Jacques Boniver

Vascular endothelial growth factor expression correlates with matrix metalloproteinases MT1-MMP, MMP-2 and MMP-9 in human glioblastomas.

Vascular endothelial growth factor (VEGF) is the major endothelial mitogen in central nervous system neoplasms and it is expressed in 64–95% of glioblastomas (GBMs). Tumour cells are the main source of VEGF in GBMs whereas VEGF receptors (VEGFR‐1, its soluble form sVEGFR‐1, VEGFR‐2 and neuropilin‐1) are expressed predominantly by endothelial cells. Infiltrating tumour cells and newly‐formed capillaries progress through the extracellular matrix by local proteolysis involving matrix metalloproteinases (MMPs). Recent studies have shown that VEGF expression and bioavailability can be modulated by MMPs. We reported previously that the expression of MT1‐MMP in human breast cancer cells was associated with an enhanced VEGF expression. We used quantitative RT‐PCR, Western blot, gelatin zymography and immunohistochemistry to study the expression of VEGF, VEGFR‐1, VEGFR‐2, sVEGFR‐1, neuropilin‐1, MT1‐MMP, MMP‐2, MMP‐9 and TIMP‐2 in 20 human GBMs and 5 normal brains. The expression of these MMPs was markedly increased in most GBMs with excellent correlation between mRNA and protein levels; activated forms of MMP‐2 and MMP‐9 were present in 8/18 and 7/18 of GBMs. A majority of GBMs (17/20) also expressed high levels of VEGF, as previously reported, with strong correlation between VEGF and MT1‐MMP gene expression levels, and double immunostaining showed that VEGF and MT1‐MMP peptides co‐localize in tumour and endothelial cells. Our results suggest that the interplay between metalloproteinases and VEGF previously described in experimental tumours may also be operative in human GBMs. Because of its dual ability to activate MMP‐2 and to up‐regulate VEGF, MT1‐MMP might be of central importance in the growth of GBMs and represent an interesting target for anti‐cancer treatments.

Increased production of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 by inflamed mucosa in inflammatory bowel disease

Inflammatory bowel diseases (IBD) are characterized by a sustained inflammatory cascade that gives rise to the release of mediators capable of degrading and modifying bowel wall structure. Our aims were (i) to measure the production of matrix metalloproteinase‐3 (MMP‐3), and its tissue inhibitor, tissue inhibitor of metalloproteinase‐1 (TIMP‐1), by inflamed and uninflamed colonic mucosa in IBD, and (ii) to correlate their production with that of proinflammatory cytokines and the anti‐inflammatory cytokine, IL‐10. Thirty‐eight patients with IBD, including 25 with Crohn’s disease and 13 with ulcerative colitis, were included. Ten controls were also studied. Biopsies were taken from inflamed and uninflamed regions and inflammation was graded both macroscopically and histologically. Organ cultures were performed for 18 h. Tumour necrosis factor‐alpha (TNF‐α), IL‐6, IL‐1β, IL‐10, MMP‐3 and TIMP‐1 concentrations were measured using specific immunoassays. The production of both MMP‐3 and the TIMP‐1 were either undetectable or below the sensitivity of our immunoassay in the vast majority of uninflamed samples either from controls or from those with Crohn’s disease or ulcerative colitis. In inflamed mucosa, the production of these mediators increased significantly both in Crohn’s disease (P < 0·01 and 0·001, respectively) and ulcerative colitis (P < 0·001 and 0·001, respectively). Mediator production in both cases was significantly correlated with the production of proinflammatory cytokines and IL‐10, as well as with the degree of macroscopic and microscopic inflammation. Inflamed mucosa of both Crohn’s disease and ulcerative colitis show increased production of both MMP‐3 and its tissue inhibitor, which correlates very well with production of IL‐1β, IL‐6, TNF‐α and IL‐10.

Influence of the mucosal epithelium microenvironment on Langerhans cells: implications for the development of squamous intraepithelial lesions of the cervix.

We have addressed the notion that the initiation and progression of human papillomavirus associated cancer of the uterine cervix are associated with alterations of Langerhans cells (LC) within the mucosal squamous epithelium. Since the transformation zone (TZ) of the cervix is the site where the majority of squamous intraepithelial lesions (SIL) are initiated, in contrast to the exocervix, we decided to investigate the influence of the local microenvironment within the TZ on the function and density of LC. We show that the TZ is associated with a significant reduction in the density of immature LC (CD1a/LAG) compared to the exocervix. In contrast, the development of SILs is attributed with a relative increased density of immature LC, compared to the TZ. Furthermore, we show that this variability in LC density is correlated with a differential expression of TNFα and MIP3α within the micro‐environment of the TZ and SILs. Both TZ and SIL epithelium‐derived LC, in the presence of allogeneic PBMC, induced lower levels of proliferation and IL2 production and higher levels of the immunosuppressive cytokine IL10 in comparison to the exocervix. Nevertheless, the epithelium‐derived LC in SILs exhibits a reduction in their functional activity, relative to the TZ. Together our studies suggest that the immunosurveillance within the epithelium of the TZ may be intrinsically perturbed due to the altered expression of chemokines/cytokines and the concomitant diminished density of LC. Furthermore, following HPV infection and the development of SILs, the function of LC may be further incapacitated by viral associated mechanisms.

Cytokine expression in squamous intraepithelial lesions of the uterine cervix: implications for the generation of local immunosuppression

We have addressed the notion that the progression of cancer of the uterine cervix is associated with a preferential constraint on the development of a type 1 cellular mediated response, which is necessary to efficiently eliminate (pre)neoplastic cells. Based on the importance of cytokines in the regulation of an appropriate immune response, we have evaluated the expression of IL‐12p40, IL‐10 and transforming growth factor‐beta 1 (TGF‐β1). Using reverse transcriptase‐polymerase chain reaction (RT‐PCR), the expression of these three cytokines was evaluated in both low‐grade (LG) and high‐grade (HG) cervical squamous intraepithelial lesions (SIL) and in normal exocervix and transformation zone biopsies. Our results show that the average level of IL‐12 increases within both the LG and HG SIL, compared with both control groups. Interestingly, the percentage of HG SIL expressing IL‐12p40 was lower compared with LG SIL. In contrast, the expression of IL‐10 increased in parallel with the severity of the lesion to a maximal level in HG SIL. Using immunohistochemistry, we ascertained the presence of IL‐12 protein in SIL and IL‐10 protein in the transformation zone and SIL biopsies. Both IL‐12‐ and IL‐10‐producing cells were localized in the stroma, not within the SIL. Furthermore, in this study we also observed that the region of the cervix the most sensitive to lesion development, the transformation zone, was associated with higher average levels of the immunosuppressive cytokines IL‐10 and TGF‐β1.

The Neurohormonal Thymic Microenvironment: Immunocytochemical Evidence that Thymic Nurse Cells Are Neuroendocrine Cells

Thymic neuroendocrine cells were identified by immunofluorescence in the murine thymus through the use of monoclonal antibody A2B5, and specific polyclonal antisera against neurophysin (NP), oxytocin (OT) and arginine vasopressin (AVP). Two reactive regions were clearly identified: the subcapsular cortex and the medulla. A close correspondence was observed between A2B5-reactive and NP-immunoreactive cells in the medulla. An important epithelial population of the subcapsular cortex, the thymic nurse cells (TNCs), were found to be A2B5-positive and to contain immunoreactive NP, OT and AVP. The neuroendocrine nature of TNCs was further substantiated by their high reactivity with an antiserum against neuron-specific enolase. These observations demonstrate the presence in the thymus gland of an original neuroendocrine microenvironment which could be of functional importance in the mediation of central influences upon T lymphocyte differentiation.

The cross-talk between dendritic and regulatory T cells: good or evil?

Immune responses against pathogens require fine regulation to avoid excessive inflammation, which could be harmful to the host. Moreover, the immune system must be tolerant to nonpathogenic antigens to prevent allergy, autoimmunity, and transplant rejection. There is accumulating evidence that interactions between dendritic cells (DC) and regulatory T (Treg) cells play a crucial role in the balance between immune response and tolerance. Communications between these cells are complex, bidirectional, and mediated by soluble or cell surface molecules. The maturation status of DC, which may be influenced by different microenvironmental factors, is considered as an important checkpoint for the induction of peripheral tolerance through modifications of the activation status of T cells. Moreover, several lines of experimental evidence suggest that different subsets or the functional status of DC are also involved in the promotion of Treg cell differentiation. A better knowledge of the regulatory mechanisms of the immune response induced or inhibited by DC via their interactions with Treg cells could be relevant for the development of new, immunotherapeutic approaches.

E-cadherin-dependent adhesion of dendritic and Langerhans cells to keratinocytes is defective in cervical human papillomavirus-associated (pre)neoplastic lesions

Although human papillomavirus (HPV) DNA is detected in the majority of squamous intraepithelial lesions (SILs) and squamous cell carcinomas (SCCs) of the uterine cervix, the persistence and progression of cervical lesions suggest that viral antigens are not adequately presented to the immune system. This hypothesis is reinforced by the observation that most SILs show quantitative and functional alterations of Langerhans cells (LCs). The aim of this study was to determine whether modulation of E‐cadherin‐mediated homophilic and heterotypic interactions between keratinocytes and LCs is involved in these abnormalities of LCs in (pre)neoplastic cervical epithelium. Cell membrane expression of E‐cadherin and the density of CD1a+ LCs were low in the epithelium of SILs and SCC biopsy specimens, compared with normal exocervical epithelium. Dendritic cells (DCs) and LCs generated in vitro were randomly distributed throughout the full thickness of organotypic cultures of E‐cadherin− HPV‐transformed cells. In contrast, these cells rapidly adhered to the keratinocyte cell layers when HPV‐transformed cells transfected with E‐cadherin were used. These data suggest that the E‐cadherin‐mediated contact between keratinocytes and LCs is potentially important for initiating or maintaining the immune response during chronic HPV infection. Copyright

Innate lymphocyte and dendritic cell cross-talk: a key factor in the regulation of the immune response

Dendritic cells (DC) are specialized in the presentation of antigens and the initiation of specific immune responses. They have been involved recently in supporting innate immunity by interacting with various innate lymphocytes, such as natural killer (NK), NK T or T cell receptor (TCR)‐γδ cells. The functional links between innate lymphocytes and DC have been investigated widely and different studies demonstrated that reciprocal activations follow on from NK/DC interactions. The cross‐talk between innate cells and DC which leads to innate lymphocyte activation and DC maturation was found to be multi‐directional, involving not only cell–cell contacts but also soluble factors. The final outcome of these cellular interactions may have a dramatic impact on the quality and strength of the down‐stream immune responses, mainly in the context of early responses to tumour cells and infectious agents. Interestingly, DC, NK and TCR‐γδ cells also share similar functions, such as antigen uptake and presentation, as well as cytotoxic and tumoricidal activity. In addition, NK and NK T cells have the ability to kill DC. This review will focus upon the different aspects of the cross‐talk between DC and innate lymphocytes and its key role in all the steps of the immune response. These cellular interactions may be particularly critical in situations where immune surveillance requires efficient early innate responses.

Silencing of E7 oncogene restores functional E-cadherin expression in human papillomavirus 16-transformed keratinocytes

Human papillomavirus (HPV) infection, particularly type 16, is causally associated with cancer of the uterine cervix. The persistence or progression of cervical lesions suggests that viral antigens are not adequately presented to the immune system. This hypothesis is reinforced by the observation that most squamous intra-epithelial lesions show quantitative and functional alterations of Langerhans cells (LCs). Moreover, E-cadherin-dependent adhesion of LC to keratinocytes (KCs) is defective in cervical HPV16-associated (pre)neoplastic lesions. The possible role of viral oncoprotein E7 in the reduced levels of cell surface E-cadherin was investigated by silencing HPV16 E7 by RNA interference (siRNA). This treatment induced an increased cell surface E-cadherin expression in HPV16-positive KC and a significant adhesion of LC to these squamous cells. The E-cadherin re-expression following HPV16 E7 silencing was associated with increased detection levels of retinoblastoma protein and the activating protein (AP)-2alpha transcription factor. These data suggest that HPV16 E7-induced alterations of LC/KC adhesion may play a role in the defective immune response during cervical carcinogenesis.

DNA methylation and cancer diagnosis: new methods and applications.

Methylation of cytosines in cytosine–guanine (CpG) dinucleotides is one of the most important epigenetic alterations in animals. The presence of methylcytosine in the promoter of specific genes has profound consequences on local chromatin structure and on the regulation of gene expression. Changes in DNA methylation play a central role in carcinogenesis. Hypermethylation and consecutive transcriptional silencing of tumor-suppressor genes has been documented in numerous cancers. The identification of target genes silenced by this modification has a great impact on diagnosis, classification, definition of risk groups and prognosis of cancer patients. Here we outline genome-wide techniques aiming at the identification of relevant methylated promoters. Methods and applications allowing clinicians to monitor the methylation of target genes will be also reviewed.