Age-specific characteristics of neutrophilic dermatoses and neutrophilic diseases in children.

BACKGROUND: Our suggested 'modern' concepts of 'neutrophilic dermatoses' (ND) and 'neutrophilic disease' were based on observations in adult patients and have not been studied in paediatric patients. Only a minority of ND occurs in children, and little is known about age-specific characteristics. OBJECTIVES: To describe age-specific characteristics of ND in children and to study whether our suggested 'modern' classification of ND may be applied to children. METHODS: We conducted a retrospective multicentre study in a French cohort of 27 paediatric patients diagnosed with pyoderma gangrenosum (PG) or Sweet's syndrome (SS). RESULTS: Demographics and distribution of typical/atypical forms were similar in patients diagnosed with PG and SS. Atypical ND were more frequent in infants (90%), when compared to young children (60%) and adolescents (33%). Neutrophilic disease was observed in 17/27 patients and was most frequent in infants. Neutrophilic disease of the upper respiratory tract, as well as cardiac neutrophilic disease, was only observed in infants, whereas other locations were similarly found in infants, young children and adolescents. In infants and young children, ND were associated with a large spectrum of general diseases, whereas in adolescents associations were limited to inflammatory bowel disease and Behçet's disease. CONCLUSIONS: Our study describes the concept of ND in paediatric patients and shows that they have some characteristics different from ND occurring in adults. ND occurring in infants can be associated with a large spectrum of general diseases. Occurrence of neutrophilic disease is frequent in children. Thus, ND occurring in young paediatric patients should incite clinicians to schedule complementary explorations in order to search for involvement of other organs and to rule out monogenetic autoinflammatory syndromes.

Pyoderma gangrenosum and Sweet syndrome: the prototypic neutrophilic dermatoses.

Pyoderma gangrenosum, a dramatic ulcerative skin disease, and Sweet syndrome, a papular dermatosis, were described independently. It was subsequently shown that they share many characteristics, including clinical overlap and the frequent association with multisystemic disorders. The group of the neutrophilic dermatoses encompasses these two dermatoses, as well as other conditions having in common an aseptic neutrophilic infiltrate predominating in the epidermis and/or the dermis and/or the subcutis. Some patients also experience neutrophilic infiltrates in other organs, defining the neutrophilic disease. Recent research suggests that the neutrophilic dermatoses could be considered as the cutaneous expression of the autoinflammation, an aberrant hyperproduction of interleukin-1. Autoinflammation is responsible for monogenic diseases, and is also involved in the mechanism of many polygenic conditions, including the neutrophilic dermatoses.

Two Plasmodium knowlesi-specific antigens on the surface of schizont-infected Rhesus monkey erythrocytes induce antibody production in immune hosts.

Purified schizonts (6--10 nuclei) and membranes of schizont-infected erythrocytes from the Malaysian and Philippine strain of Plasmodium knowlesi are analyzed immunochemically using immunoglobulin of rhesus monkey hyperimmune sera against schizonts and of sera from naturally immune monkeys. The anti-schizont Ig identifies less than 20 immune components in Triton X-100-solubilized schizonts and membranes of infected cells. Of these antigens, 9 (component 1, 3, 4, 5, 6, 10, 11, 18, and 20) are common to parasites and membranes of infected erythrocytes, and 12 (2A,B, 6, 8, 9, 12, 13p, 14, 16A,B, 19 A,Bp, 21, 22p, and 23) are predominantly found in the parasite; 4 components (13i, 19A,Bi, 22A, B, and 24) are unique to the membrane of infected erythrocytes. Only three parasite-specific components (1, 13, and 19) are exposed on the surface of parasitized erythrocytes as revealed by both lactoperoxidase-catalyzed radioiodination and extensive absorption of anti-schizont Ig using intact infected erythrocytes. Two plasmodium-specific antigens (1 and 13) on the surface of infected erythrocytes are recognized by sera of rhesus monkeys rendered naturally immune against P. knowlesi infections and, therefore, represent antigens in vivo. Analyses of schizonts and membranes of parasitized erythrocytes of the two different strains of P. knowlesi yields only some minor quantitative, but no qualitative differences when analyzed with both types of antisera. Importantly, components 1 and 13 appear identical in both strains.

Soluble cytokine receptors are present in normal human urine.

Affinity chromatography of crude human urinary proteins on either human rIL-6, human rIFN-gamma, or anti-IFN-gamma-R mAb yielded the two respective soluble receptors in significant quantities. A single sequence of 30 amino acid residues was obtained by NH2-terminal microsequencing of the protein peak purified in tandem by affinity chromatography on an IL-6 column and reversed-phase HPLC. This sequence was identical to the predicted NH2-terminal sequence of IL-6-R as previously reported. Analysis of the eluted proteins from both IFN-gamma and anti-IFN-gamma-R columns by inhibition of solid phase RIA, ELISA, SDS-PAGE, and Western blotting proved the existence of soluble IFN-gamma-R in normal urine. Our finding, together with the already known presence of urinary TNF binding proteins and a soluble IL-2-R both in plasma and in urine, indicates that release of soluble cytokine receptors into body fluids is a general phenomenon that occurs under normal physiological conditions.

Stabilization of the bioactivity of tumor necrosis factor by its soluble receptors.

The receptors for tumor necrosis factor (TNF) exist in cell-associated as well as soluble forms, both binding specifically to TNF. Since the soluble forms of TNF receptors (sTNF-Rs) can compete with the cell-associated TNF receptors for TNF, it was suggested that they function as inhibitors of TNF activity; at high concentrations, the sTNF-Rs indeed inhibit TNF effects. However, we report here that in the presence of low concentrations of the sTNF-Rs, effects of TNF whose induction depend on prolonged treatment with this cytokine are augmented, reflecting an attenuation by the sTNF-Rs of spontaneous TNF activity decay. Evidence that this stabilization of TNF activity by the sTNF-Rs follows from stabilization of TNF structure within the complexes that TNF forms with the sTNF-Rs is presented here, suggesting that the sTNF-Rs can affect TNF activity not only by interfering with its binding to cells but also by stabilizing its structure and preserving its activity, thus augmenting some of its effects.

A potential mechanism of "cross-talk" between the p55 tumor necrosis factor receptor and Fas/APO1: proteins binding to the death domains of the two receptors also bind to each other.

The p55 tumor necrosis factor (TNF) receptor and Fas/APO1 induce cell death via distinct regions in their intracellular domains. Three cytoplasmic proteins that bind to these receptor regions have been identified recently. One, MORT1 (also called FADD), binds to Fas/APO1 but not to p55-R; another, TRADD, binds to the p55 TNF receptor but not to Fas/APO1; and the third, RIP, binds weakly to both receptors. The regions within these proteins that are involved in binding to the receptors and the receptor regions to which they bind share a common sequence motif, that of the "death domain." This study shows that the death domain motifs in MORT1, TRADD, and RIP bind effectively to each other, a mode of binding that may allow "cross-talk" between the functional expression of the p55 TNF receptor and Fas/APO1.

A protein anomaly in erythrocyte membranes of patients with Duchenne muscular dystrophy.

Raman spectroscopic comparisons of erythrocyte membranes from 20 patients with Duchenne muscular dystrophy and 8 age-matched controls indicate a prominent and consistent protein anomaly in the patient samples. This was apparent in the following: (a) CH-stretching signals from control membranes reveal a thermotropic transition at 15.6 degrees C, attributable to a protein/lipid phase that is lacking in dystrophic membranes. (b) CH-stretching signals from control membranes also show a protein transition at 39 degrees C [pH 7.4] that is shifted to 45 degrees in dystrophic membranes. (c) A reduction in pH to 5.7 shifts this transition from 39 degrees C to 7 degrees C in normal membranes and from 45 degrees C to 24 degrees C in dystrophic membranes. (d) The Amide I/Amide III regions indicate a significant proportion of beta-structured peptide in dystrophic but not normal membranes. (e) Analysis of tyrosine signals indicates greater polar exposure of tyrosine hydroxyl groups in dystrophic vs normal membranes. All of the differences between dystrophic and normal membranes are highly significant (P less than 0.001).

Determinants on surface proteins of Plasmodium knowlesi merozoites common to Plasmodium falciparum schizonts.

In this report and (R. Schmidt-Ullrich, L. H. Miller, and D. F. H. Wallach. Manuscript in preparation.), we have demonstrated that malaria proteins on the surface of merozoites and infected erythrocytes cross-react between at least two primate malarias, Plasmodium knowlesi and P. falciparum. Sera from five Gambian adults who were highly immune to P. falciparum were used as a reagent to study the cross-reactivity between P. falciparum schizonts and surface proteins on P. knowlesi merozoites. Although the sera bound to the surface of viable, intact P. knowlesi merozoites, the sera did not block invasion of rhesus erythrocytes. 125I-lactoperoxidase-labeled surface proteins on merozoites formed complexes with the antibody. All major protein bands seen in the electrophoresis of the original Triton extract were bound by the immune sera. Because Gambians have never been exposed to P. knowlesi malaria, the antibodies that reacted with P. knowlesi merozoites must be directed against antigens of another parasite such as P. falciparum. We tested this hypothesis by competition for antibody in a Gambian serum between Triton-extracted antigens from P. falciparum schizont-infected erythrocytes and from surface-labeled P. knowlesi merozoites. P. falciparum inhibited the reaction, thus indicating cross-reaction between antigens in P. falciparum schizonts and P. knowlesi merozoites.

Dual role of the p75 tumor necrosis factor (TNF) receptor in TNF cytotoxicity.

Whereas there is ample evidence for involvement of the p55 tumor necrosis factor (TNF) receptor (p55-R) in the cytocidal effect of TNF, the role of the p75 TNF receptor (p75-R) in this effect is a matter of debate. In this study, we probed the function of p75-R in cells sensitive to the cytotoxicity of TNF using a wide panel of antibodies (Abs) against the receptor's extracellular domain. Two distinct Ab effects were observed. The Abs triggered signaling for cytotoxicity. This effect: (a) was correlated with the extent of p75-R expression by the cells; (b) was dependent on receptor cross-linking by the Abs; (c) occurred in HeLa cells, but not in A9 cells transfected with human p75-R or in HeLa cells expressing cytoplasmically truncated p75-R mutants, indicating that it involves cell-specific activities of the intracellular domain of the receptor; (d) was synergistic with the cytocidal effect of Abs against p55-R. Moreover, it seemed to reverse induced desensitization to the cytocidal effect of anti p55-R Abs, suggesting that it involves mechanisms different from those of the signaling by the p55 TNF-R. In addition, the Abs affected the response to TNF in a way that does not involve the signaling activity of p75-R. These effects: (a) could be observed also in cells in which only p55-R signaled for the cytocidal effect; (b) were not dependent on receptor cross-linking by the Abs; (c) varied according to the site at which the Abs bound to the receptor; and (d) were correlated inversely with the effects of the Abs on TNF binding to p75-R. That is, Abs binding to the membrane-distal part of the receptor's extracellular domain displaced TNF from the p75 receptor and enhanced cytocidal effect, whereas Abs that bind to the membrane-proximal part of the extracellular domain--a region at which a conformational change seems to take place upon TNF binding--decreased the dissociation of TNF from p75-R and inhibited its cytocidal effect. The above findings suggest that p75-R contributes to the cytocidal effect of TNF both by its own signaling and by regulating the access of TNF to p55-R.

The extrinsic cell death pathway and the élan mortel.

Early in the exploration of the chemical nature of life, it was widely believed that the molecules of living organisms, by their very nature, differ from those of inorganic material molecules and possess a vital force ('élan vital'). Similarly, early scientific thinking on the subject of cell death and its induction by cytotoxic cells of the immune system was pervaded by a sense that the molecules mediating these functions possess intrinsic deadly activity and are dedicated exclusively to death-related tasks. This impression was also reflected in the initial notions of the mode of action of intracellular proteins that signal for death. It is now gradually becoming clear, however, that proteins participating in death induction also have functions unrelated to death. Nevertheless, as exemplified by studies of the function of caspase-8 (an enzyme that signals both for activation of the extrinsic cell-death pathway and for non-death-related effects), analysis of the mechanistic basis for such heterogeneity might allow identification of distinct structural determinants in the proteins participating in death induction that do bear death specificity.

Caspase-8 deficiency facilitates cellular transformation in vitro.

Caspase-8 is frequently deficient in several kinds of human tumors, suggesting that certain effects of this enzyme restrict tumor development. To examine the nature of the cellular function whose regulation by caspase-8 contributes to its antitumor effect, we assessed the impact of caspase-8 deficiency on cell transformation in vitro. Caspase-8-deficient mouse embryonic fibroblasts immortalized with the SV40 T antigen did not survive when cultured in soft agar, and were nontumorogenic in nude mice. However, the rate of transformation of these cells during their continuous growth in culture, as reflected in the observed emergence of cells that do grow in soft agar and are able to form tumors in nude mice, was far higher than that of cells expressing caspase-8. These findings indicate that caspase-8 deficiency can contribute to cancer development in a way that does not depend on the enzyme's participation in killing of the tumor cells by host immune cytotoxic mechanisms, or on its involvement in the cell-death process triggered upon detachment of the cells from their substrate, but rather concerns cell-autonomous mechanisms that affect the rate of cell transformation.

Calcium transport of Plasmodium chabaudi-infected erythrocytes.

The calcium content and transport processes of Plasmodium chabaudi-infected rat erythrocytes were analyzed by atomic absorption spectroscopy and 45Ca2+ flux measurements. Infected erythrocytes, after fractionation on metrizamide gradients according to stage of parasite development, exhibited progressively increasing levels of Ca2+ with schizont and gametocytes containing 10- to 20-fold greater calcium levels than normal cells (0.54 +/- 0.25 nmol/10(8) cells). 45Ca2+ flux experiments showed both increased influx and decreased efflux in infected erythrocytes. Tris/NH4Cl lysis of normal erythrocytes preloaded with 45Ca2+ with the Ca2+ ionophore A23187 released less than 90% of cell calcium after incubation in ethyleneglycol bis(aminoethylether) N,N'-tetraacetic acid containing buffer, whereas lysis of the infected erythrocyte membrane resulted in release of 10-20% cell Ca2+, with the remaining portion associated with the isolated parasite fraction. This information together with the effects of various metabolic inhibitors indicates the presence of a parasite Ca2+ compartment in P. chabaudi-infected erythrocytes. Dicyclohexylcarbodiimide (DCCD) an inhibitor of proton ATPases of chloroplasts, bacteria, yeast, and mitochondria, and the proton ionophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), inhibited Ca2+ influx and stimulated efflux from infected cells. These results combined with evidence for a DCCD- and CCCP-sensitive membrane potential in P. chabaudi-infected cells (Mikkelsen et al., accompanying manuscript) suggest that Ca2+ transport of intraerythrocytic parasites is coupled to a proton-motive force across the Plasmodia plasma membrane.

Membrane potential of Plasmodium-infected erythrocytes.

The membrane potential (Em) of normal and Plasmodium chabaudi-infected rat erythrocytes was determined from the transmembrane distributions of the lipophilic anion, thiocyanate (SCN), and cation, triphenylmethylphosphonium (TPMP). The SCN- and TPMP-measured Em of normal erythrocytes are -6.5 +/- 3 mV and -10 +/- 4 mV, respectively. The TPMP-measured Em of infected cells depended on parasite developmental stage; "late" stages (schizonts and gametocytes) were characterized by a Em = -35 mV "early stages (ring and copurifying noninfected) by a low Em (-16 mV). The SCN-determined Em of infected cells was -7 mV regardless of parasite stage. Studies with different metabolic inhibitors including antimycin A, a proton ionophore (carbonylcyanide m-chlorophenylhydrazone [CCCP] ), and a H+ -ATPase inhibitor (N,N'-dicyclohexylcarbodiimide, [DCCD] ) indicate that SCN monitors the Em across the erythrocyte membrane of infected and normal cells whereas TPMP accumulation reflects the Em across the plasma membranes of both erythrocyte and parasite. These inhibitor studies also implicated proton fluxes in Em-generation of parasitized cells. Experiments with weak acids and bases to measure intracellular pH further support this proposal. Methylamine distribution and direct pH measurement after saponin lysis of erythrocyte membranes demonstrated an acidic pH for the erythrocyte matrix of infected cells. The transmembrane distributions of weak acids (acetate and 5,5-dimethyloxazolidine-2,4-dione) indicated a DCCD-sensitive alkaline compartment. The combined results suggest that the intraerythrocyte parasite Em and delta pH are in part the consequence of an electrogenic proton pump localized to the parasite plasma membrane.

Reversible, thermotropic alteration of nuclear membrane stucture and nucleocytoplasmic RNA transport in Tetrahymena.

We examine the effect of cooling upon the freeze-etch ultrastructure of nuclear membranes, as well as upon nucleocytoplasmic RNA transport in the unicellular eukaryote Tetrahymena pyriformis. Chilling produces smooth, particle-free areas on both faces of the two freeze-fractured macronuclear membranes. Upon return to optimum growth temperature the membrane-associated particles revert to their normal uniform distribution and the smooth areas disappear. Chilling lowers the incorporation of [(14)C]uridine into whole cells and their cytoplasmic RNA. Cooling from the optimum growth temperature of 28 degrees to 18 degrees C (or above) decreases [(14)C]uridine incorporation into cells more than into their cytoplasmic RNA; chilling to below 18 degrees C but above 10 degrees C causes the reverse. [(14)C]Uridine incorporation into whole cells and their cytoplasmic RNA reflects overall RNA synthesis and nucleocytoplasmic RNA transport, respectively. RNA transport decreases strongly between 20 degrees and 16 degrees C, which is also the temperature range where morphologically detectable nuclear membrane transitions occur. This suggests that the nuclear envelope limits the rate of nucleocytoplasmic RNA transport at low temperatures. We hypothesize that a thermotropic lipid phase transition switches nuclear pore complexes from an "open" to a "closed" state with respect to nucleocytoplasmic RNA transport.

Physicochemical differences between fragments of plasma membrane and endoplasmic reticulum.

Specific turbidities, densities, and refractive indices of fragments of plasma membrane (PM) and endoplasmic reticulum (ER) from Ehrlich ascites carcinoma have been measured. A spherical shell model of specified dimensions and refractive index was established for PM fragments. The ionic composition of the dispersion medium was varied systematically. Increases in Gamma/2 caused increases in the turbidity of both PM and ER suspensions, the greatest effects being observed with Ca(2+) and Mg(2+). In the case of PM this effect is attributable mainly to aggregation, whereas "structural" changes account for most of the turbidity increase with ER. The pH was also varied systematically to obtain pH- density and turbidity profiles and to establish the isoelectric pH of the two membrane types (PM-3.6; ER-4.35). Turbidity was maximum at "isoelectric" pH, which corresponds in each case to the region of minimum charge on the particle surfaces. Both PM and ER show large increases of density at the "isoelectric" pH, but only ER shows substantial structurally based turbidity increase under these conditions. Both PM and ER show operation of electrostatic attractions near "isoelectric" pH. PM has been shown to have ionically distinctive inner and outer surfaces while ER shows no such dissymmetry. The necessary theoretical background for interpretation of turbidity and density measurements is included, as well as a discussion of the limitations of our conclusions and the biological importance of our results.

Surface modification of T-lymphocytes observed during rosetting.

Scanning electron microscopy has been used to characterize alterations of lymphocyte surface topology that occur on contact with erythrocytes during the rosetting reaction. Molt-4 cells, a line of leukemic human lymphocytes, defined as T-cells through their ability to form rosettes with sheep erythrocytes, were used for this. Unreacted Molt-4 cells exhibit surfaces that are virtually smooth and carry few microvilli. In contrast, Molt-4 cells in rosettes display a time-dependent modification of surface topology, characterized by the emergence of numerous, long microvilli, particularly in areas of red cell contact.

Inside-out red cell membrane vesicles: preparation and purification.

Plasma membranes purified from human red cells were Converted into small vesicleS by disruption in alkaline buffer of low ionic strength. Most of these vesicles were inside-out. The presence of divalent cations prevented this inversion. The inside-out vesicles were separatcd from right-side-out vesicles by centrifugration to equilibrium in dextran density gradients.