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2.
Am J Transplant ; 17(10): 2591-2600, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28326672

RESUMO

BK polyomavirus (BKPyV) causes premature kidney transplant (KT) failure in 1-15% of patients. Because antivirals are lacking, most programs screen for BKPyV-viremia and, if positive, reduce immunosuppression. To evaluate the relationship of viremia and BKPyV-specific immunity, we examined prospectively cryopreserved plasma and peripheral blood mononuclear cells at the time of transplantation (T0) and at 6 mo (T6) and 12 mo (T12) after transplant from 28 viremic KT patients and 68 nonviremic controls matched for the transplantation period. BKPyV IgG seroprevalence was comparable between cases (89.3%) and controls (91.2%; p = 0.8635), but cases had lower antibody levels (p = 0.022) at T0. Antibody levels increased at T6 and T12 but were not correlated with viremia clearance. BKPyV-specific T cell responses to pools of overlapping 15mers (15mer peptide pool [15mP]) or immunodominant CD8 9mers (9mer peptide pool [9mP]) from the early viral gene region were not different between cases and controls at T0; however, clearance of viremia was associated with stronger 9mP responses at T6 (p = 0.042) and T12 (p = 0.048), whereas 15mP responses were not informative (T6 p = 0.359; T12 p = 0.856). BKPyV-specific T cells could be expanded in vitro from all patients after transplant, permitting identification of 78 immunodominant 9mer epitopes including 50 new ones across different HLA class I. Thus, 9mP-responses may be a novel marker of reconstituting CD8 T cell function that warrants further study as a complement of plasma BKPyV loads for guiding immunosuppression reduction.


Assuntos
Vírus BK/fisiologia , Linfócitos T CD8-Positivos/imunologia , Transplante de Rim , Adulto , Idoso , Vírus BK/isolamento & purificação , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Humanos , Transplante de Rim/efeitos adversos , Masculino , Pessoa de Meia-Idade , Viremia
3.
Br J Dermatol ; 175(2): 325-33, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27037558

RESUMO

BACKGROUND: KIR3DL2, an inhibitory receptor expressed by natural killer cells and a subset of normal CD8(+) T cells, is aberrantly expressed in neoplastic cells in transformed mycosis fungoides and Sézary syndrome. Anti-KIR3DL2 targeted antibody therapy has shown potent activity in preclinical models for these diseases. OBJECTIVES: To examine the expression of KIR3DL2 and its potential use as a therapeutic target in patients with primary cutaneous anaplastic large-cell lymphoma (pcALCL), the most aggressive cutaneous CD30(+) lymphoproliferative disease. METHODS: Samples from 11 patients with pcALCL and three CD30(+) lymphoproliferative disease cell lines - Mac1, Mac2a and Mac2b - were used in KIR3DL2 expression studies using immunohistochemistry, flow cytometry and reverse-transcriptase quantitative polymerase chain reaction. The effect of IPH4102, a monoclonal humanized IgG1 targeting KIR3DL2, was assessed by in vitro cytotoxicity assays against Mac1, Mac2a and Mac2b using allogeneic peripheral blood mononuclear cells as effectors. RESULTS: KIR3DL2 mRNA and protein were found in all human samples of pcALCL, and in the Mac2a and Mac2b cell lines. KIR3DL2 protein expression was present on 85·8 ± 14·0% of CD30(+) skin-infiltrating tumour cells. In vitro functional studies showed that KIR3DL2(+) Mac2a and Mac2b pcALCL lines are sensitive to antibody-derived cytotoxicity mediated by IPH4102, through activation of natural killer cells, in a concentration-dependent manner. CONCLUSIONS: pcALCL tumour cells express KIR3DL2, and we provide preclinical proof of concept for the use of IPH4102, a humanized anti-KIR3DL2 antibody, to treat patients with primary cutaneous CD30(+) ALCL.


Assuntos
Linfoma Anaplásico de Células Grandes/tratamento farmacológico , Receptores KIR2DL2/antagonistas & inibidores , Neoplasias Cutâneas/tratamento farmacológico , Adolescente , Adulto , Idoso , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Feminino , Humanos , Antígeno Ki-1/metabolismo , Células Matadoras Naturais/fisiologia , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Receptores KIR2DL2/imunologia , Receptores KIR2DL2/metabolismo , Pele/metabolismo , Células Tumorais Cultivadas , Adulto Jovem
4.
Am J Transplant ; 16(4): 1193-206, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26663765

RESUMO

Uncontrolled BK polyomavirus (BKPyV) replication in kidney transplant recipients (KTRs) causes polyomavirus-associated nephropathy and allograft loss. Reducing immunosuppression is associated with clearing viremia and nephropathy and increasing BKPyV-specific T cell responses in most patients; however, current immunoassays have limited sensitivity, target mostly CD4(+) T cells, and largely fail to predict onset and clearance of BKPyV replication. To characterize BKPyV-specific CD8(+) T cells, bioinformatics were used to predict 9mer epitopes in the early viral gene region (EVGR) presented by 14 common HLAs in Europe and North America. Thirty-nine EVGR epitopes were experimentally confirmed by interferon-γ enzyme-linked immunospot assays in at least 30% of BKPyV IgG-seropositive healthy participants. Most 9mers clustered in domains, and some were presented by more than one HLA class I, as typically seen for immunodominant epitopes. Specific T cell binding using MHC class I streptamers was demonstrated for 21 of 39 (54%) epitopes. In a prospective cohort of 118 pediatric KTRs, 19 patients protected or recovering from BKPyV viremia were experimentally tested, and 13 epitopes were validated. Single HLA mismatches were not associated with viremia, suggesting that failing immune control likely involves multiple factors including maintenance immunosuppression. Combining BKPyV load and T cell assays using immunodominant epitopes may help in evaluating risk and reducing immunosuppression and may lead to safe adoptive T cell transfer.


Assuntos
Vírus BK/imunologia , Epitopos de Linfócito T/imunologia , Antígenos HLA/imunologia , Transplante de Rim/efeitos adversos , Infecções por Polyomavirus/imunologia , Linfócitos T/imunologia , Infecções Tumorais por Vírus/imunologia , Vírus BK/fisiologia , Criança , ELISPOT , Feminino , Seguimentos , Humanos , Falência Renal Crônica/cirurgia , Falência Renal Crônica/virologia , Masculino , Pessoa de Meia-Idade , Infecções por Polyomavirus/diagnóstico , Estudos Prospectivos , Infecções Tumorais por Vírus/diagnóstico , Replicação Viral
5.
Am J Transplant ; 16(6): 1697-706, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26699950

RESUMO

Transplant patients are at increased risk of viral complications due to impaired control of viral replication, resulting from HLA mismatching between graft and host and the immunosuppression needed to avert alloimmune reactions. In the past decade, quantitative viral load measurements have become widely available to identify patients at risk and to inform treatment decisions with respect to immunosuppressive drugs and antiviral therapies. Because viral loads are viewed as the result of viral replication and virus-specific immune control, virus-specific T cell monitoring has been explored to optimize management of adenovirus, BK polyomavirus and cytomegalovirus ("ABC") in transplant patients. Although most studies are descriptive using different technologies, the overall results show that the quantity and quality of virus-specific T cells inversely correlate with viral replication, whereby strong cellular immune responses are associated with containment of viral replication. The key obstacles to the introduction of assays for virus-specific T cells into clinical practice is the definition of reliable cutoffs for clinical decision making, the poor negative predictive value of some assays, and the absence of interventional trials justifying changes of antiviral treatment or immunosuppression. More clinical research is needed using optimized assays and targets before standardization and commutability can be envisaged as achieved for viral load testing.


Assuntos
Tolerância Imunológica/imunologia , Transplante de Órgãos/efeitos adversos , Linfócitos T/imunologia , Viroses/diagnóstico , Replicação Viral/imunologia , Animais , Antivirais/uso terapêutico , Humanos , Imunologia de Transplantes , Viroses/tratamento farmacológico , Viroses/imunologia
6.
Gene Ther ; 22(2): 172-80, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25394253

RESUMO

Hepatitis C virus (HCV)-induced, end-stage liver disease is a major indication for liver transplantation, but systematic graft reinfection accelerates liver disease recurrence. Transplantation recipients may be ineligible for direct-acting antivirals, owing to toxicity, resistance or advanced liver disease. Adoptive immunotherapy with liver graft-derived, ex vivo-activated lymphocytes was previously shown to prevent HCV-induced graft reinfections. Alternatively, the applicability and therapeutic efficacy of adoptive immunotherapy may be enhanced by 'ready for use' suicide gene-modified lymphocytes from healthy blood donors; moreover, conditional, prodrug-induced cell suicide may prevent potential side effects. Here, we demonstrate that allogeneic suicide gene-modified lymphocytes (SGMLs) could potently, dose- and time-dependently, inhibit viral replication. The effect occurs at effector:target cell ratios that exhibits no concomitant cytotoxicity toward virus-infected target cells. The effect, mediated mostly by CD56+ lymphocytes, is interleukin-2-dependent, IFN-γ-mediated and, importantly, resistant to calcineurin inhibitors. Thus, post-transplant immunosuppression may not interfere with this adoptive cell immunotherapy approach. Furthermore, these cells are indeed amenable to conditional cell suicide; in particular, the inducible caspase 9 suicide gene is superior to the herpes simplex virus thymidine kinase suicide gene. Our data provide in vitro proof-of-concept that allogeneic, third-party, SGMLs may prevent HCV-induced liver graft reinfection.


Assuntos
Hepacivirus/imunologia , Hepatite C/prevenção & controle , Linfócitos/fisiologia , Caspase 9/genética , Linhagem Celular Tumoral , Terapia Genética , Humanos , Imunoterapia Adotiva , Transplante Homólogo , Replicação Viral
7.
Leukemia ; 28(4): 880-7, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24104394

RESUMO

MicroRNAs (miRs) are involved in tumorigenesis by regulating tumor suppressor genes and/or oncogenes. MiR187 was overexpressed in peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) and associated with high Ki67 expression, elevated lactate dehydrogenase, advanced International Prognostic Index and poor prognosis of patients. In vitro, ectopic expression of miR187 in T-lymphoma cell lines accelerated tumor cell proliferation, whereas treatment with miR187 inhibitor reduced cell growth. MiR187 downregulated tumor suppressor gene disabled homolog-2 (Dab2), decreased the interaction of Dab2 with adapter protein Grb2, resulting in Ras activation, phosphorylation/activation of extracellular signal-regulated kinase (ERK) and AKT, and subsequent stabilization of MYC oncoprotein. MiR187-overexpressing cells were resistant to chemotherapeutic agents like doxorubicin, cyclophosphamide, cisplatin and gemcitabine, but sensitive to the proteasome inhibitor bortezomib. Bortezomib inhibited T-lymphoma cell proliferation by downregulating miR187, dephosphorylating ERK and AKT and degrading MYC. In a murine xenograft model established with subcutaneous injection of Jurkat cells, bortezomib particularly retarded the growth of miR187-overexpressing tumors, consistent with the downregulation of miR187, Ki67 and MYC expression. Collectively, these findings indicated that miR187 was related to tumor progression in PTCL-NOS through modulating Ras-mediated ERK/AKT/MYC axis. Although potentially oncogenic, miR187 indicated the sensitivity of T-lymphoma cells to bortezomib. Cooperatively targeting ERK and AKT could be a promising clinical strategy in treating MYC-driven lymphoid malignancies.


Assuntos
Antineoplásicos/uso terapêutico , Ácidos Borônicos/uso terapêutico , Linfoma de Células T Periférico/tratamento farmacológico , MicroRNAs/fisiologia , Pirazinas/uso terapêutico , Animais , Bortezomib , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Linfoma de Células T Periférico/genética , Linfoma de Células T Periférico/patologia , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-myc/fisiologia
8.
Am J Transplant ; 13(4): 984-992, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23425311

RESUMO

Papillary renal-cell carcinoma (pRCC) is unusual for its occurrence in kidneys with chronic dysfunction, for its frequent multifocality and for its common association with papillary adenoma, a benign renal lesion morphologically indistinguishable from pRCC. Concomitant development of papillary adenoma and pRCC in five transplanted kidneys, where donor and recipient characteristics are well established, provided a unique opportunity for molecular studies of de novo pRCC carcinogenesis. We aimed to study this tumor type to determine whether or not the different papillary tumors have the same origin, and whether or not papillary adenomas are precursor lesions of pRCC. We performed XY-FISH in sex-mismatched kidney transplants, and polymorphic microsatellite DNA and high-resolution melting of mitochondrial DNA analyzes in all five patients on laser-microdissected tumor cells, then compared these molecular profiles to donor and recipient profiles. This study (i) identified the recipient origin of de novo papillary adenomas and pRCCs in a kidney transplant, (ii) demonstrated an identical origin for precursor cells of papillary adenomas and pRCCs and (iii) showed additional genetic alterations in pRCCs compared to papillary adenomas. This molecular approach of papillary tumors developed in transplanted kidney identified successive steps in carcinogenesis of human de novo papillary renal-cell carcinoma.


Assuntos
Adenoma/diagnóstico , Carcinoma de Células Renais/diagnóstico , Neoplasias Renais/diagnóstico , Transplante de Rim/efeitos adversos , Adenoma/genética , Adulto , Carcinoma de Células Renais/genética , DNA Mitocondrial/genética , Feminino , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Neoplasias Renais/genética , Transplante de Rim/métodos , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo Genético , Adulto Jovem
9.
Br J Dermatol ; 161(6): 1371-5, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19575754

RESUMO

BACKGROUND: Toxic epidermal necrolysis (TEN) and Stevens-Johnson syndrome (SJS) are characterized by extensive keratinocyte apoptosis mediated by cytotoxic proteins. Similar features have been found in another severe dysimmune syndrome, allogeneic acute graft-versus-host disease, where endothelial cell apoptosis has been recently characterized. OBJECTIVES: To determine whether endothelial cell apoptosis occurs in dermal vessels of TEN and SJS, and whether it is linked to expression of cytotoxic proteins. METHODS: Skin biopsies of eight patients with severe drug-induced bullous eruptions (four TEN, four SJS), eight with drug-induced urticaria and eight healthy controls were compared. Blood vessel damage was studied by electron microscopy and quantified by CD31 immunostaining. Apoptotic cells, characterized by electron microscopy, were quantified on terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling assay. Immunohistochemistry was also used to characterize and quantify inflammatory cells and granzyme B, tumour necrosis factor (TNF)-alpha and Fas ligand (FasL) expression. RESULTS: Endothelial cell apoptosis was observed in all TEN and SJS cases: it occurred in 85% of the vessel sections. It occurred in one case of drug-induced urticaria, in 5% of vessel sections, but not in healthy controls. Numbers of CD68+ macrophages and CD8+ T lymphocytes were significantly higher in TEN and SJS compared with both other groups; granzyme B and TNF-alpha but not FasL were expressed. CONCLUSIONS: Characterization of endothelial cell apoptosis in TEN and SJS is important to assess a factor worsening skin damage, with possible extension to other organs. It may also be useful for the development of novel therapeutic strategies.


Assuntos
Apoptose , Células Endoteliais/metabolismo , Síndrome de Stevens-Johnson/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Pele/irrigação sanguínea , Pele/metabolismo , Síndrome de Stevens-Johnson/metabolismo , Adulto Jovem
10.
J Fr Ophtalmol ; 28(10): 1052-7, 2005 Dec.
Artigo em Francês | MEDLINE | ID: mdl-16395196

RESUMO

PURPOSE: The aim of this study was to determine whether eye length measurements obtained with the IOL Master (Zeiss Humphrey) before and after phakic IOL implantation would show any changes. METHODS: In a prospective study, we used the IOL Master to measure optical biometry in 25 myopic eyes of 15 patients before and after phakic IOL implantation (PRL, ICL, Artisan). The differences between both axial length measurements were calculated and compared using a nonparametric Wilcoxon test. RESULTS: The difference between the preoperative and postoperative measurements ranged from -0.16 mm to 0.06 mm and averaged -0.016 mm, which was not statistically significant (p=0.20). Both measurements correlated in a highly positive manner (r=0.999; p<0.0001). The reproducibility of the preoperative and postoperative axial length measurements was very high (coefficient of variation=0.09% and 0.07%, respectively). The precision was 26 microm for preoperative measurements and 19 microm for postoperative measurements. CONCLUSION: Our results showed that postoperative measurements of axial length are highly comparable to preoperative measurements and that optical biometry can achieve highly precise and reliable axial length measurements in eyes with phakic IOLs. This application becomes clinically relevant in evaluating eyes with phakic IOLs that might require cataract surgery. Hence, accurate axial length measurements in eyes with phakic IOLs will be extremely important when cataract occurs in these eyes and when the preoperative measurements are no longer available.


Assuntos
Olho/anatomia & histologia , Lentes Intraoculares , Miopia/cirurgia , Adulto , Biometria , Técnicas de Diagnóstico Oftalmológico , Feminino , Humanos , Masculino , Cuidados Pós-Operatórios , Cuidados Pré-Operatórios , Estudos Prospectivos
11.
Biotech Histochem ; 78(1): 35-42, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12713140

RESUMO

Tissue processing and analysis require good preservation of both the shape and content of cells. Lowicryl resin is one of the few embedding media that allow good preservation of both tissue architecture and cellular contents. Therefore, different histochemical and immunohistochemical reactions can be applied to semithin sister sections from one biopsy. Further examination of a zone of interest can be carried out under the electron microscope. The hydrophilic property of Lowicryl resins makes possible different histochemical reactions; however, the technique used for paraffin sections must be adapted for each reaction. Antigenic preservation of cells by low temperature embedding allows immunolabeling on either semithin sections or in the zone of interest on ultrathin sections. We have shown the application and adaptation of different histochemical and immunohistochemical reactions on semithin and ultrathin sections from hepatic biopsies that were large, but thin. The variety of techniques that can be used on sister Lowicryl sections of a single biopsy makes this medium useful for extensive pathological studies of precious needle biopsies.


Assuntos
Resinas Acrílicas , Biópsia por Agulha/métodos , Nefropatias/metabolismo , Hepatopatias/metabolismo , Inclusão em Plástico/métodos , Protocolos Clínicos , Histocitoquímica , Humanos , Imuno-Histoquímica , Rim/química , Rim/patologia , Nefropatias/patologia , Fígado/química , Fígado/patologia , Hepatopatias/patologia , Inclusão em Plástico/instrumentação
12.
J Bacteriol ; 182(20): 5799-806, 2000 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11004180

RESUMO

Inactivation of ccpA in Enterococcus faecalis leads to reduction of the growth rate, derepression of the galKETR operon in the presence of a mixture of glucose and galactose, and reduction of transcription of ldh in the presence of glucose. Moreover, the E. faecalis ccpA gene fully complements a Bacillus subtilis ccpA mutant, arguing for similar functions of these two homologous proteins. Protein comparison on two-dimensional gels from the wild-type cells and the ccpA mutant cells revealed a pleiotropic effect of the mutation on gene expression. The HPr protein of the carbohydrate-phosphotransferase system was identified by microsequencing, and a modification of its phosphorylation state was observed between the wild-type and the mutant strains. Moreover, at least 16 polypeptides are overexpressed in the mutant, and 6 are repressed. Interestingly, 13 of the 16 polypeptides whose synthesis is enhanced in the mutant were also identified as glucose starvation proteins. The N-terminal amino acid sequences of four of them match sequences deduced from genes coding for L-serine dehydratase, dihydroxyacetone kinase (two genes), and a protein of unknown function from Deinococcus radiodurans.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Enterococcus faecalis/genética , Regulação Bacteriana da Expressão Gênica , Óperon , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transcrição Gênica , Bacillus subtilis/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/isolamento & purificação , Sequência de Bases , Enterococcus faecalis/crescimento & desenvolvimento , Enterococcus faecalis/fisiologia , Galactose/metabolismo , Teste de Complementação Genética , Glucose/metabolismo , Mutagênese Insercional , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Proteínas Recombinantes/metabolismo
13.
J Manipulative Physiol Ther ; 14(5): 311-6, 1991 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-1833495

RESUMO

Low back pain (LBP) affects a large proportion of the population and is an increasingly costly problem in the western world. This review highlights some of the recent theories relating to LBP and the conflicting evidence which has been brought to light. Theories relating to the causes of LBP have included single and multiple factors such as abnormal physical findings, mechanical, psychosocial and economic factors. The two lowest lumbar segments are most often afflicted in LBP sufferers, but the diagnosis of LBP is otherwise uncertain in most cases due to insufficient knowledge relating to the validity of clinical tests, inconsistent terminology and unclear symptomatic patterns. Despite a lack of understanding of the exact anatomical cause of LBP, an abundance of therapeutic models exist, most of which are purely empirical, and few methods have been shown to be clinically useful. Similarly, insufficient knowledge of the causes of LBP makes primary, secondary and tertiary prevention difficult.


Assuntos
Dor nas Costas , Dor nas Costas/epidemiologia , Dor nas Costas/prevenção & controle , Dor nas Costas/terapia , Quiroprática , Ergonomia , Humanos , Incidência , Ortopedia , Educação de Pacientes como Assunto , Prevalência , Prevenção Primária , Fatores de Risco
14.
J Manipulative Physiol Ther ; 12(2): 109-12, 1989 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-2523947

RESUMO

The subjective complaints of 41 chronic low back pain sufferers attending a chiropractic clinic were assessed twice prior to therapy with a widely used psychological self-report assessment tool, the Middlesex Hospital Questionnaire (MHQ) and a newly developed VAS Disability Scales Questionnaire (DISQ), both of which investigate various aspects of certain basic positions and activities. Reliability was generally acceptable with these two questionnaires. Subjects participating in the study were commonly found to score within the normal range on the MHQ, indicating that psychological disturbance was not a major feature of their presentation. However, mild mood disturbance was commonly reported, and a more sensitive tool may need to be developed for this type of mildly affected chronic low back pain sufferers. The DISQ generally indicated subjects were mildly to moderately affected by their low back trouble and that sitting and leisure activities were the most pain provoking. Recommendations for further development of the disability scale are made.


Assuntos
Dor nas Costas/diagnóstico , Dor nas Costas/psicologia , Quiroprática , Doença Crônica , Humanos , Inquéritos e Questionários
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