Influence of Polydatin on the Tumor Microenvironment In Vitro: Studies with a Colon Cancer Cell Model.

The tumor microenvironment of colon carcinoma, the site at which tumor cells and the host immune system interact, is influenced by signals from tumor cells, immunocompetent cells, and bacterial components, including LPS. A large amount of LPS is available in the colon, and this could promote inflammation and metastasis by enhancing tumor cell adhesion to the endothelium. Polydatin (PD), the 3-ß-D-glucoside of trans-resveratrol, is a polyphenol with anti-cancer, anti-inflammatory, and immunoregulatory effects. This study was designed to explore whether PD is able to produce antiproliferative effects on three colon cancer lines, to reduce the expression of adhesion molecules that are upregulated by LPS on endothelial cells, and to decrease the proinflammatory cytokines released in culture supernatants. Actually, we investigated the effects of PD on tumor growth in a coculture model with human mononuclear cells (MNCs) that mimics, at least in part, an in vitro tumor microenvironment. The results showed that PD alone or in combination with MNC exerts antiproliferative and proapoptotic effects on cancer cells, inhibits the production of the immunosuppressive cytokine IL-10 and of the proinflammatory cytokines upregulated by LPS, and reduces E-selectin and VCAM-1 on endothelial cells. These data provide preclinical support to the hypothesis that PD could be of potential benefit as a therapeutic adjuvant in colon cancer treatment and prevention.

How Human Papillomavirus Replication and Immune Evasion Strategies Take Advantage of the Host DNA Damage Repair Machinery.

The DNA damage response (DDR) is a complex signalling network activated when DNA is altered by intrinsic or extrinsic agents. DDR plays important roles in genome stability and cell cycle regulation, as well as in tumour transformation. Viruses have evolved successful life cycle strategies in order to ensure a chronic persistence in the host, virtually avoiding systemic sequelae and death. This process promotes the periodic shedding of large amounts of infectious particles to maintain a virus reservoir in individual hosts, while allowing virus spreading within the community. To achieve such a successful lifestyle, the human papilloma virus (HPV) needs to escape the host defence systems. The key to understanding how this is achieved is in the virus replication process that provides by itself an evasion mechanism by inhibiting and delaying the host immune response against the viral infection. Numerous studies have demonstrated that HPV exploits both the ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia and rad3-related (ATR) DDR pathways to replicate its genome and maintain a persistent infection by downregulating the innate and cell-mediated immunity. This review outlines how HPV interacts with the ATM- and ATR-dependent DDR machinery during the viral life cycle to create an environment favourable to viral replication, and how the interaction with the signal transducers and activators of transcription (STAT) protein family and the deregulation of the Janus kinase (JAK)-STAT pathways may impact the expression of interferon-inducible genes and the innate immune responses.

Downregulation of proinflammatory cytokines in HTLV-1-infected T cells by Resveratrol.

BACKGROUND: Human T-cell leukemia virus (HTLV-1) is a lymphotropic retrovirus associated to adult T cell leukemia (ATL) and to non-neoplastic inflammatory conditions affecting the central nervous system, lung or skin. The inflammatory disorders associated to HTLV-1 are mediated by different proinflammatory cytokines as IL-1α, IL-6, TNF-α. The release and the role of IL-17 is still debated. Aims of this study were to analyze IL-17 induction by HTLV-1 infection and to determine whether resveratrol (RES) is able to down regulate the pathway of cytokines production either in HTLV-1 chronically infected MT-2 cell line or in human CD4+ cells infected in vitro with HTLV-1. METHODS: MT-2 and HTLV-1 infected CD4+ cells were analyzed for proinflammatory cytokine production before or after RES treatment. The concentrations of IL-17, IL-1α, IL-6, and TNF-α were measured in cell culture supernatants by ELISA and SearchLight™ technology. The IL-17 mRNA expression was evaluated by RT-PCR. NF-kB activation was detected by non-radioactive, Electro Mobility Shift Assay (EMSA). HTLV-1 RNA expression was detected by Real-time-PCR (RQ-PCR). RESULTS: We found that RES is capable of inducing a dose-dependent inhibition of IL-1α, IL-6 and TNF-α production in vitro and can down regulate the expression of IL-17 at both mRNA and protein levels in HTLV-1 infected cells. This effect was associated with a dose-dependent inhibition of the of the nuclear factor kappa-B (NF-kB) activity. Conversely, RES did not apparently affect HTLV-1 proliferation. CONCLUSIONS: These results support the anti-inflammatory properties of RES, suggesting that it might be a useful therapeutic agent for the treatment of HTLV-1 related inflammatory diseases.

Activation of DNA Damage Response Induced by the Kaposi's Sarcoma-Associated Herpes Virus.

The human herpes virus 8 (HHV-8), also known as Kaposi sarcoma-associated herpes virus (KSHV), can infect endothelial cells often leading to cell transformation and to the development of tumors, namely Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and the plasmablastic variant of multicentric Castleman's disease. KSHV is prevalent in areas such as sub-Saharan Africa and the Mediterranean region presenting distinct genotypes, which appear to be associated with differences in disease manifestation, according to geographical areas. In infected cells, KSHV persists in a latent episomal form. However, in a limited number of cells, it undergoes spontaneous lytic reactivation to ensure the production of new virions. During both the latent and the lytic cycle, KSHV is programmed to express genes which selectively modulate the DNA damage response (DDR) through the activation of the ataxia telangiectasia mutated (ATM) pathway and by phosphorylating factors associated with the DDR, including the major tumor suppressor protein p53 tumor suppressor p53. This review will focus on the interplay between the KSHV and the DDR response pathway throughout the viral lifecycle, exploring the putative molecular mechanism/s that may contribute to malignant transformation of host cells.

Analysis of the ORFK1 hypervariable regions reveal distinct HHV-8 clustering in Kaposi's sarcoma and non-Kaposi's cases.

BACKGROUND: Classical Kaposi's Sarcoma (cKS) is a rare vascular tumor, which develops in subjects infected with Human Herpesvirus-8 (HHV-8). Beside the host predisposing factors, viral genetic variants might possibly be related to disease development. The aim of this study was to identify HHV-8 variants in patients with cKS or in HHV-8 infected subjects either asymptomatic or with cKS-unrelated cutaneous lymphoproliferative disorders. METHODS: The VR1 and VR2 regions of the ORF K1 sequence were analyzed in samples (peripheral blood and/or lesional tissue) collected between 2000 and 2010 from 27 subjects with HHV-8 infection, established by the presence of anti-HHV-8 antibodies. On the basis of viral genotyping, a phylogenetic analysis and a time-scaled evaluation were performed. RESULTS: Two main clades of HHV-8, corresponding to A and C subtypes, were identified. Moreover, for each subtype, two main clusters were found distinctively associated to cKS or non-cKS subjects. Selective pressure analysis showed twelve sites of the K1 coding gene (VR1 and VR2 regions) under positive selective pressure and one site under negative pressure. CONCLUSION: Thus, present data suggest that HHV-8 genetic variants may influence the susceptibility to cKS in individuals with HHV-8 infection.

Clinical-epidemiological features of contact dermatitis in rural and urban communities in northern Ethiopia: correlation with environmental or occupational exposure.

BACKGROUND: The widespread diffusion of low-quality products as well as the local cultural habits could be a relevant cause of allergic diseases in developing countries. In the present observational study, we explored the prevalence of allergic contact dermatitis in both rural and urban settings in northern Ethiopia, where skin diseases represent a frequent cause of morbidity. Clinical features and specific reactivities in association with environmental or occupational exposure were investigated. PATIENTS AND METHODS: We patch tested 480 consecutive patients, visited at the Mekele IDC, exhibiting symptoms of contact dermatitis. A detailed medical history of each patient was collected. RESULTS: A positive patch-test response was observed in 50% of subjects; nickel was the most frequent sensitizer (26.2%), followed by p-tert-butylphenol formaldehyde resin (10%), fragrance mix (7.1%), potassium dichromate (5.4%), cobalt chloride (4.6%), disperse blue (2.3%), and p-phenylenediamine (1.7%). Gender-related differences were analyzed for single allergen. Eczema represented the most common manifestation, affecting the head and neck as primary skin areas. While reactivity to nickel interested almost all the occupational categories, sensitization to other allergens could be ascribed to working habits or environmental exposure. CONCLUSIONS: The results gathered from this study, the first one conducted within the Tigray region in Ethiopia, confirm the need to take appropriate measures to limit the nickel rate in metal objects and may be useful to design allergenic series suitable for patch testing in those geographical settings.

Evaluation of antigen specific recognition and cell mediated cytotoxicity by a modified lysispot assay in a rat colon carcinoma model.

BACKGROUND: Antigen-specific CD8+ cytotoxic T lymphocytes represent potent effector cells of the adaptive immune response against viruses as well as tumours. Therefore assays capable at exploring the generation and function of cytotoxic T lymphocytes represent an important objective for both clinical and experimental settings. METHODS: Here we show a simple and reproducible assay for the evaluation of antigen-specific CD8+ cytotoxic T lymphocytes based on a LysiSpot technique for the simultaneous determination of antigen-specific IFN-γ production and assessment of tumor cytolysis. The assay was developed within an experimental model of colorectal carcinoma, induced by the colorectal tumor cell line DHD-K12 that induces tumors in BDIX rats and, in turn, elicits a tumor- specific immune response. RESULTS: Using DHD-K12 cells transfected to express Escherichia coli ß-galactosidase as target cells, and by the fine setting of spot colours detection, we have developed an in vitro assay that allows the recognition of cytotoxic T lymphocytes induced in BDIX rats as well as the assessment of anti-tumour cytotoxicity. The method highlighted that in the present experimental model the tumour antigen-specific immune response was bound to killing target cells in the proportion of 55%, while 45% of activated cells were not cytotoxic but released IFN-γ. Moreover in this model by an ELISPOT assay we demonstrated the specific recognition of a nonapeptide epitope called CSH-275 constitutionally express in DHD-K12 cells. CONCLUSIONS: The assay proved to be highly sensitive and specific, detecting even low frequencies of cytotoxic/activated cells and providing the evaluation of cytokine-expressing T cells as well as the extent of cytotoxicity against the target cells as independent functions. This assay may represent an important tool to be adopted in experimental settings including the development of vaccines or immune therapeutic strategies.

Underestimated clinical features of postadolescent acne.

BACKGROUND: Postadolescent acne is usually described as an inflammatory, mild-to-moderate dermatosis, frequently involving the lower third of the face, the jawline, and the neck. However, we have also frequently observed a clinical form predominantly characterized by retention lesions (microcomedones and macrocomedones), with few inflammatory lesions (comedonal postadolescent acne [CPAA]), which appears significantly correlated with cigarette smoking. OBJECTIVE: We sought to investigate the clinical features of postadolescent acne in a group of female patients affected by acne and its relationship with cigarette smoking. METHODS: A total of 226 women with acne (25-50 years) attending our department were examined by a team of 3 dermatologists, to assess the age of onset of the disease, and the number, type, and distribution of acne lesions. RESULTS: In all, 192 of 226 patients (85.0%) were classified as having CPAA and 34 as having papulopustular postadolescent acne. A smoking habit was confirmed in 150 of 226 (66.3%). Remarkably, 72.9% of patients with CPAA were smokers as compared with only 29.4% of those with papulopustular postadolescent acne (P < .0001). LIMITATIONS: Possible limitations are related to geographic area or to the prevalence of darker skin types (III and IV) (data about skin types have not been collected). Other possible aggravating factors (ie, stress and diet) have not been investigated. CONCLUSIONS: According to our results, CPAA appears as the most frequent clinical form of postadolescent acne and seems to be strictly correlated with cigarette smoking.

Immobilized HIV-1 Tat protein promotes gene transfer via a transactivation-independent mechanism which requires binding of Tat to viral particles.

BACKGROUND: Retroviral transduction of cells is improved upon virus adsorption onto immobilized fibronectin (FN) fragments. Because HIV-1 Tat possesses the same functional domains that lead to increased transduction efficiency in FN by colocalization of bound virus and cells, we hypothesized that Tat could enhance gene transfer by a similar mechanism. METHODS: Single-cycle replication retro- or lentivirus carrying green fluorescent protein or cloramphenicol acetyltransferase as reporter genes were added to wells coated with Tat or Tat peptides. Wells were extensively washed to remove unbound virus and levels of transduction were detected by measuring reporter gene expression. Virus adsorption to immobilized Tat was measured using a p24 antigen capture assay. RESULTS: Immobilized Tat efficiently binds retro- and lentiviral particles and mediates virus transmission at virus input doses that were otherwise unable to transduce susceptible cells. Virus adsorption to Tat is not mediated by envelope glycoprotein (Env) because immobilized Tat binds and retains vesicular stomatitis virus G (VSV-G) pseudotypes as well as envelope-free particles. HIV-1 Env or VSV-G are required for Tat-assisted transduction, which is abrogated by an antibody blocking the HIV-1 Env-CD4 interaction. Tat-assisted transduction is mediated by the cysteine-rich region of Tat, which is known to be essential for Tat transactivation activity. However, Tat transactivation is not required for Tat-assisted transduction, as indicated by the enhancement of transduction by transactivation-silent Tat mutants. CONCLUSIONS: Immobilized Tat promotes virus transduction by a transactiva- tion-independent mechanism, which requires binding of virus to Tat. Recombinant Tat or Tat fragments provide a new method to increase efficiency of retro- and lentiviral based gene transfer and gene therapy.

Nickel, palladium and rhodium induced IFN-gamma and IL-10 production as assessed by in vitro ELISpot-analysis in contact dermatitis patients.

BACKGROUND: Recent attempts to diminish nickel use in most industrial products have led to an increasing utilization of alternative metal compounds for destinations such as the alloys used in orthopaedics, jewellery and dentistry. The present study was undertaken with the aim to evaluate the potential for an allergic response to nickel, palladium and rhodium on the basis of antigen-specific induction of inflammatory/regulatory cytokines, and to characterize, according to the cytokine profiles, the nature of simultaneous positive patch tests elicited in vivo. Peripheral blood mononuclear cells (PBMC) from 40 patients with different patch test results were kept in short term cultures in the presence of optimized concentrations of NiSO4 x 6H2O, PdCl2 and Rh(CH3COO)2. The production of IFN-gamma and IL-10 elicited by metal compounds were analyzed by the ELISpot assay. RESULTS: We found a specific IFN-gamma response by PBMC upon in vitro stimulation with nickel or palladium in well recognized allergic individuals. All controls with a negative patch test to a metal salt showed an in vitro IL-10 response and not IFN-gamma production when challenged with the same compound. Interestingly, all subjects with positive patch test to both nickel and palladium (group 3) showed an in vitro response characterized by the release of IFN-gamma after nickel stimulation and production of IL-10 in response to palladium. CONCLUSION: These results strongly suggest that the different cytokine profiles elicited in vitro reflect different immune responses which may lead to the control of the allergic responses or to symptomatic allergic contact dermatitis. The development of sensitive and specific in vitro assays based on the determination of the cytokine profiles in response to contact allergens may have important diagnostic and prognostic implications and may prove extremely useful in complementing the diagnostic limits of traditional patch testing.

Effective therapy with anti-TNF-alpha in patients with psoriatic arthritis is associated with decreased levels of metalloproteinases and angiogenic cytokines in the sera and skin lesions.

Tumor necrosis factor-alpha (TNF-alpha) plays a central role in sustaining the inflammatory process in the skin as well as in the joints of patients with psoriasis and psoriatic arthritis. In fact, biological therapies based on monoclonal antibodies against TNF-alpha have been proven to be effective on both the arthropathy and the cutaneous symptoms of the disease. Among the several effects produced by TNF-alpha on keratinocytes there is the induction of expression of MMP-9, a matrix metalloproteinase (MMP) produced mainly by monocytes and macrophages. In this article we refer to the results of a study on the behavior of MMP-9 in the sera and in the lesional skin in association with effective therapy with infliximab. Measurements of TNF-alpha, MMP-2, vascular endothelial growth factor (VEGF), and E-selectin were also performed in the same samples. Eleven psoriatic patients included in a therapeutic protocol based on the administration of infliximab monotherapy were collected before treatment and after 6 and 12 weeks of therapy. Significant decrease of MMP-9 and MMP-2 levels in the sera was associated with clinical improvement and with the decrease of TNF-alpha, VEGF, and E-selectin, angiogenic molecules already known to be implicated in the clinical expression of psoriasis. The clinical amelioration of the cutaneous expression of psoriasis was significantly associated with the decrease of MMP-9, TNF-alpha, and E-selectin levels, spontaneously released by lesional biopsy samples before and after therapy, measured in the culture supernatants by immunoenzymatic assays. In addition, significant correlations were found between the clinical score and TNF-alpha, MMP-9, and E-selectin lesional production. MMP-9 levels were significantly correlated with those of TNF-alpha. Our findings show the existence of a direct relationship between MMP-9 and TNF-alpha production, strongly suggesting that MMP-9 may play a key role in the skin inflammatory process in psoriasis, while a different role may be attributed to MMP-2.

Human herpesvirus 8 infection in patients with cutaneous lymphoproliferative diseases.

OBJECTIVE: To investigate the prevalence of human herpesvirus 8 (HHV-8; Kaposi sarcoma-associated herpesvirus) infection in patients with lymphoproliferative skin diseases such as large-plaque parapsoriasis (LPP) and mycosis fungoides compared with inflammatory cutaneous conditions or healthy control subjects. DESIGN: A survey study was undertaken in 123 subjects with various clinical conditions. SETTING: All patients had been seen in the Dermatology Department of the San Gallicano Dermatology Institute, Rome, Italy, in the last 2 years. PATIENTS: Forty-five patients with inflammatory or autoimmune cutaneous diseases, 50 healthy control subjects, 10 patients with LPP, 12 patients with mycosis fungoides, and 6 patients with classic Kaposi sarcoma were included in the study. MAIN OUTCOME MEASURES: The prevalence of HHV-8 infection was investigated with serologic studies using the gold standard assay based on body cavity-based B-cell lymphoma-1 cells latently infected with HHV-8. The presence of HHV-8 conserved sequence, corresponding to open reading frame 26, was also assessed in the peripheral blood and lesion tissue samples from patients with lymphoproliferative cutaneous diseases with nested polymerase chain reaction. The presence and distribution of cell types infected with HHV-8 in the lesion tissues was determined with immunohistochemical staining with the monoclonal antibody directed against the latent nuclear antigen-1 of HHV-8 encoded by open reading frame 73. RESULTS: In healthy control subjects and patients with inflammatory skin diseases, 13.9% were found to have antibody against HHV-8, consistent with the seroprevalence population in Italy. A highly significant association of HHV-8 infection and LPP was found (100%) compared with mycosis fungoides (25%). The peripheral blood mononuclear cells in 8 of 10 patients with LPP were found to harbor viral sequences at nested polymerase chain reaction, whereas none of them had a detectable serum viral load. All LPP lesion tissue samples were positive for HHV-8-encoded open reading frame 26, and the presence of HHV-8-infected cells was confirmed by immunohistochemistry profiles performed on paraffin-embedded tissues from 4 of 10 patients. The positive cell types included endothelial cells and the infiltrating dermal lymphocytes, characteristic hallmarks of LPP. Analysis of T-cell receptor gamma chain rearrangements in lesion tissue from our patients confirmed the lack of a significant association between T-cell clonality and LPP. CONCLUSION: These data suggest that HHV-8 may play a role in the onset of LPP, a disease whose cause and evolution are still undefined and which has often been considered the early stage of mycosis fungoides.

Zoledronic-acid-induced circulating level modifications of angiogenic factors, metalloproteinases and proinflammatory cytokines in metastatic breast cancer patients.

BACKGROUND: To evaluate the modifications of circulating angiogenic factors, metalloproteinases and acute-phase cytokines after the first single zoledronic acid (ZA) intravenous infusion. EXPERIMENTAL DESIGN: Eighteen consecutive breast cancer patients with bone metastases were evaluated for circulating levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), metalloproteinase 1 (MMP-1), metalloproteinase 2 (MMP-2), interleukins 1beta, 6 and 8 (IL-1beta, IL-6, IL-8), interferon gamma, tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta1 just before and 2 and 7 days after ZA infusion. RESULTS: The MMP-2 basal value showed a statistically significant decrease 48 h after ZA (p = 0.01), being at 7 days higher than the day 2 value (p = 0.03). The VEGF basal value showed a statistically significant decrease 48 h after ZA infusion (p = 0.03), increasing above the basal level at 7 days (p = 0.07). The bFGF basal level almost significantly decreased 2 days after infusion (p = 0.06), being at 7 days higher than the basal value (p = 0.09). Comparing the day 2 values with basal ones, the linear regression model showed a significant positive correlation between IL-8 and bFGF (p = 0.02), IL-8 and TNF-alpha (p < 0.0001), bFGF and TNF-alpha (p = 0.01), MMP-1 and TNF-alpha (p = 0.02). CONCLUSIONS: ZA could exert an antiangiogenic activity and inhibition of tumor cell bone invasiveness by a transient reduction of VEGF, bFGF and MMP-2 circulating levels after infusion.

Antigen specific cytokine response in pediatric patients with atopic dermatitis.

The physiopathology of atopic dermatitis (AD) has still to be elucidated. T effector cells with cutaneous homing receptors or T-cell derived cytokines have been assumed to be implicated in the pathogenetic mechanisms in AD and to be responsible for the different immunologic responses of patients. In fact, the large majority of AD patients display high IgE levels while others do not develop an abnormal IgE response. Although, there are not significant clinical features characterizing the two different groups, patients with normal IgE belong to a younger age range, raising the possibility that the hypothesized dichotomy of AD might be due to age. In the present study we included 172 outpatient children attending the Pediatric Department of our institution. Serum IgE levels and percentages of peripheral T lymphocytes expressing the cutaneous homing antigen (CLA) were evaluated and results were analyzed in relation to the activity of the disease (SCORAD index) or age. In the overall patients, the IgE levels increased significantly with age (0-1 yr: 19.50 IU/ml; 1-3 yr: 62.0 IU/ml; 3-8 yr: 96.0 IU/ml; >8 yr: 148.5 IU/ml; p<0.001) and with the severity of the disease (SCORAD low: 46.80 IU/ml; medium: 42.90 IU/ml; high: 148.5 IU/ml; p=0.01). Percentages of CLA+ peripheral T lymphocytes also increased with age (0-1 yr: 3.3; 1-3 yr: 4.85; 3-8 yr: 10.6; >8 yr: 12.5; p<0.001), although they were not significantly different in patients with different SCORAD (p=0.89). We further investigated the cellular immune response to a specific antigen in 25 subjects, matched for age, SCORAD, and CLA+ T-cell percentages. Among them, 13 patients had casein serum specific IgE and 12 had no evidence of casein sensitization. Peripheral blood mononuclear cells were kept in short-term culture with endotoxin-free casein fractions and IFN-gamma, TNF-alpha, IL-5, IL-10 cytokine-producing cells were detected by ELISpot. Statistical analysis showed significant higher numbers of TNF-alpha- or IL-10-producing cultures (stimulation index >3) in the 'allergic' patients than in the milk tolerant subjects (p=0.01 and 0.05). The analysis of individual responses confirmed this finding but also provide evidence of a significant increase in IFN-gamma-producing cells (p=0.05) induced by casein stimulation in the group of 'non-allergic' children. Our data showed that immunologic parameters as IgE levels or CLA+ T cells in AD pediatric patients are influenced by the age, confirming that age could represent a bias in the analysis of immune response in those patients. Although, we demonstrated in children with AD the existence of different cytokine patterns of the lymphocyte response that could account for the different immunologic features between the two hypothesized forms of AD, which are not dependent on age.