Necrotizing fasciitis in infancy: an uncommon setting and a prognostic disadvantage.

Necrotizing fasciitis is a potentially fatal, progressive soft tissue infection that typically occurs in adults, and only rarely occurs in infants. Although adults in whom necrotizing fasciitis develops are commonly diabetic, malnourished, or otherwise immunocompromised, infants in whom the disease develops are typically healthy and without clear predisposing factors. Herein, however, the authors report the case of an infant with compromised immunity secondary to the manifestations and treatment of panhypopituitarism, in whom postoperative necrotizing fasciitis developed after bilateral inguinal herniorrhaphy. The diagnosis, pathological mechanism, and treatment of necrotizing fasciitis are reviewed and the distinguishing features in infants are highlighted. The combination of a low incidence and very high mortality rate associated with necrotizing fasciitis in this subgroup strengthens the need for hypercritical suspicion. Early diagnosis and the prompt initiation of surgical treatment are the most essential means to improve on the prognosis for necrotizing fasciitis in infants.

Augmented inflammatory responses and altered wound healing in cathepsin G-deficient mice.

BACKGROUND: Cathepsin G is a neutral serine proteinase that exists primarily in azurophilic granules of neutrophils, but also as a proteolytically active membrane-bound form. While the specificity and many in vitro biological activities have been described for cathepsin G, little is known about the role of this enzyme in neutrophil function in vivo, particularly as it applies to the wound-healing process. OBJECTIVE: To determine the role of cathepsin G in cutaneous tissue repair by examination of full-thickness incisional wound healing in mice with a null mutation for cathepsin G. METHODS: Paired, full-thickness linear incisions were made on the backs of cathepsin G +/+ and cathepsin G -/- mice, and wound tissue was harvested at days 1, 2, 3, 5, 7, 10, and 14 after wounding. Neutrophil influx, myeloperoxidase activity, and migration were examined using light microscopy, the myeloperoxidase assay, and modified Boyden chamber technique, respectively. Wound-breaking strength was measured using tensiometry. RESULTS: The absence of cathepsin G led to a 42% decrease in wound-breaking strength at day 7 after wounding (n=28; P<.002), which returned to the level of control mice by day 10 after wounding. Wound tissue sections in mice lacking cathepsin G also showed a 26% increase in neutrophil myeloperoxidase activity (n=12; P=.001) and an 18% increase in neutrophil influx (n=14; P=.002) at day 3 after wounding. Wound fluid collected on day 5 after wounding from cathepsin G-deficient mice attracted 58% more neutrophils than wound fluid collected from control mice (n=4; P<.05). CONCLUSIONS: Neutrophil cathepsin G is important during the early inflammatory stage of wound healing. Cathepsin G may be involved in processing 1 (or more) soluble mediator(s) in the wound milieu that is responsible for neutrophil chemotaxis. Our findings suggest that tight regulation of inflammation is necessary to prevent impaired healing during early tissue repair.

Inheritance of salt-dependent hypertension in the inbred Dahl rat.

The mode of inheritance of salt-dependent hypertension in the inbred Dahl salt-sensitive rat strain was examined by genetic crosses with the corresponding salt-resistant strain. The blood pressure responses to ingestion of a high NaCl (8%) diet defined three phenotypes: early onset (within 17 days) of systolic hypertension, defined as greater than or equal to 140 mm Hg, the parental salt-sensitive phenotype; late onset of systolic hypertension requiring 50 to 60 days in males and more than 200 days in females, characteristic of the F1 progeny; and normotension, less than 140 mm Hg, the parental salt-resistant phenotype. The frequencies of the phenotypes observed among 91 F2 progeny and 45 progeny of the backcross to parental salt-sensitive animals agree well with values predicted by a model in which two autosomal, unlinked, allelic loci, termed alpha and beta, determine the inheritance. For F2 male progeny, the predicted frequencies of early-onset hypertension, late-onset hypertension, and normotension are 0.1875, 0.5625, and 0.25, respectively, and the corresponding observed frequencies were 0.156, 0.50, and 0.34 (X2 = 0.48, P > .50). F1 progeny of reciprocal parental crosses were maintained on the 8% NaCl diet for 255 days. Male F1 rats developed systolic hypertension sooner than did females. From 60 to 200 days, the average blood pressure value within each group remained approximately stable; the male values exceeded those for females (P < .01); and the direction of the parental cross significantly influenced (P < .05) the levels in males and females, suggestive of genomic imprinting.

Regional differentiation in rat aorta: L-arginine metabolism and cGMP content in vitro.

Sequential segments of rat aorta incubated in vitro exhibit a characteristic activity pattern for the metabolism of L-arginine, the substrate for nitric oxide synthase, and for the content of guanosine 3',5'-cyclic monophosphate (cGMP), the mediator of nitric oxide relaxation of vascular smooth muscle. Highest values were observed just distal to the arch and diminish peripherally. Prior removal of the endothelium, treatment with ouabain, or replacement of ambient medium Na+ decreased L-arginine uptake and metabolism and eliminated the pattern of regional differentiation. Removal of endothelium reduced the cGMP content with loss of the regional pattern. A favorable extracellular/intracellular Na+ gradient is required for the moiety of L-arginine uptake destined for metabolism in the endothelial cell. Replacement of ambient Na+ or treatment with ouabain also decreases markedly the L-arginine metabolism and uptake in cultured rat aortic endothelial cells. When aortic segments were tested with five additional substances, L-leucine uptake alone followed a regional pattern similar to that for L-arginine, and no such pattern was observed for the uptake of L-alanine, alpha-aminoisobutyrate, 3-methylglucose, or Ca2+.

Immunoglobulin and renal abnormalities in Dahl genetically hypertensive rat.

The Dahl salt-sensitive rat (DS) is a model of genetically determined arterial hypertension exacerbated by dietary salt. We report two additional abnormalities of DS rats, which are both genetically determined and enhanced by salt: 1) immunoglobulin disorders and 2) renal dysfunctions. These abnormalities precede and are not the result of the arterial hypertension. In young, prehypertensive DS rats the plasma and tissue concentrations of immunoglobulin (Ig) G, but not of IgM or IgA, are decreased compared with those of the salt-resistant strain (DR). A high-salt diet (8.0% NaCl) decreases the plasma and tissue IgG levels of DS but not of DR rats. Reduction of IgG in the DS strain results from both decreased synthesis and increased urinary excretion. Renal dysfunction in young, prehypertensive DS animals is manifested by increased excretion of high molecular weight proteins, including albumin, IgG, IgA, and IgM. The high-salt diet increases the urinary excretion of these proteins within 1-2 days, and the effect is much greater in DS compared with DR rats. The urinary excretion of IgG is selectively increased relative to immunoglobulin light chains, IgA and IgM in DS compared with DR animals. The present studies provide new markers characteristic of the DS phenotype and pose the issue of possible genetic or functional interrelationships among the salt-sensitive hypertension, immunoglobulin disorders, and renal dysfunctions.

Topography and functions of sulfhydryl groups of the human erythrocyte glucose transport mechanism.

Membrane-impermeant and -permeant maleimides were applied to characterize the location and function of the sulfhydryl (SH) groups essential for the facilitated diffusion mediated by the human erythrocyte glucose transport protein. Three such classes have been identified. Type I SH is accessible to membrane-impermeant reagents at the outer (exofacial) surface of the intact erythrocyte. Alkylation of this class inhibits glucose transport; D-glucose and cytochalasin B protect against the alkylation. Type II SH is located at the inner (endofacial) surface of the membrane and is accessible to the membrane-impermeant reagent glutathione maleimide only after lysis of the erythrocyte. D-glucose enhances, while cytochalasin B reduces, the alkylation of Type II SH by maleimides. Reaction of Types I and II SH with an impermeant maleimide increases the half-saturation concentration for binding of D-glucose to erythrocyte membranes. By contrast, inactivation of Type III SH markedly decreases the half-saturation concentration for the binding of D-glucose and other transported sugars. Type III SH is inactivated by the relatively lipid-soluble reagents N-ethylmaleimide (NEM) and dipyridyl disulfide, but not by the impermeant glutathione maleimide. Type III SH is thus located in a hydrophobic membrane domain. A kinetic model constructed to explain these observations indicates that Type III SH is required for the translocation event in a hydrophobic membrane domain which leads to the dissociation of glucose bound to transport sites at the membrane surfaces.

Sulfhydryl substituents of the human erythrocyte hexose transport mechanism.

Sulfhydryl substituents of the hexose transport mechanism of human erythrocyte membranes were studied with membrane-impermeant and -permeant maleimide derivatives. Three sulfhydryl classes have been identified on the basis of their reactivity toward the reagents and their effects on the transport mechanism. Type I sulfhydryl is located at the outer (exofacial) surface of the membrane and bound covalently on treatment of intact cells with the membrane-impermeant glutathione-maleimide. This sulfhydryl is required for the transport, and it is protected from alkylation, i.e., its reactivity toward maleimides is decreased by the presence of D-glucose or cytochalasin B. Type II sulfhydryl is also required for the transport, but it differs from type I in that D-glucose (but not cytochalasin B) increases the reactivity toward maleimides. Further, it is located at the endofacial surface of the membrane, since reaction with glutathione-maleimide occurs only in leaky ghosts and not in intact cells. Alkylation by glutathione-maleimide of type I and type II sulfhydryls increases the half-saturation for the binding of D-glucose to erythrocyte membranes. In contrast, inactivation of type III sulfhydryls by N-ethylmaleimide or dipyridyl disulfide decreases the half-saturation concentration for the binding of D-glucose and other transported hexoses to the membranes; nontransported sugars are not affected similarly. Type III sulfhydryl is not inactivated by the polar reagent glutathione-maleimide and is probably located in a nonpolar domain of the transport mechanism. Inactivation of either type I or II sulfhydryls decreases or eliminates the flux asymmetry of the hexose transport mechanism.

Effects of benzyl alcohol on erythrocyte shape, membrane hemileaflet fluidity and membrane viscoelasticity.

The effects of benzyl alcohol on cell shape, hemileaflet lipid fluidity and membrane rheology of human red blood cells were studied. Membrane fluidity was assessed by determining the fluorescence anisotropy of permeant probes (1,6-diphenyl-1,3,5-hexatriene,12-(9-anthroyloxy)stearate, 2-(9-anthroyloxy)stearate) and a new impermeant probe (N-stachyosylsuccinic acid dihydrazide-2-(9-anthroyloxy)stearate). Measurements made on intact red blood cells reflected primarily the outer leaflet fluidity while measurements made on red blood cells ghosts reflected the fluidity of both leaflets. Membrane viscoelasticity was determined by micropipette aspiration. Treatment of intact red blood cells with benzyl alcohol up to 50 mM caused progressive stomatocytic shape change but no change in membrane viscoelasticity, 1,6-diphenyl-1,3,5-hexatriene anisotropy or stachyosyldihydrazide-2(9-anthroyloxy)stearate correlation time; similar treatment of leaky ghosts yielded decreases in 1,6-diphenyl-1,3,5-hexatriene anisotropy and stachyosyldihydrazide-2(9-anthroyloxy)stearate correlation time. With benzyl alcohol above 50-60 mM, intact red blood cells became echinocytic, and decreases in 1,6-diphenyl-1,3,5-hexatriene anisotropy and stachyosyldihydrazide-2(9-anthroyloxy)stearate correlation time occurred in both intact cells and ghosts; there was no change in membrane viscoelasticity. These results indicate that benzyl alcohol up to 50 mM affects primarily the inner leaflet of the red blood cell membrane and that higher concentrations affect both leaflets. These increases in membrane fluidity are not associated with changes in membrane viscoelasticity. This study illustrates the use of fluorescence techniques to monitor specifically the lipid fluidity of each hemileaflet of the erythrocyte membrane.

Membrane lipid dynamics in human promyelocytic leukemia cells sensitive and resistant to 12-O-tetradecanoylphorbol-13-acetate induction of differentiation.

A series of fluorescent probes was used to analyze membrane lipid dynamics in promyelocytic leukemic cells sensitive (HL-60) or resistant (R-55) to phorbol diester induction of cell differentiation. When examined with the probe 1,6-diphenyl-1,3,5-hexatriene, which can penetrate the plasma membrane and intercalate in the lipids of both leaflets of the plasma membrane, as well as in organellar membranes, R-55 cells were found to have higher fluorescence anisotropy values, indicative of decreased lipid fluidity, as compared to HL-60 cells. In contrast, when HL-60 and R-55 cells were compared using a series of membrane-impermeant fluorophores (stachyose derivatives of anthroyloxystearate and pyrenebutyryl hydrazide) that incorporate only into the outer hemileaflet of the plasma membrane, no difference was observed in membrane lipid fluidity. Exposure to 12-O-tetradecanoylphorbol-13-acetate (10 ng/ml) for 24 hr decreased the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene in both HL-60 and R-55 cells, whereas by 48 hr only the HL-60 cells displayed the reduction. No effect on the fluorescence anisotropy of 1-(4'-trimethylammonium phenyl)-6-phenyl-1,3,5-hexatriene, which is believed to localize in the plasma membrane, was observed in R-55 cells exposed to 12-O-tetradecanoylphorbol-13-acetate (10 or 100 ng/ml), whereas HL-60 cells treated with 12-O-tetradecanoylphorbol-13-acetate (10 ng/ml) showed a marked reduction in the fluorescence anisotropy. These observations suggest that the ability of HL-60 cells to respond to 12-O-tetradecanoylphorbol-13-acetate may be affected by the physical state of the plasma membrane lipids and that the resistant phenotype is associated with decreased fluidity of either the inner leaflet of the plasma membrane and/or of the cytosolic organellar membranes.

Lipid fluidity of the individual hemileaflets of human erythrocyte membranes.

The impermeant fluorescent probes (MIMAR reagents) described here permit the assessment of the lipid fluidity of individual membrane hemileaflets. They should also prove useful for examining the outer hemileaflets of the plasma membranes of intact cells. The observations, thus far, that normal human erythrocyte membranes have a characteristic asymmetry of fluidity, with the outer leaflet more fluid, correspond to prior findings with Mycoplasma, Newcastle Disease viral envelopes, and mouse LM cells. Hence, it is possible that the pattern is quite general in biological membranes. The particular lipid and protein components of the human-erythrocyte membrane that underly the fluidity asymmetry are unknown. The increased content of phosphatidylcholine in the outer leaflet and of the anionic phospholipids in the inner leaflet would be consonant with the fluidity difference. On the other hand, sphingomyelin, which tends to decrease fluidity, is localized mainly in the outer leaflet. Unknown at present is whether the cholesterol content of the two leaflets differs. From the results reported above, it is tempting to speculate that exogenously added cholesterol tends to localize in the outer leaflet, normally the more fluid leaflet, whereas endogenous cholesterol is more readily removed from the inner leaflet. This suggests, but clearly does not establish, that in the normal erythrocyte the cholesterol content of the inner leaflet exceeds that of the outer. Lastly, integral membrane proteins are expected to decrease lipid fluidity, and the usual pattern seen on freeze-fracture of large numbers of intra-membranous particles on the cytoplasmic face may signify a greater influence of protein in the inner leaflet. The hypothesis that perturbations of the fluidity of a given hemileaflet influence the membrane proteins (and their associated functions) in that leaflet is well-supported by the evidence described above. On the other hand, we understand less well the mechanisms by which lipid fluidity influences the proteins. For example, the decrease in sulfhydryl group reactivity of spectrin, actin, and Band 3 owing to cholesterol depletion (Table 7) may be due to a physical displacement of these proteins, as suggested by Borochov and Shinitzky. Why then does the reactivity of glyceraldehyde-phosphate dehydrogenase sulfhydryl groups increase under these conditions? There remains much to learn about membrane molecular mechanics and lipid-protein interactions. In such studies the impermeant MIMAR probes described here should prove useful.

Asymmetry of lipid dynamics in human erythrocyte membranes studied with permanent fluorophores.

The fluorescence anisotropy and mean excited-state lifetime of 1,6-diphenyl-1,3,5-hexatriene, 12-(9-anthroyloxy)stearate, 2-(9-anthroyloxy)stearate, and pyrenedecanoic acid in the membranes of intact human erythrocytes, lysate suspensions, and ghost membranes were compared. The excited-state lifetime of each lipid fluorophore, estimated by single photon counting, is significantly shorter in the intact erythrocytes as compared to the lysates, owing to nonradiative energy transfer from the lipid fluorophore donors in the membrane to heme acceptors at the endothelial surface of the intact cell. The fluorescence observed in intact cell suspensions is thus weighted in favor of outer leaflet fluorophores, and estimates of the fluorescence anisotropy by steady-state fluorescence polarization indicate that all four fluorescent probes experience greater motional freedom in the outer as compared to the inner membrane leaflet. The results are in accord with prior studies of impermeant pyrene derivatives, which also indicate that the outer leaflet lipids have greater motional freedom.

Erythrocyte membrane proteins: a modified Gorter-Grendel experiment.

The pressure-area isotherm and shear resistance of spectrin-actin monolayers indicate a close-packed structure at about 1.0 m2/mg protein. This surface area is equivalent to a thickness of about two monolayers at the erythrocyte membrane inner face. The maximum elasticity (lowest compressibility) occurs at 0.7 mg2/mg protein, indicating the limit of reversible compression. The mechanical properties of the monolayers approximate those of the intact membrane, suggesting that the structures are similar and that these monolayers may account for many of the in vivo properties.

Modulation of erythrocyte membrane proteins by membrane cholesterol and lipid fluidity.

Human erythrocyte membranes were enriched or depleted of cholesterol and effects on membrane proteins assessed with a membrane-impermeant sulfhydryl reagent, [35S]glutathione-maleimide. Reaction of the probe with intact cells quantifies exofacial sulfhydryl groups and reaction with leaky ghost membranes permits quantification of endofacial sulfhydryl groups. The mean endofacial sulfhydryl titer of cholesterol-enriched membranes exceeded that of cholesterol-depleted membrane by approximately 45 nmol/mg of protein or 64%. The corresponding exofacial titer of cholesterol-enriched cells was less than that of cholesterol-depleted cells by approximately 0.4 nmol/mg of protein, or 14%. Labeled membranes were examined by autoradiography of sodium dodecyl sulfate-polyacrylamide gel electropherograms to determine the labeling patterns of individual protein bands. Cholesterol enrichment enhanced the surface labeling of Coomassie brilliant blue stained bands 1,2,3, and 5, decreased the labeling of band 6, and did not change significantly that of band 4. The results demonstrate that changes in membrane cholesterol which influence lipid fluidity can alter the surface labeling of both intrinsic and extrinsic membrane proteins.

Impermeant maleimides. Oriented probes of erythrocyte membrane proteins.

Maleimides impermeant to human erythrocyte membranes have been synthesized and applied to studies of the sulfhydryl groups of the membrane. Reaction of radioactive dextran-maleimide and glutathione-maleimide with either intact erythrocytes or ghosts yields sulfhydryl titers for the outer (exofacial) and inner (endofacial) surfaces, respectively, of 1.5 to 1.7 and 27 to 28 amol/cell. Corresponding values for sulfhydryl groups within the membrane interior, as estimated with radioactive N-ethylmaleimide, are 16 to 22 amol/cell. After exofacial labeling of intact cells with [35S]glutathione-maleimide, autoradiography of sodium dodecyl sulfate-polyacrylamide gels demonstrates four bands (alpha, beta, gamma, and delta) containing, respectively, 13%, 63%, 11%, and 13% of the radioactivity. The major beta-band corresponds in position to polypeptides of molecular weight 40,000 to 70,000 and to Coomassie brilliant blue-stained Band 5. Selective extraction demonstrates that the major Band 5 protein is not identical with the labeled beta-band polypeptides. Following endofacial labeling of ghosts with [35S]glutathione-maleimide, autoradiography reveals radioactivity in all of the major Coomassie brilliant blue bands. The impermeant maleimides described are also applicable to studies of discrete functional proteins of the erythrocyte membrane, including the hexose transport mechanism and the major Rho antigenic site.

Impermeant maleimides. Identification of an exofacial component of the human erythrocyte hexose transport mechanism.

The facilitated diffusion of D-glucose across human erythrocyte membranes requires an exofacial (outer surface) sulfhydryl group which can be alkylated by the impermeant reagents glutathione-maleimide and dextran-maleimide. The irreversible inhibition produced by these reagents is asymmetric; inhibition of glucose efflux considerably exceeds that of influx when transport is assayed in the absence of glucose on the opposite side of the membrane. Both D-glucose and cytochalasin B protect the exofacial transport site from alkylation by the impermeant maleimides. This masking effect provides the basis for a two-step procedure for differential labeling of the outer transport site with radioactive glutathione-maleimide. The method labels clearly and consistently a component of the membrane proteins which migrates in sodium dodecyl sulfate-polyacrylamide gels between Coomassie brilliant blue-stained Bands 4.2 and 5, corresponding to an apparent molecular weight of 65,000 to 70,000. Transport studies after inhibition with N-ethylmaleimide suggest that the hexose mechanism also requires a second sulfhydryl group which is not accessible at the cell surface.