Surviving crack: a qualitative study of the strategies and tactics developed by Brazilian users to deal with the risks associated with the drug.

BACKGROUND: Due to marginalization, trafficking violence, conflicts with the police and organic and social psychological problems associated with the drug, crack is one of the most devastating drugs currently in use. However, there is evidence that some users manage to stay alive and active while using crack cocaine for many years, despite the numerous adversities and risks involved with this behavior. In this context, the aim of the present study was to identify the strategies and tactics developed by crack users to deal with the risks associated with the culture of use by examining the survival strategies employed by long-term users. METHOD: A qualitative research method was used involving semi-structured, in-depth interviews. Twenty-eight crack users fulfilling a pre-defined enrollment criterion were interviewed. This criterion was defined as the long-term use of crack (i.e., at least four years). The sample was selected using information provided by key informants and distributed across eight different supply chains. The interviews were literally transcribed and analyzed via content analysis techniques using NVivo-8 software. RESULTS: There was diversity in the sample with regard to economic and education levels. The average duration of crack use was 11.5 years. Respondents believed that the greatest risks of crack dependence were related to the drug's psychological effects (e.g., cravings and transient paranoid symptoms) and those arising from its illegality (e.g., clashes with the police and trafficking). Protection strategies focused on the control of the psychological effects, primarily through the consumption of alcohol and marijuana. To address the illegality of the drug, strategies were developed to deal with dealers and the police; these strategies were considered crucial for survival. CONCLUSIONS: The strategies developed by the respondents focused on trying to protect themselves. They proved generally effective, though they involved risks of triggering additional problems (e.g., other dependencies) in the long term.

Prostaglandin F2-alpha receptor (FPr) expression on porcine corpus luteum microvascular endothelial cells (pCL-MVECs).

BACKGROUND: The corpus luteum (CL) is a transient endocrine gland and prostaglandin F2-alpha is considered to be the principal luteolysin in pigs. In this species, the in vivo administration of prostaglandin F2-alpha induces apoptosis in large vessels as early as 6 hours after administration. The presence of the prostaglandin F2-alpha receptor (FPr) on the microvascular endothelial cells (pCL-MVECs) of the porcine corpus luteum has not yet been defined. The aim of the study was to assess FPr expression in pCL-MVECs in the early and mid-luteal phases (EL-p, ML-p), and during pregnancy (P-p). Moreover, the effectiveness of prostaglandin F2-alpha treatment in inducing pCL-MVEC apoptosis was tested. METHODS: Porcine CLs were collected in the EL and ML phases and during P-p. All CLs from each animal were minced together and the homogenates underwent enzymatic digestion. The pCL-MVECs were then positively selected by an immunomagnetic separation protocol using Dynabeads coated with anti-CD31 monoclonal antibody and seeded in flasks in the presence of EGM 2-MV (Microvascular Endothelial Cell Medium-2). After 4 days of culture, the cells underwent additional immunomagnetic selection and were seeded in flasks until the confluent stage.PCR Real time, western blot and immunodetection assays were utilized to assess the presence of FPr on pCL-MVEC primary cultures. Furthermore, the influence of culture time (freshly isolated, cultured overnight and at confluence) and hormonal treatment (P4 and E2) on FPr expression in pCL-MVECs was also investigated. Apoptosis was detected by TUNEL assay of pCL-MVECs exposed to prostaglandin F2-alpha. RESULTS: We obtained primary cultures of pCL-MVECs from all animals. FPr mRNA and protein levels showed the highest value (ANOVA) in CL-MVECs derived from the early-luteal phase. Moreover, freshly isolated MVECs showed a higher FPr mRNA value than those cultured overnight and confluent cells (ANOVA). prostaglandin F2-alpha treatment failed to induce an apoptotic response in all the pCL-MVEC cultures. CONCLUSION: Our data showing the presence of FPr on MVECs and the inability of prostaglandin F2-alpha to evoke an in vitro apoptotic response suggest that other molecules or mechanisms must be considered in order to explain the in vivo direct pro-apoptotic effect of prostaglandin F2-alpha at the endothelial level.

Heterologous expression of Brucella abortus GroEL heat-shock protein in Lactococcus lactis.

BACKGROUND: Brucella abortus is a facultative intracellular pathogen that mainly infects cattle and humans. Current vaccines rely on live attenuated strains of B. abortus, which can revert to their pathogenic status and thus are not totally safe for use in humans. Therefore, the development of mucosal live vaccines using the food-grade lactic acid bacterium, Lactococcus lactis, as an antigen delivery vector, is an attractive alternative and a safer vaccination strategy against B. abortus. Here, we report the construction of L. lactis strains genetically modified to produce B. abortus GroEL heat-shock protein, a candidate antigen, in two cellular locations, intracellular or secreted. RESULTS: Only the secreted form of GroEL was stably produced in L. lactis, suggesting a detrimental effect of GroEL protein when intracellularly produced in this bacterium. Only trace amounts of mature GroEL were detected in the supernatant fraction of induced lactococcal cultures, and the GroEL precursor remained stacked in the cell fraction. Attempts to raise the secretion yields were made, but even when GroEL was fused to a synthetic propeptide, secretion of this antigen was not improved. CONCLUSION: We found that L. lactis is able to produce, and to secrete, a stable form of GroEL into the extracellular medium. Despite the low secretion efficiency of GroEL, which suggest that this antigen interacts with the cell envelope of L. lactis, secretion seems to be the best way to achieve both production and protein yields, regardless of cellular location. The L. lactis strain secreting GroEL has potential for in vivo immunization.

Expression of endothelin-1 system in a pig model of endotoxic shock.

Endothelin (ET)-1 is a potent vasoconstrictive peptide and it is involved in the pathogenesis of septic shock. Blockade of ET-1 receptors abolishes the LPS-induced pulmonary hypertension and worsens the LPS-dependent systemic hypotension, but the role of ET-1 in sepsis remains uncertain. To determine the role of ET-1 in cardiovascular and respiratory derangement in a porcine model of endotoxemic shock we evaluated ET-1 plasma levels and ET-1 mRNA and protein levels in lung, liver, and heart as well as Endothelin Converting Enzyme-1, ET(A) and ET(B) receptors mRNA in the same tissues. Twelve piglets were randomised to sham operated or to LPS-treated (40 microg/kg/h for 4 h) groups. During the experiment, respiratory and circulatory parameters have been recorded and blood samples collected. At the end of the experiment the animals were sacrificed and tissue samples collected for real-time quantitative PCR and ELISA test. LPS infusion evokes a large increase in ET-1 plasma concentration, and in tissues mRNA levels, associated with an increase in pulmonary arterial pressure, as well as in pulmonary and systemic vascular resistances, and a decrease in stroke volume. LPS infusion caused also a derangement of respiratory mechanics, evidenced by an increase in resistance and a decrease in compliance of the respiratory system. ET(A) and ET(B) receptor mRNA levels were markedly decreased in liver and lung and slightly increased in heart, evidencing that ET receptor subtypes were differentially regulated in the major organs of endotoxin treated pigs. In conclusion our data show the presence of a continuative and differentially regulated stimulating mechanism of ET-1 expression during pig endotoxaemia as well as a fundamental role of ET-1 system in the cardiovascular and respiratory derangement.

Protein secretion in Lactococcus lactis : an efficient way to increase the overall heterologous protein production.

Lactococcus lactis, the model lactic acid bacterium (LAB), is a food grade and well-characterized Gram positive bacterium. It is a good candidate for heterologous protein delivery in foodstuff or in the digestive tract. L. lactis can also be used as a protein producer in fermentor. Many heterologous proteins have already been produced in L. lactis but only few reports allow comparing production yields for a given protein either produced intracellularly or secreted in the medium. Here, we review several works evaluating the influence of the localization on the production yields of several heterologous proteins produced in L. lactis. The questions of size limits, conformation, and proteolysis are addressed and discussed with regard to protein yields. These data show that i) secretion is preferable to cytoplasmic production; ii) secretion enhancement (by signal peptide and propeptide optimization) results in increased production yield; iii) protein conformation rather than protein size can impair secretion and thus alter production yields; and iv) fusion of a stable protein can stabilize labile proteins. The role of intracellular proteolysis on heterologous cytoplasmic proteins and precursors is discussed. The new challenges now are the development of food grade systems and the identification and optimization of host factors affecting heterologous protein production not only in L. lactis, but also in other LAB species.

Fasting influences steroidogenesis, vascular endothelial growth factor (VEGF) levels and mRNAs expression for VEGF, VEGF receptor type 2 (VEGFR-2), endothelin-1 (ET-1), endothelin receptor type A (ET-A) and endothelin converting enzyme-1 (ECE-1) in newly formed pig corpora lutea.

This study was designed to verify whether fasting influences vascular endothelial growth factor (VEGF) production and VEGF, VEGF receptor-2 (VEGFR-2) as well as endothelin (ET) system members (endothelin converting enzyme-1, ECE-1; ET-1; endothelin receptor type A, ET-A) mRNA expression in pig corpora lutea; furthermore, we wanted to assess whether fasting affects steroidogenesis in luteal cells. Eight prepubertal gilts were induced to ovulate and were randomly assigned to two groups: (A) n = 4, normally fed; and (B) n = 4, fasted for 72 h starting 3 days after ovulation. At the end of fasting, ovaries were removed from all the animals and corpora lutea (CLs) were collected. VEGF and steroid levels in luteal tissue were determined by ELISA and RIA, respectively; VEGF, VEGFR-2, ET-1, ET-A and ECE-1 mRNAs expression was measured by real-time PCR. VEGF protein levels were similar in the two groups, while all steroid (progesterone, testosterone, estradiol 17beta) concentrations were significantly (P < 0.001) higher in CLs collected from fasted animals compared with those from normally fed gilts. VEGF, VEGFR-2, ET-1 and ECE-1 (but not ET-A) mRNA expression was significantly lower (P < 0.05) in fasted versus normally fed animals. The overall conclusion is that all the parameters studied are affected by feed restriction, but the mechanisms activated at luteal level are possibly not fully adequate to compensate for nutrient shortage.

Induction of partial protection in mice after oral administration of Lactococcus lactis producing Brucella abortus L7/L12 antigen.

The Brucella abortus ribosomal protein L7/L12 is an immunodominant antigen and an interesting candidate for the development of oral live vaccines against brucellosis. Here, a recombinant Lactococcus lactis strain producing L7/L12 under the control of nisin inducible promoter was orally administered to BALB/c mice. Significant levels of anti-L7/L12 specific IgA detected in feces revealed an induced local humoral immune response. However, serum analysis did not reveal any anti-L7/L12 antibodies suggesting the absence of a systemic response. Nevertheless, the vaccinated mice showed a partial protective immunity against B. abortus virulent strain (S2308) challenged by intraperitoneal inoculation.

Heterologous protein production and delivery systems for Lactococcus lactis.

Lactic acid bacteria (LAB), widely used in the food industry, are present in the intestine of most animals, including humans. The potential use of these bacteria as live vehicles for the production and delivery of heterologous proteins of vaccinal, medical or technological interest has therefore been extensively investigated. Lactococcus lactis, a LAB species, is a potential candidate for the production of biologically useful proteins. Several delivery systems have been developed to target heterologous proteins to a specific cell location (i.e., cytoplasm, cell wall or extracellular medium). A promising application of L. lactis is its use as an antigen delivery vehicle, for the development of live mucosal vaccines. The expression of heterologous proteins and antigens as well as the various delivery systems developed in L. lactis, and its use as an oral vaccine carrier are discussed.

Inhalants as intermediate drugs between legal and illegal drugs among middle and high school students.

The aims of this study are to: (1) describe the prevalence and sociodemographic characteristics of inhalant use among middle and high school students in Brazil, and (2) test the hypothesis of inhalants being intermediate drugs between legal and illegal drug use. A representative sample of 5226 students from private schools in São Paulo, Brazil, was selected to answer a self-report questionnaire. Weighted data was analyzed through Cox proportional hazards models. In the overall sample, inhalants seems to be an intermediate drug, since prior inhalant initiation was associated with first marijuana use, adjusted for previous alcohol and tobacco initiation.

Crack cocaine craving: behaviors and coping strategies among current and former users.

OBJECTIVE: To understand crack cocaine craving among users and describe craving behaviors and coping strategies. METHODOLOGICAL PROCEDURES: Qualitative study with a non-random criterion sample consisting of 40 current and former crack cocaine users conducted in São Paulo, southeast Brazil, in 2007 and 2008. Respondents were selected using snowball sampling technique. In-depth semi-structured interviews were conducted until theoretical saturation was attained. All interviews were transcribed and content analysis was performed to construct inferences and hypotheses based on the narratives. ANALYSIS OF RESULTS: The respondents showed a similar gender distribution, were 18 to 50 years of age, and had different levels of education. Most were from low-income background. In addition to craving resulting from crack cocaine withdrawal and environmental and emotional cue effects, it was found that crack cocaine itself triggers craving. The latter appeared to be a strong trigger of binge episodes. Binge episodes made them lose their moral values, and act dangerously to get more drug. The most common ways reported to get crack cocaine or money to buy it were: prostitution, manipulation of other people, go into debt, sell personal belonging to buy drug and theft. The respondents reported strategies to overcome their cravings as well as pharmacological and behavioral approaches to prevent cravings such as eating, having sex, playing soccer, working, avoiding social situations of crack use and taking depressants. CONCLUSIONS: Crack cocaine binges are caused by a craving induced by the effects of crack cocaine itself. Users develop self-control strategies to cope with their cravings that may help improve their drug use and treatment effectiveness.

Fissura por crack: comportamentos e estratégias de controle de usuários e ex-usuários / Crack cocaine craving: behaviors and coping strategies among current and former users / Ansia de consumo por crack: conductas y estrategias de control de usuarios y ex-usuarios

OBJETIVO: Compreender a fissura do usuário de crack, bem como descrever os comportamentos desenvolvidos sob fissura e estratégias utilizadas para seu controle. PROCEDIMENTOS METODOLÓGICOS: Estudo qualitativo com amostra intencional por critérios de 40 usuários e ex-usuários de crack em São Paulo, SP, nos anos de 2007 e 2008. Os entrevistados foram recrutados pela técnica da bola de neve e submetidos à entrevista semi-estruturada em profundidade, até a saturação teórica. Após transcrição literal, seguiu-se a análise do conteúdo das entrevistas para elaboração de inferências e hipóteses alicerçadas nessas narrativas. ANÁLISE DOS RESULTADOS: Os entrevistados estavam igualmente distribuídos quanto ao sexo, possuíam idade entre 18 e 50 anos, abrangeram todos os níveis de escolaridade e a maior parte possuía poucos recursos financeiros. Além da fissura sentida na abstinência de crack e da fissura induzida por pistas ambientais e emocionais, constatou-se um tipo de fissura que faz parte do próprio efeito do crack. Esta última apareceu como forte fator mantenedor dos binges de consumo, que foram os maiores responsáveis pelo rebaixamento de valores do usuário, sujeitando-o a práticas arriscadas para a obtenção da droga. Os métodos mais citados para a obtenção de crack ou dinheiro para comprá-lo foram: prostituição, manipulação de pessoas, endividamento, troca de pertences por crack e roubo. Foram relatadas estratégias para o alívio da fissura e táticas farmacológicas e comportamentais para evitar o seu desenvolvimento, como: comer, ter relação sexual, jogar futebol, trabalhar, evitar o contexto social de uso de crack e usar drogas que causam sonolência. CONCLUSÕES: Os binges de consumo de crack são causados pela fissura induzida durante o uso da droga. As medidas criadas pelo próprio usuário para lidar com a sua fissura melhoram sua relação com o crack e podem ser ferramenta importante para o aprimoramento do tratamento.

OBJECTIVE: To understand crack cocaine craving among users and describe craving behaviors and coping strategies. METHODOLOGICAL PROCEDURES: Qualitative study with a non-random criterion sample consisting of 40 current and former crack cocaine users conducted in São Paulo, southeast Brazil, in 2007 and 2008. Respondents were selected using snowball sampling technique. In-depth semi-structured interviews were conducted until theoretical saturation was attained. All interviews were transcribed and content analysis was performed to construct inferences and hypotheses based on the narratives. ANALYSIS OF RESULTS: The respondents showed a similar gender distribution, were 18 to 50 years of age, and had different levels of education. Most were from low-income background. In addition to craving resulting from crack cocaine withdrawal and environmental and emotional cue effects, it was found that crack cocaine itself triggers craving. The latter appeared to be a strong trigger of binge episodes. Binge episodes made them lose their moral values, and act dangerously to get more drug. The most common ways reported to get crack cocaine or money to buy it were: prostitution, manipulation of other people, go into debt, sell personal belongings to buy drug and theft. The respondents reported strategies to overcome their cravings as well as pharmacological and behavioral approaches to prevent cravings such as eating, having sex, playing soccer, working, avoiding social situations of crack use and taking depressants. CONCLUSIONS: Crack cocaine binges are caused by a craving induced by the effects of crack cocaine itself. Users develop self-control strategies to cope with their cravings that may help improve their drug use and treatment effectiveness.

OBJETIVO: Comprender el ansia de consumo por el usuario de crack, así como describir las conductas desarrolladas y estrategias utilizadas para su control. PROCEDIMIENTOS METODOLÓGICOS: Estudio cualitativo con muestra intencional por criterios de 40 usuarios y ex-usuarios de crack en Sao Paulo, Sureste de Brasil, en los años de 2007 y 2008. Los entrevistados fueron reclutados por la técnica de la bola de nieve y sometidos a la entrevista semi-estructurada en profundidad, hasta la saturación teórica. Posterior a la transcripción literal, se realizó el análisis de contenido de las entrevistas para elaboración de inferencias e hipótesis cimentadas en estas narrativas. ANÁLISIS DE RESULTADOS: Los entrevistados estaban igualmente distribuidos con respecto al sexo, poseían edad entre 18 y 50 años, abarcaron todos los niveles de escolaridad y la mayor parte poseía pocos recursos financieros. Se identificó sensación de ansia de consumo en la abstinencia de crack, inducida por señales ambientales y emocionales, como efecto de la droga. Esta última apareció como fuerte factor mantenedor de los consumos excesivos, que fueron los mayores responsables por el apocamiento de los valores del usuario, sujetándolos a prácticas arriesgadas para la obtención de drogas. Los métodos más citados para la obtención de crack o dinero para comprarlo fueron: prostitución, manipulación de personas, endeudamiento, cambio de pertenencias por crack y robo. Se relataron estrategias para el alivio del ansia de consumo y tácticas farmacológicas y conductuales para evitar su desarrollo, como: comer, tener relación sexual, jugar fútbol, trabajar, evitar el contexto social de uso de crack y usar drogas que causan somnolencia. CONCLUSIONES: Los consumos excesivos de crack son causados por el ansia de consumo inducido durante el uso de la droga. Las medidas creadas por el propio usuario para lidiar con su ansiedad mejoran su relación con el crack y pueden ser herramienta importante para perfeccionar el tratamiento.

Production and targeting of the Brucella abortus antigen L7/L12 in Lactococcus lactis: a first step towards food-grade live vaccines against brucellosis.

Brucella abortus is a facultative intracellular gram-negative bacterial pathogen that infects humans and animals by entry mainly through the digestive tract. B. abortus causes abortion in pregnant cattle and undulant fever in humans. The immunogenic B. abortus ribosomal protein L7/L12 is a promising candidate antigen for the development of oral live vaccines against brucellosis, using food-grade lactic acid bacteria (LAB) as a carrier. The L7/L12 gene was expressed in Lactococcus lactis, the model LAB, under the nisin-inducible promoter. Using different signals, L7/L12 was produced in cytoplasmic, cell-wall-anchored, and secreted forms. Cytoplasmic production of L7/L12 gave a low yield, estimated at 0.5 mg/liter. Interestingly, a secretable form of this normally cytoplasmic protein via fusion with a signal peptide resulted in increased yield of L7/L12 to 3 mg/liter; secretion efficiency (SE) was 35%. A fusion between the mature moiety of the staphylococcal nuclease (Nuc) and L7/L12 further increased yield to 8 mg/liter. Fusion with a synthetic propeptide (LEISSTCDA) previously described as an enhancer for heterologous protein secretion in L. lactis (Y. Le Loir, A. Gruss, S. D. Ehrlich, and P. Langella, J. Bacteriol. 180:1895-1903, 1998) raised the yield to 8 mg/liter and SE to 50%. A surface-anchored L7/L12 form in L. lactis was obtained by fusing the cell wall anchor of Streptococcus pyogenes M6 protein to the C-terminal end of L7/L12. The fusions described allow the production and targeting of L7/L12 in three different locations in L. lactis. This is the first example of a B. abortus antigen produced in a food-grade bacterium and opens new perspectives for alternative vaccine strategies against brucellosis.

In situ DNA transfer to chicken embryos by biolistics

Ovos fertilizados de galinha foram bombardeados através da técnica de biobalística. A expressäo transiente do gene lacZ, sob o controle do promotor humano citomegalovírus, foi verificada após a transferência in situ. Diferentes níveis de pressäo de gás hélio, vácuo e tipos de partículas foram testados. A taxa de sobrevivência aumentou à medida que a velocidade das partículas diminuíram, entretanto, o nível de expressäo foi menor. Os melhores resultados, combinando taxa de sobrevivência e expressäo, foram obtidos com partículas de ouro, 600 libras por polegada ao quadrado de hélio e 600 mmHg de vácuo. Nestas condiçöes, todos os embriöes bombardeados apresentaram atividade da ß-galactosidase, indicando que esta técnica é eficiente para a transformaçäo de embriöes de galinha.