ARF6 : a potential player in T-cell biology

The ADP ribosylation factor (Arf) family of GTPases comprises of small (20-25kDa molecular mass) monomeric proteins. Among the 3 known subclasses, the highly expressed Arf6 (Class Ill) is functionally distinct, and is implicated ubiquitously in membrane trafficking, endocytosis, exocytosis in various cells and invasive activities of cancerous cells. There is very little known about the role of Arf6 towards the mechanisms of T cell signal transduction and Arf6 null embryos are lethal at midgestation which limits the role of KO models for investigating its regulatory mechanisms. We sought to study the role of Arf6 in T cell signalling. In the present piece of work, it was found that disruption of Arf6 activity leads to impairment of downstream signalling events underlying TCR signalling. This includes cell cycle progression, proliferation and expansion of T cells, IL -2 prod uction and ERK phosphorylation. These effects are mediated by cytohesins that function as regulating factors for Arf6. Moreover, it was found that Arf6 is specifically regulated in actively dividing immune cells like thymocytes, splenocytes, effector T cells. Also, regulatory T cells express high levels of Arf6 and secrete high levels of suppressive cytokine IL-10, indicating a potential role of Arf6 in inhibition of activation of T cells. Lastly, some of our pilot studies demonstrate that Arf6 has a role in the processes leading to in antigen presentation by Des. The data provides direct evidence that Arf6 constitutes an important 6omponent-of--TCR signalling- in primary Tlymphocytes and interaction between T cells and DCs. To study the mechanistic regulation of Arf6, in vivo models would prove to be an important asset. For this, I have developed a targeting vector to conditionally delete Arf6 in T cells, DCs or any other immune effector cells to elucidate the exact role of Arf6 in an in vivo setting. I anticipate that the conditional knockout model will allow a 'more precise and detailed analysis of Arf6 regulation in T cell as well as DCs and expand our understanding of human disease associated with immune dysregulation.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Influence of low cholesterol eggs enriched with vitamin-E and omega-3 fatty acid on blood lipid profile of wistar rats

601-605In the recent past, low cholesterol eggs enriched with vitamin-E and omega-3 fatty acid have been developed and are marketed under different brands claiming them as heart friendly. The influence of these eggs (smart eggs) on lipid profile of rats was evaluated in comparison to that of the standard eggs. Data of 4 week dietary treatment revealed that total plasma cholesterol, low density lipoprotein (LDL) and very low density lipoprotein (VLDL) cholesterol increased only 22% in rats fed on diet containing 4 smart eggs per kg of semi-synthetic diet in contrast to the increase of more than 100 % when fed on diet containing standard eggs. The results suggest that it is not the low cholesterol content alone but also vitamin E and omega-3 fatty acids present in smart eggs that act synergically to prevent a substantial change in blood lipid profile and impose no serious risk to the health of the consumers

Immunomodulatory plasticity of mesenchymal stem cells: a potential key to successful solid organ transplantation

Abstract Organ transplantation remains to be a treatment of choice for patients suffering from irreversible organ failure. Immunosuppressive (IS) drugs employed to maintain the allograft have shown excellent short-term graft survival, but, their long-term use could contribute to immunological and non-immunological risk factors, resulting in graft dysfunctionalities. Upcoming IS regimes have highlighted the use of cell-based therapies, which can eliminate the risk of drug-borne toxicities while maintaining efficacy of the treatment. Mesenchymal stem cells (MSCs) have been considered as an invaluable cell type, owing to their unique immunomodulatory properties, which makes them desirable for application in transplant settings, where hyper-activation of the immune system is evident. The immunoregulatory potential of MSCs holds true for preclinical studies while achieving it in clinical studies continues to be a challenge. Understanding the biological factors responsible for subdued responses of MSCs in vivo would allow uninhibited use of this therapy for countless conditions. In this review, we summarize the variations in the preclinical and clinical studies utilizing MSCs, discuss the factors which might be responsible for variability in outcome and propose the advancements likely to occur in future for using this as a “boutique/personalised therapy” for patient care

Expression, Purification and Characterization of Peanut (Arachis hypogaea) Agglutinin (PNA) from Baculovirus Infected Insect Cells

Peanut (Arachis hypogaea) seed lectin, PNA is widely used to identify tumor specific antigen (T-antigen), Gal beta 1-3GalNAc on the eukaryotic cell surface. The functional amino acid coding region of a cDNA clone, pBSH-PN was PCR amplified and cloned downstream of the polyhedrin promoter in the Autographa californica nucleopolyhedrovirus (AcNPV) based transfer vector pVL1393. Co-transfection of Spodoptera frugiperda cells (Sf9) with the transfer vector, pAcPNA and AcRP6 (a recombinant AcNPV having B-gal downstream of the polyhedrin promoter) DNAs produced a recombinant virus, AcPNA which expresses PNA. Infection of suspension culture of Sf9 cells with plaque purified AcPNA produced as much as 9.8 mg PNA per liter (2.0 x 10(6) cells/ml) of serum-free medium. Intracellularly expressed protein (re-PNA) was purified to apparent homogeneity by affinity chromatography using ECD-Sepharose. Polyclonal antibodies against natural PNA (n-PNA) crossreacted with re-PNA. The subunit molecular weight (30 kDa), hemagglutination activity, and carbohydrate specificity of re-PNA were found to be identical to that of n-PNA, thus confirming the abundant production of a functionally active protein in the baculovirus expression system

Expression, purification and characterization of peanut (Arachis hypogaea) agglutinin (PNA) from baculovirus infected insect cells

Peanut (Arachis hypogaea) seed lectin, PNA is widely used to identify tumor specific antigen (T-antigen), Galβ1-3GalNAc on the eukaryotic cell surface. The functional amino acid coding region of a cDNA clone, pBSH-PN was PCR amplified and cloned downstream of the polyhedrin promoter in the Autographa californica nucleopolyhedrovirus (AcNPV) based transfer vector pVL1393. Co-transfection of Spodoptera frugiperda cells (Sf9) with the transfer vector, pAcPNA and AcRP6 (a recombinant AcNPV having B-gal downstream of the polyhedrin promoter) DNAs produced a recombinant virus, AcPNA which expresses PNA. Infection of suspension culture of Sf9 cells with plaque purified AcPNA produced as much as 9.8 mg PNA per liter (2.0×106 cells/ml) of serum-free medium. Intracellularly expressed protein (re-PNA) was purified to apparent homogeneity by affinity chromatography using ECD-Sepharose. Polyclonal antibodies against natural PNA (n-PNA) cross-reacted with re-PNA. The subunit molecular weight (30kDa), hemagglutination activity, and carbohydrate specificity of re-PNA were found to be identical to that of n-PNA, thus confirming the abundant production of a functionally active protein in the baculovirus expression system