Do children with primary nocturnal enuresis have a retarded bone age? A cross‐sectional study

OBJECTIVE: Nocturnal enuresis is a common pediatric problem, the etiology of which is unclear. In recent years, various studies have been published stating that children with nocturnal enuresis exhibit growth and skeletal maturation retardation. METHODS: In this cross-sectional study, we included 27 patients (16 boys, 11 girls) between the ages of 6 and 14 years who had presented with primary nocturnal enuresis (PNE) complaints. We included in the evaluation 19 healthy subjects (12 boys, 7 girls), who were the siblings of the children with PNE, as the control group. RESULTS: The patients in both groups were similar in chronological age, bone age, height and weight, with no significant difference between groups (P>0.05). CONCLUSION: The two groups in our study consisted of the same genetic background. Thus, our results were found to be different from the previous studies. We have concluded that there is no direct relationship between enuresis nocturnal and skeletal maturation.     

Polymerase chain reaction--principle and application in medicine

The polymerase chain reaction, a new molecular technique, is of increasing importance in many areas of medical research and diagnosis. Within hours DNA sequences can be amplified millionfold with very high specificity, making detection and further analysis possible. Using multiple rounds of exponential amplification, even one copy of a gene of interest can be detected by agarose gel electrophoresis and other methods. Furthermore, minute amounts of DNA even from tissue damaged by embedding in paraffin can be analysed. Polymerase chain reaction methodology has already gained significance in many areas of medical research, e.g. diagnosis of inherited diseases, detection of viral DNA in clinical samples, cancer research and diagnosis, and characterization of gene expression.     

MRP8 and MRP14, S-100-like proteins associated with myeloid differentiation, are translocated to plasma membrane and intermediate filaments in a calcium-dependent manner

MRP8 and MRP14 are two Ca(2+)-binding proteins of the S-100 family expressed by myelomonocytic cells. Both proteins assemble to noncovalently associated complexes in a Ca(2+)-dependent manner. Members of the S-100 family are known to play a role in cytoskeletal- membrane interactions; therefore, we investigated the subcellular distribution of MRP8/MRP14 and their complexes in human monocytes. Using differential centrifugation and subsequent Western blot or enzyme- linked immunosorbent assay analysis, we found that MRP8/MRP14 were almost completely translocated from the cytoplasma to membrane and cytoskeletal structures in a Ca(2+)-dependent manner. Using a cross- linking technique, complexed forms of MRP8/MRP14 were found to be associated with the plasma membrane. Analysis of MRP-transfected L132 cells showed that the MRP8 as well as the MRP14 component of the MRP8/MRP14 complex may independently bind to membrane and cytoskeletal structures. Furthermore, immunogold electron microscopy showed a colocalization of MRP8/MRP14 and the intermediate filament type III protein vimentin in A23187-treated monocytes. Our data indicate that, in analogy to other S-100-like proteins, MRP8 and MRP14 play a role in Ca(2+)-dependent cytoskeletal-membrane interactions. Restriction of MRP8/MRP14 expression to distinct stages of myelomonocytic differentiation suggests that these proteins are involved in highly specific pathways of intracellular signaling in phagocytes.     

Serum 27E10 antigen: a new potential marker for staging HIV disease.

MRP8 and MRP14 are myeloic related proteins expressed by most circulating and emigrated neutrophils and monocytes. Their composite molecule MRP8/14 (27E10 antigen) was shown to exhibit striking antimicrobial properties. The aim of the present study was to assess the value of MRPs as markers for detection of the different stages of HIV infection (Centres for Disease Control and Prevention, 1993). By employing the ELISA technique we measured serum concentrations of these proteins in samples from 122 HIV patients at the various stages of disease, and the results were compared with those for healthy controls. Serum levels of the heterodimeric molecule 27E10 were significantly increased (P < 0.001) in patients with CDC stages II and III, with the highest levels being in patients with stage III and acute ongoing opportunistic infections. For the single component MRP14, significantly raised levels (P < 0.05) were only found in HIV stage III individuals with acute clinical events. Similar associations were not found for MRP8 alone. Increase was not related to CD4+ cell count. There was a significant correlation between 27E10 antigen serum concentrations and levels of neopterin in patients with HIV stages II and III without acute concurrent illness. Patients being treated with Zidovudine showed no statistically significant variation in levels of 27E10 and its single components MRP8 and MRP14 compared with untreated patients. These findings suggest that elevation of MRP14 levels occurs in HIV+ individuals at later stages post-HIV infection, after the onset of opportunistic infections. 27E10 antigen is concluded to be a potential marker for the different stages of HIV disease.     

S100A8, S100A9 and the S100A8/A9 heterodimer complex specifically bind to human endothelial cells: identification and characterization of ligands for the myeloid-related proteins S100A9 and S100A8/A9 on human dermal microvascular endothelial cell line-1 cells

The natural ligands of the S100 EF hand proteins S100A8 and A9 [myeloid-related proteins 8 and 14] have long been searched for in order to further the understanding of the role of the S100A8/A9-expressing monocyte subpopulation in progressing inflammatory processes. We demonstrate that S100A8, S100A9 and the S100A8/A9 heterodimeric complex bind to human dermal microvascular endothelial cell line (HMEC)-1 with an increasing binding capacity progressing from S100A8 < or = S100A9 < or = S100A8/A9. Similar results were obtained in the apolipoprotein E knockout mouse model, where preferably recombinant S100A9 but no S100A8 bound to the endothelium of the aorta ascendens. The binding of the S100A8/A9 heterodimer complex to activated HMEC-1 is specific as demonstrated by a dose-responding and satiable binding curve and the competition of FITC-labeled versus unlabeled protein. The protein character of the binding site was proven by treatment with trypsin. S100A8/A9 binding to HMEC-1 is inducible by lipopolysaccharide and tumor necrosis factor-alpha, and in the presence of calcium. A 163-kDa protein was isolated from a cell lysate of activated HMEC-1 cells using an affinity-chromatography protocol. The endothelial cell-associated ligand proteins isolated by the use of the S100A9 monomer and the S100A8/A9 dimer were subjected to mass spectrometry for protein identification. Clearly, alpha(2)-macroglobulin was identified as a binding partner for the S100A9 monomer, whereas no protein could be identified from the database for the ligand of the S100A8/A9 dimer.     

Identification of a novel dendritic cell surface antigen defined by carbohydrate specific CD24 antibody cross-reactivity.

Dendritic cells (DC) are characterized as leucocytes that lack mature lineage specific markers and stimulate naive T-lymphocyte proliferation in vitro and in vivo. The mouse heat stable antigen (HSA) participates in T lymphocyte co-stimulation and is expressed by DC isolated from thymus, skin and spleen. The human HSA homologue, CD24, is predominantly expressed by B lymphocytes and granulocytes, but its expression on DC has not been studied in detail. CD24 clearly participates in B-lymphocyte signalling but co-stimulatory activity for T lymphocytes has not yet been described. We have examined the expression of CD24 on human peripheral blood DC populations isolated directly or following in vitro culture. The CD24 antigen was absent from blood DC however, cross-reactive sialylated carbohydrate epitopes were detected on DC with some CD24 monoclonal antibodies (mAb). These CD24 mAb define a protein surface antigen, which is expressed by an immature or resting subpopulation of peripheral blood DC and is down-regulated following activation differentiation in vitro.     

Sequential expression of functions during macrophage differentiation in murine bone marrow liquid cultures

In the presence of a colony-stimulating factor, murine bone marrow cells proliferate and differentiate into macrophages. This culture system was taken as a model to study the expression of various functions by macrophages in the course of maturation. Several tests were performed daily and in parallel from the same batch of cells. It was found that certain functions were expressed early and were also characteristic for mature macrophages such as Fc receptors, phagocytosis of latex beads and unspecific esterase activity. Other functions appeared and disappeared in an ordered sequence, such as the response to macrophage migration inhibitory factor and chemotactic factor as well as the production of interferon and of plasminogen activator. The time course of functional expression was strongly dependent on proliferation of precursor cells as well as proliferation of differentiated macrophages. It is suggested that the phenotypic expression of functions during differentiation is the basis for the functional heterogeneity of macrophages.     

Chemical Characterization of Macrophage Cytotoxicity Factor,Macrophage Migration Inhibitory Factor,T-Helper Cell-Replacing Factor and Colony-Stimulating Factor from Culture Supernatants of Concanavalin A-Stimulated Murine Spleen Cells

Supernatants from Concanavalin A-stimulated murine spleen cells were subjected to hydrophobic interaction chromatography on phenyl-Sepharose. Macrophage cytotoxicity factor (MCF), macrophage migration inhibitory factor (MIF), T-helper cell-replacing factor (TRF) and colony-stimulating factor (CSF) were bound at high ionic strength and were released stepwise at low ionic strength. CSF thus could be separated from MCF, MIF and TRF and the bulk of other proteins. Chromatography of pools containing MCF, MIF and TRF on Sephadex did not lead to a separation of the three activities which were all found in a molecular weight range of 25.000-55.000. Isoelectric focusing of these pools in pH range from 4 to 9 gave two peaks for MCF at pH 8.2 and 7.2, whereas MIF activity focused from pH 4.5 to 5.5. TRF activity was found in a single sharp peak at pH 5.3. The results demonstrate that the four biological activities can be distinguished on a chemical basis and are accessible for purification and chemical characterization.     

Expression of calcium-binding proteins MRP8 and MRP14 in inflammatory muscle diseases

The pathophysiological role of infiltrating macrophages and their subtypes in idiopathic inflammatory myopathies such as dermatomyositis, polymyositis, and inclusion body myositis is not fully clear. Monocytes exhibit various phenotypes with different functional properties such as release of pro- or anti-inflammatory mediators. Expression of myeloid-related proteins MRP8 and MRP14, two calcium-binding S100-proteins, characterizes a proinflammatory subtype of macrophages. We immunohistochemically investigated expression of MRP8 and MRP14 in muscle biopsies of 33 patients with dermatomyositis, polymyositis, and inclusion body myositis. We found a clear association of expression of MRP8 and MRP14 by infiltrating macrophages with degeneration of myofibers. Because MRP8 and MRP14 are secreted by activated macrophages we investigated if these proteins would have direct extracellular effects on myocytes. We found that the purified MRP8/MRP14 complex inhibited proliferation and differentiation of C2C12 myoblasts and that it induced apoptosis via activation of caspase-3 in a time- and dose-dependent manner. These results indicate that in the course of inflammatory myopathies, activated macrophages can promote destruction and impair regeneration of myocytes via secretion of MRP8/MRP14.     

Immunohistochemical demonstration of migration inhibitory factor (MIF) in experimental allergic contact dermatitis.

The kinetics of appearance of MIF+ cells was investigated in experimental contact dermatitis using a monoclonal antibody (7D10) against murine MIF which was reacted with cryostat sections of tissues and detected by the indirect immunoperoxidase test. Four groups of BALB/c mice were investigated: (1) sensitized with 2,4-dinitrofluorobenzene (DNFB); (2) unsensitized controls; (3) tolerized; (4) unsensitized. A challenge dose of DNFB was applied to the ear of animals of groups 1-3 and of croton oil to those of group 4. Three phases could be distinguished in group 1: (a) an initial vascular and exudative reaction; (b) an early cellular phase; and (c) a late cellular phase. At zero time rarely any T lymphocytes (Lyt 1+; Lyt 2+) were seen in all four groups. Within less than 30 min venous endothelial cells became strongly MIF+. This was followed by an influx of monocytes/macrophages reaching a maximum of 72 h in group 1 and a slight peak at 12 h in groups 2 and 3. At 16-24 h in all groups the endothelial reaction weakened while many 7D10+ macrophages appeared in group 1. By double-labelling it was shown that lymphocytes were 7D10-. The influx of lymphocytes, part of which carried the T cell receptor, began at 12 h, reaching a maximum at 72 h in group 1. In groups 2 and 3 only a weak lymphocytic infiltrate developed which declined at 24 h. Group 4 developed an inflammatory reaction after the initial phase with similar kinetics as in group 1. The data suggest that an immune inflammatory reaction is preceded by a nonspecific reaction of the vascular endothelium and the mononuclear phagocytic system and that MIF is playing a central role in these events.