Failure of BCL-2 up-regulation in proximal tubular epithelial cells of donor kidney biopsy specimens is associated with apoptosis and delayed graft function

SUMMARY: In renal transplantation, postischemic acute renal failure (ARF) develops in more than 20% of patients. We investigated whether tubular epithelial cells obtained from donor kidneys without subsequent ARF express a different pattern of survival genes, compared with cells from kidneys exhibiting ARF. Donor kidney biopsy specimens were obtained before transplantation from eight recipients of cadaveric kidneys with primary graft function (CAD-PF), eight patients with biopsy-proven ARF without rejection (CAD-ARF), and eight recipients of living donor kidneys with primary graft function (LIV). One thousand proximal tubular epithelial cells per biopsy specimen were isolated by laser capture microdissection. Quantitative analysis of apoptosis and the apoptosis regulatory genes Bcl-2, Bcl-xL, and Bax were performed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling staining and real-time PCR, respectively. Primary cultures of human proximal tubular epithelial cells served as calibrator. The number of apoptotic cells was significantly higher in CAD-ARF compared with LIV and CAD-PF (1.5 +/- 1.1% [p < 0.05] vs. 0.3 +/- 0.2% vs. 0.4 +/- 0.2%; mean +/- SD). The apoptosis inhibitors Bcl-2 and Bcl-xL were significantly up-regulated in renal tubular cells of recipients without ARF compared with CAD-ARF. The ratios of Bcl-2/GAPDH normalized to calibrator were as follows: LIV 48 +/- 30, CAD-PF 38 +/- 55, and CAD-ARF 5 +/- 7 (p < 0.05). The corresponding ratios for Bcl-xL were as follows: LIV 6 +/- 6, CAD-PF 5 +/- 3, and CAD-ARF 1 +/- 1 (p < 0.05). No difference in the expression of the proapoptotic Bax could be observed. These data suggest that failure of proximal tubular cells to respond to injury by up-regulation of survival factors from the Bcl-2 family contributes to postischemic ARF in patients after cadaveric renal transplantation.     

Detection of perforin and granzyme A mRNA in infiltrating cells during infection of mice with lymphocytic choriomeningitis virus

The analysis of gene expression in cytotoxic T cells by in situ hybridization of serial liver and brain sections from mice infected with lymphocytic choriomeningitis virus (LCMV) and immunostaining with T cell marker- and virus-specific antibodies revealed a close histological association of infiltrating lymphocytes expressing the perforin and granzyme A genes with virally infected cells. Maximal frequency of perforin and granzyme A mRNA-containing cells on liver sections preceded by about 2 days maximal LCMV-specific cytotoxicity of the lymphoid liver infiltrating cells. These results are most consistent with an involvement of perforin and granzyme A in cell-mediated cytotoxicity in vivo.     

Favorably tipping the balance between cytopathic and regulatory T cells to create transplantation tolerance

Therapeutic application of broadly reactive anti-T cell antibodies can lead not only to potent immunosuppression but also to profound and long-lived T cell depletion. We reasoned that a strategy that almost exclusively targets activated cytopathic donor reactive T cells and spares immunoregulatory networks might prove to be an exceptionally potent and highly selective means of producing long-term engraftment and tolerance. Herein we show that the combined administration of rapamycin and agonist IL-2- and antagonist IL-15-related cytolytic fusion proteins provides for long-term engraftment/tolerance in exceptionally stringent allotransplant models by (1) limiting the early expansion of activated T cells, (2) preserving and even exaggerating their subsequent apoptotic clearance, and (3) further amplifying the depletion of these activated T cells by antibody-dependent mechanisms, while (4) preserving CD4+CD25+ T cell-dependent immunoregulatory networks.     

Kinetic fluorescence RT-PCR is highly sensitive for detection of germ-cell-tumor-specific transcripts in peripheral blood

The aim of our study was to determine whether conventional staging in patients with testicular germ-cell-tumors (GCT) could be supplemented by quantification of beta-human choriogonadotropin mRNA levels in peripheral blood using kinetic fluorescence RT-PCR. Blood samples from 41 patients with GCT of different clinical stages (CS) were pre-therapeutically examined by kinetic fluorescence RT-PCR with the LightCycler for beta-human chorionic gonadotropin (beta-HCG) mRNA expression levels. The controls comprised of samples taken from patients 3 months after treatment, from patients with inflammatory testicular diseases or non-germ-cell-tumors and from healthy males (n=66). Six positive results [cut-off level: normalized beta-HCG mRNA (Nbeta-HCG) >400 relative gene expression (RGE)] were found in controls (specificity 90.9%, 95% CI: 76.9-97.3%). The overall ratio of positive PCR results in the group of GCT patients was 82.92% (34/41) (CS I 18/23, CS IIa-b 6/7, CS >IIb 10/11) (sensitivity 82.9%, 95% CI: 65.1-91.2%). The average Nbeta-HCG level in patients with clinical stage I tumors was 63772.0+/-125720.5 (mean +/- standard deviation) relative gene expression (RGE), 35076.0+/-52253.5 RGE in those with CS IIa-b tumors and 87298.3+/-120895.3 RGE in those with CS >IIb tumors. Kinetic fluorescence RT-PCR for tumor-specific gene products is, in contrast to qualitative RT-PCR, a promising approach to improve conventional staging in clinical low-stage testicular germ-cell-tumors. With high specificity, its sensitivity is higher than that of the corresponding serum tumor marker (82.92% vs 48.72%).     

Human T lymphocyte activation induces tyrosine phosphorylation of α-tubulin and its association with the SH2 domain of the p59fyn protein tyrosine kinase

A glutathione-S-transferase-src-homology domain 2 (GST-SH2) fusion protein was employed to identify molecules interacting with the protein tyrosine kinase p59^fyn. Among several proteins which bound to the fyn SH2 domain in lysates of human Jurkat T lymphocytes, α- and β-tubulin were identified by N-terminal sequencing. Further analysis established that α-tubulin exists as a tyrosine-phosphorylated protein in Jurkat cells, where it interacts with p59^fyn, but not with p56^lck. By contrast, in untransformed resting human T lymphocytes α-tubulin is not detectable as a tyrosine phosphorylated protein. However, following T cell activation, it becomes rapidly phosphorylated on tyrosine residues and subsequently associates with the SH2 domain of fyn. Interestingly, constitutively tyrosine-phosphorylated α-tubulin that is able to interact with the fyn-SH2 domain is expressed in peripheral blood T lymphoblasts isolated from leukemic patients in the absence of external stimulation.     

Significant association of strictures and internal fistula formation in Crohn’s disease

Intestinal inflammation in Crohn’s disease (CD) may be complicated by the occurrence of strictures and fistulae. The pathogenesis of fistula formation is unknown. We therefore wanted to determine whether mechanical factors might contribute to the development of fistulae. Furthermore, we tried to define the path of internal fistulae through the muscular layer. For this purpose, surgical resection specimens from 42 consecutive patients with CD were prospectively studied. In gross examination the whole bowel was cut into circumferential cross sections 0.3 cm thick. Abnormal areas were histologically examined. Strictures were found in 38 patients (90.5%), and fistulae were observed in 27 (64.3%) patients. In 11 (40.7%) specimens fistulae were found within a stricture, in 15 (55.6%) at the proximal end, and in 1 (3.7%) no stricture was found. In 7 (25.9%) cases with fistulae, herniated mucosa was found within the muscularis propria or the subserosa. In 7 (25.9%) cases a blood vessel was identified near a fistula traversing the muscularis propria. From these findings we conclude that that mechanical factors may contribute to fistula formation. This is further supported by the fact that fistulae appear to traverse the muscular layer along piercing vessels. Received: 21 January 2000 / Accepted: 7 March 2000     

Transplantation of in vitro-generated<Emphasis Type="Italic"> Schistosoma mansoni</Emphasis> mother sporocysts into<Emphasis Type="Italic"> Biomphalaria glabrata</Emphasis>

Specific studies on schistosome gene functions require both access to the parasite stages, preferably the larvae, and to complete the life cycle. In the present study, we investigated whether short-term in vitro cultivation of sporocysts and surgical transplantation into snails could be combined to produce cercariae. Miracidia were maintained in vitro in the presence of Biomphalaria glabrata embryonic (Bge) cells or, alternatively, in Bge-cell-conditioned medium. The transformation of miracidia to mother sporocysts was observed in both cases. Two day-old sporocysts were transplanted into the cephalopedal sinus of recipient snails. Transplantation efficiencies varied between 16% and 43%, depending on the culture of the sporocysts in terms of the number of cercariae producing snails. Cercariae recovered from these snails were used to successfully infect hamsters, demonstrating that short term in vitro-generated sporocysts undergo normal cercariogenesis following transplantation. This combination of in vitro cultivation and transplantation may be useful for novel experimental approaches to investigate the genes involved in larval development or host-parasite molecular interactions.