THE UNIVERSITY EXTENSION IN THE PREVENTION OF CERVICAL CANCER IN COASTAL COMMUNITIES IN PARÁ

O câncer do colo do útero (CCU) é o câncer mais incidente em mulheres da região Norte do Brasil, excluindo o câncer de pele não melanoma. Esta região do país apresenta muitas comunidades com difícil acesso aos serviços básicos de saúde. Estratégias de prevenção do CCU em comunidades ribeirinha são importantes para que a taxa de mortalidade por esta patologia seja reduzida. Descrevemos o perfil das mulheres que realizaram o exame preventivo de Papanicolaou (PCCU), através das ações de extensão universitária em comunidades ribeirinhas atendidas pelo Programa “Luz na Amazônia” que visam à prevenção de doenças sexualmente transmissíveis e o CCU. Nas comunidades visitadas entre os anos de 2011 e 2014, um total de 154 mulheres realizou o exame preventivo do câncer de colo do útero. Destas, 24% nunca haviam realizado o exame anteriormente e 10,39% apresentaram resultados alterados. A população ribeirinha carece de serviços de saúde que ofereçam cuidados voltados à saúde da mulher e programas como o “Luz na Amazônia” são essenciais para a prevenção do CCU em mulheres destas comunidades.The cervical cancer (CC) is the most frequent cancer in women of northern Brazil, excluding non-melanoma skin cancer. This region of the country has many communities with limited access to basic health services. CC’s prevention strategies in coastal communities are important so that the death rate from this disease is reduced. We describe the characteristics of women who conducted the Pap test through the university extension programs in riverside communities served by the program “Light in the Amazon” aimed at preventing sexually transmitted diseases and cancer of the cervix. In the communities visited between 2011 and 2014, a total of 154 women conducted preventive examination of cervical cancer. Of these, 24% had never performed the test previously and 10.39% had abnormal results. The local population lacks health services that provide care focused on women's health and programs like “Light in the Amazon” are essential for the prevention of cervical cancer in women of these communities.El cáncer cervical (CC) es el cáncer más frecuente em las mujeres em el norte de Brasil, excluyendo el cáncer de piel no melanoma. Esta región del país tiene muchas comunidades com acceso limitado a los servicios básicos de salud. Estrategias de prevención del CC em las comunidades costeras son importantes por lo que la tasa de mortalidad por esta enfermedad se reduce. Se describen las características de las mujeres que se sometieron a la prueba de Papanicolaou, através de los programas de extensión universitária em las comunidades costeras atendidas por el programa “Luz em la Amazonia” destinadas a la prevención de enfermedades de transmisión sexual y el câncer del cuello del útero. Em las comunidades visitadas entre los años 2011 y 2014, un total de 154 mujeres llevó a cabo el examen de prevención del cáncer de cuello uterino. De estos, 24% nunca se había realizado la prueba com anterioridad y 10,39% tienen resultados anormales. La población local carece de servicios de salud que brindan atención focalizada em la salud de la mujer y programas tales como “Luz em la Amazonia” son esenciales para la prevención del cáncer de cuello uterino em las mujeres de estas comunidades

THE UNIVERSITY EXTENSION IN THE PREVENTION OF CERVICAL CANCER IN COASTAL COMMUNITIES IN PARÁ

O câncer do colo do útero (CCU) é o câncer mais incidente em mulheres da região Norte do Brasil, excluindo o câncer de pele não melanoma. Esta região do país apresenta muitas comunidades com difícil acesso aos serviços básicos de saúde. Estratégias de prevenção do CCU em comunidades ribeirinha são importantes para que a taxa de mortalidade por esta patologia seja reduzida. Descrevemos o perfil das mulheres que realizaram o exame preventivo de Papanicolaou (PCCU), através das ações de extensão universitária em comunidades ribeirinhas atendidas pelo Programa “Luz na Amazônia” que visam à prevenção de doenças sexualmente transmissíveis e o CCU. Nas comunidades visitadas entre os anos de 2011 e 2014, um total de 154 mulheres realizou o exame preventivo do câncer de colo do útero. Destas, 24% nunca haviam realizado o exame anteriormente e 10,39% apresentaram resultados alterados. A população ribeirinha carece de serviços de saúde que ofereçam cuidados voltados à saúde da mulher e programas como o “Luz na Amazônia” são essenciais para a prevenção do CCU em mulheres destas comunidades.The cervical cancer (CC) is the most frequent cancer in women of northern Brazil, excluding non-melanoma skin cancer. This region of the country has many communities with limited access to basic health services. CC’s prevention strategies in coastal communities are important so that the death rate from this disease is reduced. We describe the characteristics of women who conducted the Pap test through the university extension programs in riverside communities served by the program “Light in the Amazon” aimed at preventing sexually transmitted diseases and cancer of the cervix. In the communities visited between 2011 and 2014, a total of 154 women conducted preventive examination of cervical cancer. Of these, 24% had never performed the test previously and 10.39% had abnormal results. The local population lacks health services that provide care focused on women's health and programs like “Light in the Amazon” are essential for the prevention of cervical cancer in women of these communities.El cáncer cervical (CC) es el cáncer más frecuente em las mujeres em el norte de Brasil, excluyendo el cáncer de piel no melanoma. Esta región del país tiene muchas comunidades com acceso limitado a los servicios básicos de salud. Estrategias de prevención del CC em las comunidades costeras son importantes por lo que la tasa de mortalidad por esta enfermedad se reduce. Se describen las características de las mujeres que se sometieron a la prueba de Papanicolaou, através de los programas de extensión universitária em las comunidades costeras atendidas por el programa “Luz em la Amazonia” destinadas a la prevención de enfermedades de transmisión sexual y el câncer del cuello del útero. Em las comunidades visitadas entre los años 2011 y 2014, un total de 154 mujeres llevó a cabo el examen de prevención del cáncer de cuello uterino. De estos, 24% nunca se había realizado la prueba com anterioridad y 10,39% tienen resultados anormales. La población local carece de servicios de salud que brindan atención focalizada em la salud de la mujer y programas tales como “Luz em la Amazonia” son esenciales para la prevención del cáncer de cuello uterino em las mujeres de estas comunidades

Fusion of the Dhfr/Mtx and IR/MAR gene amplification methods produces a rapid and efficient method for stable recombinant protein production.

Amplification of the dihydrofolate reductase gene (Dhfr) by methotrexate (Mtx) exposure is commonly used for recombinant protein expression in Chinese hamster ovary (CHO) cells. However, this method is both time- and labor-intensive, and the high-producing cells that are generated are frequently unstable in culture. Another gene amplification method is based on using a plasmid bearing a mammalian replication initiation region (IR) and a matrix attachment region (MAR), which result in the spontaneous initiation of gene amplification in transfected cells. The IR/MAR and Dhfr/Mtx methods of gene amplification are based on entirely different principles. In this study, we combine these two methods to yield a novel method, termed the IR/MAR-Dhfr fusion method, which was used to express three proteins, the Fc receptor, GFP, and recombinant antibody. The fusion method resulted in a dramatic increase in expression of all three proteins in two CHO sub-lines, DXB-11, and DG44. The IR/MAR-Dhfr fusion amplified the genes rapidly and efficiently, and produced larger amounts of antibody than the Dhfr/Mtx or IR/MAR methods alone. While the amplified structure produced by the Dhfr/Mtx method was highly unstable, and the antibody production rate rapidly decreased with the culture time of the cells, the IR/MAR-Dhfr fusion method resulted in stable amplification and generated clonal cells that produced large amounts of antibody protein over a long period of time. In summary, the novel IR/MAR-Dhfr fusion method enables isolation of stable cells that produce larger amounts of a target recombinant protein more rapidly and easily than either the Dhfr/Mtx or IR/MAR methods alone

Efficient recombinant production in mammalian cells using a novel IR/MAR gene amplification method.

We previously found that plasmids bearing a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) efficiently initiate gene amplification and spontaneously increase their copy numbers in animal cells. In this study, this novel method was applied to the establishment of cells with high recombinant antibody production. The level of recombinant antibody expression was tightly correlated with the efficiency of plasmid amplification and the cytogenetic appearance of the amplified genes, and was strongly dependent on cell type. By using a widely used cell line for industrial protein production, CHO DG44, clones expressing very high levels of antibody were easily obtained. High-producer clones stably expressed the antibody over several months without eliciting changes in both the protein expression level and the cytogenetic appearance of the amplified genes. The integrity and reactivity of the protein produced by this method was fine. In serum-free suspension culture, the specific protein production rate in high-density cultures was 29.4 pg/cell/day. In conclusion, the IR/MAR gene amplification method is a novel and efficient platform for recombinant antibody production in mammalian cells, which rapidly and easily enables the establishment of stable high-producer cell clone

Production of antibody in suspension serum-free culture.

<p>The high producer cell clones (A, CN19-10/1A3; B, CN19-4/1F3) were adapted to suspension serum-free culture as described in the Materials and Methods section. The cell density and the antibody concentration in the medium are shown in the Graphs. The arrows indicate the time points at which the entire medium was changed.</p

Amplification and antibody expression by using plasmid set δ.

<p>Plasmids 1 (indicated) of plasmid set δ were co-transfected and selected by culture in the presence of 10 µg/ml blasticidin, the indicated concentrations of Mtx, and the absence of G418. The transfectant number (No.), Mtx concentration (nM), and the amplification method are indicated at the bottom of the figure. Transfectants CN61-3 and -1, and CN61-5 and -6, differ by whether the selection was started from 0 or 5 nM Mtx in the nucleotide-deficient medium. Cells reached confluence at the indicated number of days after transfection (Days a Trf). Cytogenetic structures were analyzed by FISH (A) (cw: chromowome width). Antibody expression was quantified by real-time PCR (B), and ELISA (C). Cells prepared for ELISA were grown in the presence (+) or absence (−) of 10 mM sodium butyrate (But). Error bars represent mean +/− S.D.</p

The integrity and the reactivity of the antibody produced by this method.

<p>SDS-PAGE of proteins produced by clone CN19-4/1F3. lane1, antibody purified from the clone CN19-4/1F3 culture; lane 2, commercial 9E10 c-MYC antibody; lane 3, immunoblot of the 50 kDa c-MYC fusion protein with the purified antibody from the CN19-4/1F3 culture (lane 3) or the commercial 9E10 c-MYC antibody (lane 4).</p

Plasmids used in this study.

<p>The schematic structures are illustrated in this Figure. The black arrow represents gene and its orientation. An IR from <i>DHFR</i> non-coding region and an AR1 MAR from <i>Ig κ</i> intron are illustrated as dashed bold line and an oval respectively in D, G and H.</p

<i>FcR</i> plasmids and expression of the <i>FcR</i> gene.

<p>CHO DXB-11 cells were transfected with the IR/MAR-positive pΔBM AR1<i>Dhfr</i>-FcR plasmid or the IR/MAR-negative pECE-FcR <i>Dhfr</i> plasmid [23_ENREF_23] by electroporation, and then cultured for 10 days in α-MEM(−). Clones were obtained by limiting dilution; the highest producer of FcR was determined by ELISA and then cultured in the presence of 25 nM Mtx for 15 days. Surviving cells were subjected to another round of limiting dilution and the highest producers were then cultured in the presence of 125 nM Mtx for 20 days. The same selection process was used for clones obtained using the conventional <i>Dhfr</i>/Mtx method; however, cells were selected and cloned in medium containing 5, 50 and 500 nM Mtx. After each round of selection, the level of FcR protein (µg/ml) was determined by ELISA.</p