Chronic ingestion of high concentrations of cholecalciferol in cats.

OBJECTIVE: To determine whether ingestion of 63 times the recommended amount of vitamin D3 (cholecalciferol) results in renal calcification or damage in cats. ANIMALS: 20 four-month-old kittens, 17 queens, and 20 kittens born to these queens. PROCEDURE: 4-month-old kittens and queens were given a purified diet with 846 microg of cholecalciferol/kg of diet (high vitamin D3 diet) or 118 microg of cholecalciferol/kg of diet (control diet) for 18 months. Kittens born to queens were weaned onto the same diet given to dams. RESULTS: There were no apparent adverse effects of the high vitamin D3 diet. Plasma cholecalciferol and 25-hydroxycholecalciferol (25-OHD3) concentrations of queens and 4-month-old kittens given the high vitamin D3 diet significantly increased with time. At 6 months, plasma cholecalciferol concentrations in these kittens and queens were 140.0+/-7.3 nmol/L and 423.6+/-26.6 nmol/L, respectively (10 times initial values). Corresponding 25-OHD3 concentration in queens was 587.5+/-59.4 nmol/L (2.5-fold increase over initial values). At 3 months of age, kittens born to queens given the high vitamin D3 diet had an increase in serum BUN and calcium concentrations and a decrease in RBC and serum total protein, albumin, and hemoglobin concentrations. By 18 months, these kittens had an increase in plasma cholecalciferol (276.0+/-22.2 nmol/L) and 25-OHD3 (1,071.9+/-115.3 nmol/L) concentrations. However, all indices of renal function and the appearance of renal tissue on histologic evaluation were normal. CONCLUSIONS AND CLINICAL RELEVANCE: These results indicate that cats are resistant to cholecalciferol toxicosis when the diet is otherwise complete and balanced.

Acute otitis media in Brazilian children: analysis of microbiology and antimicrobial susceptibility.

Between 1990 and 1995, a total of 300 children, ages 2 months to 5 years, received diagnoses of acute otitis media (AOM) in a hospital emergency room in São Paulo, Brazil, and were recruited for this study. The investigation was undertaken, first, to identify microorganisms and antimicrobial susceptibilities of pathogens from AOM in Brazilian children; next, to ascertain, by comparison, whether the isolates of Streptococcus pneumoniae have the same serotypes as those included in the new conjugated heptavalent pneumococcal vaccine; and last, to determine whether additional and/or different serotypes are needed in the vaccine to ensure an immunogenic response against pneumococcal pathogens for the indigenous children in this study. Microorganisms were isolated from ear fluid of 192 patients (64%). The 5 most prevalent pathogens were S pneumoniae (48 isolates; 16%), Haemophilus influenzae (21 isolates; 7%), Moraxella catarhalis (15 isolates; 5%), Pseudomonas aeruginosa (6 isolates; 2%), and Staphylococcus aureus (3 isolates; 1%). These 5 represented 93 of the 192 total isolates. Resistance to antibiotics was found in the 3 primary pathogens. No high-level resistance of S pneumoniae to penicillin was found; instead, there was high-level resistance to trimethoprim-sulfamethoxazole. Ten serotypes of S pneumoniae were isolated: 6B, 9V, 11A, 16, 18C, 19A, 19F, 23A, 23B, and 23F. Only 5 of the 10 serotypes isolated were included in the conjugated heptavalent pneumococcal vaccine. Therefore, the other 5 serotypes (24 of 48 strains) should be considered in selecting antigens for the new vaccine.

Metabolic conversion of retinol to retinoic acid mediates the biological responsiveness of human mammary epithelial cells to retinol.

The biological effects of vitamin A are mediated in part by retinoic acid (RA) modulation of gene transcription. In this study, we examined whether normal human mammary epithelial cells (HMECs) are biologically responsive to retinol (ROH), the metabolic precursor of RA. While both ROH and tRA resulted in time- and dose-dependent decreases in total cell number, tRA was markedly more potent. Metabolically, treatment of HMECs with physiological doses of ROH resulted in rapid uptake and subsequent production of both retinyl esters and tRA. Although a comparatively minor metabolite, tRA levels peaked at 6 h and remained above endogenous levels for up to 72 h in proportion to cellular ROH concentrations. In HMECs transfected with an RA-responsive luciferase reporter gene, treatment with 3 microM ROH resulted in an increase in luciferase activity to a level intermediate between that observed with 0.001 and 0.01 microM tRA. Citral, an RA-synthesis inhibitor, was also used to examine the biological activity of ROH. Compared to ROH alone, ROH plus citral treatment resulted in three-fold less tRA synthesis and a > 65% attentuation of RA-responsive reporter gene activity which persisted through 72 h. Citral also significantly attenuated the extent of ROH-mediated reductions in total HMEC number. Thus, treatment with physiological concentrations of ROH results in fewer total numbers of HMECs and this response is a consequence of cellular tRA synthesis which can induce RA-responsive gene expression.

Synthesis of [(14)C]pyrroloquinoline quinone (PQQ) in E. coli using genes for PQQ synthesis from K. pneumoniae.

Radiochemical forms of pyrroloquinoline quinone (PQQ) are of utility in studies to determine the metabolic role and fate of PQQ in biological systems. Accordingly, we have synthesized [(14)C]PQQ using a tyrosine auxotrophic strain of Escherichia coli (AT2471). A construct containing the six genes required for PQQ synthesis from Klebsiella pneumoniae was used to transform the auxotrophic strain of E. coli. E. coli were then grown in minimal M9 medium containing 3.7x10(9) Bq/mmol [(14)C]tyrosine. At confluence, the medium was collected and applied to a DEAE A-25 anionic exchange column; [(14)C]PQQ was eluted using a KCl gradient (0-2 M in 0.1 M potassium phosphate buffer, pH 7.0). Radioactivity co-eluting as PQQ was next pooled, acidified and passed through a C-18 column; [(14)C]PQQ was eluted with a phosphate buffer (0.1 M, pH 7.0). Reverse phase HPLC (C-18) using either the ion-pairing agent trifluoroacetic acid (0. 1%) and an acetonitrile gradient or phosphoric acid and a methanol gradient were used to isolate [(14)C]PQQ. Fractions were collected and analyzed by liquid scintillation counting. (14)C-labelled compounds isolated from the medium eluted corresponding to the elution of various tyrosine-derived products or PQQ. The radioactive compound corresponding to PQQ was also reacted with acetone to form 5-acetonyl-PQQ, which co-eluted with a 5-acetonyl-PQQ standard, as a validation of [(14)C]PQQ synthesis. The specific activity of synthesized [(14)C]PQQ was 3.7x10(9) Bq/mmol [(14)C]PQQ, equal to that of [U-(14)C]tyrosine initially added to the medium.

Correlation between respiratory alterations and respiratory diseases due to urban pollution.

BACKGROUND: Air pollution is strongly correlated with allergic and infectious diseases. Chronicity of the stimulation and immaturity of the defense system make children prone to respiratory diseases. The aim of this study was to assess the adverse effects of urban levels of air pollution, correlating children's respiratory diseases and ultrastructural studies in rats, compared to controls in a clean area. METHODS: An epidemiological survey was conducted with 2000 school children (age range 7-14 years old), divided into two groups of 1000 children each: the Red group from São Paulo city (17,000,000 inhabitants) and the Green group from a rural area around the city of Tupã with no air pollution at all. A questionnaire was given to the children's parents in order to estimate history of respiratory diseases and predisposing factors. A total of 69 rats were housed for 6 months in the center of São Paulo, and ultrastructural studies of the epithelium of the airways were done and compared to 56 control animals in the rural area. RESULTS: The Red group of children had a statistically significant (P < 0.005) high prevalence of respiratory diseases such as rhinitis, sinusitis, and upper respiratory infections (URI). Rats exposed to air pollution developed ultrastructural ciliary alterations. CONCLUSION: The results obtained in the present investigation suggest that chronic exposure to urban levels of air pollution may cause respiratory diseases in children and ultrastructural ciliary alterations in the epithelium of the airways in rats, when compared to controls in a pollution-free rural area.

[Diagnosis and therapeutic management of sinusitis in children] / Conduta diagnóstica e terapêutica na sinusite da criança.

OBJECTIVE: The authors review the main aspects related to the diagnosis and management of sinusitis in children, according to the medical literature. METHODS: Literature review. RESULTS: Clinical criteria, microbiology, image diagnosis, medical and surgical management are presented, whenever sinusitis is considered. CONCLUSIONS: The standardization of the clinical approach in children's sinusitis allows optimization of the diagnosis and the appropriate subsidiary exams, as well the correct use of antimicrobials and others drugs, or surgery in selected cases.

[Controversial drugs in otorhinolaryngology] / Medicamentos controversos em otorrinolaringologia.

OBJECTIVES: To present a review on the most important groups of drugs used to treat otorhinolaryngological disorders. METHOD: Review of the literature about treatment of pediatric upper respiratory infections and allergy, using MEDLINE and LILACS data. RESULTS AND COMMENTS: Pediatric otorhinolaryngological disorders are extremely frequent and most of the time acute. They are one of the major reasons for pediatric visits. The therapeutical management of these conditions in many cases accounts for an over use of drugs, specially antibiotics, antipyretic and drugs of doubtful value, such as decongestants and expectorants. Judicious use, correct indication, and side effects of these drugs must be better known by physicians who deal with the child's health.

[Lower airways and pollution]. / Vias aéreas inferiores e a poluição.

OBJECTIVE: The purpose of this study is to demonstrate the prevalence of lower respiratory airway diseases in children exposed to different sources of environmental pollutants. METHODS: An epidemiological survey (using a questionnaire) was conducted in São Paulo State with three groups of children (1000 in each group): in the city of São Paulo, urban pollution; in Piracicaba, sugar cane burning area; and in Tupã and Batatais, pollution-free rural zone. RESULTS: As for asthma/bronchitis and/or pneumonia, children from Piracicaba and São Paulo presented rates of 14 and 11% respectively, compared with 10% in Tupã and Batatais. Absenteeism from school was 17% in Piracicaba, 11% in São Paulo and 9% in Tupã and Batatais. Hospitalizations due to respiratory complications (asthma/ bronchitis or pneumonia) was 4% in São Paulo, 3% in Piracicaba and 2% in Tupã and Batatais. All the above results had statistical significance (p < 5). CONCLUSIONS: High prevalence of lower respiratory airways infections and their complications in children exposed to different sources of environmental air-pollutants demonstrate the need for a more efficacious control environment pollution.

Characterization of ear fluid isolates of Alloiococcus otitidis from patients with recurrent otitis media.

Nineteen isolates of Alloiococcus otitidis from ear fluid samples collected by tympanostomy from patients at four geographic locations were identified by phenotypic characterization and genetic relatedness. Initial growth of A. otitidis isolates occurred after 3 days at 37 degrees C on brain heart infusion (BHI) agar with 5% rabbit blood. Heavy growth occurred in BHI broth supplemented with 0.07% lecithin and 0.5% Tween 80 after 4 days of incubation. The isolates were gram-positive cocci that divided on an irregular plane and produced metabolic lactic acid, pyrrolidonyl arylamidase, and leucine aminopeptidase. These cocci grew sparsely in 6.5% NaCl-BHI broth, were asaccharolytic on both fermentative and oxidative bases, and were cytochrome negative by the iron-porphyrin test. The cellular fatty acid profile of A. otitidis was distinguished from those of related genera and characterized by major amounts ( > or = 14%) of 16:0, 18:2, 18:1 omega 9c, and 18:0 and smaller amounts of 14:0, 16:1 omega 7c, 17:0, and 18:1 omega 7c. Fifteen isolates demonstrated > 69% relatedness by DNA-DNA hybridization. Four isolates plus the original 15 were confirmed as A. otitidis by dot blot hybridization with a digoxigenin-labeled nucleotide probe specific for this species. The intergenic space between the genes coding for the 16S and 23S rRNAs of alloiococci was amplified by PCR, analyzed by restriction fragment length polymorphism, and determined to consist of three different genetic types. Although beta-lactamase negative, A. otitidis demonstrated intermediate levels of resistance to beta-lactams, including expanded-spectrum cephalosporins, and were resistant to trimethoprim-sulfamethoxazole and erythromycin.

Gene expression following direct injection of DNA into liver.

The liver is an attractive target tissue for gene therapy. Current approaches for hepatic gene delivery include retroviral and adenoviral vectors, liposome/DNA, and peptide/DNA complexes. This study describes a technique for direct injection of DNA into liver that led to significant gene expression. Gene expression was characterized in both rats and cats following injection of plasmid DNA encoding several different proteins. Luciferase activity was measured after injection of plasmid DNA encoding the luciferase gene (pCMVL), beta-galactosidase (beta-Gal) activity was evaluated in situ using plasmid DNA encoding Lac Z (pCMV beta), and serum concentration of secreted human alpha-1-antitrypsin was measured following injection of plasmid DNA encoding this protein (pRC/CMV-sHAT). Several variables, including injection technique, DNA dose, and DNA diluent, were investigated. Direct injection of pCMVL resulted in maximal luciferase expression at 24-48 hr. beta-Gal staining demonstrated that the majority of transfected hepatocytes were located near the injection site. Significant concentrations of human alpha-1-antitrypsin were detected in the serum of animals injected with pRC/CMV-sHAT. These findings demonstrate the general principle that direct injection of plasmid DNA into liver can lead to significant gene expression.

Dexamethasone enhancement of gene expression after direct hepatic DNA injection.

The critical physiological functions of the liver make hepatocytes important targets for therapeutic gene delivery. This study reports significant gene expression following direct injection of plasmid DNA into the livers of rats and cats. Transfection was characterized using luciferase and Lac Z expression from plasmids with the cytomegalovirus immediate early promoter/enhancer (CMV IE) or the Rous sarcoma virus long terminal repeat (RSV LTR). Dexamethasone treatment enhanced and prolonged transfected gene expression, possibly by activating gene expression. Southern analysis of total DNA extracted from liver at various times following injection detected persistent unintegrated plasmid DNA which maintained a prokaryotic methylation pattern. This study demonstrates the feasibility of direct DNA injection in the experimental analysis of hepatic gene expression in vivo.

Glycoprotein (GP) Ib beta is the critical subunit linking GP Ib alpha and GP IX in the GP Ib-IX complex. Analysis of partial complexes.

The glycoprotein (GP) Ib-IX complex is the receptor on platelet surfaces that mediates their adhesion to subendothelium. It comprises three polypeptides (GP Ib alpha, GP Ib beta, GP IX), each of which belongs to a superfamily of proteins containing conserved leucine-rich motifs. In this study, we used Chinese hamster ovary (CHO) cells expressing every combination of two GP Ib-IX complex subunits to demonstrate that GP Ib beta plays an essential role in the synthesis of the heterotrimer by associating with both of the other two subunits. Confocal microscopy demonstrated that GP Ib beta was present in the same cellular locations as GP Ib alpha in CHO alpha beta cells (cells expressing only GP Ib alpha and GP Ib beta) and as GP IX in CHO beta IX cells. The two polypeptides expressed in CHO alpha IX cells did not co-localize. Association between GP Ib alpha and GP Ib beta was demonstrated biochemically on immunoblots of detergent lysates of CHO alpha beta cells; electrophoresis under nonreducing conditions revealed the two subunits to be covalently linked through a disulfide bond. Association of GP Ib alpha and GP Ib beta was further demonstrated by the finding that immunoprecipitations with antibodies against either polypeptide precipitated both. Similarly, immunoprecipitations of lysates of CHO beta IX cells with antibodies against GP Ib beta or GP IX precipitated both polypeptides. In contrast, co-immunoprecipitation of the two polypeptides expressed in CHO alpha IX cells could not be demonstrated. Transient expression in CHO cells of GP Ib beta with GP IX yielded higher GP IX levels on the cell membrane than did expression of GP IX alone; supertransfection of CHO alpha IX cells with GP Ib beta also increased GP IX levels on the cell surface.

Association of GP Ib with actin-binding protein does not require GP IX.

GP IX is necessary for optimal expression of the GP Ib-IX complex on the surface of transfected cells, and presumably also on the surface of the platelet. The authors investigated whether increasing complex association with the cytoskeleton is one mechanism by which GP IX exerts its effect. CHO and L cell lines that express high levels of GP Ib were used to determine whether GP Ib (GPIb alpha and GPIb beta) associated with the cytoskeleton. GP Ib in these cells was found in the insoluble cytoskeletal fraction from Triton X-100 lysates in a proportion similar to that found in cells expressing the full complex. As in platelets and cells expressing the full complex, the association of GP Ib with the cytoskeleton was shown to be mediated by actin-binding protein (ABP). This was demonstrated by the observation that a monoclonal antibody against GPIb alpha precipitated ABP from GP Ib-expressing cells, and polyclonal anti-ABP antibodies specifically coprecipitated GP Ib. In addition, colocalization of the two components in intact cells was demonstrated by confocal microscopy. These data indicate that the association of GP Ib with the cytoskeleton is independent of GP IX, which therefore must increase surface expression of the complex by another mechanism.

Prophylaxis for recurrent acute otitis media: a Brazilian study.

We enrolled 60 children with recurrent acute otitis media (AOM) in a study of the effectiveness of antimicrobial prophylaxis. All children were entered into the study following an acute episode of infection treated with amoxicillin (AMX) for 10 days. Following therapy, the children were re-examined, and then randomly assigned to receive either trimethoprim-sulfamethoxazole (TMP-SMX), amoxicillin (AMX) or a placebo (PLA). Twenty children were included in each group. Each drug was administered once a day at bedtime, at 1/3 the therapeutic dose, for 3 months. Children were re-evaluated with pneumootoscopy during episodes of acute illness and with pneumootoscopy and impedance tympanometry (TYMP) at monthly intervals. We observed a significantly increased rate of recurrent AOM in children receiving placebo compared with those who received antibiotics (50% vs. 17% P < 0.005). Both prophylactic antibiotics were equally effective in preventing recurrent AOM (recurrence rate 20% TMP-SMX, 15% AMX). We also observed that recurrences in children receiving placebo occurred earlier in the study period than in those receiving antibiotics. These results suggest that antimicrobial prophylaxis in children with recurrent acute otitis media is effective in reducing subsequent disease. The similar efficacy of both antibiotics tested suggests that the less expensive agent should be used.