Increased urologic complications in children after kidney transplants for obstructive and reflux uropathy.

In the cyclosporine era, reports on pediatric kidney transplant (KTx) patients with obstructive and reflux uropathy are limited by small numbers, short follow-up, and/or lack of control groups. Our single-center study evaluated long-term outcomes (patient and graft survival, urinary tract infections [UTIs], urologic complications) in a large cohort of KTx recipients (<20 years old). We matched our 117 study patients with obstructive and reflux uropathy with 117 controls whose KTx was needed for other reasons; all 234 underwent their KTx between April 25, 1984, and October 23, 2002. The mean age was 8.0 +/- 6.2 years; mean follow-up, 133 +/- 67 months. The urologic complication rate was higher in study patients (43%) than in controls (11%) (p < 0.0001), as was the UTI rate (45% vs. 2%; p < 0.0001). The metabolic acidosis and UTI rates were higher in study patients who did (vs. did not) undergo bladder augmentation (p < 0.0001). We found no significant difference between study patients and controls in patient or graft survival, acute or chronic rejection, or mean estimated glomerular filtration rates. Unique to our study is the finding of higher metabolic acidosis and UTI rates in study patients who underwent bladder augmentation.

CD154+ graft antigen-specific CD4+ T cells are sufficient for chronic rejection of minor antigen incompatible heart grafts.

We used a defined model system to address the role of minor histocompatibility antigen-specific CD4+ T cells in chronic rejection. The coronary arteries of vascularized heart grafts expressing the model antigen ovalbumin developed intimal hyperplasia in normal recipients and those lacking CD8+ T cells but not in those lacking CD4+ T cells. Furthermore, purified ovalbumin-specific CD4+ T cells from T-cell antigen receptor transgenic mice caused intimal hyperplasia in ovalbumin-expressing heart grafts in lymphocyte-deficient mice. The graft antigen-specific CD4+ T cells only caused intimal hyperplasia when expressing CD154 and were found in the intima of the affected coronary arteries along with CD40+ cells, CD11c+ dendritic cells and CD11b+, Gr-1+ monocytes. These results show that minor histocompatibility antigen-specific CD4+ T cells are required to cause the classical vascular changes of chronic rejection. They are capable of doing so without contributions from other lymphocytes, and may cause intimal hyperplasia by using CD154 to stimulate other non-lymphoid cells in the intima.

Single-cell analysis of signal transduction in CD4 T cells stimulated by antigen in vivo.

Flow cytometry was used to study signaling events in individual CD4 T cells after antigen recognition in the body. Phosphorylation of c-jun and p38 mitogen-activated protein kinase was detected within minutes in all antigen-specific CD4 T cells in secondary lymphoid tissues after injection of peptide antigen into the bloodstream. The remarkable rapidity of this response correlated with the finding that most naive T cells are in constant contact with dendritic antigen-presenting cells. Contrary to predictions from in vitro experiments, antigen-induced c-jun and p38 mitogen-activated protein kinase phosphorylation did not depend on CD28 signals and was insensitive to inhibition by cyclosporin A. Our results highlight the efficiency of the in vivo immune response and underscore the need to verify which signaling pathways identified in vitro actually operate under physiological conditions.

CD47 (integrin-associated protein) engagement of dendritic cell and macrophage counterreceptors is required to prevent the clearance of donor lymphohematopoietic cells.

Integrin-associated protein (CD47) is a broadly expressed protein that costimulates T cells, facilitates leukocyte migration, and inhibits macrophage scavenger function. To determine the role of CD47 in regulating alloresponses, CD47(+/+) or CD47(-/-) T cells were infused into irradiated or nonconditioned major histocompatibility complex disparate recipients. Graft-versus-host disease lethality was markedly reduced with CD47(-/-) T cells. Donor CD47(-/-) T cells failed to engraft in immunodeficient allogeneic recipients. CD47(-/-) marrow was unable to reconstitute heavily irradiated allogeneic or congenic immune-deficient CD47(+/+) recipients. These data suggested that CD47(-/-) T cells and marrow cells were cleared by the innate immune system. To address this hypothesis, dye-labeled CD47(-/-) and CD47(+/+) lymphocytes or marrow cells were infused in vivo and clearance was followed. Dye-labeled CD47(-/-) cells were engulfed by splenic dendritic cells and macrophages resulting in the clearance of virtually all CD47(-/-) lymphohematopoietic cells within 1 day after infusion. Host phagocyte-depleted CD47(+/+) recipients partially accepted allogeneic CD47(-/-) T cells. Thus, dendritic cells and macrophages clear lymphohematopoietic cells that have downregulated CD47 density. CD47 expression may be a critical indicator for determining whether lymphohematopoietic cells will survive or be cleared.

Cutting edge: in vivo identification of TCR redistribution and polarized IL-2 production by naive CD4 T cells.

TCR aggregation at the point of contact with an APC is thought to play an important role in T cell signal transduction. However, this potentially important phenomenon has never been documented during an immune response in vivo. Here we used immunohistology to show that the TCR on naive Ag-specific CD4 T cells in the lymph nodes of mice injected with Ag redistributed to one side of the cell. In cases where the APC could be identified, the TCR was concentrated on the side of the T cell facing the APC. In those T cells that produced IL-2, the TCR and IL-2 localized to the same side of the cell. In vivo IL-2 production depended on costimulatory signaling through CD28, whereas TCR redistribution did not. These results show that Ag-stimulated CD4 T cells produce IL-2 in a polarized fashion and undergo CD28-independent TCR redistribution in vivo.

In vivo activation of antigen-specific CD4 T cells.

Physical detection of antigen-specific CD4 T cells has revealed features of the in vivo immune response that were not appreciated from in vitro studies. In vivo, antigen is initially presented to naïve CD4 T cells exclusively by dendritic cells within the T cell areas of secondary lymphoid tissues. Anatomic constraints make it likely that these dendritic cells acquire the antigen at the site where it enters the body. Inflammation enhances in vivo T cell activation by stimulating dendritic cells to migrate to the T cell areas and display stable peptide-MHC complexes and costimulatory ligands. Once stimulated by a dendritic cell, antigen-specific CD4 T cells produce IL-2 but proliferate in an IL-2--independent fashion. Inflammatory signals induce chemokine receptors on activated T cells that direct their migration into the B cell areas to interact with antigen-specific B cells. Most of the activated T cells then die within the lymphoid tissues. However, in the presence of inflammation, a population of memory T cells survives. This population is composed of two functional classes. One recirculates through nonlymphoid tissues and is capable of immediate effector lymphokine production. The other recirculates through lymph nodes and quickly acquires the capacity to produce effector lymphokines if stimulated. Therefore, antigenic stimulation in the presence of inflammation produces an increased number of specific T cells capable of producing effector lymphokines throughout the body.

TRANCE, a tumor necrosis factor family member, enhances the longevity and adjuvant properties of dendritic cells in vivo.

Mature dendritic cells (DCs) are powerful antigen presenting cells that have the unique capacity to migrate to the T cell zone of draining lymph nodes after subcutaneous injection. Here we report that treatment of antigen-pulsed mature DCs with tumor necrosis factor (TNF)-related activation-induced cytokine (TRANCE), a TNF family member, before immunization enhances their adjuvant capacity and elicits improved T cell priming in vivo, such that both primary and memory T cell immune responses are enhanced. By enumerating migratory DCs in the draining lymph nodes and by studying their function in stimulating naive T cells, we show that one of the underlying mechanisms for enhanced T cell responses is an increase in the number of ex vivo antigen-pulsed DCs that are found in the T cell areas of lymph nodes. These results suggest that the longevity and abundance of mature DCs at the site of T cell priming influence the strength of the DC-initiated T cell immunity in situ. Our findings have the potential to improve DC-based immunotherapy; i.e., the active immunization of humans with autologous DCs that have been pulsed with clinically significant antigens ex vivo.

Antigen-specific CD4+ T cells that survive after the induction of peripheral tolerance possess an intrinsic lymphokine production defect.

Injection of soluble foreign antigen without an adjuvant induces a state of antigen-specific immunological unresponsiveness. We investigated the cellular mechanisms that underlie this form of peripheral tolerance by physically tracking a small population of ovalbumin (OVA) peptide/I-Ad-specific, CD4+ T cell receptor (TCR) transgenic T cells following adoptive transfer into normal recipients. Injection of OVA peptide in the absence of adjuvant caused the antigen-specific T cells to proliferate for a brief period after which most of the T cells disappeared. The remaining OVA-specific T cells had converted to a memory phenotype but were poorly responsive in vivo as evidenced by a failure to accumulate in the draining lymph nodes following immunization with OVA peptide in adjuvant. These surviving T cells possessed a long-lasting, but reversible, defect in Il-2 and TNF-alpha production and in vivo proliferation, but did not gain capacity to produce Th2-type cytokines or suppress the clonal expansion of T cells specific for another antigen. Therefore, some antigen-specific T cells survive this peripheral tolerance protocol but are functionally unresponsive due to an intrinsic activation defect.

Visualization of specific B and T lymphocyte interactions in the lymph node.

Early events in the humoral immune response were visualized in lymph nodes by simultaneous tracking of antigen-specific CD4 T and B cells after immunization. The T cells were initially activated in the T cell areas when the B cells were still randomly dispersed in the B cell-rich follicles. Both populations then migrated to the edges of the follicles and interacted there, resulting in CD154-dependent B cell proliferation and germinal center formation. These results provide visual documentation of cognate T-B cell interactions and localize them to the follicular border.

In vivo detection of dendritic cell antigen presentation to CD4(+) T cells.

Although lymphoid dendritic cells (DC) are thought to play an essential role in T cell activation, the initial physical interaction between antigen-bearing DC and antigen-specific T cells has never been directly observed in vivo under conditions where the specificity of the responding T cells for the relevant antigen could be unambiguously assessed. We used confocal microscopy to track the in vivo location of fluorescent dye-labeled DC and naive TCR transgenic CD4(+) T cells specific for an OVA peptide-I-Ad complex after adoptive transfer into syngeneic recipients. DC that were not exposed to the OVA peptide, homed to the paracortical regions of the lymph nodes but did not interact with the OVA peptide-specific T cells. In contrast, the OVA peptide-specific T cells formed large clusters around paracortical DC that were pulsed in vitro with the OVA peptide before injection. Interactions were also observed between paracortical DC of the recipient and OVA peptide-specific T cells after administration of intact OVA. Injection of OVA peptide-pulsed DC caused the specific T cells to produce IL-2 in vivo, proliferate, and differentiate into effector cells capable of causing a delayed-type hypersensitivity reaction. Surprisingly, by 48 h after injection, OVA peptide-pulsed, but not unpulsed DC disappeared from the lymph nodes of mice that contained the transferred TCR transgenic population. These results demonstrate that antigen-bearing DC directly interact with naive antigen-specific T cells within the T cell-rich regions of lymph nodes. This interaction results in T cell activation and disappearance of the DC.

Use of adoptive transfer of T-cell-antigen-receptor-transgenic T cell for the study of T-cell activation in vivo.

Adoptive transfer of TCR-transgenic T cells uniformly expressing an identifiable TCR of known peptide/MHC specificity can be used to monitor the in vivo behavior of antigen-specific T cells. We have used this system to show that naive T cells are initially activated within the T-cell zones of secondary lymphoid tissue to proliferate in a B7-dependent manner. If adjuvants or inflammatory cytokines are present during this period, enhanced numbers of T cells accumulate, migrate into B-cell-rich follicles, and acquire the capacity to produce IFN-gamma and help B cells produce IgG2a. If inflammation is absent, most of the initially activated antigen-specific T cells disappear without entering the follicles, and the survivors are poor producers of IL-2 and IFN-gamma. Our results indicate that inflammatory mediators play a key role in regulating the anatomic location, clonal expansion, survival and lymphokine production potential of antigen-stimulated T cells in vivo.

Impact of age on renal graft survival in children after the first rejection episode.

Infants are thought to be more immunoreactive and at a greater risk for developing irreversible rejection compared with older children. We investigated this by analyzing patient and graft survival rates, incidence of acute rejection, reversibility of acute rejection, development of a subsequent acute rejection, and incidence of graft loss due to rejection in 154 children (< 18 years of age) after primary renal transplantation. Most patients (n = 139) were treated with quadruple immunosuppression (antibody, azathioprine, prednisone, cyclosporine). Treatment of the first acute rejection episode (ARE) consisted of antibody and increased prednisone (68%) or increased prednisone alone (30%), and was not significantly different between the age groups. Transplants were from living donors (LRD) in 80% of cases. Patients were followed for at least 1 year (mean 58 +/- 30 months); 68% (105/154) of recipients experienced 1 or more ARE. The incidence of ARE was significantly lower in patients < 2 years of age (45%) compared with patients 2-5 (76%, P = 0.01), 6-12 (78%, P = 0.005), and 13-17 (76%, P = 0.009) years of age. There was no significant difference in the 1-, 2- and 5-year patient or graft survival rates, the development of a subsequent acute rejection, or the incidence of graft loss due to acute rejection when analyzed by age group. These data suggest that the impact of an ARE is similar for younger and older children in our population receiving predominantly LRD transplants and quadruple immuno-suppression.

Focal segmental glomerulosclerosis in children.

Idiopathic focal segmental glomerulosclerosis is a histologic diagnosis that usually presents as the nephrotic syndrome but, unlike minimal change disease, often leads to renal failure in children. Standard therapies used to treat the proteinuria are often futile, and thus patients are at risk for the multiple complications resulting from persistent, severe proteinuria. Eventually, end-stage renal failure ensues, and the possibility of the disease recurring in the transplanted renal allograft is worrisome. This report reviews the clinical features and outcomes of idiopathic focal segmental glomerulosclerosis in children, the response to newer treatment options, and new insights into understanding what factors may be involved in causing the disorder.

Aggressive, long-term cyclosporine therapy for steroid-resistant focal segmental glomerulosclerosis.

Short-term cyclosporine (CsA) has been shown to reduce the proteinuria in refractory nephrotic syndrome, but the effect on disease progression has not been evaluated. This study was undertaken to evaluate whether maintenance CsA therapy in steroid-resistant focal segmental glomerulosclerosis (FSGS) will prevent progression to ESRD. Twenty-one black and Hispanic children (mean age, 8.4 +/- 4.5 yr) with biopsy-proven, steroid/cyclophosphamide-resistant FSGS were treated with CsA (initiated at 6 mg/kg per day and titrated to the serum cholesterol level to achieve a response). The mean CsA dose was 7 (4 to 20) mg/kg per day, the duration of CsA therapy was 27.5 (3 to 97) months, and the duration of follow-up was 8.5 +/- 4.7 yr. At the end of CsA therapy, the mean (+/- SE) proteinuria fell from 6.2 +/- 0.2 to 2.0 +/- 0.1 g/24 h (P < 0.001), the mean albumin rose from 1.95 +/- 0.04 to 3.41 +/- 0.04 g/dL (P < 0.001), the mean cholesterol decreased from 472 +/- 12.7 to 257 +/- 5.3 mg/dL (P < 0.005), and the mean creatinine rose from 0.79 +/- 0.02 to 1.16 +/- 0.03 mg/dL (P < 0.005). Seven children continue to receive maintenance CsA therapy, and 14 patients have had CsA stopped: 6 for an increase in serum creatinine and/or continued proteinuria, 5 for sustained remission, 2 for noncompliance, and 1 for pregnancy. Five (24%) of the 21 patients progressed to ESRD.(ABSTRACT TRUNCATED AT 250 WORDS)

An analytical review of growth hormone studies in children after renal transplantation.

With steady improvement in 1- and 5-year patient and graft survivals in the last decade, rehabilitation of the child is the major focus of the transplant physician. The notion that the elimination of the uremic milieu should enable children to grow has not been born out over time, and growth retardation continues to be a serious morbidity in many children despite a well-functioning renal allograft. In children with chronic renal failure prior to renal transplantation, recombinant human growth hormone (rhGH) has been recently shown in controlled trials to improve growth. The use of rhGH in children after renal transplantation is controversial, since uncontrolled studies have questioned its safety. Acute rejection and graft loss have been reported in children after the initiation of rhGH. This study analyzes the data regarding the safety and efficacy of rhGH in children after renal transplantation as presented in seven current published reports. The demographic characteristics of the patients, the dose and duration of rhGH therapy, the "growth" rates of the patients before and after rhGH therapy, renal function before and after rhGH therapy, and other possible complications are reviewed. Based on this analysis, suggestions for future studies are made.

Steroid withdrawal, rejection and the mixed lymphocyte reaction in children after renal transplantation.

With improved long-term graft survival after renal transplantation, cardiovascular mortality is emerging as the leading cause of death in adults and is also being reported in children. Chronic corticosteroid therapy is thought to be an important cause of post-transplant hyperlipidemia and hypertension. This study describes a steroid withdrawal protocol initiated to reduced cardiovascular risk factors in pediatric renal allograft recipients, reports on the rate of rejection observed and the use of an in vitro method to measure immunoresponsiveness and identify those patients who have not experienced a rejection episode. Thirty-six of 67 patients were able to discontinue prednisone and were maintained on cyclosporine alone. Twenty-two of the 36 patients experienced an acute rejection episode a mean of 14 months (range 1.5 to 36 months) after completion of the steroid taper. Ten of the 22 rejections occurred within 12 months after completion of the taper. Fourteen patients remain rejection free to date for a mean of 70.3 months (range 19 to 111 months) after withdrawal. Using the mixed lymphocyte culture reaction, we tested the hypothesis that steroid dependent recipients (SDR) will express donor antigen specific responsiveness and steroid independent recipients (SIR) will exhibit donor antigen-specific tolerance. Four of seven SDR were tolerant to donor specific antigens but responsive to unrelated controls, while five of six SIR were responsive to donor specific antigens. These unanticipated results highlight the complexity of allograft tolerance.

Prednisone inhibits the efficacy of recombinant human growth hormone in pediatric renal transplant recipients.

To test the efficacy and toxicity of recombinant human growth hormone (rhGH, Protropin, Genentech), we reviewed our experience of its administration of rhGH to pediatric renal transplant recipients. Endogenous growth hormone (GH) levels were measured after stimulation with L-dopa and clonidine in growth retarded children whose height (ht) was greater than or equal to 2 standard deviations (SD) below the mean for age. Criteria for receiving rhGH were either subnormal GH levels (< 10 ng/ml) or a zero growth velocity over the preceding year despite normal GH levels after stimulation. The dose of rhGH administered subcutaneously was 0.1 mg/kg/day for 6 days/week. The efficacy of rhGH was evaluated using growth velocity index (GVI) and height standard deviation (Z) score. Catch-up growth was defined as an increase in Z score (delta Z) > or = 0.4. Twenty patients (17 with subnormal GH levels and 3 with normal GH levels but zero GVI) consented to rhGH treatment. Seventeen patients (14 males, 6 pubertal) have completed one year or more of rhGH therapy and are the subjects of this analysis. All 17 patients were receiving cyclosporine and 12 were receiving prednisone during rhGH therapy. Six of the 17 patients (35%) demonstrated catch-up growth (delta Z + 1.3 +/- 0.13). By regression analysis, the only factor noted to affect rhGH response was the concurrent use of prednisone. All five patients not receiving prednisone demonstrated catch-up growth compared to only 1 of 12 patients receiving prednisone (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Synergism between the CD3 antigen- and CD2 antigen-derived signals. Exploration at the level of induction of DNA-binding proteins and characterization of the inhibitory activity of cyclosporine.

We have demonstrated earlier that the crosslinkage of the CD3/TCR complex with the CD2 antigen results in the proliferation of normal human T cells. The effect of this synergism was perceptible at the level of induction of the IL-2 gene, a process critical for T cell growth. To further understand the molecular and nuclear basis for this synergism, we have explored the induction of DNA-binding proteins in highly purified normal human T cells signaled via the CD3 and/or CD2 proteins. The effect of transmembrane signaling of T cells with ionomycin, and/or sn-1,2 dioctanoyl glycerol, was also determined. The emergence of nuclear binding proteins was investigated using interleukin-2 sequence specific oligonucleotide probes in the electrophoretic mobility shift assay. Our studies demonstrate for the first time that CD3 antigen-derived signals and CD2 antigen-derived signals are synergistic in inducing the emergence of transcription factors that bind to the NF-AT1, AP-1, and NF-kB sites located in the promoter/enhancer region of the IL-2 gene. Moreover, cyclosporine, at concentrations readily accomplished in clinical practice, was found to inhibit the emergence of these DNA-binding proteins in normal human T cells signaled via cell surface proteins implicated in antigen-dependent T cell activation and in T cells stimulated by mobilization of cellular calcium and activation of protein kinase C.