Skin differences based on age and chronicity of ultraviolet exposure: results from a gene expression profiling study.

BACKGROUND: Skin ageing represents an inevitable physiological consequence of getting older but the impact on personal health and wellbeing can be significant, and therefore considerable efforts have been made to understand the biology and pathophysiology of skin ageing to try to identify new targets that might offer therapeutic intervention and prevention. OBJECTIVES: This study was designed to identify differences at the gene expression level between young and old, sun-exposed and sun-protected skin. METHODS: We generated transcriptomic data from young and old skin from sun-exposed and sun-protected sites (10 samples of each) using HG-U133 Plus 2.0 Affymetrix GeneChips. The data were analysed using hierarchical clustering, theme analysis and interaction mapping to identify regulated pathways, processes and potential targets for therapy. RESULTS: With 54,613 probe sets on the GeneChip, 2731 significant differences would be expected by chance (at P = 0·05), but we noted that 13,640 probe sets were significantly different comparing young arm skin vs. older arm skin (photoageing), and 7215 probe sets were significant for the young buttock vs. older buttock comparison (intrinsic ageing). In both types of ageing there was reduced expression of many genes implicated in lipid biosynthesis and epidermal differentiation with functional relevance to skin barrier integrity and maintenance. Increased expression of genes contributing to oxidative stress and decreased expression of antioxidant defences were also common to both types of ageing. Differences between intrinsic ageing and photoageing were mainly noted in extracellular matrix gene expression with reduced expression of interstitial collagen genes in intrinsic ageing and increased expression of elastic tissue genes in photoageing. CONCLUSIONS: Collectively, the data identified new biomarkers of aged skin, particularly involving abnormalities of proteases, matrix proteins and inflammation. These findings offer the prospect of new and more specific targets for therapeutic development based on an improved understanding of the biology of skin ageing.

Sclerostin antibody treatment enhances bone strength but does not prevent growth retardation in young mice treated with dexamethasone.

OBJECTIVE: Exposure to supraphysiologic levels of glucocorticoid drugs is known to have detrimental effects on bone formation and linear growth. Patients with sclerosteosis lack the bone regulatory protein sclerostin, have excessive bone formation, and are typically above average in height. This study was undertaken to characterize the effects of a monoclonal antibody to sclerostin (Scl-AbI) in mice exposed to dexamethasone (DEX). METHODS: Young mice were concomitantly treated with DEX (or vehicle control) and Scl-AbI antibody (or isotype-matched control antibody [Ctrl-Ab]) in 2 independent studies. Linear growth, the volume and strength of the bones, and the levels of bone turnover markers were analyzed. RESULTS: In DEX-treated mice, Scl-AbI had no significant effect on linear growth when compared to control treatment (Ctrl-Ab). However, in mice treated with DEX and Scl-ABI, a significant increase in trabecular bone at the femoral metaphysis (bone volume/total volume +117% versus Ctrl-Ab-treated mice) and in the width and volume of the cortical bone at the femoral diaphysis (+24% and +20%, respectively, versus Ctrl-Ab-treated mice) was noted. Scl-AbI treatment also improved mechanical strength (as assessed by 4-point bending studies) at the femoral diaphysis in DEX-treated mice (maximum load +60% and ultimate strength +47% in Scl-AbI-treated mice versus Ctrl-Ab-treated mice). Elevated osteocalcin levels were not detected in DEX-treated mice that received Scl-AbI, although levels of type 5b tartrate-resistant acid phosphatase were significantly lower than those observed in mice receiving DEX and Ctrl-Ab. CONCLUSION: Scl-AbI treatment does not prevent the detrimental effects of DEX on linear growth, but the antibody does increase both cortical and trabecular bone and improves bone mechanical properties in DEX-treated mice.

The cutaneous lymphocyte antigen is a skin lymphocyte homing receptor for the vascular lectin endothelial cell-leukocyte adhesion molecule 1.

A skin-associated population of memory T lymphocytes, defined by expression of the cutaneous lymphocyte antigen (CLA), binds selectively and avidly to the vascular lectin endothelial cell-leukocyte adhesion molecule 1 (ELAM-1), an interaction that may be involved in targeting of CLA+ T cells to cutaneous sites of chronic inflammation. Here we present evidence that CLA itself is the (or a) lymphocyte homing receptor for ELAM-1. Antigen isolated with anti-CLA monoclonal antibody HECA-452 from human tonsillar lysates avidly binds ELAM-1 transfected mouse cells. Anti-CLA antibody blocks T lymphocyte binding to ELAM-1 transfectants. HECA-452 and ELAM-1 binding to lymphocytes or to isolated tonsillar HECA-452 antigen is abrogated by neuraminidase treatment implying a prominent role for sialic acid in CLA structure and function. The dominant form of CLA on T cells is immunologically distinct from the major neutrophil ELAM-1 ligand, the sialyl Lewis x (sLex) antigen (NeuAc alpha 2-3Gal beta 1-4[Fuc alpha 1-3]GlcNAc), which is absent, weakly expressed, or masked on T cells. However, neuraminidase treatment of CLA+ T cells, but not of CLA- T cells, reveals Lewis x (CD15) structures. In combination with the known requirement for terminal NeuAc alpha 2-3Gal and fucose residues attached to N-acetylglucosamine for ELAM-1 and HECA-452 binding, this finding suggests that CLA may comprise an additionally sialylated or otherwise modified form of sLex. The identification of a lymphocyte homing receptor for skin may permit novel approaches to the diagnosis and therapy of cutaneous and inflammatory disorders.

The human peripheral lymph node vascular addressin is a ligand for LECAM-1, the peripheral lymph node homing receptor.

The trafficking of lymphocytes from the blood and into lymphoid organs is controlled by tissue-selective lymphocyte interactions with specialized endothelial cells lining post capillary venules, in particular the high endothelial venules (HEV) found in lymphoid tissues and sites of chronic inflammation. Lymphocyte interactions with HEV are mediated in part by lymphocyte homing receptors and tissue-specific HEV determinants, the vascular addressins. A peripheral lymph node addressin (PNAd) has been detected immunohistologically in mouse and man by monoclonal antibody MECA-79, which inhibits lymphocyte homing to lymph nodes and lymphocyte binding to lymph node and tonsillar HEV. The human MECA-79 antigen, PNAd, is molecularly distinct from the 65-kD mucosal vascular addressin. The most abundant iodinated species by SDS-PAGE is 105 kD. When affinity isolated and immobilized on glass slides, MECA-79 immunoisolated material binds human and mouse lymphocytes avidly in a calcium dependent manner. Binding is blocked by mAb MECA-79, by antibodies against mouse or human LECAM-1 (the peripheral lymph node homing receptor, the MEL-14 antigen, LAM-1), and by treatment of PNAd with neuraminidase. Expression of LECAM-1 cDNA confers PNAd binding ability on a transfected B cell line. We conclude that LECAM-1 mediates lymphocyte binding to PNAd, an interaction that involves the lectin activity of LECAM-1 and carbohydrate determinants on the addressin.

Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro.

Inappropriate signalling through the EGFR and ErbB2/HER2 members of the epidermal growth factor family of receptor tyrosine kinases is well recognised as being causally linked to a variety of cancers. Consequently, monoclonal antibodies specific for these receptors have become increasingly important components of effective treatment strategies for cancer. Increasing evidence suggests that ErbB3 plays a critical role in cancer progression and resistance to therapy. We hypothesised that co-targeting the preferred ErbB2/ErbB3 heterodimer with a bispecific single-chain Fv (bs-scFv) antibody would promote increased targeting selectivity over antibodies specific for a single tumour-associated antigen (TAA). In addition, we hypothesised that targeting this important heterodimer could induce a therapeutic effect. Here, we describe the construction and evaluation of the A5-linker-ML3.9 bs-scFv (ALM), an anti-ErbB3/ErbB2 bs-scFv. The A5-linker-ML3.9 bs-scFv exhibits selective targeting of tumour cells in vitro and in vivo that co-express the two target antigens over tumour cells that express only one target antigen or normal cells that express low levels of both antigens. The A5-linker-ML3.9 bs-scFv also exhibits significantly greater in vivo targeting of ErbB2'+'/ErbB3'+' tumours than derivative molecules that contain only one functional arm targeting ErbB2 or ErbB3. Binding of ALM to ErbB2'+'/ErbB3'+' cells mediates inhibition of tumour cell growth in vitro by effectively targeting the therapeutic anti-ErbB3 A5 scFv. This suggests both that ALM could provide the basis for an effective therapeutic agent and that engineered antibodies selected to co-target critical functional pairs of TAAs can enhance the targeting specificity and efficacy of antibody-based cancer therapeutics.

TPD1 of Saccharomyces cerevisiae encodes a protein phosphatase 2C-like activity implicated in tRNA splicing and cell separation.

The Saccharomyces cerevisiae TPD1 gene has been implicated in tRNA splicing because a tpd1-1 mutant strain accumulates unspliced precursor tRNAs at high temperatures (W. H. van Zyl, N. Wills, and J. R. Broach, Genetics 123:55-68, 1989). The wild-type TPD1 gene was cloned by complementation of the tpd1-1 mutation and shown to encode a protein with substantial homology to protein phosphatase 2C (PP2C) of higher eukaryotes. Expression of Tpd1p in Escherichia coli results in PP2C-like activity. Strains deleted for the TPD1 gene exhibit multiple phenotypes: temperature-sensitive growth, accumulation of unspliced precursor tRNAs, sporulation defects, and failure of cell separation during mitotic growth. On the basis of the presence of these observable phenotypes and the fact that Tpd1p accounts for a small percentage of the observed PP2C activity, we argue that Tpd1p is a unique member of the PP2C family.

The Caenorhabditis elegans EGL-15 signaling pathway implicates a DOS-like multisubstrate adaptor protein in fibroblast growth factor signal transduction.

EGL-15 is a fibroblast growth factor receptor in the nematode Caenorhabditis elegans. Components that mediate EGL-15 signaling have been identified via mutations that confer a Clear (Clr) phenotype, indicative of hyperactivity of this pathway, or a suppressor-of-Clr (Soc) phenotype, indicative of reduced pathway activity. We have isolated a gain-of-function allele of let-60 ras that confers a Clr phenotype and implicated both let-60 ras and components of a mitogen-activated protein kinase cascade in EGL-15 signaling by their Soc phenotype. Epistasis analysis indicates that the gene soc-1 functions in EGL-15 signaling by acting either upstream of or independently of LET-60 RAS. soc-1 encodes a multisubstrate adaptor protein with an amino-terminal pleckstrin homology domain that is structurally similar to the DOS protein in Drosophila and mammalian GAB1. DOS is known to act with the cytoplasmic tyrosine phosphatase Corkscrew (CSW) in signaling pathways in Drosophila. Similarly, the C. elegans CSW ortholog PTP-2 was found to be involved in EGL-15 signaling. Structure-function analysis of SOC-1 and phenotypic analysis of single and double mutants are consistent with a model in which SOC-1 and PTP-2 act together in a pathway downstream of EGL-15 and the Src homology domain 2 (SH2)/SH3-adaptor protein SEM-5/GRB2 contributes to SOC-1-independent activities of EGL-15.

Leukocyte integrin Mac-1 recruits toll/interleukin-1 receptor superfamily signaling intermediates to modulate NF-kappaB activity.

The leukocyte integrin Mac-1 (alphaMbeta2, CD11b/CD18) regulates important cell functions in inflammation, including adhesion, phagocytosis, and oxidative burst. Deficiency of Mac-1 reduces vessel wall inflammation and neointimal thickening after murine carotid artery injury. Although Mac-1 has been implicated in modulating AP-1 and NF-kappaB activity, the signal transduction pathways involved are undefined. cDNA array analysis of Mac-1-clustered compared with -nonclustered monocytic THP-1 cells showed increased expression of the signal transducer TRAF6 (TNF receptor-associated factor 6), leading us to consider the possibility that Mac-1 used a Toll/IL-1 receptor family-like signaling pathway. Mac-1-dependent activation of NF-kappaB was potentiated by wild-type, and attenuated by dominant negative, TRAF6- and TGF-beta-activated kinase (TAK1) constructs. IRAK1 (IL-1 receptor associated kinase), a kinase immediately upstream of TRAF6, coimmunoprecipitated with Mac-1. Taken together, these observations indicate that Mac-1 recruits a Toll/IL-1 receptor family-like cascade to modulate NF-kappaB activity. This represents a new pathway for integrin-dependent modulation of gene expression.

Enhanced suppressor macrophage activity associated with termination of the L5178Y cell tumor-dormant state in DBA/2 mice.

Both cytolytic T-lymphocytes and cytolytic macrophages have been implicated in the long-term maintenance of L5178Y cells in a tumor-dormant state in DBA/2 mice. Eventually, however, the tumor-dormant state terminates, and all mice develop ascitic tumors. In an evaluation of the mechanisms involved in termination of the tumor-dormant state, we detected in the peritoneal cavity of many tumor-dormant mice macrophages with increased capacity to suppress the in vitro generation of a secondary anti-L5178Y cell cytolytic T-lymphocyte response. The incidence of macrophage-mediated immunosuppressive activity in individual tumor-dormant mice was related directly to the number of tumor cells in the peritoneal cavity of those mice. Furthermore, in tumor-dormant mice harboring fewer than 5 X 10(4) L5178Y cells, the detection of macrophage-mediated immunosuppressive activity was a prognostic indicator of termination of the tumor-dormant state and development of an ascitic tumor. These data suggest that peritoneal macrophage-mediated immunosuppressive activity, through inhibition of cytolytic T-lymphocyte generation in vivo, contributes to the termination of the tumor-dormant state and development of ascitic tumors.

Elimination of L5178Y cells from tumor-dormant DBA/2 mice by specific active immunotherapy.

Cytolytic T-lymphocytes (CTL) can be repeatedly stimulated in L5178Y cell tumor-dormant DBA/2 mice by the i.p. inoculation of 2 X 10(6) X-irradiated L5178Y cells. The restimulated CTL activity has the same kinetics of generation and decline and the same target cell specificity as does the CTL response generated during establishment of the L5178Y cell tumor dormant state. No increase in adherent cell-mediated cytolytic activity or cytolytic or cytophilic anti-L5178Y antibody can be detected after inoculation of irradiated L5178Y cells. The repeated stimulation of CTL activity in tumor-dormant DBA/2 mice results in the elimination of L5178Y cells from a significant number of tumor-dormant mice.

Neutrophil adhesion: a point for therapeutic intervention?

It has become increasingly clear over the last 20 years that the potential exists to modulate inflammatory responses with compounds that interfere with intercellular adhesion. This review highlights the adhesion interactions that occur during neutrophil extravasation and indicates some of the possible ways of disrupting these interactions.

Oxidant inhibition of alphaLbeta2 integrin adhesion: evidence for coordinate effects on conformation and cytoskeleton linkage.

Dithiothreitol (DTT) and other dithiol antioxidants with closely spaced thiol pairs strongly activate leukocyte function antigen-1 (LFA-1, alphaLbeta2 integrin) to bind intercellular adhesion molecule-1 (ICAM-1). Because direct biochemical modification of LFA-1 by DTT is not apparently involved, we investigated the possible role of a reduction-oxidation (redox)-sensitive adhesion-regulatory pathway. Phenylarsine oxide (PAO), an oxidant selectively reactive with closely spaced pairs of thiol groups, inhibited LFA-1-dependent adhesion of human natural killer and HSB2 T leukemia cells to murine cells expressing human ICAM-1. PAO also induced disappearance of a conformation-sensitive LFA-1 epitope recognized by KIM127 antibodies and promoted an increase in total apparent cytoskeleton-linked LFA-1 in which a novel cytochalasin D-resistant linkage was involved. Exposure of PAO-pretreated cells to DTT caused a decline in LFA-1/cytoskeleton linkages in conjunction with rapid restoration of KIM127 epitope expression and LFA-1 adhesive function. Implicating an intracellular site of action were findings that (1) an epitope-tagged PAO probe bound predominantly to intracellular proteins but not detectably to immunoprecipitation-purified LFA-1 chains, and (2) membrane permeant but not impermeant dithiol antioxidants reversed PAO adhesion-inhibitory effects. These results support the concept of a reversible redox-sensitive linkage between LFA-1 and cytoskeleton by which oxidants and antioxidants may exert profound opposing effects on LFA-1 conformation and adhesive function.

Method for estimating confidence levels for measurements by blood glucose monitoring systems.

A joint conference on self-monitoring of blood glucose (SMBG) has proposed that all glucose monitoring systems generate values that are within 10% of the actual blood glucose level 100% of the time. To estimate the confidence limits of blood glucose measurements made in a typical university ambulatory care setting and to ascertain whether they met the proposed standard, we performed duplicate determinations of blood glucose using a reflectance meter and applied to the measurements a method for calculating the closeness of measured values to a "true" mean. Based on paired measurements in 100 consecutive diabetic subjects, we were able to show that one measurement would be within 11.9% of a true mean value 95% of the time and within 8.4% of the mean 90% of the time. The 95 and 90% confidence limits for the average of two repeated measurements were calculated to be 8.4 and 5.9%, respectively. Our methodology can be applied to any set of SMBG values to calculate their confidence limits and to determine whether the measurements meet recommended standards.

Optimization of an in vitro lymphocyte blastogenesis assay for predictive assessment of immunologic responsiveness to contact sensitizers.

Current methods for the predictive and diagnostic assessment of contact sensitization rely on the visual scoring of skin reactions. Predictive animal tests, generally using guinea pigs, require a relatively large number of animals to produce a sufficient database for interpreting skin reaction scores. In vitro assays have the potential of being more quantitative than skin testing and, if so, would require fewer animals. However, although in vitro assays are commonly used to study the cellular immune response to strong contact sensitizers, there has been little effort to validate them for predictive assessment purposes. We have optimized an in vitro lymphocyte blastogenesis assay for detecting the response of mouse lymphocytes to strong contact sensitizers with the eventual objective of applying this assay to moderate and weak sensitizers as well. Lymph node lymphocytes from mice sensitized to the strong contact allergens, dinitrochlorobenzene (DNCB), dinitrofluorobenzene (DNFB), or trinitrochlorobenzene (TNCB), responded [greater than or equal to 12,000 counts per minute (CPM) above background] when cultured with water soluble chemical analogues, di- or trinitrobenzene sulfonic acid (DNBS or TNBS). However, the strong sensitizer, oxazolone (OXAZ), has no water soluble analogue and lymphocytes from mice sensitized to OXAZ responded poorly in vitro (less than 2000 CPM) to an ethanol-solubilized OXAZ preparation in spite of very strong in vivo sensitization (ear swelling assay). To increase the assay sensitivity, for OXAZ, we modified the antigen presentation conditions by using 1) solubilized antigen-modified adherent spleen cells, 2) dendritic cells from the draining lymph nodes of antigen painted mice, and 3) antigen-modified Langerhans cell-enriched cultured epidermal cells (EC). These approaches increased OXAZ-directed responses to greater than 7000, greater than 20,000, and greater than 100,000 CPM, respectively, under culture conditions optimized for cell density, responder: stimulator cell ratio, culture duration, and responder cell type. Our results represent a first attempt to directly modify cultured epidermal cells with OXAZ and use these cells to stimulate OXAZ-directed blastogenesis in microtiter plate cultures. This optimized assay is now under evaluation for predictive assessment of contact sensitizers relevant to occupational and consumer exposures.

Immunosuppressive effects of clonidine on the induction of contact sensitization in the balb/c mouse.

The clonidine transdermal therapeutic system (clonidine-TTS) has been associated with a significant incidence of allergic contact sensitization. This incidence was not predicted by premarket skin sensitization testing in animals or humans. One possible explanation lies in recent findings in guinea pigs that clonidine exposure could inhibit the elicitation of skin reactions to unrelated strong contact sensitizers. However, these studies also showed that clonidine pretreatment did not appear to affect the induction of contact sensitization. On this basis, we sought to specifically evaluate the induction phase of sensitization to clonidine as an alternative means of assessing its sensitization properties. The method selected was the assay of in situ lymphocyte proliferation in lymph nodes draining the sites of clonidine exposure, a method recently promoted as an alternative means to assess contact allergenic potential. Utilizing various induction application techniques and regimens, we were consistently unable to demonstrate clonidine's allergenic potential through such an assessment of lymphocyte proliferation. We were also unable to demonstrate sensitization by in vivo ear swelling or in vitro lymphocyte blastogenesis assay techniques. However, a subsequent assessment of the effect of clonidine exposure on the induction of sensitization to unrelated strong contact allergens demonstrated a consistent 40-70% inhibition of the proliferative response to the contact allergens oxazolone and trinitrochlorobenzene. This was similar to the degree of suppression produced by the corticosteroids fluocinonide and hydrocortisone when they were tested at 80 and 10 times lower concentrations. In addition, we observed a comparable inhibition of the ear swelling response to oxazolone. These data extend our knowledge of the immunomodulatory effects of clonidine and offer additional mechanistic insights into the failure of short-term predictive patch-test methods to detect this chemical's potential to induce allergic contact sensitization.

E-selectin binds to squamous cell carcinoma and keratinocyte cell lines.

E-selectin is an endothelial adhesion molecule that binds carbohydrate epitopes on leukocytes and has been implicated in a potential pathway of tumor metastasis. Keratinocyte cell lines express similar carbohydrate epitopes, one of which, sialyl Lewis X (SL-X) is a ligand for E-selectin and is also expressed by squamous cell carcinomas (SCC) in situ. The functional role of keratinocyte selectin ligands was investigated using a soluble E-selectin chimaeric protein (pE-sel-Ig) containing pig lectin-like and epidermal growth factor-like domains fused to human IgG. After incubation of keratinocyte cell lines (A431 and SVK14) and normal keratinocytes with pE-sel Ig, binding was quantified by flow cytometry. Frozen sections of SCC were overlaid with pE-sel Ig and binding was visualized immunoenzymatically. Immunolabeling was undertaken using monoclonal antibodies (CSLEX-1 and HECA-452), which label E-selectin ligands including sialyl Lewis X. E-selectin bound strongly to A431 and SVK14 cells; the degree of binding paralleled staining intensity with CSLEX-1 antibody. HECA-452 antibody stained A431 cells strongly but SVK14 cells only weakly. Normal keratinocytes and normal epidermis did not express CSLEX-1 or HECA-452 antigens or bind E-selectin. Serial sections of SCC revealed close correlation between fusion protein binding and antibody staining. Antibody pretreatment of tumor sections with CSLEX-1 blocked fusion protein binding, whereas HECA-452 antibody only slightly reduced fusion protein binding. pE-sel Ig pretreated with YT11.1 antibody failed to bind to A431 or SVK14 cells or to SCC. These studies provide functional evidence that SL-X/E-selectin pathways may be important in SCC metastasis and that A431 and SVK14 cells provide a good model to investigate these mechanisms.

Value of the cutaneous basophil hypersensitivity (CBH) response for distinguishing weak contact sensitization from irritation reactions in the guinea pig.

Numerous studies of the histology of allergic contact dermatitis reactions to potent allergens in guinea pigs and humans have indicated that there is significant tissue infiltration with basophilic leukocytes. In this study we determined whether this histologic finding could be of value in distinguishing weak sensitization reactions from primary irritation, thereby aiding in the predictive identification of weak or moderate contact allergens. Guinea pigs were sensitized by the Buehler test method. Skin reactions were graded 24, 48, and 72 h post-challenge with duplicate patch sites biopsied at the 24- or 72-h grading timepoints. The biopsies were fixed, embedded in glycol methacrylate, thin sectioned, and Giemsa stained. The number of basophils per 400 leukocytes were counted along the upper dermis just below the dermal/epidermal junction. Challenge patch sites from animals sensitized to a relatively low dose of the strong contact allergen, oxazolone, were compared with patch sites from animals challenged only with a strong irritant, sodium lauryl sulfate (SLS). Compared to normal skin (7.5 +/- 1.0 basophils/400 leukocytes +/- SEM) only the oxazolone patch sites showed significant basophil infiltration (36.8 +/- 6.5), despite the fact that the skin reactions to the low oxazolone challenge dose were relatively weak. SLS patch sites showed no basophil infiltration above normal skin levels (4.8 +/- 0.9). Subsequent blinded studies compared weak/moderate presumptive sensitization reactions (as defined by accepted visual skin grading criteria) to various chemicals (citronellal, vanillin, cinnamic aldehyde, and ethylenediamine) to primary irritation reactions to the same chemicals. In each case, low-challenge-dose sensitization sites on previously treated (induced) animals showed mean basophil infiltration (range, 11.9-69.2 basophils/400 leukocytes) significantly greater than higher-dose irritant reactions (range, 1.6-13.3). The range for normal skin was 0.2-10.2 and the range for strong patch reactions to higher concentrations of oxazolone was 59.8-209.3. These data strongly indicate that light-microscopic quantitation of the CBH response can be used to distinguish relatively weak to moderate contact sensitization reactions from primary irritation reactions to the same chemicals.

Increased number of dendritic cells in draining lymph nodes accompanies the generation of contact photosensitivity.

Draining lymph node cells isolated from mice 48 h after topical exposure to tetrachlorosalicylanilide (TCSA) + UVA radiation (TCSA + UVA) demonstrated a two-fivefold increase in the number of dendritic cells (DC) compared with control mice treated with vehicle + UVA. The increase in number of DC was both time and dose dependent, with the peak DC accumulation occurring at 48 h post application and at a TCSA dose of 1.0%. Photospecificity was evident in that mice irradiated prior to treatment with TCSA (UVA/TCSA) demonstrated no significant increase in DC accumulation. The accumulation of DC was followed by a significant increase in total lymph node cellularity. An in situ 3H-thymidine incorporation assay showed a significant increase in proliferative activity of cells isolated from the draining lymph nodes of mice treated with TCSA + UVA as compared to naive, vehicle, or UVA/TCSA-treated mice. Dendritic cells isolated from mice treated 24 h earlier with TCSA + UVA, but not those from naive mice or mice treated with UVA/TCSA, were capable of TCSA-specific antigen presentation. Responder lymphocytes from untreated mice or mice photosensitized with musk ambrette showed a much lower response to DC isolated from TCSA + UVA-treated mice, demonstrating the specificity of the reaction. DC-depleted lymph node cells were unable to stimulate this blastogenesis response. These results suggest that application and photoactivation of TCSA induces cellular and functional changes in the lymph node DC indicative of their involvement in the induction phase of a contact photoallergic reaction.

Dual-origin plasmid vectors whose origin of replication is controlled by the coliphage lambda promoter pL.

The insertion of an XhoI linker 30 bp from the 5' end of the RNA II primer for ColE1 plasmid replication has allowed us to replace the natural RNA II promoter by other controllable promoters, in particular lambda pL. Manipulation of this modified origin was facilitated by constructing dual-origin plasmids, stable maintenance being directed by the pSC101 origin. Experiments showed that such dual-origin plasmids could be stably maintained at approximately four copies per chromosome at 30 degrees C and readily amplified by thermal induction of strains carrying a thermolabile lambda repressor. The use of such plasmids for the construction of stable, amplifiable expression vectors is discussed.