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Biomedical subjects

H Robertson

Publications and source records attributed to H Robertson.

At least 19 recordsLinked to original sources
PubMed2006

Chronic allograft nephropathy: intraepithelial signals generated by transforming growth factor-beta and bone morphogenetic protein-7.

It has been suggested that TGFbeta can cause chronic allograft nephropathy by inducing epithelial to mesenchymal transition (EMT); some studies show a reverse transition can be produced by bone morphogenetic protein-7 (BMP7). The current study assessed the relative contribution of signals generated within tubular epithelial cells by TGFbeta and BMP7 in normal kidney and after transplantation. Epithelial cells in normal human kidneys expressed phosphorylated forms of both Smad2/3 and Smad1/5/8 within their nuclei; cell culture experiments showed that these signaling molecules were generated in response to TGFbeta and BMP7, respectively. Phospho(p)-Smad2/3 was expressed at increased levels by tubular epithelial cells during acute renal allograft rejection and chronic allograft nephropathy but pSmad1/5/8 was expressed at very low levels; this staining profile was associated with induction of the EMT marker, S100A4. Further in vitro experiments demonstrated that this pattern of Smad signaling was a consequence of the stimulation of tubular epithelial cells with TGFbeta in the absence of BMP7. Importantly, addition of BMP7 to TGFbeta-stimulated cells enhanced the expression of pSmad1/5/8 and reduced expression of S100A4. These results suggest that exogenous BMP7 could restore the homeostatic balance of pSmad signaling found in normal kidneys, thereby preventing or reversing the development of chronic allograft nephropathy.

Bone Morphogenetic Protein 7↗
PubMed2005

Study of apoptosis and heat shock protein (HSP) expression in hepatocytes following radiofrequency ablation (RFA).

BACKGROUND: Radiofrequency ablation (RFA) of the liver produces necrosis of the hepatocytes. Histological examination shows a sharp demarcation between ablated and normal liver tissue. This experiment was carried out to study the cellular injury produced by RFA in area surrounding the ablated tissue and effect of reperfusion on this zone. MATERIAL AND METHODS: Five pigs underwent RFA of liver parenchyma. Four pigs were sacrificed 30 min after RFA and one pig was sacrificed 5 days later. Ablated lesions including surrounding liver parenchyma was examined for apoptosis and HSP 70 expression. RESULTS: There was a zone of transition surrounding the necrotic ablated area that showed apoptosis as well as increased HSP 70 expression. This was more prevalent in the pig that was sacrificed 5 days later. CONCLUSION: RFA produces sub lethal injury in the zone of transition causing apoptosis and increase in HSP 70 expression. Increased HSP expression enhances immunogenicity of these cells that can have therapeutic implications for the treatment of liver.

Animals↗
PubMed2005

Phenotype of airway epithelial cells suggests epithelial to mesenchymal cell transition in clinically stable lung transplant recipients.

BACKGROUND: Obliterative bronchiolitis in chronic rejection of lung allografts is characterised by airway epithelial damage and fibrosis. The process whereby normal epithelium is lost and replaced by fibroblastic scar tissue is poorly understood, but recent findings suggest that epithelial cells can become fibroblasts through epithelial-mesenchymal transition (EMT). It is hypothesised that EMT occurs in lung allografts and plays a potential role in airway remodelling. METHODS: Sixteen stable lung transplant recipients underwent bronchoscopy with bronchoalveolar lavage (BAL), endobronchial biopsies, and bronchial brushings. Biopsy sections were stained for the fibroblast marker S100A4. Brushings were cultured on collagen, stained with anti-S100A4, and examined for further EMT markers including matrix metalloproteinase (MMP) zymographic activity and epithelial invasion through collagen coated filters. RESULTS: A median 15% (0-48%) of the biopsy epithelium stained for S100A4 in stable lung transplant recipients and MMP-7 co-localisation was observed. In non-stimulated epithelial cultures from lung allografts, S100A4 staining was identified with MMP-2 and MMP-9 production and zymographic activity. MMP total protein and activity was increased following stimulation with transforming growth factor (TGF)-beta1. Non-stimulated transplant epithelial cells were invasive and penetration of collagen coated filters increased following TGF-beta1 stimulation. CONCLUSIONS: This study provides evidence of EMT markers in lung allografts of patients without loss of lung function. The EMT process may represent a final common pathway following injury in more common diseases characterised by airway remodelling.

Adult↗
PubMed2005

Hypothesis: epithelial-to-mesenchymal transition is a common cause of chronic allograft failure.

Renal, hepatic, and lung allografts are compromised by aggressively deteriorating function. This chronic process is produced by an overall burden of organ damage, but the pathophysiology remains poorly understood. Rates of chronic rejection in the lung, for example, have not substantially improved over the last decade, despite new immunosuppressive drugs and improvements in surgical procedure. We present a hypothesis that epithelial-to-mesenchymal transition is a common cause of chronic allograft failure. Research in this area may provide insights into chronic rejection of kidney, liver, and lung allografts that impact on future therapeutic strategies.

Cell Differentiation↗
PubMed2001

Tubulitis after renal transplantation: demonstration of an association between CD103+ T cells, transforming growth factor beta1 expression and rejection grade.

BACKGROUND: Tubulitis is a defining feature for the diagnosis and management of acute renal allograft rejection. Lymphocytes extracted from rejecting renal tissue are known to express the alphaEbeta7-integrin (CD103), a receptor for E-cadherin expressed on epithelial cells. In this study, expression of CD103 was examined in situ in tubulitis associated with acute rejection. METHODS: Immuno-labeling detected CD8+ and CD103+ lymphocytes and E-cadherin on epithelial cells in cryostat sections from 34 diagnostic biopsy specimens and a limited number of transplant nephrectomies. CD8+ and CD103+ intratubular cells were enumerated as mean numbers per tubular crosssection and median values were compared between rejection grades as were median ratios of CD103+ to CD8+ cells. Active transforming growth factor (TGF) beta1 was quantified in paraffin sections by immunofluorescence and confocal microscopical analysis. A parallel in vitro study quantified CD103+ T cells after allospecific activation with and without exogenous TGFbeta1. RESULTS: CD8+ T cells were present in tubules and tubular interstitium in acute rejection. CD103+ T cells were restricted exclusively to the tubules. The numbers of intratubular CD8+ and CD103+ cells and the ratio of intratubular CD103+ to CD8+ cells increased significantly with tubulitis score (P values 0.005, 0.009, and 0.02, respectively). TGFbeta1 expression was wide-spread in tubules also increasing significantly with tubulitis score (P=0.034). In chronic rejection, CD103+ T cells and TGFbeta1 were present within both tubules and interstitial cell populations. The in vitro study demonstrated that addition of TGFbeta1 to activated, alloantigen-specific T cells increased the proportion of CD8+ cells that also expressed CD103. CONCLUSIONS: These data indicate that specific upregulation of the alphaEbeta7-integrin by activated, intratubular T cells in acute renal allograft rejection could be a consequence of exposure to high local concentrations of TGFbeta1. The capacity of CD103+ T cells to bind E-cadherin on tubular epithelial cells may be an important factor in the pathogenesis of specific tissue damage observed in acute renal allograft rejection.

Antigens, CD↗
PubMed2001

Dynamic expression of alternate splice forms of D-cbl during embryogenesis.

The Cbl family of proteins act as E3 ubiquitin-protein ligases and have been associated with the down regulation of a variety of receptor tyrosine kinases. Cbl proteins associate with many different cell signalling molecules suggesting that they may have functions outside of the RING finger-mediated ubiquitin ligase activity. The Drosophila melanogaster cbl gene (D-cbl) encodes two splice forms (Oncogene 19 (2000) 3299). Here we report on the differential expression of these isoforms during Drosophila embryogenesis. Both isoforms are maternally expressed but the long isoform of D-cbl is also transiently expressed in the invaginating mesoderm and later is specifically expressed in neurons of the central nervous system (CNS). Cbl protein is shown to be localised to axons of the longitudinal connectives and commissures in the central nervous system.

Alternative Splicing↗
PubMed2001

Distribution of lymphocytes of the alpha(E)beta(7) phenotype and E-cadherin in normal human urothelium and bladder carcinomas.

The primary aim of this work was to survey normal urothelium and transitional cell carcinoma (TCC) for the presence of T lymphocytes expressing the intraepithelial, CD103(+) phenotype. This antigen defines the alpha(E)beta(7)-integrin. The adhesive counter-receptor for alpha(E)beta(7) is E-cadherin, which is down-regulated during cancer progression. The secondary aim was to determine the pattern of distribution of CD103(+) lymphocytes in relation to E-cadherin expression in bladder cancer. Cryostat sections of normal bladder and TCC were treated with antibodies specific for human CD103, CD3, CD8 and E-cadherin. Visualization was performed by immunoperoxidase or alkaline phosphatase development with light and confocal microscopy. Dual staining and serial sections were used to assess the relationship between these antigens. Four samples of normal bladder and 26 TCC samples were assessed. Occasional T lymphocytes (CD3(+)) were seen in normal urothelium and lamina propria. In the urothelium the majority of these T lymphocytes (71%) were also CD8(+) and of these 68% expressed the CD103 marker. In the lamina propria 62% of the T lymphocytes were CD8(+) and 56% of these expressed the CD103 marker. In carcinomas significantly greater numbers of CD103(+) T lymphocytes were present in the surrounding stroma rather than infiltrating the carcinomas (P = 0.0006). Of those T lymphocytes infiltrating the tumours, 71% were CD8(+) and of these 58% expressed CD103. In the surrounding stroma 52% of lymphocytes were CD8(+) and 82% of this subset expressed CD103. Infiltration by CD103(+) lymphocytes was not related to the intensity of E-cadherin expression. T lymphocytes of the CD103(+) phenotype are present in normal urothelium where they may play a role in immunosurveillance. Rather than infiltrating into carcinomas, these cells predominate in the surrounding stroma which could suggest a failure of immune function.

Antigens, CD↗
PubMed2000

A Drosophila analogue of v-Cbl is a dominant-negative oncoprotein in vivo.

Cells rely on the ability to receive and interpret external signals to regulate growth, differentiation, and death. Positive transduction of these signals to the cytoplasm and nucleus has been extensively characterized, and genetic studies in Drosophila have made major contributions to the understanding of these pathways. Less well understood, but equally important, are the mechanisms underlying signal down-regulation. Here we report biochemical and genetic characterization of the Drosophila homologue of c-Cbl, a negative regulator of signal transduction with ubiquitin-protein ligase activity. A new isoform of D-Cbl, D-CblL, has been identified that contains SH3-binding and UBA domains previously reported to be absent. Genetic analysis demonstrates that Dv-cbl, analogous to the mammalian v-cbl oncogene, is a dominant negative mutation able to enhance signalling from the Drosophila Egfr and cooperate with activating mutations in the sevenless pathway to produce melanotic tumours. In addition, our data show genetic and biochemical links between D-Cbl and proteins involved in endocytosis and ubiquitination, suggesting that v-Cbl may exert its oncogenic effect by enhancing receptor signalling as a consequence of suppressing receptor endocytosis.

Alternative Splicing↗
PubMed2000

Intragraft proliferating T lymphocytes are associated with moderate acute pulmonary rejection.

Acute allograft rejection is characterized by infiltration of the donor organ by host lymphoid cells, predominantly T lymphocytes. However, the site of proliferation and clonal expansion of alloreactive T lymphocytes is not well defined in man. A group of normal transbronchial biopsies (TBB, n=9) from clinically well lung transplant recipients was compared to TBB showing acute rejection (at least grade A2, n=9), using CD3- and Ki67-specific antibodies to double-label proliferating T lymphocytes. Few double-labeled lymphocytes were present in the normal biopsies (range, 0-3 cells). However, five of the rejection biopsies contained significant numbers of proliferating T lymphocytes (range, 19-47; Fisher's exact test; P=0.029). Furthermore, this positive group contained all three cases of grade A3 rejection in the study, as well as a case with persistent grade A2 rejection on follow-up biopsy. These data demonstrate that T lymphocytes do proliferate in transplanted human lungs; such proliferation is associated with more severe rejection.

Acute Disease↗
PubMed2000

TGF-beta expression in renal transplant biopsies: a comparative study between cyclosporin-A and tacrolimus.

BACKGROUND: Chronic rejection is a major cause of graft dysfunction after kidney transplantation. This fibroproliferative disease may be promoted by overproduction of transforming growth factor beta (TGF-beta). Previous studies have suggested that CsA might increase production of this growth factor. The current study was designed to measure the expression of TGF-beta(b) in renal transplant biopsy specimens from patients undergoing immunosuppressive therapy with either CsA or tacrolimus (FK506). METHOD: Paraffin-embedded renal biopsy specimens were sectioned, dewaxed, and incubated with primary antibody against TGF-beta(b)1 latency-associated protein and active TGF-beta(b1). After washing, the sections were treated with secondary antibody conjugated with FITC. In each case, the sections were assessed by semi-quantitative scanning laser confocal microscopic method. RESULTS: There was no significant difference in latent TGF-beta(b) expression between biopsy specimens from patients receiving CsA and patients receiving FK506. However, biopsy specimens from patients receiving CsA expressed significantly more active TGF-beta(b1) than biopsy specimens from patients receiving FK506 (P<0.0001, Mann-Whitney test). DISCUSSION: The increased level of active TGF-beta1 expression in renal biopsy specimens of patients receiving CsA may indicate a mechanism of chronic rejection. However, these biopsies were performed to assess deranged renal function; therefore, the specimens may reflect events rather than differences in medication.

Biopsy↗
PubMed2000

Renal allograft rejection: beta-chemokine involvement in the development of tubulitis.

BACKGROUND: Tubulitis is a defining feature of renal allograft rejection. Graft dysfunction may result from damage inflicted on tubular epithelial cells by intratubular cytotoxic T lymphocytes. Graft cells are known to produce chemokines during acute rejection, but it is not known whether changes in expression of specific chemokines can influence the composition of the intratubular lymphocyte population. We examined expression of individual chemokines in biopsy sections showing different pathological rejection grades. METHODS: Sections from Banff-graded transplant biopsies were examined for the presence of beta-chemokines (MCP-1, MIP-1alpha, MIP-1beta, and RANTES) by immunofluorescence and semiquantitative confocal laser scanning microscopy. RESULTS: Beta-chemokines were expressed predominantly at the basolateral surface of tubular epithelial cells. Expression of MCP-1 and MIP-1beta was significantly higher in sections showing grade 2 rather than grade 1 acute rejection. RANTES and MIP-1alpha showed no significant variation in level of expression between rejection grades. CONCLUSIONS: Beta-chemokines are expressed by tubular epithelial cells during acute rejection. Consistent expression of RANTES and MIP-1alpha suggests a general role in recruiting T lymphocytes. However, MCP-1 and MIP-1beta may play a more subtle role in recruitment of specific T-cell subsets, such as Th1 cells, during acute cellular rejection.

Adolescent↗
PubMed2000

Active TGF-beta1 expression in kidney transplantation: a comparative study of cyclosporin-A (CyA) and tacrolimus (FK506).

Chronic rejection is a major cause of graft dysfunction following kidney transplantation. This fibroproliferative disease may be promoted by overproduction of transforming growth factor beta (TGF-beta). Previous studies have suggested that cyclosporin-A (CyA) might increase production of this growth factor. The current study was designed to measure the expression of TGF-beta in renal transplant biopsies from patients immunosuppressed with either CyA or tacrolimus. Paraffin-embedded renal biopsies were sectioned, dewaxed and incubated with primary antibody against active TGF-beta1 antibody. After washing, the sections were treated with secondary antibody conjugated with fluorescein isothiocyanate (FITC). In each case the sections were assessed by semi-quantitative scanning laser confocal microscopy. Biopsies from patients receiving CyA expressed significantly more active TGF-beta1 than biopsies from patients receiving tacrolimus (P < 0.0001, Mann-Whitney test). The increased level of active TGF-beta1 expression in renal biopsies of patients receiving CyA may indicate a mechanism of chronic rejection.

Biopsy↗
PubMed2000

Detection of cytomegalovirus (CMV) antigens in kidney biopsies and transplant nephrectomies as a marker for renal graft dysfunction.

Chronic rejection accounts for the greatest loss of renal allografts. HLA mismatching has been minimised by organ allocation and new immunosuppressive drugs have been employed, but the average cadaveric graft survival still does not exceed 12 years. Though the aetiology is multifactorial, one contributory factor for this condition is cytomegalovirus (CMV). Detection of CMV in kidney biopsies and sera can diagnose and monitor this inflammatory event and define its role in chronic nephropathy. Twenty five biopsies taken at the time of transplantation, 10 biopsies for graft dysfunction and tissue blocks from 20 explanted kidney grafts were collected and investigated for CMV antigens by immunohistochemistry. Tissue samples were snap frozen and cryostat sections were incubated with monoclonal antibodies for CMV antigens followed by immunoperoxidase staining. In 12 out of 20 transplant nephrectomies CMV antigens were found. Only two of these patients had clinical CMV disease. Time 0 biopsies from CMV seronegative donors (n = 11) and CMV seropositive donors (n = 14) were negative for CMV antigens. The prevalence of CMV antigens in grafts lost due to chronic rejection was 60%. These antigens were not found within the time 0 biopsies, but were detected in 30% of biopsies taken at the time of clinical graft dysfunction. CMV appears to contribute to chronic rejection even without clinical disease.

Antigens, Viral↗
PubMed2000

Enhancement of survival of stored dopaminergic cells and promotion of graft survival by exposure of human fetal nigral tissue to glial cell line--derived neurotrophic factor in patients with Parkinson's disease. Report of two cases and technical considerations.

The authors have studied the ability of glial cell line-derived neurotrophic factor (GDNF) to promote survival of human fetal dopaminergic tissue after a storage period of 6 days and subsequent implantation into the human putamen. The results indicate that GDNF promotes survival of stored dopaminergic cells. Cells stored without GDNF had a 30.1% decrease in survival time compared with those exposed to GDNF. Two patients with Parkinson's disease received bilateral putaminal implants of fetal dopaminergic cells exposed to GDNF for 6 days and showed enhancement of graft survival as assessed by positron emission tomography scanning. A mean increase of 107% in putaminal fluorodopa uptake from baseline values was observed 12 months postgrafting.

Aged↗