Farah Khan

Critical role of the miR-200 family in regulating differentiation and proliferation of neurons.

The generation of differentiated and functional neurons is a complex process, which requires coordinated expression of several proteins and microRNAs (miRNAs). The present study using nerve growth factor (NGF)‐differentiated PC12 cells led to the identification of miR‐200, miR‐221/222 and miR‐34 families as major up‐regulated miRNAs in fully differentiated neurons. Similar to PC12 cells, induction of miR‐200 family was observed in differentiating neural stem cells, demonstrating a direct role of miR‐200 family in neuronal differentiation. Over‐expression of miR‐200 induced neurite formation in PC12 cells and regulated neuronal markers in favour of differentiation. However, inhibition of miR‐200 induced proliferation of PC12 cells. In differentiating PC12 cells and neural stem cells, an inverse relationship was observed between expression of reprogramming transcription factors (SOX2, KLF4, NANOG, OCT4 and PAX6) and miR‐200. Over‐expression of miR‐200 in PC12 cells significantly down‐regulated mRNA and protein levels of SOX2 and KLF4. Moreover, we observed two phases of dramatic down‐regulation of miR‐200 expression in developing rat brains correlating with periods of neuronal proliferation. In conclusion, our results indicate that increased expression of the miR‐200 family promotes neuronal differentiation, while decreased expression of the miR‐200 family promotes neuronal proliferation by targeting SOX2 and KLF4.

Macrophage polarization: the link between inflammation and related diseases

ObjectiveIn the present review, we try to critically evaluate the two faces of the macrophages and their roles in relation to gene alteration in some inflammatory conditions. The pros- and cons of each type of macrophage in immunologic outcomes are discussed.IntroductionIf “Diversity is the rule of nature”, macrophages have proven to be its obedient followers. A cell type that was classically considered to be activated by Interferon-γ, under the influence of TH-1 type of response and a well-accepted warrior of cellular immunity to the intracellular pathogens is not as simple as once considered. Past decade has revolutionized this notion with the advent of TH-2 influenced alternatively activated macrophages, now established as wound repairing and tissue regenerating.MethodsLiterature survey was done to present a detailed study on this macrophage dichotomy and its relevance to immune disorders via expression of some critical genes, nuclear factor kappa-light-chain-enhancer of activated B cells, peroxisome proliferator-activated receptors and SH2-containing inositol-5′-phosphatase 1, highly implicated in a myriad of immunological emergencies like inflammation, insulin resistance, wound healing, cancer, etc.ConclusionThe evaluation of macrophage dichotomy in these disorders may prove to be the first step towards the formulation of innovative therapeutic approaches.

Brain Region Specific Monoamine and Oxidative Changes During Restraint Stress

BACKGROUND AND PURPOSE Stress-induced central effects are regulated by brain neurotransmitters, glucocorticoids and oxidative processes. Therefore, we aimed to evaluate the simultaneous alterations in the monoamine and antioxidant systems in selected brain regions (frontal cortex, striatum and hippocampus) at 1 hour (h) and 24h following the exposure of restraint stress (RS), to understand their initial response and possible crosstalk. METHODS AND RESULTS RS (150 min immobilization) significantly increased the dopamine levels in the frontal cortex and decreased them in the striatum and hippocampus, with selective increase of dopamine metabolites both in the 1h and 24h RS groups compared to control values. The serotonin and its metabolite levels were significantly increased in both time intervals, while noradrenaline levels were decreased in the frontal cortex and striatum only. The activities of superoxide dismutase, glutathione peroxidase and the levels of lipid peroxidation were significantly increased with significant decrease of glutathione levels in the frontal cortex and striatum both in the 1h and 24h RS groups. There was no significant change in the catalase activity in any group. In the hippocampus, the glutathione levels were significantly decreased only in the 1h RS group. CONCLUSIONS Our study implies that the frontal cortex and striatum are more sensitive to oxidative burden which could be related to the parallel monoamine perturbations. This provides a rational look into the simultaneous compensatory central mechanisms operating during acute stress responses which are particular to precise brain regions and may have long lasting effects on various neuropathological alterations.

Amelioration of obesity-associated inflammation and insulin resistance in c57bl/6 mice via macrophage polarization by fish oil supplementation

Enormous phenotypic plasticity makes macrophages the target cells in obesity-associated inflammatory diseases. Thus, nutritional components that polarize macrophages toward antiinflammatory phenotype can partially reverse inflammatory diseases like insulin resistance. In the present study, macrophage-polarizing and insulin-sensitizing properties of fish oil (FO) were evaluated in obese insulin-resistant c57bl/6 mice fed high-fat diet (HFD-IR) after oral supplementation with FO (4, 8 or 16mg/kg body weight) and compared to lean and HFD-IR mice. FO-supplemented HFD-IR mice exhibited reduced adiposity index, serum cholesterol and triglycerides and increased insulin sensitization and showed improved adipose tissue physiology under light and transmission electron microscopy. NF-κB/P65 expression showed a downward shift on FO supplementation. The surface marker of M1 macrophages (CD-86) and the TLR-4 expression reduced with the increased supplementation of FO. Expression of arginase 1, an important marker of M2 macrophages, increased in a dose-dependent manner in response to FO dosage, which was observed at protein level by the western blotting and at mRNA level by real-time PCR. The cytokine profile of adipose tissue macrophages showed a steep shift toward antiinflammatory ones (IL-4 and IL-10) from the inflammatory TNF-α, IFN-γ, IL-2 and IL-1β. Thus, macrophage polarization seems to be the plausible mechanism via which FO alleviates obesity-induced inflammation and insulin resistance.

Purification and some properties of galectin-1 derived from water buffalo (Bubalus bubalis) brain

An increasing number of galectins have been found in various animal species, the most abundant of which is galectin‐1. The purpose of the present study was to purify and characterize galectin‐1 from buffalo brain. We purified the galectin using a combination of ammonium sulphate fractionation and affinity chromatography and the homogeneity was determined by both native polyacrylamide gel electrophoresis (PAGE) and denaturing SDS‐PAGE. The molecular weight of the galectin as determined by SDS‐PAGE under reducing conditions and by gel filtration column under native conditions was 13.8 and 24.5 kDa, respectively, suggesting a dimeric form of galectin. The most potent inhibitor of the galectin activity was lactose, giving complete inhibition of hemagglutination at 0.8 mM. Galectin showed higher specificity towards human blood group A. Free thiol groups were estimated at a molar ratio of 2.9. The effects of alkylating reagents (iodoacetate and iodoacetamide) on saccharide binding of the galectin were studied. Both alkylating reagents significantly inactivated the activity of the galectin within 20 min. The temperature and pH stability of the galectin were determined. Our findings based on physico‐chemical properties, carbohydrate and blood group specificities of the galectin may have future implications in biological and clinical applications.

Transactivation of P53 by cypermethrin induced miR-200 and apoptosis in neuronal cells

Cypermethrin, a pyrethroid pesticide, has been shown to induce neurotoxicity in adult mammals. However, studies are also needed to explore its toxicity in developing brains and understand its mechanism of action in neurons. In our recently published study, using nerve growth factor (NGF) differentiated PC12 cells, we have identified the miR-200 family as major up-regulated miRNAs, which regulate differentiation of PC12 cells into neurons. In the present study, the toxicity of cypermethrin is compared between undifferentiated and neuron-like differentiated PC12 cells, and role of the miR-200 family is studied in cypermethrin-induced neuronal cell death. Our studies have shown that a non-cytotoxic concentration of cypermethrin selectively induces the miR-200 family and apoptosis in differentiated PC12 cells, while no significant alterations were observed in undifferentiated PC12 cells. Further, our studies have demonstrated that cypermethrin induces miR-200 by increasing P53 levels in differentiated PC12 cells and we have identified a direct correlation between the expression of miR-200 and levels of P53 in PC12 cells. Further, BCL2 is identified as a target protein of miR-200b/c, and down-regulation of the BCL2 protein regulates cypermethrin-induced apoptosis of differentiated PC12 cells. Rescue experiments carried out with inhibitors of the miR-200 family, have further confirmed the role of the miR-200 family in apoptosis of differentiated PC12 cells exposed to cypermethrin. In conclusion, our studies have shown that differentiated PC12 cells are more sensitive to cypermethrin exposure than naive and undifferentiated PC12 cells, and P53 mediated induction of the miR-200 family regulates cypermethrin-induced apoptosis of differentiated neuron-like PC12 cells.

Partial Reversal of Obesity-Induced Insulin Resistance Owing to Anti-Inflammatory Immunomodulatory Potential of Flaxseed Oil

The present study was designed to assess the potential of supplementation of diet with Flaxseed (Linum usitatissimum, L.) oil (FXO), on obesity-related inflammation and reversal of obesity-induced insulin resistance. Swiss Albino mice, C57bl/6 mice and co-culture of 3T3-L1 adipocytes – RAW 264.7 macrophages to mimick obese adipose tissue environment were used for the study. Oral gavage of FXO at concentrations of 4, 8 or 16 mg/kg body weight (bwt) for 4 weeks or high-fat diet (HFD, 60% energy as fat) supplemented with dietary FXO (4, 8 or 16 mg/kg bwt) was given to the mice. FXO was characterised using gas chromatography – mass spectrometry. FXO supplemented HFD-fed mice (4 mg/kg bwt exhibited reduced adiposity index, serum glucose levels and triglycerides (8 and 16 mg/kg bwt) and improvement in insulin sensitisation (4, 8 and 16 mg/kg bwt) when compared with HFD mice. The co-culture showed a dose-dependent shift in cytokines towards anti-inflammatory (IL-4) state, with a decrease in pro-inflammatory TNF-α (p < 0.05). For immunomodulatory studies a dose-dependent increase (p < 0.05) was observed in antigen-specific levels of Th2 (IL-4) cytokine, serum anti-ova IgG1 and IgE levels. Suppression in anti-ova IgG2a, IgG2b, and IgG3 and antigen-specific Th1 cytokines like TNF-α and IFN-γ significantly (p < 0.05) was observed at 16 mg/kg bwt dosage. The results indicate that FXO exhibits an anti-inflammatory immunomodulatory potential and may partially relieve symptoms of obesity-associated insulin resistance.

Antigen specific immune enhancement of innate and acquired immunity by pearl in ashed form.

The present study evaluated mineral compound, pearl in ashed form [PAF], for its potential as oral immunomodulator. ICP-MS, atomic absorption spectroscopy, CHNS analysis and XRD analysis were used for characterization of PAF. Surface antigen markers (TLR-2/4 and CD-80/86) were studied by flow cytometry. At dose concentration of 25, 50, 100 and 500 μg/kg body wt., administrated orally for 10 days, TLR-2 expression on murine peritoneal macrophage increased while TLR-4 expression was reduced as compared to control. There was an increase in OVA and mitogen (Con-A) specific lymphocyte proliferation in OVA immunized mice. Also, level of both Th1 (IL-2/IFN-γ) and Th2 (IL-4/IL-10) cytokines, and level and titer of total IgG, IgG1, IgG2a and IgG2b of OVA immunized mice significantly increased. The level of Inflammatory cytokines (IL-1β and TNF-α) did not increase significantly. Enhancement in T and B cell immune responses may be possibly due to significantly enhanced expression of CD-80 and CD-86 co-stimulatory signals as observed using flow cytometry. Also, enhanced phagocytic activity and DTH response exhibit stimulatory effect of PAF on innate and cell mediated immune response. Histopathological analysis of liver, kidney and spleen and analysis of other toxicity parameters, such as effect on body weight, lymphoid organ weight and cellularity, revealed PAF to exhibit no toxic effects. PAF seems to be a promising balanced Th1 and Th2 directing immunomodulator, possibly activating TLR2 through TIR domain-containing adaptor inducing interferon β (TRIF)-dependent pathway that leads to T-cell activation and promotes effective immune responses and may find useful application clinically.

Mineral pitch stimulates humoral, cellular and innate immune responses in mice

Abstract Context: Mineral pitch (MP), a traditional medicine, is proposed to boost immunity in conditions that suppress Th1 cytokines such as AIDS/HIV, tuberculosis, leishmaniasis and cancer. Objective: This study investigates the immunoregulatory mechanisms of MP in innate, humoral and cell-mediated immunity. Materials and methods: Mice given MP (100, 200, 300 or 400 mg/kg, orally) for 10 consecutive days were immunized intravenously with goat RBC or ovalbumin, and investigated for plaque-forming cells (PFC), hemagglutination titer, hypersensitivity response, lymphocyte proliferation and macrophage function. Results: MP increased PFC (330.2 versus 182.2/106 splenocytes) in mice immunized with goat RBC and elicited ovalbumin-specific IgG titer at 400 mg/kg. Increase in Th1 immunity was correlated with the increased level of IFN-γ (724 versus 470 pg/ml) and decreased IL-4 (96 versus 178 pg/ml). CD4+/CD3+ ratio and delayed-type hypersensitivity response also increased to, respectively, 20.62 ± 0.59 (versus 16.47 ± 0.72) and 1.59 ± 0.12 (versus 0.87 ± 0.10 mm) in MP-treated mice. MP increased lymphocyte proliferation (11.14 ± 0.60 versus 5.81 ± 0.40 SI) and macrophage phagocyte response (0.24 ± 0.02 versus 0.15 ± 0.009), expressed as absorbance at 570 nm, but decreased nitrite production (17.4 ± 1.10 versus 24.3 ± 1.30 µM/106 cells). We also observed an increased bone marrow cellularity (24.5 ± 1.10 versus 17.10 ± 0.70 cells/femur) and WBC count (12 667 ± 377 versus 9178 ± 213 cells/mm3) following MP treatment. There was no sign of toxicity at 400 mg/kg, 1/12th of reported LD50. Conclusion: MP elicits a dose-dependent Th1 immune response.

Balanced TH1 and TH2 immunopotentiating effects of silicates partly containing nanoparticles present in calcined serpentine.

Abstract Calcined Serpentine (CS) is used in various formulations of alternative systems of medicine as a tonic to vital organs and as an anti-inflammatory agent. The process of calcination or incineration is believed to render non-toxic, gently absorbable, adaptable and digestible properties to the mineral compounds. The present study characterized CS and also evaluated its immunostimulatory potential. CS was characterized by using transmission electron microscopy (TEM), X-ray powder diffraction, atomic absorption spectroscopy and CHNS analysis. The characterized CS was further evaluated for its immunomodulatory potential in Swiss mice. X-Ray diffraction analysis revealed that the CS contained silicates of magnesium, calcium and iron as major minerals. Elemental composition and heavy metal analyses showed a presence of various inorganic elements/heavy metals, albeit at levels well below daily permissive intake values. TEM analysis of the test CS revealed a presence of nano particles with an average size of 10–20 nm (≈ 26% of total material). Oral administration of CS to mice at 50, 75, 100 or 200 μg/kg body weight for 10 days led to enhanced levels of total IgG, IgG1, IgG2a and IgG2b in ovalbumin-immunized mice as well as ex vivo lymphocyte proliferation and levels of TH1 (IL-2, IFNγ) and TH2 (IL-4, IL-10) cytokines produced by their cultured splenocytes. Similarly, CS treatment resulted in enhanced delayed-type hypersensitivity responses in GRBC-primed hosts. CS also activated host peritoneal macrophages, as indicated by increases in phagocytic activity and in TLR-2, CD80 and CD86 expression. The CS did not affect liver, kidney and spleen histology. Taken together, the results indicated that absorbed CS was stimulatory of host cell-mediated immune responses. It is hypothesized for now that the immunomodulatory effect of CS may have been due, in part, to a presence of nanoparticles on the CS; further study is required to validate this viewpoint.