Gene expression is differently affected by pimecrolimus and betamethasone in lesional skin of atopic dermatitis.

BACKGROUND: Topical corticosteroids and calcineurin inhibitors are well-known treatments of atopic dermatitis (AD) but differ in their efficacy and side effects. We recently showed that betamethasone valerate (BM) although clinically more efficient impaired skin barrier repair in contrast to pimecrolimus in AD. OBJECTIVE: This study elucidates the mode of action of topical BM and pimecrolimus cream in AD. METHODS: Lesional AD skin samples after topical treatment with either BM or pimecrolimus were subjected to gene expression profile analysis. RESULTS: Betamethasone valerate resulted in a significant reduction in mRNA levels of genes encoding markers of immune cells and inflammation, dendritic cells, T cells, cytokines, chemokines, and serine proteases, whereas pimecrolimus exerted minor effects only. This corroborates the clinical finding that BM reduces inflammation more effectively than pimecrolimus. Genes encoding molecules important for skin barrier function were differently affected. Both BM and pimecrolimus normalized the expression of filaggrin and loricrin. BM, but not pimecrolimus, significantly reduced the expression of rate-limiting enzymes for lipid synthesis and the expression of involucrin and small proline-rich proteins, which covalently bind ceramides. This may explain the lack of restoration of functional stratum corneum layers observed after BM treatment. CONCLUSION: The gene expression profiles are consistent with our previous findings that corticosteroids may exert a more potent anti-inflammatory effect but may impair the restoration of the skin barrier. Corticosteroids are still the main treatment for severe and acutely exacerbated AD; pimecrolimus may be preferable for long-term treatment and stabilization.

Central nervous system drug development: an integrative biomarker approach toward individualized medicine.

Drug development for CNS disorders faces the same formidable hurdles as other therapeutic areas: escalating development costs; novel drug targets with unproven therapeutic potential; and health care systems and regulatory agencies demanding more compelling demonstrations of the value of new drug products. Extensive clinical testing remains the core of registration of new compounds; however, traditional clinical trial methods are falling short in overcoming these development hurdles. The most common CNS disorders targeted for drug treatment are chronic, slowly vitiating processes manifested by highly subjective and context dependent signs and symptoms. With the exception of a few rare familial degenerative disorders, they have ill-defined or undefined pathophysiology. Samples selected for treatment trials using clinical criteria are inevitably heterogeneous, and dependence on traditional endpoints results in early proof-of-concept trials being long and large, with very poor signal to noise. It is no wonder that pharmaceutical and biotechnology companies are looking to biomarkers as an integral part of decision-making process supported by new technologies such as genetics, genomics, proteomics, and imaging as a mean of rationalizing CNS drug development. The present review represent an effort to illustrate the integration of such technologies in drug development supporting the path of individualized medicine.

Determination of the effect of calcineurin inhibitors on the rat's immune system after KLH immunisation.

The calcineurin inhibitors cyclosporin A (CsA), tacrolimus (FK506) and pimecrolimus (ASM981) are on the market for the oral treatment of psoriasis and atopic dermatitis and topical treatment of atopic dermatitis, respectively. The effect of these treatments on the immune response was investigated in this study after immunisation of rats with keyhole limpet hemocyanin (KLH). Male rats (10 per group) were orally administered pimecrolimus at 10 or 30 mg/kg/day), tacrolimus at 3 mg/kg/day or CsA at 20 mg/kg/day for 4 weeks. Control animals similarly received the vehicle only. The last five animals per group were immunised and challenged with KLH on days 16 and 24, respectively. Eight days after the last injection, the immune function was investigated by detecting KLH-specific antibodies in the serum and by examination of cell infiltration at the site of the KLH-challenge. In addition, a correlation between functional and structural changes was established by quantification of lymphocyte sub-populations in the blood or residing in lymphatic tissue. In KLH-immunised rats, CsA caused complete suppression of the KLH-specific IgM and IgG production, whereas only IgG production was affected by pimecrolimus at 30 mg/kg/day and more so by tacrolimus at 3 mg/kg/day. Immunophenotyping of lymphocyte sub-populations in spleen and lymph node indicated a decrease in T lymphocytes with pimecrolimus at 30 mg/kg/day, tacrolimus and CsA, whereas these changes were marginal for pimecrolimus at 10 mg/kg/day. Immunophenotyping of peripheral white blood cells (WBC) revealed a decrease in the absolute number of T lymphocytes with all three test items. In comparison with non-immunised animals, a slight increase in absolute numbers of T lymphocytes was observed in KLH-immunised animals treated with pimecrolimus at 10 or 30 mg/kg/day. In conclusion, the ability of the immune system to respond to KLH was not affected by pimecrolimus at 10 mg/kg/day whereas a decrease in immune function was noted in the other groups as follows: pimecrolimus (30 mg/kg/day) < tacrolimus (3 mg/kg/day) < CsA (20 mg/kg/day).

Afferent and efferent phases of allergic contact dermatitis (ACD) can be induced after a single skin contact with haptens: evidence using a mouse model of primary ACD.

Allergic contact dermatitis is a T cell-mediated delayed type hypersensitivity reaction that occurs upon hapten challenge in sensitized individuals. The inflammatory response in classical allergic contact dermatitis requires both a sensitization phase and an elicitation phase responsible for the recruitment and activation of specific T cells at the site of hapten skin challenge. Conversely, previously unsensitized patients may develop a "primary allergic contact dermatitis" after the first skin contact with potent contact sensitizers leading to a skin inflammation with all the features of classical allergic contact dermatitis. In this study we used an experimental murine model, referred to as contact hypersensitivity, to study the pathophysiology of primary allergic contact dermatitis and its relationship to classical allergic contact dermatitis. We show that one epicutaneous application of a nonirritant dose of hapten (2,4-dini-trofluorobenzene, fluorescein isothiocyanate) was sufficient to induce an optimal allergic contact dermatitis reaction at the site of primary contact with the hapten without subsequent challenge. As in classical allergic contact dermatitis, the skin inflammation in primary allergic contact dermatitis was mediated by interferon-gamma producing, CD8+ effector T cells that were induced in the draining lymph nodes at day 5 postsensitization and downregulated by CD4+ T cells. Reverse transcription-polymerase chain reaction analysis revealed that the primary allergic contact dermatitis reaction was mediated by a recruitment of CD8+ T cells at the sensitization skin site at day 6 postsensitization. Analysis of the fate of the hapten fluorescein isothiocyanate applied once on the skin revealed its persistence in the epidermis for up to 14 d after skin painting. These results suggest that the development of primary allergic contact dermatitis (i.e., without secondary challenge) is associated with persistence of the hapten in the skin, which allows the recruitment and activation of CD8+ T cells at the site of the single hapten application.

Functional genomics in sarcoidosis--reduced or increased apoptosis?

BACKGROUND: A variety of studies have stressed the importance of the control of inflammatory cell longevity and the balance of pro-survival and pro-apoptotic signaling pathways. The aim of the study was to investigate the systemic activation of apoptosis pathways using cDNA array technology in patients with acute onset sarcoidosis. METHOD: We have performed a comprehensive genomic analysis, applying high-density human GeneChip probe arrays (HGU95A, Affymetrix) for RNA expression profiling from peripheral blood mononuclear cells from patients with acute pulmonary sarcoidosis and matched healthy controls. Twelve patients and 12 controls were assessed, mean age 36 +/- 12 and 33 +/- 10 years respectively. Results focus on apoptosis-related gene products. Group differences were assessed with the Mann-Whitney U-test. RESULTS: Seven patients had self-limited disease (all type I sarcoidosis) and 5 progressive disease requiring immunosuppression (all type II or III sarcoidosis). We found 53 of 112 (47%) apoptosis-related gene products dysregulated in sarcoidosis compared to controls. Particular growth factors, especially heparin-binding EGF-like GF, EGF, PDEGF, SISPDGF2 and VEGF, were upregulated in patients consistent with a pro-survival profile. The Bcl-2 family of genes also showed a net pro-survival profile in sarcoidosis patients. In contrast, alterations in the TNF-pathway were compatible with increased apoptosis signals in both, type I and type II/III sarcoidosis patients. Other cell death receptors were equally expressed, as were caspases and p53-associated genes. In contrast to patients with type I-sarcoidosis, patients with progressive type II or III disease showed an upregulation of NFKB and a leak of downregulation of inhibitor of apoptosis 1. CONCLUSION: Significant differences in the expression of apoptosis-related genes were found in peripheral blood of patients with acute onset sarcoidosis. Gene expression did not show a definite pattern that was suggestive of pro-survival or proapoptosis. However, the number of genes whose altered expression would be predicted to favour increased survival exceeded that of genes likely to reduce survival. Protein-based confirmation of the differences in the activity of apoptosis-pathways needs to be done in further studies.

Mechanism of measles virus-induced suppression of inflammatory immune responses.

Measles virus (MV) causes profound immunosuppression, resulting in high infant mortality. The mechanisms are poorly understood, largely due to the lack of a suitable animal model. Here, we report that particular MV proteins, in the absence of MV replication, could generate a systemic immunosuppression in mice through two pathways: (1) via MV-nucleoprotein and its receptor FcgammaR on dendritic cells; and (2) via virus envelope glycoproteins and the MV-hemagglutinin cellular receptor, CD46. The effects comprise reduced hypersensitivity responses associated with impaired function of dendritic cells, decreased production of IL-12, and the loss of antigen-specific T cell proliferation. These results introduce a novel model for testing the immunosuppressive potential of anti-measles vaccines and reveal a specific mechanism of MV-induced modulation of inflammatory reactions.

The popliteal lymph node response to streptozotocin is under type 1, MHC class-I restricted, CD8(+) T-cell control.

The popliteal lymph node (PLN) assay has been proposed to predict the 'autoimmunogenic' potential of xenobiotics. A better understanding of the processes involved in PLN responses is needed to establish the value of this assay for preclinical safety evaluation. In order to determine whether PLN responses involve CD4(+) or CD8(+) T-cells, the effects of streptozotocin (STZ), a prototypic immunotoxic compound, were analyzed after injection into the hind footpad of C57 BL/6 mice and major histocompatibility complex (MHC) class I or II deficient mice. The involvement of type 1 or type 2 cell control on the production of cytokine mRNAs was analyzed in lymph node cells by quantitative RT-PCR, together with the analysis of a wide range of cytokine mRNAs after STZ injection (IL-1alpha, IL-1beta, TNF-alpha, IFN-gamma, IL-2, IL-2 receptor, IL-4, IL-5, IL-6, IL-10 and IL-12). We have found that mice depleted in CD8(+) T-cells did not respond to STZ, whereas mice depleted in CD4(+) T-cells exhibited the expected positive PLN responses, with increased weight and cellularity indices. STZ induced a low production of interleukin (IL)-2 mRNAs, a mild increase in IL-1alpha and IL-6 mRNAs production, and a dramatic increase in IFN-gamma, IL-1beta, TNF-alpha, IL-12 and IL-2 receptor mRNAs, which correlated with positive PLN responses. No effects on IL-4, IL-5 and IL-10 mRNAs synthesis were noted. In CD8(+) T-cell deficient mice, there was no production of IFN-gamma or IL-6 mRNAs. These results suggest that PLN responses to STZ are under the control of type 1, MHC class-I-restricted, CD8(+) T-cells. This is in accordance to the known physiopathology of STZ-induced diabetes. Additional studies are necessary to establish the mechanism of CD8+ T-cell activation.

Oral administration of hapten inhibits in vivo induction of specific cytotoxic CD8+ T cells mediating tissue inflammation: a role for regulatory CD4+ T cells.

We investigated whether oral tolerance could block the development of an inflammatory response mediated by CD8+ T cells, using a mouse model of oral tolerance of contact sensitivity (CS) to the hapten 2, 4-dinitrofluorobenzene (DNFB). In this system, the skin inflammatory response is initiated by hapten-specific class I-restricted cytotoxic CD8+ T (CTL) cells, independently of CD4 help. Oral delivery of DNFB before skin sensitization blocked the CS response by impairing the development of DNFB-specific CD8+ effector T cells in secondary lymphoid organs. This was shown by complete inhibition of DNFB-specific CTL and proliferative responses of CD8+ T cells, lack of specific IFN-gamma-producing CD8+ T cells, and inability of CD8+ T cells to transfer CS in RAG20/0 mice. RT-PCR and immunohistochemical analysis confirmed that recruitment of CD8+ effectors of CS in the skin at the site of hapten challenge was impaired in orally tolerized mice. Sequential anti-CD4 Ab treatment showed that only depletion of CD4+ T cells during the afferent phase of CS abrogated oral tolerance induction by restoring high numbers of specific CD8+ effectors in lymphoid organs, whereas CD4 depletion during the efferent phase of CS did not affect oral tolerance. These data demonstrate that a single intragastric administration of hapten can block in vivo induction of DNFB-specific CD8+ CTL responsible for tissue inflammation and that a subset of regulatory CD4+ T cells mediate oral tolerance by inhibiting expansion of specific CD8+ effectors in lymph nodes.

Contact dermatitis II. Clinical aspects and diagnosis.

Contact dermatitis (CD) is an altered state of skin reactivity induced by exposure to an external agent. "Eczema" and "dermatitis" are often used synonymously to denote a polymorphic pattern of inflammation of the skin characterized, at least in its acute phase, by erythema, vesiculation and pruritus. Substances that induce CD after single or multiple exposures may be irritant or allergic in nature. The clinical presentation may vary depending on the identity of the triggering agent and the reactivity of the subject, but in all cases the lesions are primarily confined to the site of contact. According to the mechanism of elicitation, the following types of contact reactions may be distinguished: (1) allergic contact dermatitis (ACD); (2) irritant contact dermatitis (ICD); (3) phototoxic and photoallergic contact dermatitis, and (4) immediate type contact reactions. The present review will focus on allergic contact dermatitis. ACD is the clinical presentation of contact sensitivity in humans. The pathophysiology of the contact sensitivity reaction has been reviewed in a preceding issue of this journal [1].

Cytotoxicity is mandatory for CD8(+) T cell-mediated contact hypersensitivity.

Contact hypersensitivity (CHS) is a T cell-mediated skin inflammation induced by epicutaneous exposure to haptens in sensitized individuals. We have previously reported that CHS to dinitrofluorobenzene in mice is mediated by major histocompatibility complex (MHC) class I-restricted CD8(+) T cells. In this study, we show that CD8(+) T cells mediate the skin inflammation through their cytotoxic activity. The contribution of specific cytotoxic T lymphocytes (CTLs) to the CHS reaction was examined both in vivo and in vitro, using mice deficient in perforin and/or Fas/Fas ligand (FasL) pathways involved in cytotoxicity. Mice double deficient in perforin and FasL were able to develop hapten-specific CD8(+) T cells in the lymphoid organs but did not show CHS reaction. However, they did not generate hapten-specific CTLs, demonstrating that the CHS reaction is dependent on cytotoxic activity. In contrast, Fas-deficient lpr mice, FasL-deficient gld mice, and perforin-deficient mice developed a normal CHS reaction and were able to generate hapten-specific CTLs, suggesting that CHS requires either the Fas/FasL or the perforin pathway. This was confirmed by in vitro studies showing that the hapten-specific CTL activity was exclusively mediated by MHC class I-restricted CD8(+) T cells which could use either the perforin or the Fas/FasL pathway for their lytic activity. Thus, cytotoxic CD8(+) T cells, commonly implicated in the host defence against tumors and viral infections, could also mediate harmful delayed-type hypersensitivity reactions.

Dual role of dendritic cells in the induction and down-regulation of antigen-specific cutaneous inflammation.

We have previously reported that contact sensitivity (CS) to dinitrofluorobenzene (DNFB) in C57BL/6 mice was mediated by MHC class I-restricted CD8+ T cells and down-regulated by MHC class II-restricted CD4+ T cells. In this study, we analyzed the contribution of dendritic cells (DC) in the induction of these two T cell subsets endowed with opposite functions. Hapten-pulsed skin- and bone marrow-derived DC, obtained from either normal C57BL/6 mice or from MHC class II (I+ II-) and MHC class I (I- II+)-deficient mice, were tested for their ability to prime normal mice for CS to dinitrofluorobenzene. Expression of MHC class I molecules by transferred DC was mandatory both for the induction of CS and for the generation of hapten-specific CD8+ T cells in lymphoid organs. I+ II- DC were as potent as I+ II+ DC in priming for CS, demonstrating that activation of effector CD8+ T cells can occur independently of CD4+ T cell help. I- II+ DC could not immunize for CS, although they could sensitize for a delayed-type hypersensitivity reaction to protein Ags. Moreover, I- II+ DC injected simultaneously with cutaneous sensitization down-regulated the inflammatory response, suggesting that hapten presentation by MHC class II molecules could prime regulatory CD4+ T cells. These results indicate that DC can present haptenated peptides by both MHC class I and class II molecules and activate Ag-specific CD8+ effector and CD4+ regulatory T cell subsets, concurrently and independently.

Photoreceptor cell death by apoptosis in dogs with sudden acquired retinal degeneration syndrome.

OBJECTIVE: To examine the role of apoptosis in retinal photoreceptor degeneration in dogs with sudden acquired retinal degeneration syndrome (SARDS). SAMPLE POPULATION: Retinas from 3 dogs with SARDS and from 2 clinically normal adult dogs. PROCEDURE: Apoptosis was identified by in situ end-labeling and observation of characteristic morphologic changes by light microscopy. RESULTS: The degree of photoreceptor degeneration varied with duration of vision loss in SARDS-affected eyes. The retina of all 3 SARDS-affected eyes had numerous (34, 61, and 70) apoptotic nuclei per section that were overwhelmingly located in the outer nuclear layer. Apoptotic nuclei were not detected, or were rare in similarly sized retinal sections from normal dogs. Inflammation was not an important feature of SARDS. CONCLUSIONS: Apoptosis appears to be at least 1 mechanism of photoreceptor cell death in dogs with SARDS. CLINICAL RELEVANCE: Because apoptosis appears to be a final common pathway in many retinal degeneration syndromes, future treatment strategies that control apoptosis in other diseases may be applicable to dogs with SARDS. Halting this pathway may allow some photoreceptors to survive and, perhaps, preserve vision.

bcl-2 alters influenza virus yield, spread, and hemagglutinin glycosylation.

We previously demonstrated that expression of bcl-2 in Madin-Darby canine kidney (MDCK) cells blocks influenza virus-induced apoptosis and DNA fragmentation. We show here that expression of bcl-2 also reduces the level of infectious virus production and the spread of virus in MDCK cell cultures infected at a low multiplicity of infection. This effect is associated with modified glycosylation of the hemagglutinin protein.