Impaired regulatory T-cell homeostasis due to vitamin D deficiency in undifferentiated connective tissue disease.

OBJECTIVE: The aim of this study was to perform a quantitative and functional analysis of natural CD4+CD25(high)Foxp3+ regulatory T cells (nTregs) and CD4+IL-17+ T cells, and to assess the serum levels of proinflammatory cytokines in patients with undifferentiated connective tissue disease (UCTD) before and after 5 weeks of 0.5 µg/day alfacalcidol supplementation. METHODS: Twenty-five patients with UCTD were enrolled in an open-label trial of alfacalcidol. Plasma levels of 25-hydroxyvitamin D [25(OH)D] were assessed by a high-performance liquid chromatography (HPLC) method. Flow cytometry was used for the quantification of nTregs and the IL-17 expression of T-helper (Th)17 cells. The serum concentrations of cytokines interleukin (IL)-12, interferon (IFN)-γ, IL-23, IL-17, IL-6, and IL-10 were measured by an enzyme-linked immunosorbent assay (ELISA). RESULTS: Treatment with alfacalcidol raised 25(OH)D levels from a mean of 23.5 ± 5.6 to 34.5 ± 7.4 ng/mL (p = 0.059; NS). Alfacalcidol treatment decreased both Th1- (IL-12 and IFN-γ) and Th17-related (IL-23, IL-17, IL-6) cytokine levels in UCTD patients, while the soluble IL-10 level increased (IL-12: 156.7 ± 75.2 vs. 87.5 ± 42.1 pg/mL, p < 0.001; IFN-γ: 41.5 ± 12.0 vs. 21.7 ± 9.9 pg/mL, p < 0.001; IL-23: 385.2 ± 82.2 vs. 210.0 ± 69.3 pg/mL, p < 0.001; IL-17: 37.8 ± 9.6 vs. 17.8 ± 4.5 pg/mL, p = 0.009; IL-6: 39.4 ± 11.3 vs. 23.5 ± 6.3 pg/mL, p < 0.001, IL-10: 8.4 ± 3.0 vs. 21.4 ± 9.7 pg/mL, p < 0.001). Alfacalcidol improved the Th17/nTreg imbalance, as it inhibited the IL-17 expression of Th17 cells, and increased the number of nTregs. The alfacalcidol might increase the capacity of nTreg cells to suppress the proliferation of autologous CD4+CD25⁻ cells. CONCLUSION: Our findings support the idea that vitamin D influences the Th17/nTreg imbalance in vitamin D-insufficient patients with UCTD and could be beneficial in the management of the disease.

Urinary N-acetyl-beta-D-glucosaminidase in epileptic children treated with antiepileptic drugs.

AIM: To investigate the effect of prolonged use of antiepileptic drugs on renal function in children. METHODS: Prospective study of 72 children (aged 3-18 years) with epilepsy, on either monotherapy (n = 44) or combined therapy (n = 28). The length of treatment varied from 1 to 13 years. Drugs used were valproic acid, carbamazepine, ethosuximide, clonazepam, clobazepam, and vigabatrin. RESULTS: In 65 patients plasma concentrations of the drugs were in the therapeutic range. In the remaining seven, plasma concentrations were slightly high. In 33 patients urinary N-acetyl-beta-D-glucosaminidase (NAG) activity was raised. The incidence of pathological NAG indices was significantly higher in the combined therapy group than in the monotherapy group. There were also significant differences in the NAG indices of patients depending on the duration of therapy. CONCLUSIONS: Results suggest that chronic use of some antiepileptic drugs-in spite of normal blood concentrations-may alter tubular function, and the dysfunction may result in clinical symptoms. Therefore, we recommend screening of tubular function in these patients.

"Bloodless" treatment of a Jehovah's Witness infant with ABO hemolytic disease.

An ABO-incompatible term infant girl born to parents who are Jehovah's Witnesses was admitted to our neonatal unit with a high serum bilirubin level necessitating exchange transfusion. The parents signed a request that blood should not be administered under any circumstances. However, they authorized us to apply the possible alternative treatments of orally administered D-penicillamine (300 mg/kg per day divided in three doses for 3 days), phototherapy, intravenous fluids, and recombinant human erythropoietin (200 U/kg subcutaneously on every second day for 2 weeks). Herein, we report the outcome of this baby, who was discharged from the our unit in good condition after treatment. Her physical growth and motor milestones at 14 months of age revealed no red flags for neurodevelopmental maturation. To our knowledge, this is the first case of an infant who received such a combined alternative (and "bloodless") treatment of serious ABO hemolytic disease of the newborn.

Urinary N-acetyl-beta-D-glucosaminidase determination in newborns and children: methods and diagnostic applications.

More recently numerous papers have been written on the successful paediatric applications of some well-established urinary N-acetyl-beta-D-glucosaminidase assays by several authors. Regarding these developments we tried to collect and summarize all the available literature data published in this field thus far and, in addition, to encourage and promote the wider utilization of these effective methods for the diagnosis of very different primary and secondary renal damages. Besides the chemical and biochemical background of the assays the pathological conditions and medical treatments which may lead to tubulopathy and, therefore, to a significantly elevated urinary enzyme excretion are also discussed.

[Age-dependent urinary N-acetyl-beta-D-glucosaminidase activity in healthy children]. / Vizelet N-acetil-beta-D-glükózaminidáz aktivitás korfüggése egészséges gyermekekben.

Urinary N-acetyl-beta-D-glucosaminidase activity was measured in 123 healthy children aged between 1-14 years by two newly developed colorimetric procedures using MNP-GlcNAc and VRA-GlcNAc substrates and was compared to the well established PNP-GlcNAc assay. The enzyme activity was factored with the urinary creatinine concentration and expressed as NAG index. The applicability and the advantages of the new methods are discussed. The NAG values obtained with each substrate decreased with age as a result of a concomitant rise in the urinary creatinine concentration.

Assay of N-acetyl-beta-D-glucosaminidase in urine from neonates: comparison of two new colorimetric methods using MNP-GlcNAc and VRA-GlcNAc as substrates.

The NAG activity present in urine from newborn babies was assayed using two colorimetric procedures with either MNP-GlcNAc or VRA-GlcNAc as substrate and compared with data obtained with the well established PNP-GlcNAc procedure. Both new assays were easy to perform and reproducible. The MNP-GlcNAc method has the advantage that it is now available as a kit; however, the VRA-GlcNAc procedure is more sensitive. NAG activity, creatinine concentration and NAG-index values were determined in normal neonates and within-run imprecision calculated. Excellent correlations were found between MNP-GlcNAc-ase and VRA-GlcNAc-ase indices (r = 0.984) and between PNP-GlcNAc-ase and VRA-GlcNAc-ase indices (r = 0.952). When low molecular weight urinary components were removed by gel filtration no significant change in VRA-GlcNAc-ase activity was observed.

[D-penicillamine does not affect the liver and kidney function in newborn infants nor the in vitro function of phagocytes]. / A D-penicillamin kezelés nem befolyásolja az újszülöttek máj- és vesefunkcióját, valamint in vitro a phagocyták müködését.

D-penicillamine was introduced to treat neonatal hyperbilirubinaemia in 1973 and to prevent retinopathy of prematurity in 1980. In this study we investigated the renal and liver functions of neonates treated with DPA and the in vitro effect of the drug on superoxide anion generation and beta-glucuronidase release as well as on phagocytic and intracellular killing activation on human peripheral blood granulocytes. Our data concerning the renal and liver functions before and after 3 to 4 days DPA treatment reveal no pathological change during short-term administration in the neonatal period. Furthermore, none of the examined DPA concentrations influenced the phagocytic or killing activity of neutrophils.

[Urinary N-acetyl-beta-D-glucosaminidase activity in healthy, polycythemic and hypoxic newborn infants]. / Vizelet N-acetyl-beta-D-glucosaminidase aktivitás egészséges, polycythaemiás és hypoxiás újszülöttekben.

Urinary N-acetyl-beta-D-glucosaminidase (NAG) activity was assayed in 20 polycythemic newborns and prematures, together with 50 prematures suffering from hypoxia on the 1st, 2nd, 4th, 14th, and 28th day after birth. The enzyme was also assayed in 101 healthy newborns which provided normal reference values. NAG activity was factored by the creatinine concentration to given an index. There were significant difference in the NAG indices either between full-term and preterm babies or between appropriate for gestational age (AGA) and small for gestational age (SGA) neonates of the normal group. However, NAG excretion on the first day of life was significantly raised in the case of polycythemic newborns. Following partial plasma exchange, on the 14th day the NAG activity returned to the normal range. NAG activities of premature babies suffering from idiopathic respiratory distress syndrome (IRDS) were significantly elevated on the 1st, 2nd, 4th day but fell sharply to the 14th day. NAG activity fell to normal values by the 28th day. These results suggest that the urinary NAG index is a sensitive indicator of the renal tubular damage during the newborn period.

Urinary N-acetyl-beta-D-glucosaminidase activity in healthy, polycythemic and hypoxic neonates.

The authors investigated the urinary N-acetyl-beta-D-glucosaminidase (NAG) activity in the case of 101 normal healthy and 20 polycythemic newborns and prematures, and 50 prematures suffering from hypoxia on the 1st, 2nd, 4th, 14th, and 28th day after birth. The obtained activities were referred to the creatinine concentrations of the urine samples and given as NAG index. There were no significant differences in the NAG indices either between fullterm and preterm babies or between appropriate for gestational age (AGA) and small for gestational age (SGA) neonates of the normal group. The NAG indices on the first day of life were significantly higher in the case of polycythemic newborn in comparison with the normal group (p less than 0.01). On the 14th day, after the partial plasma exchange, the NAG indices returned to the normal range. The premature babies suffering from IRDS received an average 10.1 days oxygen supplementation. Their NAG indices were significantly (p less than 0.01) higher on the 1st, 2nd, 4th days than those of the healthy prematures of the normal group and decreased considerably up to the 14th day. Finally the NAG indices reached the normal value on the 28th day. These results support the assumption that the urinary NAG index is a suitable indicator of the renal tubular damage during the newborn period.

Erythrocyte damage in newborn babies caused by hyperbilirubinaemia and hypoxia.

Erythrocyte damage of newborn babies suffering from hyperbilirubinaemia and hypoxia was compared with a control group. In the cases of serum bilirubin level higher than physiological icterus lipid peroxidation of erythrocytes decreased probably due to the antioxidant effect of bilirubin. Moreover, an increase in potassium and protein outflow from patients' red blood cells was observed indicating a membrane damage both in hyperbilirubinaemic and hypoxic groups. Superoxide dismutase activity of serum and erythrocytes did not show significant difference in patients compared with healthy newborns. However, the low serum coeruloplasmin level in the hypoxic group and the low serum transferrin level of babies both with hypoxia and hyperbilirubinaemia suggest an insufficient antioxidant defence against free radicals.

The effect of polycythemia and hypoxia on urinary N-acetyl-beta-D-glucosaminidase activity in newborns.

Urinary N-acetyl-beta-D-glucosaminidase activity was assayed in fullterm and preterm polycythemic neonates, in preterm infants with hypoxia, and in healthy newborns. There were no significant differences between fullterm and preterm babies or between appropriate for gestational age and small for gestational age neonates in the normal group. N-acetyl-beta-D-glucosaminidase excretion on the first day of life was significantly raised in polycythemic newborns (P less than 0.01). Fourteen days after partial plasma exchange the enzyme activity returned to normal. N-acetyl-beta-D-glucosaminidase activities in preterm babies with respiratory distress syndrome were significantly (P less than 0.01) raised on the 1st, 2nd, 4th days and fell sharply to the 14th day. N-acetyl-beta-D-glucosaminidase isoenzyme studies revealed that urine samples taken from preterm babies with respiratory distress syndrome in the first week after birth contained increased amounts of intermediate and B isoenzyme forms while there was a concomitant reduction in the amount of the A form present.

[Serum cortisol and ceruloplasmin determination in neonates with intrauterine growth retardation]. / Szérum cortisol és coeruloplasmin meghatározások intrauterin retardált újszülöttekben.

The authors have shown recently that coeruloplasmin deficiency was associated with severe respiratory distress in premature neonates. In the present study they measured the serum cortisol and coeruloplasmin levels in 58 intrauterine growth retarded infants. The results were compared to the data of 44 appropriate for gestational age neonates. Both parameters showed association with the fetal distress in term babies (cortisol: 17.2 vs 12.3 ug/100 ml, coeruloplasmin: 29.7 vs 11.9 U/l). Coeruloplasmin activity was also higher in growth retarded prematures (48.5 vs 16.5 U/l), while cortisol concentration increased irrespective of intrauterine malnutrition. There was a significant positive correlation between serum cortisol and coeruloplasmin in dysmaturity. This is the first report suggesting a possible role for coeruloplasmin, an important extracellular antioxidant enzyme in the precocious development of intrauterine growth retarded infants.

The effects of D-penicillamine on the renal and liver functions in neonates and the in vitro influence on granulocytes.

D-Penicillamine (DPA) was introduced to treat neonatal hyperbilirubinemia in 1973 and to prevent retinopathy of prematurity in 1980. In this study we investigated the renal and liver functions of neonates treated with DPA and the in vitro effect of the drug on superoxide anion generation and beta-glucuronidase release as well as phagocytic and intracellular killing activation of human peripheral blood granulocytes. Our data concerning the renal and liver functions before and after 3 to 4 days DPA treatment reveal that the drug does not produce any pathological change during short-term administration in the neonatal period. Furthermore, it was found that superoxide anion generation was slightly increased, and beta-glucuronidase release markedly increased by preincubation with DPA at concentrations of 0.5-5 mM. The rise was directly proportional to the concentration in the examined range. On the other hand, none of the examined DPA concentrations influenced the phagocytic or killing activity of neutrophils.