLUSION: N. sativa seed supplementation during specific immunotherapy of allergic rhinitis may be considered a potential adjuvant therapy.

The effects of some antibiotics on polymorphonuclear leukocyte functions of elderly patients in vitro before and after zinc supplementation.

The effects of ciprofloxacin, cefodizime, rifampicine, doxycycline and cefodizime + rifampicine combination on polymorphonuclear leukocyte (PMN) functions (phagocytosis and intracellular killing activity) were investigated in vitro in elderly patients and compared with those of healthy young volunteers before and after zinc supplementation. PMNs of 13 elderly hypertensive patients and 10 healthy young volunteers were isolated by Ficoll-Hypaque gradient centrifugation method from venous blood with EDTA. The subjects were given 22 mg/daily/oral zinc supplementation for 1 month. Serum zinc levels before and after supplementation were measured by flame atomic absorbtion spectrophotometer and the effects of each drug on PMN functions at therapeutic concentrations were investigated. Ciprofloxacin significantly increased the PMN's phagocytic activity of elderly patients (p = 0.002) before zinc supplementation and significantly increased both PMN functions of elderly patients (p = 0.002) after zinc supplementation. The same antibiotic significantly increased both PMN functions of healthy young volunteers (p = 0.005 and p<0.05, respectively) before and after zinc supplementation when compared with the control (drug-free). Cefodizime significantly increased the PMN's phagocytic activity of elderly patients (p = 0.003, p = 0.002) before and after zinc supplementation when compared with the control (drug-free). It also significantly increased both PMN functions of healthy young volunteers (p = 0.005 and p<0.05, respectively) before and after zinc supplementation when compared with the control (drug-free). Doxycycline significantly increased PMN's intracellular killing activity of healthy young volunteers before zinc supplementation (p<0.05) when compared with the control (drug-free) values. Rifampicine significantly decreased PMN's phagocytic activity of elderly patients (p<0.05) after zinc supplementation. Cefodizime+rifampicine combination significantly increased PMN's phagocytic activity at therapeutic concentrations of healthy young volunteers (p = 0.005) before zinc supplementation and PMN's phagocytic activity of elderly patients (p<0.05) after zinc supplementation when compared with the control (drug-free). Consequently, in the present study from the antibiotics ciprofloxacin, cefodizime and cefodizime + rifampicine combination, which are accepted as biological response modifiers have demonstrated stimulatory effects by significantly increasing polymorphonuclear leucocyte functions (phagocytosis and/or intracellular killing activity) of elderly patients and healthy young volunteers in vitro before and after zinc supplementation. Additionally zinc supplementation has more immunostimulatory effects on PMN functions of healthy young volunteers than elderly patients.

Comparison of polymorphonuclear leukocyte functions in elderly patients and healthy young volunteers.

OBJECTIVE: To compare the polymorphonuclear leukocyte (PMN) functions (phagocytosis and intracellular killing activity) of elderly patients with healthy young volunteers. SUBJECTS AND METHODS: Fifty-nine elderly patients who had various diseases (cancer, hypertension and diabetes mellitus, DM) and 10 healthy young volunteers were included in this study. Ficoll-Hypaque gradient centrifugation was used to isolate PMNs from venous blood containing EDTA (0.1 g/ml). Phagocytosis and intracellular killing activity of neutrophils were assayed using a modification of Alexander's method, in which serum opsonins, number of neutrophils and number of microorganisms are standardized in order to detect both increases and decreases in phagocytosis and intracellular killing as well as combined abnormalities of these two functions. The least significant difference test was used to compare the results in the two groups. RESULTS: Phagocytic activity of PMNs from patients with cancer was significantly higher than that of healthy young volunteers (p < 0.05) and elderly patients with hypertension and DM (p < 0.05). There was no statistical difference between the phagocytic activity of PMNs from elderly patients with hypertension and DM and healthy young volunteers (p > 0.05). The intracellular killing activity of PMNs from elderly patients with hypertension, DM and cancer was significantly lower than that of healthy young volunteers (p = 0.001, p < 0.0001, p = 0.003, respectively). CONCLUSIONS: The intracellular killing activity of PMNs from elderly patients was significantly decreased when compared with that of healthy young volunteers. Ageing, chronic diseases and drugs used in the treatment of these elderly patients may be the cause for decreased intracellular killing activity.

The effects of allergen-specific immunotherapy on polymorphonuclear leukocyte functions in patients with seasonal allergic rhinitis.

Immunotherapy plays an important role in the therapy of allergic rhinitis and bronchial asthma. However, there is not much information about the effects of allergen-specific immunotherapy (SIT) on the polymorphonuclear leukocyte functions. The aim of this study was to investigate the effects of specific immunotherapy on phagocytic and intracellular killing activities of polymorphonuclear leukocytes (PMN) derived from patients with seasonal allergic rhinitis. Twenty-four patients with seasonal allergic rhinitis documented to be sensitive to grass pollen were included in this study. Patients were divided into 3 groups. Group 1 (n=7) received conventional immunotherapy whereas patients in Group 2 (n=7) were treated with short-term immunotherapy and the third group (n=10) were given placebo during the study process. Both phagocytic and intracellular killing activities were significantly increased (p=0.002, p<0.0001, respectively) by conventional immunotherapy when compared to the first determination. In the short-term immunotherapy group, phagocytic activity was increased very significantly (p=0.0001), whereas intracellular killing activity was not affected (p=0.252). There were no changes in these parameters in the placebo group. These results suggest that allergen-specific immunotherapy has an enhancing effect on PMNs functions in the patients with seasonal allergic rhinitis. It should be clarified by further studies whether this enhancement might be considered as another beneficial effect of the immunotherapy.