Biomedical subjects

Ravi Krishnan

Publications and source records attributed to Ravi Krishnan.

6 recordsLinked to original sources

PubMed2006

Natural killer cells prime the responsiveness of autologous CD4+ T cells to CTLA4-Ig and interleukin-10 mediated inhibition in an allogeneic dendritic cell-mixed lymphocyte reaction.

Cytotoxic T-lymphocyte antigen 4 immunoglobulin (CTLA4-Ig) and interleukin (IL)-10 are immunomodulatory molecules which target CD28 costimulation by acting either directly or indirectly on the CD80/86 receptors on dendritic cells (DCs). This study examined the effect of combined treatment with CTLA4-Ig and IL-10 on T-cell responsiveness in a dendritic cell-mixed lymphocyte reaction (DC-MLR). T cells derived from nylon wool enrichment (NWT cells) demonstrated 15% (P = 0.006) and 10% (P = 0.0015) inhibition of proliferation with suboptimal doses of IL-10 (5 ng/ml) and CTLA4-Ig (20 ng/ml), respectively. Combined treatment with both agents resulted in 38% inhibition (P = 0.004) of the MLR response compared with untreated controls. In contrast to NWT cells, which consisted of CD4+, CD8+ and CD56+ (NK) cells, purified CD4+ T cells were less responsive to immunomodulation by CTLA4-Ig and IL-10. Repletion of the CD4+ T cells with NK cells restored IL-10 and CTLA4-Ig mediated immunomodulation, suggesting a role for NK cells in the regulation of DC-T-cell interactions. The specific effect of NK cells on DC activation was demonstrated by CD80 up-regulation on DCs in the absence of T cells. However, in the absence of DCs, NK cells augmented the proliferation of autologous CD4+ T cells stimulated by anti-CD3 monoclonal antibody (mAb), which was blocked by CTLA4-Ig. It is proposed that, in the MLR, immunomodulation by suboptimal CTLA4-Ig and IL-10 is influenced by cellular interactions of NK cells with DCs and T cells involving DC lysis and costimulation. Thus, NK cells prime both DCs and T cells to low doses of CTLA4-Ig and IL-10 during alloimmune responses, providing evidence for the potential interaction between innate and adaptive immunity.

Abatacept↗

PubMed2005

Prolongation of sheep corneal allograft survival by transfer of the gene encoding ovine IL-12-p40 but not IL-4 to donor corneal endothelium.

Immunological rejection is the major cause of human corneal allograft failure. We hypothesized that local production of IL-4 or the p40 subunit of IL-12 (p40 IL-12) by the grafted cornea might prolong allograft survival. Replication-deficient adenoviral vectors encoding ovine IL-4 or p40 IL-12 and GFP were generated and used to infect ovine corneas ex vivo. mRNA for each cytokine was detected in infected corneas, and the presence of secreted protein in corneal supernatants was confirmed by bioassay (for IL-4) or immunoprecipitation (for p40 IL-12). Sheep received uninfected or gene-modified orthotopic corneal allografts. Postoperatively, untreated corneas (n = 13) and corneas expressing GFP (n = 6) were rejected at a median of 21 and 20 days, respectively. Corneas expressing IL-4 (n = 6) underwent rejection at 18.5 days (p > 0.05 compared with controls) and histology demonstrated the presence of eosinophils. In contrast, corneas expressing p40 IL-12 (n = 9) showed prolonged allograft survival (median day to rejection = 45 days, p = 0.003). Local intraocular production of p40 IL-12 thus prolonged corneal graft survival significantly, but local production of the prototypic immunomodulatory cytokine IL-4 induced eosinophilia, inflammation, and rejection. These findings have important implications for the development of novel strategies to improve human corneal graft survival.

Adenoviridae↗

PubMed2005

Advancement flaps: a basic theme with many variations.

BACKGROUND: The advancement flap involves the linear advancement of tissue in one direction. Despite its straightforwardness and simple concept, it can be used to close a variety of defects, ranging from small defects on the scalp or extremities to large, complicated defects involving cosmetic units on the face. OBJECTIVES: To provide a basic and useful review for the indications, advantages, disadvantages, and techniques for the use of advancement flaps in the reconstruction of defects in dermatologic surgery. MATERIALS AND METHODS: We performed a literature search for articles discussing advancement flaps and compiled a brief review of our findings. RESULTS: The movement of the advancement flap must be balanced by the blood supply of the flap. The excision of Burow's triangles along various aspects of the advancement flap can increase movement and improve cosmesis of the flap. The types of advancement flaps discussed include the single advancement flap, double advancement flap, A-T flap (O-T flap), Burow's triangle flap (Burow's wedge flap), crescenteric advancement flap, island pedicle flap (V-Y flap), helical rim advancement flap, and facelift flap. CONCLUSION: Advancement flaps are versatile and useful basic flaps for repairing defects.

Humans↗

PubMed2004

Ovine dendritic cells transduced with an adenoviral CTLA4eEGFP fusion protein construct induce hyporesponsiveness to allostimulation.

CTLA4 (CD152) is a transmembrane molecule expressed on activated T cells and functions as a negative regulator of T cell activation upon binding to the costimulatory molecules CD80/86. In this study, CTLA4eEGFP constructs were engineered by cloning the extracellular domains of ovine and human CTLA4 (CTLA4e) 'in frame' with the enhanced green fluorescent protein (EGFP). Recombinant adenoviral vectors were generated by incorporation of the CTLA4eEGFP sequence into the adenoviral genome using homologous recombination in Esherichia coli. The functional activity of the adenoviral vectors was shown by the secretion of the CTLA4eEGFP upon infection of ovine fibroblasts and the binding of the fusion protein to the target ovine and human dendritic cells expressing CD80/86 receptors by flow cytometry. The EGFP tag facilitated molecular size determinations and quantification of the secreted ovine CTLA4 fusion protein by immunoprecipitation and enzyme-linked immunosorbent assay (ELISA), respectively, using anti-GFP mAbs. Ovine dendritic cells obtained from pseudoafferent lymphatic cannulation of sheep were characterized based on high major histocompatibility complex (MHC) class II expression and cross-reactivity with monoclonal antibodies to the human dendritic cell markers, CD83 and CMRF-56. In addition, ovine dendritic cells (DC) were transfected with the adenoviral CTLA4eEGFP and when used as stimulators in a mixed lymphocyte reaction showed a reduced capacity to induce allogeneic lymphocyte proliferation. This study verifies that the ovine CTLA4eEGFP fusion protein functions similarly to its human homologue and that DC modified with adenoviral CTLA4-EGFP may provide an effective therapeutic approach in targeting alloreactive T cells to prolong allograft acceptance in a preclinical ovine model of renal transplantation.

Adenoviridae↗

PubMed2003

Site-specific protein modification to identify the MutL interface of MutH.

We have mapped the region for the protein interaction site of the Escherichia coli mismatch repair protein MutH for its activator protein MutL by a site-specific protein modification approach. For this purpose we generated a cysteine-free variant of MutH and 12 variants thereof, each containing a single cysteine residue at surface positions selected on the basis of available structural and sequence information for MutH. All MutH variants displayed wild type activity both in vivo and in vitro. These variants were then site-specifically modified at their cysteine residues with thiol-specific reagents and then tested for their ability to be stimulated in their DNA cleavage activity by the activator protein MutL. Thereby we were able to identify a defined region in the MutH protein that is important for interaction with MutL, and most likely represents the MutL binding site of MutH.

Adenosine Triphosphatases↗

PubMed2002

Cloning and expression of the ovine CD40 molecule and the inhibition of the mixed lymphocyte reaction by the ovine CD40(e)-EGFP fusion protein.

The CD40 molecule is a member of the tumour necrosis factor receptor (TNFR)-like supergene family and plays a major role as a co-stimulatory molecule in the activation of T cells in response to antigens presented by dendritic cells. In this study, reverse transcription-PCR cloning was used to derive the sequence encoding ovine CD40. The ovine CD40 sequence demonstrated a similarity of 97, 76 and 64% with the bovine, human and murine sequences, respectively, at the nucleic acid level. The cysteine residues characteristic of the TNFR family and N-linked glycosylation sites are conserved. Furthermore, RNA analysis confirmed expression of CD40 mRNA in both ovine dendritic cells from lymphatic drainage and dermal fibroblasts in culture. In addition, cDNA encompassing the extracellular region of ovine CD40 (CD40(e)) was fused 'in-frame' with the enhanced green fluorescent protein (EGFP) to generate a fusion protein upon the transfection of Chinese hamster ovary (CHO) cells. Immunoprecipitation with an anti-GFP monoclonal antibody of a 78 kD a protein from conditioned medium of CHO transfectants confirmed that the CD40(e)-EGFP was secreted in the supernatant. All experiments were controlled with a pEGFP-N1 vector-blank construct. Moreover, the biological activity of ovine CD40(e)-EGFP was demonstrated by its ability to inhibit a two-way mixed lymphocyte reaction. Thus these observations confirm that ovine CD40 blockade inhibits co-stimulation mediated by CD40-CD40L (CD154) interactions as has been reported in murine and human studies.

Amino Acid Sequence↗