Evaluation of Renal Gene Expression of Protein Kinase C (PKC) Isoforms in Diabetic and Nondiabetic Proliferative Glomerular Diseases

The protein kinase C (PKC) family consists of 13 members categorized as conventional or novel depending on whether diacylglycerol, calcium, or phosphatidylserine is required for activation. High glucose leads to activation of different forms of PKC across tissue types, thus determining the kind of diabetes-induced organ damage. PKC β was reported to have a positive role in B-lymphocyte activity through activation of NF-κB, leading to various immune disorders. We examined renal expression of two PKC isoforms α and β in renal biopsies of patients with diabetic nephropathy, lupus nephritis (LN) (Class 3-4), and mesangioproliferative glomerulonephritis (MPGN) to explore the role of each isoform in different glomerular diseases. PKC α and β gene expression was studied by quantitative real-time reverse transcription-PCR in 20 patients with type 2 diabetes and proteinuria (serum creatinine 2.04 ± 0.85 mg/dl, 24-h urinary protein 3.61 ± 1.75 g, eGFR 37.85 ± 17.89 ml/min/1.73 m2), 20 patients with proliferative LN (serum creatinine 1.67 ± 1.50 mg/dl, 24-h urinary protein 4.46 ± 5.01 g, eGFR 69.62 ± 40.93 ml/min/1.73 m2), and 20 patients with MPGN (serum creatinine 3.32 ± 2.79 mg/dl, 24-h urinary protein 4.65 ± 4.11 g, eGFR 32.62 ± 29.56 ml/min/1.73 m2). Normal tissues from the normal pole of four kidneys removed because of renal tumor served as controls. PKC α gene expression was significantly increased in diabetic kidneys compared to LN and MPGN (316.95 ± 152.94 µg/ml vs. 185.97 ± 32.13 and 195.46 ± 46.45 µg/ml, p < 0.05). PKC β gene expression was significantly increased in the LN and MPGN groups compared to the diabetic nephropathy group (41.01 ± 14.03 and 39.93 ± 16.41 µg/ml, respectively, vs. 18.20 ± 4.91 µg/ml, p < 0.05). Significant correlation was noted between the PKC α gene concentrations and proteinuria in diabetic patients. Renal expression of PKC α and β genes in control tissues were significantly lower compared to diabetic kidneys, LN, and MPGN groups (32.31 ± 0.36 and 4.67 ± 2.41 µg/ml, respectively, p < 0.001). The study revealed enhanced renal gene expression of both PKC isoforms α and β in diabetic kidney tissues, LN, and MPGN, but in different patterns. PKC α gene expression was significantly increased in diabetic patients with chronic kidney disease. The increased expression of the PKC β gene in LN and MPGN highlights its role in regulation of the immune system. This may represent potential therapeutic targets for prevention of progressive kidney injury in diabetic and proliferative glomerular diseases

Effect of metformin on Sirtuin-1 disorders associated with diabetes in male rats

Background: Type 2 diabetes mellitus (T2DM) is characterized by insulin resistance, hyperinsulinaemia and hyperglycaemia. Increased glucose production through abnormally elevated hepatic gluconeogenesis is central to the manifestation of hyperglycaemia in T2DM. Metformin corrects hyperglycaemia mainly through inhibition of gluconeogenesis. Sirtuin 1 (SIRT1) has been identified as regulator of gluconeogenic gene expression. The present study aimed to evaluate the effect of metformin on SIRT1 level and activity in liver and pancreas of diabetic rats. Further, the possible role of SIRT1 on metabolic disorders associated with diabetes mellitus, including serum levels of glucose, insulin, triglyceride (TG) and high density lipoprotiens (HDL), will be explored.Methods: Thirty-two male albino rats were divided into control group (GpI), diabetic (DM) group (GpII), (metformin + DM) group (GpIII) administered 120 mg/kg metformin daily for 1 month before induction of diabetes, (DM + metformin) group (GpIV) administered 250 mg/kg metformin daily for 1 month after induction of diabetes. At the end of the study, BMI%, serum levels of glucose, insulin, TG and HDL, HOMA, SIRT1 level and activity in liver and pancreas and pancreatic DNA ladder were assessed.Results: Our results showed significant decrease in serum glucose, insulin and TG levels and HOMA; significant increase in HDL level and SIRT1 level and activity in liver and pancreas beside the marked disappearance of pancreatic apoptosis in GpIII &amp; IV relative to GpII. Regarding BMI%, it showed no significant changes in GpIV relative to GpII. No significant change was recorded between GpIII and GpIV regarding all studied parameters except on serum TG.Conclusion: Lowered SIRT1 in diabetes was improved by the administration of Metformin. Consequently, the pathophysiological disorders associated with T2DM were improved.Keywords: DM, Metformin, SIRT1, Pancreatic apoptosi

Application of the Methylated Markers (Spectrin Beta and DEAD-Box Protein) for Definitive Differentiation Between Fresh and Aged Semen by evaluating Their Role in Identifying Semen From Mixed Body Fluids

Background: Semen identification is assumed a crucial proof of sexual assault. Moreover, body fluids at the crime scene of a human being, such as blood, semen, and saliva, are often mixed.Methods: Hence, in our study, we aimed to use methylation analysis targeting DNA epigenetic markers Spectrin beta chain (B_SPTB_03) and DEAD-box protein (DDX4) to differentiate between fresh semen (less than 4 hours) and aged semen (after 24 hours) as well as to differentiate between semen alone and semen mixed with other body fluids (blood and saliva) in the fresh and dried state.Results: Our findings showed statistically significant differences in the methylation patterns of the SPTB and DDX4 loci to distinguish semen from mixed body fluids in fresh and old samples. We were able to obtain two novel cutoff values to differentiate between fresh and aged semen, which are (52.25) with the SPTB marker and (70.75) with the DDX4 marker. Conclusion: It is concluded that the methylation approach based on the epigenetic markers of Spectrin beta chain and DEAD-box protein (B_SPTB_03 and DDX4) successfully identified fresh from aged semen and semen-derived alleles from mixed stains, hence it is recommended to be employed in forensic practice

The Effect Of Temperature On Age Estimation Of Semen Stains On Porous Versus Non-Porous Surfaces Using Messenger Ribonucleic Acid Measurement

Background: While mRNA can be used to identify the type of a body fluid, its degradation can also give some indication of the time interval since it was deposited. This study was conducted to evaluate the effect of temperature on the age estimation of human semen stains using mRNA deposited on porous versus non-porous surfaces at different time intervals. Methods: Ten semen samples were applied on two different media (glass and cotton) and exposed to three different temperatures (4°C, room temperature, 40°C) and examined at three time intervals (0, 45, and 90 days). The semen-specific mRNA markers PRM1 and PRM2 were quantitatively assessed along with a reference gene, beta-actin, using reverse transcription-quantitative polymerase chain reaction. Results: Mean Cq values of mRNA markers (PRM1 and PRM2) and the reference gene (beta-actin) increased with time of storage at different temperatures on both examined media. The mean quantification cycle (Cq) values of PRM2 were lower than PRM1, indicating that the levels of PRM2 marker in semen stain were higher than those of PRM1 marker. However, the mean Cq values of PRM2 at each time interval were not significantly different between temperatures, while PRM1 showed statistically significant differences in mean Cq values between temperatures at day 45 on both media. Conclusion: These results indicate that PRM2 can serve as a reliable mRNA marker to estimate the time since deposition of semen stain at different temperatures on two different media

CHOLESTATIC LIVER FIBROSIS IN A RAT MODEL OF BILE DUCT LIGATION: EVALUATING BIOCHEMICAL VERSUS HISTOPATHOLOGICAL CHANGES

Objective: Bile duct ligation (BDL), chronic liver injury model, was extensively used in studying mechanisms of fibrogenesis and antifibrotic agents. Considering the liver regenerative capacity and the diverse results from BDL, the present study aimed to evaluate the biochemical and histopathological changes over 10 weeks following BDL assessing if BDL-induced changes remain in a deterioration state or improve at a certain stage.Methods: Sham operation and BDL were conducted in Male Wistar rats. Serum AST, ALT, total bilirubin and albumin and hepatic hydroxyproline (HYP), reduced glutathione (GSH) and malondialdehyde (MDA) were measured in sham-operated (n=3) and BDL-rats (n=6) at 0, 1, 2, 4, 6, 8 and 10 weeks following operation. Liver tissue was also processed for histopathological analysis (H&amp;E and Sirus red staining).Results: Progressive liver injury (H&amp;E) and collagen deposition (Sirus red and HYP) in BDL-rats were observed starting from the first week post-operation and reached their maximum with early signs of cirrhosis on the 10th week of BDL. Severe and sustained cholestatic injury appeared in 2 weeks (increased ALT, AST, bilirubin along with decreased albumin (P&lt;0.001) compared to sham-operated rats). AST peaked on first week, however, bilirubin, ALT and MDA peaked on the 4th week (P&lt;0.001) then gradually decreased compared to their peaks.Conclusion: The relative improvement in liver function/cholestasis following their peaks in BDL model despite progression of fibrosis and hepatic injury require investigators using this model to consider not only biochemical, but also histopathological findings to guarantee an accurate interpretation of their results.Â

Homing and reparative effect of intra-articular injection of autologus mesenchymal stem cells in osteoarthritic animal model

<p>Abstract</p> <p>Background</p> <p>This work aimed to study the homing evidence and the reparative effect of mesenchymal stem cells (MSCs) in the healing process of induced osteoarthritis in experimental animal model (donkeys).</p> <p>Methods</p> <p>Twenty-seven donkeys were equally divided into 3 groups based on the observation period after induction of arthritis (3, 6 and 9 weeks) to achieve different degrees of osteoarthritis. Each group was subdivided into three subgroups of three animals each based on the follow-up period (1, 2 and 6 months) after treatment. The induction was done through intra-articular (IA) injection of 2 ml of Amphotericin-B in both carpal joints. MSCs were harvested in a separate procedure, labeled with green fluorescent protein (GFP) using monster GFP vector and suspended in hyaluronic acid for IA injection. Treatment approaches consisted of cell-treatment using MSCs suspended in 3 ml of hyaluronic acid (HA) for the right carpal joint; and using the same amount of (HA) but without MSCs for the left contralateral carpal joint to serve as a control. Animals were assessed clinically and radiologically before and after treatment. Synovial fluid was also evaluated. Histopathologically; articular cartilage structural changes, reduction of articular cartilage matrix staining, osteophyte formation, and subchondral bone plate thickening were graded. Data was summarized using median and percentile for scores of histopathologic grading. Comparison between groups was done using non-parametric Mann Whitney test.</p> <p>Results</p> <p>The reparative effect of MSCs was significant both clinically and radiologically in all treated groups (P < 0.05) compared to the control groups. Fluorescence microscopy of sections of the cell-treated joints of all animals indicated that the GFP-transduced injected cells have participated effectively in the reparative process of the damaged articular surface and have integrated within the existing articular cartilage. The cells were associated with the surface of the cartilage and, were also detected in the interior.</p> <p>Conclusions</p> <p>Homing was confirmed by the incorporation of injected GFP-labeled MSCs within the repaired newly formed cartilage. Significant recovery proves that the use of IA injection of autologous MSCs is a viable and a practical option for treating different degrees of osteoarthritis.</p

The Neuro Engraftment and Neuroregenerative effects of Hydrogen Sulphide Donor, Intracerebral MSCs, Ginko Biloba and Kefir in Attenuating Neuropathological hallmarks of Lipopolysaccharide induced Alzheimer’s disease Rat models

Background: Memory disorders have been characterized by being a devastating long term incurable diseases with a huge social impact in addition to a diminished efficient available medical treatments. Deep Brain stimulation via using neuroprotective inducers for treatment of brain structure degenerative diseases such as Alzheimer’s disease (AD) can be considered as being a promising successful therapy due to its various targets and underlying mechanisms for improving brain dysfunction. Objectives: The main aim of this study is to suggest therapeutic protocol having the potentials for restoring normal neurons diverse population and modifying neuropathological deposited hallmarks including both positive and negative lesions. Materials and Methods: Rats were divided into nine groups: (G1) control ;(G2) rats received LPS as a method of inducing nongenetically manipulated AD;(G3)AD rats received NaHS;(G4) AD rats received MSCs intracerebrally;(G5) AD rats received MSCs+NaHS;(G6)AD rats received kefir+GB;(G7)AD rats received MSCs+kefir+GB;(G8)AD rats received NaHS+kefir+GB; (G9) AD rats received MSCs+NaHS+kefir+GB. Results: AD induction resulted in down-regulation of CBS expression and GSH brain tissue level accompanied with overexpression in amyloid-? protein, MAPK, tau protein, ACAT expression and MDA brain tissue level in addition to elevated caspase-3 serum level. Conclusion: The implantation of amyloid reliving therapy that do have a wide clinical impact if initiated at benign plaques stage before irreversible brain damage occurs. The following effects have been observed following the administration of suggested medical protocol where a decrease in AD pathological deposited hallmarks has been observed with maintaining inflammatory brain factors by functioning as a potent neuroregenerative

Is there a Link between Human Herpes Virus Infection and Toll-like Receptors in the Pathogenesis of Pityriasis Rosea? A Case-control Study

Human herpesvirus (HHV) 6 and 7 are involved in the pathogenesis of pityriasis rosea (PR). Our aim was to evaluate the role of the innate immune response in PR through the detection of Toll-like receptors (TLR) 2, 3, 4, 7, 8, and 9 expression in the skin of affected patients and to detect the possibility of being induced by HHV-6 and/or HHV-7 viral coexistence in these patients. Twenty-four patients with PR and 24 healthy controls were included in this case-control study. Biopsy was obtained from the PR lesion and from the healthy skin of controls for detection of HHV-6 and 7 as well as TLRs 2, 3, 4, 7, 8, and 9 gene expression using real-time polymerase chain reaction (PCR). Significantly elevated expression of all studied TLRs and significantly higher viral load of HHV-6 and 7 in PR cases were detected. A significant higher expression of TLR2 and 4 in HHV-7 positive cases and a significant positive correlation between TLR9 and HHV-7 viral load were documented. HHV6 and 7 may also be involved in the pathogenesis of PR via TLR pathways  </p