RESUMO
Gene expression profiling of transplant recipient blood and urine can potentially be used to monitor graft function, but the multitude of protocols in use make sharing data and comparing results from different laboratories difficult. The goal of this study was to evaluate the performance of current methods of RNA isolation, reverse transcription and quantitative polymerase chain reaction (qPCR) and to test whether multiple centers using a standardized protocol can obtain the same results. Samples, reagents and detailed instructions were distributed to six participating sites that performed RNA isolation, reverse transcription and qPCR for 18S, PRF, GZB, IL8, CXCL9 and CXCL10 as instructed. All data were analyzed at a single site. All sites demonstrated proficiency in RNA isolation and qPCR analysis. Gene expression measurements for all targets and samples had correlations >0.938. The coefficient of variation of fold-changes between pairs of samples was less than 40%. All sites were able to accurately quantify a control sample of known concentration within a factor of 1.5. Collectively, we have formulated and validated detailed methods for measuring gene expression in blood and urine that can yield consistent results in multiple laboratories.
Assuntos
Perfilação da Expressão Gênica/normas , Regulação da Expressão Gênica , Transplante de Rim , RNA Mensageiro/análise , DNA Polimerase Dirigida por RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Perfilação da Expressão Gênica/métodos , Humanos , Limite de Detecção , RNA Mensageiro/genética , DNA Polimerase Dirigida por RNA/genética , Sensibilidade e Especificidade , Transplante HomólogoRESUMO
Transmembrane signaling of normal human T cells was explored with mAbs directed at TCR, CD2, CD4, CD5, or CD8 antigens and highly purified CD4+ T cells and CD8+ T cells. Our experiments explicitly show that: (a) crosslinkage of TCR with the CD2 antigen, and not independent crosslinking of TCR and of CD2 antigen or crosslinking of either protein with the CD4 or CD8 antigen induces significant proliferation independent of co-stimulatory signals (e.g., accessory cells, recombinant lymphokines, or tumor promoter), (b) F(ab')2 fragments of mAb directed at the TCR and F(ab')2 anti-CD2, crosslinked with F(ab')2 fragments of rabbit anti-mouse IgG, promote the proliferation of highly purified T cells, (c) a prompt and sustained increase in intracellular free Ca2+ concentration results from crosslinkage of TCR with the CD2 antigen, (d) T cell proliferation induced by this novel approach is curtailed by EGTA and by direct or competitive inhibitors of PKC, (e) crosslinkage of TCR with the CD2 antigen results in the transcriptional activation and translation of the gene for IL-2 and in the expression of IL-2 receptor alpha (CD25), (f) anti-CD25 mAbs inhibit T cell proliferation initiated by crosslinkage of TCR with the CD2 antigen, and recombinant IL-2 restores the proliferative response. Our first demonstration that crosslinkage of TCR with the CD2 antigen induces proliferation of normal human CD4+ T cells and CD8+ T cells, in addition to revealing a novel activation mechanism utilizable by the two major subsets of T cells, suggest that the CD2 antigen might be targeted for the regulation of antigen-specific T cell immunity (e.g., organ transplantation).
Assuntos
Antígenos de Diferenciação de Linfócitos T/imunologia , Ativação Linfocitária/imunologia , Modelos Biológicos , Receptores de Antígenos de Linfócitos T/imunologia , Receptores Imunológicos/imunologia , Transdução de Sinais , Linfócitos T/imunologia , Anticorpos Monoclonais , Sequência de Bases , Antígenos CD2 , Complexo CD3 , Antígenos CD4/imunologia , Antígenos CD8 , Cálcio/metabolismo , Reagentes de Ligações Cruzadas , DNA , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Interleucina-2/biossíntese , Interleucina-2/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteína Quinase C/metabolismo , Receptores de Interleucina-2/metabolismoRESUMO
Supernates of neuraminidase and galactose oxidase (NAGO)-treated lymphocytes induce blastogenesis in nonproliferating cells harvested 7--14 d after treatment with mitogen or alloantigen and in cells incubated with mitogen for 7--14 d but not in freshly isolated peripheral blood lymphocytes9 Virtually all the growth factor is produced by NAGO-treated cells during the first 24 h of incubation, and no increase in factor activity is detected upon further cell culture. Serum is not required for growth factor production. NAGO-primed medium induces generation of specific cytotoxic T cells from mixed lymphocyte culture (MLC) memory cells to approximately the same extent as that induced by allogeneic cells (stimulating cells in the primary MLC). NAGO-primed medium provides a useful reagent for isolation and characterization of lymphocyte growth factors and other lymphokines.
Assuntos
Galactose Oxidase/metabolismo , Substâncias de Crescimento/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/imunologia , Mitógenos , Neuraminidase/metabolismo , Adulto , Células Cultivadas , Substâncias de Crescimento/isolamento & purificação , Humanos , Memória Imunológica , Linfocinas/isolamento & purificação , Pessoa de Meia-Idade , Fito-Hemaglutininas/farmacologiaRESUMO
Advanced glycosylation endproducts (AGEs), the glucose-derived adducts that form nonenzymatically and accumulate on tissue proteins, are implicated in many chronic complications associated with diabetes and aging. We have previously described a monocyte/macrophage surface receptor system thought to coordinate AGE protein removal and tissue remodeling, and purified a corresponding 90-kD AGE-binding protein from the murine RAW 264.7 cell line. To identify AGE-binding proteins in normal animals, the tissue distribution of 125I-AGE rat serum albumin taken up from the blood was determined in rats in vivo. These uptake studies demonstrated that the liver was a major site of AGE protein sequestration. Using a solid-phase assay system involving the immobilization of solubilized membrane proteins onto nitrocellulose to monitor binding activity, and several purification steps including affinity chromatography over an AGE bovine serum albumin matrix, two rat liver membrane proteins were isolated that specifically bound AGEs, one migrating at 60 kD (p60) and the other at 90 kD (p90) on SDS-PAGE. NH2-terminal sequence analysis revealed no significant homology between these two proteins nor to any molecules available in sequence databases. Flow cytometric analyses using avian antibodies to purified rat p60 and p90 demonstrated that both proteins are present on rat monocytes and macrophages. Competition studies revealed no crossreactivity between the two antisera; anti-p60 and anti-p90 antisera prevented AGE-protein binding to rat macrophages when added alone or in combination. These results indicate that rat liver contains at least two novel and distinct proteins that recognize AGE-modified macromolecules, although p90 may be related to the previously described 90-kD AGE receptor isolated from RAW 264.7 cells. The constitutive expression of AGE-binding proteins on rat monocytes and macrophages, and the sequestration of circulating AGE-modified proteins by the liver, provides further evidence in support of a role for these molecules in the normal removal of proteins marked as senescent by accumulated glucose-derived covalent addition products, or AGEs.
Assuntos
Glicoproteínas/metabolismo , Fígado/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Envelhecimento , Sequência de Aminoácidos , Animais , Glicosilação , Ligantes , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Dados de Sequência Molecular , Peso Molecular , Ratos , Receptores de Superfície Celular/química , Receptores de Superfície Celular/imunologia , Distribuição TecidualRESUMO
The regulation of mRNA encoding transforming growth factor beta (TGF-beta) and interleukin 2 (IL-2) in normal human T cells was explored using novel competitor DNA constructs in the quantitative polymerase chain reaction and accessory cell-independent T cell activation models. Our experimental design revealed the following: (a) TGF-beta mRNA and IL-2 mRNA are regulated differentially in normal human T cells, quiescent or signaled with the synergistic combinations of: sn-1,2-dioctanoylglycerol and ionomycin or anti-CD3 monoclonal antibody (mAb) and anti-CD2 mAb; (b) the steady-state level of TGF-beta mRNA in the stimulated T cells, in contrast to that of IL-2 mRNA, is increased by the immunosuppressant cyclosporine (CsA); and (c) the paradoxical effect of CsA on TGF-beta mRNA levels is also appreciable at the level of production of functionally active TGF-beta protein. Our findings, in addition to demonstrating the utility of the competitor DNA constructs for the precise quantification of immunoregulatory cytokines, suggest a novel and unifying mechanistic basis for the immunosuppression and some of the complications (e.g., renal fibrosis) associated with CsA usage.
Assuntos
Regulação da Expressão Gênica , Interleucina-2/genética , Reação em Cadeia da Polimerase , Linfócitos T/metabolismo , Fator de Crescimento Transformador beta/genética , Antígenos de Diferenciação de Linfócitos T/fisiologia , Antígenos CD2 , Complexo CD3 , Células Cultivadas , Ciclosporina/farmacologia , Humanos , RNA Mensageiro/análise , Receptores de Antígenos de Linfócitos T/fisiologia , Receptores Imunológicos/fisiologia , Fator de Crescimento Transformador beta/biossínteseRESUMO
Expression of the pluripotent molecule TNF in a focused and antigen-restricted fashion might provide an advantage to the host organism. Given the central role of T cells in antigen-specific immunity, we examined whether activated T cells express TNF on their cell surface. FACS analysis of highly purified normal human T cells labeled with an anti-TNF mAb revealed that T cells express cell surface TNF when signaled with the synergistic combination of a calcium ionophore, ionomycin, and a protein kinase C activator, 12-o-tetradecanoyl phorbol acetate. Cell surface radioiodination studies of stimulated T cells demonstrated the presence of 26-kD transmembrane protein, a size predicted by TNF cDNA and different from that of the 17-kD secreted TNF molecule. The induced cell surface expression of TNF could be blocked with cyclosporine and/or methylprednisolone, and Northern analysis for TNF-specific transcripts revealed that this inhibitory effect occurs pretranslationally. Our demonstration for the first time that stimulated normal human T cells display cell surface TNF provides a mechanistic basis for the realization of effects of TNF in an antigen-specific fashion.
Assuntos
Ativação Linfocitária , Linfócitos T/análise , Fator de Necrose Tumoral alfa/análise , Ciclosporinas/farmacologia , Humanos , Ionomicina/farmacologia , Metilprednisolona/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genéticaRESUMO
Rat thymocyte membrane fractions have been prepared which exhibit strain-specific primary mixed-lymphocyte reaction (MLR)-stimulating and Ia (RT1-B) antigenic properties. These preparations lack the antigenicity of classical, serologically-defined RT1-A (Ag-B) antigens, as defined by in vitro serologic assays. Furthermore, after immunization of allogeneic hosts, specific anti-Ia and MLR-blocking antibodies, but not anti-AgB, alloantibodies are elaborated. Thymidine suicide experiments show that the same clones respond to whole cells and the fragments made from those cells, and the response segregates appropriately in F2 progeny as a major histocompatibility complex (RT1)-linked phenomenon. Hence, it is possible to generate Ia-related allogeneic helper signals in primary, as well as secondary, in vitro responses, using subcellular membrane fragments that have restricted expression of RT1-B-, but not RT1-A-, encoded antigens.
Assuntos
Antígenos de Superfície/análise , Isoantígenos/análise , Ativação Linfocitária , Linfócitos/imunologia , Complexo Principal de Histocompatibilidade , Animais , Reações Antígeno-Anticorpo , Membrana Celular/imunologia , Ligação Genética , Isoantígenos/genética , Teste de Cultura Mista de Linfócitos , Masculino , Ratos , Ratos Endogâmicos/imunologia , Baço/imunologia , Timo/imunologiaRESUMO
During normal aging and in chronic diabetes the excessive accumulation of reactive glucose-protein or glucose-lipid adducts known as advanced glycosylation endproducts (AGEs) has been shown to induce tissue dysfunction, in part through interaction with AGE-specific receptors on monocyte/macrophages and other cells. Recognizing that circulating lymphocytes trafficking through tissues interact with tissue AGEs, we searched for the expression of AGE-binding sites on peripheral blood T lymphocytes. Resting rat and human T cells bound 125I-AGE-albumin with an affinity of 7.8 x 10(7) M-1, whereas, after stimulation with phytohemagglutinin (PHA) for 48 h, binding affinity increased to 5.8 x 10(8) M-1. Flow cytometric analysis of resting rat T cells using polyclonal antibodies raised against rat liver AGE-binding proteins (p60 and p90) revealed the constitutive expression of both immunoreactivities. The number of resting CD4+ and CD8+ T cells positive for anti-p60 antibody binding (34.2 and 58.5%, respectively) increased to 92 and 90% of cells after 48-h stimulation with PHA. Exposure of PHA-activated T lymphocytes to AGE-albumin enhanced expression of interferon gamma (IFN-gamma) mRNA 10-fold and induced greater elaboration of the mature protein than did exposure to unmodified protein or PHA treatment alone. These data indicate that T cells contain an inducible system of surface receptors for AGE-modified proteins, and that receptor occupancy is linked to lymphokine production. This T cell AGE-receptor system might serve to target lymphocytes to AGE-rich tissues and involve them in the regulation of tissue homeostasis either by assisting in macrophage-dependent clearance of AGE-proteins, or by exerting direct antiproliferative action on mesenchymal cells. Under conditions of excessive AGE-protein and AGE lipid accumulation (e.g., aging and diabetes), enhanced production of AGE-induced IFN-gamma may accelerate immune responses that contribute to tissue injury.
Assuntos
Produtos Finais de Glicação Avançada/metabolismo , Interferon gama/biossíntese , Ativação Linfocitária , Receptores de Superfície Celular/metabolismo , Animais , Sequência de Bases , Primers do DNA , Citometria de Fluxo , Expressão Gênica , Humanos , Interferon gama/genética , Ligantes , Macrófagos/química , Masculino , Dados de Sequência Molecular , Monócitos/química , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Regulação para CimaRESUMO
The 10th Banff Conference on Allograft Pathology was held in Banff, Canada from August 9 to 14, 2009. A total of 263 transplant clinicians, pathologists, surgeons, immunologists and researchers discussed several aspects of solid organ transplants with a special focus on antibody mediated graft injury. The willingness of the Banff process to adapt continuously in response to new research and improve potential weaknesses, led to the implementation of six working groups on the following areas: isolated v-lesion, fibrosis scoring, glomerular lesions, molecular pathology, polyomavirus nephropathy and quality assurance. Banff working groups will conduct multicenter trials to evaluate the clinical relevance, practical feasibility and reproducibility of potential changes to the Banff classification. There were also sessions on quality improvement in biopsy reading and utilization of virtual microscopy for maintaining competence in transplant biopsy interpretation. In addition, compelling molecular research data led to the discussion of incorporation of omics-technologies and discovery of new tissue markers with the goal of combining histopathology and molecular parameters within the Banff working classification in the near future.
Assuntos
Anticorpos/química , Transplante de Órgãos/métodos , Biópsia , Canadá , Complemento C4b/metabolismo , Fibrose/patologia , Humanos , Nefropatias/diagnóstico , Nefropatias/patologia , Nefropatias/virologia , Transplante de Rim , Estudos Multicêntricos como Assunto , Fragmentos de Peptídeos/metabolismo , Fenótipo , Infecções por Polyomavirus/diagnóstico , Controle de QualidadeRESUMO
The ability of the monoclonal antibody directed at the T3 antigen (anti-T3) to induce cytolytic activity was investigated since several agents that can activate T cells induce the acquisition of cytolytic activity in a variety of test systems. Pretreatment of human alloimmune memory cells, generated in primary long-term mixed lymphocyte cultures, with anti-T3 resulted in the induction of statistically significant specific secondary cytolytic activity and natural killer (NK) cell-like activity. No such augmentation or induction of cytolytic activity was found with anti-T3 pretreatment when syngeneic cells or inappropriate allogeneic cells (HLA-A, B antigens different from the original priming stimulus) were used as target cells and pretreatment of memory cells with anti-T4 or anti-T8 did not induce cytolytic activity to allogeneic or syngeneic target cells. Differential effects were observed when anti-T3 was added to the cytotoxicity assay in which anti-T3 pretreated alloimmune memory cells were effectors. The addition of anti-T3 to the assay prior to the introduction of target cells resulted in 39 +/- 8% inhibition of specific secondary cytolytic activity and only 5 +/- 8% inhibition of NK cell activity. NK cell activity mediated by large granular lymphocyte-enriched fraction of peripheral blood mononuclear cells (PBM) obtained from normal individuals was significantly augmented by anti-T3 when NK-sensitive cell lines MOLT-4 or K-562 were used as target cells. This augmentation in NK cell activity was not associated with nonspecific cytotoxicity to syngeneic or allogeneic PBM, and anti-T3 failed to activate the LGL fraction depleted of T cells. The monoclonal antibodies, anti-T4 or anti-T8, did not increase NK cell activity. NK cell activity mediated by PBM from eight immunodeficient individuals (four with acquired immunodeficiency syndrome and four with renal allografts) was also significantly augmented by anti-T3 pretreatment. Our findings, in addition to providing a rationale for the frequent occurrence of re-rejection episodes in renal graft recipients treated with anti-T3, suggest that anti-T3 might be utilized to enhance the cytotoxic armamentarium of immunodeficient patients.
Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , Anticorpos Monoclonais/imunologia , Isoantígenos/imunologia , Transplante de Rim , Células Matadoras Naturais/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Citotoxicidade Imunológica , Humanos , Formação de Roseta , Ovinos , Linfócitos T/imunologia , Transplante HomólogoRESUMO
The basic immunosuppressive protocol involves the use of multiple drugs, each directed at a discrete site in the T-cell activation cascade. The prevailing paradigm regarding the mechanisms of action of immunosuppressants is that they function to prevent allograft rejection by preventing cell activation, proliferation and/or cytokine production. A new hypothesis is that some of the immunosuppressants might function by stimulating the expression of immunosuppressive molecules and/or cells.
Assuntos
Imunossupressores/farmacologia , Animais , Humanos , Ativação Linfocitária/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacosRESUMO
Forty patients with metastatic, recurrent, or unresectable renal cell carcinoma were entered into a study of the therapeutic efficacy of adoptive immunotherapy using periodate (IO4-) and interleukin-2 (IL2)-activated autologous leukocytes and continuous infusion low-dose IL2. Patient survival was also examined. The first 15 consecutive patients were enrolled in protocol A without an IL2 priming phase and the following 25 patients were entered in protocol B where a 5-day priming phase was initiated before leukapheresis. A maintenance regimen consisted of either 3 x 10(6) units of recombinant interferon-alpha (rIFN-alpha), three times per week only or together with leukapheresis and infusion of IO4-/IL2-activated cells and 2 days of continuous infusion IL2 per month. Thirty-four patients completed the protocol treatment. Four patients were removed from the study owing to rapid tumor progression and two patients died while receiving treatment. The clinical response rate was 22%: two patients had a complete response and five patients had a partial response. Among the 25 patients who had no clinical response, 11 patients had either a mixed response or stabilization. Neither response, response duration, nor site response correlated with the total dose of IL2 administered or the number of activated killer cells infused. Patients who received maintenance therapy had longer survival times than patients who did not receive such therapy. All toxicity and side effects associated with IL2 treatment were transient and resolved after discontinuation of the drug. Patients on maintenance therapy tolerated both rIFN-alpha and monthly infusions of activated killer cells and IL2 well. This study confirms the concept of adoptive immunotherapy as a new treatment approach for advanced renal cell carcinoma and suggests that maintenance therapy may prolong survival time.
Assuntos
Carcinoma de Células Renais/terapia , Interleucina-2/administração & dosagem , Neoplasias Renais/terapia , Células Matadoras Ativadas por Linfocina/imunologia , Adulto , Idoso , Esquema de Medicação , Avaliação de Medicamentos , Humanos , Imunização Passiva , Imunoterapia/efeitos adversos , Interferon Tipo I/uso terapêutico , Ativação Linfocitária/efeitos dos fármacos , Pessoa de Meia-Idade , Nefrectomia , Ácido Periódico/farmacologia , Proteínas RecombinantesRESUMO
Transforming growth factor-beta1 (TGF-beta1), a multifunctional cytokine with fibrogenic properties, has been implicated in the pathogenesis of the vascular and target organ complications of hypertension. TGF-beta1 may also regulate blood pressure via stimulation of endothelin-1 and/or renin secretion. Herein we explored the hypothesis that circulating levels of TGF-beta1 protein (quantified using a TGF-beta1-specific sandwich ELISA) are correlates of blood pressure levels. This hypothesis was tested in 98 stable end-stage renal disease (ESRD) patients. (The use of ESRD patients as the study cohort eliminates renal function-dependent alterations in circulating levels of TGF-beta1 protein.) In addition, in view of the previously reported correlation among TGF-beta1 DNA polymorphisms and systolic blood pressure, TGF-beta1 codon 25 genotype and alleles were identified in 71 hypertensive subjects and 57 normotensives using amplification refractory mutation system polymerase chain reaction. Our studies demonstrate for the first time that TGF-beta1 levels (209+/-13 ng/mL, mean+/-SEM) are positive correlates (Pearson correlation analysis) of mean arterial pressure (P=0.008), systolic pressure (P=0.02), and diastolic pressure (P=0. 01). We also report that a higher percentage of hypertensives (92%) compared with normotensives (86%) are homozygous for the arginine allele at codon 25. Our observations support the idea that genetically determined TGF-beta1 protein concentrations may play a role in blood pressure regulation in humans.
Assuntos
Pressão Sanguínea , Hipertensão/genética , Falência Renal Crônica/genética , Polimorfismo Genético , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Substituição de Aminoácidos , Códon , Estudos de Coortes , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Hipertensão/sangue , Hipertensão/fisiopatologia , Falência Renal Crônica/sangue , Falência Renal Crônica/fisiopatologia , Masculino , Pessoa de Meia-Idade , Mutação Puntual , Reação em Cadeia da Polimerase , Valores de Referência , Análise de Regressão , SístoleRESUMO
In searching for a candidate mechanism for the immunosuppressive as well as fibrogenic consequences of cyclosporine usage, we have explored the hypothesis that cyclosporine stimulates transcription of transforming growth factor-beta 1 (TGF-beta 1), a multifunctional cytokine endowed with immunosuppressive and fibrogenic properties. Our results demonstrate that cyclosporine (i) stimulates TGF-beta 1 promoter-dependent transcription of chloramphenicol acetyl transferase gene in transiently transfected human A-549 cells, (ii) stimulates the synthesis of TGF-beta 1 RNA transcripts in human T cells, and (iii) permits the expression/emergence of DNA regulatory proteins (retinoblastoma control factor-1 (RCF-1) and RCF-2) that bind and regulate TGF-beta 1 promoter activity. Our studies demonstrate for the first time that cyclosporine stimulates TGF-beta 1 gene transcription and suggest a novel mechanism of action of cyclosporine.
Assuntos
Ciclosporina/farmacologia , Transcrição Gênica/efeitos dos fármacos , Fator de Crescimento Transformador beta/genética , Sequência de Bases , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Regiões Promotoras Genéticas , Linfócitos T/metabolismoRESUMO
BACKGROUND: We have shown that measurement of mRNA for cytotoxic attack proteins perforin and granzyme B in urinary cells is a noninvasive means of diagnosing acute rejection of human renal allografts. Urinary cell mRNA studies have yielded useful information in other patient populations such as patients with cancer. The isolation of sufficient and high quality ribonucleic acid (RNA) from urinary cells however is problematic. RNAlater, an RNA stabilization solution, has been reported to optimize RNA isolation from tumor tissues stored at room temperature and from pigment-rich ocular tissues. METHODS: We explored whether the addition of RNAlater to urine cell pellets improves RNA yield, enhances purity and facilitates measurement of low abundance mRNAs. We measured, with the use of real-time quantitative polymerase chain reaction (PCR) assay, levels of expression of a constitutively expressed gene 18S rRNA and mRNA for granzyme B and transforming growth factor-beta(1) (TGF-beta(1)) in urine specimens and renal biopsies obtained from renal allograft recipients. RESULTS: RNA yield (P<0.01, Wilcoxon signed rank test) and the A260/A280 ratio (P<0.01) were both higher with urine cell pellets treated with RNAlater prior to snap freezing compared to cell pellets that were not treated with RNAlater prior to snap freezing. Levels (copy number per 1 microg of total RNA) of 18S rRNA (P<0.02), granzyme B mRNA (P=0.002) and TGF-beta(1) (P=0.02) were all higher with treated urine cell pellets compared to untreated cell pellets. Kruskall-Wallis one way analysis of variance and pair-wise comparisons with Student-Newman-Keuls test showed that the levels of mRNA for granzyme B (P<0.05) and TGF-beta(1) (P<0.05) are significantly different between renal allograft biopsies and untreated urine cell pellets but not between the biopsy specimens and RNAlater-treated urine cell pellets. CONCLUSIONS: The addition of RNAlater to urine cell pellets improves RNA isolation from urinary cells and facilitates measurement of low abundance mRNAs.
Assuntos
RNA/isolamento & purificação , Urina/citologia , Adolescente , Criança , Granzimas , Humanos , RNA/análise , RNA/urina , RNA Ribossômico 18S/urina , Serina Endopeptidases/genética , Espectrofotometria , Fator de Crescimento Transformador beta/genéticaRESUMO
Delayed hyperacute rejection, with its characteristic clinical course and histopathologic findings, occurred within one month after transplantation in five recipients of kidney transplants from HLA-A, B and D identical sibling donors. In all cases, unidirectional mixed lymphocyte cultures and immunologic studies to detect cytotoxic antibodies in the recipients against their respective donors, before kidney transplantation and after transplant nephrectomy, were unresponsive or negative. Onset of delayed hyperacute rejection was preceded by bacteremia in two of these patients. Two of these received second kidney transplants, three to six months later, from HLA-A, B and D identical sibling donors again. Although both have had an episode of acute rejection in the early postoperative period, the grafts have maintained excellent function for 21 and 25 months, respectively. Irreversible forms of transplant rejection, such as delayed hyperacute rejection, do occur even in recipients of kidney transplants from HLA-A, B and D identical sibling pairs, indicating that genetic determinants other than HLA-A, B and D loci, and perhaps other nongenetic immune mechanisms, play an important role in the ultimate results of kidney transplantation.
Assuntos
Rejeição de Enxerto , Antígenos HLA , Transplante de Rim , Adulto , Feminino , Glomerulonefrite/cirurgia , Humanos , Rim/patologia , Masculino , Pessoa de Meia-Idade , Transplante HomólogoRESUMO
In a pilot study involving 13 patients with advanced stage IV renal cell carcinoma, anti-tumor effects and toxicity of a novel form of adoptive immunotherapy were determined. The protocol utilizes infusions of autologous mononuclear leukocytes treated with the oxidizing mitogen sodium periodate (IO4-) and cultured in medium containing human recombinant interleukin-2 (IL-2), and continuous infusions of low-dose IL-2 (mean +/- SD dose = 39.5 +/- 8.6 X 10(3) U/kg/24 hours). Leukocytes (5 to 10 X 10(9] were removed by leukapheresis three times per week, mononuclear cells were separated, activated with IO4- and cultured in medium containing IL-2 (500 U/ml) for 48 to 72 hours. The cells were re-infused following the next leukapheresis procedure. IL-2 was administered five days per week. Treatment was continued for two three-week cycles. An increase in peripheral blood mononuclear cells bearing the natural killer cell (NK) surface marker, Leu 11, an increase in NK- and antibody-dependent cell-mediated cytotoxicity, and a slight increase in spontaneous cytotoxicity for non-NK targets were noted. Regressions (more than 50 percent decrease in tumor mass) of pulmonary, liver, bone, or soft tissue metastases were induced in six patients. Severe fluid retention did not develop in any patient and no patient required treatment in the intensive care unit. Five of the patients who showed a response have experienced a relapse at 5.2 +/- 1.0 (mean +/- SD) months. These observations indicate that IO4-/IL-2-activated killer cells plus continuous infusions of low-dose IL-2 can result in regression of metastatic renal cell carcinoma.
Assuntos
Carcinoma de Células Renais/terapia , Imunoterapia/métodos , Interleucina-2/imunologia , Neoplasias Renais/terapia , Leucócitos/imunologia , Ácido Periódico/imunologia , Adulto , Idoso , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/patologia , Feminino , Humanos , Imunoterapia/efeitos adversos , Interleucina-2/administração & dosagem , Neoplasias Renais/imunologia , Neoplasias Renais/patologia , Transfusão de Leucócitos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Projetos Piloto , Tomografia Computadorizada por Raios XRESUMO
Forty-six renal donors who responded to a questionnaire and two additional donors with nephrotic syndrome and renal insufficiency were studied. The mean age was 46 +/- 2.0 years (mean +/- SE). Duration of follow-up was 6 +/- 0.5 years. Serum creatinine levels increased from 1.0 +/- 0.03 mg/dl before donation to 1.2 +/- 0.04 mg/dl at follow-up. The incidence of proteinuria (more than 150 mg over 24 hours) was 39 percent. The serum creatinine level was 1.0 +/- 0.08 mg/dl and 1.2 +/- 0.06 mg/dl in the proteinuric and nonproteinuric groups, respectively. The incidence of hypertension was 31 percent with a serum creatinine level of 1.1 +/- 0.11 mg/dl and 1.2 +/- 0.07 mg/dl in the hypertensive and normotensive groups, respectively. One patient with nephrotic syndrome had proliferative glomerulonephritis. It is concluded that renal donation is associated with a minimal but statistically significant increment in serum creatinine levels. The incidence of mild hypertension and proteinuria is increased, but impact on renal function is minimal as assessed by serum creatinine determination.
Assuntos
Transplante de Rim , Doadores de Tecidos , Adolescente , Adulto , Creatinina/sangue , Feminino , Humanos , Hipertensão/etiologia , Masculino , Pessoa de Meia-Idade , Proteinúria/etiologiaRESUMO
T cell surface proteins involved in transmembrane signalling resulting in the activation of T cells were investigated utilizing as probes monoclonal antibodies directed at T cell surface antigens. Here we report that mAbs that react with a framework determinant of alpha/beta heterodimer of T cell receptor for antigen, anti-TCR-1, and those with the SRBC-binding epitope of the CD2 antigen, OKT11, are synergistic in promoting T cell proliferation. The proliferative response is dependent upon crosslinking of anti-TCR-1 and OKT11, and is associated with a significant increase in the concentration of intracellular free calcium in T cells. Moreover, EGTA and a direct (staurosporine) or a competitive (1-[5-isoquinolinylsulfonyl]-2-methyl piperazine) inhibitor of protein kinase C prevents T cell proliferation accomplished with crosslinked anti-TCR-1 and OKT11. Our findings, in addition to demonstrating the synergism between the signals initiated via the T cell receptor for antigen and the CD2 antigen, suggest a role for calcium and protein kinase C in the transduction of signals generated with crosslinked anti-TCR-1 and OKT11.
Assuntos
Antígenos de Diferenciação de Linfócitos T/imunologia , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/fisiologia , Receptores Imunológicos/imunologia , Transdução de Sinais , Linfócitos T/imunologia , Alcaloides/farmacologia , Anticorpos Monoclonais , Antígenos CD2 , Cálcio/metabolismo , Reagentes de Ligações Cruzadas , Relação Dose-Resposta Imunológica , Ácido Egtázico/farmacologia , Humanos , Ativação Linfocitária/efeitos dos fármacos , Proteína Quinase C/metabolismo , Receptores de Antígenos de Linfócitos T/efeitos dos fármacos , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/efeitos dos fármacos , Estaurosporina , Linfócitos T/metabolismo , Linfócitos T/fisiologiaRESUMO
Several polyclonal T cell activators can induce the differentiation of quiescent antigen-specific memory T cells (memory cells) into specific secondary cytotoxic T cells, in the absence of the original priming alloantigens. Such activation represents a potential pathway by which immunologically nonspecific signals can elicit specific antiallograft immunity. We therefore investigated the molecular basis for antigen-independent activation of memory cells using as probes recombinant interleukin-2 and monoclonal antibodies directed at IL-2, IL-2 receptors (IL-2R), or interferon-gamma (IFN-gamma). Antigen-specific memory cells, generated in human long-term-primary mixed lymphocyte cultures, were induced to proliferate, exhibit antigen specific secondary cytolytic activity, or produce IFN-gamma by recombinant DNA human IL-2 produced in E Coli. Monoclonal antibodies directed at the IL-2R or at IL-2 inhibited IL-2-mediated proliferation, cytolytic activity, or production of IFN-gamma. Monoclonal antibodies directed at IFN-gamma did not inhibit alloantigen or IL-2 mediated activation of memory cells. Our findings suggest that successful interaction between IL-2 and its receptor expressed on memory cells represents a plausible pathway by which an immunologically nonspecific signal might elicit specific antiallograft immunity. Moreover, therapeutic strategies that include antibodies directed at the IL-2 and/or IL-2R and not antibodies directed at IFN-gamma will inhibit IL-2-dependent alloimmunity.