尿道下裂患儿阴茎包皮内板组织成纤维细胞生长特点分析

目的 体外观察尿道下裂患儿阴茎包皮内板组织成纤维细胞生长特点,探讨成纤维细胞生长发育异常与尿道下裂发生的关系.方法 组织块法原代培养尿道下裂患儿的阴茎包皮内板组织成纤维细胞以及细胞增殖试验(CCK8法)检测其增值情况和流式细胞术分析其细胞周期.结果 在细胞原代培养实验中近端型尿道下裂组的成纤维细胞移出组织块的时间(9.5±1.0)d明显长于中远端型组(4.8±0.8)d和对照组(4.0±0.6)d(P值均＜0.05),同时细胞生长融合成束所需时间也较中远端型组和对照组明显延长(P值均＜0.001);在细胞增殖实验及细胞周期分析中显示近端型组尿道下裂受检的成纤维细胞同样表现为细胞增殖明显低于中远端型组和对照组[(17.1±2.2)d比(10.5±1.4)d比(9.0±1.1)(P值均＜0.001)],中远端型低于对照组(P＜0.05).结论 尿道下裂患儿生殖器成纤维细胞生长和增殖能力下降,可能是尿道下裂,尤其是近端型尿道下裂发病的重要因素之一,提示成纤维细胞功能越差,尿道下裂临床表现的程度可能越严重.     

Genetic deficiency of medium-chain acyl coenzyme A dehydrogenase: studies in cultured skin fibroblasts and peripheral mononuclear leukocytes

Medium-chain acyl coenzyme A (CoA) dehydrogenase deficiency was demonstrated in fibroblasts and/or mononuclear leukocytes from 14 patients, most of whom initially presented early in childhood with a Reye-like syndrome associated with hypoketotic hypoglycemia, dicarboxylic aciduria, and low levels of plasma carnitine. Parents of these patients had intermediate levels of medium-chain acyl CoA dehydrogenase activity, consistent with their being heterozygous for an autosomal recessive trait. All patients had normal levels of long-chain acyl CoA dehydrogenase activity, but had reduced short-chain acyl CoA dehydrogenase activity. Fatty acid oxidation was examined in cultured fibroblasts from five of the patients, using a series of 14C-labeled fatty acids of different chain length (palmitic, octanoic, and butyric). Oxidation of [1-14C]-octanoic acid was less than 20% of control levels: [1-14C], [6-14C]-, [16(14)C]-, and [14C(U)]-palmitic acid oxidation rates were 88, 51, 13, and 42% of control rates, respectively. [1-14C]-butyric acid was oxidized normally. These data extend our previous findings of medium-chain acyl CoA dehydrogenase deficiency in liver tissue from three of these patients. They demonstrate the value of cultured fibroblasts and leukocytes in the diagnosis and evaluation of inherited disorders of fatty acid oxidation.     

Trace metal metabolism in cultured skin fibroblasts of the mottled mouse: response to metallothionein inducers

Menkes' kinky hair syndrome is a lethal X-linked disorder marked by tissue-specific increases in copper content. An animal model of kinky hair syndrome is provided by mice mutant at the X-linked mottled locus. The basic defect is unknown. In order to discriminate among potential etiologies, we asked whether the expression of the mottled mutation causes abnormalities in the metabolism of trace metals other than copper in hemizygous mottled (blotchy) cultured skin fibroblasts, and whether we can differentiate mutant and normal cells according to their response to metal inducers of metallothionein. Blotchy fibroblasts accumulated up to 12 times more 64Cu than control (littermate) cells, over time and over a range of 64Cu concentrations. A saturable high affinity component to 64Cu accumulation over a fixed time interval was revealed in these studies. While 64Cu uptake kinetics were indistinguishable in mutant and control cells, the patterns of 64Cu exit differed. In both cell types, the rate of release of a rapidly exchangeable fraction of newly acquired 64Cu was similar. However, in mutant cells, a larger fraction of recently accumulated 64Cu is retained. In contrast to the results for 64Cu, accumulation and exit of 65Zn and 109Cd were not distinguishable in mutants and controls. With exposure to either a strong (cadmium) or weaker (zinc) inducer of metallothionein, 64Cu accumulation was increased in normal cells, while there was no change from the already elevated level of 64Cu accumulation in blotchy cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ichthyosis and steroid sulfatase: study of enzymatic activity in leukocytes and fibroblasts according to the sex and type of ichthyosis

Steroidsulfatase and arylsulfatase C were determined in fibroblasts and/or leukocytes of patients affected with different types of ichthyosis. Of the 21 patients studied, 11 showed clinical characteristics of X-linked ichthyosis (XLI) and a deficiency of these 2 enzymatic activities. Patients affected with other types of ichthyosis showed no enzymatic deficiency. In XLI families diagnosis of heterozygotes was performed by enzymatic measurements in the 5 patients' mothers studied. In 2 families enzymatic activities were studied in patients' sisters. The validity of these different enzymatic measurements is discussed.     

Multiple sulfatase deficiency (Mucosulfatidosis): Impaired degradation of labeled sulfated compounds in cultured skin fibroblasts in vivo

Skin fibroblasts from a Japanese patient with multiple sulfatase deficiency (MSD) (Mucosulfatidosis) were studied with regard to metabolism of various sulfated compounds in vivo. Several sulfatase activities (arylsulfatases A,B and C, cholesterol sulfatase, heparin N-sulfatase) were deficient in skin fibroblasts grown in F-10 CO₂ medium. The accumulation and degradation of ³⁵S-sulfatide, ³⁵S-mucopolysaccharides, ¹⁴C-cholesterol sulfate by MSD cells were also studied, comparing them to control, Hunter and metachromatic leukodystrophy cells. MSD fibroblasts accumulated and failed to degrade these compounds in vivo. Cholesterol sulfate was also incorporated into the control and pathological cells, and MSD cells were unable to hydrolyze cholesterol sulfate, though cholesterol sulfate is known to be hydrolyzed in the non-lysosomal subfraction. From these data it is clear that multiple enzyme deficiencies in MSD fibroblasts can be demonstrated in vivo.Supported by grants Nanbyo from a Ministry of Education, 1979     

Fluorescence imaging of mitochondria in cultured skin fibroblasts: a useful method for the detection of oxidative phosphorylation defects

Background:Protons are pumped from the mitochondrial matrix via oxidative phosphorylation (OXPHOS) into the intermembrane space, creating an electric membrane potential (ΔΨ) that is used for adenosine triphosphate (ATP) production. Defects in one or more of the OXPHOS complexes are associated with a variety of clinical symptoms, often making it difficult to pinpoint the causal mutation.Methods:In this article, a microscopic method for the quantitative evaluation of ΔΨ in cultured skin fibroblasts is described. The method using 5,5',6,6'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) fluorescence staining was tested in a selection of OXPHOS-deficient cell lines.Results:A significant reduction of ΔΨ was found in the cell lines of patients with either an isolated defect in complex I, II, or IV or a combined defect (complex I + complex IV). ΔΨ was not reduced in the fibroblasts of two patients with severe complex V deficiency. Addition of the complex I inhibitor rotenone induced a significant reduction of ΔΨ and perinuclear relocalization of the mitochondria. In cells with a heteroplasmic mitochondrial DNA (mtDNA) defect, a more heterogeneous reduction of ΔΨ was detected.Conclusion:Our data show that imaging of ΔΨ in cultured skin fibroblasts is a useful method for the evaluation of OXPHOS functioning in cultured cell lines.     

Adrenoleukodystrophy: impaired oxidation of very long chain fatty acids in white blood cells, cultured skin fibroblasts, and amniocytes

We compared the formation of 14CO2 from [I-14C]fatty acids in homogenates of cultured skin fibroblasts and white blood cells from 25 patients with adrenoleukodystrophy (ALD) and from 24 controls. The ALD group included 16 boys with childhood ALD, five men with adrenomyeloneuropathy (AMN), and two boys and two girls with neonatal ALD. The substrates were unbranched saturated fatty acids ranging in chain length from 16-26 carbons. From C24:0, the radioactive CO2 production by homogenates of ALD fibroblasts and white blood cells was 17% and 37% of control, respectively, and from C26:0 it was 17% of control in ALD fibroblasts. The CO2 evolution from palmitate (C16:0) in the ALD was identical to the control group; for C18:0, the value for ALD cells was 76% of control, and fatty acids with chain lengths between C18:0 and C24:0 gave intermediate results. Results for childhood ALD patients were similar to those for the AMN patients. More limited studies with cultured amniocytes of fetuses with childhood ALD gave results similar to those obtained with cultured skin fibroblasts, and results with neonatal ALD patients appeared to be the same as for childhood ALD and AMN. Studies of three women who were carriers for childhood ALD gave values intermediate between ALD and control. The total C26:0 levels in ALD cultured skin fibroblasts and white blood cells were 4-6 times that of control; the total C24:0 levels were increased 10-30%, whereas the C22:0 levels were unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute lymphoblastic leukemia in infancy: results of 5 multicenter ALL-BFM therapy studies 1970-1986

Acute lymphoblastic leukemia (ALL) of infancy has in contrast to all other age groups a less favorable prognosis. In order to determine the possible causes and biological principles for impaired outcome of infant patients in five consecutive clinical trials ALL-BFM 1970-1986, both clinical characteristics and biological features for one hundred and ninety-six patients aged two years and less have been evaluated retrospectively (forty-two infants under one year of age). The observations illustrate, that less favorable subtypes of childhood ALL are more frequent in these children, preferably in infants aged six months and less, explaining decisively the impaired prognosis. These subtypes are characterized by large tumor burden (p = less than 0.001), initial central nervous system (CNS) involvement (p = less than 0.001), and a high CNS relapse rate (p = 0.03). Phenotypically the undifferentiated leukemic blast cells show negative reactions for cALLa and often for Tdt (0-ALL and AUL; p = less than 0.001). The switch to prognostically more favorable ALL subtypes occurs about the end of the first year of life. Nevertheless, early failures by non-response or late-response seem not to influence the poorer outcome. However, compared to the preceding trials, results of studies ALL-BFM 81 and ALL-BFM 83 were significantly improved in respect to the probability of continuous complete remission for infant patients (pCCR less than 1 y: 0.44 vs. 0.28), confirming that treatment by itself is one of the major prognostic determinants.     

Enhanced adenosine 3':5' - monophosphate response to beta-adrenergic stimulation in cystic fibrosis fibroblasts after removal of conditioned medium

Skin fibroblasts derived from 6 patients with cystic fibrosis (CF), 1-6 months old, and from 6 age matched donors were investigated for their ability to accumulate cyclic adenosine 3':5'-monophosphate (c-AMP) in response to isoproterenol and prostaglandin E1 (PGE1) using strictly defined culture conditions. In order to obtain, as far as possible, constant protein content and cell number, cultures were synchronized in the early G1 phase of the cell cycle by growing them in serum free medium before adding stimulating drugs. There were no statistically significant differences both in basal c-AMP or after incubation with theophylline alone. When cultures were thoroughly washed prior to stimulation, c-AMP accumulation in response to isoproterenol was consistently higher (p less than 0.001) in CF than in normal fibroblasts, whereas response to PGE, did not differ significantly. This difference in response cannot be attributed to differences in dose- or time-response curves, or to differential escape of cAMP into the culture medium. Returning the conditioned media (CM) to the cultures after the washing procedure, or omitting the washing procedure altogether, normalized the cAMP response of CF cells. These data indicate that CF fibroblasts "delete" or "add to" the conditioned medium a substance when washed out of the cultures leave the cells hypersensitized to beta-adrenergic stimulation.     

Trends in survival of childhood malignancy for the past three decades II: Neoplasms of the respiratory,skeletal, genital,skin and endocrine organs and malignant teratomas

Survival of childhood neoplasms has improved considerably during the past three decades. Improvement in survival rate for relatively infrequent neoplasms was assessed based on data obtained from the largest children's hospital in Japan. Using the data of the cancer registry of the hospital which recorded all the patients from 1965 to 1993 totalling 1026 cases, survival rate was analyzed for each type of neoplasm categorized by the S-classification for Childhood Neoplasms, a modification of the International Classification of Diseases. Survival was assessed for three 10 year periods from 1965 for infrequent neoplasms and for two 15 year periods for rare neoplasms. Improvement of survival was considerable in the neoplasms of the respiratory organs, genital organs and germ cell tumors, moderate in bone tumors and poor in the muscle and connective tissues. In conclusion, in some types of relatively infrequent neoplasms, survival improved considerably. But in soft parts and connective tissues (mostly rhabdomyosarcoma) improvement of survival was poor.     

Radiology clinical synopsis: a simple solution for obtaining an adequate clinical history for the accurate reporting of imaging studies on patients in intensive care units

Lack of clinical history on radiology requisitions is a universal problem. We describe a simple Web-based system that readily provides radiology-relevant clinical history to the radiologist reading radiographs of intensive care unit (ICU) patients. Along with the relevant history, which includes primary and secondary diagnoses, disease progression and complications, the system provides the patients name, record number and hospital location. This information is immediately available to reporting radiologists. New clinical information is immediately entered on-line by the radiologists as they are reviewing images. After patient discharge, the data are stored and immediately available if the patient is readmitted. The system has been in routine clinical use in our hospital for nearly 2 years.     

Gastro-enteritis in Australian children: studies on the aetiology of acute diarrhoea

A prospective, year-long study of the microbiology of childhood gastro-enteritis in Perth showed that (i) Aeromonas-associated diarrhoea was the commonest recognized bacterial cause for acute diarrhoea, especially in summer, (ii) Campylobacter infections were the next most common bacterial cause, (iii) rotavirus and parasites were not significant causes of sporadic acute childhood diarrhoea, and (iv) enteric bacterial pathogens and intestinal parasites were often isolated from Aboriginal children, whether they had diarrhoea or not. Infections with enterotoxigenic bacteria, including enterotoxigenic Escherichia coli (ETEC) and enterotoxigenic Aeromonas spp., were important causes of acute diarrhoea in Aboriginal children. This is probably due to faecal pollution of their living environment and inadequate personal and community hygiene. Infections with enterotoxigenic bacteria, especially ETEC, are major causes of acute diarrhoea where living conditions are unsatisfactory, as in many developing countries. Simple and cheap methods are needed to identify these infections. A biotyping method was used to screen for ETEC which could improve the efficiency of recognizing these bacteria and help to reduce the number of tests needed to identify ETEC.