RESUMO
INTRODUCTION: Fusarium oxysporum has a high affinity for lignin and cellulose-based substrates and is known to grow in a wide range of environments. It is these properties and its ability to produce mycotoxins that have contributed to its pathogenicity in cereal crops that can affect human and animal health when ingested. OBJECTIVES: Identify the mechanisms of mycotoxin production and map the functional output of F. oxysporum under varying growth conditions. METHODS: Liquid and gas-based chromatography coupled with mass spectrometry was used to identify and map the untargeted metabolic pathway of F. oxysporum grown using nitrogen limited and organic/inorganic nitrogen supplemented media. RESULTS: Over 1300 metabolites were identified, relating to 42 metabolic pathways. Of these, 520 metabolites merged at pyruvate (glycolysis), succinate (Krebs cycle) and aspartate-glutamate metabolic pathways. CoA depletion at the growth stage triggered the initiation of fatty acid and branched amino acid degradation. This in turn activated propionyl CoA carnitine acetyltransferase enzymes, resulting in nitrogen preservation (urea, putrescine and organic acids end-products). CoA then transferred into the TCA cycle via previously unreported ß-alanine and propionyl CoA metabolic pathways, the latter likely being a novel methylmalonyl-CoA mutase activity for F. oxysporum. CONCLUSIONS: The lower supplementation of inorganic nitrogen compounds (≤ 50 mM) and the elimination of nitrates/organic nitrogen sources resulted in TCA autophagy events that boosted mycotoxin-based metabolism and decreased overall F. oxysporum growth. Such knowledge of functional mycotoxin production can be used to supplement agricultural crops and reduce the risk of mycotoxin contamination in human and animal food supplies.
Assuntos
Ciclo do Ácido Cítrico , Fusarium/metabolismo , Metaboloma , Metilmalonil-CoA Mutase/metabolismo , Micotoxinas/metabolismo , Nitrogênio/deficiência , Fusarium/crescimento & desenvolvimento , Cromatografia Gasosa-Espectrometria de Massas , Redes e Vias Metabólicas , Micotoxinas/análiseRESUMO
The measurement of vaccine-induced humoral and CD4(+) and CD8(+) cellular immune responses represents an important correlate of vaccine efficacy. Accurate and reliable assays evaluating such responses are therefore critical during the clinical development phase of vaccines. T cells play a pivotal role both in coordinating the adaptive and innate immune responses and as effectors. During the assessment of cell-mediated immunity (CMI) in subjects participating in a large-scale influenza vaccine trial, we identified the expansion of an IFN-γ-producing CD3(+)CD4(-)CD8(-) γδ (+) T cell population in the peripheral blood of 90/610 (15%) healthy subjects. The appearance of CD3(+)CD4(-)CD8(-) γδ (+) T cells in the blood of subjects was transient and found to be independent of the study cohort, vaccine group, subject gender and ethnicity, and ex vivo restimulation conditions. Although the function of this population and relevance to vaccination are unclear, their inclusion in the total vaccine-specific T-cell response has the potential to confound data interpretation. It is thus recommended that when evaluating the induction of IFN-γ-producing CD4(+) and CD8(+) immune responses following vaccination, the CD3(+)CD4(-)CD8(-) γδ (+) T cells are either excluded or separately enumerated from the overall frequency determination.
Assuntos
Leucócitos Mononucleares/imunologia , Subpopulações de Linfócitos T/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Complexo CD3/metabolismo , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Estudos de Coortes , Humanos , Imunidade Celular/imunologia , Imunofenotipagem , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Leucócitos Mononucleares/metabolismo , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Subpopulações de Linfócitos T/metabolismo , Fatores de Tempo , Vacinação , Potência de Vacina , Adulto JovemRESUMO
Selectivity of α2,6-linked human-like receptors by B hemagglutinin (HA) is yet to be fully understood. This study integrates binding data with structure-recognition models to examine the impact of regional-specific sequence variations within the receptor-binding pocket on selectivity and structure activity relationships (SAR). The receptor-binding selectivity of influenza B HAs corresponding to either B/Victoria/2/1987 or the B/Yamagata/16/88 lineages was examined using surface plasmon resonance, solid-phase ELISA and gel-capture assays. Our SAR data showed that the presence of asialyl sugar units is the main determinant of receptor preference of α2,6 versus α2,3 receptor binding. Changes to the type of sialyl-glycan linkage present on receptors exhibit only a minor effect upon binding affinity. Homology-based structural models revealed that structural properties within the HA pocket, such as a glyco-conjugate at Asn194 on the 190-helix, sterically interfere with binding to avian receptor analogs by blocking the exit path of the asialyl sugars. Similarly, naturally occurring substitutions in the C-terminal region of the 190-helix and near the N-terminal end of the 140-loop narrows the horizontal borders of the binding pocket, which restricts access of the avian receptor analog LSTa. This study helps bridge the gap between ligand structure and receptor recognition for influenza B HA; and provides a consensus SAR model for the binding of human and avian receptor analogs to influenza B HA.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza B/metabolismo , Influenza Aviária/metabolismo , Influenza Humana/metabolismo , Receptores Virais/química , Receptores Virais/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Embrião de Galinha , Galinhas , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza B/química , Vírus da Influenza B/genética , Influenza Aviária/genética , Influenza Aviária/virologia , Influenza Humana/genética , Influenza Humana/virologia , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Receptores Virais/genética , Relação Estrutura-AtividadeRESUMO
Sialic acids (SA) usually linked to galactose (Gal) in an α2,6- or α2,3-configuration are considered the main cell receptors for influenza viruses, in particular for their hemagglutinins (HA). The typing of influenza virus HA receptor selectivity is relevant for understanding the transmissibility of avian and swine viruses to the human population. In this study we developed a simple and inexpensive gel-capture assay (GCA) of the influenza virus HA receptor-binding selectivity. Its principle is the binding of soluble influenza virus to pentasaccharide analogs, representatives of receptors of human and avian influenza viruses, immobilized on a gel resin. The human and avian analogs consisted of a sialyllactose-N-tetraose c (LSTc) [Neu5Ac(α2,6)Gal(ß1-3)GlcNAc(ß1-3)Gal(ß1-4)Glc] and a sialyllactose-N-tetraose a (LSTa) [Neu5Ac(α2,3)Gal(ß1-3)GlcNAc(ß1-3)Gal(ß1-4)Glc], respectively. Following equilibration, the unbound virus is washed away and the bound one is assayed via HA by densitometry as a function of the analog concentration. Using GCA, the receptor selectivity of three influenza viruses of different HA subtype was investigated. The results showed that the egg-adapted A/California/07/2009 (H1N1) virus exhibited an avian α2,3-linked LSTa selectivity, however, it retained the ability to bind to the α2,6-linked LSTc human receptor analog. Influenza B virus B/Florida/4/2006 showed α2,6-linked LSTc selectivity and a poor α2,3-linked LSTa avidity. The H3N2 virus A/Wisconsin/15/2009 displayed almost comparable avidity for both receptor analogs with a marginally greater α2,3-linked LSTa avidity. The described assay protocol provides a simple and rapid method for the characterization of influenza virus HA receptor binding selectivity.
Assuntos
Especificidade de Hospedeiro , Vírus da Influenza A/fisiologia , Vírus da Influenza B/fisiologia , Influenza Humana/virologia , Polissacarídeos/análise , Receptores Virais/análise , Ácidos Siálicos/análise , Virologia/métodos , Animais , Embrião de Galinha , Humanos , Vírus da Influenza A/química , Vírus da Influenza B/química , Influenza Humana/metabolismo , Polissacarídeos/metabolismo , Receptores Virais/metabolismo , Ácidos Siálicos/metabolismo , Virologia/instrumentaçãoRESUMO
Extracorporeal photochemotherapy is an effective treatment for cutaneous T cell lymphoma but its mode of action is uncertain. The reduction in viability of patients' photoirradiated buffy coat lymphocytes was correlated with a 35% increase in DNA single-strand breaks and marked decreases in cellular ATP and NAD levels (to 58 and 34% of control, respectively) immediately after photoirradiation. Complementary in vitro studies were conducted with normal human peripheral blood lymphocytes using a Therakos ultraviolet A (UVA) light box. UVA light was cytotoxic on its own but was potentiated by 8-methoxysporalen. 3-aminobenzamide, a poly (ADP-ribose) synthetase inhibitor, mitigated the cytotoxic effect of ultraviolet A light in the presence of 8-methoxypsoralen in lymphocytes and reduced the amount of nucleotide depletion they caused. 10 J/cm2 of UVA light in the presence of 300 ng/ml 8-methoxypsoralen increased the poly (ADP-ribose) synthetase activity of peripheral blood lymphocytes. Exposing lymphocytes to deoxycoformycin and deoxyadenosine was found to induce biochemical and physical effects similar to those of photochemotherapy. In summary, we have shown that the lymphocytotoxic effect of extracorporeal photochemotherapy for cutaneous T cell lymphoma is apparently mediated by DNA damage, subsequent poly (ADP-ribosyl)ation and adenine nucleotide depletion. It is not known how the DNA damage and resultant biochemical effects relate to the possible immunological mechanism of extracorporeal photochemotherapy; however, it is possible that its effects can be mimicked by other DNA-damaging agents.
Assuntos
Linfócitos/efeitos da radiação , Linfoma Cutâneo de Células T/terapia , Terapia PUVA/métodos , Nucleotídeos de Adenina/metabolismo , Trifosfato de Adenosina/metabolismo , DNA/química , DNA/efeitos da radiação , Dano ao DNA , Desoxiadenosinas/toxicidade , Humanos , Técnicas In Vitro , Leucaférese/métodos , Linfócitos/efeitos dos fármacos , Metoxaleno/metabolismo , Metoxaleno/toxicidade , NAD/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
Phosphatidylinositol 3'-kinases (PI3ks) are a family of lipid kinases that play a crucial role in a wide range of important cellular processes associated with malignant behavior including cell growth, migration, and survival. We have used single-strand conformational polymorphism/heteroduplex analysis to demonstrate the presence of somatic mutations in the gene for the p85alpha regulatory subunit of PI3k (PIK3R1) in primary human colon and ovarian tumors and cancer cell lines. All of the mutations lead to deletions in the inter-SH2 region of the molecule proximal to the serine608 autoregulatory site. Expression of a mutant protein with a 23 amino acid deletion leads to constitutive activation of PI3k providing the first direct evidence that p85alpha is a new oncogene involved in human tumorigenesis.
Assuntos
Neoplasias do Colo/genética , Oncogenes/genética , Neoplasias Ovarianas/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Serina-Treonina Quinases , Idoso , Sequência de Aminoácidos , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Polimorfismo Conformacional de Fita Simples , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
Induction of c-myc gene expression is an essential response to growth promoting agents, including colony-stimulating factor 1 (CSF-1). Down regulation of c-myc expression occurs in response to a variety of negative growth regulators in many cell types. However, for many of these systems the causal link between c-myc down regulation and growth arrest remains to be established. Here we show for CSF-1-dependent BAC1.2F5 mouse macrophages that interferon-gamma (IFN gamma) results in a midlate G1 phase decrease of CSF-1-dependent c-myc mRNA and subsequent cell cycle arrest. Introduction of a deregulated c-myc gene into these cells, which prevents the IFN gamma-mediated decrease in c-myc expression, overrides the cell cycle arrest and restores CSF-1-dependent growth in the presence of the cytokine. This result contrasts with the macrophage growth arrest induced by cAMP elevation, which also suppresses c-myc expression, but is not overcome by a deregulated c-myc gene. These results show that inhibition of c-myc expression is an essential component in IFN gamma-mediated cell cycle arrest and demonstrates that distinct mechanisms contribute to IFN gamma- and cAMP-mediated growth arrest in macrophages.
Assuntos
Genes myc/fisiologia , Interferon gama/fisiologia , Macrófagos/citologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Linhagem Celular , AMP Cíclico/metabolismo , DNA/biossíntese , Regulação para Baixo , Regulação da Expressão Gênica , Genes myc/efeitos dos fármacos , Interferon gama/farmacologia , Fator Estimulador de Colônias de Macrófagos/fisiologia , Camundongos , RNA Mensageiro/metabolismoRESUMO
Identification of rearrangements in the immunoglobulin heavy chain (IgH), T cell receptor (TCR) and other genes assists in the classification of hemopoietic disorders. This study reviews a 5 year experience of genotyping as an assessment of clonality in a central referral laboratory. Patients were referred for assessment if abnormal hemopoietic populations were identified and where standard morphology, cytochemistry or immunophenotyping techniques were equivocal and unable to establish the diagnosis. Analysis of the antigen receptor genes (IgH gene and TCR gamma locus) was performed in 230 patients by either Southern blotting or the polymerase chain reaction (PCR). Clonal rearrangements of either loci could be demonstrated in 91/230 patients: 56/161 patients analyzed by Southern blotting and 48/125 analyzed by polymerase chain reaction. A subgroup of patients (n = 88) was analyzed for rearrangement of the immunoglobulin heavy chain gene by both techniques. Discordant results were observed in 18 of these patients (20%). Analysis of the TCR gamma locus in a separate group demonstrated discordant results in eight of 40 patients examined (20%). Clinical outcome could be available in 61 patients (median time: 42 months, range 1-68 months): for those in whom a rearrangement was detected approximately 80% went on to develop a lymphoproliferative disorder although this diagnosis was not able to be made at the time the sample was taken. In a subset of patients (n = 14) who presented with lymphocytosis after bone marrow transplantation (seven) or solid organ transplant (seven), clonal lymphoid populations were demonstrated in approximately 50% of cases. The majority of these cases demonstrated the presence of Epstein-Barr virus (EBV) by PCR. The clonality of EBV infection was assessed by Southern blotting and the significance of these findings are discussed. This study explores the differences between Southern blotting and PCR as applied to the study of antigen receptor rearrangement studies. We conclude that detecting a clonal population is valuable in patients where standard diagnostic techniques are equivocal. However, the inability to detect a clonal population should be treated with caution and interpreted in light of other investigations.
Assuntos
Rearranjo Gênico , Linfoma/imunologia , Transtornos Linfoproliferativos/imunologia , Southern Blotting/métodos , Medula Óssea/imunologia , Medula Óssea/patologia , Diagnóstico Diferencial , Rearranjo Gênico do Linfócito T , Rearranjo Gênico da Cadeia delta dos Receptores de Antígenos dos Linfócitos T , Rearranjo Gênico da Cadeia gama dos Receptores de Antígenos dos Linfócitos T , Genes de Imunoglobulinas , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Linfoma/genética , Transtornos Linfoproliferativos/genética , Reação em Cadeia da Polimerase/métodosRESUMO
In this paper we report on the structure and expression of the genes encoding the alpha and beta chains of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor in human leukemia. The alpha chain gene is highly polymorphic in normal individuals and no evidence for rearrangement within this locus was found in 47 hemopoietic, nine non-hemopoietic malignancies and five human cell lines. Using the polymerase chain reaction the gene for the alpha chain was shown to be expressed in 18/18 primary myeloid as well as 8/8 primary lymphoid leukemias analysed. To investigate the integrity of the mRNA, polymerase chain reactions (PCR) using a combination of oligonucleotides spanning the entire coding region of the alpha chain were performed. Normal sized fragments were generated with all combinations of oligonucleotides from all but one leukemia. One chronic lymphoid leukemia displayed an apparent alteration at the 3' end of the 3' untranslated region of the alpha chain mRNA. No polymorphisms were detected in the beta chain gene which was also not rearranged in any of the samples analysed. The beta chain mRNA was expressed in 17/18 primary myeloid and 7/8 primary lymphoid leukemias and in those leukemias there was no evidence for any lesions in the mRNA, as judged by PCR fragment size. Thus gross structural lesions in the genes encoding the GM-CSF receptor alpha and beta chains appear to be infrequent in hemopoietic neoplasms.
Assuntos
Leucemia/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Sequência de Bases , DNA de Neoplasias/genética , Expressão Gênica , Rearranjo Gênico , Genes , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Reação em Cadeia da Polimerase , RNA Neoplásico/genética , Mapeamento por Restrição , Células Tumorais CultivadasRESUMO
The SCL gene encodes a member of the helix-loop-helix (HLH) family of transcription factors and is reportedly involved in up to 25% of T-cell acute lymphoblastic leukemia (T-ALL). We have surveyed over 120 primary human tumors including melanomas, myeloid, and lymphoid leukemias, and other solid tumors without evidence of rearrangements involving SCL. These results are further supported by low level expression of SCL in these tumors (as assessed by a polymerase chain-reaction-based method). We conclude that rearrangement/translocation with subsequent activation of SCL occurs infrequently in myeloid leukemias and melanomas.
Assuntos
Proteínas de Ligação a DNA/genética , Neoplasias/genética , Proteínas Proto-Oncogênicas , Fatores de Transcrição/genética , Translocação Genética , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Linfoma de Burkitt/genética , Expressão Gênica , Rearranjo Gênico , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Mieloide/genética , Leucemia-Linfoma de Células T do Adulto/genética , Melanoma/genética , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Proteína 1 de Leucemia Linfocítica Aguda de Células TRESUMO
The human erythroleukemia cell line K562 can be induced to differentiate along the erythroid and megakaryocytic lineages. Here we demonstrate that hexamethylene bisacetamide (HMBA) induced K562 cells to differentiate along a third pathway. This was accompanied by downregulation of two transcription factors normally expressed in erythroid, mast and megakaryocyte lineages. Northern analysis demonstrated coordinate downregulation of alpha globin and gamma globin in addition to the two lineage-restricted transcription factors, SCL and GATA-1. Proliferation of the K562 cells was also suppressed. Clonal assay showed that the suppression was irreversible and appeared analogous to the commitment of murine erythroleukemia (MEL) cells to terminal differentiation. In contrast to MEL cells, however, K562 cells acquired a macrophage-like morphology and exhibited a complete failure to generate benzidine-positive cells. Electron microscopy revealed a marked increase in granules resembling those specific for eosinophils. Surface marker analysis showed that HMBA-induced cells expressed reduced levels of glycophorin A, CD5, CD7 and CD11b. No upregulation of megakaryocyte or lymphoid markers occurred. Thus the response of K562 cells to HMBA may provide a useful experimental system for studying the molecular mechanisms responsible for downmodulation of lineage-restricted transcription factors during hemopoietic lineage commitment.
Assuntos
Eritrócitos/citologia , Hematopoese/efeitos dos fármacos , Megacariócitos/citologia , Fatores de Transcrição/fisiologia , Acetamidas/farmacologia , Animais , Antígenos de Superfície/análise , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Glicoforinas/metabolismo , Humanos , Leucemia Eritroblástica Aguda/patologia , Macrófagos/citologia , Camundongos , Fatores de Tempo , Células Tumorais CultivadasRESUMO
We have determined the molecular structure of the gene encoding the murine SCL protein (helix-loop-helix transcription factor). The gene consists of seven exons spanning approx. 20 kb. The intron/exon structure, coding region sequences and sequences present at the splice junctions were highly conserved between mouse and human. The 5' flanking sequence contains CCAAT and TATA consensus motifs with several putative binding sites for SP-1, AP-1 and GATA-1. Multiple mRNA transcripts were generated by alternate exon usage. The transcripts differed primarily in the 5' untranslated region (UTR), but potentially also encode a smaller SCL protein. Despite the high degree of conservation between species, the heptamer/nonamer signal sequences in the 5' region of the human SCL gene (the frequent site of SCL disruption in human leukemia) were poorly represented in the murine sequence. In keeping with this, structural abnormalities of murine SCL were uncommon in murine leukemias that express the SCL transcript.
Assuntos
Proteínas de Ligação a DNA/genética , Sequências Hélice-Alça-Hélice/genética , Camundongos/genética , Proteínas Proto-Oncogênicas , Fatores de Transcrição , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Sequência Conservada , Éxons , Hominidae/genética , Humanos , Íntrons , Dados de Sequência Molecular , Splicing de RNA , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Transcrição GênicaRESUMO
Methylation of the p16 gene was studied in 16 oesophageal tumours. Five (31%) of the tumours were found to be methylated in exon 1 and eight (50%) were methylated in exon 2. The loss of p16 protein correlated with methylation of exon 1 (P = 0.005). However, methylation of exon 2, but not exon 1, was found to be associated with late stage tumours (P = 0.01). We conclude that the methylation of exon 2 of p16 may have effects on the progression of oesophageal tumours that are independent of the expression of the p16 protein.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Metilação de DNA , Neoplasias Esofágicas/metabolismo , Éxons , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Inibidor p16 de Quinase Dependente de Ciclina/genética , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Estadiamento de NeoplasiasRESUMO
This case is of an unusual florid reactive CD4+ T Cell lymphocytosis involving lymph node (LN) and overshadowing residual B chronic lymphocytic leukemia (CLL). A 65 year old female with a 9 year history of untreated B-CLL presented with weight loss, splenomegaly and lymphadenopathy. B-CLL was confirmed on the basis of peripheral blood lymphocytosis, bone marrow trephine findings and flow cytometry analysis. However, the LN biopsy showed appearances of a diffuse small lymphocytic population mimicking a leukemic T-cell infiltrate. Immunophenotyping and molecular analysis demonstrated the major cell population to be reactive CD4 positive T lymphocytes.
Assuntos
Linfócitos T CD4-Positivos , Leucemia Linfocítica Crônica de Células B/patologia , Linfonodos/patologia , Linfocitose/etiologia , Idoso , Biomarcadores Tumorais , Medula Óssea/patologia , DNA de Neoplasias/análise , Feminino , Rearranjo Gênico , Genes de Imunoglobulinas , Humanos , Hiperplasia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias kappa de Imunoglobulina/genética , Imunofenotipagem , Leucemia Linfocítica Crônica de Células B/complicações , Linfocitose/patologia , PescoçoRESUMO
The clinical, and laboratory features of 9 patients presenting with chronic proliferations of large granular lymphocytes (LGL) are described. The median patient age was 61 years (33-80) and median patient follow up was 3.5 years (28 mo-10 years) with all patients surviving. Clinical features and blood and bone marrow findings are documented. Immunophenotypic analysis showed lymphocytes from 4 patients were CD3 negative and 5 were CD3 positive with natural killer associated cell surface antigens expressed in both these groups. Analysis of the T-cell receptor (TCR) loci revealed a clonal rearrangement in 4 samples including one CD3 negative sample. Clonality did not correlate with immunophenotype or clinical or haematological features. We conclude that patients with persistent LGL have a wide diversity of cell surface marker expression and that whilst some patients with CD3 negative LGL proliferations have cells which are most likely of natural killer (NK) cell origin, in others TCR rearrangements can be demonstrated suggesting these cells are possibly of T-cell, not NK cell, origin.
Assuntos
Antígenos CD/sangue , Complexo CD3/sangue , Linfócitos/imunologia , Transtornos Linfoproliferativos/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Southern Blotting , DNA de Neoplasias/análise , DNA de Neoplasias/sangue , Feminino , Seguimentos , Hemoglobinas/análise , Humanos , Imunofenotipagem , Contagem de Leucócitos , Transtornos Linfoproliferativos/sangue , Transtornos Linfoproliferativos/genética , Masculino , Pessoa de Meia-Idade , Contagem de Plaquetas , Mapeamento por Restrição , Linfócitos T/imunologia , Fatores de TempoRESUMO
Expression of C3 receptors (CR1 and CR3) on neutrophils (PMN) was measured in 26 patients with IgA nephropathy (IgA N), 17 normal persons, and 8 patients with non-IgA glomerulonephritis (non-IgA GN) by fluorescence activated cell sorting after labeling with monoclonal antibodies. Mean channel fluorescence for both CR1 and CR3 on PMN was significantly higher in IgA N patients than in either control group (p less than 0.01). (CR1 mean +/- SD 42.5 +/- 10.4 in IgA N, 31.7 +/- 11.5 in normal controls, 27.8 +/- 9.0 in non-IgA GN; CR3 94.0 +/- 16.5, 75.0 +/- 16.6 and 76.7 +/- 15.6, respectively.) No differences were found between the two control groups or between IgA N patients with normal and impaired renal function. These results imply that PMN are activated in IgA N patients. The expression of CR1 and CR3 of PMN may be upregulated by immune complexes (ICs), enhancing both phagocytosis of C3b- or iC3b-coated particles, and absorptive pinocytosis of soluble ICs containing C3b or iC3b. Erythrocyte CR1 and CR3 expression was measured by ELISA and found to be slightly but significantly lower in IgA N patients than in the other 2 groups (CR1 85.3 +/- 28.4 in IgA N, 113.1 +/- 28.8 in normal controls, 109.4 +/- 28.2 in non-IgA GN, p less than 0.02; CR3 80.9 +/- 20.0, 100.4 +/- 19.9, 101.2 +/- 24.9, respectively, p less than 0.05).
Assuntos
Eritrócitos/análise , Glomerulonefrite por IGA/sangue , Neutrófilos/análise , Receptores de Complemento/análise , Adulto , Idoso , Ensaio de Imunoadsorção Enzimática , Feminino , Glomerulonefrite por IGA/etiologia , Glomerulonefrite por IGA/imunologia , Humanos , Antígeno de Macrófago 1 , Masculino , Pessoa de Meia-Idade , Fagocitose/imunologiaRESUMO
Full-length cDNA clones for the pig, cow and sheep mucosal addressin cellular adhesion molecule (MAdCAM)-1 homologues were isolated from Peyer's patches by a combination of reverse transcription (RT)-polymerase chain reaction and 5' and 3' RACE strategies. Degenerate primers based on conserved amino acid (aa) sequences within the N-terminal immunoglobulin (Ig)-like domains of the human and rodent MAdCAM-1 molecules were used for initial sequencing of the Ig-like domains. MAdCAM-1 transcripts of 1425 bp, 1525 bp and 1510 bp obtained for the pig, cow and sheep contained an open-reading frame for proteins of 390, 424 and 418 aa, respectively. The pig and ruminant MAdCAM-1 had two N-terminal Ig-like domains, a mucin-like region and a third Ig-like domain found in rodent but not human MAdCAM-1. Antibodies raised against bacterially expressed N-terminal Ig-like domains of pig, human and sheep MAdCAM-1 demonstrated the existence of cross-reactive epitopes, raising the possibility of producing monoclonal antibodies which can be used as multi-species MAdCAM-1-targeting reagent for the development of mucosal vaccines.
Assuntos
Bovinos/imunologia , Moléculas de Adesão Celular/imunologia , Imunoglobulinas/imunologia , Mucoproteínas/imunologia , Carneiro Doméstico/imunologia , Suínos/imunologia , Sequência de Aminoácidos , Animais , Bovinos/genética , Moléculas de Adesão Celular/genética , Clonagem Molecular , Reações Cruzadas/imunologia , DNA Complementar/genética , Epitopos/genética , Epitopos/imunologia , Imunoglobulinas/genética , Dados de Sequência Molecular , Mucoproteínas/genética , Carneiro Doméstico/genética , Suínos/genéticaRESUMO
The increasing availability of fluorescence-activated cell sorters (FACS) and flow cytometers makes it reasonable to consider the routine use of these machines for the evaluation of the granulocyte immunofluorescence test (GIFT). A comparison of microscopic reading and FACS reading of the standard GIFT demonstrated equivalent simplicity, specificity, sensitivity, reproducibility and duration of test procedure. Differentiation of a negative GIFT from a weakly positive GIFT using the FACS reading method was, however, made difficult by considerable variation in background fluorescence for different neutrophil donors. However, we demonstrate that the FACS reading method was superior for the interpretation of immunofluorescence on chloroquine-treated neutrophils used in the differentiation of HLA from neutrophil-specific antibodies. The systematic study highlighted the fact that any laboratory contemplating conversion from microscopic reading of the GIFT should carefully evaluate and standardize their interpretation of FACS results with their manual reference method.
Assuntos
Separação Celular/instrumentação , Citometria de Fluxo/instrumentação , Imunofluorescência/instrumentação , Granulócitos/análise , Antígenos HLA/análise , Cloroquina , Estudos de Avaliação como Assunto , Feminino , Humanos , Isoanticorpos , MasculinoRESUMO
BACKGROUND & AIMS: The factors controlling the proliferation and differentiation of the colonic mucosa are unknown and have proved difficult to identify mainly because of a lack of in vitro methods for studying the proliferative cells of the mucosa. METHODS: We have developed a novel method of preparing a viable single-cell suspension from isolated crypts and cloning these single cells. RESULTS: We have obtained clonogenic growth from this single-cell suspension with an average of 1 colony per 10(5) cells in control cultures. Addition of conditioned medium from the LIM1863 colon carcinoma cell line increased the mean colony number to 11 +/- 3 per 10(5) cells. The cells forming the colonies are still viable after 4 weeks in culture. The epithelial nature of the cells was confirmed by ultrastructural and immunohistochemical methods with staining for keratin 8 and 18 and anti-human epithelial membrane-specific antigen and a positive result on polymerase chain reaction for keratin 19. CONCLUSIONS: We have successfully cloned single cells from disaggregated colonic crypts from both human and murine colonic mucosa. We have also demonstrated the presence of an active clonogenic factor in the conditioned medium of a colon carcinoma cell line. Assays show that the clonogenic activity in the conditioned medium is not caused by the presence of any of the epidermal growth factor family of growth factors. This is the first report of a clonogenic assay for epithelial cells of normal colonic mucosa.
Assuntos
Colo/citologia , Mucosa Intestinal/citologia , Animais , Divisão Celular , Células Clonais/citologia , Colo/efeitos dos fármacos , Colo/ultraestrutura , Ensaio de Unidades Formadoras de Colônias , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Substâncias de Crescimento/farmacologia , Humanos , Imuno-Histoquímica , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Valores de ReferênciaRESUMO
The term cutaneous T-cell lymphoma (CTCL) encompasses the spectrum of diseases characterized by a monoclonal proliferation of malignant T lymphocytes predominantly of the mature T-helper cell type. These include mycosis fungoides, which is usually localized to the skin for many years, and the Sezary syndrome, the leukemic variant in which characteristic, bizarre lymphatic cells with deeply indented or cerebriform nuclei, the Sezary cells, infiltrate lymph glands and internal organs such as the spleen, liver, lungs, heart, and bone marrow. The condition is more common in men after their fourth decade. The treatment of CTCL varies depending on the stage of the disease. The skin of the patient is the primary index of effectiveness of therapy. Options range from topical steroids, topical nitrogen mustard, X-irradiation, electron beam irradiation, and 8-methoxypsoralen (8-MOP) combined with ultraviolet A photochemotherapy (PUVA), to leukapheresis and systemic chemotherapy. More recently, extracorporeal photochemotherapy (ECPC) has been introduced. The safety and efficacy of this modality is further investigated in six patients who fulfilled the criteria of diagnosis of CTCL. The problems encountered in objectively evaluating the clinical responses in the patients are outlined and improvements in the protocol to overcome these are suggested. The proposed mechanism of action is an immunostimulatory one and a procedure that is relatively free from side effects; it offers promise as a potential treatment for a difficult cancer.