Disseminated histoplasmosis associated with hemophagocytic lymphohistiocytosis in kidney transplant recipients.

Transplant patients are susceptible to infectious complications due to chronic immunosuppression. We present two cases of persistent fever, weight loss and pancytopenia in kidney transplant recipients (originally concerning for posttransplant lymphoproliferative disease) that were later diagnosed with disseminated histoplasmosis on bone marrow and lymph node biopsy. In both patients, pancytopenia was due to hemophagocytic lymphohistiocytosis (HLH) which has rarely been described in association with histoplasmosis and not previously reported in kidney transplant recipients with this fungal infection. The diagnosis of histoplasmosis can be complex due to nonspecific symptomatology, delays in isolating histoplasma by fungal culture and false-negative antibody titers in immunocompromised patients. A review of the literature including the clinical features of histoplasmosis in immunosuppressed patients (prevalence, current diagnostic testing and treatment options) as well as the association of HLH in immunocompromised states are discussed.

Porcine neural xenografts in rats and mice: donor tissue development and characteristics of rejection.

Embryonic ventral mesencephalic tissue from the pig is a potential alternative donor tissue for neural transplantation to Parkinson's disease patients. For stable graft survival, the host immune response has to be prevented. This study was performed in order to analyze the mechanisms and dynamics of neural xenograft rejection, as well as neurobiological properties of the donor tissue. Adult normal mice and rats, and cyclosporin A-treated rats, received intrastriatal transplants of dissociated embryonic ventral mesencephalic pig tissue that was 27 or 29 embryonic days of age (E27 and E29). The animals were perfused at 2, 4, 6, and 12 weeks after grafting and the brains were processed for immunohistochemistry of dopaminergic (tyrosine hydroxylase positive) neurons, CD4(+) and CD8(+) lymphocytes, natural killer cells, macrophages, microglia, and astrocytes. Thirty-five rats received daily injections of BrdU for 5 consecutive days at different time points after transplantation and were perfused at 6 weeks. These animals were analyzed for proliferation of cells in the donor tissue, both in healthy and in rejecting grafts. No tyrosine hydroxylase-positive cells proliferated after grafting. Our results demonstrated that E27 was superior to E29 donor tissue for neurobiological reasons. Cyclosporin A immunosuppression was protective only during the first weeks and failed to protect the grafts in a long-term perspective. Grafts in mice were invariably rejected between 2 and 4 weeks after transplantation, while occasional grafts in untreated rats survived up to 12 weeks without signs of an ongoing rejection process. CD8(+) lymphocytes and microglia cells are most likely important effector cells in the late, cyclosporin A-resistant rejection process.

Effects of immunosuppressive treatment on host responses against intracerebral porcine neural tissue xenografts in rats.

BACKGROUND: Embryonic xenogeneic neural tissue is an alternative for transplantation in Parkinson's disease, but immune responses limit the application. The aims of this study were to enhance the in vitro viability rates by donor tissue pretreatment; to compare the efficacy of cyclosporine A (CsA) and tacrolimus (FK) in inhibiting xenograft rejection in rats; to evaluate additional inductive therapy with prednisolone (PRE) or mycophenolate mofetil (MMF). METHODS: Tirilazad (a lipid peroxidase inhibitor) or FK and acYVAD-cmk (a caspase inhibitor), were added to embryonic porcine ventral mesencephalic tissue and viability was assessed in vitro. Tirilazad-treated tissue was grafted to the striatum of rats that were either left untreated or immunosuppressed with FK (1 mg/kg) or CsA (15 mg/kg) alone or in combination with a 2-week PRE (20 mg/kg) or MMF (40 mg/kg) induction course. Xenograft survival and host responses were determined using immunohistochemistry. RESULTS: Pretreatment with tirilazad enhanced tissue survival in vitro. After transplantation into untreated controls, there was no graft survival at twelve weeks. Neural cell counts were significantly improved in immunosuppressed recipients, but there were no differences between the treatment groups. Additional inductive treatment reduced the infiltration with CD4+ and CD8+ cells, and macrophage infiltration was reduced compared with animals given CsA or FK alone. CONCLUSION: Pretreatment of the donor tissue with free-radical scavengers reduces cell loss caused by tissue trauma. Porcine neural tissue xenografts survive significantly better in animals immunosuppressed with either FK or CsA. Additional inductive treatment with PRE or MMF reduced the infiltration of host cells into the xenografts.

Intrastriatal ventral mesencephalic xenografts of porcine tissue in rats: immune responses and functional effects.

Transplantation of neural tissue from other species has the potential to improve function in patients with neurodegenerative disorders. We investigated the functional effects of embryonic porcine dopaminergic neurons transplanted in a rat model of Parkinson's disease and the immune responses to the grafts in immunosuppressed and nonimmunosuppressed hosts. Twenty-three rats with unilateral 6-hydroxydopamine lesions received dissociated, 27-day-old embryonic porcine ventral mesencephalic tissue in the right striatum. Eighteen rats received cyclosporine (10 mg/kg, IP, daily) during the whole period of 14 weeks, in combination with prednisolone (20 mg/kg, IP, daily) the first 4 days. Five rats served as nonimmunosuppressed controls. All rats were tested for amphetamine-induced rotational behavior at 3-week intervals. Two immunosuppressed rats were excluded due to severe side effects of the treatment. Functional recovery was seen in 9 of 16 immunosuppressed rats at 12 weeks. Six animals remained functionally recovered at 14 weeks and contained an average of 5750+/-1450 (SEM) dopaminergic neurons. Between 9 and 14 weeks, three immunosuppressed rats rejected their grafts, based on rotation scores and immunohistochemical demonstration of cell infiltrates. One additional immunosuppressed rat showed evidence of ongoing rejection at 14 weeks. The striata in animals with ongoing or recent rejection contained large numbers of CD4- and CD8-positive lymphocytes, NK cells, macrophages, and microglia cells, whereas scar tissue was found in rats with grafts rejected at earlier time points (n = 11). Embryonic porcine ventral mesencephalic tissue matures in the adult rat striatum, reinnervates the host brain, and restores behavioral defects. Immunosuppressive treatment was necessary for long-term graft survival and functional recovery, but did not sufficiently protect from rejection mechanisms. Porcine neural tissue is an interesting alternative to embryonic human tissue for intracerebral transplantation in neurodegenerative diseases. However, to achieve stable graft survival in discordant xenogeneic combinations, an appropriate immunosuppressive treatment or donor tissue modifications are needed.

Discordant neural tissue xenografts survive longer in immunoglobulin deficient mice.

BACKGROUND: The immune response against discordant xenografts in the brain is incompletely understood and remains a major obstacle for future clinical applications of xenogeneic neural tissue transplants in neurodegenerative disorders. To determine the role of antibodies in the rejection process, we compared graft survival and immune reactions between immunoglobulin deficient (IgKO) and normal mice. METHODS: A cell suspension of embryonic porcine ventral mesencephalon was injected into the striatum of adult normal and IgKO mice. Graft sizes and number of infiltrating CD4- and CD8-positive lymphocytes were determined by stereological methods at 4 days and 2, 4, and 6 weeks after the transplants. Microglial accumulation was determined using the optical densitometrical method. Intraparenchymal deposition of IgG was investigated at 4 days and 2 weeks. RESULTS: The majority of IgKO mice had surviving grafts for up to 4 weeks, whereas survival was minimal in control mice beyond 4 days. Graft sizes differed significantly between IgKO and control mice at 2 weeks (P<0.01, Kruskal Wallis ANOVA, followed by Mann Whitney test). The majority of infiltrating lymphocytes were CD4-positive in control mice but CD8-positive in IgKO mice. Microglial accumulation was strong around surviving grafts in IgKO mice at 4 weeks. Prominent staining of IgG, diffuse in the transplanted hemisphere and specific on grafted neurons, was found in control mice. CONCLUSIONS: Our results suggest that immunoglobulins play an initiating role in rejection of discordant neural xenografts. After a prolonged graft survival of approximately 4 weeks, a cellular response with a large proportion CD8-positive cells leads to rejection in IgKO mice.

Human natural antibodies cytotoxic to pig embryonic brain cells recognize novel non-Galalpha1,3Gal-based xenoantigens.

Transplantation of porcine embryonic brain cells, including dopaminergic neurons, from ventral mesencephalon (VM) is considered a potential treatment for patients with Parkinson's disease. In the present study, we characterized the distribution among VM cells of the major porcine endothelial xenoantigen, the Galalpha1,3Gal epitope, and evaluated the cytotoxic effect of anti-Galalpha1,3Gal antibody-depleted and nondepleted human AB serum on VM cells. Overall levels of Galalpha1,3Gal-epitope expression was very low on the VM cell population using Bandeiraea simplicifolia IB(4) lectin staining of resuspended VM cells in flow cytometric analyses or staining of SDS-PAGE-separated, solubilized VM cell membrane proteins in Western blot analyses. Lectin-histochemical staining of sections of pig embryonal VM regions with BSA IB(4) lectin showed staining restricted to endothelial cells and microglia. In the presence of complement, both nondepleted and anti-Galalpha1,3Gal antibody-depleted AB sera were shown to be cytotoxic to VM cells as assessed in microcytotoxicity- and flow cytometry-based cytotoxicity assays. Purified IgM and IgG were both cytotoxic in the presence of complement. Three major VM cell membrane antigens of approximately 210, 105, and 50 kDa were reactive with natural IgM antibodies present in pooled human AB sera. Thus, antibody-dependent cytotoxicity may contribute to pig to human brain cell xenorejection, necessitating donor tissue modifications prior to a more widespread utilization of neural tissue xenografting.

One-year chromaffin cell allograft survival in cancer patients with chronic pain: morphological and functional evidence.

The control of chronic pain through transplantation of chromaffin cells has been reported over the past few years. Analgesic effects are principally due to the production of opioid peptides and catecholamines by chromaffin cells. Clinical trials have been reported with allografts consisting of whole-tissue fragments implanted into the subarachnoid space of the lumbar spinal cord (14,19,36). In the present study, allogeneic grafts were successfully used to control chronic pain in two patients over a period of 1 yr based on patient reported pain scores, morphine intake, and CSF levels of Met-enkephalin. Macroscopic examination at autopsy located the transplanted tissue fragments in the form of multilobulated nodules at the level of the spinal axis and cauda equina. Immunocytochemical microscopy showed neuroendocrine cells are positive for chromagranin A (CGA), and enzymes tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DbetaH). The results suggest that there is a relationship between analgesic effect, Met-enkephalin levels in CSF, and the presence of chromaffin cells surviving in spinal subarachnoid space.

The influence of xenotransplant immunogenicity and immunosuppression on host MHC expression in the rat CNS.

During the early stages following neural transplantation, host immune responses are initiated that are not normally found in the CNS including the induction of major histocompatibility antigens (MHC I and II). Previous laboratory findings have demonstrated prolonged survival of bovine chromaffin cells (BCC) in the rat CNS following transient immunosuppression with cyclosporin A (CSA) providing chromaffin cells are isolated from highly immunogenic passenger cells. To assess the influence of passenger and chromaffin cells on host MHC I and II expression, either BCC, nonchromaffin cell adrenal constituents (NCC), or adrenal medullary endothelial cells (EC) were implanted into the host. At 2 weeks postimplantation, robust BCC survival was obtained in CSA-treated animals. This correlated with low expression of MHC I at the host-graft border and the virtual absence of MHC II. Good BCC survival with reduced MHC I expression only was seen at 6 weeks postimplantation in animals transiently immunosuppressed (4 weeks). In contrast, poor survival was seen in the EC group (even with CSA treatment). In addition, marked MHC I and II expression was found in and around these grafts at 2 weeks, and was particularly intense in EC implanted animals. The results of this study suggest that nonchromaffin passenger cells in BCC preparations, most notably endothelial cells, can induce strong immune responses even in the presence of immunosuppression. Based on MHC staining, removal of these passenger cells can reduce host responses and improve long term survival of xenogeneic chromaffin cells in the CNS.

Bovine chromaffin cells for CNS transplantation do not elicit xenogeneic T cell proliferative responses in vitro.

Adrenal chromaffin cells have been utilized for several neural grafting applications, but limitations in allogeneic donor availability and dangers inherent in autografting limit the widespread use of this approach clinically. While xenogeneic donors offer promise as a source for cell transplantation in the central nervous system (CNS), immunologic responses to cellular components of the adrenal medulla have not been well characterized. To further study the host T cell xenogeneic response to chromaffin and passenger cells of the adrenal medulla, an in vitro lymphocyte proliferation assay was used. Lymphocyte proliferation was determined by mixing rat lymphocytes with potential stimulator cell subpopulations of the bovine adrenal medulla: isolated chromaffin cells, isolated endothelial cells, or passenger nonchromaffin cells, which include a mixture of fibroblasts, smooth muscle cells, and endothelial cells. As a positive control, bovine aortic endothelial cells were also used. 3[H]-thymidine incorporation, corresponding to lymphocyte proliferation, was measured. Results indicated that isolated bovine chromaffin cells produce only a mild, statistically insignificant stimulation of rat lymphocytes. In contrast, there was a significant response to passenger nonchromaffin cells of the adrenal medulla, especially endothelial cells. The inclusion of low levels of cyclosporin A in the cultures completely eliminated the mild proliferative response to isolated bovine chromaffin cells, while near toxic levels were necessary to abrogate the response to endothelial cells. Immunocytochemical analysis revealed that routine chromaffin cell isolation procedures result in the inclusion of a small percentage of endothelial cells, which may be responsible for the slight lymphocyte stimulation. The results of this study indicate that isolated chromaffin cells possess low immunogenicity, and suggest that passenger cells in the adrenal medulla, particularly endothelial cells, may be primarily responsible for progressive rejection in CNS grafts. Thus, removal of passenger nonchromaffin cells from xenogeneic donor tissues prior to transplantation may produce a more tolerated graft in rodent models of neural transplantation.

Update on cellular transplantation into the CNS as a novel therapy for chronic pain.

The transplantation of cells that secrete neuroactive substances with analgesic properties into the CNS is a novel method that challenges current approaches in treating chronic pain. This review covers pre-clinical and clinical studies from both allogeneic and xenogeneic sources. One cell source that has been utilized successfully is the adrenal chromaffin cell, since such cells constitutively release catecholamines, opioid peptides, and neurotrophic factors; release can be augmented with nicotine. Other graft sources include AtT-20 and B-16 cell lines which release enkephalins and catecholamines, respectively. For grafting in rodents, adrenal medullary tissue pieces are transplanted to the subarachnoid space. Chromaffin cell transplants can decrease pain sensitivity in normal rats using standard acute pain tests (paw-pinch, hot-plate, and tail-flick). In addition, transplants can restore normal pain thresholds in rodent models of chronic pain (formalin, adjuvant-induced arthritis, and sciatic-nerve tie) which closely similate the pathologies of human chronic pain conditions. Xenografts have been studied due to concerns that future application for human pain may be limited by donor availability. Despite immune privileges of the CNS, xenografts require at least short-term immunosuppression to obtain a viable graft. Cell encapsulation is one method of sustaining a xenograft (in rat and human hosts) while circumventing the need for immunosuppression. Clinical studies have been initiated for terminal cancer patients with promising results as assessed by markedly reduced narcotic intake, visual analog scale ratings, and increased CSF levels of catecholamines and met-enkephalin.