Adriforant is a functional antagonist of histamine receptor 4 and attenuates itch and skin inflammation in mice.

BACKGROUND: Histamine has been postulated to play a role in atopic dermatitis via histamine receptor 4, mediating pruritic and inflammatory effects. The H4R antagonist adriforant (PF-3893787 or ZPL389) indicated clinical efficacy in a Ph2a study in atopic dermatitis. Preclinical investigations of adriforant had been scarce as experiments in transfectants with H4R from several species suggested partial agonism, not seen in human cells. OBJECTIVE: During the Ph2b trial in AD, we performed experiments to understand the pharmacology of adriforant in primary murine cells and in vivo models. We assessed its effects on ERK phosphorylation and transcriptional changes in bone marrow-derived mast cells, histamine-dependent Ca2+ flux in neurons and histamine-induced itch response. In addition, its impact on MC903-induced skin inflammation was evaluated. RESULTS: We show that, contrary to transfectants, adriforant is a competitive antagonist of the murine histamine receptor 4, antagonizes histamine-induced ERK phosphorylation, normalizes histamine-induced transcriptional changes in mast cells and reduces histamine-dependent Ca2+ flux in neurons. Administration to mice reduces acute histamine-induced itch response. In addition, adriforant ameliorates inflammation in the mouse MC903 model. CONCLUSIONS: Our results suggest that functional inhibition of histamine receptor 4 by adriforant reduces itch and inflammation in vivo. The effects observed in mice, however, did not translate to clinical efficacy in patients as the Ph2b clinical trial with adriforant did not meet pre-specified efficacy endpoints. Given the complex pathogenesis of AD, antagonism of histamine receptor 4 alone appears insufficient to reduce disease severity in AD patients, despite the effects seen in mouse models.

Disease Association of Anti‒Carboxyethyl Lysine Autoantibodies in Hidradenitis Suppurativa.

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease characterized by recurring suppurating lesions of the intertriginous areas, resulting in a substantial impact on patients' QOL. HS pathogenesis remains poorly understood. An autoimmune component has been proposed, but disease-specific autoantibodies, autoantigens, or autoreactive T cells have yet to be described. In this study, we identify a high prevalence of IgM, IgG, and IgA antibodies directed against Nε-carboxyethyl lysine (CEL), a methylglyoxal-induced advanced glycation end-product, in the sera of patients with HS. Titers of anti-CEL IgG and IgA antibodies were highly elevated in HS compared with those in healthy controls and individuals with other inflammatory skin diseases. Strikingly, the majority of anti-CEL IgG was of the IgG2 subclass and correlated independently with both disease severity and duration. Both CEL and anti-CEL‒producing plasmablasts could be isolated directly from HS skin lesions, further confirming the disease relevance of this autoimmune response. Our data point to an aberration of the methylglyoxal pathway in HS and support an autoimmune axis in the pathogenesis of this debilitating disease.

Requirement for O-linked N-acetylglucosaminyltransferase in lymphocytes activation.

The dynamic modification of nuclear and cytoplasmic proteins with O-linked beta-N-acetylglucosamine (O-GlcNAc) by the O-linked N-acetylglucosaminyltransferase (OGT) is a regulatory post-translational modification that is responsive to various stimuli. Here, we demonstrate that OGT is a central factor for T- and B-lymphocytes activation. SiRNA-mediated knockdown of OGT in T cells leads to an impaired activation of the transcription factors NFAT and NFkappaB. This results in a reduction of IL-2 production consistent with prevention of T-cell activation. OGT is also required for the early activation of B cells mediated by stimulation of the B-cell receptor. Mechanistically, we demonstrate that NFkappaB as well as NFAT are glycosylated with O-GlcNAc after direct binding to OGT. Moreover, kinetic experiments show that O-GlcNAc modification prominently increased shortly after activation of lymphoid cells and it might be required for nuclear translocation of the transcription factors NFkappaB and NFAT.

Genetic, biochemical, and transcriptional responses of Saccharomyces cerevisiae to the novel immunomodulator FTY720 largely mimic those of the natural sphingolipid phytosphingosine.

Sphingolipids are signaling molecules that influence diverse cellular functions from control of the cell cycle to degradation of plasma membrane proteins. The synthetic sphingolipid-like compound FTY720 is an immunomodulating agent in clinical trials for transplant graft maintenance. In this report, we compare the effects of the natural yeast sphingolipid phytosphingosine with FTY720 in Saccharomyces cerevisiae. We show that the multicopy suppressor genes that induce growth resistance to FTY720 also confer resistance to growth-inhibitory concentrations of phytosphingosine. In addition, mutants for ubiquitination pathway proteins are shown to be resistant to the growth-inhibiting effect of both FTY720 and phytosphingosine. We observe fewer similarities between sphingosine and FTY720 than between FTY720 and phytosphingosine as revealed by genetic studies. Yeast cells lacking the specific sphingosine kinase LCB4 are sensitive to phytosphingosine and FTY720 but resistant to sphingosine, suggesting that FTY720 and phytosphingosine have a more related mechanism of action. Gene expression profile comparisons of sensitive and resistant yeast cells exposed to FTY720 and phytosphingosine highlight a number of similarities. In response to treatment with these compounds, approximately 77% of the genes that are regulated >2-fold by FTY720 also respond to phytosphingosine in the same direction in the parent strain. In addition, a close inspection of TAT1 and TAT2 transporters following exposure to phytosphingosine indicates that TAT1 protein is degraded in a similar fashion upon treatment with FTY720 and phytosphingosine. There were differences, however, with respect to the TAT2 protein level and the expression profiles of a subset of genes. The genetic, transcriptional, and biochemical data together indicate that FTY720 and phytosphingosine influence similar pathways in yeast cells. These findings offer further insights into the physiological pathways influenced by these compounds in all eukaryotic cells and help us to understand the therapeutic consequences of FTY720 in humans.

Ubiquitin pathway proteins influence the mechanism of action of the novel immunosuppressive drug FTY720 in Saccharomyces cerevisiae.

FTY720 is an immunosuppressive drug in clinical development for transplant graft protection in humans. This agent is of particular interest because, unlike currently available regimes, it acts to sequester lymphocytes without causing cytotoxicity or blocking differentiation and growth potential. In an effort to elucidate the mechanism of action of FTY720, and identify its downstream effectors, we have screened genomic libraries and spontaneous mutants of the model system Saccharomyces cerevisiae for resistance to FTY720. We identified several proteins and pathways as being involved in the mechanism of action of FTY720. We show specifically that the two amino acid transporters TAT1 and TAT2, the two ubiquitin proteases UBP5 and UBP11, and the heat shock protein CAJ1 confer growth resistance to FTY720 when overexpressed. Another amino acid transporter, GNP1, and the ubiquitin structural gene UBI4 as well as the ubiquitin ligase RSP5, and its binding protein BUL1 confer growth resistance in a mutated form. Supporting the importance of amino acid transport in the growth resistance phenotype of S. cerevisiae to the immunosuppressive agent FTY720, a prototrophic strain was more resistant to FTY720 than the isogenic auxotroph. To further explore these results, the effects on amino acid uptake and protein degradation were measured in the presence of FTY720. Due to the high conservation of these proteins and pathways between yeast and humans, these results may provide valuable insights into the mechanism of action of FTY720 in lymphocyte sequestration in humans.