Effects of water gradients and use of urea on skin ultrastructure evaluated by confocal Raman microspectroscopy.

The rather thin outermost layer of the mammalian skin, stratum corneum (SC), is a complex biomembrane which separates the water rich inside of the body from the dry outside. The skin surface can be exposed to rather extreme variations in ambient conditions (e.g. water activity, temperature and pH), with potential effects on the barrier function. Increased understanding of how the barrier is affected by such changes is highly relevant for regulation of transdermal uptake of exogenous chemicals. In the present study we investigate the effect of hydration and the use of a well-known humectant, urea, on skin barrier ultrastructure by means of confocal Raman microspectroscopy. We also perform dynamic vapor sorption (DVS) microbalance measurements to examine the water uptake capacity of SC pretreated with urea. Based on novel Raman images, constructed from 2D spectral maps, we can distinguish large water inclusions within the skin membrane exceeding the size of fully hydrated corneocytes. We show that these inclusions contain water with spectral properties similar to that of bulk water. The results furthermore show that the ambient water activity has an important impact on the formation of these water inclusions as well as on the hydration profile across the membrane. Urea significantly increases the water uptake when present in skin, as compared to skin without urea, and it promotes formation of larger water inclusions in the tissue. The results confirm that urea can be used as a humectant to increase skin hydration.

Effects of water activity and low molecular weight humectants on skin permeability and hydration dynamics - a double-blind, randomized and controlled study.

OBJECTIVES: The mammalian skin is a barrier that effectively separates the water-rich interior of the body from the normally dryer exterior. Changes in the external conditions, for example ambient humidity, have been shown to affect the skin barrier properties. The prime objective of this study was to evaluate the effect of water activity of a topical formulation on skin hydration and permeability. A second objective was to gain more understanding on how two commonly used humectants, urea and glycerol, affect skin barrier function in vivo. METHODS: Simple aqueous formulations were applied under occlusion to the volar forearm of healthy volunteers. Following 4-h exposure, skin water loss (by transepidermal water loss measurements), skin hydration (by Corneometry) and skin permeability (by time to vasodilation due to benzyl nicotinate exposure) were monitored. RESULTS: The results demonstrate that a relatively small change in the water activity of a topical formulation is sufficient to induce considerable effects on stratum corneum hydration and permeability to exogenous substances. Exposing the skin to high water activity leads to increased skin hydration and also increased permeability. Furthermore, urea and glycerol promote skin hydration and permeability even at reduced water activity of the applied formulation. CONCLUSION: These results highlight the importance of considering the water activity in topically applied formulations and the potential benefit of using humectants. The results may impact formulation optimization in how to facilitate skin hydration and to modify skin permeability by temporarily open and close the skin barrier.

The complexity of HLA-DS molecules. A homozygous cell line expresses multiple HLA-DS alpha chains.

Ia molecules expressed by an HLA-DRw6 homozygous cell line were immunoprecipitated with anti-Ia allosera and monoclonal antibodies and analyzed by 2-D gel electrophoresis. The DRw6 homozygous cell line was shown to express two DS beta chains; this observation extends our previous finding that a DR5 homozygous cell line expresses two DS beta chains and suggests that the expression of at least two DS beta chains by DR homozygous cell lines is a generalized phenomenon. The data presented here document for the first time that a DR homozygous cell line expresses at least two DS alpha chains. Therefore, this cell line expresses at least two DS molecules with the potential for the expression of four DS molecules. In agreement with previous reports, the cell line was shown to express two DR beta chains and one DR alpha chain that combine to form two DR molecules. The molecular specificities of two MB1 allosera and two MB1 -like monoclonal antibodies were also compared in these studies. Both MB1 allosera isolated a single DS molecule, while the MB1 -like monoclonal antibodies isolated at least two DS molecules. Therefore, these studies document for the first time that anti-Ia reagents which are specific for the MB1 or MB1 -like determinants in population studies do not recognize the same Ia molecules in immunochemical studies. The data presented here for the expression of at least two DS alpha chains and the location of the MB1 allodeterminant on only one of multiple DS molecules are in agreement with recent studies at the gene level.

Identification of residues involved in polymorphic antibody binding epitopes on HLA-DR molecules.

Based on previous studies it was predicted that amino acids 4 or 25 of the DR4 beta 1 and DR7 beta 1 chains are involved in polymorphic antibody binding epitopes on DR4 or DR7 molecules. These predictions were tested by analyzing monoclonal antibody (mAb) binding to transfectants expressing mutant DR4 beta 1 or DR7 beta 1 chains with single amino acid substitutions at positions 4 or 25. Antibody binding to transfectants expressing additional DR4/7 beta 1 hybrids was also analyzed to assess further the contributions of four segments of the DR4 beta 1 or DR7 beta 1 chains: amino acids 1-20, 21-40, 41-97, and the beta 2 domain. Single amino acid substitutions at positions 4 and 25 of the DR4 beta 1 chain or DR7 beta 1 chain eliminate binding of several mAb to DR4 or DR7 molecules, documenting that these residues are involved in antibody epitopes. However, the data with the hybrid DR4/7 beta 1 chains indicate that some of these epitopes require contributions from both segments 1-20 and 21-40 of these DR beta chains, whereas other epitopes can be generated by placing the appropriate segment in the context of the other DR beta chain. In addition, the data with other mAb indicate that their epitopes are determined primarily by sequences within the 41-97 segment or in the beta 2 domain.

Diverse locations of amino acids in HLA-DR beta chains involved in polymorphic antibody binding epitopes on DR(alpha, beta 1*0101), DR(alpha, beta 1*1101), and DR(alpha,beta 3*0202) molecules.

In a previous study, we used transfectants expressing hybrid HLA-DR(beta 1*0403)/DR(beta 1*0701) chains to map sequences involved in polymorphic antibody binding epitopes on DR(alpha, beta 1*0403) or DR(alpha, beta 1*0701) molecules. Amino acids 1-40 of the beta 1 domain were found to make the major contributions to most of the antibody binding epitopes studied. To begin to localize sequences that contribute to polymorphic antibody epitopes on DR(alpha,beta 1*0101), DR(alpha,beta 1*1101) and DR(alpha,beta 3*0202) molecules, we used indirect immunofluorescence and flow cytometry to assess the binding of mAb to transfectants expressing hybrid DR(beta 1*0101)/DR(beta 1*1101) or DR(beta 1*1101)/DR(beta 3*0202) chains that divide the DR beta chain into three segments: amino acids 1-40, 41-97, and the beta 2 domain. The results indicate that amino acids 41-97 of the beta 1 domain on DR(beta 1*0101), DR(beta 1*1101), or DR(beta 3*0202) are critical in most of the epitopes, including those recognized by human antibodies MP4 and MP12, and mouse mAb GS88.2, I-LR1, 21r5, and 7.3.19.1, whereas amino acids 1-40 of DR(beta 1*1101) are critical in the epitope recognized by the MCS-7 mAb, and both segments 1-40 and 41-97 of DR(beta 1*1101) are important in the epitopes recognized by the I-LR2 and UL-52 mAbs. Based on these data and comparison of DR beta allelic protein sequences, the residues that may play critical roles in these antibody binding epitopes are predicted.

Acidic residues in the DR beta chain third hypervariable region are required for stimulation of a DR(alpha, beta 1*0402)-restricted T-cell clone.

We investigated the minimal requirements for stimulation of an antigen-specific HLA-DR(alpha, beta 1*0402)-restricted T-cell clone (Een217) by using transfectants expressing mutant DR beta chains as APCs. Antigen-specific stimulation of Een217 was induced with transfectants expressing DR(alpha, beta 1*0402) and DR(alpha, beta 1*0403) but not other DR4 subtypes that also bind the peptide recognized by this clone. Analysis of the effects of single amino acid substitutions in the beta chains of each of the DR4 subtypes revealed a requirement for acidic residues in the third HVR, particularly amino acid 71, in stimulation of clone Een217. Functional class II mutants were generated from nonstimulatory DR4 subtype beta chains by acidic residue substitutions within the third HVR. These data define the requirement for negatively charged residues in this region for peptide-induced stimulation of this T-cell clone. The required acidic residues can be located at either position 70, 71, or 74 in the DR beta chain. The negative charge in this segment of the DR beta chain alpha-helix may be required for direct interactions with the T-cell receptor of Een217 or may affect peptide conformation.

TH1 cytokine response of CD57+ T-cell subsets in healthy controls and patients with alcoholic liver disease.

Patients with chronic inflammatory diseases, including Crohn's disease and rheumatoid arthritis, as well as those with certain viral infections, and patients who are transplant recipients or who have certain hematologic malignancies have been observed to have CD57+ T cell expansion in both CD4+ and CD8+ subsets. We have reported previously that alcoholic patients also have CD57+ T cell expansion. Because many alcoholics become seriously deficient in cell-mediated immunity, it is of interest to determine whether the expanded CD57+ subsets can respond to stimulation with normal T helper cell subtype 1 (TH1) cytokine production. We report evaluation of the CD57 T-cell subsets of patients with alcoholic liver disease (ALD) with the use of cytoplasmic staining after stimulation through the T-cell receptor (TCR). The CD57+ subsets of the T cells of both healthy individuals and patients with ALD express significantly higher amounts of cytoplasmic tumor necrosis factor-alpha (TNF-) and interferon-gamma (IFN-) after 6 h of stimulation than do the CD57- subsets. This increased production can persist up to 46 h of continuous stimulation. Under these assay conditions, very little cytoplasmic interleukin (IL)-4 is observed in the T cells of either healthy control subjects or patients with ALD. Measurement of cytokine secretion by sort-purified CD57 T-cell subsets with the use of enzyme-linked immunosorbent assay (ELISA) shows that the CD57+ T-cell subset produces 18- to 30-fold more TNF- and IFN-, respectively, than does the CD57- subset in the first 12 h of stimulation. This response requires only stimulation through the TCR for the CD57+ subset, whereas significant secretion by the CD57- subset requires added IL-2 or anti-CD28 antibody. These results are consistent with the concept of the CD57+ T-cell subset as a differentiated effector cell and demonstrate that patients with ALD who are not drinking at the time of evaluation have normal or increased immediate TH1 T-cell responses.

Obsessive-compulsive disorder due to neuroacanthocytosis treated with citalopram. A case report.

Neuroacanthocytois is a rare hereditary disease, which causes a degeneration of the striatum. Patients develop a choreatic movement disorder and also complex psychiatric symptoms, such as psychosis or Tourette's syndrome. We report a case of obsessive-compulsive disorder due to neuroacanthocytosis. The striatum plays an important role in the pathophysiology of obsessive-compulsive disorder. In Huntington's disease we also find obsessive-compulsive disorders, due to impairment of the fronto-striatal loop. Encouraged by similar pathophysiology and promising reports of selective serotonin reuptake inhibitors in this disease, we started a treatment with citalopram to which the patient responded very well.

[The patients' perception of the anaesthetist in a Swiss university hospital]. / Das Berufsbild des Anästhesisten Eine Untersuchung aus Patientensicht in einem Schweizer Universitätsspital.

OBJECTIVES: The role or recognition of the anaesthetist as an independent medical specialist has been the subject of many studies. Since most of this work was performed in English speaking countries, only few data are available for Germany, Austria, or Switzerland. The goal of this study was to determine how much knowledge patients had of the training and activities of anaesthetists. The study included patients ( n=685) who underwent elective operations in all surgical subspecialties at the University Hospital of Basel. METHODS: The data were collected using a questionnaire distributed at the end of the preoperative visit, which included 14 different questions examining the role of the anaesthetist. RESULTS: Surprisingly, and in contrast to previous studies, almost all patients (99%) knew that the anaesthetist is a qualified physician. In addition, 75% of the patients were aware that the anaesthetist is also engaged in activities outside the operating room; these percentages compare favourably with previous results. However, when asked about the specifics of these activities or about how long it takes to train an anaesthetist, the Swiss patients knew little more than patients from other countries. Only one fifth of all patients estimated the duration of postgraduate training correctly and 45% thought that the anaesthesia team worked under the supervision of the surgical team. Previous anaesthetic experiences as well as additional informational material such as a booklet or videofilm did not improve the patients' knowledge with respect to the training or activities of anaesthetists. DISCUSSION: Since other even more elaborate and expensive methods such as large exhibitions, national anaesthesia days, or increased coverage on radio and television also failed to enhance patients' knowledge, the focus is once again on the relationship between the patient and anaesthetist. If we wish to improve the role and recognition of anaesthetists for patients and/or the public, the anaesthetist must be visible for the patients as a true physician in the pre- and postoperative period. To improve this important patient-anaesthetist relationship, we have begun a training program in communication skills for all physicians in our department.

A newly characterized HLA-DP beta-chain allele. Evidence for DP beta heterogeneity within the DPw4 specificity.

cDNA clones corresponding to the DPw4 alpha- and DPw4 beta-chains were isolated from a cDNA library prepared from a DPw4 homozygous cell line, their nucleotide sequences were determined, and the corresponding amino acid sequences were deduced. This DPw4 alpha-chain is identical to the conserved DP alpha-chains from DPw4 and DPw2 haplotypes, although the DPw4 beta-chain (referred to as DPw4b beta) differs from all reported DP beta-chain sequences. The DPw4b beta-chain differs from the reported DPw4 beta sequence (referred to as DPw4a beta) at three amino acid positions in the first domain (36, 55, and 56). The DPw4b beta-chain sequence differs from the DPw2 beta-chain sequence only at position 69 in the first domain, suggesting that the lysine at position 69 in DPw4b beta and the glutamic acid at position 69 in DPw2 beta contribute to the epitopes that define "DPw4-ness" and "DPw2-ness," respectively. In addition, the patterns of sequence identities and differences among the DPw4b beta-, DPw4a beta-, DPw2 beta-, and DPw3 beta-chains suggest that the DPw4b beta sequence arose via a gene conversion event or a point mutation. The I-LR1 mAb, which was previously found to bind only to DPw2, DPw3, and DR5 molecules, binds to an L cell transfectant expressing the DPw4 alpha:DPw4b beta molecule. The DPw4b beta sequence provides the first evidence for structural heterogeneity within the DPw4 specificity.

Ia molecular localization of the DRw52 allodeterminant and the DRw52-like determinants defined by monoclonal antibodies.

DRw52 (formerly MT2) is a human Ia alloantigen that is expressed in linkage disequilibrium with DR3, 5, w6, and w8. Although there is general agreement that the DRw52 determinant resides on biochemically defined DR molecules, conflicting evidence exists regarding whether DRw52 resides on one or both DR molecules, DQ and DR molecules, or DR and BR molecules. Six anti-DRw52 allosera and three DRw52-like monoclonal antibodies were used to identify the Ia molecules that bear the DRw52 and DRw52-like determinants from DR5 and DRw6 homozygous cells. Based on these two-dimensional gel studies, the DRw52 allodeterminant appears to reside on a subset of DR molecules from DR5 and DRw6 cells. In contrast, the determinants defined by the three anti-DRw52-like monoclonal antibodies were found to reside on one DR molecule, on the second DR molecule, or on both DR molecules, respectively. Therefore, there is considerable complexity of Ia antigenic determinants that are associated with DR3, 5, w6, and w8 at the population level.

Ontogeny of thymic NK1.1+ cells.

Thymic NK1.1+ cells are a recently described lymphocyte subset whose biologic function is not well defined. There is some controversy as to whether thymic NK1.1+ cells mature in a thymic or an extrathymic pathway. In this study, we examined the ontogeny of murine thymic NK1.1+ cells utilizing direct examination of freshly obtained fetal thymi as well as fetal thymi established in organ cultures (FTOC). We found a reproducible peak (5-40%) of NK1.1+ cells, demonstrable in day 15 to 16 freshly obtained fetal thymi, which was markedly decreased by day 17 of gestation; this peak preceded the appearance of the CD4+ CD8+ thymocytes by 12 to 24 h. Reverse-transcriptase PCR analysis of NK1.1 demonstrated its presence as early as day 9 of gestation, thus placing it as one of the earliest lymphocytic genes to be transcribed. Utilizing FTOC, we found that: 1) day 12 fetal thymi contained a progenitor that can differentiate into an NK1.1+ CD4+ CD8+ lymphocyte; 2) NK1.1+ cells dwindle to <5% in FTOC established from day 14 thymi; 3) NK1.1+ cells dominate in FTOC supplemented with IL-2; and 4) most of the NK1.1+ cells seen in FTOC did not express CD3epsilon on their surface, except for the FTOC supplemented with IL-12. These findings suggest that NK1.1+ cells may play an important role in thymic maturation. Moreover, these findings suggest that fetal thymi contain several novel lymphocyte subsets that can be induced to overgrow the normal thymocytes upon exposure to certain cytokines.

Hepatic acylcoenzyme A: cholesterol acyltransferase activity during diet-induced hypercholesterolemia in cynomolgus monkeys.

Acylcoenzyme A: cholesterol acyltransferase (ACAT) activity was studied in hepatic microsomes of cynomolgus monkeys fed either commercial chow or an atherogenic diet of high cholesterol and saturated fat content. ACAT activity (pmol/min per mg protein) was 35 in liver microsomes from control monkeys, and 142 and 161 at 10 and 100 days, respectively, after starting the high cholesterol diet. The cholesterol-fed monkeys had about 1.5-fold increase in cholesterol content of hepatic microsome was compared to control monkeys (94 nmol/mg protein in controls versus 142 nmol/mg protein in the cholesterol fed group). There was no difference between the two groups in microsomal fatty acids in saturated, monoenoic, or polyenoic acid classes. However, the cholesterol-fed monkeys had relatively lower amounts of linoleic acid and higher amounts of arachidonic acid in the microsomes. To determine whether the increased microsomal cholesterol content might be responsible for the increase in ACAT activity, liver microsomes from control monkeys were incubated for 15-120 min with liposomes composed of cholesterol and dipalmitoyl phosphatidylcholine, 2:1 (mol/mol). The microsomal cholesterol content increased from 90 to 128 nmol/mg protein as the incubation progressed. There was a corresponding increase in ACAT activity from 80 to 240 pml/min per mg protein. This observation is consistent with the view that the high hepatic ACAT activity in the cholesterol-fed monkeys is due to the larger amount of cholesterol contained in the microsomes. The increase in hepatic ACAT activity occurs soon after cholesterol feeding is started; this response may be involved in the production of cholesteryl ester-rich lipoprotein by the liver, and thereby may be related to the atherogenic process in these primates.

Analysis of DR beta and DQ beta chain cDNA clones from a DR7 haplotype.

A cDNA library was constructed from a DR7, DRw53, DQw2 homozygous cell line, cDNA clones corresponding to DR beta and DQ beta chains were isolated, and the nucleotide sequences of the polymorphic first domains of these chains were determined. A novel screening strategy allowed rapid and simple identification of cDNA clones corresponding to both DR beta chains (DR7 beta1 and DR7 beta2): DR7 beta2 clones have a recognition site for the enzyme BssHII, whereas DR7 beta1 clones do not. The DR7 beta 1 sequence differs significantly from all previously described DR beta chains. As predicted by the presence of the BssHII site in DR7 beta 2 clones, the DR7 beta 2 sequence differs from the DR7 beta 1 sequence. The sequence of the DRw53-associated DR7 beta 2 chain is identical to the reported sequence of the DRw53-associated DR4 beta 2 chain. In addition, the sequence of the DQ beta chain from the DR7, DQw2 cell line is identical to the reported sequence of a DQ beta chain from a DR3, DQw2 cell. These findings raise interesting questions about the evolution of the DR3, DR4, and DR7 haplotypes.

Ethanol and natural killer cells. II. Stimulation of human natural killer activity by ethanol in vitro.

A single ethanol ingestion of 1 g/kg by healthy individuals under controlled conditions does not inhibit and may stimulate fresh natural killer (NK) activity measured 16 hr later. However, ethanol inhibits fresh human NK activity when added to the lytic assay medium, as reported previously by other investigators. In contrast, using the same target (K562 erythroleukemia cells), peripheral blood mononuclear cells cultured 3 days with 50 units/ml of interleukin-2 are no longer inhibited significantly by the same concentration of ethanol that inhibited the fresh cells by 80%. When freshly isolated peripheral blood mononuclear cells, monocyte-depleted lymphocytes, or partially purified NK cells are pre-exposed to ethanol in vitro for 1 to 7 days, washed, and assayed for lytic activity against K562, the lytic activity is increased compared with nonethanol-exposed cells incubated concurrently. This increase is not dependent on accessory cells, added cytokines, or cell growth, and seems to be an intrinsic response of the NK subset to ethanol exposure. The finding of NK stimulation by ethanol, considered together with the observation of NK cell loss in some chronic alcoholics, suggests that loss of NK activity in the chronic alcoholic may result from cell loss rather than direct ethanol inhibition of NK activity.

Analysis of the molecular specificities of anti-class II monoclonal antibodies by using L cell transfectants expressing HLA class II molecules.

Expressible HLA class II alpha- and beta-chain cDNA were used for DNA-mediated gene transfer to produce L cell transfectants expressing single types of human class II molecules. Cloned transfectants expressing nine different class II molecules were isolated: DR alpha: DR1 beta I, DR alpha: DR4 beta I, DR alpha: DR5 beta I, DR alpha: DR5 beta III (DRw52), DR alpha: DR7 beta I, DR alpha: DR4/7 beta IV (DRw53), DQ7 alpha: DQw2 beta, DQ7 alpha: DQw3 beta, and DPw4 alpha: DPw4 beta. These class II-expressing transfectants were used to analyze by flow cytometry the molecular specificities of 20 anti-class II mAb. These analyes indicate that some mAb are more broadly reactive than was previously thought based on immunochemical studies. In contrast, the narrow molecular specificities of other anti-class II mAb were confirmed by this approach. Transfectants expressing human class II molecules should be valuable reagents for studies of B cell and T cell defined epitopes on these molecules.

Multiple regions of HLA-DR beta 1 chains determine polymorphic epitopes recognized by monoclonal antibodies.

To investigate the locations of antibody binding epitopes on HLA class II molecules, four DR4/7 beta 1 hybrid cDNA were constructed by exchanging the DNA encoding the NH2-terminal portions (amino acids 1 to 40) or the COOH-terminal portions (amino acids 41 to 94) of the first domains of DR4 beta 1- and DR7 beta 1-chains, in association with DNA encoding either the DR4 beta 1 or DR7 beta 1 second domains. Transfectants expressing a DR alpha cDNA and a wild-type DR4 beta 1 or DR7 beta 1 cDNA or one of four hybrid DR4/7 beta 1 cDNA were produced, and the binding to the transfectants of anticlass II mAb, which detect polymorphic epitopes on either DR4 or DR7 molecules, was analyzed. Four different patterns of mAb binding to the transfectants were observed, indicating that multiple regions of DR beta 1-chains play the predominant roles in the contributions of these chains to polymorphic epitopes recognized by mAb on intact molecules. The relevant regions of these chains and the number of mAb that recognize the associated polymorphic epitopes are: 1) the COOH-terminal portion of the first domain of DR4 beta 1; a DR4-specific mAb, 2) the NH2-terminal portion of the first domain of DR7 beta 1; two mAb, including a DR7-specific mAb, 3) the NH2-terminal portion of the first domain of DR4 beta 1; seven mAb, and 4) the second domain of DR4 beta 1; one mAb.