Apoptosis-associated proteins p53 and APO-1/Fas (CD95) in brains of adult patients with Down syndrome.

In Down syndrome (DS), enhanced apoptosis (programmed cell death) may play a role in the pathogenesis of characteristic mental retardation and precocious dementia of Alzheimer-type. Upregulation of p53 and APO-1/Fas (CD95) precedes apoptosis in many cell types, and a potential role for these molecules has already been demonstrated in Alzheimer's disease (AD) and several other neurodegenerative diseases. We measured p53 and APO-1/Fas (CD95) protein in four different regions of cerebral cortex and cerebellum in nine adult DS patients with Alzheimer-like neuropathologic lesions compared to nine controls. Quantitative ELISA demonstrated higher frontal lobe (mean+/-SD: 0.10+0.035 vs. 0.041+/-0.016 ng/mg protein), temporal lobe (0.062+/-0.021 vs. 0.032+/-0.019 ng/mg protein) and cerebellar levels (0.078+/-0.030 vs. 0.039+/-0.032 ng/mg protein) of p53 protein, and higher temporal lobe (mean+/-SD: 12.3+/-4.3 vs. 5.3+/-2.0 U/mg protein) and cerebellar levels (5.9+/-1.4 vs. 2.9+/-1.1 U/mg protein) of APO-1/Fas (CD95) protein. The results suggest that p53- or APO-1/Fas (CD95)-associated apoptosis may be an important feature of neurodegeneration in DS.

Evidence for apoptosis in the fetal Down syndrome brain.

In Down syndrome, enhanced apoptosis (programmed cell death) may play a role in the pathogenesis of characteristic early mental retardation and precocious neurodegeneration of Alzheimer type. Various apoptosis-associated proteins (Bax, Bcl-2, Fas, p53, Hsp70, neuronal apoptosis inhibitory protein-like immunoreactivity) were investigated in four different cortical regions and the cerebellum of one fetal Down syndrome (35 weeks' gestation) postmortem brain sample compared with a control brain sample. The most impressive finding was an at least fivefold elevation of Bax protein together with decreased Bcl-2 values in all Down syndrome cerebral regions investigated. In addition, antiapoptotic, presumably caspase-inhibitory, principles like heat shock protein 70 and neuronal apoptosis inhibitory protein were also reduced. Whereas Fas protein, an important member of receptor-mediated apoptosis, was inconsistently altered, a rather surprising finding was reduced proapoptotic, regulatory protein p53 in four of five regions. The findings are in good agreement with the proposed role of the Bcl-2 protein family in regulating developmental (naturally occurring) apoptotic neuronal death and further suggest that developmental apoptosis may be inappropriately commandeered by so far undefined pathologic processes in Down syndrome.

Timing of peritoneal dialysis catheter removal after pediatric renal transplantation.

BACKGROUND: A peritoneal dialysis (PD) catheter is in place at the time of kidney transplantation in children receiving PD. Removal of the catheter eliminates the risk of catheter-related infections. However, the patient benefits from leaving the catheter in place if dialysis is necessary posttransplantation. There is currently no consensus on the proper timing of PD catheter removal after kidney transplantation in children. OBJECTIVE: To identify the risks and benefits of an in-dwelling PD catheter after renal transplantation in children. DESIGN: Retrospective single-center study of infectious complications and posttransplantation PD catheter use in 31 renal transplantations in 26 children. RESULTS: Peritoneal dialysis catheters were used postoperatively in 13 of the 31 transplantations. In 12 instances the catheter was needed during the first month after transplantation, and 2 of the patients involved did not have a catheter in place when needed. Six catheter-related infections occurred in 5 patients posttransplantation, with only 1 infection taking place within 1 month after transplantation. CONCLUSION: Our data suggest that the need for catheter use occurs predominantly during the first month, while infectious complications usually happen later. This strongly suggests that PD catheters should not be removed until approximately 1 month after kidney transplantation.

Heat shock protein-70 repairs proximal tubule structure after renal ischemia.

BACKGROUND: Recent studies have suggested a role of heat shock protein (HSP)-70 in cytoskeletal repair during cellular recovery from renal ischemia. The aim of this study was to test the hypothesis that HSP-70 interacts in vitro with cytoskeletal elements obtained from rat renal cortex during early reflow after renal ischemia. METHODS: Cellular proteins were fractionated into cytoskeletal pellets and noncytoskeletal supernatants by Triton X-100 extraction of rat renal cortex obtained after 15 minutes or 18 hours of reflow after 45 minutes of renal ischemia, or from controls. Aliquots of isolated pellets were coincubated with aliquots of isolated supernatants in different combinations. A repeat Triton extraction was performed, and differential distribution of Na, K-ATPase or HSP-70 was assessed by Western blots and densitometric analysis. RESULTS: Coincubation of cytoskeletal pellets obtained during early reflow after renal ischemia (exhibiting severe injury of the cytoskeletal anchorage of Na,K-ATPase) and noncytoskeletal supernatant obtained during later reflow (showing high HSP expression) resulted in specific translocation of HSP-70 from the supernatant into the pellet, functionally associated with dose-dependent stabilization of Na,K-ATPase within this cytoskeletal fraction. These effects could be reproduced by incubation with purified HSP-70 and were abolished by the addition of anti-HSP-70 antibodies. CONCLUSION: These data support the hypothesis that HSP-70 interacts with cytoskeletal elements during the restoration of proximal tubule cell structure and polarity after renal ischemia. This experimental approach represents a new in vitro assay to study further the role of HSP in cellular repair.

Low serial serum neopterin does not predict low risk for chronic renal graft rejection.

Research has provided new and potent immunosuppressants which can potentially stop ongoing rejection. Subclinical rejection is a particular problem in the pediatric age group and early identification of children at risk is of the utmost importance. Neopterin has been previously shown to be a non-specific but sensitive marker for immunologic activity. In this study we hypothesized that low serum neopterin in the 1st year after transplantation predicts a low risk of chronic rejection. We retrospectively analyzed serial neopterin data obtained beyond the early postoperative period in 21 children and correlated the peak and average with glomerular filtration rate (GFR) loss during the subsequent years (P = 0.63, NS, r = 0.10). Our results show that serum neopterin did not differ between the majority of children who developed chronic transplant dysfunction and children with stable transplant function beyond the early post-transplant period. Thus serum neopterin failed to delineate a low-risk population who might be spared more invasive diagnostic procedures such as protocol biopsy.

Reduction of delayed renal allograft function using sequential immunosuppression.

Previous data suggested that outcome in small children with cadaveric renal transplantation might be improved with sequential therapy. This protocol combines augmented immunosuppression [by including antibody induction (ATG)] with avoidance of nephrotoxic medication in the immediate postoperative phase (by delayed start of cyclosporin therapy). In this report, we describe effects of this approach in 12 consecutively transplanted small children of less than 5 years of age (mean 3.2 years) who received a cadaveric renal graft at our institution between 1991 and 1998. Up to 1996 triple therapy (prednisolone, azathioprine, cyclosporin) and since 1997 sequential therapy (prednisolone, azathioprine, ATG until serum creatinine <2 mg/dl, then cyclosporin) was used for immunosuppression. Five children had delayed graft function (45.4%), all of whom were treated with triple therapy including cyclosporin from the very beginning, whereas children treated by the sequential protocol gained immediate graft function (P<0.05). There was no statistical difference between the two protocols concerning frequency or severity of rejections (67% vs. 60%, all steroid responsive), difference in the incidence of either bacterial or viral infections, or between the incidence of hypertension. Although not reaching statistical significance, 1-year graft survival rates also increased from 60% for triple therapy to 80% for sequential therapy. In conclusion, our findings confirm previous studies showing that outcome in small children undergoing renal transplantation may be improved by specially tailored treatment protocols such as sequential therapy.

Reproducible erythroid aplasia caused by mycophenolate mofetil.

Anemia secondary to mycophenolate mofetil (MMF) was recently described in experimental animals. A clinical association between MMF and anemia has been observed, but there are no proven reports. We describe a girl with chronic graft failure who developed erythroid aplasia under immunosuppression with MMF. She showed prompt resolution when MMF was discontinued and a recurrence of this clinical course when MMF was restarted. As re-challenge with a medication is the most definitive approach for showing a direct relationship between the drug and the side effect, this case clearly demonstrates that MMF can cause erythroid aplasia.

Deficient brain snRNP70K in patients with Down syndrome.

The small nuclear ribonucleoprotein 70K (snRNP 70K; U1-70 kDa) is an integral part of the spliceosome, a large RNA-protein complex catalyzing the removal of introns from nuclear pre-mRNA. snRNP is one of the best-studied essential subunits of snRNPs, is highly conserved and its inactivation was shown to result in complete inhibition of splicing. Applying subtractive hybridization, we found a sequence with 100% identity to snRNP absent in fetal Down syndrome (DS) brain. This observation made us determine snRNP-mRNA steady-state levels and protein levels in brains of adult patients with DS. snRNP-mRNA and protein levels of five individual brain regions of DS and controls each, were determined by blotting techniques. snRNP-mRNA steady state levels were significantly decreased in DS brain. Performing Western blots with monoclonal and human antibodies, snRNP protein levels were decreased in several regions of DS brain, although one monoclonal antibody did not reveal different snRNP-immunoreactivity. Although decreased snRNP-protein could be explained by decreased mRNA-steady state levels, another underlying mechanism might be suggested: snRNP is one of the death substrates rapidly cleaved during apoptosis by interleukin-1-beta-converting enzyme-like (ICE) proteases, which was well-documented by several groups. As apoptosis is unrequivocally taking place in DS brain leading to permanent cell loses, decreased snRNP-protein levels may therefore reflect decreased synthesis and increased apoptosis-related proteolytic cleavage.

Peritoneal dialysate fluid composition determines heat shock protein expression patterns in human mesothelial cells.

BACKGROUND: Low biocompatibility of peritoneal dialysis fluids (PDF) contributes to mesothelial injury. We investigated whether the heat shock proteins (HSP)-27, HSP-72, and HSP-90 are differentially induced upon exposure of mesothelial cells to PDF and whether this was affected by selective modulation of the physicochemical properties of PDF. METHODS: Human mesothelial cells (Met5A and primary human mesothelial cells) were exposed to acidic lactate and glucose-monomer based PDF (CAPD2 and CAPD3), to control culture media, or to a neutral lactate and glucose-monomer-based PDF with reduced levels of glucose degradation products (BALANCE). Expression of HSP-27, HSP-72, and HSP-90 and cellular distribution of HSP-72 were assessed by Western blotting and immunocytochemistry. RESULTS: Mesothelial cells exhibited strong constitutive expression of HSP-27 and to a lesser extent HSP-72 and HSP-90. Exposure of the cells to CAPD2 and CAPD3 resulted in strong up-regulation of HSP-72. HSP-27 levels were slightly increased, but HSP-90 levels were unchanged upon exposure to CAPD2 or CAPD3. In contrast, exposure of the cells to BALANCE did not affect HSP-27 or HSP-72 expression. The acidic pH and glucose degradation products were found to be principal in mediating increased HSP-72 expression upon exposure to PDF. CONCLUSIONS: Analysis of HSP expression represents a novel tool to assess biocompatibility of PDF. Among the HSP investigated, HSP-72 is the most predictive and accurate parameter to assess mesothelial cell injury in the early phase of exposure to PDF.