Exploration of new drug delivery pathways: I. Mechanism of folate uptake in cultured human cells. II. Role of nuclear localization signal peptides in the delivery of oligonucleotide into reconstituted nuclei

The roles of the folate receptor and an anion carrier in the uptake of 5- methyltetrahydrofolate (5-MeH_4folate) were studied in cultured human (KB) cells using radioactive 5-MeH_4folate. Binding of the 5-MeH_4folate was inhibited by folic acid, but not by probenecid, an anion carrier inhibitor. The internalization of 5-MeH_4folate was inhibited by low temperature, folic acid, probenecid and methotrexate. Prolonged incubation of cells in the presence of high concentrations of probenecid appeared to inhibit endocytosis of folatereceptors as well as the anion carrier. The V_(max) and K_M values for the carrier were 8.65 ± 0.55 pmol/min/mg cell protein and 3.74 ± 0.54µM, respectively. The transport of 5-MeH4folate was competitively inhibited by folic acid, probenecid and methotrexate. The carrier dissociation constants for folic acid, probenecid and methotreate were 641 µM, 2.23 mM and 13.8 µM, respectively. Kinetic analysis suggests that 5-MeH_4folate at physiological concentration is transported through an anion carrier with the characteristics of the reduced-folate carrier after 5-MeH_4folate is endocytosed by folate receptors in KB cells. Our data with KB cells suggest that folate receptors and probenecid-sensitive carriers work in tandem to transport 5-MeH_4folate to the cytoplasm of cells, based upon the assumption that 1 mM probenecid does not interfere with the acidification of the vesicle where the folate receptors are endocytosed.

Oligodeoxynucleotides designed to hybridize to specific mRNA sequences (antisense oligonucleotides) or double stranded DNA sequences have been used to inhibit the synthesis of a number of cellular and viral proteins (Crooke, S. T. (1993) FASEB J. 7, 533-539; Carter, G. and Lemoine, N. R. (1993) Br. J. Cacer 67, 869-876; Stein, C. A. and cohen, J. S. (1988) Cancer Res. 48, 2659-2668). However, the distribution of the delivered oligonucleotides in the cell, i.e., in the cytoplasm or in the nucleus has not been clearly defined. We studied the kinetics of oligonucleotide transport into the cell nucleus using reconstituted cell nuclei as a model system. We present evidences here that oligonucleotides can freely diffuse into reconstituted nuclei. Our results are consistent with the reports by Leonetti et al. (Proc. Natl. Acad. Sci. USA, Vol. 88, pp. 2702-2706, April 1991), which were published while we were carrying this research independently. We also investigated whether a synthetic nuclear localization signal (NLS) peptide of SV40 T antigen could be used for the nuclear targeting of oligonucleotides. We synthesized a nuclear localization signal peptide-conjugated oligonucleotide to see if a nuclear localization signal peptide can enhance the uptake of oligonucleotides into reconstituted nuclei of Xenopus. Uptake of the NLS peptide-conjugated oligonucleotide was comparable to the control oligonucleotide at similar concentrations, suggesting that the NLS signal peptide does not significantly enhance the nuclear accumulation of oligonucleotides. This result is probably due to the small size of the oligonucleotide.

Model systems for the study of drug hypersensitivity

I. It was not possible to produce anti-tetracycline antibody in laboratory animals by any of the methods tried. Tetracycline protein conjugates were prepared and characterized. It was shown that previous reports of the detection of anti-tetracycline antibody by in vitro-methods were in error. Tetracycline precipitates non-specifically with serum proteins. The anaphylactic reaction reported was the result of misinterpretation, since the observations were inconsistent with the known mechanism of anaphylaxis and the supposed antibody would not sensitize guinea pig skin. The hemagglutination reaction was not reproducible and was extremely sensitive to minute amounts of microbial contamination. Both free tetracyclines and the conjugates were found to be poor antigens.

II. Anti-aspiryl antibodies were produced in rabbits using 3 protein carriers. The method of inhibition of precipitation was used to determine the specificity of the antibody produced. ε-Aminocaproate was found to be the most effective inhibitor of the haptens tested, indicating that the combining hapten of the protein is ε-aspiryl-lysyl. Free aspirin and salicylates were poor inhibitors and did not combine with the antibody to a significant extent. The ortho group was found to participate in the binding to antibody. The average binding constants were measured.

Normal rabbit serum was acetylated by aspirin under in vitro conditions, which are similar to physiological conditions. The extent of acetylation was determined by immunochemical tests. The acetylated serum proteins were shown to be potent antigens in rabbits. It was also shown that aspiryl proteins were partially acetylated. The relation of these results to human aspirin intolerance is discussed.

III. Aspirin did not induce contact sensitivity in guinea pigs when they were immunized by techniques that induce sensitivity with other reactive compounds. The acetylation mechanism is not relevant to this type of hypersensitivity, since sensitivity is not produced by potent acetylating agents like acetyl chloride and acetic anhydride. Aspiryl chloride, a totally artificial system, is a good sensitizer. Its specificity was examined.

IV. Protein conjugates were prepared with p-aminosalicylic acid and various carriers using azo, carbodiimide and mixed anhydride coupling. These antigens were injected into rabbits and guinea pigs and no anti-hapten IgG or IgM response was obtained. Delayed hypersensitivity was produced in guinea pigs by immunization with the conjugates, and its specificity was determined. Guinea pigs were not sensitized by either injections or topical application of p-amino-salicylic acid or p-aminosalicylate.

A Delphi Process to Optimize Quality and Performance of Drug Evaluation in Neonates

Background: Neonatal trials remain difficult to conduct for several reasons: in particular the need for study sites to have an existing infrastructure in place, with trained investigators and validated quality procedures to ensure good clinical, laboratory practices and a respect for high ethical standards. The objective of this work was to identify the major criteria considered necessary for selecting neonatal intensive care units that are able to perform drug evaluations competently. Methodology and Main Findings: This Delphi process was conducted with an international multidisciplinary panel of 25 experts from 13 countries, selected to be part of two committees (a scientific committee and an expert committee), in order to validate criteria required to perform drug evaluation in neonates. Eighty six items were initially selected and classified under 7 headings: "NICUs description - Level of care'' (21), "Ability to perform drug trials: NICU organization and processes (15), "Research Experience'' (12), "Scientific competencies and area of expertise'' (8), "Quality Management'' (16), "Training and educational capacity'' (8) and "Public involvement'' (6). Sixty-one items were retained and headings were rearranged after the first round, 34 were selected after the second round. A third round was required to validate 13 additional items. The final set includes 47 items divided under 5 headings. Conclusion: A set of 47 relevant criteria will help to NICUs that want to implement, conduct or participate in drug trials within a neonatal network identify important issues to be aware of. Summary Points: 1) Neonatal trials remain difficult to conduct for several reasons: in particular the need for study sites to have an existing infrastructure in place, with trained investigators and validated quality procedures to ensure good clinical, laboratory practices and a respect for high ethical standards. 2) The present Delphi study was conducted with an international multidisciplinary panel of 25 experts from 13 countries and aims to identify the major criteria considered necessary for selecting neonatal intensive care units (NICUs) that are able to perform drug evaluations competently. 3) Of the 86 items initially selected and classified under 7 headings - "NICUs description - Level of care'' (21), "Ability to perform drug trials: NICU organization and processes (15), "Research Experience'' (12), "Scientific competencies and area of expertise'' (8), "Quality Management'' (16), "Training and educational capacity'' (8) and "Public involvement'' (6) - 47 items were selected following a three rounds Delphi process. 4) The present consensus will help NICUs to implement, conduct or participate in drug trials within a neonatal network.

Biofunctionalization of alginate microcapsules: advances in cell-based drug delivery systems

172 p.

Undertreatment of human immunodeficiency virus in psychiatric inpatients: a cross-sectional study of seroprevalence and associated factors

Background: The aims of this study were to evaluate the prevalence of HIV and its associated demographic and clinical factors among psychiatric inpatients of a general hospital. Methods: This was a single-center, observational, cross-sectional study that included patients consecutively admitted to our unit aged 16 years or older and with no relevant cognitive problems. The patients were evaluated using a semistructured interview and an appropriate test for HIV infection. Results: Of the 637 patients who were screened, 546 (86%) who consented to participate were included in the analyses. Twenty-five (4.6%, 95% confidence interval [CI] 3.0-6.8) patients were HIV-positive. The prevalence was higher among patients with substance misuse (17.4%, 95% CI 9.7-28.8). All except one of the 25 patients knew of their seropositive condition prior to participation in the study. Only 14 (56%) of the 25 seropositive patients had previously received pharmacological treatment for their infection. According to the multiple logistic regression analysis, the likelihood of HIV infection was lower in patients with higher levels of education and higher among patients who were single, had history of intravenous drug use, and had an HIV-positive partner, particularly if they did not use condoms. Among the patients with HIV infection, 18 (72%) had a history of suicide attempts compared with 181 (34.7%) of the patients without HIV infection (relative risk 2.1, 95% CI 1.6-2.7; P<0.001). Conclusion: HIV infection is highly prevalent in patients admitted to a psychiatric unit, especially those with a diagnosis of substance misuse. Seropositive patients show very poor treatment adherence. The risk of suicide seems to be very high in this population. Implementing interventions to reduce the suicide risk and improve adherence to antiretroviral therapy and psychotropic medications seems crucial.

Modular Multimodal Iron Oxide-Based Nanocarriers for Image-Guided dsRNA Immunostimulation and Platinum Anticancer Drug Design

237 p.

Exploring new labelling strategies for boronated compounds: towards fast development and efficient assessment of BNCT drug candidates

208 p.

Violência entre parceiros íntimos nos primeiros cinco meses de pós-parto em usuárias de unidades básicas de saúde do Rio de Janeiro

A Violência entre Parceiros Íntimos (VPI) tem sido reconhecida como um importante problema de saúde pública e um fator de risco para agravos a saúde de mulheres e crianças. Os serviços de saúde desempenham importante papel na detecção precoce da VPI, especialmente em momentos da vida nos quais se preconiza o atendimento sistematizado, como na gestação e primeira infância. Esta dissertação tem como objetivo principal estimar a probabilidade de ocorrência de Violência Física entre Parceiros Íntimos (VFPI) durante a gestação e/ou pós-parto em população atendida em Unidades Básicas de Saúde (UBS), segundo diferentes características sócio-econômicas e demográficas da clientela. Trata-se de um estudo transversal, realizado com usuárias de 5 UBS da cidade do Rio de Janeiro no ano de 2007. Foram entrevistadas 811 mães de crianças de até cinco meses de idade, que não possuíam nenhuma contra-indicação formal para a amamentação e que relataram ter tido pelo menos uma relação amorosa um mês ou mais durante a gestação ou no período do pós-parto. Condições socioeconômicas e demográficas e relativas aos hábitos de vida do casal foram consideradas como potenciais preditores de violência. Utilizou-se a versão em português da CTS2 para identificar as situações de VPI. A variável de desfecho foi analisada em três níveis: ausência de VFPI, presença de VFPI no período da gestação ou do pós-parto e presença de VFPI em ambos os períodos. Utilizou-se um modelo logito-multinomial para as projeções de prevalências segundo os descritores selecionados. Os fatores que mais aumentaram a probabilidade de ocorrência de violência durante a gestação e/ou nos primeiros cinco meses de vida da criança foram: idade materna < 20 anos, escolaridade materna inferior ao 2 grau completo, ter 2 ou mais filhos menores de cinco anos, tabagismo materno, uso inadequado de álcool pela mãe e/ou companheiro, uso de drogas pela mãe e/ou companheiro e percepção materna sobre a saúde do bebê aquém da esperada. Entre mães com todas estas características, a estimativa de prevalência projetada de VFPI na gestação e/ou no pós-parto chegou a 96,4%, sendo 59,4% a estimativa de ocorrência em apenas um dos dois períodos e 37% em ambos. Por outro lado, a probabilidade de ocorrência de VFPI cai a 3,6% em famílias sem estas características. Os resultados indicam que a presença de certas características da criança e de sua família aumenta enormemente a probabilidade de ocorrência de VFPI, devendo ser levadas em consideração ao se estabelecer estratégias de intervenção que visem à detecção precoce e uma efetiva intervenção.

Use of homeo drug for curing ulcerative syndrome in fishes

Fungal infection was observed in Catla catla and Labeo rohita cultured in two private fish farms. The later stage of the infection resulted in ulcerations followed by haemorrhage on the dorsal surface of the body. Initially, usual treatments of copper sulphate, potassium permanganate and common salt solution were tried, but no improvement was observed. Then repeated intramuscular injections of homeopathic drug Heaper Sulpher and Arnica spray were given with encouraging results. Infection reported in another farm was also successfully controlled using a similar treatment.

Limit of drug residues in aquatic products (NY5071-2002)

Weimin, ZOU, lexian YANG, lan JIANG, Shuqin WU, Qi YI, Jianli WU

Distribution of drug resistant luminous bacteria in penaeid shrimp grow-out ponds

Distribution of luminous bacteria (LB) in penaeid shrimp grow-out pond water in semiintensive seawater farming system and their resistance to 15 antibacterials were investigated. Total viable counts and luminous bacterial counts in pond water ranged from 2.00xl03 to 1.35xl04/ml and l.OOxl01 to 8.00Xl02/ml, respectively. The percentage composition of LB in the total viable population increased significantly with period of culture. Five species of LB such as Vibrio fischeri, V. harveyi, V. orientalis, V. splendidus 1 and Photobacterium leiognathi were encountered. V. harveyi was the dominant species, constituting >80% of the total LB. Multiple antibiotic resistance was more common in these LB. Pond water isolates showed resistance to at least four antibacterial agents.