RESUMO
PURPOSE: The primary objective of this phase I dose-escalation study was to identify the maximum tolerated dose (MTD) of sunitinib plus pemetrexed in patients with advanced cancer. METHODS: Using a 3 + 3 dose-escalation design, patients received oral sunitinib qd by continuous daily dosing (CDD schedule; 37.5 or 50 mg) or 2 weeks on/1 week off treatment schedule (Schedule 2/1; 50 mg). Pemetrexed (300-500 mg/m(2) IV) was administered q3w. At the proposed recommended phase 2 dose (RP2D), additional patients with non-small cell lung cancer (NSCLC) were enrolled. RESULTS: Thirty-five patients were enrolled on the CDD schedule and seven on Schedule 2/1. MTDs were sunitinib 37.5 mg/day (CDD/RP2D) or 50 mg/day (Schedule 2/1) with pemetrexed 500 mg/m(2). Dose-limiting toxicities included grade (G) 5 cerebral hemorrhage, G3 febrile neutropenia, and G3 anorexia. Common G3/4 drug-related non-hematologic adverse events (AEs) at the CDD MTD included fatigue, anorexia, and hand-foot syndrome. G3/4 hematologic AEs included lymphopenia, neutropenia, and thrombocytopenia. No significant drug-drug interactions were identified. Five (24%) NSCLC patients had partial responses. CONCLUSIONS: In patients with advanced solid malignancies, the MTD of sunitinib plus 500 mg/m(2) pemetrexed was 37.5 mg/day (CDD schedule) or 50 mg/day (Schedule 2/1). The CDD schedule MTD was tolerable and demonstrated promising clinical benefit in NSCLC.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Administração Oral , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Estudos de Coortes , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Glutamatos/administração & dosagem , Glutamatos/efeitos adversos , Glutamatos/farmacocinética , Glutamatos/uso terapêutico , Guanina/administração & dosagem , Guanina/efeitos adversos , Guanina/análogos & derivados , Guanina/farmacocinética , Guanina/uso terapêutico , Humanos , Indóis/administração & dosagem , Indóis/efeitos adversos , Indóis/farmacocinética , Indóis/uso terapêutico , Neoplasias Pulmonares/metabolismo , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Pemetrexede , Pirróis/administração & dosagem , Pirróis/efeitos adversos , Pirróis/farmacocinética , Pirróis/uso terapêutico , Sunitinibe , Resultado do TratamentoRESUMO
BACKGROUND: Islet xenotransplantation will most likely be performed in diabetic patients treated with immunosuppressive drugs. The importance of the galactosyl alpha(1-3) galactose (Galalpha1-3Gal) antigen in immunosuppressed islet xenograft recipients has not been studied. METHODS: Fetal porcine islet-like cell clusters (ICCs) were transplanted into the renal subcapsular space of both Gal-knockout mice and ordinary mice. Transplantations were performed in untreated mice and mice immunosuppressed with cyclosporine A (CsA) plus 15-deoxyspergualin (DSG). Studies were also performed in immunosuppressed Gal-knockout mice that had been actively immunized against Galalpha1-3Gal. Evaluation was performed 12 days after transplantation using morphologic techniques. The levels of serum immunoglobulin (Ig)G and IgM to the Galalpha1-3Gal antigen or to the ICCs were determined. RESULTS: No difference in the morphologic appearance could be seen between ordinary mice and Gal-knockout mice. No deposits of IgG, IgM, or C3 could be detected. Almost no difference could be seen between immunosuppressed Gal-knockout mice and immunosuppressed ordinary mice. In immunosuppressed, immunized Gal-knockout mice, the results were similar. In ordinary mice treated with CsA+DSG, the levels of anti-Gal IgM were lower than they were in untreated mice, whereas the levels of anti-Gal IgG were similar. In Gal-knockout mice (including immunized animals) treated with CsA+DSG, the levels of anti-Gal IgG and IgM were lower than they were in untreated Gal-knockout mice. CONCLUSIONS: After renal subcapsular transplantation, antibodies against Galalpha1-3Gal have no major influence on islet xenograft rejection in the pig-to-mouse model. Immunosuppression, which inhibits rejection in the pig-to-mouse model, is equally effective when transplantation is performed across the Galalpha1-3Gal barrier.
Assuntos
Antígenos Heterófilos , Dissacarídeos/imunologia , Transplante das Ilhotas Pancreáticas/imunologia , Animais , Anticorpos Heterófilos/sangue , Autoanticorpos/sangue , Galactosiltransferases/deficiência , Galactosiltransferases/genética , Rejeição de Enxerto/etiologia , Rejeição de Enxerto/imunologia , Humanos , Imunização , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Nus , Sus scrofa , Transplante HeterólogoRESUMO
BACKGROUND: Arsenic trioxide (As2O3) is an anticancer drug that has been reported to induce apoptosis and inhibit differentiation in human plasmacytoma and normal plasma/B cells without significant myelosuppression. We assessed the ability of As2O3 as single therapy or in combination with an anti-CD20 monoclonal antibody (mAb) and whole body irradiation (WBI) to deplete B and plasma cells, both in vitro and in vivo, and to reduce the level of anti-alphaGal1-3Gal antibody (anti-Gal Ab) in baboons. METHODS: In vitro the effect of As2O3 on antibody secretion (anti-Gal IgM, total IgG and IgM) was measured by enzyme-linked immunospot assay (ELISPOT). Its inhibition of proliferation of baboon splenocytes and the NCI-H929 human plasmacytoma cell line was measured by tritiated thymidine uptake. In vivo: all baboons (n=7) had undergone splenectomy. The effects of As2O3 (0.18 to 0.36 mg/kg) on B/plasma cell depletion and anti-Gal Ab production were assessed in three baboons. For comparison, three baboons received either WBI (2 x 150 cGy) or anti-CD20 mAb (20 mg/kg x 4 doses), or both WBI and anti-CD20 mAb. A final baboon received As2O3 + WBI (150 cGy) + anti-CD20 mAb. Anti-Gal Ab levels were measured daily by ELISA. Depletion of B cells from blood and bone marrow (BM) was monitored by flow cytometry and by histology of lymph nodes (LN). Autopsy was performed in three baboons. RESULTS: In vitro: As2O3 (at 5 x 10-6 mol/l) reduced anti-Gal IgM and total IgM secretors by 76% (P=0.53) and 95% (P < 0.001), respectively, but did not reduce total IgG secretors. As2O3 inhibited in a dose-dependent manner the proliferation of activated splenocytes and of the NCI-H929 plasmacytoma cell line; complete inhibition was achieved at a dose of 1 x 10-5 mol/l. In vivo: As2O3 was found to be toxic at the doses given and was associated with the deaths of two of the four baboons that received it. Daily intravenous therapy with As2O3 alone reduced B cells (CD20+) in the blood (by 50 to 90%), BM (40%) and LN (20 to 30%), but anti-Gal Ab levels were not significantly decreased. Anti-CD20 mAb therapy alone or WBI alone depleted B cells by 100% in the blood and BM, and 80 to 100% in the LN. The combination of anti-CD20 mAb + WBI led to depletion of B cells in blood, BM and LN for 3 months, but reduction of anti-Gal Ab remained marginal. The combination of As2O3 + anti-CD20 mAb + WBI did not reduce anti-Gal Ab levels further. At autopsy in the latter baboon, B cells remained present in Peyer's patches and tonsils. CONCLUSIONS: In vitro: As2O3 reduced B/plasma cell numbers and suppressed IgM secretors, but not IgG secretors. In vivo: As2O3 was not as effective as either anti-CD20 mAb or WBI in depleting B/plasma cells, and was largely ineffective in reducing anti-Gal Ab levels. Its administration was associated with considerable toxicity. Autopsy in one baboon suggested that B cells in Peyer's patches and tonsils may be resistant to therapy and remain a source of continuing production of anti-Gal Ab.
Assuntos
Antineoplásicos/toxicidade , Dissacarídeos/imunologia , Óxidos/toxicidade , Plasmócitos/efeitos dos fármacos , Transplante Heterólogo/imunologia , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Antígenos CD20/imunologia , Arsênio/sangue , Trióxido de Arsênio , Arsenicais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Técnicas In Vitro , Linfonodos/citologia , Papio , Plasmócitos/imunologia , Baço/citologia , Irradiação Corporal TotalRESUMO
The successful establishment of tolerance toward pig tissues in primates through hematopoietic progenitor cell engraftment is restricted by the rapid disappearance of these cells in the recipient following infusion. We developed and tested the hypothesis that phagocytes of the reticuloendothelial system are responsible for the rapid clearance of infused pig hematopoietic cells using a mouse model. Mice received non-myeloablative conditioning and, on various days, were injected with medronate-encapsulated liposomes (M-L) or control blank liposomes, followed by the intravenous infusion of miniature swine hematopoietic cells. M-L were well-tolerated in mice (n=100) at levels that deplete mononuclear phagocytes. Depletion of mononuclear phagocytes in normal Balb/c mice as well as in severe combined immune deficient mice increased the accumulation of pig hematopoietic cells in the bone marrow (BM) by 10-fold when measured 24 h after the infusion of the cells. Colony-forming unit analysis showed an increased accumulation of pig hematopoietic progenitors in the BM of mice that were infused with medronate-liposomes. We conclude that depletion of mononuclear phagocytes by M-L has the potential to lower the barrier to the establishment of mixed chimerism and tolerance induction in xenotransplantation.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Leucócitos Mononucleares/imunologia , Fagócitos/imunologia , Quimeras de Transplante/imunologia , Transplante Heterólogo/imunologia , Animais , Sobrevivência Celular/imunologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Tolerância Imunológica/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , PapioRESUMO
The treatment of primary central nervous system lymphoma (PCNSL) with chemo- and radiotherapy is efficient in terms of tumor response. However, time to tumor progression often is of short duration and leptomeningeal relapse is common. We present a 66-year-old man in third relapse of a CD20-positive PCNSL. After treatment with intravenous and intraventricular administration of the chimeric anti-CD20 monoclonal antibody rituximab, a total clearing of lymphoma cells in the cerebrospinal fluid (CSF) was achieved. There was no change in the size of the parenchymal tumor mass but there was slight improvement of clinical symptoms after therapy. Rituximab infusions (375 mg/m2) were first given systemically on days 1 and 8. Intraventricular injections of rituximab via Ommaya reservoir were given on days 16 (10 mg), 17 (40 mg), 24 (25 mg) and 25 (25 mg). Reversible side effects such as nausea, chills and hypotension were observed only immediately after intraventricular administration of 40 mg rituximab. Antibody levels in CSF were measured at 7 timepoints during and after the treatment period. These data suggest that intraventicular treatment with rituximab is safe and feasible with a potential activity on leptomeningeal tumor manifestation. Efficacy and pharmacokinetics of rituximab in PCNSL should be investigated in future trials.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Antineoplásicos/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Linfoma de Células B/tratamento farmacológico , Idoso , Anticorpos Monoclonais Murinos , Antígenos CD20/metabolismo , Circulação Sanguínea/imunologia , Neoplasias Encefálicas/líquido cefalorraquidiano , Neoplasias Encefálicas/patologia , Humanos , Infusões Intravenosas , Injeções Intraventriculares , Linfoma de Células B/líquido cefalorraquidiano , Linfoma de Células B/patologia , Imageamento por Ressonância Magnética , Masculino , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/imunologia , Indução de Remissão , RituximabRESUMO
The effect of CD154 blockade and macrophage depletion or inhibition on baboon humoral and cellular immune responses to pig antigens was studied in a pig-to-baboon peripheral blood mobilized progenitor cell (PBPC) transplantation model aimed at inducing tolerance. We infused pig PBPCs in baboons pretreated with a nonmyeloablative regimen along with murine anti-human CD154 monoclonal antibody (mAb) and macrophage-depleting or -inhibiting agents. Group 1 baboons (n=2) underwent a nonmyeloablative regimen and immunoadsorption of anti-Gal(alpha)1,3Gal (Gal) antibody (Ab) before intravenous infusion of high doses (1.3-4.6 x 10(10)cells/kg) of PBPCs. In group 2 (n=5), cyclosporine was replaced by 8 doses of anti-CD154 mAb over 14 days. Group 3 (n=3) received the group 2 regimen plus medronate liposomes (n=2) or commercially available human intravenous immunoglobulin G depleted of anti-Gal Ab (n=1) to deplete/inhibit recipient macrophages. Group 1 developed sensitization to Gal and also developed new Ab to non-Gal porcine antigens within 10 to 20 days. In group 2, no sensitization to Gal or non-Gal determinants was seen, but Gal-reactive antibodies did return to their preleukocyte transplantation levels. CD154 blockade, therefore, induced humoral unresponsiveness to pig cells. In group 3, sensitization to Gal was seen in all three baboons at 20 days, and Abs against new porcine determinants developed in one baboon. The depletion or inhibition of host macrophages, therefore, prevented the induction of humoral unresponsiveness by CD154 blockade. These results suggest that CD154 blockade induces humoral unresponsiveness by a mechanism that involves the indirect pathway of antigen presentation. In vitro investigation of baboon anti-pig mixed lymphocyte reaction confirmed that only the indirect pathway is efficiently blocked by anti-CD154 mAb. The mechanism in which blockade of the CD40-CD154 pathway induces its effect remains to be determined, but it could involve the generation of regulatory cells capable of suppressing the direct pathway.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos CD40/imunologia , Ligante de CD40/imunologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/imunologia , Macrófagos/imunologia , Transplante Heterólogo/imunologia , Animais , Anticorpos/análise , Formação de Anticorpos , Fluorescência , Látex , Teste de Cultura Mista de Linfócitos , Microesferas , Papio , Suínos , Porco MiniaturaRESUMO
INTRODUCTION: Attempts to achieve immunological tolerance to porcine tissues in nonhuman primates through establishment of mixed hematopoietic chimerism are hindered by the rapid clearance of mobilized porcine leukocytes, containing progenitor cells (pPBPCs), from the circulation. Eighteen hours after infusing 1-2 x 10(10) pPBPC/kg into baboons that had been depleted of circulating anti-alphaGal and complement, these cells are almost undetectable by flow cytometry. The aim of the present study was to identify mechanisms that contribute to rapid clearance of pPBPCs in the baboon. This was achieved by depleting, or blocking the Fc-receptors of, cells of the phagocytic reticuloendothelial system (RES) using medronate liposomes (MLs) or intravenous immunoglobulin (IVIg), respectively. METHODS: Baboons (preliminary studies, n=4) were used in a dose-finding and toxicity study to assess the effect of MLs on macrophage depletion in vivo. In another study, baboons (n=9) received a nonmyeloablative conditioning regimen (NMCR) aimed at inducing immunological tolerance, including splenectomy, whole body irradiation (300 cGy) or cyclophosphamide (80 mg/kg), thymic irradiation (700 cGy), T-cell depletion, complement depletion with cobra venom factor, mycophenolate mofetil, anti-CD154 monoclonal antibody, and multiple extracorporeal immunoadsorptions of anti-alphaGal antibodies. The baboons were divided into three groups: Group 1 (n=5) NMCR+pPBPC transplantation; Group 2 (n=2) NMCR+ML+pPBPC transplantation; and Group 3 (n=2) NMCR+IVIg+pPBPC transplantation. Detection of pig cells in the blood was assessed by fluorescence-activated cell sorter and polymerase chain reaction (PCR). PRELIMINARY STUDIES: ML effectively depleted macrophages from the circulation in a dose-dependent manner. Group 1: On average, 14% pig cells were detected 2 hr postinfusion of 1 x 10(10) pPBPC/kg. After 18 hr, there were generally less than 1.5% pig cells detectable. Group 2: Substantially higher levels of pig cell chimerism (55-78%) were detected 2 hr postinfusion, even when a smaller number (0.5-1 x 10(10)/kg) of pPBPCs had been infused, and these levels were better sustained 18 hr later (10-52%). Group 3: In one baboon, 4.4% pig cells were detected 2 hr after infusion of 1 x 10(10) pPBPC/kg. After 18 hr, however, 7.4% pig cells were detected. A second baboon died 2 hr after infusion of 4 x 10(10) pPBPC/kg, with a total white blood cell count of 90,000, of which 70% were pig cells. No differences in microchimerism could be detected between the groups as determined by PCR. CONCLUSIONS: This is the first study to report an efficient decrease of phagocytic function by depletion of macrophages with MLs in a large-animal model. Depletion of macrophages with MLs led to initial higher chimerism and prolonged the survival of circulating pig cells in baboons. Blockade of macrophage function with IVIg had a more modest effect. Cells of the RES, therefore, play a major role in clearing pPBPCs from the circulation in baboons. Depletion or blockade of the RES may contribute to achieving mixed hematopoietic chimerism and induction of tolerance to a discordant xenograft.
Assuntos
Células-Tronco Hematopoéticas/fisiologia , Sistema Fagocitário Mononuclear/fisiologia , Fagocitose/fisiologia , Animais , Contagem de Células Sanguíneas , Relação Dose-Resposta a Droga , Transplante de Células-Tronco Hematopoéticas , Imunoglobulinas Intravenosas/farmacologia , Contagem de Leucócitos , Lipossomos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Papio , Receptores Fc/antagonistas & inibidores , Suínos , Fatores de Tempo , Condicionamento Pré-Transplante/métodos , Transplante HeterólogoRESUMO
E-, P-, and L-selectin counterreceptor activities, leukocyte trafficking, and lymphocyte homing are controlled prominently but incompletely by alpha(1,3)fucosyltransferase FucT-VII-dependent fucosylation. Molecular determinants for FucT-VII-independent leukocyte trafficking are not defined, and evidence for contributions by or requirements for other FucTs in leukocyte recruitment is contradictory and incomplete. We show here that inflammation-dependent leukocyte recruitment retained in FucT-VII deficiency is extinguished in FucT-IV(-/-)/FucT-VII(-/-) mice. Double deficiency yields an extreme leukocytosis characterized by decreased neutrophil turnover and increased neutrophil production. FucT-IV also contributes to HEV-born L-selectin ligands, since lymphocyte homing retained in FucT-VII(-/-) mice is revoked in FucT-IV(-/-)/FucT-VII(-/-) mice. These observations reveal essential FucT-IV-dependent contributions to E-, P-, and L-selectin ligand synthesis and to the control of leukocyte recruitment and lymphocyte homing.
Assuntos
Fucosiltransferases/fisiologia , Leucócitos/fisiologia , Linfócitos/fisiologia , Selectinas/fisiologia , Animais , Movimento Celular , Mapeamento Cromossômico , Feminino , Fucosiltransferases/genética , Humanos , Contagem de Leucócitos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBARESUMO
Anti-Galalpha1-3Gal antibodies (antialphaGal Ab) are a major barrier to clinical xenotransplantation as they are believed to initiate both hyperacute and acute humoral rejection. Extracorporeal immunoadsorption (EIA) with alphaGal oligosaccharide columns temporarily depletes antialphaGal Ab, but their return is ultimately associated with graft destruction. We therefore assessed the ability of two immunotoxins (IT) and two monoclonal antibodies (mAb) to deplete B and/or plasma cells both in vitro and in vivo in baboons, and to observe the rate of return of antialphaGal Ab following EIA. The effects of the mouse anti-human IT anti-CD22-ricin A (proportional to CD22-IT, directed against a B cell determinant) and anti-CD38-ricin A (proportional to CD38-IT, B and plasma cell determinant) and the mouse anti-human anti-CD38 mAb (proportional to CD38 mAb) and mouse/human chimeric anti-human anti-CD20 mAb (proportional to CD20 mAb, Rituximab, B cell determinant) on B and plasma cell depletion and antialphaGal Ab production were assessed both in vitro and in vivo in baboons (n = 9) that had previously undergone splenectomy. For comparison, two baboons received nonmyeloablative whole body irradiation (WBI) (300 cGy), and one received myeloablative WBI (900 cGy). Depletion of B cells was monitored by flow cytometry of blood, bone marrow (BM) and lymph nodes (LN), staining with anti-CD20 and/or anti-CD22 mAbs, and by histology of LN. EIA was carried out after the therapy and antialphaGal Ab levels were measured daily. In vitro proportional to CD22-IT inhibited protein synthesis in the human Daudi B cell line more effectively than proportional to CD38-IT. Upon differentiation of B cells into plasma cells, however, less inhibition of protein synthesis after proportional to CD22-IT treatment was observed. Depleting CD20-positive cells in vitro from a baboon spleen cell population already depleted of granulocytes, monocytes, and T cells led to a relative enrichment of CD20-negative cells, that is plasma cells, and consequently resulted in a significant increase in antialphaGal Ab production by the remaining cells, whereas depleting CD38-positive cells resulted in a significant decrease in antialphaGal Ab production. In vivo, WBI (300 or 900 cGy) resulted in 100% B cell depletion in blood and BM, > 80% depletion in LN, with substantial recovery of B cells after 21 days and only transient reduction in antialphaGal Ab after EIA. Proportional to CD22-IT depleted B cells by > 97% in blood and BM, and by 60% in LN, but a rebound of B cells was observed after 14 and 62 days in LN and blood, respectively. At 7 days, serum antialphaGal IgG and IgM Ab levels were reduced by a maximum of 40-45% followed by a rebound to levels up to 12-fold that of baseline antialphaGal Ab by day 83 in one baboon. The results obtained with proportional to CD38-IT were inconclusive. This may have been, in part, due to inadequate conjugation of the toxin. Cell coating was 100% with proportional to CD38 mAb, but no changes in antialphaGal Ab production were observed. Proportional to CD20 mAb resulted in 100% depletion of B cells in blood and BM, and 80% in LN, with recovery of B cells starting at day 42. Adding 150cGy WBI at this time led to 100% depletion of B cells in the BM and LN. Although B cell depletion in blood and BM persisted for > 3 months, the reduction of serum antialphaGal IgG or IgM Ab levels was not sustained beyond 2 days. Proportional to CD20 mAb + WBI totally and efficiently depleted CD20- and CD22-positive B cells in blood, BM, and LN for > 3 months in vivo, but there was no sustained clinically significant reduction in serum antialphaGal Ab. The majority of antibody secretors are CD38-positive cells, but targeting these cells in vitro or in vivo with proportional to CD38-IT was not very effective. These observations suggest that CD20-and CD22-positive B cells are not the major source of antialphaGal Ab production. Future efforts will be directed towards suppression of plasma cell function.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos CD20/imunologia , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Linfócitos B/imunologia , Moléculas de Adesão Celular , Dissacarídeos/imunologia , Lectinas , Depleção Linfocítica , Plasmócitos/imunologia , Animais , Formação de Anticorpos , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Humanos , Imunoterapia , Camundongos , Papio , Ricina/imunologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Transplante Heterólogo/imunologiaAssuntos
Anticorpos Monoclonais/uso terapêutico , Ligante de CD40/imunologia , Antígenos CD55/imunologia , Transplante de Rim/imunologia , Transplante Heterólogo/imunologia , Animais , Ciclofosfamida/uso terapêutico , Coagulação Intravascular Disseminada/etiologia , Sobrevivência de Enxerto , Humanos , Imunoglobulina M/imunologia , Imunossupressores/uso terapêutico , Papio , Suínos , Porco Miniatura , Irradiação Corporal TotalRESUMO
Human natural Abs against Galalpha1-3Galbeta1-4GlcNAc (Gal) epitopes are a major barrier to xenotransplantation. Studies in this report, which use combined multiparameter flow cytometric sorting and enzyme-linked immunospot assay, demonstrate that anti-Gal IgM-producing cells are found exclusively in a small B cell subpopulation (i.e., CD21(-/low) IgM(high) B220(low) CD5(-) Mac-1(-) 493(-) cells) in the spleens of alpha1, 3-galactosyltransferase-deficient mice. All IgM-producing cells were detected in a similar splenic subpopulation of alpha1, 3-galactosyltransferase-deficient and wild-type mice. A higher frequency of B cells with anti-Gal surface IgM receptors was observed in the peritoneal cavity than in the spleen, but these did not actively secrete Abs, and showed phenotypic properties of B-1b cells (CD21(-/low) IgM(high) CD5(-) CD43(+) Mac-1(+)). However, these became Mac-1(-) and developed anti-Gal Ab-producing activity after in vitro culture with LPS. The splenic B cells with anti-Gal receptors consisted of both Mac-1(+) B-1b cells and Mac-1(-) B-1b-like cells. The latter comprised most anti-Gal IgM-producing cells. Our studies indicate that anti-Gal natural IgM Abs are produced by a B1b-like, Mac-1(-) splenic B cell population and not by plasma cells or B-1a cells. They are consistent with a model whereby B-1b cells lose Mac-1 expression upon Ag exposure and that these, rather than plasma cells, become the major IgM Ab-producing cell population.
Assuntos
Subpopulações de Linfócitos B/enzimologia , Subpopulações de Linfócitos B/imunologia , Dissacarídeos/imunologia , Epitopos de Linfócito B/imunologia , Galactosiltransferases/deficiência , Galactosiltransferases/genética , Antígeno de Macrófago 1/biossíntese , Animais , Células Produtoras de Anticorpos/enzimologia , Células Produtoras de Anticorpos/imunologia , Células Produtoras de Anticorpos/metabolismo , Subpopulações de Linfócitos B/metabolismo , Antígenos CD5/metabolismo , Dissacarídeos/metabolismo , Epitopos de Linfócito B/metabolismo , Imunidade Celular , Imunoglobulina M/biossíntese , Imunofenotipagem , Antígenos Comuns de Leucócito/biossíntese , Antígeno de Macrófago 1/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cavidade Peritoneal/citologia , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Complemento 3d/biossíntese , Baço/citologia , Baço/enzimologia , Baço/imunologiaAssuntos
Linfócitos B/imunologia , Moléculas de Adesão Celular , Dissacarídeos/imunologia , Lectinas , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Animais , Antígenos CD/imunologia , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Epitopos/imunologia , Galactosiltransferases/deficiência , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Glicoproteínas de Membrana , Camundongos , Camundongos Knockout , NAD+ Nucleosidase/imunologia , Papio , Plasmócitos/imunologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Baço/imunologiaAssuntos
Anticorpos Monoclonais/farmacologia , Antígenos de Diferenciação/imunologia , Antígenos CD2/imunologia , Imunoconjugados , Imunossupressores/farmacologia , Teste de Cultura Mista de Linfócitos , Glicoproteínas de Membrana/imunologia , Linfócitos T/imunologia , Abatacepte , Animais , Antígenos CD/imunologia , Ligante de CD40 , Antígeno CTLA-4 , Humanos , Imunidade Celular , Suínos , Porco Miniatura , Transplante Heterólogo/imunologiaAssuntos
Linfócitos B/imunologia , Moléculas de Adesão Celular , Galactosídeos/imunologia , Imunossupressores/farmacologia , Lectinas , Depleção Linfocítica , Plasmócitos/imunologia , Animais , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Cladribina/farmacologia , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Metotrexato/farmacologia , Papio , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Timo/efeitos da radiação , Irradiação Corporal Total , Zidovudina/farmacologiaAssuntos
Transplante de Células-Tronco Hematopoéticas , Terapia de Imunossupressão/métodos , Glicoproteínas de Membrana/imunologia , Transplante Heterólogo/imunologia , Animais , Formação de Anticorpos , Antígenos CD40/imunologia , Ligante de CD40 , Proteínas Inativadoras do Complemento/farmacologia , Ciclosporina/uso terapêutico , Venenos Elapídicos/farmacologia , Imunidade Celular , Leucaférese , Teste de Cultura Mista de Linfócitos , Depleção Linfocítica , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/farmacologia , Papio , Esplenectomia , Suínos , Linfócitos T/imunologia , Irradiação Corporal TotalAssuntos
Transplante de Células-Tronco Hematopoéticas , Terapia de Imunossupressão/métodos , Quimeras de Transplante , Transplante Heterólogo/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Antígenos CD40/imunologia , Ligante de CD40 , Proteínas Inativadoras do Complemento/farmacologia , Venenos Elapídicos/farmacologia , Imunoglobulina G/análise , Imunossupressores/farmacologia , Interleucina-3/farmacologia , Depleção Linfocítica , Glicoproteínas de Membrana/imunologia , Papio , Fator de Células-Tronco/farmacologia , Suínos , Linfócitos T/imunologia , Irradiação Corporal TotalAssuntos
Anticorpos Anti-Idiotípicos/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Transplante de Células-Tronco Hematopoéticas , Transplante Heterólogo/imunologia , Animais , Dissacarídeos/imunologia , Epitopos/imunologia , Fator Estimulador de Colônias de Granulócitos/imunologia , Humanos , Técnicas de Imunoadsorção , Interleucina-3/imunologia , Papio , Fator de Células-Tronco/imunologia , SuínosRESUMO
P-selectin glycoprotein ligand-1 (PSGL-1) and E-selectin ligand-1 (ESL-1) are the two major selectin ligands on mouse neutrophils. Transfection experiments demonstrate that each ligand requires alpha1,3-fucosylation for selectin-binding. However, the relative contributions made by the two known myeloid alpha1, 3-fucosyltransferases Fuc-TVII or Fuc-TIV to this alpha1, 3-fucosylation are not yet clear. To address this issue, we have used mice deficient in Fuc-TIV and/or Fuc-TVII to examine how these enzymes generate selectin-binding glycoforms of PSGL-1 and ESL-1 in mouse neutrophils. Selectin binding was analyzed by affinity isolation experiments using recombinant, antibody-like forms of the respective endothelial selectins. We observe essentially normal binding of E- or P-selectin to PSGL-1 expressed by Fuc-TIV-deficient neutrophils but find that PSGL-1 expressed by Fuc-TVII-deficient neutrophils is not bound by E- or P-selectin. By contrast, E-selectin binds with normal efficiency to ESL-1 on Fuc-TVII-deficient neutrophils but exhibits an 80% reduction in its ability to bind ESL-1 isolated from Fuc-TIV-deficient neutrophils. The same specificity with which Fuc-TVII and Fuc-TIV generate selectin-binding forms of PSGL-1 and ESL-1 was found in transfection experiments with CHO-Pro(-)5 cells. In contrast, each fucosyltransferase alone could generate selectin-binding glycoforms of each of the two ligands in CHO-DUKX-B1 cells. Our data imply that in mouse neutrophils and their precursors, Fuc-TVII exclusively directs expression of PSGL-1 glycoforms bound with high affinity by P-selectin. By contrast, Fuc-TIV preferentially directs expression of ESL-1 glycoforms that exhibit high affinity for E-selectin. This substrate specificity can be mimicked in CHO-Pro(-)5 cells.
Assuntos
Fucosiltransferases/metabolismo , Glicoproteínas de Membrana/metabolismo , Neutrófilos/enzimologia , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Animais , Biotinilação , Células CHO , Cromatografia de Afinidade , Cricetinae , Fucosiltransferases/genética , Imunoglobulina G/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Testes de Precipitina , Ligação Proteica , Isoformas de Proteínas , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Selectinas/metabolismo , Sialoglicoproteínas , TransfecçãoRESUMO
BACKGROUND: In pig-to-primate organ transplantation, hyperacute rejection can be prevented, but the organ is rejected within days by acute vascular rejection, in which induced high-affinity anti-Gal alpha1-3Gal (alphaGal) IgG and possibly antibodies directed against new porcine (non-alphaGal) antigenic determinants are considered to play a major role. We have explored the role of an anti-CD40L monoclonal antibody in modifying the humoral response to porcine hematopoietic cells in baboons pretreated with a nonmyeloablative regimen. METHODS: Porcine peripheral blood mobilized progenitor cells obtained by leukapheresis from both major histocompatibility complex-inbred miniature swine (n=7) and human decay-accelerating factor pigs (n=3) were transplanted into baboons. Group 1 baboons (n=3) underwent whole body (300 cGy) and thymic (700 cGy) irradiation, T cell depletion with ATG, complement depletion with cobra venom factor, short courses of cyclosporine, mycophenolate mofetil, porcine hematopoietic growth factors, and anti-alphaGal antibody depletion by immunoadsorption before transplantation of high doses (2-4 x 10(10)/cells/kg) of peripheral blood mobilized progenitor cells. In group 2 (n=5), cyclosporine was replaced by eight doses of anti-CD40L monoclonal antibodies over 14 days. The group 3 baboons (n=2) received the group 1 regimen plus 2 doses of anti-CD40L monoclonal antibodies (on days 0 and 2). RESULTS: In group 1, sensitization to alphaGal (with increases in IgM and IgG of 3- to 6-fold and 100-fold, respectively) and the development of antibodies to new non-alphaGal porcine antigens occurred within 20 days. In group 2, no sensitization to alphaGal or non-alphaGal determinants was seen, but alphaGal-reactive antibodies did return to their pre- peripheral blood mobilized progenitor cells transplant levels. In group 3, attenuated sensitization to alphaGal antigens was seen after cessation of cyclosporine and mycophenolate mofetil therapy at 30 days (IgM 4-fold, IgG 8-30-fold), but no antibodies developed against new porcine determinants. In no baboon did anti-CD40L monoclonal antibodies prevent sensitization to its own murine antigens. CONCLUSIONS: We believe these studies are the first to consistently demonstrate prevention of a secondary humoral response after cell or organ transplantation in a pig-to-primate model. The development of sensitization to the murine elements of the anti-CD40L monoclonal antibodies suggests that nonresponsiveness to cell membrane-bound antigen (e.g., alphaGal) is a specific phenomenon and not a general manifestation of immunological unresponsiveness. T cell costimulatory blockade may facilitate induction of mixed hematopoietic chimerism and, consequently, of tolerance to pig organs and tissues.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Glicoproteínas de Membrana/antagonistas & inibidores , Papio/imunologia , Porco Miniatura/imunologia , Transplante Heterólogo/imunologia , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Formação de Anticorpos/efeitos dos fármacos , Sangue/imunologia , Ligante de CD40 , Citotoxicidade Imunológica , Humanos , Glicoproteínas de Membrana/imunologia , Camundongos , Morbidade , Mortalidade , Suínos , Condicionamento Pré-Transplante/métodos , Transplante Heterólogo/mortalidadeRESUMO
Despite increasing use of swine in transplantation research, the ability to block costimulation of allogeneic T cell responses has not been demonstrated in swine, and the effects of costimulatory blockade on xenogeneic human anti-porcine T cell responses are also not clear. We have compared the in vitro effects of anti-human CD154 mAb and human CTLA4IgG4 on allogeneic pig T cell responses and xenogeneic human anti-pig T cell responses. Both anti-CD154 mAb and CTLA4IgG4 cross-reacted on pig cells. While anti-CD154 mAb and CTLA4IgG4 both inhibited the primary allogeneic pig MLRs, CTLA4IgG4 (7.88 microg/ml) was considerably more inhibitory than anti-CD154 mAb (100 microg/ml) at optimal doses. Anti-CD154 mAb inhibited the production of IFN-gamma by 75%, but did not inhibit IL-10 production, while CTLA4IgG4 completely inhibited the production of both IFN-gamma and IL-10. In secondary allogeneic pig MLRs, CTLA4IgG4, but not anti-CD154 mAb, induced Ag-specific T cell anergy. CTLAIgG4 completely blocked the indirect pathway of allorecognition, while anti-CD154 mAb blocked the indirect response by approximately 50%. The generation of porcine CTLs was inhibited by CTLA4IgG4, but not by anti-CD154 mAb. Human anti-porcine xenogeneic MLRs were blocked by CTLA4IgG4, but only minimally by anti-CD154 mAb. Finally, CTLA4IgG4 prevented secondary xenogeneic human anti-porcine T cell responses. These data indicate that blockade of the B7-CD28 pathway was more effective than blockade of the CD40-CD154 pathway in inhibiting allogeneic pig T cell responses and xenogeneic human anti-pig T cell responses in vitro. These findings have implications for inhibiting cell-mediated immune responses in pig-to-human xenotransplantation.