Receptor-mediated attenuation of insulin-like growth factor-1 activity by galactose-1-phosphate in neonate skin fibroblast cultures: Galactosemia pathogenesis.

BACKGROUND: The pathogenesis of classical galactosemia, a rare metabolic disorder associated with developmental complications in neonates and children due to inherited deficiency of galactose-1-phosphate (Gal-1-P) uridylyltransferase (GALT), is known to be mediated by elevated Gal-1-P levels and involves a cascade of cytokines, reactive oxygen species (ROS) and growth factors. OBJECTIVES: To examine ex vivo the effect of Gal-1-P on the mitogenic activity of different growth factors, particularly insulin-like growth factor-1 (IGF-1), known to regulate growth and development from the fetal stage to adulthood. MATERIAL AND METHODS: Fibroblasts derived from the foreskin of 3-8-day-old healthy neonates were cultured for 1-14 days with 0-20 mM galactose or 0-10 mM Gal-1-P and then stimulated with 5% fetal bovine serum (FBS) or 50 ng/mL of platelet-derived growth factor (PDGF) or fibroblast growth factor (FGF) or IGF-1 for 24 h. DNA synthesis was measured and protein expression of PDGFR, FGFR and IGF-1R was assessed with western blotting. RESULTS: Supra-physiological concentrations of galactose significantly decreased FBSand IGF-1-induced BrdU incorporation. The presence of Gal-1-P (5-10 mM) in culture medium for 7-14 days significantly (p < 0.01) decreased IGF-1-, PDGFand FBS-stimulated DNA synthesis. While treatment with Gal-1-P selectively and significantly (p < 0.01) reduced the protein expression of IGF-1 receptor, galactose treatment did not have any marked effect on examined growth factor receptors. CONCLUSIONS: This study demonstrates that Gal-1-P impairs IGF-1 activity through IGF-1-receptor impairment, thereby providing a new insight into the molecular mechanisms of galactosemia pathogenesis.

Selective receptor-mediated impairment of growth factor activity in neonatal- and X-linked adrenoleukodystrophy patients.

Background Neonatal adrenoleukodystrophy (n-ALD) and X-linked ALD (X-ALD) patients present with demyelination, poor growth and progressive mental retardation. Growth factors are known to play a vital role in the development of children. Objective To examine the mitogenic activity of various growth factors in skin fibroblasts from n-ALD and X-ALD patients. Methods Skin fibroblast cultures from n-ALD and X-ALD patients, and controls were treated with 50 ng/mL of platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF) or insulin-like growth factor-1 (IGF-1) to examine DNA synthesis by 5-bromo-2'-deoxyuridine (BrdU) incorporation. Expression of receptors for PDGF, bFGF and IGF-1 was measured by western blotting. Serum levels of IGF-1 were assayed by enzyme-linked immunosorbent assay (ELISA). Results Fibroblasts from n-ALD and X-ALD patients had significantly (p < 0.01) less BrdU incorporation in response to fetal bovine serum (FBS). The mitogenic effect of PDGF, bFGF and IGF-1 was significantly lower in n-ALD as compared to control and X-ALD cells. X-ALD cells showed significant impairment in IGF-1-induced DNA synthesis. Expression of the FGF receptor (FGF-R) was significantly reduced in n-ALD cells. PDGF receptor remained unaffected, and IGF-1 receptor (IGF-1R) expression and serum IGF-1 levels were significantly (p < 0.01) reduced in n-ALD and X-ALD patients as compared to controls. Conclusions Growth factor activity differs in n-ALD and X-ALD patients, with marked impairment of IGF-1 function through receptor down-regulation.

Correction: Arterial tortuosity syndrome: 40 new families and literature review.

In the published version of this paper the author Neus Baena's name was incorrectly given as Neus Baena Diez. This has now been corrected in both the HTML and PDF versions of the paper.

Arterial tortuosity syndrome: 40 new families and literature review.

PURPOSE: We delineate the clinical spectrum and describe the histology in arterial tortuosity syndrome (ATS), a rare connective tissue disorder characterized by tortuosity of the large and medium-sized arteries, caused by mutations in SLC2A10. METHODS: We retrospectively characterized 40 novel ATS families (50 patients) and reviewed the 52 previously reported patients. We performed histology and electron microscopy (EM) on skin and vascular biopsies and evaluated TGF-ß signaling with immunohistochemistry for pSMAD2 and CTGF. RESULTS: Stenoses, tortuosity, and aneurysm formation are widespread occurrences. Severe but rare vascular complications include early and aggressive aortic root aneurysms, neonatal intracranial bleeding, ischemic stroke, and gastric perforation. Thus far, no reports unequivocally document vascular dissections or ruptures. Of note, diaphragmatic hernia and infant respiratory distress syndrome (IRDS) are frequently observed. Skin and vascular biopsies show fragmented elastic fibers (EF) and increased collagen deposition. EM of skin EF shows a fragmented elastin core and a peripheral mantle of microfibrils of random directionality. Skin and end-stage diseased vascular tissue do not indicate increased TGF-ß signaling. CONCLUSION: Our findings warrant attention for IRDS and diaphragmatic hernia, close monitoring of the aortic root early in life, and extensive vascular imaging afterwards. EM on skin biopsies shows disease-specific abnormalities.

Downregulation of Insulin-Like Growth Factor-1 via Nitric Oxide Production in a Hypergalactosemic Model of Neonate Skin Fibroblast Cultures.

BACKGROUND: Galactosemia is a severe metabolic disorder known to cause hepatosplenomegaly, jaundice and cataracts in neonates, and many patients develop later complications such as mental retardation, disorders of motor function or speech, and hypergonadotrophic hypogonadism. The pathogenetic mechanisms of classical galactosemia are unclear; however, nitric oxide (NO) has been suggested to play a role. OBJECTIVES: Insulin-like growth factor-1 (IGF-1) is important for the growth and development of children, and the aim of this study was to examine the association of NO production with IGF-1 gene expression under galactosemic conditions. METHODS: Serum levels of IGF-1 and nitrite were measured in 15 galactosemia patients and 15 age- and gender-matched healthy controls. Fibroblast cultures established from postcircumcision foreskin of 3- to 8-day-old healthy neonates were treated for 72 h with 0-10 mM of galactose or 0-5 mM of galactose-1-phosphate (Gal-1-P) in the presence or absence of NO synthase inhibitor (L-NAME), and inducible NO synthase (iNOS) protein was measured using Western blot analysis. RT-PCR was performed to assess the IGF-1 gene expression. RESULTS: Galactosemia patients were observed to have significantly (p < 0.01) elevated serum nitrites and markedly decreased levels (p < 0.01) of serum IGF-1 as compared to healthy controls. The cotreatment of neonate skin fibroblast cultures with galactose and Gal-1-P significantly (p < 0.01) increased cellular levels of NO and iNOS protein expression, and decreased (p < 0.01) IGF-1 mRNA levels. Treatment with L-NAME, a NOS inhibitor, significantly (p < 0.05) alleviated a galactose/Gal-1-P-induced decrease in IGF-1 mRNA levels. CONCLUSION: These results suggest that NO mediates the downregulation of IGF-1 by Gal-1-P/galactose, thereby providing a new molecular mechanism and possible therapeutic insight for galactosemia-related complications.

Use of palivizumab with other infection control measures to control respiratory syncytial virus outbreaks in neonatal care units.

OBJECTIVE: No guidelines exist on the use of palivizumab during outbreaks of Respiratory Syncytial Virus (RSV) in Neonatal Intensive Care Units (NICUs). We aimed to describe an outbreak of RSV in NICU settings and the role of palivizumab in controlling the outbreak. METHODS: The index case was a 30-day-old premature infant. During the outbreak, 13 cases of RSV were confirmed by RT-PCR. All infants in the NICU received palivizumab after RSV diagnosis. RESULTS: Of the 13 cases, seven were male; and the median (interquartile) of birth weight was 1585 (IQR: 1480-1705) g. All cases were premature under 34-weeks-gestation. Age at onset of disease varies between 10 and 160 days. Only four cases occurred after administering palivizumab and applying other infection control measures. CONCLUSION: During nosocomial outbreaks of RSV, administration of palivizumab to all infants in NICU appears to be rational and may help contain outbreaks.

Impaired NADPH oxidase activity in peripheral blood lymphocytes of galactosemia patients.

Galactosemia is an autosomal recessive disorder with a wide range of clinical abnormalities. Cellular oxidative stress is considered as one of the pathogenic mechanisms of galactosemia. In this study, we examined the activity of NADPH oxidase (NOX), a major superoxide-generating enzyme system, in peripheral blood lymphocytes (PBL) from galactosemia patients. PBL were isolated from galactosemia patients and healthy control subjects and used for cell culture studies and biochemical assays. PBL were cultured in the presence or absence of galactose or galactose-1-phosphate (Gal-1-P), and enzyme activities and/or gene expression of NOX, catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx) were measured in the cell homogenates. PBL isolated from galactosemia patients showed significantly reduced (P < 0.01) activities of catalase and GPx; however SOD activity remained unaltered. Galactosemia patients were found to have significantly (P < 0.01) increased levels of malondialdehyde (MDA) in blood lymphocytes. Enzymatic activity of NOX was significantly (P < 0.001) reduced in galactosemia patients; however, Western blotting revealed that NOX-1 protein was not significantly altered. Interestingly, levels of NOX activity in lymphocytes isolated from galactosemia patients significantly increased but remained subnormal when cultured in galactose-deficient medium for two weeks, indicating a galactose-mediated inhibition of NOX. Lymphocytes isolated from control subjects were found to have significantly (P < 0.01) reduced NOX activity when cultured in the presence of galactose or Gal-1-P for two weeks. These results show that galactose-induced cellular oxidative stress is not NOX mediated. However, impairment of the NOX system might be responsible for some of the clinical complications in galactosemia patients.

Very-long-chain fatty acids activate lysosomal hydrolases in neonatal human skin tissue.

OBJECTIVE: The aim of this study was to examine the in vitro effect of peroxisomal dysfunction on lysosomal enzymes, the autophagic machinery in the cell, in order to understand the mechanisms of pathogenesis of peroxisomal disorders. MATERIALS AND METHODS: Foreskin samples were obtained immediately after circumcision of 1- to 2-day-old infants at the Maternity Hospital, Kuwait. Skin tissues were cleaned, cut into slices of 1-2 mm2 in size and treated with lignoceric acid (1-20 microg/ml), a very-long-chain fatty acid (VLCFA), in the presence or absence of 1-5 mM aminotriazole (ATZ). A battery of lysosomal enzymes were assayed following treatment of dermal tissue with VLCFA or ATZ. RESULTS: Treatment of skin slices with lignoceric acid significantly increased (p < 0.001) the enzymic activities of acid lipase, acid phosphatase, alpha-glucosidase, alpha-galactosidase, N-acetyl-alpha-D-glucosaminidase (NAGA) and N-acetyl-alpha-D-galactosaminidase (NAGTA). ATZ (1-5 mM), an inhibitor of key peroxi somal enzyme catalase, also markedly increased the enzymic activities of acid phosphatase, alpha-glucosidase (23%) and alpha-galactosidase (18%) without any significant effect on NAGA or NAGTA. Western blot analysis further revealed that both VLCFA and ATZ significantly increased the protein expression of lysosomal enzymes, beta-galactosidase and beta-glucuronidase. CONCLUSION: Experimen tal dysfunction of peroxisomes mimicked by elevated VLCFA or ATZ-mediated catalase inhibition significantly increased the activities of lysosomal hydrolases in human dermal tissue, suggesting that activation of the lysosomal system could be one of the factors responsible for cellular damage during pathogenesis of peroxisomal diseases.

Double-catheter technique for the proper insertion of umbilical venous catheters in newborns.

OBJECTIVES: To document the usefulness and safety of inserting a second umbilical venous catheter in ill neonates, while a previously misplaced first catheter was still in its place. SUBJECTS AND METHODS: The case series involved 25 newborn babies who were admitted to the Neonatal Intensive Care Unit, Maternity Hospital, Kuwait, over a 3-year period from 1999 to 2002. The umbilical venous catheter of the babies was misplaced and diverted to the liver, necessitating insertion of a second catheter while the previous one was still in place. The characteristics of the babies and possible catheter-related complications were recorded. RESULTS: Of the 25 babies, 19 had the second catheter properly placed in the right atrium, while in the remaining 6 neonates, the catheter was still misplaced. Misplacement occurred mostly in full-term babies or the catheter was inserted at a later stage. No life-threatening complication was observed during the procedure. CONCLUSION: Insertion of a second umbilical venous catheter with the misplaced first catheter in situ is a useful and safe procedure.

Detection of thiopental in the steroid fraction of serum from neonates following maternal exposure.

To follow up an investigation which studied effects of antenatal dexamethasone therapy on neonatal respiratory performance in multifetal gestations, neonatal serum steroids were determined by HPLC. A major peak (X) whose retention time coincided with that of dexamethasone was observed in many, but not all, serum samples. However, there was no correlation between the neonates whose serum samples displayed this X-peak and the mothers who had actually received the steroid therapy, indicating that the X-substance was not dexamethasone. An alternate mobile phase was employed which separated the X-substance and dexamethasone validating the indication. Among ten clinical conditions of the neonate birth, the X-substance was found to correlate only with the mothers who had the cesarean operation for delivery, suggesting that the substance was not necessarily a steroid. Four anesthetic agents used for cesarean operations were studied; the X-substance was identified as thiopental using a LC/MS technique. This was based on the same retention times, the same negative ions at m/z 240.9 and the same daughter ions at m/z 100.8 between the two substances. Thus, caution must be exercised when HPLC is employed to study serum steroids of patients who have previously been exposed to thiopental. Moreover, recent reports have shown that thiopental affects certain metabolic reactions in the rat; the present findings also suggest a need for further investigations of thiopental effect on neonates.

Carnitine prevents cyclic GMP-induced inhibition of peroxisomal enzyme activities.

Peroxisomes, also termed as microbodies, are now known to carry out several specialized metabolic activities that are vital to cellular function. A defect in peroxisomal function leads to development of a fatal human disease, and a number of peroxisomal disorders are now linked to inherited peroxisomal enzyme abnormalities. Peroxisomal enzyme activities are also altered during pathophysiological conditions through various endogenously produced bio-molecules such as nitric oxide (NO). NO produced by cytokines or NO-donors is known to modulate peroxisomal functions, and these effects of NO are mediated through cGMP. We are reporting for the first time that L-carnitine (1-5 mm) prevents cGMP-mediated impairment of peroxisomal enzyme activities. Cyclic GMP (250-1000 muM) significantly inhibited (p < 0.01) the specific activities of catalase, acyl CoA oxidase and dihydroxyacetone-phosphate acyltransferase (DHAPATase) in human dermal fibroblasts, and treatment of cells with 1-5 mM of carnitine significantly (p < 0.001) reduced the inhibitory effects of cGMP on peroxisomal enzyme activities. These findings suggest that carnitine, previously thought to participate only in fatty acid oxidation, may in fact be regulating other cellular events including oxidative stress, and could possibly be used to correct cytokine-impaired peroxisomal functions.