Guidelines for the management of postoperative soiling in children with Hirschsprung disease.

Although most children with Hirschsprung disease ultimately achieve functional and comfortable stooling, some will experience a variety of problems after pull-through surgery. The most common problems include soiling, obstructive symptoms, enterocolitis, and failure to thrive. The purpose of this guideline is to present a rational approach to the management of postoperative soiling in children with Hirschsprung disease. The American Pediatric Surgical Association Hirschsprung Disease Interest Group engaged in a literature review and group discussions. Expert consensus was then used to summarize the current state of knowledge regarding causes, methods of diagnosis, and treatment approaches to children with soiling symptoms following pull-through for Hirschsprung disease. Causes of soiling after pull-through are broadly categorized as abnormalities in sensation, abnormalities in sphincter control, and "pseudo-incontinence." A stepwise algorithm for the diagnosis and management of soiling after a pull-through for Hirschsprung disease is presented; it is our hope that this rational approach will facilitate treatment and optimize outcomes.

Induction of chimerism in rhesus macaques through stem cell transplant and costimulation blockade-based immunosuppression.

A strategy for producing high-level hematopoietic chimerism after non-myeloablative conditioning has been established in the rhesus macaque. This strategy relies on hematopoietic stem cell transplantation after induction with a non-myeloablative dose of busulfan and blockade of the IL2-receptor in the setting of mTOR inhibition with sirolimus and combined CD28/CD154 costimulation blockade. Hematopoietic stem cells derived from bone marrow and leukopheresis products both were found to be successful in inducing high-level chimerism. Mean peripheral blood peak donor chimerism was 81% with a median chimerism duration of 145 days. Additional immune modulation strategies, such as pre-transplant CD8 depletion, donor-specific transfusion, recipient thymectomy or peritransplant deoxyspergualin treatment did not improve the level or durability of chimerism. Recipient immunologic assessment suggested that chimerism occurred amidst donor-specific down-regulation of alloreactive T cells, and the reappearance of vigorous T-mediated alloreactivity accompanied rejection of the transplants. Furthermore, viral reactivation constituted a significant transplant-related toxicity and may have negatively impacted the ability to achieve indefinite survival of transplanted stem cells. Nevertheless, this chimerism-induction regimen induced amongst the longest-lived stem cell chimerism reported to date for non-human primates and thus represents a platform upon which to evaluate emerging tolerance-induction strategies.

Characterization of virus-mediated inhibition of mixed chimerism and allospecific tolerance.

Simultaneous blockade of the CD28 and CD40 T cell costimulatory pathways has been shown to effectively promote skin allograft survival in mice. Furthermore, blockade of one or both of these pathways has played a central role in the development of strategies to induce mixed hematopoietic chimerism and allospecific tolerance. It has recently been observed that the beneficial effects of CD40 blockade and donor splenocytes in prolonging skin graft survival can be abrogated by some viral infections, including lymphocytic choriomeningitis virus (LCMV). In this study, we show that LCMV infection prevents prolonged allograft survival following CD28/CD40 combined blockade. We further show that LCMV prevents the induction of allospecific tolerance and mixed hematopoietic chimerism, while delay of infection for 3-4 wk posttransplant has no effect on tolerance induction. Because of reports of anti-H-2(d) activity following LCMV infection, we assayed the ability of LCMV-specific T cells to respond to alloantigen at a single cell level. Although we confirm that LCMV infection induces the generation of alloreactive cells, we also demonstrate that LCMV-specific T cells do not divide in response to alloantigen. The alloresponse suppressed by costimulation blockade is restored by LCMV infection and correlates with increased dendritic cell maturation. We hypothesize that the costimulation blockade-resistant rejection mediated by LCMV could be partly attributable to the up-regulation of alternative costimulatory pathways subsequent to LCMV-induced dendritic cell maturation.

Aggressive skin allograft rejection in CD28-/- mice independent of the CD40/CD40L costimulatory pathway.

CD28-/- mice have been utilized to study the role of B7/CD28 and B7-CTLA4 interactions. There is evidence that CTLA4 ligation may be critical for tolerance induction. The aim of the current study is to further investigate rejection responses of CD28-/- mice and to define the role of B7-CTLA4 interactions in the absence of the CD40 and CD28 pathways. Balb/c skin allografts were transplanted onto C57BL/6 (B6) wild type or CD28-/- mice treated with anti-CD40L, CTLA4-Ig, or combination blockade. To investigate the cellular mechanism of rejection in CD28-/- recipients, mice were treated with anti-CD4 or anti-CD8 antibodies prior to treatment with costimulation blockade. The fluoroscein dye CFSE was utilized to study T cell expansion in vivo. Surprisingly, treatment of B6 CD28-/- mice with CTLA4-Ig alone (MST 12d), anti-CD40L alone (MST 13d), or combined blockade (MST 13d) had no effect on allograft survival compared to untreated B6 CD28 mice (MST 11d). CD28-/- recipients depleted of CD4+ cells and treated with CTLA4-Ig, anti-CD40L, or combination blockade also did not have prolonged survival compared with untreated mice (MST 10d). In contrast, CD28-/- recipients depleted of CD8+ cells had markedly prolonged allograft survival when treated with either anti-CD40L alone (MST 49d) or with combination blockade (MST 57d). Studies utilizing CFSE demonstrated that CD28-/- CD8+ T cells are not defective in in vivo proliferation responses compared with wild type CD8 cells. Thus, CD28-/- CD8+ T cells are responsible for aggressive rejection responses of CD28-/- mice independent of the CD40 pathway. In addition, CD40L blockade does not result in CD4+ T cell tolerance in CD28 recipients, despite an intact B7-CTLA4 pathway.

Analysis of the CD40 and CD28 pathways on alloimmune responses by CD4+ T cells in vivo.

BACKGROUND: Blockade of the CD40 and CD28 pathways is a powerful strategy to inhibit CD4-mediated alloimmune responses. In this study, we examine the relative roles of the CD40 and CD28 pathways on CD4-mediated allograft rejection responses, and further characterize the role of these pathways on CD4+ T-cell activation, priming for cytokine production, and cell proliferation in response to alloantigen in vivo. METHODS: BALB/c skin allografts were transplanted onto C57BL/6 Rag 1-/- recipients reconstituted with CD4 cells from CD28-/- or CD40L-/- donors. The popliteal lymph node assay was used to study the role of these pathways on CD4-cell activation and priming in vivo. To investigate the role of CD40 and CD28 blockade on CD4-cell proliferation, the fluorescein dye carboxyfluorescein diacetate succinimidyl ester was used. We performed heterotopic cardiac transplantation using CD40-/- mice to evaluate the role of CD40 on donor versus recipient cells in CD4-mediated rejection. RESULTS: B6 Rag 1-/- recipients reconstituted with CD28-/- CD4+ T cells acutely rejected allografts (median survival time 15 days), whereas recipients reconstituted with CD40L-/- CD4+ T cells had significantly prolonged survival of BALB/c skin grafts (MST 71 days). CD40L blockade was equivalent to or inferior to CD28 blockade in inhibition of in vivo CD4-cell activation, priming for cytokine production, and proliferation responses to alloantigen. BALB/c recipients depleted of CD8 cells promptly rejected donor B6 CD40-/- cardiac allografts, whereas B6 CD40-/- recipients depleted of CD8 cells had significantly prolonged survival of BALB/c wild-type cardiac allografts. CONCLUSIONS: The CD40/CD40L pathway, but not the CD28/B7 pathway, is critical for CD4-mediated rejection responses, however, the responsible mechanisms remain unclear.

Costimulation blockade, busulfan, and bone marrow promote titratable macrochimerism, induce transplantation tolerance, and correct genetic hemoglobinopathies with minimal myelosuppression.

Mixed hemopoietic chimerism has the potential to correct genetic hemological diseases (sickle cell anemia, thalassemia) and eliminate chronic immunosuppressive therapy following organ transplantation. To date, most strategies require either recipient conditioning (gamma-irradiation, depletion of the peripheral immune system) or administration of "mega" doses of bone marrow to facilitate reliable engraftment. Although encouraging, many issues remain that may restrict or prevent clinical application of such strategies. We describe an alternative, nonirradiation based strategy using a single dose of busulfan, costimulation blockade, and T cell-depleted donor bone marrow, which promotes titratable macrochimerism and a reshaping of the T cell repertoire. Chimeras exhibit robust donor-specific tolerance, evidenced by acceptance of fully allogeneic skin grafts and failure to generate donor-specific proliferative responses in an in vivo graft-versus-host disease model of alloreactivity. In this model, donor cell infusion and costimulation blockade without busulfan were insufficient for tolerance induction as donor-specific IFN-gamma-producing T cells re-emerged and skin grafts were rejected at approximately 100 days. When applied to a murine beta-thalassemia model, this approach allows for the normalization of hemologic parameters and replacement of the diseased red cell compartment. Such a protocol may allow for clinical application of mixed chimerism strategies in patients with end-stage organ disease or hemoglobinopathies.

Genetic characterization of strain differences in the ability to mediate CD40/CD28-independent rejection of skin allografts.

Simultaneous blockade of the CD40 and CD28 T cell costimulatory pathways effectively promotes skin allograft survival in C3H/HeJ mice, extending median survival times (MSTs) beyond 100 days. This strategy is markedly less effective in C57BL/6 mice, with MSTs ranging between 20 and 30 days. In this study, we investigate the underlying genetic causes of these distinct phenotypes. Using H-2 congenic mice, we show that the genetic basis for the varied responses between these two strains is independent of the H-2 locus and T cell precursor frequency. C57BL/6 mice treated with costimulation blockade are able to generate allospecific CTL- and IFN-gamma-producing T cells within 3-4 wk posttransplant, whereas mice with a C3H background generate neither CTL- nor IFN-gamma-producing cells. Thus, differences appear to be in the generation of the immune response and not T cell homing. Strain differences in costimulation blockade-induced hyporesponsiveness persist in the absence of CD4(+) T cells, implying a direct effect on CD8(+) T cells. We demonstrate that genetic differences are important in cells of hemopoietic origin and that the costimulation blockade-resistant phenotype is dominant. Analysis of BXH recombinant inbred strains indicates that multiple loci contribute to the phenotype, and that the blockade resistance loci are preliminarily linked to 17 markers on four chromosomes. We conclude that strain variation in allograft MSTs following CD40/CD28 blockade results from the ability of CD8(+) T cells in some strains to use alternative modes of costimulation to mount an effective alloresponse.

Cutting edge: administration of anti-CD40 ligand and donor bone marrow leads to hemopoietic chimerism and donor-specific tolerance without cytoreductive conditioning.

Transplantation tolerance, defined as allograft acceptance by an immunocompetent recipient in the absence of long-term immunosuppression, has remained an elusive goal in clinical transplantation. Robust experimental tolerance induction strategies have in common methods to induce mixed hemopoietic chimerism. To date, however, chimerism induction across allogeneic barriers has required recipient conditioning with irradiation or cytoablative agents. In this paper we show that B6 recipients of fully allogeneic BALB/c skin grafts treated with repeated doses of donor bone marrow and anti-CD40 ligand (CD40L) develop durable (>300 days), readily detectable (6-12%) multilineage hemopoietic chimerism, indefinite allograft acceptance (>300 days), and donor-specific tolerance to secondary skin grafts. Analysis of the TCR repertoire of treated mice indicates that the underlying mechanisms of tolerance are in part mediated by deletion of donor-reactive T cells. These data demonstrate that durable hemopoietic chimerism and robust transplantation tolerance can be achieved without cytotoxic conditioning using a potentially clinically applicable regimen.

Vigorous allograft rejection in the absence of danger.

Tolerance to self is a necessary attribute of the immune system. It is thought that most autoreactive T cells are deleted in the thymus during the process of negative selection. However, peripheral tolerance mechanisms also exist to prevent development of autoimmune diseases against peripheral self-Ags. It has been proposed that T cells develop tolerance to peripheral self-Ags encountered in the absence of inflammation or "danger" signals. We have used immunodeficient Rag 1-/- mice to study the response of T cells to neo-self peripheral Ags in the form of well-healed skin and vascularized cardiac allografts. In this paper we report that skin and cardiac allografts without evidence of inflammation are vigorously rejected by transferred T cells or when recipients are reconstituted with T cells at a physiologic rate by nude bone graft transplantation. These results provide new insights into the role of inflammation or "danger" in the initiation of T cell-dependent immune responses. These findings also have profound implications in organ transplantation and suggest that in the absence of central deletional tolerance, peripheral tolerance mechanisms are not sufficient to inhibit alloimmune responses even in the absence of inflammation or danger.

Asialo GM1(+) CD8(+) T cells play a critical role in costimulation blockade-resistant allograft rejection.

Simultaneous blockade of the CD40 and CD28 costimulatory pathways is an effective treatment strategy to promote allograft acceptance but does not lead to indefinite allograft survival. The immune mechanisms responsible for costimulation-independent rejection are not defined. Here we have studied the rejection responses of murine C57BL/6 recipients, which we show to be relatively resistant to inhibition by combined CD40/CD28 blockade. We demonstrate that asialo GM1(+) CD8(+) cells play a critical role in this costimulation blockade-resistant rejection. These results provide new insights into the costimulatory requirements for T-cell subsets and demonstrate for the first time that combined blockade of the CD40 and CD28 pathways does not adequately inhibit CD8-mediated skin allograft rejection. Furthermore, we provide evidence that asialo GM1 is a potentially important therapeutic target for CD8-dependent immune responses.

Neonatal gastric perforation and necrosis with Hunt-Lawrence pouch reconstruction.

This report describes a newborn infant girl who presented with abdominal distension and pneumoperitoneum. At operation, near total necrosis of the stomach was observed. The esophagus was ligated, the stomach resected. The baby was fed by an transpyloric feeding tube. At 8 weeks, an esophagojejunal anastomosis was performed with a Hunt-Lawrence Pouch. A good outcome was achieved.

Presacral neuroenteric fistula presenting with meningitis and vaginal fistula: a case report.

Neuroenteric fistulas are rare congenital malformations arising from a persistent connection between the embryonic neural and intestinal tissues; they are extremely rare in the lumbosacral area. Neuroenteric fistulas should be suspected whenever children present with anaerobic meningitis in the absence of brain abscesses or respiratory infections. This patient is the first patient who presented with a presacral cyst that separately connected to the nervous system and the vagina. It is thought that the rape of this 12-year-old patient resulted in soilage of the spinal fluid through dual fistulae, causing anaerobic meningitis without an abscess.