Development of efficient and cost effective vaccines have been recognized as the primary concern to improve the overall healthcare in a country. In order to achieve this goal, more improved and ...powerful adjuvants need to be developed. Lacking in the self-adjuvanting immuno-modulatory constituents, vaccines exhibit lower immunogenicity. Combining potent adjuvants with vaccines is the most appropriate method to enhance the efficacy of the vaccines. Hence, this review is focussed on the most potent adjuvants for the formulation of vaccines. Areas covered: This review focuses on Oil-based emulsions, Mineral compounds, Liposomes, Bacterial products, ISCOMs and most recently used nanomaterials as adjuvants for enhancing the antigenicity of vaccines. Furthermore, this review explains the immunological response elicited by various particles. Moreover, case studies are incorporated providing an in depth analyses of various adjuvant-containing vaccines which are currently used. Expert commentary: Enhanced fundamental knowledge about the adjuvants and their immuno-stimulatory capabilities and delivery mechanisms will facilitate the rational designing of prophylactic vaccines with better efficacy.
The tyrosine phosphatase Src homology 2-containing phosphatase 1 (SHP-1) is a key negative regulator of TCR-mediated signaling. Previous studies have shown that in T cells a fraction of SHP-1 ...constitutively localizes to membrane microdomains, commonly referred to as lipid rafts. Although this localization of SHP-1 is required for its functional regulation of T cell activation events, how SHP-1 is targeted to the lipid rafts was unclear. In this study, we identify a novel, six-amino acid, lipid raft-targeting motif within the C terminus of SHP-1 based on several biochemical and functional observations. First, mutations of this motif in the context of full-length SHP-1 result in the loss of lipid raft localization of SHP-1. Second, this motif alone restores raft localization when fused to a mutant of SHP-1 (SHP-1 DeltaC) that fails to localize to rafts. Third, a peptide encompassing the 6-mer motif directly binds to phospholipids whereas a mutation of this motif abolishes lipid binding. Fourth, whereas full-length SHP-1 potently inhibits TCR-induced tyrosine phosphorylation of specific proteins, expression of a SHP-1-carrying mutation within the 6-mer motif does not. Additionally, although SHP-1 DeltaC was functionally inactive, the addition of the 6-mer motif restored its functionality in inhibiting TCR-induced tyrosine phosphorylation. Finally, this 6-mer mediated targeting of SHP-1 lipid rafts was essential for the function of this phosphatase in regulating IL-2 production downstream of TCR. Taken together, these data define a novel 6-mer motif within SHP-1 that is necessary and sufficient for lipid raft localization and for the function of SHP-1 as a negative regulator of TCR signaling.
Introduction of magnetisable solid phase extraction procedures have provided various advantages over spin-column based extraction techniques. Although certain methods for magnetic bead based ...extraction of DNA from human saliva already exist, there is still a need to address the inadequate purity profile and low yield which occur due to the inefficiency of extraction methods. Hence, an improved method for DNA extraction from human saliva using uncoated magnetic nanoparticles (MNPs) intended to resolve the issues mentioned above is described here. The uncoated magnetic nanoparticles used in this study facilitate reversible binding of DNA and due to the absence of surface coating the particle size remains small thereby providing higher surface area to volume ratio for binding DNA. Another objective of this study was to develop a saliva preservation buffer (SPB) to solve the major challenges associated with storage and easy transportation of saliva sample at room temperature. Human saliva samples stored in the saliva preservation buffer were stable up to 160 days at room temperature without any bacterial or fungal growth and the quality of genomic DNA was intact.
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•Saliva preservation buffer formulation for long term storage of saliva samples at room temperature.•No deterioration in the quality of stored DNA and is compatible with downstream applications.•Magnetic nanoparticles based DNA extraction: quick and robust method.•High yield and purity for DNA compared to other extraction methods.•Co-extraction and detection of human and microbial DNA.
A method for immobilization of functional proteins by chemical cross-linking of the protein of interest and uncoated iron oxide nanoparticles in the presence of Epichlorohydrin is described. As a ...result of the cross-linking, the proteins form a matrix in which the particles get entrapped. The optimum concentration of Epichlorohydrin that facilitates immobilization of protein without affecting the functional properties of the protein was determined. This method was used to immobilize several functional proteins and the development and functional activity of Protein A-magnetic nanoparticles (MNPs) is described here in detail. The Protein A-MNPs possess high binding capacity due to the increased surface area of uncoated nanoparticles and robust magnetic separation due to the absence of polymeric coating materials. Protein A-MNPs were successfully used for purification of antibodies and also for immunoprecipitation. We also immobilized enzymes such as horse radish peroxidase and esterase and found that by providing the optimum incubation time, temperature and protein to nanoparticle ratio, we can retain the activity and improve the stability of the enzyme. This study is the first demonstration that Epichlorohydrin can be used to entrap nanoparticles in a cross-linked matrix of protein without impairing the activity of immobilized protein.
The importance of regulatory T cells (Tregs) for immune tolerance is well recognized, yet the signaling molecules influencing their suppressive activity are relatively poorly understood. In this ...article, through in vivo studies and complementary ex vivo studies, we make several important observations. First, we identify the cytoplasmic tyrosine phosphatase Src homology region 2 domain-containing phosphatase 1 (SHP-1) as an endogenous brake and modifier of the suppressive ability of Tregs; consistent with this notion, loss of SHP-1 expression strongly augments the ability of Tregs to suppress inflammation in a mouse model. Second, specific pharmacological inhibition of SHP-1 enzymatic activity via the cancer drug sodium stibogluconate potently augmented Treg suppressor activity both in vivo and ex vivo. Finally, through a quantitative imaging approach, we directly demonstrate that Tregs prevent the activation of conventional T cells and that SHP-1-deficient Tregs are more efficient suppressors. Collectively, our data reveal SHP-1 as a critical modifier of Treg function and a potential therapeutic target for augmenting Treg-mediated suppression in certain disease states.
The homeodomain factor Pdx-1 regulates an array of genes in the developing and mature pancreas, but whether regulation of each specific gene occurs by a direct mechanism (binding to promoter elements ...and activating basal transcriptional machinery) or an indirect mechanism (via regulation of other genes) is unknown. To determine the mechanism underlying regulation of the insulin gene by Pdx-1, we performed a kinetic analysis of insulin transcription following adenovirus-mediated delivery of a small interfering RNA specific for pdx-1 into insulinoma cells and pancreatic islets to diminish endogenous Pdx-1 protein. insulin transcription was assessed by measuring both a long half-life insulin mRNA (mature mRNA) and a short half-life insulin pre-mRNA species by real-time reverse transcriptase-PCR. Following progressive knock-down of Pdx-1 levels, we observed coordinate decreases in pre-mRNA levels (to about 40% of normal levels at 72 h). In contrast, mature mRNA levels showed strikingly smaller and delayed declines, suggesting that the longer half-life of this species underestimates the contribution of Pdx-1 to insulin transcription. Chromatin immunoprecipitation assays revealed that the decrease in insulin transcription was associated with decreases in the occupancies of Pdx-1 and p300 at the proximal insulin promoter. Although there was no corresponding change in the recruitment of RNA polymerase II to the proximal promoter, its recruitment to the insulin coding region was significantly reduced. Our results suggest that Pdx-1 directly regulates insulin transcription through formation of a complex with transcriptional coactivators on the proximal insulin promoter. This complex leads to enhancement of elongation by the basal transcriptional machinery.
The homeodomain factor Pdx-1 regulates an array of genes in the developing and mature pancreas, but whether regulation of each specific gene occurs by a direct mechanism (binding to promoter elements ...and activating basal transcriptional machinery) or an indirect mechanism (via regulation of other genes) is unknown. To determine the mechanism underlying regulation of the insulin gene by Pdx-1, we performed a kinetic analysis of insulin transcription following adenovirus-mediated delivery of a small interfering RNA specific for pdx-1 into insulinoma cells and pancreatic islets to diminish endogenous Pdx-1 protein. insulin transcription was assessed by measuring both a long half-life insulin mRNA (mature mRNA) and a short half-life insulin pre-mRNA species by real-time reverse transcriptase-PCR. Following progressive knock-down of Pdx-1 levels, we observed coordinate decreases in pre-mRNA levels (to about 40% of normal levels at 72 h). In contrast, mature mRNA levels showed strikingly smaller and delayed declines, suggesting that the longer half-life of this species underestimates the contribution of Pdx-1 to insulin transcription. Chromatin immunoprecipitation assays revealed that the decrease in insulin transcription was associated with decreases in the occupancies of Pdx-1 and p300 at the proximal insulin promoter. Although there was no corresponding change in the recruitment of RNA polymerase II to the proximal promoter, its recruitment to the insulin coding region was significantly reduced. Our results suggest that Pdx-1 directly regulates insulin transcription through formation of a complex with transcriptional coactivators on the proximal insulin promoter. This complex leads to enhancement of elongation by the basal transcriptional machinery.
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While the importance of regulatory T (Treg) cells for immune tolerance has been increasingly appreciated, our mechanistic and molecular understanding of their function is still very ...limited. We have previously shown that mice lacking the SHP‐1, so‐called
motheaten
(
me/me
) mice, have increased numbers of Treg cells. SHP‐1 is a well‐recognized negative regulator of TCR‐mediated signaling. We therefore asked whether SHP‐1 also plays a role in Treg function. A comparison of the suppressive capabilities of wild type and
me/me
Treg cells demonstrated increased suppressive activity of
me/me
Treg cells in
in vitro
suppression assays. While this suppression is dependent on stimulation via the TCR, our data indicate that additional factors, which are critical for an effective Treg function, are regulated by SHP‐1. Interestingly when comparing wild type and
me/me
conventional T cells, we observed that SHP‐1 deficient CD4+CD25‐ T cells are more resistant to Treg‐mediated suppression than wild type T cells, indicating a role for SHP‐1 both in suppression as well as in resistance to suppression. We are currently addressing at the molecular level how SHP‐1 regulates these processes. We expect from these studies to not only better understand the regulatory role of SHP‐1 in conventional and Treg T cells but to also gain further insights into the mechanism of suppression by Treg cells.
This work was supported by the NIH grant RO1 AI48672.
Abstract
CD4+CD25+Foxp3+ regulatory T (Treg) cells are well recognized as mediators of peripheral immune tolerance. However, their function remains poorly understood at the molecular and mechanistic ...level. We have previously shown that mice, which are deficient in the protein tyrosine phosphatase SHP-1 ('motheaten' or me/me mice), have increased numbers of Treg cells. Since SHP-1 is a negative regulator of TCR-mediated signaling in conventional T cell, we investigated whether SHP-1 also plays a regulatory role in Treg cells.
A comparison of the suppressive activities demonstrated that Treg cells derived from me/me or me/+ mice suppressed lymphocyte proliferation in vitro more efficiently than Treg cells from wild type mice indicating that SHP-1 is a negative regulator of Treg cell-mediated suppression. Since based on micro array analyses, the differences in suppressive activities are not due to developmental disparities, our data suggest that SHP-1 directly regulates the suppressive activity of Treg cells. To gain a better mechanistic understanding of Treg cell-mediated suppression, flow cytometric and in particular ImageStream100 (Amnis) analyses were performed. These studies showed that in the absence of SHP-1, Treg cells express different levels of a number of surface and intracellular molecules. Moreover, SHP-1 deficiency promoted increased conjugate formation between Treg cells and antigen presenting cells as visualized by ImageStream100. Conjugate formation with SHP-1-deficient Treg cells caused a more efficient down-modulation of the co-stimulatory molecules CD80/CD86 on the dendritic cells. Taken together, our data indicate that SHP-1 regulates the function and activation status of Treg cells and that under conditions of SHP-1 deficiency, Treg cells become more potent.
Source: NIH, RO1 AI 048672
Abstract
The mechanistic and molecular understanding about factors that regulate the susceptibility and/or resistance of T cells to suppression by regulatory T cells (Treg) is very limited. The ...protein tyrosine phosphatase SHP-1 is a negative regulator of TCR-mediated signaling. SHP-1-deficient T cells hyper-proliferate in response to antigen stimulation. We therefore asked whether SHP-1 affects the susceptibility of CD4+ T cells to suppression by Treg cells. Our in vitro suppression assays showed that heterozygous (me/+) and homozygous (me/me) SHP-1-deficient CD4+ T cells were more resistant to Treg cell-mediated suppression than +/+ CD+ T cells, indicating that SHP-1 modulates the resistance to Treg cell suppression.
In order to gain a better understanding of the mechanism that controls the resistance to suppression, we examined the conjugate formation between CD4+ T cells and the antigen presenting cells (APCs). Using flow cytometric and ImageStream100 (Amnis) analyses, we observed that me/me and me/+ T cells form more conjugates with APCs than +/+ T cells both in the presence or absence of Treg cells. Moreover, +/+ T cells treated with an inhibitor of SHP-1 showed a comparable increased conjugate formation as SHP-1-deficient T cells indicating that the observed phenotype is T cell intrinsic and not due to developmental differences. Furthermore, SHP-1-deficient T cells exhibited elevated surface levels of ICAM-1. Ongoing studies are addressing whether ICAM-1 directly affects the resistance to suppression. Taken together, the increased resistance to suppression displayed by SHP-1-deficient T cells and the enhanced capability of forming conjugates with APCs compared to wild type T cells suggest that the ability of forming T cell-APC conjugates influences to the susceptibility and/or resistance of a T cells to suppression.
Source: NIH, RO1 AI 048672
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