Evaluación de la Escala EFD-66 como herramienta para predecir la adherencia al tratamiento en pacientes con enfermedades crónicas no transmisibles

Objetivo. Los objetivos del presente estudio son validar la construcción de la escala de fases de duelo (EFD-66) como instrumento para medir las etapas de duelo (ED) y evaluar su utilidad para discriminar entre pacientes con enfermedad crónica no transmisible (ECNT) que cumplen con adherencia al tratamiento farmacológico (ATF) en la consulta médica ambulatoria. Métodos. Se realizó un estudio transversal para determinar la asociación entre las ED y la ATF, en los meses de abril a octubre de 2015. La recogida de datos se realizó mediante un diseño prolectivo. Se aplicaron tres instrumentos: una cédula sociodemográfica, la escala EFD-66 y el cuestionario de Morisky-Green. Se reclutaron pacientes con antecedente de ECNT procedentes de la Clínica de Medicina Familiar “Gustavo A. Madero”, en la Ciudad de México. Los datos se analizaron con las pruebas estadísticas apropiadas. Resultados. Se incluyó un total de 165 pacientes. Se observó que altos puntajes en las subescalas de negación (razón de momios [RM]: 1,124; intervalo de confianza de 95% [IC95%]: 1,066-1,186; P < 0,001), de ira (RM: 1,157; IC95%: 1,080-1,240; P < 0,001) y de depresión (RM: 1,071; IC95%: 1,029-1,116; P = 0,001) se asocian con falta de ATF; sin embargo, un puntaje elevado en la subescala de aceptación (RM: 0,913; IC95%: 0,880-0,948; P < 0,001) se asocia con ATF. La mayor sensibilidad entre las subescalas se observó en las etapas de negación e ira (área bajo la curva [ABC]: 0,597 en ambas). Conclusiones. La EFD-66 es un instrumento que presenta una buena validez de construcción como instrumento para medir las ED y permite detectar pacientes con ECNT que cumplen con ATF, por lo que recomendamos su aplicación en la consulta médica ambulatoria. Además, nuestros hallazgos indican que el duelo es un factor de riesgo que incrementa la falta de ATF.

Objective. The objectives of this study are to validate the construct of the stages of grief scale (FD-66) as an instrument for measuring the stages of grief and to evaluate its usefulness in discriminating among patients with chronic non-communicable diseases in terms of adherence to the pharmacological treatment prescribed. Methods. A cross-sectional study was conducted from April to October 2015 to determine the association between the stages of grief and treatment adherence. Data were collected using a prolective design. Three instruments were applied: a sociodemographic document, the FD-66 scale, and the Morisky-Green questionnaire. Patients with a history of CNCDs were recruited from the Gustavo A. Madero Family Medicine Clinic in Mexico City. The data were analyzed using the appropriate statistical tests. Results. A total of 165 patients were included. It was observed that high scores on the subscales of denial (odds ratio [OR]: 1.124; confidence interval of 95% [CI95%]: 1.066-1.186; P < 0,001); anger (OR: 1.157; CI95%: 1.080-1.240; P < 0.001), and depression (OR: 1.071; CI95%: 1.029-1.116; P = 0.001) are associated with poor treatment adherence; however, a high score on the acceptance subscale (OR: 0.913; CI95%: 0.880-0.948; P < 0.001) is associated with good treatment adherence. The greatest sensitivity among the subscales was observed in the denial and anger stages (area under the [ABC] curve: 0.597 in both). Conclusions. The FD-66 is an instrument with good construct validity as a tool for measuring the stages of grief and makes it possible to identify patients with CNCD that will adhere to treatment. We therefore recommend its use in outpatient medical consultations. Furthermore, our findings indicate that grief is a risk factor that increases poor treatment adherence.

Objetivo. Validar a construção da Escala facial de dor (EFD-66) como instrumento para medir as fases da dor e avaliar sua utilidade para discriminar, na consulta médica ambulatorial, os pacientes com doenças crônicas não transmissíveis (DCNT) que aderem ao tratamento medicamentoso. Métodos. Estudo transversal para determinar a associação entre as fases da dor e o tratamento medicamentoso conduzido de abril a outubro de 2015. A coleta de dados foi realizada prospectivamente. Foram aplicados três instrumentos: uma ficha sociodemográfica, a escala EFD-66 e o teste de Morisky-Green. Foram recrutados pacientes com história de DCNT provenientes da Clínica de Medicina da Família “Gustavo A. Madero”, na Cidade do México. Os dados foram analisados com os testes estatísticos apropriados. Resultados. Foi estudada uma amostra de 165 pacientes. Observou-se que uma pontuação alta nas subescalas de negação (odds ratio [OR] 1,124; intervalo de confiança de 95% [IC95%] 1,066–1,186; P < 0,001), raiva (OR 1,157; IC95% 1,080–1,240; P < 0,001) e depressão (OR 1,071; IC95% 1,029–1,116; P = 0,001) está associada à não adesão ao tratamento medicamentoso. No entanto, uma pontuação alta na subescala de aceitação (OR 0,913; IC95% 0,880–0,948; P < 0,001) está associada à adesão ao tratamento medicamentoso. As subescalas com maior sensibilidade foram as fases de negação e raiva (área sob a curva [ASC]: 0,597 em ambas). Conclusões. A escala EFD-66 tem boa validade de construção como instrumento para medir as fases da dor e permite discriminar os pacientes com DCNT que aderem ao tratamento medicamentosos. Assim, recomendamos que esta escala seja aplicada em consultas médicas ambulatoriais. Além disso, nossos achados indicam que a dor é um fator de risco que contribui para a não adesão ao tratamento medicamentoso.

[Evaluation of the FD-66 Scale as a tool for predicting treatment adherence in patients with chronic non-communicable diseasesAvaliação da escala EFD-66 como instrumento para predizer a adesão ao tratamento em pacientes com doenças crônicas não transmissíveis]. / Evaluación de la Escala EFD-66 como herramienta para predecir la adherencia al tratamiento en pacientes con enfermedades crónicas no transmisibles.

OBJECTIVE: The objectives of this study are to validate the construct of the stages of grief scale (FD-66) as an instrument for measuring the stages of grief and to evaluate its usefulness in discriminating among patients with chronic non-communicable diseases in terms of adherence to the pharmacological treatment prescribed. METHODS: A cross-sectional study was conducted from April to October 2015 to determine the association between the stages of grief and treatment adherence. Data were collected using a prolective design. Three instruments were applied: a sociodemographic document, the FD-66 scale, and the Morisky-Green questionnaire. Patients with a history of CNCDs were recruited from the Gustavo A. Madero Family Medicine Clinic in Mexico City. The data were analyzed using the appropriate statistical tests. RESULTS: A total of 165 patients were included. It was observed that high scores on the subscales of denial (odds ratio [OR]: 1.124; confidence interval of 95% [CI95%]: 1.066-1.186; P < 0,001); anger (OR: 1.157; CI95%: 1.080-1.240; P < 0.001), and depression (OR: 1.071; CI95%: 1.029-1.116; P = 0.001) are associated with poor treatment adherence; however, a high score on the acceptance subscale (OR: 0.913; CI95%: 0.880-0.948; P < 0.001) is associated with good treatment adherence. The greatest sensitivity among the subscales was observed in the denial and anger stages (area under the [ABC] curve: 0.597 in both). CONCLUSIONS: The FD-66 is an instrument with good construct validity as a tool for measuring the stages of grief and makes it possible to identify patients with CNCD that will adhere to treatment. We therefore recommend its use in outpatient medical consultations. Furthermore, our findings indicate that grief is a risk factor that increases poor treatment adherence.

OBJETIVO: Validar a construção da Escala facial de dor (EFD-66) como instrumento para medir as fases da dor e avaliar sua utilidade para discriminar, na consulta médica ambulatorial, os pacientes com doenças crônicas não transmissíveis (DCNT) que aderem ao tratamento medicamentoso. MÉTODOS: Estudo transversal para determinar a associação entre as fases da dor e o tratamento medicamentoso conduzido de abril a outubro de 2015. A coleta de dados foi realizada prospectivamente. Foram aplicados três instrumentos: uma ficha sociodemográfica, a escala EFD-66 e o teste de Morisky-Green. Foram recrutados pacientes com história de DCNT provenientes da Clínica de Medicina da Família "Gustavo A. Madero", na Cidade do México. Os dados foram analisados com os testes estatísticos apropriados. RESULTADOS: Foi estudada uma amostra de 165 pacientes. Observou-se que uma pontuação alta nas subescalas de negação (odds ratio [OR] 1,124; intervalo de confiança de 95% [IC95%] 1,066­1,186; P < 0,001), raiva (OR 1,157; IC95% 1,080­1,240; P < 0,001) e depressão (OR 1,071; IC95% 1,029­1,116; P = 0,001) está associada à não adesão ao tratamento medicamentoso. No entanto, uma pontuação alta na subescala de aceitação (OR 0,913; IC95% 0,880­0,948; P < 0,001) está associada à adesão ao tratamento medicamentoso. As subescalas com maior sensibilidade foram as fases de negação e raiva (área sob a curva [ASC]: 0,597 em ambas). CONCLUSÕES: A escala EFD-66 tem boa validade de construção como instrumento para medir as fases da dor e permite discriminar os pacientes com DCNT que aderem ao tratamento medicamentosos. Assim, recomendamos que esta escala seja aplicada em consultas médicas ambulatoriais. Além disso, nossos achados indicam que a dor é um fator de risco que contribui para a não adesão ao tratamento medicamentoso.

Evaluación de la Escala EFD-66 como herramienta para predecir la adherencia al tratamiento en pacientes con enfermedades crónicas no transmisibles / Evaluation of the FD-66 Scale as a tool for predicting treatment adherence in patients with chronic non-communicable diseases / Avaliação da escala EFD-66 como instrumento para predizer a adesão ao tratamento em pacientes com doenças crônicas não transmissíveis

RESUMEN Objetivo Los objetivos del presente estudio son validar la construcción de la escala de fases de duelo (EFD-66) como instrumento para medir las etapas de duelo (ED) y evaluar su utilidad para discriminar entre pacientes con enfermedad crónica no transmisible (ECNT) que cumplen con adherencia al tratamiento farmacológico (ATF) en la consulta médica ambulatoria. Métodos Se realizó un estudio transversal para determinar la asociación entre las ED y la ATF, en los meses de abril a octubre de 2015. La recogida de datos se realizó mediante un diseño prolectivo. Se aplicaron tres instrumentos: una cédula sociodemográfica, la escala EFD-66 y el cuestionario de Morisky-Green. Se reclutaron pacientes con antecedente de ECNT procedentes de la Clínica de Medicina Familiar "Gustavo A. Madero", en la Ciudad de México. Los datos se analizaron con las pruebas estadísticas apropiadas. Resultados Se incluyó un total de 165 pacientes. Se observó que altos puntajes en las subescalas de negación (razón de momios [RM]: 1,124; intervalo de confianza de 95% [IC95%]: 1,066-1,186; P < 0,001), de ira (RM: 1,157; IC95%: 1,080-1,240; P < 0,001) y de depresión (RM: 1,071; IC95%: 1,029-1,116; P = 0,001) se asocian con falta de ATF; sin embargo, un puntaje elevado en la subescala de aceptación (RM: 0,913; IC95%: 0,880-0,948; P < 0,001) se asocia con ATF. La mayor sensibilidad entre las subescalas se observó en las etapas de negación e ira (área bajo la curva [ABC]: 0,597 en ambas). Conclusiones La EFD-66 es un instrumento que presenta una buena validez de construcción como instrumento para medir las ED y permite detectar pacientes con ECNT que cumplen con ATF, por lo que recomendamos su aplicación en la consulta médica ambulatoria. Además, nuestros hallazgos indican que el duelo es un factor de riesgo que incrementa la falta de ATF.

ABSTRACT Objective The objectives of this study are to validate the construct of the stages of grief scale (FD-66) as an instrument for measuring the stages of grief and to evaluate its usefulness in discriminating among patients with chronic non-communicable diseases in terms of adherence to the pharmacological treatment prescribed. Methods A cross-sectional study was conducted from April to October 2015 to determine the association between the stages of grief and treatment adherence. Data were collected using a prolective design. Three instruments were applied: a sociodemographic document, the FD-66 scale, and the Morisky-Green questionnaire. Patients with a history of CNCDs were recruited from the Gustavo A. Madero Family Medicine Clinic in Mexico City. The data were analyzed using the appropriate statistical tests. Results A total of 165 patients were included. It was observed that high scores on the subscales of denial (odds ratio [OR]: 1.124; confidence interval of 95% [CI95%]: 1.066-1.186; P < 0,001); anger (OR: 1.157; CI95%: 1.080-1.240; P < 0.001), and depression (OR: 1.071; CI95%: 1.029-1.116; P = 0.001) are associated with poor treatment adherence; however, a high score on the acceptance subscale (OR: 0.913; CI95%: 0.880-0.948; P < 0.001) is associated with good treatment adherence. The greatest sensitivity among the subscales was observed in the denial and anger stages (area under the [ABC] curve: 0.597 in both). Conclusions The FD-66 is an instrument with good construct validity as a tool for measuring the stages of grief and makes it possible to identify patients with CNCD that will adhere to treatment. We therefore recommend its use in outpatient medical consultations. Furthermore, our findings indicate that grief is a risk factor that increases poor treatment adherence.

RESUMO Objetivo Validar a construção da Escala facial de dor (EFD-66) como instrumento para medir as fases da dor e avaliar sua utilidade para discriminar, na consulta médica ambulatorial, os pacientes com doenças crônicas não transmissíveis (DCNT) que aderem ao tratamento medicamentoso. Métodos Estudo transversal para determinar a associação entre as fases da dor e o tratamento medicamentoso conduzido de abril a outubro de 2015. A coleta de dados foi realizada prospectivamente. Foram aplicados três instrumentos: uma ficha sociodemográfica, a escala EFD-66 e o teste de Morisky-Green. Foram recrutados pacientes com história de DCNT provenientes da Clínica de Medicina da Família "Gustavo A. Madero", na Cidade do México. Os dados foram analisados com os testes estatísticos apropriados. Resultados Foi estudada uma amostra de 165 pacientes. Observou-se que uma pontuação alta nas subescalas de negação (odds ratio [OR] 1,124; intervalo de confiança de 95% [IC95%] 1,066-1,186; P < 0,001), raiva (OR 1,157; IC95% 1,080-1,240; P < 0,001) e depressão (OR 1,071; IC95% 1,029-1,116; P = 0,001) está associada à não adesão ao tratamento medicamentoso. No entanto, uma pontuação alta na subescala de aceitação (OR 0,913; IC95% 0,880-0,948; P < 0,001) está associada à adesão ao tratamento medicamentoso. As subescalas com maior sensibilidade foram as fases de negação e raiva (área sob a curva [ASC]: 0,597 em ambas). Conclusões A escala EFD-66 tem boa validade de construção como instrumento para medir as fases da dor e permite discriminar os pacientes com DCNT que aderem ao tratamento medicamentosos. Assim, recomendamos que esta escala seja aplicada em consultas médicas ambulatoriais. Além disso, nossos achados indicam que a dor é um fator de risco que contribui para a não adesão ao tratamento medicamentoso.

Bcl-3 regulates UVB-induced apoptosis.

B cell leukemia-3 (Bcl-3) has been defined as an anti-apoptotic gene; however, the exact mechanisms through which Bcl-3 influences apoptosis have been elusive. To determine the specific role of Bcl-3 in apoptosis, we evaluated the effect of its silencing on the expression of proteins involved in either the extrinsic or intrinsic apoptotic pathways induced by ultraviolet light B-mediated DNA damage. We found that, in Bcl-3-silenced cells, caspase-3, caspase-8 and caspase-9 activation is accelerated and tBid mitochondrial content is increased. It is important to note that, although mitochondrial Smac levels were reduced after UV exposure, the rate of reduction was slightly higher in Bcl-3 silenced cells than in control cells. Additionally, p53 levels diminished in Bcl-3 silenced cells compared to control cells, as did those of DNA-PK, a DNA repair protein. Altogether, our data indicate that Bcl-3 protects cells from apoptosis by regulating both apoptotic pathways.

Multimolecular signaling complexes enable Syk-mediated signaling of CD36 internalization.

CD36 is a versatile receptor known to play a central role in the development of atherosclerosis, the pathogenesis of malaria, and the removal of apoptotic cells. Remarkably, the short cytosolically exposed regions of CD36 lack identifiable motifs, which has hampered elucidation of its mode of signaling. Using a combination of phosphoprotein isolation, mass spectrometry, superresolution imaging, and gene silencing, we have determined that the receptor induces ligand internalization through a heteromeric complex consisting of CD36, ß1 and/or ß2 integrins, and the tetraspanins CD9 and/or CD81. This receptor complex serves to link CD36 to the adaptor FcRγ, which bears an immunoreceptor tyrosine activation motif. By coupling to FcRγ, CD36 is able to engage Src-family kinases and Syk, which in turn drives the internalization of CD36 and its bound ligands.

Class I and class III phosphoinositide 3-kinases are required for actin polymerization that propels phagosomes.

Actin polymerization drives the extension of pseudopods that trap and engulf phagocytic targets. The polymerized actin subsequently dissociates as the phagocytic vacuole seals and detaches from the plasma membrane. We found that phagosomes formed by engagement of integrins that serve as complement receptors (CR3) undergo secondary waves of actin polymerization, leading to the formation of "comet tails" that propel the vacuoles inside the cells. Actin tail formation was accompanied by and required de novo formation of PI(3,4)P(2) and PI(3,4,5)P(3) on the phagosomal membrane by class I phosphoinositide 3-kinases (PI3Ks). Although the phosphatidylinositide phosphatase Inpp5B was recruited to nascent phagosomes, it rapidly detached from the membrane after phagosomes sealed. Detachment of Inpp5B required the formation of PI(3)P. Thus, class III PI3K activity was also required for the accumulation of PI(4,5)P(2) and PI(3,4,5)P(3) and for actin tail formation. These experiments reveal a new PI(3)P-sensitive pathway leading to PI(3,4)P(2) and PI(3,4,5)P(3) formation and signaling in endomembranes.

Cathepsin D is the primary protease for the generation of adenohypophyseal vasoinhibins: cleavage occurs within the prolactin secretory granules.

Vasoinhibins are a family of N-terminal prolactin (PRL) fragments that inhibit blood vessel growth, dilation, permeability, and survival. The aspartyl endoprotease cathepsin D is active at acidic pH and can cleave rat PRL to generate vasoinhibins. We investigated whether and where vasoinhibins could be generated by cathepsin D in the adenohypophysis of rats and mice and whether their production could be gender dependent. Vasoinhibins were detected in primary cultures of rat adenohypophyseal cells by Western blot with antibodies directed against the N terminus of PRL but not the C terminus. Ovariectomized, estrogen-treated females show greater levels of adenohypophyseal vasoinhibins than males. Peptide sequencing analysis revealed that the cleaved form of PRL in rat adenohypophyseal extracts contains the PRL N terminus and a second N terminus starting at Ser(149), the reported cleavage site of cathepsin D in rat PRL. In addition, cathepsin D inhibition by pepstatin A reduced vasoinhibin levels in rat adenohypophyseal cell cultures. Confocal and electron microscopy showed the colocalization of cathepsin D and PRL within rat adenohypophyseal cells and secretory granules, and a subcellular fraction of rat adenohypophysis enriched in secretory granules contained cathepsin D activity able to generate vasoinhibins from PRL. Of note, vasoinhibins were absent in the adenohypophysis of mice lacking the cathepsin D gene but not in wild-type mice. These findings show that cathepsin D is the main protease responsible for the generation of adenohypophyseal vasoinhibins and that its action can take place within the secretory granules of lactotrophs.

CD36 and TLR interactions in inflammation and phagocytosis: implications for malaria.

CD36 participates in macrophage internalization of a variety of particles, and has been implicated in inflammatory responses to many of these ligands. To what extent CD36 cooperates with other receptors in mediating these processes remains unclear. Because CD36 has been shown to cooperate with TLR2, we investigated the roles and interactions of CD36 and TLRs in inflammation and phagocytosis. Using Ab-induced endocytosis of CD36 and phagocytosis of erythrocytes displaying Abs to CD36, we show that selective engagement and internalization of this receptor did not lead to proinflammatory cytokine production by primary human and murine macrophages. In addition, CD36-mediated phagocytosis of Plasmodium falciparum malaria-parasitized erythrocytes (PEs), which contain parasite components that activate TLRs, also failed to induce cytokine secretion from primary macrophages. Furthermore, we demonstrate that CD36-mediated internalization did not require TLR2 or the TLR-signaling molecule IRAK4. However, macrophage pretreatment with TLR agonists markedly stimulated particle uptake via CD36. Similarly, PE uptake was unaffected by TLR deficiency, but in wild-type cells was increased by pretreatment with purified P. falciparum glycosylphosphatidylinositols, which activate TLR2. Our findings indicate that CD36 must cooperate with other receptors such as TLRs to participate in cytokine responses. Although purified P. falciparum components activate TLRs, CD36-mediated internalization of intact PEs is not inflammatory. Further, CD36 mediates internalization of particles, including PEs, independently of TLR signaling, but can functionally cooperate with TLRs to enhance internalization.

Antimicrobial mechanisms of phagocytes and bacterial evasion strategies.

Professional phagocytes have a vast and sophisticated arsenal of microbicidal features. They are capable of ingesting and destroying invading organisms, and can present microbial antigens on their surface, eliciting acquired immune responses. To survive this hostile response, certain bacterial species have developed evasive strategies that often involve the secretion of effectors to co-opt the cellular machinery of the host. In this Review, we present an overview of the antimicrobial defences of the host cell, with emphasis on macrophages, for which phagocytosis has been studied most extensively. In addition, using Mycobacterium tuberculosis, Listeria monocytogenes, Legionella pneumophila and Coxiella burnetii as examples, we describe some of the evasive strategies used by bacteria.

Analysis of phosphoinositide dynamics during phagocytosis using genetically encoded fluorescent biosensors.

Phosphoinositide signaling is essential for successful phagocytosis. Phosphoinositides regulate processes such as actin assembly and the recruitment of molecular motors required for ingestion, as well as fusion events required for the maturation of the phagosome. Phosphoinositides not only serve as substrates for the generation of second messengers, but also function to anchor to the membrane cytosolic proteins that contain phosphoinositide-binding motifs. Conventional methods for the detection of phosphoinositides involve their extraction from the cells and separation by chromatographic procedures. These approaches are laborious and expensive and fail to provide spatio-temporal information, which is critical when analyzing localized and transient phenomena like phagocytosis. In this chapter we describe a method to monitor phosphoinositides dynamically by transfection of fluorescently tagged probes (biosensors) into cultured macrophages. These biosensors are based on the fusion of phosphoinositide-binding protein domains with fluorescent proteins. Some specifications for live cell imaging of such phosphoinositide-specific probes are also provided.

Role of CrkII in Fcgamma receptor-mediated phagocytosis.

Phagocytosis of IgG-opsonized pathogens by Fcgamma receptors requires extensive remodeling of the actin cytoskeleton, a process regulated by the small GTPase Rac. Vav was thought to be the guanine nucleotide exchange factor responsible for the activation of Rac, but recent evidence indicates that Fcgamma receptor-mediated phagocytosis is unaffected in macrophages lacking all three isoforms of Vav. We therefore tested whether another GEF, DOCK180, participates in Fcgamma receptor-initiated phagocytosis. DOCK180 associates with the adaptor protein Crk, which mediates recruitment of the GEF to sites of tyrosine phosphorylation. CrkII and DOCK180 were found to accumulate at the phagocytic cup. Knockdown of Crk or DOCK180 in murine macrophages using small interfering RNA inhibited phagocytosis of IgG-opsonized particles. Moreover, transfection of dominant negative CrkII prevented both recruitment of DOCK180 and the activation of Rac at the phagocytic cup. This is the first report of a role for either Crk or DOCK180 in Fcgamma receptor-mediated phagocytosis. The Crk-DOCK180 complex is involved in the clearance of apoptotic cells, which unlike the ingestion of IgG-opsonized particles, is an anti-inflammatory process. The finding that CrkII-DOCK180 is also responsible, at least in part, for the effects of Fcgamma receptors implies that additional, parallel pathways must account for the associated pro-inflammatory effect.

Bacterial expression, purification and functional characterization of a recombinant chimeric Fab derived from murine mAb BCF2 that neutralizes the venom of the scorpion Centruroides noxius hoffmann.

The murine monoclonal antibody BCF2 is able to neutralize the venom of the scorpion Centruroides noxius Hoffmann. A chimeric Fab of BCF2 (chFab-BCF2) comprising the variable regions of murine BCF2 and human constant regions was assembled. chFab-BCF2 was expressed as a soluble and functional protein in the periplasmic space of Escherichia coli. An expression yield of 1 mg/l was reached by combination of late-log-phase induction, rich culture medium, low expression temperature and addition of sucrose (0.3 M) to the culture medium. The addition of sucrose induced secretion of 60% of the protein into the medium. After expression for 23 h, a novel process was used to release the remaining periplasmic protein in situ consisting in the addition of lysozyme and sucrose up to 0.6 M (20%) directly to the culture medium. chFab-BCF2 was recovered by ammonium sulfate precipitation and purified in a single step by affinity chromatography using anti-human anti-F(ab')(2) IgG coupled to Sepharose-proteinG. Pure chFab-BCF2 maintained a similar nanomolar affinity as BCF2 to its cognate antigen, the Na(+)-channel-affecting toxin Cn2. Recombinant chFab-BCF2 was able to neutralize Cn2 in vivo even at a molar ratio of 1:1, as well as the whole venom of C. noxius. Thus, it is a promising candidate to be used as a specific and efficient recombinant antidote against scorpion stings.

Hypoxia inhibits expression of prolactin and secretion of cathepsin-D by the GH4C1 pituitary adenoma cell line.

Diminished oxygen concentration within growing tumors may stimulate neovascularization by inducing both up-regulation of angiogenic factors and down-regulation of antiangiogenic agents. A potentially important molecule in the growth of pituitary adenomas is prolactin (PRL), which can be cleaved by cathepsin-D to yield a 16-kDa form (16K-PRL) with potent antiangiogenic effects. We examined the expression of PRL in cultured GH4C1 pituitary adenoma cells after exposure to hypoxia (0.1% oxygen) for periods of 12 to 36 hours. In contrast to increased expression of the angiogenic factor vascular endothelial growth factor in hypoxic cells, PRL mRNA and levels of intracellular and secreted PRL were significantly reduced under hypoxia. The reduction was not attributable to a general suppression of either transcription or protein synthesis. Although 16K-PRL was not evident in conditioned medium at physiologic pH, lowering the pH to mimic the acidic tumor microenvironment resulted in generation of 16K-PRL, which was sharply reduced in medium drawn from hypoxic cells. Production of 16K-PRL was blocked by the cathepsin-D inhibitor pepstatin-A, and the reduced 16K-PRL formation in hypoxic-conditioned medium correlated with a decrease in secretion of cathepsin-D and its precursor, procathepsin-D. Thus, hypoxia acts upon GH4C1 cells to increase vascular endothelial growth factor expression, decrease PRL synthesis, and suppress conversion of PRL to 16K-PRL via inhibition of cathepsin-D proteolysis. These mechanisms may act in concert to stimulate angiogenesis in prolactinomas.