Preparative enantiomeric separation of potent AMP-activated protein kinase activator by HPLC on amylose-based chiral stationary phase. Determination of enantiomeric purity and assignment of absolute configuration

The development of high performance liquid chromatography method on amylose-based stationary phases (Chiralpak AD) has permitted to achieve the preparative enantioseparation of one benzimidazole derivative, potent-AMP-kinase (AMPK) activator with satisfactory yields. Analytical enantioseparation method was optimized and validated to determine the enantiomeric purity. Using the UV detection, repeatability, limits of detection (LD) and quantification (LQ) were determined. Single-crystal X-ray analysis was successful to determine the absolute configuration of the individual enantiomers. A relation between the retention order and the absolute configuration of the enantiomers was established.     

Interferon-β inhibits inflammatory responses mediators via suppression of iNOS signaling pathway in PBMCs from patients with primary Sjögren’s syndrome

Background

Primary Sjögren’s syndrome (pSS) represents a chronic, systemic autoimmune disorder, characterized by lymphocytic infiltration of exocrine glands, inducing compromised secretory function and tissue destruction. Increasing evidence had revealed that inflammatory mediators, such as nitric oxide (NO) and pro-inflammatory cytokines, are critical in the development and perpetuation of pSS systemic manifestations. In our current study, we aimed to investigate the ex vivo immunomodulatory effect of interferon (IFN)-β on iNOS expression, as well as on pro-inflammatory (tumor necrosis factor (TNF)-α, interleukin (IL)-6) and immunoregulatory (IL-10) cytokine production. Furthermore, we examined potential associations between the influence of IFN-β treatment on NO production, and pSS clinical and serological manifestations.

Methods

In 41 pSS patients documented for their clinical and serological features, NO and cytokines levels were measured by the Griess method and enzyme-linked immunosorbent assay, respectively. Inducible nitric oxide synthase expression was analyzed by fluorescence immunostaining assay, using peripheral blood mononuclear cells (PBMCs) isolated from healthy controls and pSS patients.

Results

Our results revealed a strong down-modulating effect of IFN-β in the secretion of pro-inflammatory mediators including TNF-α, IL-6, and NO production. Interestingly, IFN-β exerts an increase in IL-10 levels. The most suppressive effect exerted by IFN-β on NO production was importantly reported for patients with neurological manifestation. This immunomodulatory effect of IFN-β on NO production is highly related to the decrease of inducible nitric oxide synthase (iNOS) expression.

Conclusion

Our findings highlight a consistent ex vivo inhibitory effect of IFN-β on pro-inflammatory cytokine production and NO pathway in pSS patients. Our data suggest that IFN-β could represent a potential candidate for targeting inflammation during pSS.

     

Morphological and ultrastructural changes in the placenta of the diabetic pregnant Egyptian women

Diabetes mellitus (DM) is a chronic metabolic disease in which the body fails to produce enough insulin or increased tissue resistance to insulin. The diabetes may have profound effects on placental development and function. This study was designed to detect the placental changes in pregnancy associated with DM comparing these changes with normal placenta. The study was carried out on sixty full-term placentae; divided into three equal groups; control group (group I): placentae of normal pregnancy, uncontrolled diabetes (group II): placentae from pregnant women whose blood glucose is poorly controlled during pregnancy. Controlled diabetes (group III): includes placentae from diabetic women whose blood glucose is controlled during pregnancy. The placentae from group II tend to be heavier and exhibited immaturity of villi, villous edema, fibrosis, excessive syncytial knots formation and infarctions. In addition to, fibrinoid necrosis, increased thickness of vasculosyncytial membrane, syncytial basement membrane, microvillous abnormalities and vascular endothelial changes were demonstrated. The syncytial multivesicular knots were present in placentae of group II. The nuclei within these syncytial knots display condensed chromatin, either dispersed throughout the nucleus or in the form of dense peripheral clumps with and numerous cytoplasmic vacuoles. The syncytial basement membrane showed focal areas of increase in its thickness and irregularity. Villous cytotrophoblasts showed increased number and activity in the form of numerous secretory granules, abundant dilated RER, larger distorted mitochondria. Villous vessels showed various degrees of abnormalities in the form of endothelial cell enlargement, folding, thickening and protrusion of their luminal surfaces into vascular lumen making it narrower in caliber. In placentae of group III, most of these abnormalities decreased. In most of placentae of group III, the VSM appeared nearly normal in thickness and showed nearly normal composition of one layer of syncytiotrophoblastic cells, one layer of smooth, regular capillary endothelium and the space between them. Mild microvillous abnormalities were noted in few placentae as they appeared short and blunted with mild decrease in their number per micron. The electron picture of syncytial knots appeared nearly normal containing aggregations of small, condensed hyperchromatic nuclei, minimal vacuoles could be seen in the cytoplasm of syncytial knots. Syncytial basement membrane appeared regular and nearly normal in its thickness and composition coming in direct contact with fetal blood capillaries but mild abnormalities were noted in the basement membrane in few placentae as increased its thickness and deposition of fibers or fibrinoid. Regarding cytotrophoblasts in the terminal villi of placentae with controlled diabetes, these cells appeared nearly normal. They were scattered beneath the syncytium and were active containing mitochondria, rough endoplasmic reticulum, free ribosomes and a large nucleus with fine dispersed chromatin. The vascular ultrastructural pattern in terminal villi of placentae of this group showed no significant abnormalities and was normally distributed in the villous tree. The luminal surface of the vascular endothelium appeared regular smooth in the majority of placentae of this group. The endothelial cells appeared connected to each other with tight junctions. It could be concluded that whether if long-term diabetes is controlled or not, placentae of diabetic mother showed a variety of significant histological structural changes seen more frequently than in the placentae of pregnant women without diabetes.     

Hepatoprotective and antioxidant activity of Melaleuca styphelioides on carbon tetrachloride-induced hepatotoxicity in mice

Context: Liver disease is a serious problem. Polyphenolic compounds have marked antioxidant effect and can prevent the liver damage caused by free radicals. In vitro studies have revealed the strong antioxidant activity of an ellagitannin-rich plant, namely, Melaleuca styphelioides Sm. (Myrtaceae).

Objective: In view of the limited therapeutic options available for the treatment of liver diseases, the hepatoprotective potential of the methanol extract of M. styphelioides leaves (MSE) was investigated against CCl₄-induced liver injury in mice.

Materials and methods: MSE was administered (500 and 1000?mg/kg/d p.o.) along with CCl₄ for 6 weeks. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were determined in the serum. Glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione transferase (GST), and malondialdehyde (MDA) were estimated in the liver homogenate. The bioactive components of MSE were identified by NMR, UV and HRESI-MS/MS data.

Results: MSE treatment (500 and 1000?mg/kg/d) markedly inhibited the CCl₄-induced increase in the levels of AST (31 and 38%), ALT (29 and 32%), ALP (13 and 19%), and MDA (22 and 37%) at the tested doses, respectively. MSE treatment markedly increased the levels of GSH (29 and 57%) and antioxidant enzymes compared with the CCl₄-treated group. MSE was more effective than silymarin in restoring the liver architecture and reducing the fatty changes, central vein congestion, Kupffer cell hyperplasia, inflammatory infiltration, and necrosis induced by CCl₄. The LD₅₀ of MSE was more than 5000?mg/kg.

Conclusion: MSE confers potent antioxidant and hepatoprotective effects against CCl₄-induced toxicity.     

Phase II Trial: Undifferentiated Versus Differentiated Autologous Mesenchymal Stem Cells Transplantation in Egyptian Patients with HCV Induced Liver Cirrhosis

The study was aimed to evaluate the effect of autologous transplantation of BM-derived undifferentiated and differentiated MSCs in cirrhotic patients following chronic hepatitis C virus infection. Twenty-five patients with Child C liver cirrhosis, MELD score >12 were included. They were divided into 2 groups. Group I, the MSCs group (n=15), this group was subdivided into two subgroups: Ia & Ib (undifferentiated and differentiated respectively). Group II (control group; n=10) involved patients with cirrhotic liver under conventional supportive treatment. Ninety ml BM was aspirated from the iliac bone for separation of MSCs. Surface expression of CD271, CD29 and CD34 were analyzed using flowcytometry. Hepatogenesis was assessed by immunohistochemical expression of OV6, AFP and albumin. Finally approximately 1 million MSCs/Kg were suspended in saline and were placed in blood bag and injected slowly intravenously over 15 min at a rate of 5 drops/min in one session. Follow up of patients at 3 and 6 months postinfusion revealed partial improvement of liver function tests with elevation of prothrombin concentration and serum albumin levels, decline of elevated bilirubin and MELD score in MSCs group. Statistical comparisons between the two subgroups (group Ia & Ib) did not merit any significant difference regarding clinical and laboratory findings. In conclusion: Bone marrow MSCs transplantation either undifferentiated or differentiated can be used as a potential treatment for liver cirrhosis.     

Evaluation of circulating zonulin as a potential marker in the pathogenesis of nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is a spectrum of liver disorders ranging from simple hepatic steatosis up to nonalcoholic steatohepatitis (NASH) evolving to cirrhosis and hepatocellular carcinoma (HCC). Liver biopsy is still the gold standard modality for diagnosing and staging NAFLD. The linkage between intestinal microbiota and NAFLD, might suggest a potential role of serum zonulin in NAFLD diagnosis. To appraise the role of circulating zonulin in NAFLD pathogenesis, 56 subjects with proved NAFLD by ultrasonography and liver biopsy, as well as 20 healthy controls were tested. Liver function tests, serum glucose, fasting insulin, C peptide, lipid profile, homeostasis model assessment of insulin resistance (HOMA‐IR), IL‐6, and circulating zonulin were performed to all subjects. Aspartate transaminase (AST), alanine transaminase (ALT), gamma‐glutamyl transferase (GGT), triglycerides, HDL‐c, fasting insulin, C peptide, HOMA‐IR, IL‐6, and serum zonulin were higher in NAFLD group than in controls (p < 0.05), and in NASH patients than those with simple steatosis (p < 0.05). Zonulin was positively correlated with body mass index (BMI), ALT, triglycerides, fasting insulin, HOMA‐IR, liver histopathology, and serum IL‐6 (p < 0.05), with inverse correlation to HDL‐C (p < 0.05). At cut off 8.3 pc/mL, serum zonulin was found to be of diagnostic value of NASH occurrence with 100% sensitivity and specificity (AUR = 1.000, p‐value = <0.001). The increasing zonulin levels in NAFLD patients with steep rise in NASH group denotes a possible role in pathogenesis of NAFLD occurrence and progression. This could open a new avenue of implicating zonulin antagonists as targeted therapies in NAFLD prevention.