Vascular endothelial growth factor expression in human chronic renal allograft rejection.

BACKGROUND: Chronic renal allograft rejection is characterized by interstitial fibrosis and vasculopathy. Vascular endothelial growth factor (VEGF) is an endothelial mitogen with increased expression in inflammation and vasculopathy. METHODS: Renal tissue from 17 patients with chronic rejection was examined for VEGF protein and the presence of CD 68-positive macrophages, and compared to biopsies from patients with temporary allograft dysfunction, acute rejection, and native kidneys with thin membrane disease. RESULTS: In the chronic rejection group, there was markedly increased expression of VEGF protein in the interstitium (P<0.0001). In serial sections, VEGF colocalized with the expression of CD 68-positive macrophages. Significantly more macrophages were in the tubulointerstitium in tissue with chronic rejection than in those with temporary allograft dysfunction (P<0.005). Additionally, VEGF protein expression in the glomeruli and the vascular compartment of patients with chronic rejection was increased. CONCLUSION: The up-regulation of VEGF in chronic renal allograft rejection may be important in inflammation and development of fibrosis.

Early up-regulation of macrophages and myofibroblasts: a new marker for development of chronic renal allograft rejection.

BACKGROUND: Increased numbers of macrophages and myofibroblasts are observed to occur in chronic renal allograft rejection (CR). The aim of this study was to examine the expression of cellular markers for the macrophage and myofibroblast in early renal transplant biopsy specimens and correlate these findings with allograft outcome. METHODS: The first postengraftment biopsy specimens from 53 patients who underwent renal transplantation between January 1993 and December 1995 were studied using immunohistochemistry with antibodies to alpha-smooth muscle actin, which identifies myofibroblasts and CD68, a marker for monocytes and macrophages. Patients were followed until December 1998 (mean follow-up 4.7+/-1.2 years). RESULTS: Nine patients had progressed to CR by the time of the study, whereas 44 patients continued to have stable renal function. A marked increase in both macrophages (P=0.02) and myofibroblasts (P=0.04) was noted in the first biopsy specimen obtained after engraftment in the patients who developed CR compared with those with stable allograft function. There was a positive correlation between alpha-smooth muscle actin and collagen expression (P=0.0001). CONCLUSION: Significant increases in macrophages and myofibroblasts occur in the first renal biopsy specimen in those patients who later develop CR.

Time course of upregulation of fibrogenic growth factors and cellular infiltration in a rodent model of chronic renal allograft rejection.

BACKGROUND: Chronic Rejection (CR) is the leading cause of renal allograft dysfunction. Upregulation of growth factors has been shown in CR but the time point at which this occurs in not known. The aim of this study was to examine the time course of upregulation of growth factors and correlate this with the macrophage and myofibroblast interstitial infiltrate. METHODS: Using a rat model of CR (F344 kidney donor to Lewis recipient), infiltration by ED1 + macrophages and proliferation of alpha-smooth muscle actin (alpha-SMA) and desmin-expressing cells was examined using immunohistochemistry. In addition, expression of mRNA for interferon-gamma (IFN-gamma), transforming growth factor-beta (TGF-beta), basic-fibroblast growth factor (b-FGF) and vascular endothelial growth factor (VEGF) was studied using a semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) technique. Native Lewis rat kidney and Lewis-Lewis isografts were used as controls. RESULTS: Immunohistochemical staining of ED1 + cells showed a marked increase in the macrophage infiltrate of allografts compared to isografts at all time periods (P = 0.0002) peaking at weeks 8-12 after transplantation. Expression of alpha-SMA was also increased in allografts (P = 0.002). RT-PCR analysis showed that mRNA for TGF-beta was maximally upregulated in allografts in comparison to isografts at week 8 after engraftment (P = 0.05) and declined thereafter, although remained at elevated levels compared to controls. IFN-gamma and b-FGF gene expression was increased in allografts late in the post-transplantation period. CONCLUSION: Early infiltration of macrophages and production of TGF-beta1 was followed by later upregulation of fibrogenic growth factors and myofibroblasts associated with interstitial fibrosis and organ dysfunction.

Anergic self-reactive B cells present self antigen and respond normally to CD40-dependent T-cell signals but are defective in antigen-receptor-mediated functions.

B-cell tolerance to soluble protein self antigens such as hen egg lysozyme (HEL) is mediated by clonal anergy. Anergic B cells fail to mount antibody responses even in the presence of carrier-primed T cells, suggesting an inability to activate or respond to T helper cells. To investigate the nature of this defect, B cells from tolerant HEL/anti-HEL double-transgenic mice were incubated with a membrane preparation from activated T-cell clones expressing the CD40 ligand. These membranes, together with interleukin 4 and 5 deliver the downstream antigen-independent CD40-dependent B-cell-activating signals required for productive T-B collaboration. Anergic B cells responded to this stimulus by proliferating and secreting antibody at levels comparable to or better than control B cells. Furthermore, anergic B cells presented HEL acquired in vivo and could present the unrelated antigen, conalbumin, targeted for processing via surface IgD. In contrast, the low immunoglobulin receptor levels on anergic B cells were associated with reduced de novo presentation of HEL and a failure to upregulate costimulatory ligands for CD28. These defects in immunoglobulin-receptor-mediated functions could be overcome in vivo, suggesting a number of mechanisms for induction of autoantibody responses.

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for measurement of cytokine and growth factor mRNA expression with fluorogenic probes or SYBR Green I.

Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) is the method of choice for rapid and reproducible measurements of cytokine or growth factor expression in small samples. Fluorescence detection methods for monitoring real-time PCR include fluorogenic probes labelled with reporter and quencher dyes, such as Taqman probes or Molecular Beacons and the dsDNA-binding dye SYBR Green I. Fluorogenic (Taqman) probes for a range of human and rat cytokines and growth factors were tested for sensitivity and compared with an assay for SYBR Green I quantification using real-time fluorescence monitoring (PE Applied Biosystems Model 7700 sequence detector). SYBR Green I detection involved analysis of the melting temperature of the PCR product and measurement of fluorescence at the optimum temperature. Fluorogenic probes provided sensitive and reproducible detection of targets that ranged from low (<10 copies/reaction) to high (>107 copies/ reaction) expression. SYBR Green I gave reproducible quantification when the target gene was expressed at moderate to high levels (> or =1000 copies/reaction), but did not give consistently reproducible quantification when the target gene was expressed at low levels. Although optimization of melting temperature improved the specificity of SYBR Green I detection, in our hands it did not equal the reproducible sensitivity and specificity of fluorogenic probes. The latter method is the first choice for measurement of low-level gene expression, although SYBR Green I is a simple and reproducible means to quantify genes that are expressed at moderate to high levels.