Estudo epidemiológico de pacientes com esclerose múltipla diagnosticados e acompanhados no ambulatório de neurologia do Hospital Universitário da UFJF

-Introdução e Objetivo: A esclerose múltipla (EM) é uma doença auto-imune de natureza inflamatória, apresentando heterogeneidade clínica, patológica e imunológica que atinge o sistema nervoso central, causando incapacidade funcional em cerca de 50% dos pacientes. Essa doença apresenta maior prevalência entre mulheres, onde os sintomas clínicos geralmente surgem entre os 20 e 40 anos de idade. O objetivo desse trabalho avaliou o perfil epidemiológico de pacientes com EM e acompanhados no ambulatório de Neurologia do Hospital Universitário da UFJF e avaliar o perfil de citocinas e quimiocinas em um caso de esclerose Múltipla familiar. Metodologia e Resultados: Inicialmente, realizou-se um levantamento dos dados dos prontuários dos pacientes atendidos no HU-UFJF no período de 1996 a 2007. Desses, foram selecionados 26 pacientes, os quais contemplavam os critérios de Mc Donald para diagnóstico de EM. A razão de gênero foi de 56 mulheres para cada homem. A apresentação clínica surto-remitente foi identificada em 50% dos pacientes, a forma progressiva-secundária em 30% e a forma progressiva-primária em 20%. Observou-se que 73.6 % utilizavam inunomoduladores; 58% já se submeteram a pulsoterapia; 32% foram tratados com imunossupressores; 30,7% apresentaram sintomas adversos, tais como febre, mialgia e hipersensibilidade local, devido ao uso de imunomoduladores. Adicionalmente, foram avaliados os perfis de citocinas e quimiocinas em três irmãs. Dentre essas, duas são gêmeas monozigóticas discordantes para esclerose múltipla e, outra irmã fraterna, também portadora da doença e em tratamento com IFN-beta1a. Os níveis de citocinas e quimiocinas presentes no sobrenadante de células mononucleares do sangue periférico das pacientes após estímulo com PHA (phytohaemagglutinin) por 48 horas foram avaliados pelo método CBA ( Cytometric Bead Array). Os níveis de IFN-gama e TNF-alfa detectados nas irmãs afetadas por EM foram menores que aqueles obtidos na irmã gêmea saudável. Já os níveis de IL-4, IL-6 e IL-10 foram maiores na irmã fraterna efetada àqueles detectados na gêmea afetada. Esses dados sugerem que essas citocinas têm papel importante na progressão da doença. Além disso, a utilização do tratamento com IFN-beta1a pela irmã fraterna afetada pode estar proporcionando o aumento dos níveis dessas citocinas. Com relação às quimiocinas, valores detectáveis foram observados apenas no sobrenadante de PBMC da irmã saudável, sugerindo que na EM estas quimiocinas podem estar diminuídas. Conclusão: Em conjunto, estes dados sugerem que a terapia com IFN-beta1a interfere na progressão da EM, pela indução de mecanismos imunorregulatórios mediados principalmente por IL-10

An Efficient Low Cost Method for Gene Transfer to T Lymphocytes

<div><p></p><p>Gene transfer to T lymphocytes has historically relied on retro and lentivirus, but recently transposon-based gene transfer is rising as a simpler and straight forward approach to achieve stable transgene expression. Transfer of expression cassettes to T lymphocytes remains challenging, being based mainly on commercial kits.</p> <p>Aims</p><p>We herein report a convenient and affordable method based on <i>in house</i> made buffers, generic cuvettes and utilization of the widely available Lonza nucleofector II device to promote efficient gene transfer to T lymphocytes.</p> <p>Results</p><p>This approach renders high transgene expression levels in primary human T lymphocytes (mean 45%, 41–59%), the hard to transfect murine T cells (mean 38%, 36–42% for C57/BL6 strain) and human Jurkat T cell line. Cell viability levels after electroporation allowed further manipulations such as <i>in vitro</i> expansion and Chimeric Antigen Receptor (CAR) mediated gain of function for target cell lysis.</p> <p>Conclusions</p><p>We describe here an efficient general protocol for electroporation based modification of T lymphocytes. By opening access to this protocol, we expect that efficient gene transfer to T lymphocytes, for transient or stable expression, may be achieved by an increased number of laboratories at lower and affordable costs.</p> </div

Electroporation of the Jurkat T cell line.

<p>(<b>A</b>) Jurkat cells were electroporated with pT2-GFP in the presence of each of the 7 different buffers. Viability and GFP expression were evaluated by flow cytometry after 24 h. Cell viability is expressed as % of the control mock electroporated condition (100%). Electroporation scores were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060298#s2" target="_blank">materials and methods</a>. Statistical analysis was performed using One Way ANOVA and Tukey post test (* = P<0.05). (<b>B</b>) Jurkat cells were electroporated with buffer 3P. Cell viability and GFP expression were observed until day 20. Values in this figure are the average of two separate experiments in triplicate and are expressed as mean±SEM. Data were analyzed by unpaired Student t test; p<0.05 (*);p<0.01 (**).</p

Electroporation of resting vs activated mouse primary T lymphocytes.

<p>Total lymphocytes from lymph nodes of C57BL/6 mice were isolated and either directly electroporated or activated for 24 h with anti-CD3/anti-CD28 and then electroporated. Buffer 2S and 4 µg of pT2-GFP plasmid were used. Cell viability and GFP expression were analyzed after 24 h by flow cytometry. Cell viability is normalized to control (not electroporated) cells. Data are representative of two experiments in triplicate and expressed as mean±SEM. Data were analyzed by unpaired Student t test; p<0.0001 (**) or p = 0.002 (*).</p

Electroporation of transposon and transposase maintains transgene expression after cell viability recovery.

<p>PBMCs from two healthy donors were electroporated using 1SM buffer, 20 µg of pT2-GFP plasmid and 2 µg of SB100x transposase. Cell viability and GFP expression were analyzed by flow cytometry on day 1 and 9. Values are the average of two donors in triplicate and are expressed as mean±SEM.</p

Efficiency of murine T lymphocyte electroporation is dependent on different buffer and mouse strain.

<p>(<b>A</b>) Total lymphocytes from lymph nodes of C57BL/6 mice were isolated and electroporated using in house buffers and 4 µg of pT2-GFP plasmid. Cell viability and GFP expression were analyzed after 24 h by flow cytometry. Electroporation scores were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060298#s2" target="_blank">materials and methods</a>. Statistical analysis was performed using One Way ANOVA and Tukey post test. P<0.05 (*). (<b>B</b>) Total lymphocytes from lymph nodes of C57BL/6 mice were isolated and electroporated using 2S buffer or Lonza buffer and 4 µg of pT2-GFP. Analysis was performed 24 h later by flow cytometry. Data were analyzed by unpaired Student t test p<0,05 (*). (<b>C</b>) Total lymphocytes from lymph nodes of C57BL/6, Balb/c and B10A mice were isolated and electroporated using 2S buffer and 4 µg of pT2-GFP plasmid. Cell viability was normalized to control (not electroporated) cells. All values in this figure represent the average of two experiments in triplicate and are expressed as mean±SEM.</p

Electroporation efficiency of different buffers in primary human T lymphocytes.

<p>(<b>A</b>) PBMCs from two healthy donors were electroporated using in house buffers and 4 µg of pT2-GFP plasmid. Cell viability and GFP expression were analyzed after 24h by flow cytometry. Cell viability is normalized to control (not electroporated) cells. Electroporation scores were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060298#s2" target="_blank">materials and methods</a>. Values are the average of two donors in triplicate and are expressed as mean±SEM. Statistical analysis was performed using One Way ANOVA and Tukey post test (* = P<0.05). (<b>B</b>) Representative dot plots of GFP (pT2-GFP; 4 µg) expression 24 h after electroporation with 1SM buffer. Numbers in plots represents the percentage of cells in the gate. Gray line = negative control; black line = cells electroporated with pT2-GFP. (<b>C</b>) Electroplated lymphocytes were stained for CD4 and CD8 24 h after electroporation with 1SM buffer and 4 µg pT2-GFP plasmid. Data are representative of two donors. Numbers in plots represents the percentage of cells in the gate.</p

T cell electroporation with Chimeric Antigen Receptor (CAR) results in stable gene expression conferring target-specific cytotoxicity.

<p>PBMCs from one donor were electroporated using 1SM buffer, 20 µg of pT3-20z plasmid and 0,5 µg of SB100x transposase plasmid. One day later, lymphocytes were stimulated with irradiated L388 cells and CAR expression was evaluated until d+30 by flow cytometry analysis using an anti-fab antibody. (A) Representative histograms of 20z CAR expression at d+1, d+10, d+20 and d+30 after electroporation (gray line = non electroporated cells stained anti-Fab; black line = lymphocytes electroporated with pT3-20z). (B) Analysis of 20z CAR expression until d+30 summarizing data of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060298#pone-0060298-g008" target="_blank">Figure 8A</a>. (C) Kinetics of expansion of control mock electroporated (Neg) or 20z+ lymphocytes after stimulation with L388 cells. (D) Expanded cells were used in different effector/target (E/T) ratios in a cytotoxic assay against the CD19+/CD20+ Nalm-6 GFP+target cell line; Neg = CTLs not electroporated.</p

Long term expression of GFP after electroporation with buffer 1SM.

<p>PBMCs from two healthy donors were electroporated using 1SM buffer and 4 µg of pT2-GFP plasmid. After 24 h cells were activated with anti-CD3/anti-CD28 and GFP expression was evaluated for 7 days by flow cytometry.</p

An efficient low cost method for gene transfer to T lymphocytes.

UNLABELLED: Gene transfer to T lymphocytes has historically relied on retro and lentivirus, but recently transposon-based gene transfer is rising as a simpler and straight forward approach to achieve stable transgene expression. Transfer of expression cassettes to T lymphocytes remains challenging, being based mainly on commercial kits. AIMS: We herein report a convenient and affordable method based on in house made buffers, generic cuvettes and utilization of the widely available Lonza nucleofector II device to promote efficient gene transfer to T lymphocytes. RESULTS: This approach renders high transgene expression levels in primary human T lymphocytes (mean 45%, 41-59%), the hard to transfect murine T cells (mean 38%, 36-42% for C57/BL6 strain) and human Jurkat T cell line. Cell viability levels after electroporation allowed further manipulations such as in vitro expansion and Chimeric Antigen Receptor (CAR) mediated gain of function for target cell lysis. CONCLUSIONS: We describe here an efficient general protocol for electroporation based modification of T lymphocytes. By opening access to this protocol, we expect that efficient gene transfer to T lymphocytes, for transient or stable expression, may be achieved by an increased number of laboratories at lower and affordable costs