RESUMO
Systemic administration of mesenchymal stem cells (MSCs) has been shown to be safe and efficacious in humans with Crohn's disease. The aim of this study was to evaluate the safety of an intravenous (IV) infusion of adipose tissue-derived mesenchymal stem cells (ASCs) and to assess macroscopic and histological effects in the digestive tract of dogs with inflammatory bowel disease (IBD). Eleven dogs with confirmed IBD received a single ASC infusion (2 × 10(6) cells/kg bodyweight). Full digestive endoscopic evaluation was performed pre-treatment and between 90 and 120 days post-treatment with mucosal changes being assessed using a fit-for-purpose endoscopic scale. Endoscopic biopsies from each digestive section were evaluated histologically according to the World Small Animal Veterinary Association (WSAVA) Gastrointestinal Standardization Group criteria. The pre- and post-treatment canine IBD endoscopic index (CIBDEI) and histological score (HS) were calculated and compared using the Wilcoxon test. Remission was defined as a reduction of >75% of the CIBDEI and HS compared with pre-treatment. No acute reactions to ASC infusion or side effects were reported in any dog. Significant differences between pre- and post-treatment were found in both the CIBDEI (P = 0.004) and HS (P = 0.004). Endoscopic remission occurred in 4/11 dogs with the remaining dogs showing decreased CIBDEI (44.8% to 73.3%). Histological remission was not achieved in any dog, with an average reduction of the pre-treatment HS of 27.2%. In conclusion, a single IV infusion of allogeneic ASCs improved gastrointestinal lesions as assessed macroscopically and slightly reduced gastrointestinal inflammation as evaluated by histopathology in dogs with IBD.
Assuntos
Tecido Adiposo/citologia , Doenças do Cão/terapia , Doenças Inflamatórias Intestinais/veterinária , Transplante de Células-Tronco Mesenquimais/veterinária , Células-Tronco Mesenquimais , Animais , Doenças do Cão/patologia , Cães , Endoscopia Gastrointestinal/veterinária , Feminino , Doenças Inflamatórias Intestinais/patologia , Doenças Inflamatórias Intestinais/terapia , Masculino , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Resultado do TratamentoRESUMO
Mesenchymal stem cells (MSCs) have shown immunomodulatory and anti-inflammatory effects in experimental colitis, and promising clinical results have been obtained in humans with Crohn's disease and ulcerative colitis. The aim of this study was to determine the safety and feasibility of adipose tissue-derived MSC (ASC) therapy in dogs with inflammatory bowel disease (IBD). Eleven dogs with confirmed IBD received one ASC intravascular (IV) infusion (2 × 10(6) cells/kg bodyweight). The outcome measures were clinical response based on percentage reduction of the validated Clinical Inflammatory Bowel Disease Activity Index (CIBDAI) and Canine Chronic Enteropathy Clinical Activity Index (CCECAI), as well as normalisation of C-reactive protein (CRP), albumin, folate and cobalamin serum concentrations at day 42 post-treatment. The Wilcoxon test was used to compare variables before and after treatment. No acute reaction to ASC infusion and no side effects were reported during follow-up in any dog. Six weeks post-treatment, the CIBDAI and CCECAI decreased significantly and albumin, cobalamin and folate concentrations increased substantially. Differences in CRP concentrations pre- and post-treatment were not significant (P = 0.050). Clinical remission (defined by a reduction of initial CIBDAI and CCECAI >75%) occurred in 9/11 dogs at day 42. The two remaining dogs showed a partial response with reduction percentages of 69.2% and 71.4%. In conclusion, a single IV infusion of allogeneic ASCs was well tolerated and appeared to produce clinical benefits in dogs with severe IBD.
Assuntos
Tecido Adiposo/citologia , Doenças do Cão/terapia , Doenças Inflamatórias Intestinais/veterinária , Transplante de Células-Tronco Mesenquimais/veterinária , Células-Tronco Mesenquimais , Animais , Células Cultivadas , Cães , Feminino , Doenças Inflamatórias Intestinais/terapia , Masculino , Transplante Homólogo/veterinária , Resultado do TratamentoRESUMO
In this work, we will present some attempts to analyze tyrosine and nitrotyrosine using capillary electrophoresis and either UV-Visible detection or laser-induced fluorescence (LIF) detection. An argon ion (488 nm) laser is used for fluorescein isothiocyanate (FITC) and 7-fluoro-4-nitro-2,1,3-benzoxadiazole (NBD-F). A near infrared (780 nm) laser is used for NIR 780 derivatives. The UV-Visible limit of detection is 2.5 microM whereas it is in the range of 30 nM for LIF detection.
Assuntos
4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/química , Fluoresceína-5-Isotiocianato/química , Corantes Fluorescentes/química , Tirosina/análogos & derivados , Tirosina/química , Eletroforese Capilar , Espectrofotometria Ultravioleta , Espectroscopia de Luz Próxima ao InfravermelhoRESUMO
In recent papers, we presented a new analytical method for thiol quantification in serum. This method was developed with capillary electrophoresis (CE) and laser-induced fluorescence (LIF) to analyze thiol-iodoacetamidofluoresceine (IAF) derivatives. Quantitative results for homocysteine, glutathione, cysteinylglycine, and cysteine were presented (Caussé E., et al., Clin. Chem. 45 (1999) 412). An exhaustive comparison of the quantitation of homocysteine in plasma, using high-performance liquid chromatography with either conventional fluorescence detection or fluorescence polarization immunoassay was also reported (Caussé E., et al., Electrophoresis 21 (2000) 2074). Sample preparation prior to derivatization with IAF had never been investigated. Recently we studied protein precipitation in serum with different organic agents (Caussé E., et al., J. Chromatogr. A 895 (2000) 173). In this work, we evaluated the conditions of protein precipitation in function of the amounts of acetonitrile and their influence on quantitation and quality of the electropherograms. Then, we looked at the variation of thiol concentrations in the haemolysis states and studied the thiol stability of blood samples cooled on ice.
Assuntos
Eletroforese Capilar/métodos , Homocisteína/sangue , Espectrometria de Fluorescência/métodos , Compostos de Sulfidrila/sangue , Adulto , Centrifugação , Feminino , Hemólise , Humanos , Lasers , Masculino , Pessoa de Meia-IdadeAssuntos
Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Leucemia Mieloide/induzido quimicamente , Segunda Neoplasia Primária/induzido quimicamente , Neoplasias da Bexiga Urinária/tratamento farmacológico , Doença Aguda , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Cisplatino/administração & dosagem , Cisplatino/efeitos adversos , Doxorrubicina/administração & dosagem , Doxorrubicina/efeitos adversos , Humanos , Masculino , Metotrexato/administração & dosagem , Metotrexato/efeitos adversos , Neoplasias da Bexiga Urinária/radioterapia , Vimblastina/administração & dosagem , Vimblastina/efeitos adversosAssuntos
Cromossomos Humanos Par 21 , Cromossomos Humanos Par 8 , Granulócitos/patologia , Hematopoese , Translocação Genética , Doença Aguda , Contagem de Células , Criança , Pré-Escolar , Diagnóstico Diferencial , Feminino , Humanos , Leucemia Mieloide/classificação , Leucemia Mieloide/diagnóstico , Leucemia Mieloide/genética , Leucemia Mieloide/patologia , Masculino , Síndromes Mielodisplásicas/classificação , Síndromes Mielodisplásicas/diagnósticoRESUMO
LC-fluorescence and LC-MS methods have been previously reported for use in decoding bead-based combinatorial libraries. We present the use of capillary electrochromatography (CEC) for highly selective decoding in combination with laser-induced fluorescence (LIF) detection for high sensitivity. The results are compared to prior data obtained using HPLC with fluorescence detection. The use of CEC shows promise for miniaturization and multiplexing for future applications, and the use of LIF detection can allow for detection at sub-pmol amounts.
Assuntos
Aminas/análise , Cromatografia Capilar Eletrocinética Micelar/métodos , Técnicas de Química Combinatória , Compostos de Dansil/química , Espectrometria de Fluorescência/métodos , Aminas/química , Lasers , Sensibilidade e EspecificidadeRESUMO
Previous studies demonstrated that ex vivo IL-2- activated PBSC could generate cytotoxic effectors without impairing haematopoietic reconstitution. Clinical experience and previous studies indicated that children with solid tumours could benefit from high-dose chemotherapy with immune modulation. We studied the generation of cytotoxic effectors from growth-factor +/- chemotherapy-mobilised PBSC from 10 patients (five adults and five children) with different solid tumours. Cells were placed in culture in serum-free culture medium supplemented with IL-2 1000 U/ml +/- IL-12 for 1, 2, 4 or 7 days. Anti-tumour cytotoxicity was tested against K562, Daudi and two neuroblastoma cell lines (Gau, NB91). Cultured adult PBSC in the presence of IL-2 (1000 U/ml) showed marked cytotoxicity against all the cell lines tested from day 1. At day 2, with an E:T ratio of 25:1, cytotoxicity was 53% +/- 10.4, 63.2% +/- 23.8, 38% +/- 9.1, and 39% +/- 15.7 against K562, Daudi, Gau and NB91, respectively. Cytotoxic activity of child PBSC was significantly lower (P < 0.05) and was displayed after longer culture times (day 4). No difference was found in the phenotype analysis of lymphoid subsets before and after IL-2 activation between adult and child PBSC. Haematological properties of the graft were not significantly impaired by IL-2 activation.
Assuntos
Interleucina-2/farmacologia , Neoplasias/patologia , Adulto , Fatores Etários , Contagem de Células Sanguíneas , Células Sanguíneas/citologia , Células Sanguíneas/imunologia , Técnicas de Cultura de Células/métodos , Criança , Testes Imunológicos de Citotoxicidade , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Humanos , Imunofenotipagem , Interleucina-12/farmacologia , Neoplasias/terapia , Células-Tronco/citologia , Células-Tronco/imunologia , Transplante Autólogo , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
In this work we present some applications of near-UV laser-induced fluorescence (LIF) with micro-HPLC (microHPLC) and HPLC. To test the sensitivity of the detection, we used pyrene and aflatoxins, because both of these molecules exhibit native fluorescence. Then we studied catecholamines derivatized with 1,2-diphenylethylenediamine. The results show that we were able to reach better sensitivity levels than previously described in LIF studies. For catecholamines, a 50-fold increase in sensitivity compared to conventional fluorescence was obtained. These results indicate that LIF detection associated with HPLC or microHPLC can be used to detect very low concentrations of substances that can be excited in the near-UV range after labeling at nanomolar concentrations.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Fluorescência/métodos , Raios Ultravioleta , Aflatoxinas/análise , Animais , Catecolaminas/análise , Lasers , Camundongos , Sensibilidade e EspecificidadeRESUMO
In this publication we present results on the determination of enantiomers of amino acids at very low concentrations. A fluoresceine-based chiral dye was synthesized to allow the separation of diastereoisomers of D- and L-amino acids. We used capillary electrophoresis with different non-ionic surfactants (Brij). The separation parameters were optimized and separations of D- and L-isovaline, an unusual terrestrial amino acid, were obtained. The sensitivity limits were also determined using a commercial laser-induced fluorescence detector. The quantitation of these amino acids is very important to understand the process of chiral selection on Earth.
Assuntos
Aminoácidos/análise , Eletroforese Capilar/métodos , Fluorescência , Lasers , Sensibilidade e Especificidade , Espectrometria de Fluorescência , EstereoisomerismoRESUMO
In recent papers, we presented a new analytical method for thiol quantification in serum. It is based on the use of capillary electrophoresis and laser-induced fluorescence to analyze thiol 6-iodoacetamidofluoresceine (IAF) derivatives. Quantitative results of homocysteine, glutathione, cysteine-glycin, and cysteine were shown (Clin. Chem. 45 (1999) 412). A comprehensive comparison of the quantitation of homocysteine in serum, using high-performance liquid chromatography/conventional fluorescence detection and fluorescence polarization immunoassay was also used (E. Caussé et al., Electrophoresis 21 (2000) 2074). Sample preparation prior to derivatization with IAF had never been investigated. In this work we present the results of quantitation of thiols in serum and plasma with three different anticoagulants widely used: ethylenediaminetetraacetic acid (EDTA), heparin, and sodium citrate. We show that serum and EDTA plasma gave the same results. Then serum protein precipitations by acetonitrile, acetone, sulfosalicylic acid, perchloric acid and trichloracetic acid, prior to derivatization by IAF, were also investigated. Their influence on the concentrations of the thiols were determined. Sulfosalicylic acid and acetonitrile precipitations are well adapted, whereas acetone cannot be used.
Assuntos
Eletroforese Capilar/métodos , Homocisteína/sangue , Compostos de Sulfidrila/sangue , Adulto , Anticoagulantes/farmacologia , Feminino , Imunoensaio de Fluorescência por Polarização , Humanos , Lasers , Masculino , Pessoa de Meia-Idade , Espectrometria de FluorescênciaRESUMO
We present a new analytical method for thiol quantification in plasma, based on the use of capillary electrophoresis (CE) and laser-induced fluorescence (LIF) to analyze 6-iodoacetamidofluorescein derivatives. Quantitative results of homocysteine, glutathione, cysteinylglycine, and cystationine are presented. A comparison of the quantitation of homocysteine in plasma, using high performance liquid chromatography/fluorescence detection and fluorescence polarization immunoassay is proposed. The results indicate that these techniques for plasma total homocysteine (tHcy) determination can be used interchangeably. The major advantage of CE-LIF is that it can quantitate the thiols in one run while keeping the price of consumables reasonable.
Assuntos
Homocisteína/sangue , Compostos de Sulfidrila/sangue , Cromatografia Líquida de Alta Pressão/métodos , Eletroforese Capilar/métodos , Polarização de Fluorescência/métodos , Humanos , Imunoensaio/métodos , Sensibilidade e Especificidade , Espectrometria de Fluorescência/métodosRESUMO
Asymmetric dimethyl-L-arginine (ADMA) is a naturally occurring analogue of L-arginine (L-Arg), the substrate of nitric oxide synthase (NOS). ADMA is a potent endogenous inhibitor of NOS and accumulates in the plasma of patients with renal failure, with peripheral arterial occlusive disease or with clinically asymptomatic hypercholesterolemia. We measured circulating concentrations of L-arginine, symmetric and asymmetric dimethylarginine (SDMA and ADMA, respectively) in human serum. We developed a new method for the rapid determination of these molecules using capillary electrophoresis and laser-induced fluorescence (CE-LIF). All methylated arginines were labeled with fluorescein isothiocyanate (FITC) prior to analysis. Under the capillary electrophoresis (CE) conditions used, methylated arginine derivatives were well separated, with a migration time of around 10 min. These migration times were smaller than the ones of other amino acids which do not have the same charge at pH 10. Consequently, such basic amino acids were well separated from most of the other amines or amino acids. Moreover, CE allowed one to separate all the analogues of fluorescein thiocarbamyl-arginine. The results indicated that CE-LIF is useful as a selective, rapid, cheap and sensitive tool for the determination of methylated arginine products. This new technology might appreciate the endogenous substrate for NO synthase and facilitate the knowledge of the physiological and pathophysiological regulation of NO synthesis.
Assuntos
Arginina/análogos & derivados , Eletroforese Capilar/métodos , Arginina/sangue , Calibragem , Fluorescência , Humanos , Falência Renal Crônica/sangue , Lasers , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Through two clinical studies, tumor cells were searched for in the bone biopsies and cytapherisis of patients suffering from inflammatory tumors and who had undergone intensive therapy and autografts (Pegase 2 program). In these studies we used immunocytochemical test with two monoclonal antibodies. The results have shown the presence of tumor cells in 14% of the patients (respectively 18%), with no correlation to the appearance of metastases after 4 years in the first study. Nevertheless, the presence of these tumor cells seems to be an important factor in the number of relapses. It seems important to develop research into tumor contamination especially in the selection of grafts over the next few years.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Neoplasias/análise , Osso e Ossos/patologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Adulto , Anticorpos Antineoplásicos/imunologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/fisiopatologia , Neoplasias da Mama/terapia , Terapia Combinada , Citaferese , Feminino , Humanos , Pessoa de Meia-Idade , Metástase Neoplásica , RecidivaRESUMO
We report four cases of a rare subtype of CD30-positive anaplastic large cell lymphoma (ALCL) with a predominant small cell component (small cell variant of ALCL) presenting with a leukaemic feature. Lymph node biopsy showed malignant cells of varying size with a predominant population of small to medium-sized malignant cells associated with large anaplastic cells strongly positive for CD30 and epithelial membrane antigen (EMA). Both large and small cells were reactive with antibody ALK1, which recognizes the chimaeric NPM-ALK protein associated with the t(2;5)(p23;q35). All patients presented with hyperleucocytosis with atypical small lymphocytes. Bone marrow involvement was detected on both aspirate and bone marrow trephine where scattered malignant cells were only demonstrated by immunostaining for CD30 and ALK protein. Atypical cells in peripheral blood, lymph node and skin biopsies showed a T or null cell phenotype. Cytogenetic analysis of blood, bone marrow and/or lymph node revealed the t(2:5)(p23;q35) characteristic of ALCL. The patients responded to chemotherapy but showed early relapse without abnormal cells in peripheral blood. This report shows that the small cell variant of ALCL may have a leukaemic presentation with peripheral blood involvement by atypical lymphocytes and provides evidence that, in the small cell variant of ALCL, the small cell component is a part of the malignant clone.
Assuntos
Linfoma Anaplásico de Células Grandes/patologia , Adolescente , Tamanho Celular , Criança , Cromossomos Humanos Par 2/genética , Cromossomos Humanos Par 5/genética , Evolução Fatal , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Lactente , Leucocitose/patologia , Linfócitos/patologia , Linfoma Anaplásico de Células Grandes/genética , Masculino , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Translocação GenéticaRESUMO
CD(34+)-enriched peripheral blood stem cells (PBSC) are increasingly being used as an autograft in patients with multiple myeloma (MM). The rationale for the use of the CD34+-enriched fraction in MM is the ability to obtain a graft with a significant reduction of contamination by plasma cells. However, the effect of such a manipulation on the proliferating potential of the engrafted cells is not known. We wished to study, as part of a randomized trial comparing the outcome in MM patients transplanted with either CD(34+)-enriched cells or unfractionated PBSC, the primitive hematopoietic cell content of the autografts using long-term culture initiating cell (LTC-IC) assays in 7 MM patients. In 3 patients CD(34+)cell-enriched fraction was compared to unfractionated PBSC whereas in the remaining 4 patients the LTC-IC assay was performed on total PBSC. The mean percentage of CD34+ cells of the CD34+ selected fraction in three patients was 82% (range 71%-96%) whereas the same percentage in PBSC varied from 0.6% to 10% in 4 patients (mean: 4.2%). Out of three patients transplanted with CD34+ cell fraction, two patients were found to have a very similar LTC-IC generating potential in their CD34+ versus PBSC fractions as this was assessed by the clonogenic cell output at week+5 per 10(4) CD34+ cells initiating the culture (PBSC: 92 and 168 and CD34+ fraction: 102 and 16, respectively) whereas one patient had a slightly different values (PBSC: 51 and CD34+ fraction: 103). When the PBSC fraction was compared in all 7 patients, the LTC-IC generation potential was very heterogenous, varying from 1.4 to 168. To determine if the selection procedure influences the numbers of LTC-IC's in both fractions, we have performed limiting dilution assays to determine both the frequency of distribution of hematopoietic colonies and the frequency of LTC-IC's in two patients. The frequency of distribution of hematopoietic colonies was linear in both CD34+ and PBSC fractions as was the frequency of LTC-IC when the corrections were made with regard to the CD34+ cell-content of the cultures (1/20). Our results indicate that the CD34+ selection procedure used in all three patients (Ceprate) is not deleterious for the generation of LTC-IC's and these findings support the rationale for the use of this procedure in multiple for the purposes of tumor depletion.
Assuntos
Antígenos CD34/sangue , Citaferese/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Mieloma Múltiplo/terapia , Adulto , Antígenos CD34/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Terapia Comportamental/métodos , Técnicas de Cultura de Células , Divisão Celular , Células Clonais , Dexametasona/administração & dosagem , Doxorrubicina/administração & dosagem , Feminino , Mobilização de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Humanos , Masculino , Pessoa de Meia-Idade , Transplante Autólogo/métodos , Vincristina/administração & dosagemRESUMO
PURPOSE: Burkitt-like lymphoma (BLL) is a tumor with morphologic features intermediate between Burkitt's lymphoma (BL) and large-cell lymphoma, but its relationship with these lymphomas is currently unclear. We have therefore analyzed its characteristics within a large series of human immunodeficiency virus (HIV)-associated lymphomas. MATERIALS AND METHODS: Clinical, histologic, immunophenotypic, and molecular analyses were performed on 103 patients with AIDS lymphomas. RESULTS: Nineteen cases (18.4%) were identified as BLL. They were monoclonal B-cell proliferations, as evaluated by immunoglobulin (Ig) gene rearrangement analyses, and had rearrangement of the c-myc oncogene in 68% of cases but not the bcl-2 gene, in contrast to a previous study on non-HIV-associated BLL. This molecular pattern was therefore identical to that of typical BL, suggesting that they represented tumors of similar origin. However, some features could clearly differentiate BLL from BL and were similar to those seen in the diffuse large-cell immunoblastic lymphomas (DLC-IBL) group. These included a greater frequency of Epstein-Barr Virus (EBV) infection (79% v 48%, P = .04), an upregulation of CD39 (50% v 0%, P = .0007) and CD70 (75% v 15%, P = .003) activation antigens and of the CD11a/LFA-1 adhesion molecule (83% v30%, P = .05), and, finally, a lower CD4 count (mean, 119/microL v 270/microL, P = .04). CONCLUSION: BLL is a frequent entity among AIDS lymphomas and should be considered as a morphologic variant of BL in the context of severe immunodepression that occurs in HIV-infected patients.