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Systemic administration of autologous regulatory dendritic cells (DCreg; unpulsed or pulsed with donor antigen [Ag]), prolongs allograft survival and promotes transplant tolerance in rodents. Here, we demonstrate that nonhuman primate (NHP) monocyte-derived DCreg preloaded with cell membrane vesicles from allogeneic peripheral blood mononuclear cells induce T cell hyporesponsiveness to donor alloantigen (alloAg) in vitro. These donor alloAg-pulsed autologous DCreg (1.4-3.6 × 106 /kg) were administered intravenously, 1 day before MHC-mismatched renal transplantation to rhesus monkeys treated with costimulation blockade (cytotoxic T lymphocyte Ag 4 immunoglobulin [CTLA4] Ig) and tapered rapamycin. Prolongation of graft median survival time from 39.5 days (no DCreg infusion; n = 6 historical controls) and 29 days with control unpulsed DCreg (n = 2), to 56 days with donor Ag-pulsed DCreg (n = 5) was associated with evidence of modulated host CD4+ and CD8+ T cell responses to donor Ag and attenuation of systemic IL-17 production. Circulating anti-donor antibody (Ab) was not detected until CTLA4 Ig withdrawal. One monkey treated with donor Ag-pulsed DCreg rejected its graft in association with progressively elevated anti-donor Ab, 525 days posttransplant (160 days after withdrawal of immunosuppression). These findings indicate a modest but not statistically significant beneficial effect of donor Ag-pulsed autologous DCreg infusion on NHP graft survival when administered with a minimal immunosuppressive drug regimen.

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Transforming growth factor ß1 (TGFß1) plays a key role in T cell homeostasis and peripheral tolerance. We evaluated the influence of a novel human mutant TGFß1/Fc (human IgG4 Fc) fusion protein on memory CD4+ and CD8+ T cell (Tmem) responses in vitro and their recovery following antithymocyte globulin (ATG)-mediated lymphodepletion in monkeys. TGFß1/Fc induced Smad2/3 protein phosphorylation in rhesus and human peripheral blood mononuclear cells and augmented the suppressive effect of rapamycin on rhesus Tmem proliferation after either alloactivation or anti-CD3/CD28 stimulation. In combination with IL-2, the incidence of CD4+ CD25hi Foxp3hi regulatory T cells (Treg) and Treg:Th17 ratios were increased. In lymphodepleted monkeys, whole blood trough levels of infused TGFß1/Fc were maintained between 2 and 7 µg/mL for 35 days. Following ATG administration, total T cell numbers were reduced markedly. In those given TGFß1/Fc infusion, CD8+ T cell recovery to predepletion levels was delayed compared to controls. Additionally, numbers of CD4+ CD25hi CD127lo Treg increased at 4-6 weeks after depletion but subsequently declined to predepletion levels by 12 weeks. In all monkeys, CD4+ CD25hi Foxp3hi Treg/CD4+ IL-17+ cell ratios were reduced, particularly after stopping TGFß1/Fc infusion. Thus, human TGFß1/Fc infusion may delay Tmem recovery following lymphodepletion in nonhuman primates. Combined (low-dose) IL-2 infusion may be required to improve the Treg:Th17 ratio following lymphodepletion.

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The ability of regulatory T cells (Treg) to prolong allograft survival and promote transplant tolerance in lymphodepleted rodents is well established. Few studies, however, have addressed the therapeutic potential of adoptively transferred, CD4(+) CD25(+) CD127(-) Foxp3(+) (Treg) in clinically relevant large animal models. We infused ex vivo-expanded, functionally stable, nonselected Treg (up to a maximum cumulative dose of 1.87 billion cells) into antithymocyte globulin-lymphodepleted, MHC-mismatched cynomolgus monkey heart graft recipients before homeostatic recovery of effector T cells. The monkeys also received tacrolimus, anti-interleukin-6 receptor monoclonal antibodies and tapered rapamycin maintenance therapy. Treg administration in single or multiple doses during the early postsurgical period (up to 1 month posttransplantation), when host T cells were profoundly depleted, resulted in inferior graft function compared with controls. This was accompanied by increased incidences of effector memory T cells, enhanced interferon-γ production by host CD8(+) T cells, elevated levels of proinflammatory cytokines, and antidonor alloantibodies. The findings caution against infusion of Treg during the early posttransplantation period after lymphodepletion. Despite marked but transient increases in Treg relative to endogenous effector T cells and use of reputed "Treg-friendly" agents, the host environment/immune effector mechanisms instigated under these conditions can perturb rather than favor the potential therapeutic efficacy of adoptively transferred Treg.

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Increasing evidence from small animal models shows that myeloid-derived suppressor cells (MDSCs) can play a crucial role in inhibiting allograft rejection and promoting transplant tolerance. We identified CD3(-)CD20(-)HLA-DR(-)CD14(+)CD33(+)CD11b(+) cells in peripheral blood of healthy rhesus macaques. These putative monocytic MDSCs constituted 2.1% ± 1.7% of lin(-)HLA-DR(-) peripheral blood mononuclear cells. Administration of granulocyte-macrophage colony-stimulating factor (CSF) and granulocyte CSF increased their incidence to 5.3% ± 3.4%. The total number of MDSCs that could be flow sorted from a single whole rhesus leukapheresis product was 38 ± 13 × 10(6) (n = 10 monkeys). Freshly isolated or cryopreserved MDSCs from mobilized monkeys incorporated in cultures of anti-CD3- and anti-CD28-stimulated autologous T cells markedly suppressed CD4(+) and CD8(+) T cell proliferation and cytokine secretion (interferon γ, IL-17A). Moreover, these MDSCs enhanced CD4(+)CD25(hi)Foxp3(+) regulatory T cell (Treg) expansion while inhibiting proliferation of activated memory T cells and increasing Treg relative to effector and terminally differentiated memory T cells. Inhibition of arginase-1, but not inducible nitric oxide synthase activity, partially reversed the inhibitory effect of the MDSCs on CD8(+) T cell proliferation. Consequently, functional MDSCs can be isolated from nonhuman primates for prospective use as therapeutic cellular vaccines in transplantation.

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Since the discovery of Rapamycin (RAPA) and its immunosuppressive properties, enormous progress has been made in characterizing the mechanistic target of rapamycin (mTOR). Use of RAPA and its analogues (rapalogs) as anti-rejection agents has been accompanied by extensive investigation of how targeting of mTOR complex 1 (mTORC1), the principal target of RAPA, and more recently mTORC2, affects the function of immune cells, as well as vascular endothelial cells, that play crucial roles in regulation of allograft rejection. While considerable knowledge has accumulated on the function of mTORC1 and 2 in T cells, understanding of the differential roles of these complexes in antigen-presenting cells, NK cells and B cells/plasma cells is only beginning to emerge. Immune cell-specific targeting of mTORC1 or mTORC2, together with use of novel, second generation, dual mTORC kinase inhibitors (TORKinibs) have started to play an important role in elucidating the roles of these complexes and their potential for targeting in transplantation. Much remains unknown about the role of mTOR complexes and the consequences of mTOR targeting on immune reactivity in clinical transplantation. Here we address recent advances in understanding and evolving perspectives of the role of mTOR complexes and mTOR targeting in immunity, with extrapolation to transplantation.

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Ex vivo-expanded cynomolgus monkey CD4(+)CD25(+)CD127(-) regulatory T cells (Treg) maintained Foxp3 demethylation status at the Treg-specific demethylation region, and potently suppressed T cell proliferation through three rounds of expansion. When carboxyfluorescein succinimidyl ester- or violet proliferation dye 450-labeled autologous (auto) and nonautologous (non-auto)-expanded Treg were infused into monkeys, the number of labeled auto-Treg in peripheral blood declined rapidly during the first week, but persisted at low levels in both normal and anti-thymocyte globulin plus rapamycin-treated (immunosuppressed; IS) animals for at least 3 weeks. By contrast, MHC-mismatched non-auto-Treg could not be detected in normal monkey blood or in blood of two out of the three IS monkeys by day 6 postinfusion. They were also more difficult to detect than auto-Treg in peripheral lymphoid tissue. Both auto- and non-auto-Treg maintained Ki67 expression early after infusion. Sequential monitoring revealed that adoptively transferred auto-Treg maintained similarly high levels of Foxp3 and CD25 and low CD127 compared with endogenous Treg, although Foxp3 staining diminished over time in these nontransplanted recipients. Thus, infused ex vivo-expanded auto-Treg persist longer than MHC-mismatched non-auto-Treg in blood of nonhuman primates and can be detected in secondary lymphoid tissue. Host lymphodepletion and rapamycin administration did not consistently prolong the persistence of non-auto-Treg in these sites.

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The mechanistic/mammalian target of rapamycin (mTOR) is inhibited clinically to suppress T cell function and prevent allograft rejection. mTOR is the kinase subunit of two mTOR-containing complexes, mTOR complex (mTORC) 1 and 2. Although mTORC1 is inhibited by the macrolide immunosuppressant rapamycin (RAPA), its efficacy may be limited by its inability to block mTORC1 completely and its limited effect on mTORC2. Adenosine triphosphate (ATP)-competitive mTOR inhibitors are an emerging class of mTOR inhibitors that compete with ATP at the mTOR active site and inhibit any mTOR-containing complex. Since this class of compounds has not been investigated for their immunosuppressive potential, our goal was to determine the influence of a prototypic ATP-competitive mTOR inhibitor on allograft survival. AZD8055 proved to be a potent suppressor of T cell proliferation. Moreover, a short, 10-day course of the agent successfully prolonged murine MHC-mismatched, vascularized heart transplant survival. This therapeutic effect was associated with increased graft-infiltrating regulatory T cells and reduced CD4(+) and CD8(+) T cell interferon-γ production. These studies establish for the first time, that ATP-competitive mTOR inhibition can prolong organ allograft survival and warrant further investigation of this next generation mTOR inhibitors.

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Liver interstitial dendritic cells (DC) have been implicated in immune regulation and tolerance induction. We found that the transmembrane immuno-adaptor DNAX-activating protein of 12 kDa (DAP12) negatively regulated conventional liver myeloid (m) DC maturation and their in vivo migratory and T cell allostimulatory ability. Livers were transplanted from C57BL/6(H2(b) ) (B6) WT or DAP12(-/-) mice into WT C3H (H2(k) ) recipients. Donor mDC (H2-K(b+) CD11c(+) ) were quantified in spleens by flow cytometry. Anti-donor T cell reactivity was evaluated by ex vivo carboxyfluorescein diacetate succinimidyl ester-mixed leukocyte reaction and delayed-type hypersensitivity responses, while T effector and regulatory T cells were determined by flow analysis. A threefold to fourfold increase in donor-derived DC was detected in spleens of DAP12(-/-) liver recipients compared with those given WT grafts. Moreover, pro-inflammatory cytokine gene expression in the graft, interferon gamma (IFNγ) production by graft-infiltrating CD8(+) T cells and systemic levels of IFNγ were all elevated significantly in DAP12(-/-) liver recipients. DAP12(-/-) grafts also exhibited reduced incidences of CD4(+) Foxp3(+) cells and enhanced CD8(+) T cell IFNγ secretion in response to donor antigen challenge. Unlike WT grafts, DAP12(-/-) livers failed to induce tolerance and were rejected acutely. Thus, DAP12 expression in liver grafts regulates donor mDC migration to host lymphoid tissue, alloreactive T cell responses and transplant tolerance.

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Mammalian target of rapamycin kinase inhibitor (mTORi) rapamycin (RAPA) use in transplantation can lead to inflammatory complications in some patients. Our goal was to better understand how mTORi-exposed human monocyte-derived dendritic cells (DC) stimulated with pro-inflammatory cytokines shape T cell allo-immunity. RAPA-conditioned-DC (RAPA-DC) displayed a more immature phenotype than untreated, control (CTRL)-DC. However, subsequent exposure of RAPA-DC to an inflammatory cytokine cocktail (ICC) plus IFN-γ induced a mature Type-1 promoting phenotype, consisting of elevated HLA-DR and co-stimulatory molecules, augmented IL-12p70 and IL-27 production, but decreased IL-10 secretion compared to CTRL-DC. Co-culture of mature (m)RAPA-DC with allogeneic peripheral blood mononuclear cells resulted in significantly increased Type-1 (IFN-γ) responses by T cells. Moreover, NK cells acted as innate modulators that conveyed activating cell-to-cell contact signals in addition to helper (IFN-γ) and/or regulatory (IL-10) soluble cytokines. We conclude that production of IL12-p70, IL-27 and low IL-10 by RAPA-DC allowed us to elucidate how these cytokines as well as NK-DC interaction shapes T cell allo-immunity. Thus, lack of inhibitory NK cell function during allo-specific T cell activation by human ICC + IFN-γ-stimulated RAPA-DC may represent an unwanted effector mechanism that may underlie RAPA-induced inflammatory events in transplant patients undergoing microbial infection or allograft rejection.

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INTRODUCTION: Patients with amygdala dysfunction generally have behavioral impairment. Temporal lobe surgery might be a model of study of unilateral amygdala resection. The objective of this study was to evaluate behavioral flexibility in epileptic patients who undergo amygdala resection for epilepsy surgery and evaluate its relationship with their neuropsychiatric symptoms. MATERIAL AND METHODS: Ten epileptic patients who underwent amygdala and hippocampal resection (6 left and 4 right) matched by age and educational level with 10 healthy controls were tested with an extensive neuropsychological and neuropsychiatric battery. Psychiatric symptomatology was measured with the positive and negative syndrome scale (PANSS) and the Beck depression inventory. To assess behavioral flexibility the emotion-related visual reversal-learning task (O'Doherty et al., 2001) and the gambling task (Bechara et al., 1994) were used. RESULTS: Patient's mean scores were: Beck: 8 +/- 1.5; PANSS positive: 10 +/- 1.3, and negative: 14.4 +/- 2.2; intellectual quotient (IQ): 101.4 +/- 6.3; category number in Wisconsin card sorting test: 4.6 +/- 2.4. The emotion-related visual reversal-learning task showed significance differences in the number of reversion: healthy controls: 9.3; epileptic patients: 4.23 (p < 0.001); in the number of trials to the first reversion: healthy controls: 5; epileptic patients: 23.42 (p < 0.05). There was no correlation between reversion and depression, PANSS and IQ. CONCLUSIONS: Patients with epilepsy who undergo unilateral hippocampal and amygdala resection appear to have alterations in the reversion capacity with an emotional component that would explain the lack of behavior flexibility that they sometimes have and that are not related with either the isolated presence of executive alterations or low intellectual quotient.

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Dendritic cells (DC) are highly specialized antigen-presenting cells (APC) derived from precursors within the bone marrow (BM). They are distributed ubiquitously throughout the body, and are few in number (1). They are classified as lymphoid-related or myeloid DC depending on their developmental lineage. Lymphoid-related DC develop from very immature T-cell precursors or BM progenitors, which are also the source of future natural killer cells and B cells (2,3). In vitro, these lymphoid-derived DC can be generated in the absence of granulocyte macrophage colony stimulating factor (GM-CSF), and are CD8(+) and Fas ligand(+) (CD95L(+)). In the presence of GM-CSF, DC develop directly from myeloid committed precursors, which also give rise to monocytes and granulocytes, or from myelomonocytic cells, which are also precursors of monocytes (4,5). DC can be propagated from progenitors in BM (6), blood (7), or secondary lymphoid tissues (8). In addition, peripheral blood mononuclear cells (PBMC) can develop into DC-like cells if cultured in the presence of GM-CSF and interleukin-4 (IL-4) (9,10).

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This study examines the performance on executive function--classically considered as purported by the frontal lobes--in patients with temporal lobe epilepsy (TLE). Two groups of patients were evaluated: one consisted of 16 TLE patients and the other comprised 12 patients with primary generalised epilepsy (PGE). The Wisconsin Card Sorting Test (WCST) was used as a measure of executive function. Results demonstrated that performance on the WCST was remarkably defective in the TLE group, showing a pattern suggestive of frontal like executive dysfunction in a 75% of the patients, against the 17% of the PGE group (p < 0.001). Impairment was evident when number of categories achieved (p < 0.05), perseverative errors (p < 0.001) and perseverative responses (p < 0.001) were considered. Clinical and theoretical significance of these findings may reflex the executive dysfunction of the mesial temporal lobe region as part of the temporo-frontal circuit.

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This study examines the performance on executive function--classically considered as purported by the frontal lobes--in patients with temporal lobe epilepsy (TLE). Two groups of patients were evaluated: one consisted of 16 TLE patients and the other comprised 12 patients with primary generalised epilepsy (PGE). The Wisconsin Card Sorting Test (WCST) was used as a measure of executive function. Results demonstrated that performance on the WCST was remarkably defective in the TLE group, showing a pattern suggestive of frontal like executive dysfunction in a 75

of the patients, against the 17

of the PGE group (p < 0.001). Impairment was evident when number of categories achieved (p < 0.05), perseverative errors (p < 0.001) and perseverative responses (p < 0.001) were considered. Clinical and theoretical significance of these findings may reflex the executive dysfunction of the mesial temporal lobe region as part of the temporo-frontal circuit.

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INTRODUCTION: Cognitive dysfunction in mesial temporal lobe epilepsy (MTLE) is still a controversial subject. Since refractory MTLE is considered a surgically remediable syndrome, the neuropsychological assessment to establish the presence of cognitive impairment in the surgical candidate became an important issue, given its possible relevance in predicting outcome after surgery. OBJECTIVE: Our aim was to study the cognitive profile of MTLE patients and to correlate the cognitive status with the presence or absence of hippocampal sclerosis. PATIENTS AND METHODS: Fifty patients with MTLE and 20 control subjects were matched by age and educational level. All subjects were studied with EEG, MRI scan, SPECT, and a comprehensive neuropsychological battery that included measures of language (naming and verbal fluency), visuospatial function (Block Design Test), memory (Signoret Battery), attention (Digit Span and Trail Making Test A) and executive abilities (Trail Making Test B and Wisconsin Card Sorting Test). RESULTS: MTLE patients, particularly those with hippocampal sclerosis, were found to have a considerable lower performance in learning (p < 0.01), naming (p < 0.05), attention (p < 0.05) and executive functions (p < 0.001). Among the MTLE patients a considerable number of subjects showed results within the normal range (n = 27) while others evidenced laterality specific cognitive impairments (n = 13). Material specific memory effects were seen in some patients but not all. CONCLUSIONS: Our results showed the neuropsychological heterogeneity of temporal lobe epilepsy, thus ruling out the existence of a single specific cognitive pattern of impairment in all MTLE patients, and suggesting the need of a thorough pre-surgical neuropsychological evaluation to be used with post-surgical prognosis purposes.

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During development the gastrointestinal tract undergoes marked changes in many physiological and anatomic properties. The remarkable degree of coordination between the development of the gastrointestinal function suggests that the processes may be signalled by some factors, such as weaning, nutrient intake, growth and hormones. The interactions between nutrition and intestinal development begin when fetuses start swallowing amniotic fluid and extend past weaning. Hormonal control plays a major role in the ontogeny of the small intestine. There are late effects of early nutrition, and the normal progress of ontogeny may be important to ensure that the intestine is capable of adaptation in later life.

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Fulminant hepatic failure (FHF) is characterized by massive necroinflammation of the liver tissue and is associated with high mortality. Serum concentrations of IL-1 beta, tumour necrosis factor-alpha (TNF-alpha), IL-6 and IL-1 receptor antagonist (IL-1Ra) were measured in 30 patients with FHF and in 23 patients with acute hepatitis (AH) before start of treatment and in 23 healthy controls. Levels of all four molecules were increased significantly in FHF compared with AH, in which values were higher than in the healthy controls. High serum levels of IL-1 beta and a significantly reduced ratio of IL-1Ra to IL-1 beta (IL-1Ra/IL-1 beta) were observed in FHF patients who subsequently died compared with subjects who survived. TNF-alpha and IL-6 concentrations were correlated with levels of human hepatocyte growth factor (hHGF), an index of hepatocyte regeneration. Although serum cytokine levels varied considerably between patients within each group studied, it is suggested that the striking elevation in proinflammatory cytokine levels in FHF may reflect both the insufficiency of hepatitis virus elimination and a failure to control a vicious cytokine cascade leading to overwhelming hepatocyte destruction rather than regeneration. The high cytokine levels observed in these patients and the significantly elevated IL-1Ra/IL-1 beta ratio in FHF patients who survived compared with those who did not suggest the possible therapeutic use of cytokine antagonists for the control of this life-threatening disease.

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A study of the histopathological abnormalities in a case of alopecia universalis was accompanied by immunohistochemical analysis of the expression of intercellular adhesion molecule-1 (ICAM-1) and E-selectin (formerly known as endothelial leukocyte adhesion molecule-1) within the skin. ICAM-1 expression on follicular epithelium co-localized with intraepithelial mononuclear cells (MNC) positive for the interleukin-2 receptor alpha-chain (IL-2R) or HLA-DR. Aberrant expression of E-selectin was observed on dermal endothelium. Although restricted to one case, these new observations concerning the expression of E-selectin and IL-2R in alopecia universalis are consistent with the view that extravascular trafficking of MNC into follicular epithelium may play a key role in the pathogenesis of alopecia universalis and that use of agents that interfere with this process may be an effective therapeutic strategy.

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