Vitamin B6-responsive and -unresponsive cystathioninuria: two variant molecular forms.

Cystathionase activity in a lymphoid cell line extracts from a vitamin B6-responsive patient with cystathioninuria was increased strikingly by pyridoxal phosphate. Immunodiffusion with antiserum to human hepatic cystathionase showed identity between this cystathionase protein and cystathionase from an extract of normal lymphoid cells. Neither an increase in cystathionase activity nor immunochemical identity was found using extract of cells from a B6-unresponsive patient.

Characterization of the molecular defect in infantile and adult acid alpha-glucosidase deficiency fibroblasts.

Different clinical expressions of acid alpha-glucosidase deficiency have been described. The present study was undertaken to investigate the basic metabolic defect in the infantile and adult forms of the disease. Acid alpha-glucosidase (EC 3.2.1.20) was purified from normal and from adult acid alpha-glucosidase deficiency fibroblasts. The pH optimum; Michaelis constant; electrophoretic mobility in starch; thermal denaturation at pH 4.0 and 7.0; and inhibition by turanose, alpha-methylglucoside and trehalose were the same in purified enzyme from normal and mutant cells. Placental acid alpha-glucosidase was purified to, or near, homogeneity. Monospecific antibodies raised against the enzyme in each of three enzyme peaks obtained from the last purification step were found to cross-react with the enzyme of all three peaks, and with purified, normal fibroblast enzyme. Cross-reacting material (CRM) also was identified in fibroblast lysates from normal subjects and from both forms of acid alpha-glucosidase deficiency. The amount of CRM in the adult form appeared to be significantly less than in normal cells or cells from the infantile form. Enzyme activity was demonstrated in the immune complexes of the normal and adult acid alpha-glucosidase deficiency fibroblasts, but not of the infantile form. Competition for antibody binding sites was observed between normal and both types of mutant enzymes. The findings indicate that this case of infantile acid alpha-glucosidase deficiency is the result of a structural gene mutation which causes the synthesis of a catalytically inactive (CRM-positive) enzyme protein. It appears that in the adult form, the mutation causes a reduction in the amount of the enzyme protein present in the cells.

Cell-specific differences in membrane beta-glucosidase from normal and Gaucher cells.

Two isozymes of membrane-bound beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) with activity towards 4-methylumbelliferyl-beta-D-glucopyranoside have been identified in human cells. One of these isozymes was found to have a pH optimum of 5.0, a Km of 0.4 mM and to be rapidly inactivated at pH 4.0 ("acid-labile"). The second isozyme had a pH optimum of 4.5, a Km of 0.8 mM and was stable at pH 4.0 ("acid-stable"). Cultured long-term lymphoid lines and peripheral blood leukocytes contained both isozymes while cultured skin fibroblasts contained only the "acid-stable" form in detectable amounts. The specific activity of the "acid-stable" isozyme was severely reduced in cultured skin fibroblasts, cultured long-term lines and peripheral leukocytes from patients with Gaucher's disease. The specific activity of the "acid-labile" enzyme in the latter two cell types was apparently unaffected. The beta-glucosidase activity in all three cell types examined was predominantly particulate but the enzyme could be solubilized with low concentrations of Triton X-100. The solubilized enzyme required sodium taurocholate (0.2%) for maximum activity. Solubilized beta-glucosidase did not exhibit the cell-specific differences in pH optimum and Km shown by the membrane-bound enzyme.

Increased activity of lysosomal enzymes in the peritoneal fluid of patients with gynecologic cancers and pelvic inflammatory disease.

PURPOSE: To investigate whether the activity of lysosomal enzymes is increased in the peritoneal fluid of patients with gynecologic cancers compared to activity in the peritoneal fluid from normal subjects and those with pelvic inflammatory disease, and fluid from benign ovarian cysts. PATIENTS AND METHODS: beta-glucuronidase, beta-galactosidase, and alpha-mannosidase activity was measured in the peritoneal fluid from patients with gynecologic cancer, pelvic inflammatory disease, and normal subjects, and fluid from benign ovarian cysts. RESULTS: The mean+/-SD of beta-glucuronidase, beta-galactosidase, and alpha-mannosidase activity in the gynecologic cancers was 120+/-50 nmol, 203+/-86 nmol, and 240+/-119 nmol 4-methylumbelliferone/ml/h, respectively; in the normal control subjects it was 22+/-9 nmol, 46+/-10 nmol, and 80+/-23 nmol, respectively (P=0.00003, 0.0001, and 0.0001, respectively). The activity was increased even in cases without malignant cells in the peritoneal fluid. In pelvic inflammatory disease it was 148+/-82 nmol, 278+/-112 nmol, and 291+/-140 nmol, respectively. The activity in the fluid of the ovarian cysts was similar to that of the normal peritoneal fluid. There was a significant positive correlation between enzyme activity and stage of cancer, that was stronger for beta-glucuronidase (r=0.889, P=0.003). CONCLUSION: The increased lysosomal enzyme activity in gynecologic cancers, without overlapping between patients and normal subjects or benign ovarian cyst fluid, indicates that such measurements might be applied for diagnostic purposes.

Effect of birth weight and maternal smoking on cord blood leptin concentrations of full-term and preterm newborns.

Prematurity, maternal smoking, and low birth weight each result in neuroendocrine dysfunction and increased perinatal morbidity and mortality. Leptin, an adipocyte-secreted protein, has provided the first physiological link to the regulatory system controlling starvation-induced neuroendocrine changes in rodents. This study investigated whether leptin concentrations were detectable in cord blood of newborns, and assessed the effect of birth weight, prematurity, and maternal smoking on cord blood leptin concentrations. Fifty consecutively enrolled full-term and 12 preterm newborns born to mothers who smoked during pregnancy were compared to 50 full-term and 12 preterm newborns born to parents who were nonsmokers. RIA for leptin was performed using cord blood samples collected immediately after birth. Leptin concentrations were detectable in newborns and correlated positively with obesity (full-term, r = 0.30, P < 0.01; preterm, r = 0.47, P < 0.05). Maternal smoking during pregnancy was associated with decreased leptin concentrations in the cord blood of both full-term and preterm newborns. This effect was independent of obesity (full-term newborns: 5.25 +/- 2.48 vs. 4.21 +/- 2.71 ng/ml, P = 0.01) and was more pronounced in premature newborns (5.67 +/- 3.6 vs. 2.46 +/- 2.03, P = 0.02), and its magnitude in full-term newborns was directly related to the reported number of cigarettes the mothers of the full-term newborns smoked per day (r = -0.438, P < 0.001). Thus, low birth weight and maternal smoking are both associated with decreased leptin concentrations, and these effects are more pronounced in premature newborns. Future studies will be needed to determine whether administration of leptin might reverse the neuroendocrine dysfunction caused by maternal smoking.

Accumulation of ganglioside Gm2 in cerebrospinal fluid of a patient with the variant AB of infantile Gm2 gangliosidosis.

Brain biopsy has been used for the diagnosis of the variant AB of infantile GM2 gangliosidosis. Accumulation of ganglioside GM2 (300 ng of neuraminic acid per milliliter) was observed in the CSF of a patient with this disorder. GM2 was found also in the CSF of a patient with classic Tay-Sachs disease. Normal CSF did not contain any measurable amounts of GM2. In addition, a glycolipid with a mobility, by thin-layer chromatography, similar to that of paragloboside was observed in the CSF of the patient with the variant AB of GM2 gangliosidosis. These findings indicate that the variant AB can be diagnosed by demonstrating accumulation of GM2 in the CSF of patients with normal hexosaminidase activity.

Fucosidosis type 2.

Two siblings, 9 and 4 1/2 years old, had alpha-L-fucosidase deficiency, angiokeratoma, progressive psychomotor retardation, neurologic signs, coarse facila features, and dysostosis multiplex. It appears that genetic heterogeneity is present in fucosidosis; there are at least two types. In type 1, patients have no vascular lesions, but have rapid psychomotor regression, severe and rapidly progressing neurologic signs, elevated sodium and chloride excretion in the sweat, and fatal outcome before the sixth year. In type 2, patients have angiokeratoma, milder psychomotor retardation and neurologic signs, longer survival, and normal salinity in the sweat. Quantitative studies on erythrocytes and in saliva disclosed severely increased expressions of Lea and Leb. Biopsies of skin and gingiva showed alterations as seen in angiokeratoma. There was also evidence of lysosomal storage in vascular endothelium, eccrine sweat gland epithelium, and fibroblasts of the skin.

Alpha-L-fucosidase in cultured bone marrow fibroblasts from fucosidosis patients.

Bone marrow fibroblasts were cultured from two patients with fucosidosis type 2, six control subjects, and three patients with other lysosomal disorders. Optimal conditions for measuring alpha-L-fucosidase activity in lysates of these cells with the fluorogenic substrate 4-methylumbelliferyl-alpha-L-fucoside were established. The pH profile of normal bone marrow fibroblasts showed three peaks and a shoulder of enzymatic activity, with maximum activity at pH 4.75. In cells derived from fucosidosis patients two peaks of apparent alpha-L-fucosidase activity were obtained; the pH optimum was 4.5. alpha-L-Fucosidase activity (mean +/- SD) in the fucosidosis and control bone marrow fibroblasts was 2.5 and 312.4 +/- 10.9 nmoles 4-methylumbelliferone per milligram protein per hour, respectively. A reduction in the apparent specific enzymatic activity in the fucosidosis cells was observed by using increasing concentrations of cellular protein in the assay system. Mixing experiments between normal and fucosidosis cells gave the expected activities. These findings indicate that cultured bone marrow fibroblasts can be used for the diagnosis and study of fucosidosis.

Ureaplasma urealyticum colonization of full term infants: perinatal acquisition and persistence during early infancy.

In a prospective study 225 (35%) of 640 pregnant women who delivered at term had vaginal colonization with Ureaplasma urealyticum at the time of delivery. One hundred ninety-three full term infants born to U. urealyticum-colonized mother were cultured from the throat, eyes and vagina within the first 3 days of life. One hundred seven infants (55%) had at least one culture site positive for U. urealyticum (throat 41%, eyes 20%, vagina 40%). Rupture of membranes for greater than or equal to 12 hours and the mode of delivery did not affect vertical transmission of U. urealyticum. We were able to follow 108 infants during the first 3 months of life. Sixty-eight, 33 and 37% of the infants who were initially colonized with U. urealyticum in the throat, eyes and vagina, respectively, were still colonized when the follow-up cultures were obtained 3 months later. Fourteen of the 108 infants whom we followed developed a lower respiratory tract illness. In the pharyngeally colonized infants there was no increased risk for lower respiratory tract illness during early infancy compared with the pharyngeally noncolonized infants.

Sterile cerebrospinal fluid pleocytosis in young infants with urinary tract infection.

BACKGROUND: During the first 3 months of life febrile infants are subjected to sepsis workup, which includes evaluation for urinary tract infection (UTI) and meningitis. We investigated the existence of concomitant meningeal inflammation in infants younger than 90 days old affected with UTI. METHODS: We reviewed the medical records of all infants younger than 90 days old, who were hospitalized for UTI from January, 1990, to January, 2001. For the diagnosis of sterile cerebrospinal fluid (CSF) pleocytosis, the child's age, the CSF total white blood cell (WBC) count and the CSF absolute neutrophil count were taken into consideration. CSF pleocytosis was defined as the presence of > or = 35, > or = 21 and > or = 15 WBC/mm3 of CSF during the first, second and third month of life, respectively. The CSF Gram-stained smear, latex agglutination test and bacterial culture were negative. RESULTS: Sterile CSF pleocytosis was found in 15 (12.8%) of 117 infants with UTI who had had a lumbar puncture included in their initial laboratory evaluation. The 15 infants had a median age +/- semiinterquartile range of 40 +/- 25 days (range, 4 to 75 days). In these infants the median CSF WBC count +/- semiinterquartile range was 55 +/- 125/mm3 (range, 21 to 1,270/mm3). CONCLUSIONS: Sterile CSF pleocytosis was found in 12.8% of infants younger than 90 days old with UTI. The pathogenesis of this meningeal inflammation is not fully understood. Although bacterial infection of the subarachnoid space, with low bacterial seeding, cannot be excluded, at least in some cases, it is possible that CSF pleocytosis in some of the infants with UTI is mainly caused by the endotoxin of Gram-negative or other inflammation-inducing molecules of Gram-positive urine pathogens.

High prevalence of erythromycin resistance of Streptococcus pyogenes in Greek children.

BACKGROUND: Macrolide resistance among Streptococcus pyogenes strains is increasing in many European countries. Greece was not considered a country with high prevalence of macrolide-resistant S. pyogenes strains, and until now the genetic mechanism of resistance was unknown. METHODS: During the 25-month period from December, 1998, to December, 2000, pharyngeal cultures for S. pyogenes were performed on 743 Greek children with the clinical diagnosis of pharyngitis. The children were 1 to 16 years old (median age, 7 years) and were living in Central and Southern Greece. S. pyogenes isolates were tested for their susceptibility to erythromycin, clarithromycin, azithromycin, clindamycin, penicillin G, amoxicillin/clavulanate and cefprozil. The erythromycin-resistant isolates were further studied for their genetic mechanism of resistance by means of PCR. RESULTS: Of a total of 275 S. pyogenes isolates recovered, 105 (38%) were erythromycin-resistant (MIC > or = 1 microgram/ml) [corrected], with 54, 45 and 1% of them carrying mef(A), erm(A) [subclass erm(TR)] and erm(B) gene, respectively. The prevalence of erythromycin-resistant strains was 29 and 42% during the time periods December, 1998, to December, 1999, and January, 2000, to December, 2000, respectively. All erythromycin-resistant isolates were also resistant to clarithromycin and azithromycin. The isolates carrying the erm(A) gene were inducibly resistant to clindamycin. The 275 S. pyogenes isolates had ceprozil MICs < or = 0.032 microgram/ml. CONCLUSIONS: The current high (38%) prevalence of erythromycin-resistant S. pyogenes in Central and Southern Greece requires continuous surveillance and careful antibiotic policy.

Ocular abnormalities in patients with beta thalassemia.

We examined 29 patients with homozygous beta thalassemia. The mean age of the patients was 15.6 +/- 8.9 years. Twelve patients (mean age, 02.0 +/- 10.4 years) had one or more ocular abnormalities. Five patients had degeneration of the retinal pigment epithelium, one had lens opacities, two had lens opacities and degeneration of the retinal pigment epithelium, one had vascular abnormalities and degeneration of the retinal pigment epithelium, one had angioid streaks, lens opacities, and degeneration of the retinal pigment epithelium, and two had angioid streaks and degeneration of the retinal pigment epithelium. These abnormalities were observed in patients with both forms of beta thalassemia, major and intermedia. The frequency of the ocular abnormalities increased with age. The youngest patient with an ocular abnormality was 6 1/2 years old. There was no correlation between the abnormalities observed and the serum ferritin level, the mean hematocrit value, and the dose of deferoxamine given to the patients.

Acid alpha-glucosidase deficiency in cultured fibroblasts with phenotype 2 of acid alpha-glucosidase.

The enzyme acid alpha-glucosidase shows a polymorphism determined by two alleles, namely phenotypes 1, 2-1 and 2. We report that the activity of acid alpha-glucosidase, measured in fibroblasts homozygous for isozyme 2 is significantly reduced. In fibroblasts harvested at early confluency the activity in the strain with phenotype 2 of acid alpha-glucosidase was 23% and 5% compared to the levels in normal phenotype 1 strains using maltose and glycogen as substrate, respectively. At late confluency, the same fibroblasts showed an increase in activity to 60% and 15% of normal, respectively. At both early and late confluency, two fibroblast strains with the adult form of acid alpha-glucosidase deficiency had activity of approximately 7% of the activity in normal phenotype 1 fibroblasts with both substrates. In general, an increased amount of glycogen was measured in deficient fibroblasts, including the strain with phenotype 2, but overlapping was observed with normal cells. These findings show that fibroblasts homozygous for the isozyme 2 allele have a reduced ability to hydrolyse the natural substrate, glycogen.

Correlation of leucocyte count and erythrocyte sedimentation rate with the day of illness in presumed bacterial pneumonia of childhood.

We investigated the effect of the duration of illness on the white blood cell (WBC) and total neutrophil counts and the erythrocyte sedimentation rate (ESR) in untreated children with clinical and roentgenographic findings compatible with bacterial pneumonia. According to the duration of illness before admission, the patients were divided into: Group I, 48 patients ill for < 24 h; Group II, 39 patients ill for 24-48 h; Group III, 21 patients ill for 48-72 h; and Group IV, eight patients ill for 72-96 h. In children with presumably bacterial pneumonia the number of the WBC was greater during the first 2 days of illness. Thereafter, the leucocyte count declined, reaching the lowest levels on the fourth day. A similar course was followed by the absolute number of total neutrophils. During the second day of illness, 92% and 72% of the patients had leucocyte counts > 10,000 and > 15,000/mm3, respectively, whereas on the fourth day of illness only half of the patients had > 10,000 and one-quarter > 15,000 WBC/mm3. The ESR followed an opposite course to that of the WBC. During the first day of illness it was normal or mildly elevated, increasing steadily thereafter. The validity of the WBC and total neutrophil counts in conjunction with the ESR in the evaluation of bacterial pneumonia is augmented when the day of illness is taken into consideration.

Incidence of scoliosis in beta-thalassemia and follow-up evaluation.

STUDY DESIGN: One hundred fifteen of 120 patients with beta-thalassemia followed in the thalassemia unit were studied for the presence of scoliosis. Forty-nine of these patients were reevaluated 1 year later. OBJECTIVES: To determine the frequency and the course of scoliosis in beta-thalassemia and to compare the findings with those of patients with idiopathic scoliosis. SUMMARY AND BACKGROUND DATA: There is only one report indicating increased frequency of scoliosis in a limited number of patients with thalassemia. In this study, the authors assessed the frequency of scoliosis in a large sample of patients and followed the evolution of this spinal deformity. METHODS: Patients with beta-thalassemia aged 3-35 years were examined clinically and radiologically for scoliosis. Forty-nine of them were reexamined 1 year later for determination of the evolution of scoliosis. RESULTS: Lateral curves of at least 5 degrees Cobb were found in 77 patients (67%), with a male-to-female ratio of 0.9. Scoliosis of at least 10 degrees was found in 21.7% of the male and 20% of the female patients with thalassemia. The ratio was 1.18 for curves of at least 10 degrees and 0.77 for curves of a smaller magnitude. The most common curve pattern was the left lumbar (35.1%), followed by the double-curve pattern (16.9%). Forty-nine randomly selected patients (42.6%) of the 115 included in the study were reexamined 1 year later. Seven male and 7 female patients (total, 28.6%) showed a progression of at least 5 degrees. Six patients (12.2%) experienced spontaneous improvement of less than 6 degrees. The pattern and the evolution of scoliosis observed in patients with beta-thalassemia differ from those found in Greek children with idiopathic scoliosis. CONCLUSIONS: The findings of this study show that the incidence, evolution, and etiology of scoliosis in beta-thalassemia differ from those of idiopathic scoliosis, indicating that the spinal deformities in patients with beta-thalassemia represents a distinct type of scoliosis. Longer follow-up is needed to investigate the natural history of this type of scoliosis.

Cefuroxime axetil in the treatment of acute otitis media in children.

Cefuroxime axetil was evaluated for clinical efficacy and tolerance in the treatment of acute otitis media in children. Fifty-five children, 5.0 to 10.8 years, were randomly assigned to receive 250mg cefuroxime axetil every 12 hours or 50mg/kg/day amoxicillin in three divided doses. Both treatment schemes lasted for 10 days. Acute otitis media was diagnosed by the presence of erythema and/or opacity with bulging of the tympanic membrane. A tympanogram was obtained upon enrollment to the study, as well as within 2 days after completion of therapy. Fifty-one children completed the treatment protocol. The cure rate was 74.1% for cefuroxime axetil and 75.0% for amoxicillin. Clinical improvement was noted in 25.9% of children treated with cefuroxime axetil and in 25.0% of those treated with amoxicillin. There was no clinical failure in the two treatment groups. None of the children experienced relapse of acute otitis media during the month following completion of therapy. The findings indicate that cefuroxime axetil given twice daily has comparable efficacy to amoxicillin given three times daily in the treatment of children with acute otitis media.

Maternal smoking during pregnancy improves the anatomy of the hip joint in the female neonate.

OBJECTIVE: Because the greater frequency of developmental dysplasia of the hip in the female could have an endocrine etiology, and because maternal smoking in pregnancy causes fetal endocrine disequilibrium, we investigated the anatomy of the hip in neonates of smoking and non-smoking mothers. METHODS: Clinical and sonographic examination was performed on 2066 hips of 521 male and 512 female neonates. In 48 male and 53 female neonates, the mothers smoked during pregnancy. RESULTS: The mean +/- SD alpha angle in the male neonates of the non-smoking mothers was 62.3 degrees +/- 5.1 degrees and of the smoking mothers 62.1 degrees +/- 4.9 degrees (p = 0.7). In the female neonates of the non-smoking and the smoking mothers, it was 60.7 degrees +/- 5.3 degrees and 61.9 degrees +/- 4.8 degrees, respectively (p = 0.02). The difference between the male and the female neonates of the non-smoking mothers was significant (p < 0.000001), but there was no significant difference between the female neonates of the smoking mothers and the male neonates (p = 0.5). Among the female neonates whose mothers were non-smokers, the number of those with hip type IIa or worse was significantly greater than among the female neonates whose mothers were smokers. The clinical findings were in agreement with the sonographic findings. CONCLUSIONS: Maternal smoking during pregnancy reduces the frequency of sonographic and possibly clinically detected hip dysplasia in female but not in male neonates. Nonetheless, because smoking causes numerous adverse effects on the fetus and child, it is contraindicated during pregnancy.

alpha-L-Fucosidase in normal and deficient individuals.

Deficiency of the enzyme alpha-L-fucosidase has been demonstrated in peripheral leukocytes, cultured skin fibroblasts and long-term lymphoid lines from two sibs with fucosidosis 2. Reliable identification of heterozygotes for this disease was accomplished by enzyme assay of mononuclear cells isolated from peripheral blood. Isoelectric focusing of alpha-L-fucosidase isozymes in leukocyte extracts from normal individuals revealed a common polymorphism which was shown to result from two autosomal alleles at a single, autosomal locus. Evidence was obtained for a third, "silent" allele segregating in the family of the two affected children. It is probable that the disease fucosidosis type 2 results from homozygosity for this "silent" allele. Preliminary experiments have shown that cultured skin fibroblasts from deficient individuals can incorporate and retain purified alpha-L-fucosidase added to the culture medium.