Beyond the concept of cold and hot tumors for the development of novel predictive biomarkers and the rational design of immunotherapy combination.

Immunotherapy has revolutionized the management of cancers. At the end of 2018, 1,716 clinical trials assessed regimen that combine program death-1 (PD-1)/program death ligand-1 (PD-L1) blockers with other cancer therapies (tyrosine kinase inhibitor, chemotherapy and radiotherapy). There is a contrast between these clinical dynamics and the difficulty of identifying biomarkers to better select patients that could benefit from immunotherapy. In this context, different tumor classifications have been proposed to try to better stratify patients. They rely on the characteristics of the tumor microenvironment and led first to divide them into hot and cold tumors. In this review, we aim to demonstrate the limitations of this classification focusing on the differential significance of subpopulations of intratumor CD8 + T cells. We also underline novel mechanisms of resistance to anti-PD-1/PD-L1 blockade, focusing on myeloid cells, hypoxia and tumor immunoediting under treatment. Understanding the mechanisms of resistance to immune-checkpoint inhibitor is indeed a powerful research driver that allows further identification of novel biomarkers, drug development and bring a rational to innovative therapeutic combinations.

Gingival fibroblasts protect against experimental abdominal aortic aneurysm development and rupture through tissue inhibitor of metalloproteinase-1 production.

AIMS: Abdominal aortic aneurysm (AAA), frequently diagnosed in old patients, is characterized by chronic inflammation, vascular cell apoptosis and metalloproteinase-mediated extracellular matrix destruction. Despite improvement in the understanding of the pathophysiology of aortic aneurysm, no pharmacological treatment is yet available to limit dilatation and/or rupture. We previously reported that human gingival fibroblasts (GFs) can reduce carotid artery dilatation in a rabbit model of elastase-induced aneurysm. Here, we sought to investigate the mechanisms of GF-mediated vascular protection in two different models of aortic aneurysm growth and rupture in mice. METHODS AND RESULTS: In vitro, mouse GFs proliferated and produced large amounts of anti-inflammatory cytokines and tissue inhibitor of metalloproteinase-1 (Timp-1). GFs deposited on the adventitia of abdominal aorta survived, proliferated, and organized as a layer structure. Furthermore, GFs locally produced Il-10, TGF-ß, and Timp-1. In a mouse elastase-induced AAA model, GFs prevented both macrophage and lymphocyte accumulations, matrix degradation, and aneurysm growth. In an Angiotensin II/anti-TGF-ß model of aneurysm rupture, GF cell-based treatment limited the extent of aortic dissection, prevented abdominal aortic rupture, and increased survival. Specific deletion of Timp-1 in GFs abolished the beneficial effect of cell therapy in both AAA mouse models. CONCLUSIONS: GF cell-based therapy is a promising approach to inhibit aneurysm progression and rupture through local production of Timp-1.

Inhibition of the differentiation of monocyte-derived dendritic cells by human gingival fibroblasts.

We investigated whether gingival fibroblasts (GFs) can modulate the differentiation and/or maturation of monocyte-derived dendritic cells (DCs) and analyzed soluble factors that may be involved in this immune modulation. Experiments were performed using human monocytes in co-culture with human GFs in Transwell® chambers or using monocyte cultures treated with conditioned media (CM) from GFs of four donors. The four CM and supernatants from cell culture were assayed by ELISA for cytokines involved in the differentiation of dendritic cells, such as IL-6, VEGF, TGFß1, IL-13 and IL-10. The maturation of monocyte-derived DCs induced by LPS in presence of CM was also studied. Cell surface phenotype markers were analyzed by flow cytometry. In co-cultures, GFs inhibited the differentiation of monocyte-derived DCs and the strength of this blockade correlated with the GF/monocyte ratio. Conditioned media from GFs showed similar effects, suggesting the involvement of soluble factors produced by GFs. This inhibition was associated with a lower stimulatory activity in MLR of DCs generated with GFs or its CM. Neutralizing antibodies against IL-6 and VEGF significantly (P<0.05) inhibited the inhibitory effect of CM on the differentiation of monocytes-derived DCs and in a dose dependent manner. Our data suggest that IL-6 is the main factor responsible for the inhibition of DCs differentiation mediated by GFs but that VEGF is also involved and constitutes an additional mechanism.

Endoluminal gingival fibroblast transfer reduces the size of rabbit carotid aneurisms via elastin repair.

OBJECTIVE: Matrix metalloproteinase-9 is considered to play a pivotal role in aneurismal formation. We showed that gingival fibroblasts (GF) in vitro reduced matrix metalloproteinase-9 activity via increased secretion of tissue inhibitor of metalloproteinase 1. We aimed to evaluate in vivo the efficacy of GF transplantation to reduce aneurism development in a rabbit model. METHODS AND RESULTS: Seventy rabbit carotid aneurisms were induced by elastase infusion. Four weeks later, GF, dermal fibroblast, or culture medium (DMEM) were infused into established aneurisms. Viable GF were abundantly detected in the transplanted arteries 3 months after seeding. GF engraftment resulted in a significant reduction of carotid aneurisms (decrease of 23.3% [P<0.001] and 17.6% [P=0.01] of vessel diameter in GF-treated arteries, 1 and 3 months after cell therapy, respectively), whereas vessel diameter of control DMEM and dermal fibroblast-treated arteries increased. GF inhibited matrix metalloproteinase-9 activity by tissue inhibitor of metalloproteinase 1 overexpression and matrix metalloproteinase-9/tissue inhibitor of metalloproteinase 1 complex formation, induced elastin repair, and increased elastin density in the media compared with DMEM-treated arteries (38.2 versus 18.0%; P=0.02). Elastin network GF-induced repair was inhibited by tissue inhibitor of metalloproteinase 1 blocking peptide. CONCLUSIONS: Our results demonstrate that GF transplantation results in significant aneurism reduction and elastin repair. This strategy may be attractive because GF are accessible and remain viable within the grafted tissue.

Impact of photodynamic therapy on inflammatory cells during human chronic periodontitis.

The aim of this study was to evaluate the effects of the photodynamic therapy (PDT) on the inflammatory infiltrate and on the collagen network organization in human advanced chronic periodontitis. Two different drug delivery systems (DDS) were tested (liposomes and nanoemulsions) to determine if the effects of PDT could differ according to the DDS used. Sixteen patients presenting two teeth with chronic advanced periodontitis and important tooth mobility with clinical indication of extraction were included in the group liposomes (group L, n=8) or in the group nanoemulsions (group N, n=8) in order to compare the effects of each DDS. Seven days before extractions one tooth of each patient was treated with PDT using phthalocyanine derivatives as photosensitizers and the contralateral tooth was taken as control. In group L the density of gingival collagen fibers (66±19%) was significantly increased (p<0.02) when compared to controls (35±21%). Concerning the antigen-presenting cells, PDT had differential effects depending on the drug delivery system; the number of macrophages was significantly decreased (p<0.05) in group L while the number of Langerhans cells was significantly decreased in group N (p<0.02). These findings demonstrate that PDT presents an impact on gingival inflammatory phenomenon during chronic periodontitis and leads to a specific decrease of antigen-presenting cells populations according to the drug delivery system used.

Fusiform aneurysm model in rabbit carotid artery.

AIMS: To develop a reproducible and accessible model of elastase-induced fusiform aneurysm in carotid rabbit arteries. METHODS: Elastase, at a concentration of 1-30 U, was incubated into the lumen of carotid rabbit arteries. Four weeks later, angiography, histomorphometry, immunohistochemistry and zymography were performed. RESULTS: The optimal concentration of elastase in this model was 3 U according to the balance between mortality and thrombosis rates. Indeed, at 3 U, external carotid diameter increased from 1.9 +/- 0.1 to 3.1 +/- 0.4 mm (p < 0.0001) associated with degradation of elastic fibers, matrix metalloproteinase-9 secretion, apoptosis and macrophage infiltration. CONCLUSIONS: Our study underlines that abdominal aortic aneurysm can be reliably duplicated in an elastase-induced aneurysm in carotid artery, a much more accessible vessel.

Tissular senescence and modifications of oral ecosystem in the elderly: risk factors for mucosal pathologies.

The aim of this present review is to describe the pathogenesis and mechanisms behind mucosal pathologies in the elderly including a description of the risk factors for these pathologies. The oral cavity - and particularly oral mucosae - is exposed to many stresses as well as physical, chemical, thermic and pathogenic agents. In the elderly, mucosae are less resistant to the insults, and this increases the occurrence of diseases. Several factors contribute to the prevalence of mucosal pathologies with aging. There are two categories: intrinsic factors linked to the senescence of the tissues and functions, and extrinsic factors related to the older people general health status. The intrinsic factors are: 1) mucosal senescence which induces fragility 2) immunosenescence which causes a decrease in the host response against micro-organisms and an increase in the autoimmune diseases and 3) senescence of salivary glands and reduction of the saliva protective function. Furthermore, there are extrinsic factors which contribute to change the oral ecosystem during aging, such as polypathologies and polymedications, malnutrition, degradation of oral hygiene, pathogen proliferation (mainly bacteria and Candida species) and old or ill-fitted removable dentures. In the elderly several diseases occur on the oral mucosae: inflammation, bacterial infections or candidiasis, ulcerations, autoimmune dermatosis, tumoral processes. This review describes some common oral mucosal pathologies in the older people, which illustrate the impact of different risk factors described in the first part.

Increase of gingival matured dendritic cells number in elderly patients with chronic periodontitis.

OBJECTIVE: The purpose of this study was to evaluate the inflammatory cell subset proportions in the upper gingival connective tissue, including mature dendritic cells (DC) in elderly and younger patients with generalized chronic periodontitis in order to further understand the effect of aging on gingival inflammatory phenomenon. METHODS: Gingival tissue specimens presenting chronic periodontitis from 8 elderly patients aged >75 (test group, group T) and from 8 younger patients aged 50-60 (considered as controls, group C) were analysed by immunohistochemistry using monoclonal antibodies against CD45RB, CD4, CD8, CD19, CD68, DC-SIGN, DC-LAMP molecules. The number of each immunolabelled cells subset was counted using image analysis. RESULTS: The difference in the number of CD45RB+leucocytes in the upper gingival connective tissue between groups was not significant permitting to use it as reference. As compared to group C, the lymphocyte subsets/CD45RB+leucocytes ratios tended to decrease in group T but the decrease was significant only for CD4+T lymphocytes/CD45RB+cells ratio (p<0.03). On the opposite, the ratios of antigen-presenting cells DC-SIGN+cells/CD45RB+cells and DC-LAMP+cells/CD45RB+cells were significantly increased (p<0.03 and <0.0001, respectively) in group T. Moreover, in group T the DC-LAMP+cells/DC-SIGN+cells ratio was significantly increased (p<0.05) showing an increased number of matured dendritic cells. CONCLUSION: During chronic periodontitis in elderly patients, our results show a decrease in the ratio of gingival CD4+lymphocyte subset associated with an increase in the ratios of antigen-presenting cells subsets and more particularly maturated DC-LAMP+dendritic cells.

Long-term follow-up of 61 coeliac patients diagnosed in childhood: evolution toward latency is possible on a normal diet.

BACKGROUND/AIMS: Whether a life-long gluten-free diet (GFD) is necessary in all children with diagnosed coeliac disease (CD) remains debated. To address this question, a retrospective analysis of the clinical and biological status of adult coeliac patients diagnosed in childhood, who remained on a normal diet after gluten challenge and were clinically silent, was carried out. METHODS: Patients aged 18-65 years with CD diagnosed in childhood were included. Clinical status, gluten intake, biological parameters of malabsorption, bone mineral density, human leucocyte antigen (HLA) genotype, serological markers of CD, and histological and immunohistochemical parameters in duodenal biopsies were recorded. RESULTS: Sixty-one patients had resumed a normal diet and were asymptomatic. Forty-eight showed different degrees of villous atrophy (silent CD), while 13 had no detectable atrophy (latent CD) on duodenal biopsies. Latent CD patients had significantly less osteopenia/osteoporosis (1/9 (11%) vs 23/33 (70%), p<0.001)), and lower T cell receptor (TCR) alphabeta+ intraepithelial T cell counts (38+/-20 vs 55+/-15, p<0.01) than silent CD patients. The mean age at diagnosis and first GFD was lower in latent than in silent patients (14.4+/-5 vs 40.1+/-47 months, p<0.05). Latent patients did not differ significantly from the seven control patients on a long-term GFD, except for a higher frequency of CD-specific serum antibodies. However, two latent patients relapsed clinically and histologically during subsequent follow-up. CONCLUSIONS: Long-term latency developed in about 20% of CD patients who remained symptom free after gluten reintroduction. This latency can be transient and thus a regular follow-up is mandatory. In silent patients, the increased risk of osteoporosis substantiates the need for a GFD.

Epigallocatechin gallate's protective effect against MMP7 in recessive dystrophic epidermolysis bullosa patients.

The analysis of phenotype-genotype correlations of patients suffering from recessive dystrophic epidermolysis bullosa (RDEB) evidenced intrafamilial and interfamilial phenotype variability occurring for the same mutation of COL7A1; this underscores the role of other genetics environmental factors in the expressivity of the disease. In this work, we checked whether matrilysin 1 (matrix metalloproteinase (MMP)7) could take part in the epidermal detachment in RDEB. Furthermore, we investigated epigallocatechin 3 gallate (EGCG) to determine whether it could inhibit matrilysin activities on collagen type VII and fibrillin 1 known to be associated with the dermo-epidermal junction. In this work, matrilysin 1 was detected in affected and unaffected skins of the three RDEB patients; furthermore, MMP7 was shown to degrade ex vivo on healthy normal skin collagen VII and fibrillin 1. Thus, we suspect that MMP7 could take an active part in the epidermal detachment occurring during RDEB. We evidenced that EGCG in in vitro as well as in ex vivo experiments was a good inhibitor of MMP7 and developed a good protection of collagen type VII and fibrillin 1 susceptible of being degraded by MMP7. We therefore propose that EGCG could be used beneficially in patients suffering from RDEB.

Do Langerhans cells behave similarly in elderly and younger patients with chronic periodontitis?

OBJECTIVE: The purpose of this study was to compare the number, the distribution and the expression of markers of maturation of Langerhans cells (LC) in elderly and younger patients with chronic periodontitis in order to evidence the effect of aging on LC in inflammatory gingival tissue. METHODS: Gingival tissue specimens presenting chronic periodontitis from 8 elderly patients aged >75 (group E) and from 8 younger patients aged 50-60 (considered as controls, group C) were used for immunohistochemistry with monoclonal antibodies against CD45RB (leucocytes), CD1a (LC), markers of LC maturation (DC-LAMP, CD83) and number of immunolabelled cell subsets was evaluated using image analysis. RESULTS: The difference in the number of CD45RB+ leucocytes in the upper connective tissue between groups was not significant. In group E, the number of CD1a+ LC was significantly decreased (P<0.002) in the epithelium and significantly increased (P<0.0004) in the upper connective tissue. Furthermore, in group E, intraepithelial CD1a+ LC are more often observed in the upper epithelium and their dendritic processes were shorter and less numerous. Concerning the expression of markers of maturation, the numbers of intraepithelial DC-LAMP+ cells and CD83+ cells were significantly increased (P<0.0007 and P<0.02, respectively) in group E. CONCLUSION: During chronic periodontitis in elderly patients, the decrease in the number of intraepithelial LC and the alteration of dendritic processes could be balanced by a cellular distribution often observed in the upper epithelium associated with changes in cell maturation in response to bacterial elements.

Skin expression of metalloproteinases and tissue inhibitor of metalloproteinases in sibling patients with recessive dystrophic epidermolysis and intrafamilial phenotypic variation.

A number of COL7A1 mutations have now been reported in recessive dystrophic epidermolysis bullosa patients, and the analysis of phenotype-genotype correlations showed evidence for interfamilial and intrafamilial phenotypic variability, occurring for the same mutation. Collagenase and stromelysin activities have been found to be overexpressed in skin cultures of some recessive dystrophic epidermolysis bullosa patients, and tissue destruction in the disease process might result from an imbalance of metalloproteinases (MMP) over tissueinhibitor of metalloproteinases (TIMP). So we suspected that the phenotypic variability for the same mutation could be linked to other genetic or environmental factors, as a particular balance between MMP and TIMP. Organ cultures were performed using explants from the skin of three patients from the same family with recessive dystrophic epidermolysis bullosa to reveal and quantify the expression of MMP-1 (collagenase 1), MMP-2 and MMP-9 (gelatinases A and B), MMP-3 (stromelysin 1), TIMP-1, and TIMP-2, and to compare the results with those obtained with two human control skins, with the same experimental conditions. Increased amounts of all metalloproteinases investigated were observed in the skin of the three recessive dystrophic epidermolysis bullosa affected sibling brothers, both in lesioned and in apparently nonlesioned skin, compared with controls. The amounts of MMP-1, MMP-2, MMP-3, and MMP-9 increased particularly in the skin of the more clinically affected patient. Furthermore for this patient we evidenced higher amounts of MMP-1 and also a lower TIMP-1 amount in his unlesioned and lesioned skin compared with the other two affected patients and with healthy control donors. So we can suspect that recessive dystrophic epidermolysis bullosa phenotypic variability could be related to patients' collagenase activity heterogeneity, linked to imbalance between MMP-1 and TIMP-1.

Langerin+ versus CD1a+ Langerhans cells in human gingival tissue: a comparative and quantitative immunohistochemical study.

Langerhans cells (LC) are dendritic cells of the immune system able to capture intraepithelial pathogens and migrate to regional lymph nodes to present them to naive T cells. Up to now immunohistological studies on human gingival LC have been carried out using antibodies against HLA-DR or CD1a molecules. A new marker of LC called Langerin (CD207) and described, among other subcellular localisations, in the Birbeck granules is now available in immunohistochemistry. The purpose of this in situ study was to quantify and to compare Langerin+ versus CD1a+ LC number in order to show differences in the expression of these molecules, if any, and to determine which marker is the most specific. The present study was conducted using nine frozen healthy gingival samples. Double immunofluorescence procedures were performed with an anti-Langerin antibody revealed by FITC and with an anti-CD1a-PE antibody. Mounted slides were analysed by fluorescence microscopy and quantifications were performed on projected slides associated with a grid of 0.015 mm(2). Our results have shown that 1/ the number of CD1a+ LC was significantly increased (P=0.01) when compared with Langerin+ LC 2/ 92% of Langerin+ LC co-expressed CD1a 3/ only 82% of CD1a+ cells co-expressed Langerin 4/ a positive correlation was noted between CD1a+ and Langerin+ LC numbers. The present study has revealed the heterogeneity in the phenotype of gingival LC population and shown that Langerin seems the most specific marker for the study of LC.