Validated HPLC determination of 4-dimethylaminoantipyrine in different suppository bases.

Suppositories are important tools for individual therapy, especially in paediatrics, and an instrumental assay method has become necessary for the quality control of dosage units. The aim of this work was to develop a rapid, effective high-performance liquid chromatography method to assay aminophenazone in extemporaneous suppositories prepared with two different suppository bases, adeps solidus and massa macrogoli. With a novel sample preparation method developed by the authors, 4-dimethylaminoantipyrine was determined in these suppository bases with 95-105% recovery. The measurements were carried out on a Shimadzu Prominence ultra high-performance liquid chromatography system equipped with a 20 µl sample loop. The separation was achieved on a Hypersil ODS column, with methanol, sodium acetate buffer (pH 5.5±0.05, 0.05 M, 60:40, v/v) as the mobile phase at a flow rate of 1.5 ml/min. The chromatograms were acquired at 253 nm. The chromatographic method was fully validated in accordance with current guidelines. The presented data demonstrate the successful development of a rapid, efficient and robust sample preparation and high-performance liquid chromatography method for the routine quality control of the dosage units of suppositories containing 4-dimethylaminoantipyrine.

Evaluation of HIV-1 resistance to antiretroviral drugs among 150 patients after six months of therapeutic interruption.

Most of the antiretroviral (ARV) studies in Brazil have been reported in treatment-experienced and naive patients rather than in the setting of treatment interruption (TI). In this study, we analysed reasons given for TI and resistance mutations occurring in 150 HIV-1-infected patients who underwent TI. Of the patients analysed, 110 (73.3%) experienced TI following medical advice, while the remaining patients stopped antiretroviral therapy (ART) of their own accord. The main justifications for TI were: ARV-related toxicities (38.7%), good laboratory parameters (30%) and poor adherence (20%). DNA sequencing of the partial pol gene was successful in 137 (91.3%) patients, of whom 38 (27.7%) presented mutations conferring ARV resistance. A higher viral load prior to TI correlated with drug resistance (P < 0.05). Our results demonstrate that there are diverse rationales for TI and that detection of resistant strains during TI most likely indicates a fitter virus than the wild type. High viral loads coupled with unprotected sex in this group could increase the likelihood of transmission of drug-resistant virus. Thus, treating physicians should be alerted to this problem when the use of ARVs is interrupted.

Paracoccidioidomycosis: an epidemiologic survey in a pediatric population from the Brazilian Amazon using skin tests.

Since Paracoccidioides brasiliensis and Histoplasma capsulatum are known to be present in similar environments, there have been many epidemiologic investigations regarding the prevalences of these two organisms. However, cross-reactivity can occur in paracoccidioidin and histoplasmin skin tests, and this usually results in the overestimation of the prevalence of P. brasiliensis. The prevalence of infection with P. brasiliensis was evaluated in a cross-sectional study of 298 asymptomatic school children in the Brazilian Amazon region (Mato Grosso State). In this investigation, the reactivity of children to two different P. brasiliensis antigen preparations, paracoccidioidin and a purified 43-kD glycoprotein (gp43), was compared with or without the co-administration of histoplasmin. In the group of individuals receiving paracoccidioidin who had a positive histoplasmin skin test result, the prevalence of exposure to P. brasiliensis was 44% (16 of 36). This reactivity to P. brasiliensis was significantly higher than that observed in other groups, which ranged from 4% to 6% (P < 5 x 10(-4) for each). Overall prevalence was 4.6% (95% confidence interval = 2.5-7.7%). These data suggest that gp43 provides a better estimate of exposure to P. brasiliensis when the co-administration of histoplasmin is desired.

A simple method for the simultaneous separation of peripheral blood mononuclear and polymorphonuclear cells in the dog.

A simple method for the simultaneous separation and purification of peripheral blood mononuclear cells (PBMC) and polymorphonuclear neutrophil cells (PMNC) was developed for comparative and functional studies in the immune system of the dog. Purity and cell viability were > 95%, yields were similar to those obtained by other techniques but without red blood cell contamination. Differential blood cell count studies of the isolated cells in blood samples of beagle dogs and German shepherd dogs demonstrated that the 1.077/1.119 double density centrifugation is an effective method of acquiring both highly purified blood mononuclear cells and polymorphonuclear cells as separate entities from the same sample. The interface between plasma and 1.077 contained an average 97% blood mononuclear cells vs. 3% polymorphonuclear cells, and the interface between 1.077 and 1.119 an average 96% polymorphonuclear cells vs. 4% blood mononuclear cells. These data indicate that Histopaque 1.077/1.119 double density gradient allows the purification and physical separation of lymphocytes and phagocytes from a blood sample in the dog, enabling the investigator to examine both cell types from the same sample simultaneously.

Local changes in vascular architecture following partial spinal cord lesion in the rat.

Lesions of the CNS induce a complex cascade of tissue reactions. The purpose of this study was to determine the response of the vasculature to partial spinal cord transection. Adult rat spinal cords were lesioned and then examined during acute, subacute, and chronic periods for the presence of endothelial cells and blood vessels at the lesion site. The association of endothelial cells and astrocytes was examined immunohistochemically (RECA-1 and glial fibrillary associated protein, respectively). During the first 48 h following an incision lesion of the dorsal spinal cord, the vasculature was significantly decreased, concurrently with the tissue loss due to primary and secondary degeneration. Subsequently, at 4 days postlesion, vasculature repair processes were evidenced by a significant increase in the number of vessels present at the lesion center. Blood vessels even formed in areas densely packed with macrophages and tissue debris. After 1 week, the number of blood vessels declined in the lesion center and at the place of the forming caverns. These results show significant initial attempts at repair of the vasculature which do not, however, lead to the restoration of a compact tissue and cannot prevent the subsequent formation of caverns.

Radioassay of soluble immune complexes using their uptake by macrophage Fc receptors.

Sera from patients were tested for the presence of soluble immune complexes (IC) by a method based on the observation that IC inhibit uptake of aggregated IgG by the Fc receptors of macrophages. From the saturation curve of macrophages with 125I-labelled aggregates the optimal inhibitory effect of a measured amount of IC was determined. This radioassay is highly sensitive, being capable of detecting 20 ng of IC, is reproducible and covers a wide range of measurement (20-2000 ng).

Familial differences in antibody response of broiler chickens to vaccination with attenuated and inactivated Newcastle disease virus vaccine.

Genetic differences in immune response to Newcastle disease virus (NDV) were studied in 4-week-old broilers, vaccinated with attenuated (live) or inactivated NDV. The experiment included 370 chicks from two farms distributed among 22 sire families and 60 dam families. Results in chicks from both farms were similar. Survival after challenge was closely related to titer level. The genetic correlation between day-7 and day-12 titers (attenuated virus) was 1.0. Significant differences were found between sire families in both sorts of vaccinations. Heritabilities based on the sire variance components for attenuated and inactivated virus vaccinations were respectively 0.31 and 0.60. The genetic correlation between them was 0.49. Nevertheless, it is concluded that selection for response to NDV based on inactivated virus may be most effective in improving response to attenuated NDV vaccinations.

Isolation and properties of the replicase of encephalomyocarditis virus.

The RNA-dependent RNA polymerase (replicase) of encephalomyocarditis (EMC) virus was found to be closely associated with the smooth membranes of infected BHK-21 cells. An RNA-dependent EMC replicase was extracted from the membranes with 0.15% sodium dodecyl sulfate (SDS) and 1,1,2-trichlorotri-fluoroethane (Genetron 113) and further purified by high-salt dextran-polyethylene glycol phase separation, sievorptive chromatography, and glycerol gradient sedimentation. The enzyme does not manifest strict specificity toward EMC RNA template. It can use also Qbeta RNA, rRNA of BHK cells, or poly(C). SDS-polyacrylamide gel electrophoresis of purified EMC replicase labeled with radioactive methionine revealed that, of all the stable EMC proteins, the enzyme contains predominantly the 56,000-dalton (E) polypeptide.

The assay of gosling hepatitis virus and antibody by spermagglutination and spermagglutination-inhibition. I. Spermagglutination.

Agglutination of bovine spermatozoa by gosling hepatitis virus grown in embryonated eggs of 3 different hosts and from several different passage levels was demonstrated. A good agreement was found between the egg-infective and spermagglutinating titres of the virus. The reaction closely resembles that previously described for Newcastle disease virus, influenza virus and Mycoplasma. Gosling hepatitis virus differs however from the other spermagglutinating agents by its inability to agglutinate erythrocytes. Optimal physico-chemical requirements for the reaction were determined. The spermagglutination technique provides a rapid and simple method for assaying the virus. The method is also convenient and inexpensive owing to the availability of spermatozoa in pelleted form which can be stored frozen for long periods.

The assay of gosling hepatitis virus and antibody by spermagglutination and spermagglutination-inhibition. II. Spermagglutination-inhibition.

Spermagglutination by gosling hepatitis virus was specifically inhibited by sera from sick and convalescent goslings exposed to experimental and natural infection, by hyperimmune horse serum and by sera from vaccinated geese. When compared with passive protection of goslings, neutralization of egg infectivity and precipitation techniques, the spermagglutination-inhibition technique was found to be a highly sensitive serological procedure for antibody detection and assay.