Immune cell function assay does not identify biopsy-proven pediatric renal allograft rejection or infection.

Management of pediatric renal transplant patients involves multifactorial monitoring modalities to ensure allograft survival and prevent opportunistic infection secondary to immunosuppression. An ICFA, which utilizes CD4 T-cell production of ATP to assess immune system status, has been used to monitor transplant recipients and predict susceptibility of patients to rejection or infection. However, the validity of this assay to reflect immune status remains unanswered. In a two-yr retrospective study that included 31 pediatric renal transplant recipients, 42 patient blood samples were analyzed for immune cell function levels, creatinine, WBC (white blood cell) count, immunosuppressive drug levels, and viremia, concurrent with renal biopsy. T-cell ATP production as assessed by ICFA levels did not correlate with allograft rejection or with the presence or absence of viremia. ICFA levels did not correlate with serum creatinine or immunosuppressive drug levels, but did correlate with WBC count. The ICFA is unreliable in its ability to reflect immune system status in pediatric renal transplantation. Further investigation is necessary to develop methods that will accurately predict susceptibility of pediatric renal transplant recipients to allograft rejection and infection.

Haemodialysis is an effective treatment in acute metabolic decompensation of maple syrup urine disease.

Acute metabolic decompensation in maple syrup urine disease can occur during intercurrent illness and is a medical emergency. A handful of reports in the medical literature describe the use of peritoneal dialysis and haemodialysis as therapeutic inventions. We report the only patient from our centre to have haemodialysis performed in this setting. Combined with dietary BCAA restriction and calorific support, haemodialysis allows rapid reduction in plasma leucine concentrations considerably faster than conservative methods.

Rapid nonradioactive in situ hybridization for interleukin-2 mRNA with riboprobes generated using the polymerase chain reaction.

In situ hybridization is a technique with widespread application. However, its usefulness has been limited by the need for radioactive materials and the requirement for the DNA to be cloned onto an appropriate vector. We have utilized the polymerase chain reaction to directly incorporate a T7 RNA polymerase promoter sequence onto the cDNA for interleukin-2. Digoxigenin-labelled riboprobes were then synthesized using this PCR product as a template. The digoxigenin-labelled riboprobes were then used in non-radioactive in situ hybridization to detect messenger RNA for interleukin-2 in mitogen stimulated peripheral blood mononuclear cells. This methodology has the potential for widespread application in immunology and cytokine research.

Low frequency of infiltrating cells intensely expressing T cell cytokine mRNA in human renal allograft rejection.

Immunosuppressive drugs used in clinical transplantation block cytokine mRNA transcription in vitro, but clinical rejection episodes are common. An understanding of what cytokine message is transcribed would be helpful in determining what contributes to the success of immunosuppression and provide directions for further research aimed at targeting specific cytokines. Previous studies have examined cytokine mRNA in rejecting solid organ biopsies by the reverse transcriptase polymerase chain reaction (RT-PCR) with variable results. We used nonradioactive in situ hybridization with cytokine-specific riboprobes to determine the frequency of cells expressing cytokine mRNA in the allograft infiltrate. Kidney biopsies were obtained from patients receiving protocol biopsies and with clinical evidence of rejection. Fourteen biopsies with a pathologic diagnosis of rejection were studied. Eight showed no cytokine staining, 2 expressed IL-2, and 3 expressed IL-4 and IFN-gamma. The positive cells were present at a low frequency (mean 2, range 1-5 per 10 high-power fields). The proportion of kidney biopsies expressing detectable message for interleukin-2 (IL-2), interleukin-4 (IL-4), and interferon-gamma (IFN-gamma) by in situ hybridization were similar to those reported using RT-PCR. The novel finding is that these cytokines are expressed in a few strongly positive cells in the allograft infiltrate. The vast majority of infiltrating cells are negative. This suggests that either the biopsies were performed when cytokine message was not expressed at a high level or that in human allograft recipients the sustained expression of the cytokines IL-2, IL-4, and IFN-gamma may not be necessary for graft rejection.

Immune-activation gene expression in clinically stable renal allograft biopsies: molecular evidence for subclinical rejection.

BACKGROUND: A significant percentage of biopsies from stable, well-functioning renal allografts have histologic findings consistent with acute rejection or borderline rejection. The implication of this finding is not yet fully understood. We analyzed immune-activation gene transcripts in stable protocol biopsies to determine the extent of immunologic activity of graft-infiltrating cells in this setting. Histologic classification of the biopsies was based on the Banff criteria. To emphasize that the tissue samples were procured from grafts with no clinical evidence of impaired function, we interjected the term "subclinical" into the Banff terminology. This produced three histologic categories: normal, borderline subclinical rejection, and acute subclinical rejection. METHODS: We used competitive template polymerase chain reaction techniques to quantify transcript amounts for the constant region of the T-cell receptor beta chain; the cytokines, tumor necrosis factor alpha, interleukin (IL)-1beta, transforming growth factor beta, interferon gamma, IL-2, IL-4, IL-10, and IL-15; and the cytotoxic T lymphocyte effector molecules, granzyme B, perforin, and Fas ligand. RESULTS: We found that histologically normal biopsies were typically devoid of gene transcripts or had very low amounts. Conversely, biopsies with acute subclinical rejection by histologic examination had heightened amounts of transcripts for many of the genes assayed. Borderline subclinical rejection samples showed an intermediate amount of expression. CONCLUSIONS: These results demonstrate that histologic features of rejection are often accompanied by enhanced expression of pro-inflammatory gene transcripts, despite the absence of clinically overt graft dysfunction. As this state of subclinical rejection could prove detrimental to long-term graft function, a role for surveillance biopsies of stable grafts with intent to treat subclinical rejection should be considered.

Matching for private or public HLA epitopes reduces acute rejection episodes and improves two-year renal allograft function.

BACKGROUND: The current role of HLA matching in renal transplantation is controversial. Public HLA epitope matching has been suggested to be as advantageous as private HLA matching, with the added benefit of increasing recipients' access to well-matched grafts. METHODS: In this single-center study of 105 renal transplant recipients, we examined the association of HLA matching with early (0-3 months) and late (4-6 months) rejection episodes (RE), as well as renal allograft function up to 2 years after transplant. RESULTS: Poor HLA-DR, but not HLA-A or -B, matching was associated with early RE (0 DR matches, RE=2.7+/-0.19, 1 DR match, RE=2.37+/-0.18, vs. 2 DR matches, RE=1.5+/-0.38; P < 0.01). In contrast, poor HLA-B, but not HLA-A or -DR, matching was associated with late rejections (0 HLA-B matches, RE=1.1+/-0.51 vs. 1-2 HLA-B matches, RE=0.51+/-0.1; P < 0.004). HLA-B matching was also associated with a significantly lower serum creatinine (SCr) level at 24 months (0 HLA-B matches, SCr=178+/-20 micromol/L vs. SCr=132+/-6 micromol/L for 1-2 HLA-B matches; P < 0.025). Matching for 10 supertypic HLA-A and -B cross-reactive groups was associated with reduced late graft rejection (0-2 residue matches, RE=1.15+/-0.18 vs. RE=0.62+/-0.12 for 3 to 7 residue matches; P < 0.013) as well as a significantly lower SCr level at 24 months (0-2 residue matches, SCr=205+/-29 micromol/L vs SCr=131+/-6 micromol/L for 3 to 7 residue matches; P < 0.001) after transplantation. CONCLUSIONS: HLA-DR matching was associated with a reduced frequency of early rejection episodes, whereas HLA-B or residue/cross-reactive group matching was associated with a reduced frequency of late rejection episodes and improved graft function at 2 years.

Renal tubule Na,K-ATPase polarity in glucocorticoid-induced polycystic kidney disease.

Cyst formation in polycystic kidney disease (PKD) involves proliferation of cyst lining epithelial and changes in trans-epithelial fluid and electrolyte transport. In vitro studies have suggested that mislocation of Na,K-ATPase to the apical tubular surface may be an important component of cyst fluid transport. We undertook in vivo studies of Na,K-ATPase location using the "threshold" murine model of glucocorticoid-induced PKD (GIPKD). Using histological, immunohistochemical, and densitometric techniques, we compared cyst formation and the cellular location of Na,K-ATPase in suckling C3H (low threshold for GIPKD) and DBA (high threshold) mice given an inducing dose of 200 mg/kg methylprednisolone acetate. As expected, C3H mice demonstrated greater cyst formation as measured by proportion of section area occupied by the tubule lumen (26.7% vs 15.5%; p < 0.001). Cyst formation was associated with increased Na,K-ATPase staining and increased apical Na,K-ATPase location. MPA treatment in C3H mice resulted in apical staining that exceeded basolateral staining (35.3% of reference window vs 29.8%; p < 0.001). The relatively GIPKD-resistant DBA mice did not show such change in Na,K-ATPase location. These immunohistochemical studies suggest a role for Na,K-ATPase in renal cyst formation.

Enhanced immunohistochemical detection of autonomic nerve fibers, cytokines and inducible nitric oxide synthase by light and fluorescent microscopy in rat spleen.

We have developed enhanced immunohistochemical protocols for detecting autonomic nerve fibers and splenocyte-associated proteins in rat spleen. This includes norepinephrine-synthesizing enzymes (dopamine-beta hydroxylase (DBH) and tyrosine hydroxylase (TH)), neuropeptide Y (NPY), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), c-fos protein, inducible nitric oxide synthase (iNOS), and the macrophage cell marker ED1. Animals were divided into sham-operated and splenic nerve-sectioned groups for detection of DBH, TH, and NPY. For immunodetection of TNF-alpha, iNOS, IFN-gamma and c-fos, animals were injected IV with saline or 100 microg of lipopolysaccharide (LPS) and were sacrificed at various time intervals post injection. Rats were perfused with 4% paraformaldehyde, spleens removed and cryoprotected, and 50-microm floating sections were cut on a freezing microtome. Immunodetection was performed with various detection systems and substrate/chromogen solutions, and in some cases using pretreatment with proteinase K (PK) for antigen unmasking. PK pretreatment increased immunostaining for DBH, TH, NPY, IFN-gamma, iNOS, and ED1, and the improvement was concentration-dependent. Using NPY immunostaining to index the signal-to-noise ratio for various substrates and detection systems, we found that an alkaline phosphatase detection system with NBT/BCIP as a substrate was the best procedure for light microscopy, whereas the CY3-labeled secondary antibody technique proved optimal for fluorescent microscopy. Surgical transection of the splenic nerve eliminated all nerve fiber staining for DBH, TH, and NPY. TNF-alpha, IFN-gamma, c-fos, and iNOS proteins were observed in the spleen in a time-dependent manner after LPS stimulation. Fluorescent double labeling, visualized with fluorescent confocal scanning laser microscopy, revealed many NPY fibers distributed among the ED1-labeled macrophages. These results demonstrate that immunohistochemistry can be used to index the activational effects of an immune challenge on splenocytes in situ and verifies that splenic immune cells are innervated by the sympathetic nervous system.

Production of digoxigenin-labelled RNA probes and the detection of cytokine mRNA in rat spleen and brain by in situ hybridization.

Non-radioactive in situ hybridization is a sensitive method for determining the site of production for secretory molecules such as cytokines. We report here on the central and peripheral induction of proinflammatory cytokines by endotoxin, and outline procedures for the generation and application of rat-specific digoxigenin (Dig)-labelled RNA probes for the localization of mRNA by in situ hybridization. Rats were injected either intravenously (i.v.) or intracerebroventricularly (i.c.v.) with vehicle or lipopolysaccharide (LPS) and sacrificed at various time intervals post-injection. Rats were then perfused with 4% paraformaldehyde and the spleens and brains were removed and cryoprotected in 30% sucrose. Dig-labelled, rat-specific, antisense and sense RNA probes were generated by in vitro transcription from PCR-derived templates. Positive staining with all the antisense probes was cytoplasmic, whereas the sense probes showed no staining. Numerous tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) mRNA positive cells were observed in the marginal zone and in the red pulp of the spleen after iv LPS injections, whereas sections from saline-treated animals showed minimal cytokine mRNA expression. Cells positive for TNF-alpha and IL-1beta mRNA were detectable in the brain after i.c.v. injections of LPS, but not after icv injection of vehicle. An antisense probe for c-fos was utilized in these studies as a positive control for our procedure due to its anatomically specific expression in the rat brain after LPS. In conclusion we have demonstrated that in situ hybridization with Dig-labelled RNA probes is an efficient, sensitive and reliable tool to localize cytokine mRNA production in rat tissue.

Banff '05 Meeting Report: differential diagnosis of chronic allograft injury and elimination of chronic allograft nephropathy ('CAN').

The 8th Banff Conference on Allograft Pathology was held in Edmonton, Canada, 15-21 July 2005. Major outcomes included the elimination of the non-specific term "chronic allograft nephropathy" (CAN) from the Banff classification for kidney allograft pathology, and the recognition of the entity of chronic antibody-mediated rejection. Participation of B cells in allograft rejection and genomics markers of rejection were also major subjects addressed by the conference.

Pediatric renal biopsy in the ambulatory care environment.

The use of pediatric ambulatory care facilities to perform invasive procedures that have low morbidity is increasingly popular. Over a 2-year period, 46 pediatric renal biopsies were performed in an ambulatory care setting at the Winnipeg Children's Hospital, with the patient discharged the same day. There was no serious complications and adequate tissue was obtained in 45 cases. Renal biopsy may be safely performed on an outpatient basis on carefully selected patients by experienced operators in properly equipped facilities.

Nifedipine, verapamil and cyclosporin A pharmacokinetics in children.

We report two paediatric renal transplant patients in whom interaction with the calcium channel blocking agent verapamil resulted in reduced cyclosporin A (CyA) elimination. Prior therapy with another calcium channel blocking agent, nifedipine, did not affect CyA pharmacokinetics. We speculate that verapamil reduced CyA elimination in children via an effect upon the microsomal oxidase system that is independent of calcium channel activity.

Sensitized B lymphocytes contribute to acute allograft rejection.

The contribution of sensitized B lymphocytes to second-set allograft rejection has been relatively ignored despite their regular appearance in rejecting allografts. This study presents evidence that adoptively transferred sensitized B lymphocytes accelerate the rate of acute allograft rejection in a sublethally irradiated rat cardiac allograft model. Donors of reconstituting B lymphocytes were sensitized with three consecutive ACI skin grafts. Transplantation of a heart from an ACI strain donor into a Lewis strain recipient (complete RT1 mismatch) results in rejection in 6.8 +/- 0.3 days. When the allograft donor and recipient are irradiated with 650 cGy prior to transplantation, rejection occurs at 31.5 +/- 3.0 days. Irradiated recipients reconstituted with 10(6) syngeneic sensitized splenic B cells reject their grafts in 20.1 +/- 2.0 days, while reconstitution with 10(6) unsensitized syngeneic B cells has no effect on the rate of rejection (P = 0.0007). These data strongly suggest that sensitized B lymphocytes have a marked accelerating effect on the tempo of allograft rejection.