Frank Antonicelli

Annexin A1 processing is associated with caspase-dependent apoptosis in BZR cells

Annexins are widely distributed and have been described in lung as well as in other cells and tissues. Annexin I (ANX AI) is a member of the calcium‐dependent phospholipid binding protein family. Besides its anti‐inflammatory function, ANX AI has been involved in several mechanisms such as the Erk repression pathway or apoptosis. To investigate the role of ANX AI on apoptosis in broncho‐alveolar cells, we have constructed a plasmid containing the ANX AI full length cDNA. Transfected BZR cells displayed a higher level of both forms of ANX AI (37 and 33 kDa) as well as a decrease in cell viability (two‐fold versus cells transfected with an empty vector). In order to analyse the endogenous ANX AI processing during stimulus‐induced apoptosis, BZR cells were treated with a commonly used inducer, i.e. C2 ceramides. In these conditions, microscopic analysis revealed chromatin condensation in dying cells and the Bcl‐2, Bcl‐xL/Bax mRNA balance was altered. Caspase‐3 is one of the key executioners of apoptosis, being responsible for the cleavage of many proteins such as the nuclear enzyme poly(ADP‐ribose) polymerase (PARP). We demonstrate that caspase‐3 was activated after 4 h treatment in the presence of ceramide leading to the cleavage of PARP. Dose–response experiments revealed that cell morphology and viability modifications following ceramide treatment were accompanied by an increase in endogenous ANX AI processing. Interestingly, in both ceramide and transfection experiments, the ANX AI cleaved form was enhanced whereas pre‐treatment with the caspase inhibitor Z‐VAD‐fmk abolished ANX AI cleavage. In conclusion, this study demonstrates a complex regulatory role of caspase‐dependent apoptosis where ANX AI is processed at the N‐terminal region which could give susceptibility to apoptosis upon ceramide treatment.

Usefulness of BP230 and BP180-NC16a enzyme-linked immunosorbent assays in the initial diagnosis of bullous pemphigoid: a retrospective study of 138 patients.

OBJECTIVEnTo investigate the diagnostic value of commercially available BP230 and BP180-NC16a enzyme-linked immunosorbent assays (ELISAs) in routine practice in patients with bullous pemphigoid (BP).nnnDESIGNnSingle-center retrospective study.nnnSETTINGnFrench academic dermatology department.nnnPATIENTSnThe study population comprised 138 patients, who were admitted from January 1998 through December 2008.nnnINTERVENTIONSnSera samples were analyzed by ELISA; clinical and immunopathological data were recorded from the patients medical charts.nnnMAIN OUTCOME MEASURESnBP230 and BP180-NC16a ELISA scores were evaluated with respect to clinical characteristics (number of blisters, mucosal involvement, localized or generalized disease, and outcome) and routine indirect immunofluorescence (IF).nnnRESULTSnOf the 138 study patients, 81 (59%) had a positive BP230 ELISA result and 119 (86%) had a positive BP180 ELISA result. There was no relationship between a positive ELISA BP230 result and the disease extent at diagnosis or the presence of mucosal involvement. Serum anti-basement membrane zone autoantibodies (indirect IF) were more frequently detected when the BP230 ELISA result was positive (P < .001). The median anti-basement membrane autoantibody titer as detected by indirect IF was higher in patients with a positive BP230 result (P < .001). The BP180 ELISA result was associated with disease extent at diagnosis as estimated by both the percentage of patients with extensive BP (P = .01) and the mean number of blisters (P = .03) but was not associated with mucosal involvement.nnnCONCLUSIONSnThe currently available BP230 ELISA is a reliable although less-sensitive test than BP180 ELISA in BP, and its diagnostic added value compared with BP180 ELISA alone is approximately 5%. Our results support the predominant contribution of the BP230-specific autoantibodies to anti-basement membrane zone antibody titer as detected by indirect IF.

Elastin-Derived Peptides Induce a T-Helper Type 1 Polarization of Human Blood Lymphocytes

Objective—Increased level of elastin-derived peptides (EDPs) is observed in the serum of patients with manifestations of arterial diseases. We here investigated whether EDPs might exert, at systemic level, a regulatory role for the T-helper type 1 (Th-1)/Th-2 cellular immune response by human peripheral blood lymphocytes (PBLs) expressing the spliced-galactosidase (S-gal)–elastin receptor. Methods and Results—Using flow cytometry and Western blot analysis, we demonstrated that EDPs led to an activation of the S-gal-elastin receptor associated with cytokine production on PBLs and CD4+ T cell subpopulations. The constitutive expression of the S-gal–elastin receptor at the surface of human PBLs was upregulated at the mRNA (RT-PCR) and protein (ELISA) levels on cell activation. In nonactivated and phytohemagglutinin-activated conditions, expressions of the predominant Th-2 cytokine interleukin-5 (IL-5) and IL-10 were reduced, whereas those of the major Th-1 cytokines interferon-γ and IL-2 were enhanced by EDPs. Furthermore, we evidenced that EDPs could not only potentiate the IL-12–induced Th-1 profile but also could reverse the Th-2 (over Th-1) profile induced by IL-4. Finally, Th-1 cytokine upregulation was associated to an increased activator protein-1 DNA binding and enhanced pro–matrix metalloproteinase-9 secretion. Conclusions—This study highlights the importance of EDPs as stimuli for Th-1 differentiation, whether T cells are in an inactivated state or already orientated toward a Th-1 (IL-12) or Th-2 (IL-4) response.

Innate immune cell-produced IL-17 sustains inflammation in bullous pemphigoid

Bullous pemphigoid (BP) is an autoimmune skin disease characterized by the binding of autoantibodies to components of the hemidesmosome structure resulting in an inflammatory response and subepidermal blister formation. To investigate the role of immune orientation in the inflammatory processes associated to disease progression, blister fluid, serum and biopsy specimens were collected from thirty one consecutive BP patients. Blister fluids displayed high level of IL-6, IL-17, IL-22, IL-23, whereas TGF-β was increased in BP sera. However neither immunocytochemistry on a trans-differentiation model of IL-17-producing PBMCs nor immunohistochemistry on BP biopsy specimens could demonstrate the presence of Th17 lymphocytes. Instead innate immune cells, especially neutrophils, produced IL-17 at the skin lesional site. Of note, superpotent topical corticosteroid application quickly and dramatically reduced both IL-17 expression and clinical signs of BP. Consistently, IL-17 upregulated MMP-9 and neutrophil elastase expression, two proteases involved in blister formation, thereof further demonstrating its role in the progress of BP. Finally IL-17-induced matrix degradation originated from neutrophil activation, initiated the formation of an amplification loop of the inflammatory response that could represent the underlying phenomenon leading to the maintenance and even disease extent. Thus, our results could open new therapeutic strategies for BP patients.

Control by thyrotropin of the production by thyroid cells of an inositol phosphate-glycan

The increased turnover of phosphatidylinositol promoted by thyrotropin (TSH) in pig thyroid tissue does not seem to be caused by an increased production of inositol tris-phosphate. We have explored another possibility, the synthesis of an inositol phosphate-glycan (IP-gly). Our results show that thyroid cells in culture produced this substance from a precursor phosphatidylinositol-glycan (Gly-PI). The obtained IP-gly seemed, by its analytical and biological properties, to be identical, or similar, to the previously described insulin mediator.

The elastin peptides-mediated induction of pro-collagenase-1 production by human fibroblasts involves activation of MEK/ERK pathway via PKA- and PI3K-dependent signaling

Elastin peptides, such as κ‐elastin (kE), bind to the elastin receptor at the cell surface of human dermal fibroblasts and stimulate collagenase‐1 expression at the gene and protein levels. Using specific inhibitors and phosphospecific antibodies, we show here that the binding of elastin peptides to their receptor activates the extracellular signal‐regulated kinase (ERK) pathway; this activation is essential for the induction of pro‐collagenase‐1 production. Moreover, protein kinase A (PKA) and phosphatidylinositol 3‐kinase (PI3K) signaling were found to participate in ERK activation. Concomitantly, we demonstrate that stimulation by elastin peptides leads to enhanced DNA binding of activator protein‐1 (AP‐1). Our data indicate that the up‐regulation of collagenase‐1 following treatment of fibroblasts with elastin peptides results from a cross‐talk between PKA, PI3K and the ERK signaling pathways and that this regulation is accompanied by activation of AP‐1 transcription factors.

Role of the elastin receptor complex (S-Gal/Cath-A/Neu-1) in skin repair and regeneration.

Impaired elastic fiber assembly constitutes one major problem in skin wound healing. Recent data indicate that a ternary complex involving a splicing form of β‐galactosidase associated with cathepsin‐A and neuraminidase‐1 directs the transport of tropoelastin to the fibroblast plasma membrane and participates in the deposition of the elastin precursor onto a microfibrillar scaffold. In addition, this elastin receptor complex is ubiquitously expressed and also acts as a true receptor for elastin‐derived peptides produced during the initial stage of wound repair following elastase‐mediated proteolysis action. Among the peptides generated, those having a x.G.x.x.P.G. motif upregulate (i) keratinocyte migration, (ii) endothelial cell angiogenic phenotype, (iii) fibroblast proliferation, and (iv) induction of the expression of matrix metalloproteinases, type I collagen, and tropoelastin. All of these properties could accelerate the different stages of wound repair. Elastin‐derived peptides from a chemical or a proteolytic digest of insoluble elastin alone or linked to the collagen scaffold significantly improve skin wound healing and dermal regeneration in vivo in several animal models. Such a beneficial influence has been recently extended to the treatment of burn patients. In this respect, recent investigations have focused on the design of elastin‐derived peptides or elastin‐building blocks, as obtained from peptide chemistry or by genetic engineering, to elaborate biocompatible elastin peptides, which are considered as ideal biomaterials for “catalyzing” skin repair and regeneration following injury.

Prevalence and Clinical Significance of Anti–Laminin 332 Autoantibodies Detected by a Novel Enzyme-Linked Immunosorbent Assay in Mucous Membrane Pemphigoid

IMPORTANCEnA rare variant of mucous membrane pemphigoid (MMP) is characterized by circulating anti-laminin 332 (Lam332) autoantibodies and seems to be associated with concurrent malignant neoplasms.nnnOBJECTIVEnTo determine the prevalence and clinical significance of anti-Lam332 autoantibody detection from a large series of patients with MMP.nnnDESIGNnMulticenter retrospective study.nnnSETTINGnFour French national centers for autoimmune bullous diseases.nnnPARTICIPANTSnOne hundred fifty-four patients with MMP and 89 individuals serving as controls were included.nnnINTERVENTIONSnSerum samples were analyzed by a new Lam332 enzyme-linked immunosorbent assay (ELISA); clinical and immunopathologic data were obtained from the patients medical records.nnnMAIN OUTCOME MEASURESnThe Lam332 ELISA scores were evaluated with respect to clinical characteristics, standard and salt-split indirect immunofluorescence, and bullous pemphigoid (BP) 230 and BP180-NC16A ELISAs.nnnRESULTSnThe Lam332 ELISA score was positive (≥9 U/mL) in 20.1% of serum samples from patients with MMP, 1 of 50 patients with bullous pemphigoid (BP), none of 7 with pemphigus, and 3 of 32 other controls. No relationship was evidenced between a positive ELISA Lam332 score and age; sex ratio; oral, ocular, genital, skin, or esophageal/laryngeal involvement; internal malignant neoplasm; or BP180 ELISA score. Salt-split skin indirect immunofluorescence and ELISA BP230 results were more frequently positive when Lam332 ELISA results were positive (P = .04 and .02, respectively). Patients with a positive Lam332 ELISA score frequently had more severe MMP (67.8% vs 47.2%; P = .04).nnnCONCLUSIONS AND RELEVANCEnResults of this novel ELISA showed that serum anti-Lam332 autoantibodies are detected in 20.1% of patients with MMP. Anti-Lam332 autoantibodies are mainly detected in patients with severe MMP but not preferentially in those with a malignant neoplasm. The association between anti-Lam332 and anti-BP230 autoantibodies might arise from an epitope-spreading phenomenon.

CXCL10 reduces melanoma proliferation and invasiveness in vitro and in vivo

Backgroundu2002 Melanoma is often infiltrated by inflammatory and immune cells that might either maintain chronic inflammation, therefore promoting tumour growth, or mount an antitumour response to control tumour outcome. In this setting, Th1‐oriented lymphocyte infiltration is associated with a better outcome in melanoma. Although the interferon‐induced protein CXCL10 is expressed by Th1 immune cells, its receptor was also shown to be involved in melanoma progression and metastasis.

Targeting the Tumor Microenvironment: The Protumor Effects of IL-17 Related to Cancer Type.

The inflammatory process contributes to immune tolerance as well as to tumor progression and metastasis. By releasing extracellular signals, cancerous cells constantly shape their surrounding microenvironment through their interactions with infiltrating immune cells, stromal cells and components of extracellular matrix. Recently, the pro-inflammatory interleukin 17 (IL-17)-producing T helper lymphocytes, the Th17 cells, and the IL-17/IL-17 receptor (IL-17R) axis gained special attention. The IL-17 family comprises at least six members, IL-17A, IL-17B, IL-17C, IL-17D, IL-17E (also called IL-25), and IL-17F. Secreted as disulfide-linked homo- or heterodimers, the IL-17 bind to the IL-17R, a type I cell surface receptor, of which there are five variants, IL-17RA to IL-17RE. This review focuses on the current advances identifying the promoting role of IL-17 in carcinogenesis, tumor metastasis and resistance to chemotherapy of diverse solid cancers. While underscoring the IL-17/IL-17R axis as promising immunotherapeutic target in the context of cancer managing, this knowledge calls upon further in vitro and in vivo studies that would allow the development and implementation of novel strategies to combat tumors.