Induction of emphysema and bronchial mucus cell hyperplasia by intratracheal instillation of lipopolysaccharide in the hamster.

The aim of this study was to determine whether lipopolysaccharide-induced elastase release from recruited neutrophils in the hamster lung would induce emphysema, measured by mean linear intercept (Lm) and bronchial mucus cell hyperplasia (BMCH), scored in tissue sections stained with periodic acid-Schiff. Lipopolysaccharide (LPS) was instilled transorally twice a week for up to 5 weeks in hamsters. At 4 weeks after seven LPS instillations, Lm amounted to 87.6 +/- 1.2 microns, while it was 68.3 +/- 1.5 microns after seven saline instillations (P less than 0.01). At 6 months after the sixth LPS instillation, the Lm of these lungs was 83.3 +/- 1.6 microns, indicating irreversible tissue destruction. LPS-treated hamsters showed marked to severe BMCH, which was most evident in large intrapulmonary airways. Instillations of highly selective inhibitor of hamster PMN elastase resulted in 50 per cent inhibition of LPS-induced emphysema. The development of BMCH was inhibited by approximately 35 per cent by this agent. To study the response in time of cellular infiltration after a single LPS instillation, the lungs of groups of four hamsters were lavaged at different time points. PMN recruitment showed peak values at 4 and 48 h after LPS instillation and returned to baseline values at 96 h. Simultaneous intratracheal instillation of LPS and anti-TNF alpha antiserum resulted in a considerable reduction of neutrophil influx into bronchoalveolar spaces in the first 6 h after instillation.


Impact of within-person variability on identifying children with hypercholesterolemia: Framingham Children's Study.

The National Cholesterol Education Program guidelines recommending that children be screened to detect abnormal levels of blood lipids depend on accurate assignment to risk categories. Our objective was to assess the impact of within-person variability of blood lipids on such risk classification. We measured total cholesterol (TC) and high-density lipoprotein-cholesterol (HDL-C) at three successive weekly visits in 24 children aged 6 to 9 years. Using within-person variance estimates, we constructed 95% confidence intervals to evaluate how well one, two, and three lipid measurements per child allowed assignment of risk as recommended by the National Cholesterol Education Program. The mean, overall, and within-person standard deviations were 145.7, 24.5, and 9.2 mg/dl, respectively, for TC, and 42.8, 10.1, and 4.6 mg/dl, respectively, for HDL-C. One TC measurement allowed confident assignment to the "acceptable" (less than 170 mg/dl) category only if less than 154.9 mg/dl, and to the "high" (greater than or equal to 200 mg/dl) category only if greater than 215.1 mg/dl. The average of two TC values allowed assignment to these categories if less than 159.3 or greater than 210.7 mg/dl, respectively. For the average of three TC values, these ranges were less than 161.3 and greater than 208.7 mg/dl, respectively. With one TC measurement, no value allowed assignment to the "borderline" (170 to 199 mg/dl) category. The average of two TC values allowed assignment to the "borderline" category if between 180.7 and 189.3 mg/dl. Using the average of three values enlarged this range by 4 mg/dl. For HDL-C, multiple measurements improved risk assignment in a similar fashion. The magnitude of within-person variability of TC and HDL-C limits the ability to classify children into risk categories recommended by the National Cholesterol Education Program.


Improved outcome of respiratory syncytial virus infection in a high-risk hospitalized population of Canadian children. Pediatric Investigators Collaborative Network on Infections in Canada.

To determine the outcomes in children at high risk for death or complications from respiratory disease who are hospitalized with respiratory syncytial virus (RSV) infection. Retrospective chart review. Twelve pediatric tertiary care centers. All hospitalized children with an RSV infection diagnosed by a positive antigen detection test result or viral isolation during the study period from 1988 to 1991, encompassing three winter seasons. Charts from patients in the following high-risk groups were reviewed in detail: (1) congenital heart disease, (2) chronic lung disease, (3) immunodeficiency, (4) age less than 6 weeks, (5) gestational age less than 36 weeks, and (6) hypoxia (defined as oxygen saturation less than 90% or arterial oxygen pressure less than 60 mm Hg). The age of all children, the date of RSV identification, and the use of oxygen supplementation, intensive care, and ventilatory support. In addition, the duration of these treatments and the duration of hospitalization were noted. Left-to-right shunting and pulmonary hypertension before RSV infection were determined in those children with congenital heart disease. The nature of the chronic lung disease was noted. Death within 2 weeks of RSV identification was recorded, and the use of ribavirin, bronchodilators, and corticosteroids was determined. Significant year-to-year variation in the frequency of RSV infection was confirmed, with a peak during the 1989-1990 winter noted by the majority of centers (p = 0.0001). Of the 1584 patients in the study, 260 had underlying cardiac disease, 200 had chronic lung disease, 35 had compromised immune function, 378 had been premature, 373 were less than 6 weeks of age, and 338 had hypoxia. Seventeen patients died within 2 weeks (mortality rate 1%); significantly more patients with underlying cardiac disease (3.4%) or lung disease (3.5%) died. Immunocompromised patients had the longest hospital stay (median 39 days), followed by those patients with underlying cardiac or pulmonary disease (11 days); patients less than 6 weeks of age (5 days) and those with hypoxia (6 days) had the shortest hospital stays. Patients with underlying cardiac and pulmonary disease also required oxygen supplementation for a significantly longer period. The year-to-year variation in frequency of RSV infection was confirmed in this study. Morbidity and mortality rates associated with RSV infection in a high-risk population in Canada were significantly lower than previously reported.


Efficacy and safety of a Haemophilus influenzae type b capsular polysaccharide-tetanus protein conjugate vaccine.

A polyribosylribitol phosphate (polysaccharide)-tetanus protein conjugate vaccine (PRP-T) against Haemophilus influenzae type b (Hib) was evaluated for safety and efficacy after vaccination of more than 100,000 infants. No major side effects were attributed to the vaccine. Immunogenicity studies showed an antibody response in 70% to 100% of infants after two doses, and in 98% to 100% of infants after three doses, within the first 6 months of life. Antibodies persisted in 90% of recipients, in whom significant anamnestic responses developed after a booster dose at 18 months of age. In comparison with other available Hib vaccines, PRP-T induces equal or higher mean titers after three doses. Although licensure of other vaccines interrupted controlled efficacy trials, up to that point five cases of Hib disease in those trials had occurred in placebo recipients, and no Hib disease has been reported in the more than 100,000 vaccinated infants who have received more than one dose of PRP-T. Thus PRP-T combined immunogenicity early in life with induction of immunologic memory.


Protection against infection with Giardia lamblia by breast-feeding in a cohort of Mexican infants.

To determine whether breast-feeding protects infants against symptomatic and asymptomatic infection by Giardia lamblia, we followed 197 infants in a poor area of Mexico City from birth to 18 months of age; symptoms and feeding status were recorded weekly. Stool specimens were collected every 1 to 2 weeks and tested for Giardia by enzyme-linked immunosorbent assay. A mean of 1.0 Giardia infection per child-year was detected; 94 infants had a total of 139 infections; 17% of infections were symptomatic. Ninety-one percent of infants were breast fed from birth and 38% were breast fed at 1 year of age. Lack of breast-feeding was a significant risk factor for first Giardia infection at all ages. The adjusted incidence rate ratio for first Giardia infection for none versus complete breast-feeding was 5.0 (confidence interval (CI) 1.5 to 16.9; p = 0.009), and for none versus any breast-feeding, 1.8 (CI 1.1 to 2.8; p = 0.013). Symptomatic Giardia infection was also associated with lack of breast-feeding (none vs any: incidence rate ratio = 2.5; CI 0.9 to 6.8; p = 0.077), but breast-feeding did not protect against chronic carriage of Giardia. Other significant risk factors for Giardia infection were presence of animals in the household (p = 0.005) and the use of water or nonmilk liquid for infant feedings (p = 0.035). We conclude that breast-feeding protects infants against Giardia by mechanisms that include preventing the establishment of infection.


Immunologic changes associated with the development of tolerance in children with cow milk allergy.

The purpose of this study was to determine whether cow milk-specific antibody responses correlated with the development of clinical tolerance in cow milk-allergic children. Double-blind, placebo-controlled food challenges were performed annually in 29 patients with cow milk allergy. Clinical reactivity was lost in 11 (38%) of 29 patients. The median age for all patients at the time of diagnosis by these food challenges was 3 years; more than 80% of patients in each group had atopic dermatitis as part of their presenting symptoms. Casein-specific and beta-lactoglobulin-specific IgE, IgG, IgG1, and IgG4 antibody concentrations were analyzed in all patients at regular intervals. In the patients becoming clinically tolerant to cow milk, the IgE-specific antibody concentrations and IgE/IgG-specific ratios for both milk proteins were lower initially and decreased significantly with time, in comparison with those in the group who retained clinical sensitivity. The concentrations of IgG1- and IgG4-specific antibody to casein and the IgE/IgG1 and IgE/IgG4 ratios for both casein and beta-lactoglobulin were significantly less in the patients losing clinical reactivity. No differences in the IgG-specific concentrations were observed in either group at any of the evaluation times noted above. Monitoring similar casein-specific and beta-lactoglobulin-specific IgE concentrations and IgE/IgG ratios may help predict which patients will ultimately lose their clinical reactivity to cow milk.


Renovascular disease in childhood.

Fifty-four children referred for investigation of hypertension had renovascular disease. In eight patients it was associated with neurofibromatosis, in three with idiopathic hypercalcemia of infancy, and in five cases it followed an arteritic illness. Fibromuscular dysplasia was the underlying abnormality in the majority of cases (46%). Twenty-six patients (48%) were first seen with accelerated hypertension; 38 children (70%) had bilateral renal arterial disease, and in 41 (76%), disease of the small intrarenal vessels was found. Renal vein renin ratios indicated unilateral disease in 31 cases; the results correlated with arteriography findings in 32 (62%) of 51 patients. Eleven children also had the middle aortic syndrome, and 9 of 16 patients, investigated by cerebral arteriography because of cranial bruits or focal neurologic signs, had cerebral vascular abnormalities. Twenty patients were treated surgically--10 by reconstructive procedures, 11 by nephrectomy or heminephrectomy, and 6 by transluminal angioplasty. Of these, 9 (45%) are normotensive with no treatment, 10 have a decreased requirement for antihypertensive drugs, and 1 had no improvement. Thirty-four patients were treated medically because of the extent of their disease; two patients have died of hypertensive complications. We conclude that renal vascular disease in children is often widespread, may be associated with intracerebral vascular disease, frequently affects both kidneys, including both intrarenal and extrarenal vessels, and is therefore not always amenable to surgical intervention and cure.


Use of a molecular genetic approach to diagnosing the fragile X genotype.

We report the direct molecular detection of the fragile X genotype in 111 individuals from 17 families with a total of 31 cases of fragile X syndrome. Comparison of our molecular data with our previous cytogenetic and linkage data from these same families indicates the effectiveness of the direct molecular analysis. We have been able to assign a genotype unambiguously in 100% of the persons tested, and in all cases the molecular data correlated with the cytogenetic or linkage findings or both. Two of the three families presented in this study represent inheritance of this gene through normal transmitting males, and the third is strongly suggestive of this mode of inheritance. Our data show that the direct molecular approach will be of great utility for confirmation of the diagnosis and for the detection of female carriers and normal transmitting males who are at high risk for having affected children or grandchildren.


Hypercapnic and hypoxic ventilatory and cardiac responses in school-aged siblings of sudden infant death syndrome victims.

Siblings of sudden infant death syndrome (SIDS) victims have been shown to have abnormal ventilatory patterns and altered responses to respiratory stimuli during infancy. To evaluate whether these abnormalities persist, we studied ventilatory responses in 20 older SIDS siblings (9.8 +/- 0.9 (mean +/- SEM) years of age) and 20 control subjects (10.2 +/- 0.9 years of age). To evaluate hypercapnic ventilatory responses, we had subjects rebreathe 5% carbon dioxide and 95% oxygen until end-tidal carbon dioxide tension reached 65 mm Hg. Instantaneous minute ventilation, mean inspiratory flow, and respiratory rate were calculated breath by breath. Hypercapnic responses did not differ between SIDS siblings (2.08 +/- 0.14 L/min per mm Hg) and control subjects (1.90 +/- 0.10 L/min per mm Hg; not significant). To assess hypoxic ventilatory responses, we asked subjects to rebreathe 13% oxygen and 7% carbon dioxide, with the balance nitrogen, at mixed-venous end-tidal carbon dioxide tension, until arterial oxygen saturation by pulse oximetry fell to 75%. No differences in hypoxic ventilatory responses were found between the SIDS siblings (-1.39 +/- 0.15 L/min/% saturation) and the control subjects (-1.22 +/- 0.17 L/min/% saturation; not significant). The mean inspiratory flow, tidal volume, respiratory rate, and heart rate responses to hypercapnia and hypoxia were also similar in the two groups. We conclude that there is no difference in hypercapnic and hypoxic ventilatory and cardiac responses, as assessed by rebreathing techniques, between school-aged SIDS siblings and control subjects. We speculate that in SIDS siblings the control of breathing is immature during infancy and that they achieve maturity of control and resolution of breathing abnormalities with time.


Ataxia-telangiectasia in a child with vaccine-associated paralytic poliomyelitis.

Vaccine-acquired poliomyelitis developed in a nonimmunized 10-month-old boy. At age 4 years, ataxia-telangiectasia was recognized. We conclude that the occurrence of vaccine-related poliomyelitis warrants a detailed assessment of immunity, and that, in patients with ataxia-telangiectasia, the use of live vaccines may be hazardous, even in those with apparently normal immunity.


Familial hypotonia of childhood caused by isolated 3-methylcrotonyl-coenzyme A carboxylase deficiency.

Hypotonia was the initial symptom in four siblings from a nonconsanguineous Tunisian-Jewish family. Plasma carnitine was severely deficient, and urinary organic acid analysis revealed increased excretion of 3-hydroxyisovaleric acid and 3-methylcrotonylglycine. 3-Methylcrotonyl-coenzyme A carboxylase activity was reduced in skin fibroblasts; pyruvate carboxylase and serum biotinidase activities were normal. We conclude that 3-methylcrotonyl-coenzyme A carboxylase deficiency should be added to the list of metabolic causes of familial hypotonia of childhood.


Variability of breath hydrogen excretion in breast-fed infants during the first three months of life.

Breath hydrogen excretion was measured serially in breast-fed infants. There was marked variability in H2 excretion, both within and between infants. The findings indicate that unabsorbed food is not the only substrate, and that breath H2 may not be an effective method to assess carbohydrate absorption in young infants.


Bentiromide test using liquid-chromatographic measurement of p-aminobenzoic acid and its metabolites for diagnosing pancreatic insufficiency in childhood.

We assessed the diagnostic capability of the bentiromide test using a high-pressure liquid-chromatography method to analyze p-aminobenzoic acid and its metabolites in plasma as an indirect measure of exocrine pancreatic function. Mean total amine concentration in pancreatic-insufficient subjects was significantly lower than in control subjects. There were 3 of 15 false-negative results and no false-positive results. We conclude that this chromatographic method is an effective means of analyzing p-aminobenzoic acid and its metabolites after ingestion of bentiromide.


High synovial immunoglobulin E levels in eosinophilic synovitis.

Eosinophilic synovitis occurred in a 7-year-old boy. Synovial fluid leukocytes were mostly eosinophils; the peripheral blood showed only mild eosinophilia. The level of eosinophil-derived neurotoxin in the synovial fluid was higher than that in the serum, suggesting intraarticular eosinophil degranulation. The IgE level was also elevated in the synovial fluid (3854 ng/ml) but normal in the serum (408 ng/ml), suggesting a localized immediate hypersensitivity immune response.


Trial of vitamin A supplementation in very low birth weight infants at risk for bronchopulmonary dysplasia.

We performed a randomized, double-blind, controlled trial to determine whether vitamin A supplementation in a group of very low birth weight infants would reduce the incidence of bronchopulmonary dysplasia. Forty-nine infants (birth weight 700 to 1100 gm) requiring mechanical ventilation and supplemental oxygen at 96 hours age were randomly assigned to receive either 2000 IU retinyl palmitate (n = 27) or saline placebo (n = 22) intramuscularly every other day for up to 14 doses. There were no differences between treatment groups in the incidences of bronchopulmonary dysplasia at 31 days of postnatal age (vitamin A group 48%, placebo group 55%; p = 0.776), supplemental oxygen requirement at 34 weeks of postconceptional age, or other complications of prematurity. The vitamin A group had higher mean plasma vitamin A concentrations than the placebo group, but mean plasma vitamin A concentrations were greater than 20 micrograms/dl (suggesting sufficiency) in both groups after the first study week. By study day 28, only one fourth of the infants in either group had plasma vitamin A concentrations less than 20 micrograms/dl. In contrast to an earlier report, we found no change in the incidence of BPD with vitamin A supplementation. Our findings may reflect a low baseline incidence of vitamin A deficiency in the study population and recent changes in the respiratory care of very low birth weight infants. The latter may have lessened the potential impact of vitamin A deficiency on lung disease.


Early-onset group B streptococcal sepsis: a current assessment.

Group B streptococcus (GBS) is a common cause of early-onset sepsis in neonates. The most recent reviews describing incidence, diagnosis, treatment, and outcome evaluated data on patients from the early 1980s. To obtain current information about this disease, we retrospectively evaluated data on neonates with GBS early-onset sepsis from nine hospitals in the United States between Jan. 1, 1987, and Dec. 31, 1989. There were 245 infants with GBS bacteremia identified among 61,809 live births, resulting in an incidence of 0.32%. Ninety-six infants (39%) were preterm (less than 38 weeks of gestational age). Maternal risk factors for infected preterm and term infants were similar. Antibiotics were administered during parturition in 10% of infants with bacteremia. Mothers of preterm infants received antibiotics up to 48 hours before delivery; mothers of term infants received antibiotics less than 4 hours before delivery. All preterm infants with bacteremia had symptoms; 22% of term infants with bacteremia had no symptoms. Group B streptococcal meningitis was confirmed in 6.3% of infants. Although 86% survived, GBS sepsis increased the birth weight-specific mortality rate up to eightfold in preterm infants and more than 40-fold in term infants. Although the incidence of GBS early-onset sepsis is not changing, we speculate that the improved birth weight-specific survival rate and the changing clinical presentation are due to improved intrapartum and neonatal management.


Intravenous immune globulin therapy for early-onset sepsis in premature neonates.

Newborn infants may have IgG deficiencies that increase their susceptibility to bacterial infection. To determine whether intravenous immune globulin (IVIG) therapy improves survival rates in early-onset sepsis, we prospectively entered 753 neonates (birth weight 500 to 2000 gm, gestation less than or equal to 34 weeks, age less than or equal to 12 hours) into a multicenter, double-blind, controlled trial. Blood culture specimens were obtained and infants randomly assigned to receive 10 ml (per kilogram) intravenously of a selected IVIG (500 mg/kg) or albumin (5 mg/kg) preparation. Maternal and neonatal risk factors were not different between groups. Thirty-one babies (4.2%) had early-onset sepsis; the causative organisms were group B streptococcus (12 babies), Escherichia coli (6), and others (13). Of these 31 neonates, 7 (23%) died. Total serum IgG was higher for 7 days after IVIG therapy than after albumin treatment (p less than 0.05). During these 7 days, 5 (30%) of 17 albumin-treated and none of 14 IVIG-treated patients died (p less than 0.05). The survival rate at 56 days of age, however, was not significantly improved. Group B streptococcus type-specific IgG antibody was significantly increased after IVIG treatment and appeared to be related to the amount of IVIG specific antibody. Infusion-related adverse reactions were less frequent in patients receiving IVIG therapy (0.5%) than in those receiving albumin. The IVIG therapy in neonates with early-onset sepsis, while reducing the early mortality rate, did not significantly affect the overall survival rate. Further studies are necessary to confirm these findings and to determine more effective therapeutic regimens.


Effects of type of dietary protein on acid-base status, protein nutritional status, plasma levels of amino acids, and nutrient balance in the very low birth weight infant.

To determine the effect of the type of dietary protein (3.3 gm/kg per day) on acid-base status, protein nutritional status, plasma amino acid concentrations, and nutrient (nitrogen, fat, mineral, trace element) balance. Preterm infants (birth weight less than or equal to 1250 gm, gestational age less than or equal to 32 weeks) with no evidence of systemic disease, who had achieved a minimal enteral intake of 110 kcal/kg per day by 21 days of age. Each infant was fed three study formulas that differed only with respect to the ratio of whey to casein (60:40, 35:65, 18:82). Each formula was given for 1 week. At the end each week, blood was drawn and a 48-hour balance was determined. Late metabolic acidosis, uremia, and hyperammonemia were not observed. No differences in pH or serum bicarbonate were noted. Base excess was greater with the casein-predominant formula (18:82 greater than 35:65, 60:40) but remained within normal limits for the preterm infant. Plasma concentrations of threonine (60:40 greater than 35:65 greater than 18:82), phenylalanine, and tyrosine (18:82 greater than 35:65 greater than 60:40) differed. Nitrogen absorption (60:40 less than 35:65, 18:82), nitrogen retention (60:40 less than 35:65, 18:82), fat absorption (60:40, 35:65 greater than 18:82), and phosphorus absorption (60:40 less than 35:65, 18:82) also differed. At an intake of 3.3 gm/kg per day, the type of dietary protein had little effect on metabolic status. Differences in plasma amino acid concentrations and nutrient balance suggest that a formula containing protein with a whey/casein ratio of 35:65 may be preferable to that with a whey/casein ratio of 60:40 or 18:82 for the very low birth weight infant.


Mortality and morbidity rates among lower birth weight infants (2000 to 2500 grams) treated with extracorporeal membrane oxygenation.

To assess the validity of the currently accepted lower weight limit of 2 kg for treatment of neonates with extracorporeal membrane oxygenation (ECMO), we reviewed the outcome of lower birth weight (2.0 to 2.5 kg, n = 29) and higher birth weight (n = 235) for infants treated with venoarterial ECMO at our institution from 1984 through 1990. Newborn infants with congenital diaphragmatic hernia were not included. The mortality rate was significantly greater after venoarterial ECMO in lower than in higher birth weight infants (relative risk 3.45; confidence interval = (1.68, 5.79)). For infants with the diagnosis of respiratory distress syndrome, the mortality rate was 56% (5/9) for lower and 8% (2/25) for higher birth weight infants (p less than 0.01). The most frequent cause of death in lower birth weight infants was intracranial hemorrhage (7/10 deaths). The overall incidence of any neuroimaging abnormality was significantly greater for lower birth weight infants (p = 0.044), primarily because of the higher incidence of major intracranial hemorrhage. Finally, the risk of developmental delay (development quotient less than 70 at 1 to 2 years of age) among survivors available for follow-up was significantly higher among the lower than the higher birth weight infants. These outcome data suggest that further reduction of the current lower weight limit for ECMO should not become standard without prospective research or technologic advances.


Cefpodoxime proxetil compared with amoxicillin-clavulanate for the treatment of otitis media.

In a multicenter, randomized, investigator-blinded trial, patients were randomly selected to receive either cefpodoxime proxetil or amoxicillin-clavulanate potassium orally for the treatment of acute suppurative otitis media. Patients were seen before, during, and at the end of therapy, and 2 to 3 weeks after completion of therapy. A total of 229 patients, 153 receiving cefpodoxime and 76 receiving amoxicillin-clavulanate were entered into the study; all patients were examined to determine drug safety. A total of 146 patients, 98 in the cefpodoxime group and 48 in the amoxicillin-clavulanate group, completed the study and were examined to determine drug efficacy. End-of-therapy microbiologic eradication rates in assessable patients were 92% for cefpodoxime and 86% for amoxicillin-clavulanate (p = 0.14; 95% confidence interval (CI) on difference: -4.4%, 19.2%). End-of-therapy clinical response rates for assessable patients were as follows: cured, 68% for cefpodoxime and 65% for amoxicillin-clavulanate; improved, 24% for cefpodoxime and 23% for amoxicillin-clavulanate; and failed, 8% for cefpodoxime and 13% for amoxicillin-clavulanate (p = 0.57; 95% CI: -8.4%, 16.5%). Recurrence rates at long-term follow-up were 24% for cefpodoxime-treated patients and 25% for those given amoxicillin-clavulanate. Both drugs were well tolerated; 20.9% of those given cefpodoxime and 31.6% of amoxicillin-clavulanate-treated patients had drug-related adverse medical events (p = 0.102; 95% CI: -23.9%, 2.6%). Gastrointestinal complaints were the most frequently reported drug-related side effect in both groups: 11.8% of cefpodoxime-treated patients and 21.1% of those given amoxicillin-clavulanate (p = 0.076; 95% CI: -20.8%, 2.2%). Drug-related dermatologic side effects (e.g., diaper rash, pruritus, urticaria) were reported in 7.8% of cefpodoxime-treated patients and 14.5% of those who received amoxicillin-clavulanate (p = 0.160; 95% CI: -16.6%, 3.3%). Our findings suggest that clinical efficacy for cefpodoxime administered twice daily is equivalent to that of amoxicillin-clavulanate administered three times a day.


Lack of effect of respiratory syncytial virus infection on theophylline disposition in children.

Conflicting reports raise a question about decreased plasma clearance (Clp) of theophylline in man during viral infections. Thus a dilemma exists concerning requisite dose adjustments. We examined this issue by retrospectively evaluating theophylline Clp in children infected with respiratory syncytial virus (RSV). Two pharmacokinetic approaches were applied to a one-compartment open model to fit theophylline concentrations during 83 hospitalizations of 76 children, 6 to 48 months of age, who received intravenous theophylline therapy and were tested for RSV infection. Iterative linear regression analyses of all theophylline data were used to estimate apparent volume of distribution, elimination rate constant, plasma half-life, and Clp in 39 of the hospitalizations. When insufficient data were available to distinguish apparent volume of distribution and elimination rate constant (n = 44), steady-state estimates of Clp were calculated. An age-matched and percentile body weight-matched cohort design presented RSV as the primary covariate. Theophylline Clp was similar in 29 matched RSV-infected and -uninfected pairs (1.32 +/- 0.14 and 1.25 +/- 0.05 ml/kg per minute, respectively), as were other pharmacokinetic values. Unexpectedly, a significant, inverse linear relationship was found for Clp and percentile body weight. Additionally, children born prematurely and hospitalized in the neonatal intensive care unit had significantly higher theophylline Clp; this did not affect findings regarding RSV infection. Theophylline Clp was not decreased in RSV-infected children. Current theophylline dosing recommendations for young children infected with RSV should not be altered, but careful monitoring of plasma theophylline levels should be continued.


Management of neonatal Graves disease with iopanoic acid.

An infant with thyrotoxicosis whose mother had Graves disease was treated with sodium iopanoate. Euthyroid status was achieved rapidly and maintained with minimal effort in comparison with the conventional multidrug regimen. There were no toxic effects, and hypothyroidism did not occur.


Double-blind, controlled trial of short-term prednisone therapy in immunoglobulin A glomerulonephritis.

Twenty children and adolescents with IgA glomerulonephritis were enrolled in a crossover trial. Each received 12 weeks of prednisone therapy and 12 weeks of placebo dosing. Urinary protein and erythrocyte excretion were monitored during both courses. There was no evidence that, under the conditions of the study, corticosteroid therapy was effective in IgA nephropathy.


Effect of long-term calcitonin therapy by injection and nasal spray on the incidence of fractures in osteogenesis imperfecta.

The effect of calcitonin therapy by injection or nasal spray on the incidence of bone fractures was studied in patients with osteogenesis imperfecta. In contrast to injection of calcitonin, intranasal administration of calcitonin twice a week for 2 weeks, followed by 2 weeks of no therapy, was simple and seemed beneficial for decreasing bone fractures in patients with osteogenesis imperfecta.


Tibia vara: a complication of adolescent obesity.

Tibia vara is characterized by inhibited growth of the medial portion of the proximal tibial growth plate, leading to progressive bowleg deformity. Twenty-nine adolescent patients with this condition were reviewed: all were black, 27 (93%) were male, and 19 (66%) had only one side affected. Progressive deformity rather than knee discomfort was the most common presenting complaint. The deformity reportedly developed rapidly during the adolescent growth spurt. The body weights of these patients exceeded the 95th percentile for age and gender by an average of 43 kg. The absence of significant symptoms and a body habitus that obscured the deformity often resulted in delayed diagnosis. Physicians involved in the care of obese black male adolescents should carefully examine them for tibia vara, which has a reported prevalence of 2% to 3% in this population. Treatment options are severely restricted if the condition is not diagnosed early.


Recurrent genitourinary chlamydial infections in sexually active female adolescents.

To determine the recurrence rate of chlamydial infections, we initially screened an urban population of 1308 sexually active female adolescents for chlamydial infection at the urethral and endocervical sites; these young women were followed and had additional examinations for infection. Chlamydial infection was documented by tissue culture in 31.1% (407) of them at some time during the study. After appropriate antibiotic treatment, 68.3% (278/407) returned for test-of-cure cultures within 3 months of their initial infection; of those 278, a total of 254 had sterile cultures. These patients were followed to determine the recurrence rate of chlamydial infections. Of these 254 patients, 177 (69.7%) had one or more follow-up visits; 38.4% (68/177) had a recurrent chlamydial infection. The majority of recurrent infections were documented within 9 months of the initial infection. Recurrent infections with the same serovar were frequent, suggesting reinfection by untreated partners or possible relapse of the initial chlamydial infection. This high rate of recurrent infection suggests that female adolescents should be rescreened frequently for genitourinary chlamydial infections.


Short bowel syndrome.

With utilization of the techniques discussed in this review, patients with short bowel syndrome can be expected to live prolonged lives, even after fairly massive resection. Advances in parenteral and home parenteral nutrition and changes in the long-term management of chronic complications have altered the lifestyles of patients with short bowel syndrome. Intestinal transplantation may become an effective mode of therapy within a short period of time. Many surgeons faced with the possibility of leaving a child with only 10-15 cm of bowel in the past have closed the abdomen with the dead bowel and let the child die. These recent advances raise interesting ethical dilemmas. Research into the process of intestinal adaptation as well as greater experience with intestinal transplantation and other modes of therapy may further improve prognosis.


Ultrasonography and gastric emptying: evaluation in infants with gastroesophageal reflux.

We have applied ultrasonography to the evaluation of gastric emptying in children. Two different populations have been investigated: normal children and children with gastroesophageal reflux. All the patients were less than 6 months of age. The diagnosis of gastroesophageal reflux was defined by 24-h pH measurement. The technique, used to measure gastric emptying, is the one described by Bolondi et al. In this research we used the simplified method. All children had been submitted to the examination after 4-h fasting. The standard meal was the usual milk formula, 300 ml/m2 body surface area (BSA). A cross-section area of the gastric antrum was determined before a meal and every 15 min for 2 h. The examination was concluded after two measurements were equal to the basal one. The normal gastric emptying curve was determined by a control group. Patients with gastroesophageal reflux showed three different kind of gastric emptying: (a) normal gastric emptying in 20% of cases, (b) abnormal gastric emptying in 15% of cases, and (c) intermediate cases in which the plateau curve is abnormal but the end time of gastric emptying is normal. We defined these three kinds of curves as type I or normal, type III or abnormal, and type II or intermediate. The estimate of frequency in patients with gastroesophageal reflux is similar to the reported data of the literature.


Efficacy of domperidone in infants and children with gastroesophageal reflux.

This study sought to define the therapeutic efficacy of domperidone in infants and children with gastroesophageal reflux. A double-blind, placebo-controlled trial was performed in seventeen children (ages 5 months to 11.3 years) with moderate to severe gastroesophageal reflux who had not responded to standard nonpharmacological therapy. Subjective and objective measures (weight gain, esophageal pH probe study, radionuclide gastric emptying scan) of gastroesophageal reflux were evaluated. Therapy with domperidone for 4 weeks was effective only in reducing the total number of reflux episodes in the two-hour postprandial period (p less than 0.01); however, it did not result in symptomatic improvement or significant improvement in other measures of gastroesophageal reflux or gastric emptying. After therapy for 8 weeks symptomatic improvement was reported in some patients who had denied improvement after 4 weeks of therapy, suggesting that more than 4 weeks of therapy may be required for some patients to obtain a clinical response. Mild self-limited diarrhea was reported by six patients (four domperidone, two placebo). We conclude that domperidone is tolerated by most infants and children with gastroesophageal reflux; however, 4 weeks of therapy was only minimally effective in producing objective improvement of gastroesophageal reflux and did not result in symptomatic improvement. Further studies of longer duration are needed to resolve the question raised by this study: that domperidone may be beneficial for patients with gastroesophageal reflux when given for more than four weeks.


Effect of diosmectite on intestinal permeability changes in acute diarrhea: a double-blind placebo-controlled trial.

The effect of diosmectite on intestinal permeability changes in acute diarrhea was measured during a double-blind placebo-controlled trial carried out in 59 Gabonese children aged 5-35 months. Intestinal permeability tests (IPTs), measuring the urinary elimination of orally administered lactulose and mannitol at a dosage of 1 g/10 kg each, were performed during the morning following admission and repeated 2 days later after treatment by diosmectite or placebo. During the first IPT, urine volume and lactulose and mannitol urinary recoveries were comparable in the diosmectite and in the placebo groups: 50 vs. 35.5 ml (median; p = 0.21), 1.01 vs. 1.27% (p = 0.35), and 2.20 vs. 2.87% (p = 0.12). As a result, the lactulose/mannitol (L/M) ratio was similar in the two groups: 44.44 vs. 35.33% (p = 0.98). During the second IPT, the urinary lactulose recovery decreased similarly in both groups (-0.18 vs. -0.29%; p = 0.76), whereas the urinary mannitol recovery exhibited opposite variations, the increase in the diosmectite group (+ 1.43%) contrasting significantly with the decrease in the placebo group (-0.47%; p = 0.01). When comparing the first and the second IPT, the decrease of the L/M ratio was significant in the diosmectite group (44.44 vs. 28.32%; p = 0.02) and not in the placebo group (35.33 vs. 48.23%; p = 0.91). During gastroenteritis, diosmectite appears to enhance absorption of mannitol, a marker of intestinal absorptive area.


Helicobacter pylori infection in children: clinical, endoscopic, and histologic correlations.

We have assessed 270 consecutive patients (age range 0.8-20 years) referred for endoscopic study because of abdominal pain during 32 months. Helicobacter pylori (HP) was detected by culture in 91 cases (33.7%). HP colonization increased significantly with age (p less than 0.01). Nine patients less than 5 years of age were colonized by HP. A previous history of peptic ulcer disease in first-degree relatives was significantly more frequent in the HP-positive group (p less than 0.001). The frequency of HP positiveness as related to diagnosis was: normal, 3.3%; nonactive chronic gastritis, 100%; active chronic gastritis, 97.2%; gastric ulcer, 75%; and duodenal ulcer, 90.9% (p less than 0.001). Endoscopic nodular antritis was a frequent (67%) and specific finding; this presence was associated with that of lymphoid follicles in the histopathological study. Signs of histological activity were observed in 55.9% of the HP-positive patients. The histological colonization by HP was assessed semiquantitatively, and a significantly greater HP colonization score was observed in patients with signs of histological activity (p less than 0.001). A significant correlation was found between HP colonization score and histological score (rs = 0.574), with a significant association between the degree of HP colonization and the histologic categories (p less than 0.001). The present study suggests a pathogenic role of HP in the development of gastroduodenal disease in children.


Hepatitis B virus DNA in liver tissue of chronic HBsAg carriers in childhood and its relationship to other viral markers.

The aim of the study was to examine the state of hepatitis B virus (HBV) DNA in liver tissue of 103 children with chronic hepatitis B aged 0.5-18 years to detect free and integrated viral sequences by Southern blot hybridization. HBV DNA was found in 74 patients. Seventy-two were seropositive for hepatitis B e antigen (HBeAg) and two had anti-HBe antibodies. Integrated sequences could be demonstrated in two children. One of them had only integrated HBV DNA and was anti-HBe seropositive. The other one presented both free and integrated viral sequences and developed seroconversion from HBeAg to anti-HBe 5 months after biopsy. In 29 hepatitis B surface antigen (HBsAg) carriers, no HBV DNA could be detected in the liver. Ten were HBeAg and 19 anti-HBe seropositive. HBV DNA in serum was found in 65 of the 74 Southern blot-positive and only in two cases of the Southern blot-negative patients. In conclusion, most of the HBeAg-positive children had free HBV DNA in their liver tissue and all patients with anti-HBe except one were negative. According to our results, HBV DNA integration into the liver cell genome can occur at an early stage of chronic disease but is not a frequent event.


Serum primary bile acids in the course of celiac disease in children.

Serum concentrations of primary bile acids (BAs) were determined in the course of celiac disease and in healthy age-matched controls. Determinations of BAs were made by radioimmunoassays after an overnight fasting and for 4 h postprandially after intake of a standardized test meal. In untreated celiac disease presenting with subtotal villous atrophy, peak concentrations of primary BAs were significantly delayed and serum levels were significantly higher than in controls. Small but significant abnormalities persisted after a period of gluten-free diet despite a normalization of mucosal morphology. Slightly pathological standard liver function tests in five patients were related to increased fasting levels but not to the postprandial pattern of BAs, indicating that the observed abnormalities postprandially were not related to liver disease. Further studies simultaneously focusing on deconjugation mechanisms, absorption, and pool sizes of BAs are required to explain the postprandial BA pattern under the course of celiac disease.


Milk protein quality in low birth weight infants: effects of protein-fortified human milk and formulas with three different whey-to-casein ratios on growth and plasma amino acid profiles.

Growth rates (weight, length, and head circumference) and selected biochemical indexes of protein metabolism (serum urea, acid-base status, and plasma amino acid concentrations) were determined in low birth weight (LBW) infants appropriate for gestational age (birth weight less than 1,650 g) fed three formulas differing only in the whey-to-casein ratios: 60/40, 50/50, and 35/65. A group of infants fed exclusively human milk protein (HMP)-fortified human milk was used as a control. All diets provided similar daily protein and energy intakes, which were 3.5 g/kg and 122 kcal/kg in the human milk-fed infants and 3.3 g/kg and 121 kcal/kg in the formula-fed infants. Neither weight gain nor rate of growth in length and head circumference differed between the feeding groups and reached intrauterine or better rates in all groups. Values for serum urea and acid-base status were normal and also did not differ among the groups. At the end of the study, plasma threonine concentrations were significantly higher in all formula-fed infants than in the infants fed human milk. The highest plasma threonine concentration was found in the infants receiving the whey-predominant formula. Plasma concentrations of valine, methionine, and phenylalanine were also significantly higher in all formula-fed groups when compared with the human milk group. Plasma total essential amino acid concentrations were also significantly higher in the formula-fed infants than in the human milk fed. The results show that protein quality does not affect growth rate or biochemical indexes of metabolic tolerance in LBW infants fed adequate protein and energy intakes.(ABSTRACT TRUNCATED AT 250 WORDS)


Tryptophan fortification of adapted formula increases plasma tryptophan concentrations to levels not different from those found in breast-fed infants.

Several recent studies have demonstrated significantly lower plasma total tryptophan concentrations in formula-fed than in breast-fed infants. We have measured preprandial plasma amino acid concentrations in infants breast-fed or fed a formula with a protein concentration of 1.57 g/dl and with a whey/casein ratio of 60:40 or a formula with a protein concentration of 1.37 g/dl and a whey/casein ratio of 40:60 and fortified with 10 mg/dl (15 mg/100 kcal) of tryptophan. Healthy term infants (10 per group) were either breast-fed from birth or randomly assigned to one of the two study formulas. At 4 and 12 weeks of age, anthropometric measurements were performed and blood samples were obtained. During the study period of 12 weeks, all infants showed normal growth (weight, length, and head circumference) and there were no statistically significant differences between the groups. The plasma concentrations of the essential amino acids phenylalanine, threonine, valine, and lysine were significantly lower in the breast-fed group than in both formula-fed groups. For tyrosine, methionine, leucine, histidine, isoleucine, and arginine, no significant differences could be found between the feeding groups. Concentration of total plasma tryptophan was significantly higher in the breast-fed group than in the group fed the tryptophan-unfortified formula, but no statistically significant difference could be found between the plasma tryptophan concentration in the breast-fed group versus the group fed the tryptophan-fortified formula. The results indicate that tryptophan fortification of adapted formula is necessary to achieve plasma total tryptophan concentrations similar to those found in breast-fed infants.(ABSTRACT TRUNCATED AT 250 WORDS)


Development of pulmonary fibrosis in mice during infection with Paracoccidioides brasiliensis conidia.

Pulmonary fibrosis is a feared sequelae of paracoccidioidomycosis. We sought to determine if mice exposed to Paracoccidioides brasiliensis conidia would develop pulmonary fibrosis. BALB/c mice were infected intranasally with P. brasiliensis conidia and sacrificed at regular intervals. One lung was sectioned for histopathology and sections were stained with haematoxylin and eosin, trichromic and argentic stains; the other lung was homogenized and cultured to determine the viability of the fungus. One week post-challenge, only small peribronchial foci were apparent. After 4 weeks, reticular fibres appeared disorganized and disrupted. Six to 8 weeks later peribronchial infiltrates were larger and appeared surrounded by reticular fibres; thick collagen I fibres were noticed in the infiltrated areas at this time. On weeks 10-12, infiltrates were confluent and reticular fibres were concentrated around the inflammatory foci; collagenization was apparent. Observations up to 16 weeks revealed diffuse involvement of the lung parenchyma with extensive collagenization. Lung cultures were always positive. We suggest that inhalation of P. brasiliensis conidia induces adverse lung responses leading to changes in the proportion of collagen fibres I and III.


Effects of calcium ions on the germination of Sporothrix schenckii conidia.

Sporothrix schenckii conidia were induced to germinate in varying concentrations of added calcium. Calcium had a stimulatory effect on the process and a calcium concentration of 1 mM was found to be optimal. Measurements of radioactive calcium uptake confirmed that uptake was taking place during the germination of conidia to the mycelial form of the fungus. Three calcium uptake peaks were observed, the first occurred 10 min after inoculation and the other two peaks coincided with the first 2 cycles of DNA synthesis in these cells. Ionophore A23187 and cobalt sulfate inhibited germination of S. schenckii conidia and altered the normal calcium uptake pattern in these cells. On the other hand, phorbol 12-myristate-13-acetate, a stimulator of protein kinase C (PKC) activity, stimulated germination in medium without calcium. This suggests that PKC activation might account for the germination of conidia in the absence of added calcium. The effects of increasing the calcium concentration on RNA and protein synthesis were also studied. A stimulation of RNA synthesis was found at all times tested with maximum stimulation 6 h after inoculation. A stimulation in protein synthesis was also observed but only after 12 h incubation.


Synergistic postantifungal effect of flucytosine and fluconazole on Candida albicans.

The in vitro efficacy of flucytosine and fluconazole, separately and in combination, with respect to induction of a postantifungal effect (PAFE) on Candida albicans was studied. PAFE refers to the persistent suppression of fungal cell growth following a short period of exposure to an antifungal agent. A turbidometric method was used to measure cell growth and to quantitate the PAFE following exposure of C. albicans yeast cells to different concentrations of the two agents for 2 h. The PAFE was determined by the difference in time (h) required for growth of the control and test cultures to increase to the 0.5 absorbance level following removal of the drug by dilution. Minimum (MIC) and fractional inhibitory concentration determinations were made and the data used for selecting the concentrations used in the PAFE evaluations. A synergistic interaction of the two drugs at concentrations well below their individual MICs was evidenced. Flucytosine:fluconazole ratios of 1:16-1:32 at concentrations ranging from 0.024-0.098 micrograms ml-1 and from 0.78-1.56 micrograms ml-1, with flucytosine and fluconazole, respectively, induced PAFEs which persisted for 2.5 h longer than those achieved when each of the two agents was assayed separately.


Fatal systemic candidiasis of gastrointestinal origin: an experimental model in mice compromised by anti-cancer treatment.

An experimental model of fatal systemic candidiasis originating from the gastrointestinal (GI) tract of compromised mice is presented. ICR female mice were compromised by a single anti-cancer treatment: irradiation (4 or 6 Greys i.e. 400-600 rads), methotrexate (MTX) (3 mg per mouse, intraperitoneally) or 5-fluorouracil (5FU) (200 mg kg-1, intravenously). Three days later, compromised and non-treated control mice were exposed to Candida albicans administered orally. Morbidity and mortality due to candidiasis were monitored for 30 days post-candidal inoculation. Increased and longer GI colonization was noted among the MTX and 5FU treated mice, or 6 Greys irradiated mice (up to 92.3% for over 30 days in anti-cancer treated mice). The stomach was found to be the major part of the GI tract involved in fungal colonization. A significant number (53.8-83.3%) of the anti-cancer treated mice developed systemic candidiasis originating from the GI tract, which was fatal in 30-80% of the infected animals. In systemically infected animals, candidal antigen was demonstrated in the serum, and fungal abscesses containing C. albicans were observed in the liver, kidneys and spleen. C. albicans was isolated from the infected organs. The severity of the infection, as reflected by the number of fungi in visceral organs, and by mortality during the 30 days post-candidal inoculation, indicated differences in the course and nature of the infection among the three treatment groups (i.e. MTX, 5FU, 6 Greys).(ABSTRACT TRUNCATED AT 250 WORDS)


Disseminated African histoplasmosis in a Congolese patient with AIDS.

We report the first Congolese case of African histoplasmosis in a patient with AIDS which represents the third case to be described in the literature. This contrasts with the now frequent occurrence of Histoplasma capsulatum histoplasmosis in HIV-infected subjects in North America; the difference is likely to be due to differences in exposure to the fungus rather than to differences in behaviour of the fungus or in the epidemiology of HIV.


Coccidioides immitis presenting as a mycelial pathogen with empyema and hydropneumothorax.

A previously healthy Caucasian male developed hydropneumothorax and a pleural peel filled with pleomorphic, septate hyphae. The only organism grown from cultures of the lung and pleural fluid was Coccidioides immitis, confirmed by exoantigen testing. Spherule-endospore forms were produced, however, following injection of the arthroconidial tissue isolate into BALB/c mice. The patient had a positive immunodiffusion complement-fixation test and developed a positive coccidioidin skin test during therapy. He recovered following thoracotomy and wedge resection of the ruptured coccidioidal cavity, and therapy with amphotericin B followed by fluconazole. The sole presence of the mycelial form of the dimorphic fungus C. immitis in the pleural space may have been due to a low CO2 partial pressure at that site secondary to a bronchopleural fistula. The case shows a distinctive and uncommon presentation of coccidioidomycosis which demonstrates the specificity of both the immunodiffusion complement-fixation assay in diagnosing this disease and the exoantigen test in confirming culture results.


The source of the growth-promoting factor(s) affects the plating efficiency of Paracoccidioides brasiliensis.

We studied the influence of the growth factor (GF) source, concentration and production time on the plating efficiency of Paracoccidioides brasiliensis yeast cells. The highest plating efficiencies were achieved when the GF was derived from a fast growing P. brasiliensis isolate which was not homologous to the plated samples.


Sulfonic acid dyes: inhibition of the human immunodeficiency virus and mechanism of action.

Over 50 different commercially available sulfonic acid-containing dyes were analyzed for their ability to prevent HIV-1-induced cell killing and in inhibiting HIV-1 replication. Compounds of remarkably similar structure, but with differing patterns of sulfonic acid group substitutions, had a wide range of potency in inhibiting HIV-1. Chicago sky blue (CSB) was highly effective in the inhibition of HIV-1 with less toxicity to CEM-SS cells than most of the other sulfonated dyes tested. Synthesis of CSB was undertaken to produce a product greater than 98% pure and this compound was used to elucidate the possible mechanisms by which this class of structurally related compounds inhibits HIV-1. Addition of CSB to cells infected at high multiplicity at any time up to 24 h after infection, unlike dideoxycytidine (ddC) or oxathiin carboxanilide (OC), inhibited HIV-1-induced cell killing. Other postinfection time course studies revealed that CSB had to be present for 24 h or longer immediately after infection to be protective. Virus binding to cells occurred in the presence of CSB, but the requirement for virion envelope-cell membrane fusion was delayed. CSB was a potent inhibitor of the reverse transcriptase (RT) of both HIV-1 and HIV-2, although it was less active against HIV-2 in a cell killing-based assay. CSB also inhibited Rauscher and LP-BM5 murine leukemia viruses. CSB appears to disrupt the interaction between viral proteins and cell membranes, both in the fusion step early in the infection cycle and in the development of syncytia in the late stages of virus infection.


Predictors of HIV-1 disease progression in early- and late-stage patients: the U.S. Army Natural History Cohort. Military Medical Consortium for Applied Retrovirology.

HIV-infected individuals in both early and late stages of HIV disease were evaluated over 2 years to assess temporal trends and determinants of disease progression. The Walter Reed (WR) staging system was used to categorize patients into an early-stage cohort (WR Stages 1 and 2, N = 1183) and a late-stage cohort (WR Stage 5, N = 260) based on the initial clinical evaluation. Progression was defined as the occurrence of Stage 5 disease or beyond for the early cohort and Stage 6 disease or beyond for the late cohort. The cumulative incidence of progression was 15.7% (137 events) for the early-stage cohort, and 53.7% (85 events) for the late-stage cohort. Baseline CD4+ T lymphocyte (T4) count was the most significant marker of progression: 26% of WR Stage 1 or 2 patients with T4 lymphocytes below 500/mm3 progressed, compared with 12% with T4 lymphocytes at or above 500/mm3. In late-stage individuals, 83% with T4 lymphocytes under 200/mm3 progressed, compared with 27% with T4 lymphocytes at or above 200/mm3. Older age was associated with progression in both early- and late-stage groups. Differences in the rates of disease progression were not significant between blacks and whites or between men and women. Two-year rates of progression among the late-stage patients dropped from 78 to 47% between 1986 and 1988. This contrasted with progression rates in the early-stage cohort, which remained stable: 18% for those entering follow-up in 1986 and 17% for those entering follow-up in 1988. These data indicate a significant slowing of HIV disease progression rates and mortality rates among individuals with late-stage disease that is temporally associated with the increased availability and use of therapies. With control of T4 lymphocyte count, age, and calendar time, neither gender nor race was significantly associated with progression in either early- or late-stage patients.


HIV infection in American Indians and Alaska Natives: surveys in the Indian Health Service.

A network of surveys of HIV seroprevalence in American Indians and Alaska Natives (AI/AN) was begun in 1989. From July 1, 1989 through June 30, 1991, 37,681 serologic specimens were collected from prenatal and sexually transmitted disease patients in 58 facilities operated or funded by the Indian Health Service. Specimens from AI/AN women receiving initial prenatal care showed an overall HIV prevalence of 0.3/1,000, while specimens obtained during the third trimester of pregnancy showed an overall prevalence of 1.0/1,000. The rate for rural third trimester prenatal patients (0.9/1,000) was similar to that for urban patients (1.1/1,000). HIV rates among third trimester AI/AN patients in three western states were 4 to 8 times higher than rates observed in childbearing women of all races in those states. The overall HIV seroprevalence in AI/AN seeking care for sexually transmitted diseases was 4.5/1,000 for males (urban 10.8/1,000; rural 2.0/1,000) and 0.7/1,000 for females (urban 0.9/1,000; rural 0.6/1,000). Approximately 1,210 to 4,250 (midpoint of range = 2,730) AI/AN in the U.S. are projected from survey findings to be currently infected with HIV. The presence of HIV in multiple specimens from rural areas and the similarity of HIV infection rates for female patients from rural and urban locations provides evidence of diffusion of the HIV epidemic to rural AI/AN, and emphasizes the need for effective HIV prevention for this population.


HTLV-I DNA sequences in CNS tissue of a patient with tropical spastic paraparesis and HTLV-I-associated myelopathy.

Human T cell lymphotrophic virus type I (HTLV-I) is the etiologic agent of adult T cell lymphoma/leukemia (ATLL) and tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM). We studied an HTLV-I-seropositive, white man diagnosed in 1977 with ATLL and 10 years later, 6 months prior to his death, with TSP/HAM. Sections of brain, spinal cord, and visceral tissues were examined histologically, immunohistochemically, by in situ hybridization, and by the polymerase chain reaction (PCR). PCR amplification of a region of the polymerase (pol) gene of HTLV-I from visceral tissue demonstrated the presence of proviral HTLV-I DNA in paraffin-embedded sections from the liver and in DNA extracted from frozen sections of kidney and spleen, but failed to demonstrate viral sequences in paraffin sections of the lung and a lymph node. PCR analysis of CNS tissue demonstrated viral sequences in regions of the brain including frozen samples from cerebellum and cerebral cortex and paraffin sections of the thoracic spinal cord, but failed to detect proviral DNA in sections from a region in the lumbar cord. These results map the distribution of HTLV-I DNA sequences in the CNS of a patient with TSP/HAM for 3 months.


Envelope-specific antibodies in the saliva of individuals vaccinated with recombinant HIV-1 gp160.

HIV-1-specific antibodies have been detected in the saliva of seropositive individuals and may play a role in preventing oral transmission of the virus. We have analyzed saliva samples obtained from HIV-1-seronegative individuals who were immunized with various dosages of a recombinant HIV-1 envelope glycoprotein (gp160) vaccine for the presence of antibodies to HIV-1. Antibodies specific for envelope glycoproteins were detected in saliva from all of the volunteers, with those vaccinated with the higher doses of 640 and 1,280 micrograms showing the strongest responses. Peak salivary antibody titers were obtained 4-14 weeks after vaccination; they then gradually dropped in parallel with serum antibody titers. These envelope-specific antibodies were detected in whole saliva and in submandibular saliva but not in parotid saliva, suggesting that the source of antibodies in saliva is from serum transudation. The class of reactive antibodies was found to be IgG. The HIV-1-specific antibodies in the saliva of vaccinated individuals may offer local protection against HIV-1 infection.


HIV detection in culture of peripheral blood mononuclear cells: a relevant in vitro correlate of the antiviral effect of ZDV in vivo.

To better assess the antiviral effect of zidovudine (ZDV) in vivo and understand its limitations, we have studied human immunodeficiency virus (HIV) replication in peripheral blood mononuclear cell (PBMC) cultures from 25 ZDV-treated patients and 20 untreated controls. Three months after initiation of therapy, 9 of the 25 treated cases became negative for HIV isolation (36%). Untreated cases never converted to a negative culture. In patients treated by ZDV and who remained culture positive, the kinetics of HIV replication in PBMC culture was found to vary with time. A statistically significant delay in the production of HIV in PBMC cultures from treated cases could be demonstrated after 3 months of ZDV therapy, when compared with untreated patients. By contrast, in such untreated patients the time required to reach the peak of reverse transcriptase activity in culture decreased during the follow-up period. These changes of in vitro HIV replication kinetics as well as the change to negative culture during ZDV therapy probably reflected the reduction of the number of infected cells in vivo. These results as well as the decrease of p24 antigenemia do indicate that ZDV indeed inhibits HIV replication in vivo. However, the effect of ZDV on HIV replication kinetics in PBMC fails to reach significance at 6 months, suggesting that the antiviral effect of ZDV may decrease over time. Our results suggest that ZDV is most effective when the intensity of HIV replication in vivo is still low.


Family physician attitudes about HIV screening.

This study examined why and for whom family physicians are likely to require HIV screening according to general policies or procedures, based on a national survey of 2,660 family practice physicians. Of those contacted, 1,678 responded, yielding a response rate of 63.7%. The study also examined whether attitudes toward screening differed for physicians in different types of practices, of varying educational backgrounds, and with differing opinions regarding treating persons with AIDS (PWAs). The results showed that required screening was most strongly endorsed for pregnant women who had other risk factors and for i.v. drug users. Those physicians who most favored mandatory screening were also most likely to favor the mandatory reporting of AIDS cases to public health officials. They also had the least formal medical training (were not residency trained or board certified) and expressed the greatest apprehension regarding their own and their staff's preparedness and willingness to treat PWAs. A major implication of the findings is that family physicians and others who do mandatory testing should be provided opportunities, through residency training, board-certification preparation, or continuing medical education, to learn how to care for those patients they test who turn out to be seropositive.


[Synthesis of 1-trans-cinnamoyl- and 1-[trans-3-(pyridyl)acryloyl]-2-pyrrolidinone derivatives and their effect on hemicholinium-induced impairment of water maze learning in mice].

Various 1-trans-cinnamoyl-2-pyrrolidinones (6a-k) and 1-[trans-3-(pyridyl)acryloyl]-2-pyrrolidinones (9a-c) were prepared as analogues of the notropic agent, aniracetam (2), and their effects on hemicholinium-induced impairment of water maze learning in mice were examined. 1-(trans-3-Methoxycinnamoyl)-, 1-(trans-4-methoxycinnamoyl)- and 1-(trans-4-chlorocinnamoyl)-2-pyrrolidinones (6c, d, h) and 1-[trans-3-(3-pyridyl)acryloyl]-2-pyrrolidinone (9c) were found to be more active than aniracetam in these tests. In addition, these 2-pyrrolidinones (6a-k, 9a-c) were apparently observed to lengthen mouse survival time following hemicholinium injection into the brain.


[Protective effects of the fractions extracted from the callus of Acer nikoense Maxim. on alpha-naphthylisothiocyanate induced liver injury].

It has been reported that the methanol extract from the bark of Acer nikoense MAXIM. (AN) and the fractions separated from this extract showed the protection for liver injuries induced by carbontetrachloride (CCl4) in rat. On the other hand, the evidence manifested that the fractions of the callus of young branches from AN were recognized to have protective effects on the CCl4-induced liver injuries in rat. In this study, the protective activities of the fractions extracted from the callus of AN were investigated on the alpha-naphthylisothiocyanate (ANIT)-induced liver injuries in rat. The protectively active principles on the ANIT-induced liver injuries were found in the ether soluble fraction. The components of the protective effect were purified by repeated silica gel chromatographies. One of the protective substances in this fraction was identified to be beta-sitosterol. The protective fractions on the ANIT-induced liver injuries showed the protective effects on ANIT-induced cholestasis, but did not show cholagogic effects.


[Antipyretic effects of traditional Chinese medicines in bacterial endotoxin-induced febrile rabbits].

The antipyretic effects of oral administration of eight traditional Chinese medicines (dried extracts) were tested in febrile rabbits injected with bacterial pyrogen, lipopolysaccharide (LPS) 0.05 micrograms/kg (i.v.). The traditional Chinese medicines were given 0.6-2 g/10 ml/kg (p.o.) simultaneously with LPS. The most potent antipyretic effect was observed in Dai-jyoki-to (Ta-chen-chi-tang). The moderate antipyretic effects were observed in Toki-syakuyaku-san (Tang-kuei-shao-yao-san) and Syo-saiko-to (Hssiao-chai-hu-tang). Koso-san (Hsiang-su-san), Oren-gedoku-to (Huang-lien-chieh-tu-tan), Gorei-san (Wu-ling-san), Kakkon-to (Ko-ken-tang) and Byakkoka-ninjin-to (Pai-hu-chia-jen-sheng-tang) showed no effects.


[The depression of hepatic drug-metabolizing enzyme activity in rats with carrageenan-induced inflammation. II. The effect on the status of plasma antipyrine concentration in rats].

The variation of hepatic drug-metabolism was investigated in male Wistar rats bearing hind paw edema induced by carrageenan (1%, 0.1 ml, s.c.). The content of cytochrome P-450 and the biotransformation of aminopyrine in the hepatic 9000 x g supernatant (S-9) decreased after the subcutaneous injection of carrageenan to rat hind paw. In carrageenan-treated rats the plasma concentration of antipyrine after the administration of antipyrine (20 mg/kg, i.v.) was higher than that of control rats. In comparison with the control groups, the pharmacokinetic parameters of antipyrine in carrageenan-treated rats was altered. AUC0-200 and t 1/2 significantly increased and CLtot and ke significantly decreased. On the other hand, no change of the volume of distribution (Vd) was observed. The results indicate that the hepatic drug metabolism under the carrageenan-induced inflammation is inhibited both in vitro and in vivo.


[Determination and stability studies of urea in urea creams by high-performance liquid chromatography].

A method was developed for determining urea in urea creams by high-performance liquid chromatography using strong acidic cation exchange gel. Elution was carried out with 0.05 M phosphate buffer (pH 3.4) at 40 degrees C, and detection was made with a UV-spectrometer at 200 nm. This method was used for studying and comparing the stability of urea creams produced by ourselves and by other manufactures. The assay showed that there were differences in the stability of the tested creams.


[Study on the development of biological-active compounds after the model of natural products].

In reviewing my lifework, I wish to summarize the results of studies on the development of new biological-active compounds. The following subjects are discussed: 1) syntheses and biological activities of benzoxazole derivatives, 2) structure-activity relationships of phyllodulcin, a natural sweetner, and 3,4-dihydroisocoumarins for sweetness, 3) syntheses and structure-activity relationships of spiro[isocoumarin-piperidine] analogs for antiallergic activity, 4) design, structure-activity relationships, and a mechanism of antitumor action of bistropolone derivatives, 5) design, structure-activity relationships of fused tetracyclic quinolines for antitumor activity.


Action agenda for the 1990s. Reflections on directions for podiatric medical education.

The author reflects upon his experiences within podiatric medical education and shares his thoughts about the directions podiatric medical education should take before the end of the century. The author envisions a collaborative effort between the practitioners and educators within the profession in areas such as curriculum, research, academic health science centers, accreditation, and other important issues. The author calls for an education system relevant to and tested upon the anvil of patient care.


CCPM and podiatric medical education. Preparing for the 21st century.

There are many changes on the horizon that will affect how we teach the future practitioners of podiatric medicine. The author describes the processes undertaken to date at the California College of Podiatric Medicine (CCPM), as well as the vision of tomorrow, for podiatric medical education. The educational system that will result from these changes will be more efficient; it will better meet the needs of students; and, it will broaden their base of knowledge, all to improve care given to the patient.


Curriculum at the Scholl College. Toward mainstream medical education.

The Dr. William M. Scholl College of Podiatric Medicine in Chicago recently affiliated with a teaching hospital, the Illinois Masonic Medical Center, and used this alliance as a catalyst to effect a change in the clinical curriculum. The affiliation set up a joint venture to operate two clinics, one on Scholl College's traditional campus and one at the teaching hospital. At the hospital site, Scholl College students rotate through clinical externships in areas such as internal medicine, emergency medicine, and podiatric elective; podiatric and general medical residents assist in the tutelage of the students. At the Scholl College campus, beginning clinical students learn basic skills in a teaching clinic, then refine and further their skills in a comprehensive clinic under the guidance of faculty members. The faculty and administration at Scholl College have embraced the concept of mainstream medical education, and are striving to prepare podiatric physicians to practice 21st century medicine.


College of Podiatric Medicine and Surgery in Des Moines.

The College of Podiatric Medicine and Surgery was established in 1981 as a fully integrated college of the University of Osteopathic Medicine and Health Sciences, Des Moines, Iowa, becoming the only school in the profession to be part of an academic health science center. Thus, this college provides a unique opportunity for the students and the podiatric medical profession to receive a multidisciplinary education, preparing them for podiatric medical practice as an integral part of total health care.


Independent studies program in the basic sciences. A curricular innovation.

The Independent Studies Program was initiated at the New York College of Podiatric Medicine during the early months of 1987, and it has achieved continued success and acceptance from the administration, faculty, and students. The program emphasizes competence as the constant within a relatively time-independent framework. This paper presents, in a longitudinal fashion, the Independent Studies Program as a program model that combines criterion-referencing concepts, mastery-based learning combinations, and self-learning strategies. As a result, students have taken the initiative for diagnosing their learning needs, formulating their learning goals, identifying their human and material resources, and evaluating their learning outcomes.


Challenges and opportunities facing the Ohio College of Podiatric Medicine.

This article presents the problems and challenges facing the Ohio College of Podiatric Medicine in the view of its President and its Chief Academic Officer. It explores the progress made and the challenges facing the Ohio College overall and in the areas of education. It presents an exciting new vision of the style of podiatric medical education, and the methods that are and will be used to assess the quality of the educational program.


The future of podiatric medical education. Cautious optimism.

The leadership of the Pennsylvania College of Podiatric Medicine sets forth the following treatise on the outlook for podiatric medical education into the 21st century. Despite the seemingly impossible challenges facing the profession and its students, it is their opinion that the future is bright and with dedicated effort the profession will become stronger in the years ahead.


Accreditation and the enhancement of the quality of professional education.

The postsecondary accreditation process is a unique system that attempts to evaluate and enhance the quality of higher and professional education in the United States. Critics of accreditation see the process as coercive and a deterrent to academic freedom while others call for accreditors to exercise greater authority. The origination of the process was a result of a number of coinciding events involving the development of educational and professional standards and concern over access. The current focus on assessment of educational outcomes has further established the credibility of postsecondary accreditation and its role in protecting the public interest.


Relationship between fertilizing ability of frozen human spermatozoa and capacity for heparin binding and nuclear decondensation.

Nuclear decondensation of spermatozoa induced by heparin, reduced glutathione (GSH) or a mixture of heparin and GSH was studied using frozen-thawed human spermatozoa. The percentages of decondensed spermatozoa in controls and after treatment for 60 min with 30 mumol heparin l-1, 5 mmol GSH l-1, or heparin-GSH mixture were 1.5, 22.1, 4.3 and 37.6%, respectively. Most of the decondensed spermatozoa were eliminated by Percoll gradient centrifugation of samples previously treated with heparin or heparin-GSH mixture. However, comparable numbers of motile spermatozoa were recovered in the control and in each treated sample, demonstrating that a major proportion of motile spermatozoa was resistant to heparin (or heparin-GSH) effects on nuclear decondensation of spermatozoa. Fertilization of hamster oocytes was attempted using spermatozoa recovered in the 90% Percoll fraction and resistant to heparin-GSH decondensing mixture. Although insemination used a constant number of motile spermatozoa, fertilization rates were higher after treatments with heparin and GSH alone than in control or heparin-GSH-treated samples. In addition the number of spermatozoa that attached to the oocyte plasma membrane was a sixth or a half for sperm pretreated with heparin-GSH or heparin alone, respectively compared with untreated values. However, there was no evidence for induced acrosomal reaction by heparin and GSH, at least at the concentrations used. Qualitative analyses of heparin-binding sites were performed on untreated spermatozoa recovered in the 90% Percoll fraction by incubating spermatozoa in the presence of heparin covalently linked to albumin and coupled to colloidal gold (5 nm). Among this population of spermatozoa, 40.5% bound heparin-gold and labelling was mainly observed on the sperm head surface (88% of labelled spermatozoa) with (59.5%) or without (28.5%) tail labelling. Only a small proportion (23%) of spermatozoa that attached to the oocyte plasma membrane bound heparin-gold conjugate and only weak labelling was observed on the sperm head. Moreover, the proportion of spermatozoa that bound heparin-gold conjugate decreased (r = -0.77, P less than 0.0001) in relation to increasing concentrations of motile spermatozoa in the sample.(ABSTRACT TRUNCATED AT 400 WORDS)


Endogenous circannual rhythms and photorefractoriness of testis activity, moult and prolactin concentrations in mink (Mustela vison).

Mink are seasonal photosensitive breeders; testis activity is triggered when days have less than 10 h light. Increasing and decreasing plasma concentrations of prolactin induce the spring and autumn moults. In a 5 year experiment, males were maintained under short days (8 h light:16 h dark) at 13 degrees C or long days (16 h light:8 h dark) at 21 degrees C, winter and summer conditions, respectively. Under winter and summer conditions, circannual cycles of prolactin secretion and moulting were observed at intervals of about 11 months. Recurrence of testis cycles was not evident. In a second experiment, males were maintained under an 8 h light:16 h dark cycle from the winter solstice or under 10 h light:14 h dark, 12 h light:12 h dark or 14 h light:10 h dark cycles from 10 February. Under 8 h light:16 h dark cycle, testis regression was slightly later than under natural conditions, indicating photorefractoriness. However, mink remained sensitive to light: the longer the photoperiod, the faster the testis regression. In a third experiment, males were transferred under 8 h light:16 h dark or 16 h light:8 h dark from 15 May (group 1), 12 June (group 2) or 4 July (group 3); males submitted to long days received melatonin capsules on the day of transfer. Increasing concentrations of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and testis volume were shown by half the males in group 2 and nearly all the males in group 3; the constant release of melatonin from implants was more efficient than short days; but in the three groups, prolactin concentrations decreased in the few days after short-day or melatonin treatment. Overall, the results demonstrate endogenous circannual rhythms of prolactin secretion, body weight and moulting. Although a refractory period to short days was observed, the annual cycle of testis activity totally relies on the annual changes in daylength.


Role of the embryonic vesicle and progesterone in embryonic loss in mares.

Characteristics of spontaneous embryonic loss in 21 mares were compared with those of 52 contemporary mares that maintained pregnancy. Embryonic losses were, in retrospect, grouped according to day of loss and length of the interovulatory interval, respectively, as follows: group 1, less than or equal to day 20 and less than or equal to 30 days (n = 10); group 2, less than or equal to day 20 and greater than 30 days (n = 3); and group 3, greater than day 20 and greater than 30 days (n = 8); ovulation was day 0. Mean diameter of the embryonic vesicle in group 1 was smaller (P less than 0.05) on days 12-18 than in the pregnancy-maintained group, but among the pregnancy-maintained group and the embryonic-loss groups, the mean individual growth rates of vesicles was similar (no significant difference). A more frequent (P less than 0.05) location of the vesicles in the uterine body on day 13 in group 1 was due to a greater proportion of small vesicles and for day 18 was due to a greater incidence of fixation failure. Luteal regression occurred at the expected time in 77% of the mares with loss sooner than day 20. Low concentration of progesterone on days 12, 15 and 18, a detected decrease in diameter of the corpus luteum on days 15 and 18, and an interovulatory interval of less than or equal to 30 days indicated that luteolysis was not prevented by the embryonic vesicle in group 1.(ABSTRACT TRUNCATED AT 250 WORDS)


Factors influencing the rate of preantral and antral growth of mouse ovarian follicles in vitro.

The development of a culture system for individual mouse ovarian follicles using a low concentration of homologous serum, human follicle-stimulating hormone (hFSH) and a simple combination of growth factors is reported. Preantral follicles, 150 microns in diameter, with thecal cells attached were isolated mechanically. After 6-7 days on a Millicell membrane, a high proportion of the preantral follicles cultured individually with hFSH grew to morphologically normal large antral follicles (400-500 microns in diameter) with high oestradiol secretion. Without hFSH, the follicles grew to approximately 275 microns diameter in 6 days, but did not form antra or secrete oestradiol. The growth trajectory (overall pattern of growth formed by daily measurements of diameter) of each follicle was recorded and used as a measurement of response to experimental variation of culture conditions. The rapidly growing follicles were morphologically normal, but those that grew more slowly showed some abnormality or atresia and secreted less oestradiol. Follicles cultured in groups without being in direct contact with each other showed much poorer growth than those grown individually, but the inhibition was not uniform and some follicles grew larger than others in the group. Follicles that contacted each other directly in culture tended to fuse into one mass and their growth was substantially inhibited. Even under these conditions, one follicle often continued to grow slowly while the others degenerated. Such alteration of growth patterns suggests interfollicular paracrine control and may be a means of three-dimensional spacing of follicle growth within the ovary, as well as part of the mechanism of follicle selection. The dose-response curve based on the mean growth trajectory of follicles cultured individually, produced increasing rates of growth with 12.5-100 miu hFSH ml-1. Higher concentrations of hFSH did not increase growth rate further, but oestradiol secretion continued to increase with increasing hFSH up to the maximum used (2000 miu ml-1).


Pregnancy rates and births after unilateral or bilateral transfer of bovine embryos produced in vitro.

Late morulae and blastocysts produced in vitro were nonsurgically transferred to heifers by unilateral (n = 184) or bilateral (n = 94) transfer. Of the recipients, 58% had serum progesterone values greater than 1.4 ng ml-1 on day 21 and rectal palpation on day 35 showed that 50% (138 of 278) were pregnant. The embryonic mortality rate between days 21 and 35 was estimated to be about 14% and between days 36 and 90 to be about 12%. Of the animals, 8% aborted between days 91 and 250 of pregnancy. No difference was observed in pregnancy rates between unilateral transfer of one (47%) or two embryos (49%) and bilateral transfer (53%), or in the twinning rate between bilateral transfer (42%) and unilateral transfer of two embryos (33%). The pregnancy rate was 54% with embryos evaluated as morphologically excellent or good, 51% with fair embryos and 26% with poor ones. A higher pregnancy rate (60%) was obtained after embryo transfer when the synchrony between recipient and embryo was -1 day.


Follicular steroidogenesis and oocyte maturation after superovulation of goats (Capra hircus) with gonadotrophins.

Follicles were sampled at three different times after treatment with 1200 iu pregnant mares' serum gonadotrophin (PMSG) or 12 mg ovine follicle-stimulating hormone (FSH), and from untreated control animals. The meiotic status and protein synthesis of the oocyte from each follicle was determined and correlated with the intrafollicular concentration of oestradiol and progesterone. Significantly higher amounts of oestradiol were present in PMSG-treated animals at sponge withdrawal than in FSH-treated and control goats. Twenty hours later, both oestradiol and progesterone concentrations in the PMSG group were higher than those in the FSH group, and were equivalent to control animals at the onset of oestrus. At 18 h after the administration of human chorionic gonadotrophin (hCG), oestradiol decreased markedly in all three treatment groups, whereas progesterone remained significantly higher in PMSG-treated follicles. Although these high concentrations of intrafollicular steroids were associated with a higher incidence of premature condensation of chromatin in oocytes, the two events were not causally related. Moreover, cytoplasmic maturation was not prematurely activated in these oocytes and a changed pattern of protein synthesis was observed in oocytes from all three treatment groups after the hCG injection. Whereas disturbances in follicular steroidogenesis of oestradiol and progesterone occur in vivo in goats superovulated with PMSG, they do not underlie the premature activation of the initial stages of nuclear maturation in oocytes but are associated with normal cytoplasmic maturation.


Polarized functions and permeability properties of rat epididymal epithelial cells in vitro.

Cultured rat caput and cauda epididymidal epithelial cells are shown to exhibit polarized properties characteristic of functioning epithelia. When grown on plastic substrates coated with reconstituted basement membrane, confluent monolayers of cells from both regions formed domes characteristic of other transporting epithelia. Immunocytochemical localization of three proteins characteristically associated with epithelial junctional complexes revealed that uvomorulin, zonula occludens 1 and cingulin were present in cultured epididymal epithelial cells and that their distribution was similar to that in the epididymal epithelium in vivo. These three molecules were not found in epididymal stromal cells. Cells from both regions growing in two compartment chambers developed an electrical resistance across the monolayer with a magnitude characteristic of high resistance epithelia. The optimal plating density of cells was 0.75 x 10(6) cells cm-2. The presence of reconstituted basement membrane on the filters did not affect the resistance of the cells. Inulin passage from basal to apical chambers was less than 2% over 24 h. These results show that several polarized functions of epididymal epithelial cells can be maintained in culture and that this type of culture system is useful for studying the function of the epididymis in vitro.


Evidence for immunoreactive relaxin in boar seminal vesicles using combined light and electron microscope immunocytochemistry.

Light-microscope immunocytochemistry using the peroxidase-antiperoxidase technique and a polyclonal rabbit antiserum raised against purified porcine relaxin showed that cytoplasmic immunostaining for relaxin could be visualized in the epithelial cells of the seminal vesicle. No relaxin immunoreactivity was seen in the testis, epididymis, ductus deferens, prostate or bulbo-urethral gland. A ten times higher concentration of porcine relaxin antiserum was necessary to achieve immunostaining in the seminal vesicle comparable to that in the corpora lutea of pregnant sows. Ultrastructural examination showed that the epithelial cells of the boar seminal vesicle resembled typical protein-secreting cells with prominent rough endoplasmic reticulum and well-developed Golgi apparatus. The most striking feature of these cells was the accumulation of granules with a limiting membrane, which ranged from 200 to 600 nm in diameter and contained flocculent material of moderate electron density. Electron-microscope immunocytochemistry using the protein A-gold technique and relaxin antiserum demonstrated that the granules were the only intracellular organelles that showed immunoreactivity for relaxin. These results indicate that a relaxin-like substance is present in boar seminal vesicles and that the subcellular site of its localization is the granules, suggesting that the seminal vesicle produces and stores a relaxin-like substance, but that it is present at much lower concentrations than in the corpora lutea of pregnant sows.


Development of rabbit preimplantation blastocysts cultured with precultured endometrial tissue.

Endometrial fragments were explanted from pseudopregnant rabbits 4.5 days after injecting with human chorionic gonadotrophin and were precultured for 2 days in suspension culture in the presence of oestradiol and progesterone equivalent to concentrations in rabbit serum at that stage. Preimplantation blastocysts were obtained at day 6.5 of pregnancy and cultured in the presence or absence of precultured endometrial fragments. Attachment of the trophoblast to the endometrium was prevented by continuous agitation. After 2 and 3 days, specimens were monitored for development in vitro using light and scanning electron microscopy. Although the development of blastocysts was slower in vitro than in vivo in both groups, development was clearly superior in the presence of precultured synchronous endometrial fragments. In the absence of endometrium, the embryonic anlage appeared disordered, particularly in the caudal region, but in the presence of uterine tissue the blastocysts developed much better. Up to nine somites were differentiated; the neural tube had started to close and the various parts of the brain anlage showed incipient differentiation. Syncytiotrophoblast differentiated in the presence or absence of endometrium in the embryonic and abembryonic hemispheres, but typical patterns were maintained better and cell degeneration was less frequent during co-culture. Although the culture model described here has not been optimized using criteria of blastocyst differentiation, the results suggest that culture of blastocysts with precultured synchronous endometrial fragments is advantageous.


Role of androgens in survival of spermatozoa in epididymis of tammar wallaby (Macropus eugenii).

Studies of undiluted micropuncture samples of luminal fluid from the cauda epididymidis of the tammar indicated that spermatozoa are immotile in situ and spontaneously activate during collection or subsequent incubation in vitro. The suppression of sperm motility was related to the androgen status of the tammars and when this was increased by the use of Silastic implants of testosterone propionate, the spontaneous activation of samples was delayed for up to 2 h during incubation in vitro. Spermatozoa survived for up to 9 weeks when isolated in the cauda epididymidis between ligatures around the ductus. However, even after isolation for 3 weeks their viability was reduced compared with samples from the contralateral, unligated duct. Isolation of a length of ductus between ligatures also reduced the concentration of spermatozoa in the lumen of the duct and reduced the concentration of some proteins in the epididymal plasma. However, it did not affect the electrophoretic pattern of detergent extracts of spermatozoa. A study of the effects of orchidectomy and testosterone therapy indicated that sperm survival in the epididymis is androgen dependent. Orchidectomy reduced the concentration of spermatozoa in the luminal fluid and the volume of luminal fluid, and resulted in an increase in the concentration and a change in the electrophoretic pattern of protein in the fluid. The effects of orchidectomy were reduced or prevented by testosterone therapy. It is concluded that the cauda epididymidis of the tammar is at least as well adapted for sperm storage as it is in the eutherian mammals that have been studied.


Changes in the properties and composition of zona pellucida of pigs during fertilization in vitro.

Several hundred fertilized pig eggs were prepared by an in vitro fertilization (IVF) technique in which follicular phase ovarian eggs were matured in vitro to metaphase II before incubation with capacitated epididymal spermatozoa for 12 h at 39 degrees C. Parthenogenetic eggs were also prepared by stimulation of the mature eggs with an electric pulse. The zonae were solubilized with 0.2% pronase/phosphate-buffered saline (PBS) or lactic acid/PBS. The time taken for solubilization was 30-40% shorter than for unfertilized eggs, indicating that zona hardening was induced during fertilization. At the same time, the sperm receptor activity of the zona was reduced. Electrophoretic analyses of zona glycoproteins from the ovarian, mature and fertilized eggs revealed that the amount of 90 kDa proteins decreased substantially during fertilization. This fraction could barely be detected in the zonae from parthenogenetic eggs. However, modification with a fluorescent probe showed that the general architecture of the zona remained unchanged during fertilization. These results suggest that the minor 90 kDa proteins are specifically degraded by the protease(s) released from the oocyte at fertilization, thereby leading to the block to polyspermy.


Hormonal correlates of 'masculinization' in female spotted hyaenas (Crocuta crocuta). 1. Infancy to sexual maturity.

This report is concerned with hormone concentrations accompanying sexual maturation in a highly 'masculinized' female mammal, the spotted hyaena, Crocuta crocuta. Plasma concentrations of testosterone, androstenedione and oestrogen were determined by radioimmunoassay in a longitudinal study of 12 female and eight male hyaenas 2.5-62.5 months old. Concentrations of testosterone were significantly higher in males than in females after 26.5 months of age, but earlier measurements did not differ between sexes. Mean testosterone concentrations in adult female hyaenas (0.4-0.5 ng ml-1) were similar to those in several other female mammals that do not display a 'masculine' profile, but mean concentrations of androstenedione (2.5-5.5 ng ml-1) in female hyaenas were significantly higher than in males (1.0-2.0 ng ml-1), at most ages. Oestrogen could not be detected (less than 0.03 ng ml-1) in females until about 14 months of age and then increased (to approximately 0.13 ng ml-1) between 18 and 30 months; oestrogen remained undetectable in males. This rise in oestrogen in females corresponded to nipple enlargement and to changes in the size and elasticity of the urogenital meatus, permitting copulation and parturition through the clitoris. Gonadectomy (two males and four females) at 4-7 months resulted in nondetectable concentrations of testosterone and oestrogen and a marked attenuation in androstenedione (to approximately 0.39 ng ml-1), indicating that the gonads are the major source of these three steroids. Gonadectomy also eliminated sex differences in weight, nipple development and elasticity of the urogenital meatus.


Hormonal correlates of 'masculinization' in female spotted hyaenas (Crocuta crocuta). 2. Maternal and fetal steroids.

Concentrations of androgens (androstenedione, testosterone, 5 alpha-dihydrotestosterone), oestrogen and progesterone were measured in relation to pregnancy in the spotted hyaena (Crocuta crocuta). The gestation period was estimated to be about 110 days. There was a marked progressive rise in all the steroids starting in the first third of gestation. Chromatographic separation of plasma showed that much of the oestrogen is not oestradiol (only 12% of total measured) and that a significant fraction of the 'testosterone' may be dihydrotestosterone. In the final third of pregnancy, concentrations of androgen (especially testosterone plus dihydrotestosterone) in the female circulation reached the maximal values of adult males; the percentage of dihydrotestosterone relative to total testosterone plus dihydrotestosterone was higher in females (44 +/- 3.9%, n = 20) than in males (29.5 +/- 3.5%, n = 17). Plasma androstenedione was also significantly higher in females, but the increment was less than for oestrogen, testosterone and progesterone, and the temporal pattern was less clear. Samples from the maternal uterine and ovarian circulation showed that androstenedione is largely of ovarian origin and metabolized by the placenta, while testosterone, progesterone and oestrogen are primarily of placental or uterine origin. Fetal samples were taken from two mixed-sex sets of twins and one male singleton. Gradients across the placenta measured in the fetal circulation confirmed that the placenta metabolizes androstenedione and is a source of testosterone for the female fetus; there were no consistent differences in androgens between male and female fetuses. It is suggested that the conspicuous masculinization of the female spotted hyaena, especially evident in the external genitalia at birth, is a result, at least in part, of high placental production of testosterone or dihydrotestosterone derived from the metabolism of high maternal androstenedione.