A geriatric re-evaluation clinic is associated with fewer unplanned returns in the Emergency Department: an observational case-control study.

PURPOSE: The increasing share of older adults is associated with heavier Emergency Health Services utilization. In this context, a significant problem is the rate of unplanned revisits of geriatric patients after discharge from the Emergency Department (ED). We aimed to evaluate whether the referral to a dedicated Geriatric Revaluation Clinic (GRC) after discharge from the ED is associated with fewer early unplanned returns. METHODS: We conducted an observational, retrospective, case-control study comparing patients 65 years or older evaluated in a GRC after an ED visit and a control group of same age subjects accessing the ED during the study period and discharged with one of the ICD-9-CM diagnoses used for the cases, for whom defined post-ED assessment was not arranged. The intervention at the GRC consisted of a comprehensive geriatric evaluation. We calculated unadjusted and adjusted OR for unplanned ED revisits within 30 days from ED discharge using two logistic regression models including the variables with statistically significant differences among study groups at univariate analysis. RESULTS: During the study period, 121 eligible patients were evaluated at the GRC and were matched to 242 subjects included in the control group. The median age of the study population was 85 years. The adjusted OR for unplanned return after ED discharge and unplanned hospital admission after ED discharge were 0.44 (CI 0.20-0.89) and 0.52 (CI 95% 0.18-1.74), respectively. CONCLUSIONS: In a population of older patients discharged from the ED, the referral to a GRC is associated with fewer early unplanned revisits.

In vitro dermal intoxication by bis(chloroethyl)sulfide. Effect on secondary epidermization.

Skin intoxication by bis(beta-chloroethyl)sulfide (BCES; sulfur mustard) induces cutaneous lesions similar to thermal burns, characterized by slowness of skin healing. We have developed an in vitro model of skin equivalent to investigate mechanisms involved in this delay. Direct intoxication of dermal equivalent produced dose- and time-dependent cytotoxicity. A decrease of macroscopic retraction of collagen gels was observed, parallel to the toxic concentration with, at histological level, absence of collagen fiber reorganization. Fibroblast synthesis of fibronectin was also inhibited by intoxication, as demonstrated at an immunobiochemical and immunohistochemical level. These dermal alterations were correlated with secondary modifications of epithelial maturation of nonintoxicated normal human keratinocytes. Cellular adhesion was perturbed, as visualized by a delay in expression and reorganization of basement membrane components, laminen, collagen IV, and fibronectin. Epidermal terminal differentiation was also affected, as shown by the absence of profilaggrin/filaggrin biosynthesis. We demonstrated in vitro, that direct dermal alterations secondarily induce disturbance of epithelial maturation. Taken together, these data show the fundamental role of dermal-epidermal interactions in a normal skin reconstruction. Clinical slowness of wound healing observed after cutaneous intoxication by BCES may thus be explained by direct alkylation of some structures and further disturbances of their biosynthesis.

CD40 ligation of human keratinocytes inhibits their proliferation and induces their differentiation.

While CD40-CD40 ligand interactions are known to regulate B cell proliferation and differentiation, much less is known about the role this receptor plays on other cell types, especially those of nonhemopoietic origin. We report here that CD40 is expressed in normal human epidermis in situ, especially on the basal cell layer, and that it is maintained on cultured epidermal basal cells. Immunoprecipitation and SDS-PAGE analysis confirms that CD40 expressed by epidermal basal cells is immunologically related to the B cell CD40. IFN-gamma up-regulates CD40 expression on cultured keratinocytes, whereas other proinflammatory cytokines, such as IL-1 or TNF-alpha, have little effects. Using CD40-ligand-transfected L cells (CD40Lc), we demonstrated that CD40 triggering results in an enhanced secretion of both IL-8 and TNF-alpha by cultured epidermal basal cells, suggesting that CD40-CD40L interactions may play a role in amplifying the cutaneous inflammatory reactions. More importantly, we found that keratinocyte proliferation was significantly inhibited when the cells were grown on CD40Lc, as compared with CD32-transfected, or nontransfected, L cells. This inhibitory effect can be reversed substantially by pretreatment of keratinocytes with anti-CD40 mAb. In addition, inhibition of proliferation could be obtained by adding a soluble form of CD40 ligand to the keratinocyte cultures. Interestingly, inhibition of keratinocyte proliferation on CD40Lc correlates with differentiation of the cells, as assessed by morphologic analysis and increased profilaggrin content. Collectively, these results demonstrate that CD40 is expressed and functional on human epidermal basal cells and that, on these cells, CD40 ligation may be a signal for limitation of cell growth and induction of differentiation.

Interferon gamma-induced PNA-binding glycoproteins as markers of human keratinocyte differentiation: biological evidence using protein kinase C agonists, antagonists and retinoic acid.

Membrane glycoproteins (gps) play an important role in cell-cell interactions during epidermal maturation, and we have previously shown an up-regulation of PNA-binding gps in cultured human keratinocytes treated with interferon gamma (IFN-gamma). The protein kinase C (PKC) pathway is known to play a key role in the regulation of proliferation and differentiation of keratinocytes and is also reported to be involved in some IFN-gamma-mediated effects. In order to evaluate the cellular mechanisms and whether PNA-binding gp expression is related to the differentiative activity of the lymphokine, we studied the effects of PKC agonists and antagonists and the role of retinoic acid (RA), in the induction of these gps in cultured human keratinocytes stimulated with IFN-gamma and processed for protein analysis. The expression of PNA-binding gps was revealed by incubation of SDS-polyacrylamide gels with 125I-PNA. The PKC antagonists (H7, sphingosine) as well as RA downregulated the IFN-gamma-induced PNA-reactive gps, whereas staurosporine and TPA upregulated their expression. These results provide evidence that PNA-reactive gps are late highly IFN-gamma-sensitive markers of keratinocyte differentiation, drastically modulated through selective isoforms of PKC.

A longitudinal study of a harlequin infant presenting clinically as non-bullous congenital ichthyosiform erythroderma.

Over the past 8 years, we have followed a child born as a harlequin baby, who survived due to treatment with retinoids. His condition evolved clinically towards the erythrodermic form of lamellar ichthyosis (non-bullous congenital ichthyosiform erythroderma, NBCIE). According to ultrastructural and biochemical criteria, our patient originally presented with type II harlequin ichthyosis. Investigations showed an abnormal keratinosome structure and extrusion, a keratin pattern characteristic for epidermal hyperproliferation, and an absence of conversion of profilaggrin to filaggrin. Persisting keratinocyte hyperproliferation, associated with the presence of a dermal infiltrate, is in agreement with the present clinical picture of severe NBCIE. However, abnormal lamellar body production and defective filaggrin processing, which is not one of the diagnostic criteria of NBCIE, persist in the patient's skin. Further studies of the epidermal lipid composition, and of possible mutations of the keratinocyte transglutaminase gene performed on epidermal cell cultures of harlequin ichthyosis, will be necessary before type II harlequin ichthyosis can be accepted as an extremely severe form of NBCIE.

Major antigenic epitopes of bullous pemphigoid 230 kDa antigen map within the C-terminal end of the protein. Evidence using a 55 kDa recombinant protein.

In order to obtain greater insight into the nature of B-cell epitopes in bullous pemphigoid (BP), we generated a BP recombinant protein of 55 kDa M(r) (rBP 55) from a cDNA sequence encoding for the carboxyterminal region of the 230 kDa BP antigen. Serum IgG from guinea-pigs immunized with rBP 55 stained the basement membrane zone of normal human skin and immunoprecipitated the rBP 55 protein, and also the 230 kDa BP antigen recovered from extracts of cultured keratinocytes, thus confirming that the rBP 55 amino acid sequence is present in native BP antigen. The reactivity of sera from 60 patients with BP was analysed using an immunoblot assay on epidermal protein extracts and on the rBP 55 protein. Forty of the 60 BP sera (66%) contained autoantibodies to the 230 kDa polypeptide in an epidermal extract, and 37 of these 40 sera (92%) recognized the rBP 55 protein. In contrast, no reactivity against rBP 55 was detected with 20 BP sera devoid of autoantibodies against the 230 kDa antigen. Likewise, sera from patients with autoimmune blistering skin disorders other than BP (epidermolysis bullosa acquisita or pemphigus vulgaris), and control sera, were unreactive to rBP 55. These results clearly demonstrate the immunogenicity and antigenicity of the C-terminal end of the 230 kDa BP antigen. They confirm that this 555 amino acid segment, corresponding to rBP 55, contains major epitopes which can bind BP patients' autoantibodies, and suggest that the rBP 55 protein could be useful for further characterization of these B-cell epitopes.

Effects of cytokines on the gamma interferon-induced tryptophanyl-tRNA synthetase expression by human keratinocytes.

Incubation of human keratinocytes with gamma interferon (gamma-IFN) has been shown to potently induce the synthesis of a 53 kDa protein which was recently identified as tryptophanyl-tRNA synthetase (TRS). However, in spite of the high sensitivity of cultured keratinocytes to TRS induction by gamma-IFN, the study of inflammatory skin lesions has allowed the detection of the protein only in a few cases, suggesting regulatory mechanisms from soluble endogenous mediators with antagonistic activity on the induction of TRS by gamma-IFN. Among these mediators, we wondered whether cytokines selected for possible anti-inflammatory activity and potentially derived from activated resident skin cells, such as IL-4, IL-10, TNF-alpha and TGF-beta, may be involved in the modulation of the keratinocyte TRS expression. To assess this possibility, we investigated the modulation of the synthesis of TRS by human cultured keratinocytes upon stimulation by various gamma-IFN/cytokine combinations. The effects were evaluated by immunoblotting assay revealed by enhanced chemiluminescence, with the aid of a specific antibody to the TRS protein. Results failed to demonstrate any effect of the tested cytokines, whether on the basal level of the TRS, or on the gamma-IFN-induced enzyme expression in keratinocytes. It is thus unlikely that such cytokines can account for the infrequency of the TRS detection in inflammatory skin processes. Further investigations of alternative working hypothesis should help elucidate the regulation of TRS in human keratinocytes.

Human monoclonal autoantibodies specific for the bullous pemphigoid antigen 1 (BPAg 1).

Bullous pemphigoid (BP) is an acquired blistering skin disease associated with the production of IgG autoantibodies to the 230-kDa BP Ag (BPAg1). To better characterize the epitopes of BPAg1, we generated immortalized B cell lines secreting human mAbs (HumAbs) to BPAg1 from two BP patients whose sera reacted with native BPAg1 but not with a recombinant BP55 carboxyl-terminal peptide. Ab-producing B cell lines were established by EBV infection of CD40-activated PBMCs. Three independent clonal lines were obtained that secreted IgG HumAbs, including one IgG1 kappa (BP3) and two distinct IgG4 kappa (BP1 and BP2). These three HumAbs immunoprecipitated BPAg1. Blocking immunofluorescence experiments and phylogenetic studies showed that these Abs recognize different epitopes of BPAg1. This analysis with HumAbs further extends the serologic demonstration of the wide variety of epitopes recognized by BPAg1 autoantibodies which contrasts with the limited number of epitopes recognized by thyroid peroxidase monoclonal autoantibodies.

[The desmosome: structure, function and acquired pathology]. / Le desmosome: structure, fonction et pathologie acquise.

The essential function of the skin is that of a barriere against the outside. The epidermis is the tissue which assures the solidity and the resistance of the skin against forces of traction. The desmosomes, allowing the adhesion of epidermal cells, the keratinocytes, between them, are the structures of resistance of the epidermis. This adhesion function is possible thanks to the interactions between the desmosomial molecules and cytosqueletal filaments. Currently fifteen desmosomal proteins implicated in this process have been characterized. The role of desmosome in integrity of epidermis is demonstrated by the existence of the auto immune dermatosis, the pemphigus, in which the inflammatory reaction against desmosomal proteins results in the loss of the cohesion of keratinocytes.

Gamma interferon potently induces tryptophanyl-tRNA synthetase expression in human keratinocytes.

Incubation of human keratinocytes with gamma interferon (gamma-IFN) induces the synthesis of a 53-kDa protein of unknown nature and function. We report the identification of this protein through amino acid microsequencing. The NH2-terminal amino acid sequence of the 53-kDa antigen demonstrated that this protein was tryptophanyl-tRNA synthetase (Frolova et al, Gene 109:291-296, 1991, Genbank accession number 61715). This result was validated by the sequencing of tryptic peptides. Identification of the 53-kDa gamma-IFN-induced protein was confirmed by immunoblotting with an antiserum directed against beef pancreas tryptophanyl-tRNA synthetase. Northern blot analysis using a synthetic oligonucleotidic 32P-labeled probe evidenced a 3.1-kb transcript in gamma-IFN-treated cells indicating that the gene was regulated at the pre-translational level. These data show that gamma-IFN potently induces in keratinocytes the expression of an enzyme directly involved in protein biosynthesis. Elevated levels of tryptophanyl-tRNA synthetase in treated cultured keratinocytes might be involved in the cell-growth-inhibitory activity of gamma interferon.

Identification of late differentiation antigens of human cornified epithelia, expressed in re-organized desmosomes and bound to cross-linked envelope.

Little is known about the process leading to desquamation in cornified epithelia. We describe late differentiation antigens (Ag) specific for human cornified squamous epithelia, defined by two murine monoclonal antibodies (MoAb), G36-19 and B17-21, produced after immunization with plantar stratum corneum (SC). Histologically, in epidermis both Ag are cytoplasmic in the lower stratum granulosum (SG), become pericellular in the upper SG, and progressively disappear in the lower SC. In contrast, they persist up to the desquamating corneocytes in the palmoplantar epidermis and hard palate epithelium, as well as in the three cornified epithelial components of the inner root sheath (IRS) of the hair follicle (HF). Cytologically, both Ag are expressed as surface spots only on rough corneocytes. They are largely preserved on cross-linked envelopes (CLE) of the fragile type. Ultrastructurally, both Ag appear in keratinosome-like cytoplasmic vesicles in the upper stratum spinosum (SS) and the SG keratinocytes, then are found in both the regular and reorganizing desmosomes of the SG keratinocytes, and lastly in the corneocyte-specific reorganized desmosomes we propose to name corneodesmosomes. On CLE, the Ag are located on fibrils gathered over the external side of the envelope. Immunochemically, the G36-19--defined epitope is sequential and shared by five non-cytokeratin protein antigens of molecular weight 33.5, 36.5, 40, 49, and 52 kD, the higher molecular weight polypeptides being possibly precursors of the 33.5-kD protein. In contrast, the B17-21 epitope, unaccessible by immunoblotting, is probably conformational. In long-term cultured keratinocytes, the Ag are only expressed when epidermal sheets are morphologically differentiated. The expression is enhanced in the absence of fetal calf serum (FCS) and of epidermal growth factor (EGF). G36-19 and B17-21 Ag participate in a corneodesmosome-CLE superstructure that is probably involved in corneocyte cohesiveness and partly responsible for the mechanical resistance of the SC. These Ag are relevant markers for studying desmosomal maturation during epidermal differentiation and desquamation.

GP37 is different from filaggrin.

The identity of two differentiation markers of human epidermis, filaggrin and a Concanavalin A (Con-A) reactive glycoprotein of 37 kD, has been studied. Human epidermis was extracted in Nonidet P-40 buffer, and the soluble proteins were separated by two-dimensional electrophoresis. Con-A reactive glycoproteins were identified by incubating gels with iodinated lectin followed by autoradiography. Identical, parallel gels were electrophoretically transferred to nitrocellulose paper and filaggrin-related molecules labeled by the specific monoclonal antibody AKH1. We found that the 37-kD Con-A reactive component was resolved by two-dimensional gel electrophoresis into several glycoproteins and that the lectin Con-A does not bind to filaggrin. Under these conditions, the anti-GP37 serum failed to identify any component. However, when applied to human keratinocyte culture extract, AKH1 and the anti-GP37 serum reacted in a similar way. These data show 1) that the 37-kD band is not homogeneous but contains distinct markers of differentiation (filaggrin and Con-A reactive glycoproteins) and 2) that the GP37 antibody's specificity is for the filaggrin precursor.

Expression of cell surface sialic acid and galactose by normal adult human melanocytes in culture.

Normal adult human melanocytes grown either in the presence of phorbol ester or dialyzed hypothalamic extract were analyzed for their cell surface sialic acid and galactose content. In both cases, cells expressed large amounts of sialic acid, whereas they differed in their terminal nonreducing beta-D-galactosyl residues linked to N-acetyl galactosamine; such residues were accessible to peanut agglutinin and Bauhinia purpurea lectin on cells grown in phorbol ester and inaccessible on cells grown with dialyzed hypothalamic extract. In addition, striking differences in morphology and growth characteristics were observed between adult melanocytes grown with phorbol ester or with dialyzed hypothalamic extract. Thus, pure cultures of normal adult human melanocytes grown in the presence of dialyzed hypothalamic extract displayed cell surface properties different from those of melanocytes grown with phorbol ester. Cultures of melanocytes with dialyzed hypothalamic extract are likely to reflect known cell surface characteristics of human melanocytes in the skin. Such cultures could represent a useful model to study normal behavior and tumor progression of pigmented cells.

Effect of gamma-interferon on lectin-binding glycoproteins in cultured human keratinocytes.

We report the effect of exposure of human keratinocyte cultures to human recombinant gamma-interferon (g-IFN) on the expression of glycoproteins. Concanavalia ensiformis agglutinin (Con-A), and Arachis hypogaea agglutinin (PNA) were used to investigate expression of glycoproteins. NP-40 extracts from cultures grown with or without 100 U/ml g-IFN were analyzed by incubation of SDS-polyacrylamide gels with 125I-labeled lectins. Comparison of Con-A binding glycoprotein profiles showed both qualitative and quantitative changes related to the effect of g-IFN. Differences were also apparent after labeling of the gels with PNA. A limited number of components were labeled, with most of the reactivity falling within a couple of diffuse bands with high molecular weight (300 to 360 kDa). These components were strongly labeled in extracts from cells grown in the presence of g-IFN, but weakly reactive in control cultures. Neuraminidase treatment unmasked a 205 kDa PNA binding molecule only when cells were cultured in the absence of g-IFN. These changes are interpreted in terms of increased keratinocyte differentiation induced by g-IFN and demonstrate that glycoproteins bearing carbohydrate residues available to lectins Con-A and PNA have to be taken into account to better understand the complex action of this lymphokine. In inflammatory lesions, such changes in the glycoproteins of keratinocytes expressing HLA-DR antigens remain to be explored.

Modulation of bullous pemphigoid antigens by gamma interferon in cultured human keratinocytes.

We report the effects of human recombinant gamma interferon (gamma-IFN) on the expression of bullous pemphigoid (BP) antigens by human cultured keratinocytes. Secondary epidermal cell cultures were grown on 3T3 mouse fibroblasts; when confluent, some cultures were maintained in control medium while others were exposed to various concentrations of gamma-IFN (100, 200, 400 U/ml) for 14 days. The expression of BP antigens was analyzed by indirect immunofluorescence on epithelial sheets and immunoblotting of Tris, SDS, beta-mercaptoethanol culture extracts using different BP sera. Our results show that gamma-IFN alters the expression of BP antigens in a way varying according to the skin donor: we observed results ranging from complete loss and decreased expression to unchanged reactivity patterns. Thus, gamma-IFN modifies BP antigen expression; this behavior has been previously shown for other adhesion molecules such as fibronectin and thrombospondin. However, the observed variability of the expression of BP antigens according to the skin donor suggests an unexpected variability in keratinocyte sensitivity to gamma interferon, which remains to be explored both in vitro and in vivo.

Vimentin expression in normal human keratinocytes grown in serum-free defined MCDB 153 medium.

We have shown that subcultured keratinocytes derived from normal human skin and grown on plastic in serum-free defined medium (MCDB 153, with 0.1 mM Ca2+ concentration) synthesize vimentin. A fibrillar vimentin expression was observed in the cell cytoplasm by immunohistochemistry with different monoclonal antibodies to vimentin in a high proportion of cultured cells. Two dimensional gel electrophoresis of cytoskeletal proteins from keratinocyte subcultures and immunoblotting reaction with different monoclonal antibodies to vimentin showed a specific reaction in a position corresponding to vimentin. No cross-reaction with keratin polypeptides was obtained. This expression of vimentin may be related to the adaptation of cells to in vitro growth conditions.

Epithelial cell heterogeneity in mammalian thymus: monoclonal antibody to high molecular weight keratins exclusively binds to Hassall's corpuscles.

Hassall's corpuscles represent a subset of medullary thymic epithelial cells whose origin and function within the thymus still remain largely unknown. The present study shows that Hassall's corpuscles can be defined by their intracellular content in specific keratin subunits. Two monoclonal anti-keratin antibodies were used: KL1, directed to high molecular weight keratins, and KL4, specific for high and medium molecular weight polypeptides. In vivo, KL1 exclusively binds to Hassall's corpuscles of five mammalian species including mouse, rat, guinea-pig, rabbit and pig. Thus KL1 appears as an exclusive marker of Hassall's corpuscles in a large number of mammals. In vitro, thymic epithelial cells gave rise in certain species to Hassall's corpuscles. In contrast to its in vivo reactivity, KL1 never labelled Hassall's corpuscles developed in vitro. These data strongly support the following conclusions: (1) Hassall's corpuscles derive from medullary epithelial cells; (2) they represent advanced stages of thymic epithelial maturation; (3) thymic epithelial cell differentiation is impaired in vitro. Furthermore, this study provides additional evidence that thymic epithelium heterogeneity reflects different stages in epithelial maturation.

Comparative expression of bullous pemphigoid antigens in normal human epidermis and cultured keratinocytes.

A heterogeneity of the bullous pemphigoid (BP) antigen initially described as a 220- to 240-kDa polypeptide doublet expressed in normal human skin was recently demonstrated. The aim of our study was to compare the heterogeneity of BP antigen by the immunoblotting technique in extracts of both human epidermis and keratinocyte cultures. Extracts of epidermal tissues obtained from plastic surgery skin samples and secondary keratinocyte cultures were analyzed for their immunologic content defined by 30 BP sera. Twenty-six out of 30 tested sera showed similar binding reactivities with one or several polypeptide bands in both extracts. These sera defined seven antigens of molecular weights ranging from 240 to 97 kDa. Three groups of sera reacting with three, two, or only one antigen could be distinguished in which 180, 200, and 220 kDa polypeptides represented the major BP antigens both in vivo and in vitro. The binding reactivity of most immunofluorescent negative sera demonstrated the sensitivity of the immunoblotting technique to evidence the heterogeneity of BP antigens and antibodies. Keratinocyte cultures represent a reproducible substrate for such analysis and offer a standardization attempt in comparative investigations in BP.