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.

Assessment of the phototoxic potential of compounds and finished topical products using a human reconstructed epidermis.

The goal of this study was to design a model system for the assessment of phototoxic potential using a human reconstructed epidermis (HRE, SkinEthic Laboratories, Nice, France), by testing some representative phototoxic (P) and non-phototoxic (NP) compounds and finished topical products. The tissue response to 24-h application of 5-5000 microg/mL of the test agents in the presence and absence of UVA light was analyzed in terms of viability (Lactate Dehydrogenase release), pro-inflammatory activity (IL-8 release and mRNA expression) and morphology (histopathology). 8-Methoxypsoralen (P) and promethazin (P), but not sodium lauryl sulfate (NP) produced cytotoxicity concentration-response curves significantly different between irradiated and nonirradiated tissues. Only irradiated tissues showed morphological damage. Application of tetracyclin (P) in the culture medium, but not topically, induced similar signs of phototoxicity. 6-Methylcoumarine (weak P) was not cytotoxic, yet it increased IL-8 release and mRNA expression only following UVA irradiation. PUVA therapy creams containing 1% 8-Methoxy-psoralen (P) or coal tar (P) decreased viability and induced histologic damage in UVA-exposed tissues. In conclusion, the phototoxic potential of the tested agents was correctly predicted by using a tiered strategy that involves determining cytotoxicity, production of IL-8, and morphological damage following exposure of the HRE to the compounds and UVA light.

Use of human skin equivalent Apligraf for in vitro assessment of cumulative skin irritation potential of topical products.

The main goal of the present study was to investigate the response of the human skin equivalent Apligraf in vitro to the application of irritant substances and its predictivity as a screening tool for cumulative skin irritant potential in humans. Vaseline, calcipotriol, trans-retinoic acid, and sodium lauryl sulfate were applied to Apligraf in vitro for 24 h. Cell viability (lactate dehydrogenase leakage), release and mRNA expression of the proinflammatory cytokines IL-1alpha and IL-8, and morphological changes were assessed. The same products were applied to 30 healthy volunteers in a double-blind, randomized, vehicle-controlled within-subject study. The skin reactions after repeated 24-h applications over 3 weeks under Finn chamber patches were monitored by visual scoring and biophysical methods (trans-epidermal water loss, chromametry, and blood flow). Sodium lauryl sulfate was cytotoxic to Apligraf, and increased the release and expression of cytokines at low (0.2%, 0. 4%), but not at high (0.8%, 1%) concentrations. It induced severe irritancy in vivo. Trans-retinoic acid increased the expression and release of cytokines with no detectable cytotoxicity and showed moderate irritancy in humans. Although calcipotriol did neither affect cell viability nor the production of cytokines, it induced morphological signs of irritation and was mildly irritant for healthy volunteers. Vaseline was innocuous in vivo and induced no changes in Apligraf. In conclusion, the cumulative skin irritation potential of the tested products could be predicted with Apligraf in a sensitive and specific manner, by monitoring cytotoxicity, proinflammatory cytokines, and morphological changes.

Evidence for the impairment of the vitamin D activation pathway by cyclosporine A.

Cyclosporine A (CsA) is a potent immunosuppressant with the drawback of renal side effects. We reported that CsA markedly decreases calcium-binding protein calbindin-D28k mRNA levels in rat kidneys, and showed that this decrease is associated with its adverse renal effects. The transcription of the calbindin-D28k gene is activated via the vitamin D pathway. In this work, the potential CsA-mediated impairment of the vitamin D pathway was investigated. Wistar rats were treated for 12 days with 50 mg/kg/day CsA or for 20 days with 50 mg/kg/day of the non-immunosuppressant and non-nephrotoxic SDZ PSC 833, which had been previously shown not to affect calbindin-D28k mRNA levels. The expression of the three vitamin D-regulated genes calbindin-D28k, 1,25-dihydroxyvitamin D3-24-hydroxylase (24-OHase), and vitamin D receptor (VDR) were quantified in rat kidney homogenates by real-time reverse transcription-polymerase chain reaction. Plasma parathyroid hormone (PTH) as well as plasma and kidney 1,25 dihydroxyvitamin D3 (calcitriol) levels were monitored in all animals. CsA induced a 85% decrease in calbindin-D28k mRNA levels as well as a 40% and 69% decrease in VDR and 24-OHase mRNA levels, respectively. Plasma and kidney 1,25 dihydroxyvitamin D3 as well as plasma PTH levels were increased by CsA, but not by SDZ PSC 833. The treatment with SDZ PSC 833 did not affect calbindin-D28k or VDR expression, but did cause a 73% decrease in 24-OHase mRNA levels. Taken together, these results indicate an association between CsA-mediated down-regulation of rat renal calbindin-D28k mRNA and the decrease in other 1,25 dihydroxyvitamin D3-regulated genes, suggesting an impairment of the vitamin D pathway by CsA which may be related to its adverse renal side effects and its immunosuppressive activity.