Prevalence of malnutrition and 12-month incidence of mortality in two Sydney teaching hospitals.

AIMS: The objectives of the present study were to determine: (i) the prevalence of malnutrition in two Sydney teaching hospitals using Subjective Global Assessment (SGA), (ii) the effect of malnutrition on 12-month mortality and (iii) the proportion of patients previously identified to be at nutritional risk. METHODS: A prospective study using SGA to assess nutritional status of eligible inpatients, from April to September 1997, with a 12-month follow-up to assess mortality. A total of 819 patients was systematically selected from 2,194 eligible patients. Patients were excluded if they were under the age of 18, had dementia or communication difficulties, or were under obstetric or critical care. The main outcome measures were prevalence of malnutrition, 12-month incidence of mortality, proportion of patients identified with malnutrition, and hospital length of stay (LOS). RESULTS: The prevalence rate of malnutrition was 36%. The proportion of malnourished patients was not significantly different between the two hospitals (P = 0.4). The actuarial incidence of mortality at 12 months after assessment was 29.7% in malnourished subjects compared with 10.1% in well-nourished subjects (P < 0.0005). Malnourished subjects had a significantly longer median LOS (17 days vs 11 days, P< 0.0005) and were significantly older (median 71 years vs 63 years, P < 0.0005) than well-nourished subjects. Only 36% of the malnourished patients had been previously identified as being at nutritional risk. CONCLUSIONS: Malnutrition in Australian hospitals is a continuing health concern and is associated with increased LOS and decreased survival after 12 months. The present study revealed that malnourished patients were not regularly identified. Further studies are required to determine whether routine identification of malnutrition and subsequent nutritional intervention are effective in improving clinical outcomes in these individuals.

Human keratinocytes maintain reversible anti-apoptotic defenses in vivo and in vitro.

Human keratinocytes proliferate and differentiate in an epidermal environment where induction of apoptosis can be triggered by ultraviolet radiation (UVR), activated lymphocytes and cytokines. The purpose of this study was to determine whether keratinocytes were susceptible to apoptosis induced by ionophore, ultra-violet radiation, cytokines or crosslinking of CD95 (Fas/APO-1). In normal human skin exposed to two minimal erythema doses of ultraviolet radiation, suprabasal cells were the first keratinocytes to demonstrate apoptotic nuclei, and by 48 h apoptotic cells were identified throughout the mid to upper epidermis. However, most keratinocytes resisted apoptosis and UVR-induced apoptosis was not observed in basal cells, or in the most differentiated epidermis. Human keratinocytes and keratinocyte cell lines cultured in vitro developed maximal apoptosis 48 h after radiation. Human keratinocytes cultured in full growth factor supplements were resistant to UVR-induced apoptosis compared to keratinocyte cell lines or to a lymphoid cell line (HL60) susceptible to apoptosis. Keratinocyte cell lines were completely resistant to apoptosis induced by interferon-gamma, interferon-alpha, IL-2, IL-6, TNF-alpha, IL-1Ra, and GM-CSF. A subset of the cells in cultures of keratinocytes and transformed keratinocyte cell lines died by apoptosis in response to anti-Fas, IL-1alpha and TNF-alpha plus IFN-gamma and ionophore. Second passage freshly isolated human keratinocytes were much more resistant to apoptosis induced by ionophore, anti-Fas and cytokines than were transformed keratinocyte cell lines. Calcium shift to induce differentiation in second-passage keratinocyte cultures made keratinocytes even more resistant to UVR-induced apoptosis. This parallels the lack of UVR-induced apoptosis observed in the most differentiated keratinocytes in irradiated human skin. Both keratinocytes and keratinocyte cell lines express rather low levels of the anti-apoptotic proteins bcl-2 and bcl-x compared to other apoptosis-resistant cell types. The differences between keratinocytes and keratinocyte cell lines in susceptibility to apoptosis are not explained by difference in expression of bcl-2 or bcl-x. Finally, withdrawal of growth factors from keratinocytes decreased cell survival following UVR and increased the induction of apoptosis. Inhibition of protein synthesis with cyclo-heximide also made keratinocytes more susceptible to UVR-induced apoptosis, indicating that anti-apoptotic defences in cultured keratinocytes are dependent on active protein synthesis. These experiments show that the strong keratinocyte defences against apoptosis are stratified within the epidermis, and can be altered by differentiation and growth factor withdrawal.

Staphylococcal toxins and protein A differentially induce cytotoxicity and release of tumor necrosis factor-alpha from human keratinocytes.

It has been proposed that toxins and other bacterial protein products of Staphylococcus aureus can act as triggers or persistence factors in several inflammatory skin diseases. In this study, we examined the S. aureus isolates from the skin of patients with atopic dermatitis and psoriasis. We found that the bacterial isolates from these patients exhibited either characteristic superantigenic toxins or thermolabile toxins believed to be staphylococcal alpha-toxin. All of these staphylococcal strains also secreted extracellular staphylococcal protein A. We found significant differences in the action of these toxins on human keratinocytes and keratinocyte cell lines. The superantigenic toxins toxic shock syndrome toxin-1, staphylococcal enterotoxins A and B, and exfoliative toxin-A, as well as staphylococcal protein A, did not induce significant cytotoxic damage in the keratinocyte cell line HaCaT, whereas the staphylococcal alpha-toxin produced profound cytotoxicity. Keratinocyte cytotoxicity induced by staphylococcal alpha-toxin was time and concentration dependent and demonstrated the morphologic and functional characteristics of necrosis, not apoptosis. Addition of alpha-toxin to keratinocytes simultaneously induced cell lysis and tumor necrosis factor-alpha release into the medium within 30 min; apparently, it was constitutive tumor necrosis factor-alpha. On the other hand, superantigenic toxins and, in particular, protein A showed stimulation of tumor necrosis factor-alpha secretion in keratinocytes and release of this cytokine after 6-12 h of incubation. Thus, staphylococcal protein A, alpha-toxin, and superantigenic toxins found in S. aureus isolates from patients with psoriasis and atopic dermatitis can produce direct pro-inflammatory effects on keratinocytes through the release of tumor necrosis factor-alpha. We propose that these effects may be relevant to the induction and persistence of lesions in these two diseases.

Expression of E and P-cadherin by melanoma cells decreases in progressive melanomas and following ultraviolet radiation.

The purpose of our study was to determine whether the degree of E- and P-cadherin expression in melanomas correlates with the invasive behavior of the clinical lesions from which the cell lines were derived. Cadherins comprise a family of calcium-dependent cellular adhesion molecules expressed on most cell types that form solid tissues. In the human epidermis, melanocyte cadherin expression may function to maintain the integrity of the epidermal-melanin unit. Employing both immunofluorescence microscopy and fluorescence-activated cell sorter analysis, we localized and quantitated E- and P-cadherin expression on melanoma cell lines derived from primary or metastatic lesions using the monoclonal antibodies HECD-1 and NNC-CAD-299, respectively. Human epidermal melanocytes isolated from neonatal foreskin were evaluated by similar techniques and served as a biologic control. Melanoma cell lines were isolated from primary or metastatic lesions of patients described as having "early," "intermediate," or "advanced disease." Melanoma E- and P-cadherin immunofluorescence, as quantified by fluorescence-activated cell sorter, varied inversely with disease progression. Selected log mean ratios of E-cadherin fluorescence, as compared to human epidermal melanocytes (arbitrarily = 1), ranged from 1.04 in the WM 35 melanoma cell line (low invasive potential) to 0.1 and 0.02 in the WM 983A and 1361A melanoma cell lines (derived from primary lesions with metastases), respectively. Although values for P-cadherin fluorescence were less, the trend of decreasing cadherin amounts with more advanced disease was observed. Melanoma cells appear to express E- and P-cadherin levels inversely related to disease progression. Ultraviolet radiation significantly decreased E- and P-cadherin expression in the human epidermal melanocytes and P-cadherin expression in the WM 35 melanoma cell line (p < 0.05). Although not statistically significant, E-cadherin expression in the WM 35 melanoma cell line decreased substantially. Thus, ultraviolet radiation may have a direct effect on human epidermal melanocytes and melanoma cell attachment through cadherins within the epidermis or tumor nodules.

In three types of interface dermatitis, different patterns of expression of intercellular adhesion molecule-1 (ICAM-1) indicate different triggers of disease.

We found distinct patterns of intercellular adhesion molecule-1 (ICAM-1) expression in three diseases characterized by interface dermatitis with mononuclear infiltrates and keratinocyte cytotoxicity: lichen planus (LP), subacute cutaneous lupus erythematosus (SCLE), and erythema multiforme (EM). In LP, basal keratinocytes show strong ICAM-1 expression associated with a dermal infiltrate, but ICAM-1 expression in the rest of the epidermis is minimal. In SCLE, there is diffuse epidermal ICAM-1 expression, sometimes with accentuation on the cell surface of basal cells. In EM, there is strong basal cell expression of ICAM-1 with evident cell surface accentuation, and also pockets of suprabasal expression with cell surface accentuation. These patterns are associated with different factors that trigger cytokine release in different locations. Both tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) produce greater relative ICAM-1 expression in basal keratinocytes than in more differentiated keratinocytes. In LP, the pure basal keratinocyte expression of ICAM-1 appears to be caused by cytokines, predominantly IFN-gamma, released by dermal lymphocytes. The pattern of ICAM-1 in SCLE corresponds to the pattern induced by ultraviolet radiation (UVR): diffuse epidermal ICAM-1 expression, sometimes with basal accentuation. Some individuals are "responders" to TNF-alpha or UVR, showing high levels of ICAM-1 expression following UVR or TNF-alpha stimulation in vitro or UVR stimulation in vivo. We propose that the pattern of ICAM-1 induction in SCLE is dependent on UVR-induced TNF-alpha release. EM is associated with apparent latent Herpes simplex virus, and Herpes simplex virus (HSV)-infected keratinocytes show enhanced ICAM-1 expression. We propose that in EM suprabasal ICAM-1 expression may be induced directly by HSV infection or indirectly through TNF-alpha release induced by HSV reactivation. Induction of ICAM-1 within the epidermis is stratified and individually variable. Basal keratinocytes show maximal induction of ICAM-1 expression due to innate sensitivity to TNF and IFN-gamma stimulation, and to location adjacent to dermal sources of cytokines. Suprabasal ICAM-1 can be induced by UVR and epidermal TNF-alpha release, and by factors such as viral infection. Different triggers of cytokine release and adhesion molecule induction may influence the different patterns of inflammation seen in diverse inflammatory skin diseases.

Cytokine-induced ICAM-1 expression in human keratinocytes is highly variable in keratinocyte strains from different donors.

Induction of intercellular adhesion molecule 1 (ICAM-1) expression in the epidermis is felt to be an important initiator of leukocyte/keratinocyte interactions in many inflammatory skin diseases. The purpose of this project was to determine the individual variability of cytokine-induced ICAM-1 expression in human keratinocytes obtained from different donors. In 55 different keratinocyte strains, there was significant individual variability in ICAM-1 expression by either tumor necrosis factor alpha (TNF-alpha) or interferon-gamma. There was no correlation (r = 0.266, p = 0.06) in response of the same strain to either TNF-alpha or interferon-gamma. Multiple (n = 22) keratinocyte strains showed no significant induction of ICAM-1 expression to IL-1. The level of ICAM-1 expression in response to TNF-alpha and ultraviolet radiation (UVR) in individual strains was highly correlated in three different comparisons: level of stimulated response versus baseline (TNF-alpha and UVR both p < 0.0001); stimulation index TNF-alpha versus UVR (p = 0.00947); and variability of stimulated response versus variability of baseline (TNF p < 0.001; UVR p = 0.002). UVR-induced release of TNF from keratinocytes also showed variability among different keratinocyte strains. The UVR-induced ICAM-1 response in human keratinocytes and transformed epithelial cell was variably blocked with anti-TNF antibodies. The release of TNF from keratinocytes by UVR and the individually variable but linked characteristics of UVR and TNF-alpha stimulated ICAM-1 expression support the hypothesis that TNF-alpha is a major mediator of UVR-induced ICAM-1 expression.

Ultraviolet radiation can either suppress or induce expression of intercellular adhesion molecule 1 (ICAM-1) on the surface of cultured human keratinocytes.

Interactions of the ligand/receptor pair LFA-1(CD11a/CD18) and ICAM-1(CD54) initiate and control the cell-cell interactions of leukocytes and interactions of leukocytes with parenchymal cells in all phases of the immune response. Induction of the intercellular adhesion molecule 1 (ICAM-1) on the surface of epidermal keratinocytes has been proposed as an important regulator of contact-dependent aspects of cutaneous inflammation. Ultraviolet radiation (UVR) also modifies cutaneous inflammation, producing both up- and down-regulation of contact hypersensitivity. We have found that UVR has a biphasic effect on the induction of keratinocyte CD54. Using immunofluorescence and FACS techniques to quantitate cell-surface CD54 staining, we have shown that UVR (100 mJ/cm2 of UVB) significantly (p less than 0.01) inhibits keratinocyte CD54 induction by gamma interferon 24 h after irradiation. However, at 48, 72, and 96 h after UVR (10 to 100 mJ/cm2), CD54 expression is significantly induced (p less than 0.01 to p less than 0.001) to levels even greater than are induced by gamma interferon (20 U/ml). In addition, at 48, 72, or 96 h following UVR (30-100 mJ/cm2), the gamma-interferon-induced CD54 expression on human keratinocytes is also strongly (p less than 0.05 to p less than 0.001) enhanced. In this cell-culture system, gamma interferon and TNF-alpha are both strong CD54 inducers and are synergistic, but GM-CSF, TFG-beta, and IL-1 have no direct CD54-inducing effects. Thus the effects of UVR on CD54 induction are biphasic, producing inhibition at 24 h and induction at 48, 72, and 96 h. This effect on CD54 may contribute to the biphasic effects of UVR on delayed hypersensitivity in vivo. The early inhibition of ICAM-1 by UVR may also contribute to the therapeutic effects of UVR. We also speculate that the late induction of ICAM-1 by UVR might be an important step in the induction of photosensitive diseases such as lupus erythematosus.

Inhibition of antibody formation by receptor cross-linking: the molecular characteristics of inhibitory haptenated polymers.

Previous studies from this laboratory have reported on the characteristics of a molecule which make it inhibitory. These findings were based on studies using the T cell-independent haptenated polymer, 2,4-dinitrophenyl-polyacrylamide (DNP-PA). The present study was undertaken to determine whether the same molecular properties which defined the inhibitory potentials of DNP-PA were characteristic of other haptenated polymers as well. The molecules studied here consisted of a series of five diverse fluoresceinated polymers with varying molecular mass and hapten valence. In agreement with the previous findings on DNP-PA, definable molecular forms of fluoresceinated dextrans. Ficolls, polyacrylamides, carboxymethyl celluloses and polyvinyl alcohols were each found to be capable of inhibiting the anti-fluorescein response to immunogenic forms of haptenated polymers of either the same or different carrier chemistry. This inhibitory ability was relatively independent of the chemical composition and conformation of the carrier polymer. These studies allow some logical generalizations to be made as to which quantitative molecular properties of haptenated type 2 T cell-independent antigens determine whether they will be stimulatory or inhibitory of an anti-hapten immune response.

The immunogenicity of soluble haptenated polymers is determined by molecular mass and hapten valence.

T cell-independent Ag are believed to stimulate antibody formation in the relative absence of Ag processing and T cell help. Previous studies on the type 2 T cell independent (TI-2) Ag DNP-polyacrylamide, have shown that when one systematically varies the molecular mass and hapten valence, the immunogenic potential of this type of molecule depends on definable molecular characteristics. It was found that to be immunogenic, these molecules had to exceed a threshold molecular mass of 100,000 Da and a threshold hapten valence of 20. The present study was undertaken to determine whether such findings could be generalized to other molecules belonging to the TI-2 class of Ag. The molecular characteristics of five chemically different fluoresceinated (FL)-polymers were systematically varied, and their ability to stimulate an IgM antihapten immune response was measured. The polymers used as carriers were carefully size-fractionated and consisted of one natural polymer (dextran), one modified natural polymer (carboxymethyl cellulose), and three synthetic polymers (Ficoll, polyvinyl alcohol, and polyacrylamide). The carriers varied in physical structure from the highly cross-linked Ficoll, to the somewhat branched dextran, to the linear polyacrylamide, carboxymethyl cellulose, and polyvinyl alcohol. Polymers were haptenated with FL and size-fractionated so as to yield a panel of molecules with varying molecular mass, hapten valence, and hapten density. Anti-FL IgM response to these haptenated polymers was measured in vivo after i.p. injection of the FL-polymer in saline, and measured in vitro after culture with unfractionated spleen cells from naive mice. In agreement with the previous studies on DNP-polyacrylamide, it was found that to be immunogenic, each of the FL-polymers had to exceed a comparable threshold value of molecular mass and of hapten valence. Optimal immunogenicity occurred when the FL-polymers had values of mass and hapten density lying within a predictable range. Immunogenicity decreased when these optimal parameters were substantially increased or decreased. We conclude that the immunogenicity of soluble haptenated polymers depends on predictable physical molecular characteristics, and is relatively independent of the chemical composition and conformation of the carrier polymer.

Recombinant mouse interferon-gamma is not chemotactic for macrophages or neutrophils.

Recent evidence implicates interferon-gamma (IFN-gamma) in resistance to infection with the facultative intracellular bacterium, Listeria monocytogenes. Reports showing that inflammatory macrophages and neutrophils are highly bactericidal for listeria suggest that IFN-gamma might act by directly or indirectly causing recruitment of these cells. The purpose of the experiments described here was to test whether recombinant mouse IFN-gamma is chemotactic for macrophages or neutrophils in vivo or in vitro. In vivo experiments showed rIFN-gamma to have no ability to cause recruitment of inflammatory neutrophils or macrophages into the peritoneal cavities of mice. When tested in vitro, rIFN-gamma also did not induce migration of inflammatory neutrophils or macrophages through cellulose nitrate filters in a chemotaxis assay. The data indicate that rIFN-gamma has no direct or indirect chemotactic activity in vivo or in vitro for mouse macrophages or neutrophils.

Tolerogen-mediated suppression of the immune response. Suppressor cells as passive transfer agents.

The anti-dinitro-phenyl (Dnp)IgM antibody response in mice was inhibited by administration of either a non-immunogenic form of Dnp-polyacrylamide (Dnp-Pa) or an excess amount of an immunogenic form of Dnp-Pa. Spleen cells, alive or heat-killed, from mice tolerized in vivo by either method, inhibited the anti-Dnp response of naive spleen cells co-cultured in vitro with antigen. Conversely, donor cells tolerized in vivo by a high dose of immunogenic Dnp-Pa, when titrated into a naive cell culture which contained no antigen, produced a stimulatory dose-response curve. Both the dose-dependent inhibition and stimulation correlated strongly with the amount of 125I-labelled Dnp-Pa carried over by tolerized spleen cells into the naive cell culture system. Because the doses and experimental procedures used were comparable to those commonly used for suppressor cell generation and assay, it is suggested that antigen-specific suppressor cells may produce their effects through passive transfer of antigen and/or tolerogen.

Inhibition of anti-DNP antibody formation by high doses of DNP-polyacrylamide molecules; effects of hapten density and hapten valence.

High-dose inhibition of anti-DNP antibody formation by a series of DNP-polyacrylamide molecules of varying hapten density and hapten valence was measured. It was found that a molecule's inhibitory ability correlated directly with its hapten density, but not with its hapten valence nor with its own ability at optimal dose to stimulate an anti-DNP response.

Studies on the immunogenicity and tolerogenicity of T-independent antigens.

The T-cell independent immunological responses of naive mice to a series of size-fractionated dinitrophenylpolyacrylamide molecules were measured. These responses were correlated in vitro and in vivo with hapten number per molecule and hapten density per unit length. We found that to be immunogenic, a T-independent molecule must contain a threshold number (approximately 20) of appropriately spaced haptens or epitopes. We infer that this condition is necessary for simultaneous B-cell receptor binding and clustering. Molecules with less than this number of haptens are not immunogenic at any dose. Some nonimmunogenic molecules are capable of inhibiting the response to the immunogenic ones; this capability increases with increasing hapten density. In order for a molecule to accommodate 20 or more appropriately spaced haptens, it must be above a threshold size. Increasing hapten density in a molecule at or above threshold size and hapten number increases its immunogenicity. Increasing hapten density in a molecule below threshold size increases its tolerogenicity. Conclusions concerning immunogenicity and tolerogenicity of T-independent antigens may not be warranted unless the molecular weight and hapten or epitope number per molecule are well characterized.

Expression of cloned herpesvirus genes. I. Detection of nuclear antigens from herpes simplex virus type 2 inverted repeat regions in transfected mouse cells.

Three different recombinant plasmids containing the entire 15-kilobase L and S inverted repeat sequence of herpes simplex virus type 2 DNA have been introduced into cultured Ltk- or BSC cells by both the calcium and DEAE-dextran transfection procedures. In each case, after 24 h approximately 1% of the cells gave strongly positive nuclear staining when assayed by immunofluorescence with hyperimmune antisera made against early and immediate-early infected-cell polypeptides. The nuclear fluorescence pattern and intensity mimicked that observed within 2 to 3 h after infection of Ltk- cells with either herpes simplex virus type 1 or type 2 wild-type virus. Herpes simplex virus type 1 (KOStsB2)-infected Ltk- cells under nonpermissive conditions did not express these antigens in the nucleus. Therefore, we conclude that either one or both of the 185,000- and 110,000-molecular-weight immediate early proteins, or some other as yet unknown gene product encoded entirely within the inverted repeats, can be transiently expressed in large amounts in transfected cells in the absence of other viral genes or accompanying virion components. Permanent mouse cell lines derived from transfection with these plasmids by using the thymidine kinase coselection procedure did not express sufficient nuclear antigen to be detectable by immunofluorescence.