Ravi Krishnan - Academia.edu (original) (raw)

Papers by Ravi Krishnan

Research paper thumbnail of Improved clinical outcome measures of knee pain and function with concurrent resolution of subchondral Bone Marrow Edema Lesion and joint effusion in an osteoarthritic patient following Pentosan Polysulphate Sodium treatment: a case report

BMC Musculoskeletal Disorders

Background: At present, there are no registered products for the treatment of subchondral Bone Ma... more Background: At present, there are no registered products for the treatment of subchondral Bone Marrow Edema Lesion (BML) and associated knee pain. Patients who do not respond to current anti-inflammatory therapies are left with limited treatment options, and may resort to operative management with Total Knee Arthroplasty (TKA). We report the use of Pentosan Polysulphate Sodium (PPS) for the treatment of BMLs of the knee. Case presentation: We report the case of a 70-year-old female with knee osteoarthritis presenting with a high level of knee pain, scoring 8 on the Numerical Rating Scale (NRS), and functional limitation demonstrating a poor Lysholm Knee Score of 37. MRI scans of the knee revealed subchondral BML in the medial femoral condyle and medial tibial plateau. The patient was administered a course of Pentosan Polysulphate Sodium (PPS) intramuscularly twice weekly, for 3 weeks. MRI scans 2 weeks post-treatment showed complete resolution of the bone marrow edema at the medial femoral condyle and medial tibial plateau with concomitant recovery from pain (NRS pain score of 0), and a 43% improvement of the Lysholm Knee Score. In addition, marked reduction in joint effusion was also demonstrated in the MRI scan post PPS therapy. Conclusion: The MRI interpretations demonstrate improved clinical outcome measures ensuing therapeutic intervention with PPS, and warranting further investigation into the efficacy of PPS in the treatment of BML associated pain and dysfunction in the osteoarthritic population via randomized controlled trial, or equivalent rigorous methodological technique.

Research paper thumbnail of Broad Th2 neutralization and anti-inflammatory action of pentosan polysulfate sodium in experimental allergic rhinitis

Immunity, inflammation and disease, Jan 12, 2017

Th2 cytokines like interleukin-4, -5, and -13 are regarded as important drivers of the immunopath... more Th2 cytokines like interleukin-4, -5, and -13 are regarded as important drivers of the immunopathology underlying allergic rhinitis (AR) and asthma. The present study explores the capacity of pentosan polysulfate sodium (PPS), a semi-synthetic heparin-like macromolecular carbohydrate, to bind Th2 cytokines and exert biological neutralization in vitro, as well as anti-inflammatory actions in vivo. The capacity of PPS to bind recombinant Th2 cytokines was tested with surface plasmon resonance (SPR) technology and biological Th2 neutralization was assessed by Th2-dependent proliferation assays. The in vivo anti-inflammatory action of PPS was studied using a validated Guinea-pig model of AR. Binding studies revealed a strong and specific binding of PPS to IL-4, IL-5, and IL-13 with IC values suggesting as stronger cytokine binding than for heparin. Cytokine binding translated to a biological neutralization as PPS dose dependently inhibited Th2-dependent cell proliferation. Topical admin...

Research paper thumbnail of ASCIA-P33: TH2 Neutralization and in Vivo Anti-Inflammatory Action of Pentosan Polysuphate Sodium (PPS) in an Allergic Rhinitis Model

Internal Medicine Journal, 2016

Outcome: Consumer feedback has been positive regarding the improved booking system, particularly ... more Outcome: Consumer feedback has been positive regarding the improved booking system, particularly by consumers awaiting multiple food allergy challenges. Data demonstrated increased utilisation of booking capacity by 10%. We project that over a year that would mean 32 more patients receive their booked provocative challenges rather that remaining on the Waiting list.

Research paper thumbnail of Anti-ovine VCAM-1 monoclonal antibodies inhibit adhesion and proliferation between sheep endothelial and mononuclear cells in vitro

Immun Cell Biol, 1997

This paper reports the production and characterization of three monoclonal antibodies (mAb) recog... more This paper reports the production and characterization of three monoclonal antibodies (mAb) recognizing ovine vascular cell adhesion molecule-1 (VCAM-1). The mAb were raised against sheep umbilical vein endothelial cells (ShUVEC) and flow cytometric analysis demonstrated that one mAb, QE4G9, was cross-reactive with human VCAM-1 expressed on Chinese hamster ovary cell transfectants. Protein modulation studies on ShUVEC and immunoperoxidase staining of inflamed renal tissue further indicated the reactivity of the other two ovine mAb, QE1F3 and QE2G4, with ovine VCAM-1. The flow cytometric profile of the three mAb on stimulated ShUVEC was identical to that observed with the cross-reactive anti-human VCAM- mAb, HAE2-1. Peak expression occurred between 6-12 h after stimulation, followed by a slight decrease to a plateau extending beyond 48 h. In functional assays, all mAb inhibited adhesion of PMA-activated sheep PBMC to stimulated ShUVEC. In addition, QE4G9 inhibited proliferation of sheep PBMC in the mixed lymphocyte-endothelial cell reaction (MLER) by 56%. The results demonstrate that the three anti-ovine VCAM-1 mAb recognize functional epitopes on sheep vascular endothelial cells. These mAb will be valuable tools in the investigation of VCAM-1 expression in various pathophysiological conditions using sheep models, and in the study of VCAM-1-mediated leucocyte-endothelial cell interactions, both in vitro and in vivo.

Research paper thumbnail of Prolongation of Sheep Corneal Allograft Survival by Ex Vivo Transfer of the Gene Encoding INTERLEUKIN-101

Transplantation, May 1, 2001

Modification of a donor cornea by gene therapy ex vivo has potential to modulate irreversible rej... more Modification of a donor cornea by gene therapy ex vivo has potential to modulate irreversible rejection, the major cause of corneal graft failure. Our aim was to transfer the gene encoding mammalian IL-10 to ovine donor corneas and to determine subsequent orthotopic corneal allograft survival in an outbred sheep model. The replicative capacity of ovine corneal endothelium was determined by autoradiography after deliberate injury. A replication-defective adenovirus was used to deliver the lacZ reporter gene to ovine corneas and transfected corneas were organ-cultured in vitro to allow transfection efficiency, duration of reporter gene expression, and toxicity attributable to the vector to be determined. A cDNA encoding full-length ovine IL-10 was cloned into an adenoviral vector that was used to transfect donor corneas ex vivo before transplantation. Orthotopic penetrating corneal transplantation was performed in outbred sheep. Sheep corneal endothelium was found to be essentially amitotic. Transfection of > 70% corneal endothelial cells was achieved with the viral vector and expression was maintained for 28 days in vitro. IL-10 mRNA was detectable in transfected, organ-cultured corneas for 21 days in vitro. Donor corneas transfected with cDNA encoding IL-10 showed significantly prolonged survival after penetrating keratoplasty (median 55 days, range 19 > or =300 days) compared with control corneas (median 20.5 days, range 18-32 days, P=0.011). Local gene therapy-mediated expression of the immunomodulatory cytokine IL-10 has the potential to reduce the incidence of corneal graft rejection and to prolong corneal allograft survival.

Research paper thumbnail of Method for Treating or Preventing a Pancreatic Dysfunction

Research paper thumbnail of Methods for treating obesity and/or metabolic syndrome

Research paper thumbnail of Methods for Increasing Osteoblastic Function

Research paper thumbnail of Some Biochemical Studies on the Adaptation Associated with Xylitol Ingestion in Rats

Immun Cell Biol, 1980

The mechanism of adaptation to dietary carbohydrates was investigated by examining cellular metab... more The mechanism of adaptation to dietary carbohydrates was investigated by examining cellular metabolism in the liver and gut lumen. The inclusion of 10% (w/w) glucose, fructose, sucrose, xylose, sorbitol, xylitol or arabitol in the diet of rats for 7 days had essentially no elTect on tbe ability of liver homogenates to produce '''COj from labelled glucose, fructose, xylose, sorbitol or xylitol. Moreover, no major changes were observed in the activities of hepatic enzymes. In these studies, diarrhoea and caeca! distension were only observed in those rats receiving dietary sugar alcohols.

Research paper thumbnail of Interleukin-17A-Induced Human Mesenchymal Stem Cells Are Superior Modulators of Immunological Function

Research paper thumbnail of Prolongation of Sheep Corneal Allograft Survival by Transfer of the Gene Encoding Ovine IL12-p40 but Not IL4 to Donor Corneal Endothelium1

Modification of a donor cornea by gene therapy ex vivo has potential to modulate irreversible rej... more Modification of a donor cornea by gene therapy ex vivo has potential to modulate irreversible rejection, the major cause of corneal graft failure. Our aim was to transfer the gene encoding mammalian IL-10 to ovine donor corneas and to determine subsequent orthotopic corneal allograft survival in an outbred sheep model. The replicative capacity of ovine corneal endothelium was determined by autoradiography after deliberate injury. A replication-defective adenovirus was used to deliver the lacZ reporter gene to ovine corneas and transfected corneas were organ-cultured in vitro to allow transfection efficiency, duration of reporter gene expression, and toxicity attributable to the vector to be determined. A cDNA encoding full-length ovine IL-10 was cloned into an adenoviral vector that was used to transfect donor corneas ex vivo before transplantation. Orthotopic penetrating corneal transplantation was performed in outbred sheep. Sheep corneal endothelium was found to be essentially amitotic. Transfection of > 70% corneal endothelial cells was achieved with the viral vector and expression was maintained for 28 days in vitro. IL-10 mRNA was detectable in transfected, organ-cultured corneas for 21 days in vitro. Donor corneas transfected with cDNA encoding IL-10 showed significantly prolonged survival after penetrating keratoplasty (median 55 days, range 19 > or =300 days) compared with control corneas (median 20.5 days, range 18-32 days, P=0.011). Local gene therapy-mediated expression of the immunomodulatory cytokine IL-10 has the potential to reduce the incidence of corneal graft rejection and to prolong corneal allograft survival.

Research paper thumbnail of Endothelium Not IL4 to Donor Corneal Gene Encoding Ovine IL12-p40 but Allograft Survival by Transfer of the Prolongation of Sheep Corneal

Research paper thumbnail of Cloning and expression of the ovine CD40 molecule and the inhibition of the mixed lymphocyte reaction by the ovine CD40e–EGFP fusion protein

Veterinary Immunology and Immunopathology, 2002

The CD40 molecule is a member of the tumour necrosis factor receptor (TNFR)-like supergene family... more 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.

Research paper thumbnail of Combined Anti-Vascular Cell Adhesion MOLECULE-1 and Anti-Leukocyte Function-Associated MOLECULE-1 Monoclonal Antibody Therapy Does Not Prolong Allograft Survival in an Ovine Model of Renal TRANSPLANTATION1

Transplantation, 1998

phoramidon abolishes the increases in endothelin-1 release induced by ischemia-hypoxia in isolate... more phoramidon abolishes the increases in endothelin-1 release induced by ischemia-hypoxia in isolated perfused guinea pig lungs. J Pharmacol Exp Ther 1992; 262: 1062. 9. Ruetten H, Thiemermann C, Vane JR. Effects of the endothelin receptor antagonist, SB 209670, on circulatory failure and organ injury in endotoxic shock in the anaethetized rat. Br J Pharmacol 1996; 118: 198. 10. Willette RN, Ohlstein EH, Mitchell MP, et al. Nonpeptide endothelin receptor antagonists: attenuation of acute hypoxiainduced pulmonary hypertension in the dog.

Research paper thumbnail of Dendritic cells, tolerance and transplantation

Nephrology, 2000

... Dendritic cells, tolerance and transplantation. Toby Coates,; Ravi Krishnan,; Graeme R. Russ.... more ... Dendritic cells, tolerance and transplantation. Toby Coates,; Ravi Krishnan,; Graeme R. Russ. Article first published online: 25 DEC 2001. DOI: 10.1046/j.1440-1797.2000.00520.x. Issue. Nephrology. Volume 5, Issue 1-2, pages 125–131, February/May 2000. Additional Information ...

Research paper thumbnail of Prolongation of Sheep Corneal Allograft Survival by Transfer of the Gene Encoding Ovine IL-12-p40 but Not IL-4 to Donor Corneal Endothelium

The Journal of Immunology, 2005

Research paper thumbnail of A simple technique for evaluation of methods of cell separation

Journal of Immunological Methods, 1985

A simple method is described for labelling cells with fluorescein and using them in artificial mi... more A simple method is described for labelling cells with fluorescein and using them in artificial mixtures to assess cell separation procedures. The method facilitates the examination of the variables in a separation procedure. It is thus possible to tailor a separation procedure (for example panning with monoclonal antibody) to suit the specific requirements of the experiment.

Research paper thumbnail of Effects of enalapril and hydralazine treatment and withdrawal upon cardiovascular hypertrophy in stroke-prone spontaneously hypertensive rats

Journal of Hypertension, 1992

To test the hypothesis that effects of angiotensin converting enzyme (ACE) inhibitors upon resist... more To test the hypothesis that effects of angiotensin converting enzyme (ACE) inhibitors upon resistance vessel structure are responsible for their ability to cause long-term reduction in blood pressure. Stroke-prone spontaneously hypertensive (SHRSP) and Wistar-Kyoto (WKY) rats were treated with enalapril or hydralazine from 4 to 15 weeks of age. Effects upon tail-cuff blood pressure, left ventricular hypertrophy and structural indices of the superior mesenteric artery (SMA) and its resistance vessels were assessed at 11 weeks of treatment and up to 11 weeks post-treatment. Left ventricular hypertrophy was assessed by left ventricular weight:body weight ratios. Evidence of vascular structural change was obtained from tissue weight:body weight ratios, levels of RNA, DNA and expression of alpha-actin and elastin messenger (m)RNA. The effects of enalapril and hydralazine upon left ventricular hypertrophy in SHRSP were consistent with their respective effects upon blood pressure. Both drugs prevented the development of medial hypertrophy in SMA and resistance vessels. This was accompanied by substantial reductions in RNA:DNA ratios. Alpha-actin mRNA levels were not affected by either drug but elastin mRNA levels were reduced by both drugs. During the first 12 days post-treatment there was evidence of structural change in SMA accompanying the increases in blood pressure but importantly not in the resistance vessels. The effects of enalapril upon left ventricular hypertrophy and mesenteric arterial hypertrophy are totally consistent with responses to blood pressure and the persistence of structural changes post-treatment does not underlie the ability of the ACE inhibitors to persistently suppress hypertension.

Research paper thumbnail of Rapamycin downregulates the inhibitory receptors ILT2, ILT3, ILT4 on human dendritic cells and yet induces T cell hyporesponsiveness independent of FoxP3 induction

Immunology Letters, 2008

Dendritic cells (DC) mediate potent alloimmune responses through the positive costimulatory molec... more Dendritic cells (DC) mediate potent alloimmune responses through the positive costimulatory molecules CD40, CD80 and CD86 while the negative costimulatory molecules, immunoglobulin-like transcript 2 (ILT2), ILT3 and ILT4 have been associated with tolerogenic DC function. Due to the pivotal role played by DC in immunity the effect of the immunosuppressive agent rapamycin (RAPA) on the expression profile of both positive and negative costimulatory molecules during human DC differentiation and maturation was investigated. During monocyte differentiation to DC an increase in the mean fluorescence intensity (MFI) for CD40, CD80, CD83 and CD86 was observed in association with a concomitant downregulation of ILT2, ILT3, ILT4 and HLA-G expression. While DC differentiation in the presence of RAPA (10nM) showed a reduction in the MFI for CD40, CD80 and CD86 expressing cells, treatment after differentiation had no effect on the expression of these costimulatory molecules. The inhibitory receptors were also downregulated by RAPA only when added during differentiation. In comparison to RAPA, Cyclosporin A (CsA) had relatively minor effects on DC phenotype whereas IFN-alpha showed induction of CD80, CD86 and HLA-G when added prior to differentiation. Functionally, RAPA-treated DC used as stimulators in a DC-T cell mixed lymphocyte reaction (MLR) showed 40% inhibition of T cell proliferation relative to untreated DC (P=0.001) whereas CsA-treated DC showed no difference, and IFN-alpha-treated DC stimulated T cell proliferation. Nevertheless, the induction of T cell hyporesponsiveness by coculture of RAPA-treated DC with T cells was not associated with the generation of increased numbers of FoxP3 positive T regulatory cells. In conclusion, although RAPA downregulated ILT2, ILT3 and ILT4 expression in DC, the inhibition of T cell proliferation by RAPA-treated DC is predominantly due to the reduction of CD40, CD80 and CD86 expression rather than the propensity to generate FoxP3 expressing regulatory cells.

Research paper thumbnail of IFN-γ generates maturation-arrested dendritic cells that induce T cell hyporesponsiveness independent of Foxp3+ T-regulatory cell generation

Immunology Letters, 2010

Interferon-gamma (IFN-gamma) is a proinflammatory cytokine that induces the proliferation of T-he... more Interferon-gamma (IFN-gamma) is a proinflammatory cytokine that induces the proliferation of T-helper 1 cells that contribute to allograft rejection. Surprisingly, allografts transplanted in IFN-gamma deficient mice are rapidly rejected, suggesting that this cytokine has a paradoxical role in regulating alloimmune responses. Since dendritic cells (DC) play an essential role in initiating allograft rejection the effect of IFN-gamma on DC differentiation, maturation and function in vitro were investigated. DC were differentiated with IL4/GMCSF and treated with IFN-gamma at day 0 (IFN-gamma-DC(0)) or day 5 (IFN-gamma-DC(5)) during maturation and compared with untreated DC (UT-DC). Flow cytometric analysis of IFN-gamma-DC(0) demonstrated a downregulation in the DC maturation marker CD83 by 90% whereas the expression of the inhibitory molecules ILT2, ILT3 and ILT4 were upregulated. Inhibition of relB mRNA expression (79%; p=0.01) and IL-12 (97%; p=0.02) compared to UT-DC further confirmed that IFN-gamma-DC(0) were 'maturation-arrested'. Moreover, IFN-gamma-DC(0) inhibited allogeneic T cell proliferation by 33% (p=0.02) compared to UT-DC. However, induction of T cell hyporesponsiveness by IFN-gamma-DC(0) was not regulated by the generation of CD4(+)Foxp3(+) T cells nor due to IFN-gamma induced inhibitory molecules, HLA-G and IDO. In contrast, IFN-gamma-DC(5) expressed higher levels of costimulatory molecules and MHC class II compared to UT-DC and did not cause T cell hyporesponsiveness. Thus, the timing of IFN-gamma treatment of monocytes prior to their differentiation to DC is critical for generating DC that regulate T cell function. IFN-gamma may therefore play a regulatory role in alloimmunity by acting on DC precursors.

Research paper thumbnail of Improved clinical outcome measures of knee pain and function with concurrent resolution of subchondral Bone Marrow Edema Lesion and joint effusion in an osteoarthritic patient following Pentosan Polysulphate Sodium treatment: a case report

BMC Musculoskeletal Disorders

Background: At present, there are no registered products for the treatment of subchondral Bone Ma... more Background: At present, there are no registered products for the treatment of subchondral Bone Marrow Edema Lesion (BML) and associated knee pain. Patients who do not respond to current anti-inflammatory therapies are left with limited treatment options, and may resort to operative management with Total Knee Arthroplasty (TKA). We report the use of Pentosan Polysulphate Sodium (PPS) for the treatment of BMLs of the knee. Case presentation: We report the case of a 70-year-old female with knee osteoarthritis presenting with a high level of knee pain, scoring 8 on the Numerical Rating Scale (NRS), and functional limitation demonstrating a poor Lysholm Knee Score of 37. MRI scans of the knee revealed subchondral BML in the medial femoral condyle and medial tibial plateau. The patient was administered a course of Pentosan Polysulphate Sodium (PPS) intramuscularly twice weekly, for 3 weeks. MRI scans 2 weeks post-treatment showed complete resolution of the bone marrow edema at the medial femoral condyle and medial tibial plateau with concomitant recovery from pain (NRS pain score of 0), and a 43% improvement of the Lysholm Knee Score. In addition, marked reduction in joint effusion was also demonstrated in the MRI scan post PPS therapy. Conclusion: The MRI interpretations demonstrate improved clinical outcome measures ensuing therapeutic intervention with PPS, and warranting further investigation into the efficacy of PPS in the treatment of BML associated pain and dysfunction in the osteoarthritic population via randomized controlled trial, or equivalent rigorous methodological technique.

Research paper thumbnail of Broad Th2 neutralization and anti-inflammatory action of pentosan polysulfate sodium in experimental allergic rhinitis

Immunity, inflammation and disease, Jan 12, 2017

Th2 cytokines like interleukin-4, -5, and -13 are regarded as important drivers of the immunopath... more Th2 cytokines like interleukin-4, -5, and -13 are regarded as important drivers of the immunopathology underlying allergic rhinitis (AR) and asthma. The present study explores the capacity of pentosan polysulfate sodium (PPS), a semi-synthetic heparin-like macromolecular carbohydrate, to bind Th2 cytokines and exert biological neutralization in vitro, as well as anti-inflammatory actions in vivo. The capacity of PPS to bind recombinant Th2 cytokines was tested with surface plasmon resonance (SPR) technology and biological Th2 neutralization was assessed by Th2-dependent proliferation assays. The in vivo anti-inflammatory action of PPS was studied using a validated Guinea-pig model of AR. Binding studies revealed a strong and specific binding of PPS to IL-4, IL-5, and IL-13 with IC values suggesting as stronger cytokine binding than for heparin. Cytokine binding translated to a biological neutralization as PPS dose dependently inhibited Th2-dependent cell proliferation. Topical admin...

Research paper thumbnail of ASCIA-P33: TH2 Neutralization and in Vivo Anti-Inflammatory Action of Pentosan Polysuphate Sodium (PPS) in an Allergic Rhinitis Model

Internal Medicine Journal, 2016

Outcome: Consumer feedback has been positive regarding the improved booking system, particularly ... more Outcome: Consumer feedback has been positive regarding the improved booking system, particularly by consumers awaiting multiple food allergy challenges. Data demonstrated increased utilisation of booking capacity by 10%. We project that over a year that would mean 32 more patients receive their booked provocative challenges rather that remaining on the Waiting list.

Research paper thumbnail of Anti-ovine VCAM-1 monoclonal antibodies inhibit adhesion and proliferation between sheep endothelial and mononuclear cells in vitro

Immun Cell Biol, 1997

This paper reports the production and characterization of three monoclonal antibodies (mAb) recog... more This paper reports the production and characterization of three monoclonal antibodies (mAb) recognizing ovine vascular cell adhesion molecule-1 (VCAM-1). The mAb were raised against sheep umbilical vein endothelial cells (ShUVEC) and flow cytometric analysis demonstrated that one mAb, QE4G9, was cross-reactive with human VCAM-1 expressed on Chinese hamster ovary cell transfectants. Protein modulation studies on ShUVEC and immunoperoxidase staining of inflamed renal tissue further indicated the reactivity of the other two ovine mAb, QE1F3 and QE2G4, with ovine VCAM-1. The flow cytometric profile of the three mAb on stimulated ShUVEC was identical to that observed with the cross-reactive anti-human VCAM- mAb, HAE2-1. Peak expression occurred between 6-12 h after stimulation, followed by a slight decrease to a plateau extending beyond 48 h. In functional assays, all mAb inhibited adhesion of PMA-activated sheep PBMC to stimulated ShUVEC. In addition, QE4G9 inhibited proliferation of sheep PBMC in the mixed lymphocyte-endothelial cell reaction (MLER) by 56%. The results demonstrate that the three anti-ovine VCAM-1 mAb recognize functional epitopes on sheep vascular endothelial cells. These mAb will be valuable tools in the investigation of VCAM-1 expression in various pathophysiological conditions using sheep models, and in the study of VCAM-1-mediated leucocyte-endothelial cell interactions, both in vitro and in vivo.

Research paper thumbnail of Prolongation of Sheep Corneal Allograft Survival by Ex Vivo Transfer of the Gene Encoding INTERLEUKIN-101

Transplantation, May 1, 2001

Modification of a donor cornea by gene therapy ex vivo has potential to modulate irreversible rej... more Modification of a donor cornea by gene therapy ex vivo has potential to modulate irreversible rejection, the major cause of corneal graft failure. Our aim was to transfer the gene encoding mammalian IL-10 to ovine donor corneas and to determine subsequent orthotopic corneal allograft survival in an outbred sheep model. The replicative capacity of ovine corneal endothelium was determined by autoradiography after deliberate injury. A replication-defective adenovirus was used to deliver the lacZ reporter gene to ovine corneas and transfected corneas were organ-cultured in vitro to allow transfection efficiency, duration of reporter gene expression, and toxicity attributable to the vector to be determined. A cDNA encoding full-length ovine IL-10 was cloned into an adenoviral vector that was used to transfect donor corneas ex vivo before transplantation. Orthotopic penetrating corneal transplantation was performed in outbred sheep. Sheep corneal endothelium was found to be essentially amitotic. Transfection of > 70% corneal endothelial cells was achieved with the viral vector and expression was maintained for 28 days in vitro. IL-10 mRNA was detectable in transfected, organ-cultured corneas for 21 days in vitro. Donor corneas transfected with cDNA encoding IL-10 showed significantly prolonged survival after penetrating keratoplasty (median 55 days, range 19 > or =300 days) compared with control corneas (median 20.5 days, range 18-32 days, P=0.011). Local gene therapy-mediated expression of the immunomodulatory cytokine IL-10 has the potential to reduce the incidence of corneal graft rejection and to prolong corneal allograft survival.

Research paper thumbnail of Method for Treating or Preventing a Pancreatic Dysfunction

Research paper thumbnail of Methods for treating obesity and/or metabolic syndrome

Research paper thumbnail of Methods for Increasing Osteoblastic Function

Research paper thumbnail of Some Biochemical Studies on the Adaptation Associated with Xylitol Ingestion in Rats

Immun Cell Biol, 1980

The mechanism of adaptation to dietary carbohydrates was investigated by examining cellular metab... more The mechanism of adaptation to dietary carbohydrates was investigated by examining cellular metabolism in the liver and gut lumen. The inclusion of 10% (w/w) glucose, fructose, sucrose, xylose, sorbitol, xylitol or arabitol in the diet of rats for 7 days had essentially no elTect on tbe ability of liver homogenates to produce '''COj from labelled glucose, fructose, xylose, sorbitol or xylitol. Moreover, no major changes were observed in the activities of hepatic enzymes. In these studies, diarrhoea and caeca! distension were only observed in those rats receiving dietary sugar alcohols.

Research paper thumbnail of Interleukin-17A-Induced Human Mesenchymal Stem Cells Are Superior Modulators of Immunological Function

Research paper thumbnail of Prolongation of Sheep Corneal Allograft Survival by Transfer of the Gene Encoding Ovine IL12-p40 but Not IL4 to Donor Corneal Endothelium1

Modification of a donor cornea by gene therapy ex vivo has potential to modulate irreversible rej... more Modification of a donor cornea by gene therapy ex vivo has potential to modulate irreversible rejection, the major cause of corneal graft failure. Our aim was to transfer the gene encoding mammalian IL-10 to ovine donor corneas and to determine subsequent orthotopic corneal allograft survival in an outbred sheep model. The replicative capacity of ovine corneal endothelium was determined by autoradiography after deliberate injury. A replication-defective adenovirus was used to deliver the lacZ reporter gene to ovine corneas and transfected corneas were organ-cultured in vitro to allow transfection efficiency, duration of reporter gene expression, and toxicity attributable to the vector to be determined. A cDNA encoding full-length ovine IL-10 was cloned into an adenoviral vector that was used to transfect donor corneas ex vivo before transplantation. Orthotopic penetrating corneal transplantation was performed in outbred sheep. Sheep corneal endothelium was found to be essentially amitotic. Transfection of > 70% corneal endothelial cells was achieved with the viral vector and expression was maintained for 28 days in vitro. IL-10 mRNA was detectable in transfected, organ-cultured corneas for 21 days in vitro. Donor corneas transfected with cDNA encoding IL-10 showed significantly prolonged survival after penetrating keratoplasty (median 55 days, range 19 > or =300 days) compared with control corneas (median 20.5 days, range 18-32 days, P=0.011). Local gene therapy-mediated expression of the immunomodulatory cytokine IL-10 has the potential to reduce the incidence of corneal graft rejection and to prolong corneal allograft survival.

Research paper thumbnail of Endothelium Not IL4 to Donor Corneal Gene Encoding Ovine IL12-p40 but Allograft Survival by Transfer of the Prolongation of Sheep Corneal

Research paper thumbnail of Cloning and expression of the ovine CD40 molecule and the inhibition of the mixed lymphocyte reaction by the ovine CD40e–EGFP fusion protein

Veterinary Immunology and Immunopathology, 2002

The CD40 molecule is a member of the tumour necrosis factor receptor (TNFR)-like supergene family... more 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.

Research paper thumbnail of Combined Anti-Vascular Cell Adhesion MOLECULE-1 and Anti-Leukocyte Function-Associated MOLECULE-1 Monoclonal Antibody Therapy Does Not Prolong Allograft Survival in an Ovine Model of Renal TRANSPLANTATION1

Transplantation, 1998

phoramidon abolishes the increases in endothelin-1 release induced by ischemia-hypoxia in isolate... more phoramidon abolishes the increases in endothelin-1 release induced by ischemia-hypoxia in isolated perfused guinea pig lungs. J Pharmacol Exp Ther 1992; 262: 1062. 9. Ruetten H, Thiemermann C, Vane JR. Effects of the endothelin receptor antagonist, SB 209670, on circulatory failure and organ injury in endotoxic shock in the anaethetized rat. Br J Pharmacol 1996; 118: 198. 10. Willette RN, Ohlstein EH, Mitchell MP, et al. Nonpeptide endothelin receptor antagonists: attenuation of acute hypoxiainduced pulmonary hypertension in the dog.

Research paper thumbnail of Dendritic cells, tolerance and transplantation

Nephrology, 2000

... Dendritic cells, tolerance and transplantation. Toby Coates,; Ravi Krishnan,; Graeme R. Russ.... more ... Dendritic cells, tolerance and transplantation. Toby Coates,; Ravi Krishnan,; Graeme R. Russ. Article first published online: 25 DEC 2001. DOI: 10.1046/j.1440-1797.2000.00520.x. Issue. Nephrology. Volume 5, Issue 1-2, pages 125–131, February/May 2000. Additional Information ...

Research paper thumbnail of Prolongation of Sheep Corneal Allograft Survival by Transfer of the Gene Encoding Ovine IL-12-p40 but Not IL-4 to Donor Corneal Endothelium

The Journal of Immunology, 2005

Research paper thumbnail of A simple technique for evaluation of methods of cell separation

Journal of Immunological Methods, 1985

A simple method is described for labelling cells with fluorescein and using them in artificial mi... more A simple method is described for labelling cells with fluorescein and using them in artificial mixtures to assess cell separation procedures. The method facilitates the examination of the variables in a separation procedure. It is thus possible to tailor a separation procedure (for example panning with monoclonal antibody) to suit the specific requirements of the experiment.

Research paper thumbnail of Effects of enalapril and hydralazine treatment and withdrawal upon cardiovascular hypertrophy in stroke-prone spontaneously hypertensive rats

Journal of Hypertension, 1992

To test the hypothesis that effects of angiotensin converting enzyme (ACE) inhibitors upon resist... more To test the hypothesis that effects of angiotensin converting enzyme (ACE) inhibitors upon resistance vessel structure are responsible for their ability to cause long-term reduction in blood pressure. Stroke-prone spontaneously hypertensive (SHRSP) and Wistar-Kyoto (WKY) rats were treated with enalapril or hydralazine from 4 to 15 weeks of age. Effects upon tail-cuff blood pressure, left ventricular hypertrophy and structural indices of the superior mesenteric artery (SMA) and its resistance vessels were assessed at 11 weeks of treatment and up to 11 weeks post-treatment. Left ventricular hypertrophy was assessed by left ventricular weight:body weight ratios. Evidence of vascular structural change was obtained from tissue weight:body weight ratios, levels of RNA, DNA and expression of alpha-actin and elastin messenger (m)RNA. The effects of enalapril and hydralazine upon left ventricular hypertrophy in SHRSP were consistent with their respective effects upon blood pressure. Both drugs prevented the development of medial hypertrophy in SMA and resistance vessels. This was accompanied by substantial reductions in RNA:DNA ratios. Alpha-actin mRNA levels were not affected by either drug but elastin mRNA levels were reduced by both drugs. During the first 12 days post-treatment there was evidence of structural change in SMA accompanying the increases in blood pressure but importantly not in the resistance vessels. The effects of enalapril upon left ventricular hypertrophy and mesenteric arterial hypertrophy are totally consistent with responses to blood pressure and the persistence of structural changes post-treatment does not underlie the ability of the ACE inhibitors to persistently suppress hypertension.

Research paper thumbnail of Rapamycin downregulates the inhibitory receptors ILT2, ILT3, ILT4 on human dendritic cells and yet induces T cell hyporesponsiveness independent of FoxP3 induction

Immunology Letters, 2008

Dendritic cells (DC) mediate potent alloimmune responses through the positive costimulatory molec... more Dendritic cells (DC) mediate potent alloimmune responses through the positive costimulatory molecules CD40, CD80 and CD86 while the negative costimulatory molecules, immunoglobulin-like transcript 2 (ILT2), ILT3 and ILT4 have been associated with tolerogenic DC function. Due to the pivotal role played by DC in immunity the effect of the immunosuppressive agent rapamycin (RAPA) on the expression profile of both positive and negative costimulatory molecules during human DC differentiation and maturation was investigated. During monocyte differentiation to DC an increase in the mean fluorescence intensity (MFI) for CD40, CD80, CD83 and CD86 was observed in association with a concomitant downregulation of ILT2, ILT3, ILT4 and HLA-G expression. While DC differentiation in the presence of RAPA (10nM) showed a reduction in the MFI for CD40, CD80 and CD86 expressing cells, treatment after differentiation had no effect on the expression of these costimulatory molecules. The inhibitory receptors were also downregulated by RAPA only when added during differentiation. In comparison to RAPA, Cyclosporin A (CsA) had relatively minor effects on DC phenotype whereas IFN-alpha showed induction of CD80, CD86 and HLA-G when added prior to differentiation. Functionally, RAPA-treated DC used as stimulators in a DC-T cell mixed lymphocyte reaction (MLR) showed 40% inhibition of T cell proliferation relative to untreated DC (P=0.001) whereas CsA-treated DC showed no difference, and IFN-alpha-treated DC stimulated T cell proliferation. Nevertheless, the induction of T cell hyporesponsiveness by coculture of RAPA-treated DC with T cells was not associated with the generation of increased numbers of FoxP3 positive T regulatory cells. In conclusion, although RAPA downregulated ILT2, ILT3 and ILT4 expression in DC, the inhibition of T cell proliferation by RAPA-treated DC is predominantly due to the reduction of CD40, CD80 and CD86 expression rather than the propensity to generate FoxP3 expressing regulatory cells.

Research paper thumbnail of IFN-γ generates maturation-arrested dendritic cells that induce T cell hyporesponsiveness independent of Foxp3+ T-regulatory cell generation

Immunology Letters, 2010

Interferon-gamma (IFN-gamma) is a proinflammatory cytokine that induces the proliferation of T-he... more Interferon-gamma (IFN-gamma) is a proinflammatory cytokine that induces the proliferation of T-helper 1 cells that contribute to allograft rejection. Surprisingly, allografts transplanted in IFN-gamma deficient mice are rapidly rejected, suggesting that this cytokine has a paradoxical role in regulating alloimmune responses. Since dendritic cells (DC) play an essential role in initiating allograft rejection the effect of IFN-gamma on DC differentiation, maturation and function in vitro were investigated. DC were differentiated with IL4/GMCSF and treated with IFN-gamma at day 0 (IFN-gamma-DC(0)) or day 5 (IFN-gamma-DC(5)) during maturation and compared with untreated DC (UT-DC). Flow cytometric analysis of IFN-gamma-DC(0) demonstrated a downregulation in the DC maturation marker CD83 by 90% whereas the expression of the inhibitory molecules ILT2, ILT3 and ILT4 were upregulated. Inhibition of relB mRNA expression (79%; p=0.01) and IL-12 (97%; p=0.02) compared to UT-DC further confirmed that IFN-gamma-DC(0) were 'maturation-arrested'. Moreover, IFN-gamma-DC(0) inhibited allogeneic T cell proliferation by 33% (p=0.02) compared to UT-DC. However, induction of T cell hyporesponsiveness by IFN-gamma-DC(0) was not regulated by the generation of CD4(+)Foxp3(+) T cells nor due to IFN-gamma induced inhibitory molecules, HLA-G and IDO. In contrast, IFN-gamma-DC(5) expressed higher levels of costimulatory molecules and MHC class II compared to UT-DC and did not cause T cell hyporesponsiveness. Thus, the timing of IFN-gamma treatment of monocytes prior to their differentiation to DC is critical for generating DC that regulate T cell function. IFN-gamma may therefore play a regulatory role in alloimmunity by acting on DC precursors.