Transcriptomic analysis of pancreatic adenocarcinoma specimens obtained from Black and White patients.

In pancreatic cancer clinical trials, Black patients are under-represented while having higher morbidity and mortality rates as compared to other racial groups. Multiple factors, including socioeconomic and lifestyle factors may contribute to this disparity, but genomic contributions remain unclear. In an exploratory project to identify genes that may contribute to differences in survival between Black (n = 8) and White (n = 20) patients with pancreatic cancer, transcriptomic sequencing of over 24,900 genes was performed in human pancreatic tumor and non-tumor tissue obtained from Black and White patients. Over 4,400 genes were differentially expressed in tumor and non-tumor tissue, irrespective of race. To validate these results, the expression of four genes (AGR2, POSTN, TFF1, and CP) reported to be up-regulated in pancreatic tumor tissue as compared to non-tumor tissue were confirmed using quantitative PCR. Transcriptomic analysis that compared pancreatic tumor tissue from Black and White patients revealed differential expression in 1,200 genes, while a comparison of the non-tumor and tumor gene expression differences within each race revealed over 1,500 tumor-specific differentially expressed genes in pancreatic tumor and non-tumor tissue from Black patients. We identified TSPAN8 as a potential tumor-specific gene significantly overexpressed in pancreatic tumor tissue in Black patients as compared to White patients. Using Ingenuity Pathway Analysis software to compare the race-associated gene expression profiles, over 40 canonical pathways were identified to be potentially impacted by the gene expression differences between the races. Heightened expression of TSPAN8 was associated with poor overall survival, suggesting TSPAN8 as one potential genetic factor contributing to the differential outcomes in Black patients with pancreatic cancer, supporting the potential utility of larger genomic studies to further explore the role of TSPAN8 in pancreatic cancer.

In search of autophagy biomarkers in breast cancer: Receptor status and drug agnostic transcriptional changes during autophagy flux in cell lines.

Autophagy drives drug resistance and drug-induced cancer cell cytotoxicity. Targeting the autophagy process could greatly improve chemotherapy outcomes. The discovery of specific inhibitors or activators has been hindered by challenges with reliably measuring autophagy levels in a clinical setting. We investigated drug-induced autophagy in breast cancer cell lines with differing ER/PR/Her2 receptor status by exposing them to known but divergent autophagy inducers each with a unique molecular target, tamoxifen, trastuzumab, bortezomib or rapamycin. Differential gene expression analysis from total RNA extracted during the earliest sign of autophagy flux showed both cell- and drug-specific changes. We analyzed the list of differentially expressed genes to find a common, cell- and drug-agnostic autophagy signature. Twelve mRNAs were significantly modulated by all the drugs and 11 were orthogonally verified with Q-RT-PCR (Klhl24, Hbp1, Crebrf, Ypel2, Fbxo32, Gdf15, Cdc25a, Ddit4, Psat1, Cd22, Ypel3). The drug agnostic mRNA signature was similarly induced by a mitochondrially targeted agent, MitoQ. In-silico analysis on the KM-plotter cancer database showed that the levels of these mRNAs are detectable in human samples and associated with breast cancer prognosis outcomes of Relapse-Free Survival in all patients (RSF), Overall Survival in all patients (OS), and Relapse-Free Survival in ER+ Patients (RSF ER+). High levels of Klhl24, Hbp1, Crebrf, Ypel2, CD22 and Ypel3 were correlated with better outcomes, whereas lower levels of Gdf15, Cdc25a, Ddit4 and Psat1 were associated with better prognosis in breast cancer patients. This gene signature uncovers candidate autophagy biomarkers that could be tested during preclinical and clinical studies to monitor the autophagy process.

Achyrocline satureioides compounds, achyrobichalcone and 3-O-methylquercetin, induce mitochondrial dysfunction and apoptosis in human breast cancer cell lines.

Natural products are a valuable source of new molecules and are important for drug discovery. Many chemotherapeutics currently in clinical use were originated from natural sources and are effective cytotoxic agents. In this study, we investigated the cytotoxic and pro-apoptotic effects of achyrobichalcone (ACB) and 3-O-methylquercetin (3OMQ), two novel compounds isolated from the Achyrocline satureioides plant. Because extracts from this plant have been shown to have anticancer activity in vitro, we evaluated ACB and 3OMQ using a human breast cancer cell line, MDA-MB-231, and a nontumorigenic human breast epithelial cell line, MCF-12A. We found that ACB demonstrates cytotoxic effects on MDA-MB-231 cells, but not MCF-12A cells. 3OMQ also demonstrated cytotoxic effects on MDA-MB-231 cells, but with lower selectivity compared to treated MCF-12A cells. Cell death by both compounds was associated with caspase-9 and caspase-3/7 activation. Using high-resolution respirometry, we found that ACB and 3OMQ were able to cause acute mitochondrial dysfunction in MDA-MB-231-treated cells. These results suggest that apoptosis in MDA-MB-231 cells is induced through the activation of the mitochondrial-dependent pathway. Collectively, these findings suggest that ACB is a strong candidate for further anticancer in vivo tests.

Intracellular Insulin-like growth factor binding protein 2 (IGFBP2) contributes to the senescence of keratinocytes in psoriasis by stabilizing cytoplasmic p21.

Psoriasis is a chronic Th1/Th17 lymphocytes-mediated inflammatory skin disease, in which epidermal keratinocytes exhibit a peculiar senescent state, resistance to apoptosis and the acquisition of senescence-associated secretory phenotype (SASP). SASP consists of the release of soluble factors, including IGFBPs, that exert extracellular and intracellular functions in IGF-dependent or independent manner.In this report, we investigated the expression and function of IGFBP2 in senescent keratinocytes isolated from the skin of patients with plaque psoriasis. We found that IGFBP2 is aberrantly expressed and released by these cells in vivo, as well as in vitro in keratinocyte cultures undergoing progressive senescence, and it associates with the cyclin-dependent kinase inhibitors p21 and p16 expression. For the first time, we provide evidence for a dual action of IGFBP2 in psoriatic keratinocytes during growth and senescence processes. While extracellular IGFBP2 counter-regulates IGF-induced keratinocyte hyper-proliferation, intracellular IGFBP2 inhibits apoptosis by interacting with p21 and protecting it from ubiquitin-dependent degradation. Indeed, we found that cytoplasmic p21 sustains anti-apoptotic processes, by inhibiting pro-caspase 3 cleavage and JNK phosphorylation in senescent psoriatic keratinocytes. As a consequence, abrogation of p21, as well as that of IGFBP2, found to stabilize cytoplasmic p21 levels, lead to the restoration of apoptosis mechanisms in psoriatic keratinocytes, commonly observed in healthy cells.

Doxorubicin-induced cardiotoxicity is suppressed by estrous-staged treatment and exogenous 17ß-estradiol in female tumor-bearing spontaneously hypertensive rats.

BACKGROUND: Doxorubicin (DOX), an anthracycline therapeutic, is widely used to treat a variety of cancer types and known to induce cardiomyopathy in a time and dose-dependent manner. Postmenopausal and hypertensive females are two high-risk groups for developing adverse effects following DOX treatment. This may suggest that endogenous reproductive hormones can in part suppress DOX-induced cardiotoxicity. Here, we investigated if the endogenous fluctuations in 17ß-estradiol (E2) and progesterone (P4) can in part suppress DOX-induced cardiomyopathy in SST-2 tumor-bearing spontaneously hypersensitive rats (SHRs) and evaluate if exogenous administration of E2 and P4 can suppress DOX-induced cardiotoxicity in tumor-bearing ovariectomized SHRs (ovaSHRs). METHODS: Vaginal cytology was performed on all animals to identify the stage of the estrous cycle. Estrous-staged SHRs received a single injection of saline, DOX, dexrazoxane (DRZ), or DOX combined with DRZ. OvaSHRs were implanted with time-releasing pellets that contained a carrier matrix (control), E2, P4, Tamoxifen (Tam), and combinations of E2 with P4 and Tam. Hormone pellet-implanted ovaSHRs received a single injection of saline or DOX. Cardiac troponin I (cTnI), E2, and P4 serum concentrations were measured before and after treatment in all animals. Cardiac damage and function were further assessed by echocardiography and histopathology. Weight, tumor size, and uterine width were measured for all animals. RESULTS: In SHRs, estrous-staged DOX treatment altered acute estrous cycling that ultimately resulted in prolonged diestrus. Twelve days after DOX administration, all SHRs had comparable endogenous circulating E2. Thirteen days after DOX treatment, SHRs treated during proestrus had decreased cardiac output and increased cTnI as compared to animals treated during estrus and diestrus. DOX-induced tumor reduction was not affected by estrous-staged treatments. In ovaSHRs, exogenous administration of E2 suppressed DOX-induced cardiotoxicity, while P4-implanted ovaSHRs were partly resistant. However, ovaSHRs treated with E2 and P4 did not have cardioprotection against DOX-induced damage. CONCLUSIONS: This study demonstrates that estrous-staged treatments can alter the extent of cardiac damage caused by DOX in female SHRs. The study also supports that exogenous E2 can suppress DOX-induced myocardial damage in ovaSHRs.

Cell autonomous or systemic EGFR blockade alters the immune-environment in squamous cell carcinomas.

Targeting mutations and amplifications in the EGFR has been successful precision therapy for cancers of the lung, oral cavity and gastrointestinal track. However, a systemic immune reaction manifested by dose-limiting inflammation in the skin and gut has been a consistent adverse effect. To address the possibility that intra-tumoral immune changes contribute to the anti-cancer activity of EGFR inhibition, squamous cancers were produced by syngeneic orthografts of either EGFR null or wildtype mouse primary keratinocytes transduced with an oncogenic H-ras retrovirus. Flow cytometric, RNA and Bioplex immunoassay analyses of the tumor immune milieu were performed. Cancers forming from keratinocytes genetically depleted of EGFR were smaller than wildtype cancers and had fewer infiltrating FoxP3 Treg cells, lower Foxp3 RNA and a lower percentage of CD4 PD1 positive cells indicating a tumor cell autonomous regulation of its microenvironment. Hosts bearing wildtype cancers treated with gefitinib for 1 week showed a trend for smaller tumors. In this short term pharmacological model, there was also a trend to reduced FoxP3 cells and FoxP3 RNA in the tumors of treated mice as well as a substantial increase in the ratio of IL-1A/IL-1RA transcripts. These results suggest that relatively brief systemic inhibition of EGFR signaling alters the immune environment of the targeted cancer. Together these data imply that an EGFR dependent Treg function supports the growth of squamous cancers and is a target for the therapeutic activity of EGFR inhibition.

Genetic ablation of epidermal EGFR reveals the dynamic origin of adverse effects of anti-EGFR therapy.

Cancer patients treated with anti-EGFR (epidermal growth factor receptor) drugs often develop a dose-limiting pruritic rash of unknown etiology. The aims of our study were to define causal associations from a clinical study of cutaneous and systemic changes in patients treated with gefitinib and use these to develop and characterize a mouse model that recapitulates the human skin rash syndrome caused by anti-EGFR therapy. We examined the patients' plasma before and after treatment with gefitinib and documented changes in chemokines and leukocyte counts associated with the extent of rash or the presence of pruritus. We established a parallel mouse model by ablating EGFR in the epidermis. These mice developed skin lesions similar to the human rash. Before lesion development, we detected increased mRNA expression of chemokines in the skin associated with early infiltration of macrophages and mast cells and later infiltration of eosinophils, T cells, and neutrophils. As the skin phenotype evolved, changes in blood counts and circulating chemokines reproduced those seen in the gefitinib-treated patients. Crossing the mutant mice with mice deficient for tumor necrosis factor-α (TNF-α) receptors, MyD88, NOS2, CCR2, T cells, or B cells failed to reverse the skin phenotype. However, local depletion of macrophages provided partial resolution, suggesting that this model can identify targets that may be effective in preventing the troublesome and dose-limiting skin response to anti-EGFR drugs. These results highlight the importance of EGFR signaling in maintaining skin immune homeostasis and identify a macrophage contribution to a serious adverse consequence of cancer chemotherapy.

Gene from a psoriasis susceptibility locus primes the skin for inflammation.

Psoriasis is a common complex genetic disease characterized by hyperplasia and inflammation in the skin; however, the relative contributions of epidermal cells and the immune system to disease pathogenesis remain unclear. Linkage studies have defined a psoriasis susceptibility locus (PSORS4) on 1q21, the epidermal differentiation complex, which includes genes for small S100 calcium-binding proteins. These proteins are involved in extracellular and intracellular signaling during epithelial host defense, linking innate and adaptive immunity. Inflammation-prone psoriatic skin constitutively expresses elevated concentrations of S100A7 (psoriasin) and S100A15 (koebnerisin) in the epidermis. Here, we report that genetically modified mice expressing elevated amounts of doxycycline-regulated mS100a7a15 in skin keratinocytes demonstrated an exaggerated inflammatory response when challenged by exogenous stimuli such as abrasion (Koebner phenomenon). This immune response was characterized by immune cell infiltration and elevated concentrations of T helper 1 (T(H)1) and T(H)17 proinflammatory cytokines, which have been linked to the pathogenesis of psoriasis and were further amplified upon challenge. Both inflammation priming and amplification required mS100a7a15 binding to the receptor of advanced glycation end products (RAGE). mS100a7a15 potentiated inflammation by acting directly as a chemoattractant for leukocytes, further increasing the number of inflammatory cells infiltrating the skin. This study provides a pathogenetic psoriasis model using a psoriasis candidate gene to link the epidermis and innate immune system in inflammation priming, highlighting the S100A7A15-RAGE axis as a potential therapeutic target.

EGFR regulates the expression of keratinocyte-derived granulocyte/macrophage colony-stimulating factor in vitro and in vivo.

Recent advances in the knowledge of the EGFR pathway have revealed its contribution to distinct immune/inflammatory functions of the epidermis. The purpose of our study was to evaluate the role of EGFR in the regulation of keratinocyte GM-CSF expression. In cultured human keratinocytes, proinflammatory cytokines synergized with TGF-alpha to induce GM-CSF expression. Accordingly, high epidermal levels of EGFR activation are associated with enhanced expression of GM-CSF in lesional skin of patients with psoriasis or allergic contact dermatitis. In cultured keratinocytes, pharmacological inhibition of EGFR activity reduced GM-CSF promoter transactivation, whereas genetic inhibition of AP-1 reduced expression of GM-CSF. Furthermore, EGFR activation enhanced TNF-alpha-induced c-Jun phosphorylation and DNA binding, whereas c-Jun silencing reduced GM-CSF expression. Using two different mouse models, we showed that the lack of a functional EGFR pathway was associated with reduced cytokine-induced phosphorylation of ERK1/2, JNK1/2, c-Jun and reduced keratinocyte-derived GM-CSF expression both in vitro and in vivo. Finally, the analysis of GM-CSF expression in the skin of cancer patients treated with anti EGFR drugs showed an association between ERK activity, c-Jun phosphorylation, and epidermal GM-CSF expression. These data demonstrate that the EGFR pathway is critical for the upregulation of keratinocyte GM-CSF expression under conditions of cytokine stimulation.

The epidermal growth factor receptor system in skin repair and inflammation.

The epidermal growth factor (EGF) family comprises multiple mediators such as transforming growth factor-alpha, amphiregulin, heparin binding-EGF, and epiregulin, which are crucially involved in the tissue-specific proliferation/differentiation homeostasis. Typically, they act in an autocrine and paracrine manner on their specific cell membrane receptor and mount an effective reparative response to any attack to biophysical integrity. In addition, the EGFR can be activated by transactivation from a variety of G-protein-coupled receptors, integrins, and cytokine receptors, so that it acts as the major transducer of disparate cell functions, including changes in proliferation rate, cellular shape, attachment and motility, and regulation of proinflammatory activation. However, numerous experimental observations indicate that the different EGFR ligands are not redundant, but may rather provide distinct and specific contributions to keratinocyte functions. Importantly, increasing evidence now suggests that the EGFR pathway has a major impact on the inflammatory/immune reactions of the skin, in the apparent effort of enhancing innate immune defense while opposing overactivation of keratinocyte pro-inflammatory functions. This review covers the molecular mechanisms and functions activated by this major growth factor system in the regulation of keratinocyte biology and focuses on the complex contribution of EGFR signaling to the inflammatory processes in the skin.

Stimulation of purinergic receptors modulates chemokine expression in human keratinocytes.

ATP is abundantly released from stressed or damaged cells in response to mechanical stimulation, bacteria, or noxious agents. In this study, we have investigated the possible involvement of P2 receptors (receptor for extracellular nucleotides) in the expression and release of inflammatory mediators by human keratinocytes. Notably, extracellular ATP displayed a complex regulation of IFN-gamma-stimulated chemokine expression, with upregulation of CC chemokine ligand 2 (CCL2), CCL5 and CXC chemokine ligand 8 (CXCL8), and suppression of the receptor CXC chemokine receptor 3 (CXCR3), CXCL9, CXCL10, and CXCL11. The effect of ATP was mimicked by ADP and adenosine-5'-O-3-thiotriphosphate, whereas 2',3'-O-(4-benzoylbenzoyl) ATP (BzATP) downmodulated all chemokines investigated. UTP had no effect on IFN-gamma-stimulated chemokine secretion. The broad-spectrum P2 receptor antagonist suramin and the selective P2Y1 inhibitor adenosine 3'-phosphate 5'-phosphosulfate counteracted the effect of ATP on secretion of all the chemokines examined, whereas pyridoxal phosphate 6-azophenyl 2',4'-disulfonic acid and KN62 (1-[N,O-bis(5-isoquinoline sulfonyl)-N-methyl-L-tyrosyl] 4 phenylpiperazine) partially prevented the inhibitory effect of ATP on CXCL10 secretion, but on the other hand potentiated the ATP-stimulatory effect on CCL5, CCL2, and CXCL8 release. In lesional skin of psoriasis and atopic dermatitis patients, intense P2X7 reactivity was confined to the cell membrane of the basal layer, whereas diffuse P2Y1 immunostaining was found throughout the epidermis. Collectively, our data suggest that the orchestrated activation of distinct P2Y and P2X receptors modulates skin inflammation.

CXCR2 ligands and G-CSF mediate PKCalpha-induced intraepidermal inflammation.

Transgenic mice overexpressing PKCalpha in the epidermis (K5-PKCalpha mice) exhibit an inducible severe intraepidermal neutrophilic inflammation and systemic neutrophilia when PKCalpha is activated by topical 12-O-tetradecanoylphorbol-13-acetate (TPA). This inducible model of cutaneous inflammation was used to define mediators of skin inflammation that may have clinical relevance. Activation of cutaneous PKCalpha increased the production of the chemotactic factors cytokine-induced neutrophil chemoattractant (KC) and macrophage inflammatory protein 2 (MIP-2) in murine plasma. TPA treatment of cultured K5-PKCalpha keratinocytes also released KC and MIP-2 into culture supernatants through an NF-kappaB-dependent pathway. MIP-2 and KC mediated the infiltration of neutrophils into the epidermis, since this was prevented by ablating CXCR2 in K5-PKCalpha mice or administering neutralizing antibodies against KC or MIP-2. The neutrophilia resulted from PKCalpha-mediated upregulation of cutaneous G-CSF released into the plasma independent of CXCR2. These responses could be inhibited by topical treatment with a PKCalpha-selective inhibitor. Inhibiting PKCalpha also reduced the basal and TNF-alpha- or TPA-induced expression of CXCL8 in cultured psoriatic keratinocytes, suggesting that PKCalpha activity may contribute to psoriatic inflammation. Thus, skin can be the source of circulating factors that have both local and systemic consequences, and these factors, their receptors, and possibly PKCalpha could be therapeutic targets for inhibition of cutaneous inflammation.

GDNF family ligand receptor components Ret and GFRalpha-1 in the human trigeminal ganglion and sensory nuclei.

The occurrence of Ret and GFRalpha-1 receptors is shown by immunohistochemistry in the human trigeminal sensory system at pre-, postnatal and adult age. Receptor-labeled neurons occur in both trigeminal ganglion and mesencephalic nucleus. In adult trigeminal ganglion, about 75% of Ret- and 65% of GFRalpha-1-labeled neurons are small- and medium-sized. The proportion of Ret+ and GFRalpha-1+ trigeminal ganglion neurons in the adult is about 25 and 60%, respectively. The majority of Ret+ are double labeled for GFRalpha-1 and glial cell line-derived neurotrophic factor (GDNF). Most of the GFRalpha-1+ cells contain GDNF and about 50% of them contain Ret. Triple labeling shows many Ret+/GDNF+/GFRalpha-1+ neurons, but also a number of Ret-/GDNF+/GFRalpha-1+ and Ret+/GDNF-/GFRalpha-1+ cells. Both Ret+ and GFRalpha-1+ neuronal subpopulations overlap with that containing calcitonin gene-related peptide. Ret+ pericellular basket-like nerve fibers occur in the adult trigeminal ganglion. Centrally, immunoreactivity is restricted to the spinal nucleus pars caudalis and pars interpolaris and to the mesencephalic nucleus. In adult specimens, Ret+ nerve fibers and puncta gather in the inner substantia gelatinosa. Ret+ neurons occur in the spinal nucleus and are more frequent in newborn than in adult subjects. Central GFRalpha-1+-labeled neurons and punctate elements are sparse. These findings support the involvement of GDNF and possibly other cognate ligands in the trophism of human trigeminal primary sensory neurons from prenatal life to adulthood, indicating a selective commitment to cells devoted to protopathic and proprioceptive sensory transmission. They also support the possibility that receptor molecules other than Ret could be active in transducing the ligand signal.

Ret, GFRalpha-1, GFRalpha-2 and GFRalpha-3 receptors in the human hippocampus and fascia dentata.

The immunohistochemical occurrence and localization of the receptor components of the glial cell line-derived neurotrophic factor (GDNF) family ligands, the Ret receptor tyrosine kinase and GDNF family receptor (GFR) alpha-1 to -3, is described in the human post-mortem hippocampal formation at pre- and full-term newborn, and adult age. Two different antibodies for each of the four-receptor molecules were used. Western blot analysis indicates that the availability of GFRalpha receptor proteins may vary with age and post-mortem delay. The immunohistochemical detectability of GFRalpha-1, GFRalpha-2, GFRalpha-3 and Ret receptor molecules is shown in the rat up to 72 h post-mortem. In the human specimens, labelled neuronal perikarya were detectable for each receptor protein at all examined ages, with prevalent localization in the pyramidal layer of the Ammon's horn and hilus and granular layer of the fascia dentata. In the adult subjects, abundant punctate-like structures were also present. Labelled glial elements were identifiable. Comparison of the pattern of immunoreactive elements among young and adult subjects suggests that the intracellular distribution of the GDNF family ligands may vary between pre- and perinatal life and adult age. The results obtained suggest the involvement of the Ret and GFRalpha receptors signalling in processes subserving both the organization of this cortical region during development and the functional activity and maintenance of the mature hippocampal neurons.

ERK1/2 regulates epidermal chemokine expression and skin inflammation.

Resident cell populations of the skin contribute to the inflammatory response by producing an array of chemokines, which attract leukocytes from the circulation. TNF-alpha is a major inducer of proinflammatory mediators in keratinocytes. We have recently observed that epidermal growth factor receptor (EGFR) signaling affects TNF-alpha-driven chemokine expression in epidermal keratinocytes, and its functional impairment increases the levels of crucial chemoattractants such as CCL2/MCP-1, CCL5/RANTES, and CXCL10/IFN-gamma-inducible protein-10. In this study, we report evidence that EGFR-dependent ERK1/2 activity is implicated in this mechanism. Abrogation of ERK1/2 activity with specific inhibitors increased chemokine expression in keratinocytes by enhancing mRNA stabilization. In mouse models, inflammatory response to irritants and T cell-mediated contact hypersensitivity were both aggravated when elicited in a skin area previously treated with an EGFR or a MAPK kinase 1/2 inhibitor. In contrast, impairment of p38alpha beta MAPK phosphorylation markedly attenuated these responses. Our data indicate that EGFR-dependent ERK1/2 activity in keratinocytes takes part to a homeostatic mechanism regulating inflammatory responses, and emphasize the distinct role of MAPKs as potential targets for manipulating inflammation in the skin.

Regulatory role of nitric oxide on monocyte-derived dendritic cell functions.

Nitric oxide (NO) has an established role in the defense against bacterial infections and exerts multiple modulatory activities on both inflammatory and immune responses. However, the relevance of NO on dendritic cell (DC) functions has been poorly investigated. In this study, we found that addition of the NO donor S-nitrosoglutathione (GSNO) to monocyte-derived DCs matured by lipopolysaccharide (LPS) or soluble CD40 ligand led to a decreased capacity to activate naive allogeneic T cells but a more prominent Th1 polarization, with increased interferon-gamma (IFN-gamma) secretion and reduced interleukin-5 (IL-5) release. The presence of GSNO during maturation of DCs caused a reduced expression of surface CD86, whereas CD80, CD83, and MHC molecule expression was not affected. Moreover, GSNO induced a dose-dependent decrease of IL-10 and enhancement of tumor necrosis factor-alpha (TNF-alpha) release from mature DCs. In parallel, a marked reduced production of IL-12 p40 subunit but no significant perturbation of the bioactive IL-12 p70 production was observed. Finally, GSNO significantly reduced the release of IP-10/CXCL10 and RANTES/CCL5 but not IL-8/CXCL8 by mature DCs. Although GSNO can strengthen the capacity of mature DCs to induce type 1 polarization of T lymphocytes, our data suggest that it elicits distinct anti-inflammatory functions, eventually reducing T lymphocyte proliferation and recruitment.

Positive regulation of GABA(B) receptors dually coupled to cyclic AMP by the allosteric agent CGP7930.

The ability of 2,6 Di-tert-butyl-4-(-hydroxy-2,2-dimethyl-propyl)-phenol (CGP7930), a positive allosteric modulator of GABA(B) receptors, to regulate GABA(B) receptor-induced stimulation and inhibition of adenylyl cyclase activity in rat brain was investigated. In olfactory bulb granule cell layer and in frontal cortex, CGP7930 potentiated the stimulatory effects of (-)-baclofen and gamma-aminobutyric acid (GABA) on basal and corticotropin-releasing hormone-stimulated adenylyl cyclase activities, respectively. In these stimulatory responses, CGP7930 enhanced both agonist potencies and maximal effects. When GABA(B) receptor-mediated inhibition of forskolin-stimulated adenylyl cyclase activity of frontal cortex was examined, CGP7930 increased the agonist potencies but failed to affect the maximal effect of (-)-baclofen and modestly increased that of GABA. Similar results were obtained for the inhibition of Ca(2+)/calmodulin-stimulated adenylyl cyclase in striatum and cerebellum. Western blot analysis of each membrane preparation showed the presence of GABA(B2) receptor subunit, a putative site of action of CGP7930. These data indicate that CGP7930 positively modulates brain GABA(B) receptors coupled to either stimulation or inhibition of cyclic AMP signalling.

Blockade of the EGF receptor induces a deranged chemokine expression in keratinocytes leading to enhanced skin inflammation.

During inflammatory skin disorders such as psoriasis, atopic dermatitis, and allergic contact dermatitis, epidermal keratinocytes overexpress large amounts of soluble epidermal growth factor receptor ligands in response to tumor necrosis factor alpha and interferon gamma. These cytokines also promote de novo synthesis of numerous chemokines, including CCL2/MCP-1, CCL5/RANTES, CXCL10/IP-10, and CXCL8/IL-8, in turn responsible for the recruitment of different leukocyte populations. This study demonstrates that stimulation of EGFR down-regulates CCL2, CCL5, and CXCL10, while it increases CXCL8 expression in keratinocytes. Conversely, EGFR signaling blockade produces opposite effects, with increased CCL2, CCL5, and CXCL10, and reduced CXCL8 expression. In a mouse model of contact hypersensitivity, a single topical administration of a selective EGFR kinase blocker before antigen challenge results in a markedly enhanced immune response with increased chemokine expression and heavier inflammatory cell infiltrate. Targeting EGFR on epithelial cells may thus have profound impact on inflammatory and immune responses.

House dust mite allergen exerts no direct proinflammatory effects on human keratinocytes.

BACKGROUND: Dermatophagoides pteronyssinus is a trigger of atopic dermatitis. Many D pteronyssinus allergens are proteases that can elicit airway inflammation by stimulating the release of cytokines and chemokines by bronchial epithelial cells. OBJECTIVE: We sought to investigate whether D pteronyssinus allergens can exert a similar activity on skin keratinocytes. METHODS: Primary cultures of keratinocytes from healthy subjects or patients with atopic dermatitis and normal human bronchial epithelial cells were compared for cytokine production in response to D pteronyssinus extract. RESULTS: Keratinocytes, but not bronchial epithelial cells, displayed a modest dose-dependent release of IL-1alpha and IL-1 receptor antagonist but no induction of their mRNA after exposure to D pteronyssinus. However, D pteronyssinus also degraded these cytokines. On the other hand, D pteronyssinus extract induced bronchial epithelial cells, but not keratinocytes, to increased expression of IL-8/CXCL8 and GM-CSF mRNA and protein. These effects were efficiently abrogated by a mixture of cysteine and serine protease inhibitors. Both IL-8 and GM-CSF were fully resistant to D pteronyssinus proteolytic attack. No induction of monocyte chemoattractant protein 1/CCL2, RANTES/CCL5, or IFN-gamma-induced protein of 10 kd/CXCL10 was detected in either cell type. Only bronchial epithelial cells expressed protease-activated receptor (PAR) 4 mRNA, whereas PAR-1, PAR-2, and PAR-3 mRNA was found in both cell types. D pteronyssinus did not affect PAR mRNA signals. CONCLUSIONS: Although D pteronyssinus can cause proteolysis-dependent release of cytokines from keratinocytes, it appears incapable of activating de novo expression of cytokines and chemokines, arguing against a direct proinflammatory activity of house dust mite on the skin.