Phosphorylated CXCR4 expression has a positive prognostic impact in colorectal cancer.

BACKGROUND: The CXCL12-CXCR4 chemokine axis plays an important role in cell trafficking as well as in tumor progression. In colorectal cancer (CRC), the chemokine receptor CXCR4 has been shown to be an unfavorable prognostic factor in some studies, however, the role of its activated (phosphorylated) form, pCXCR4, has not yet been evaluated. Here, we aimed to investigate the prognostic value of CXCR4 and pCXCR4 in a large cohort of CRC patients. PATIENTS AND METHODS: A tissue microarray (TMA) of 684 patient specimens of primary CRCs was analyzed by immunohistochemistry (IHC) for the expression of CXCR4 and pCXCR4 by tumor cells and tumor-infiltrating immune cells (TICs). RESULTS: The combined high expression of CXCR4 and pCXCR4 showed a favorable 5-year overall survival rate (68%; 95%CI = 59-76%) compared to tumors showing a high expression of CXCR4 only (48%; 95%CI = 41-54%). High expression of pCXCR4 was significantly associated with a favorable prognosis in a test and validation group (p = 0.015 and p = 0.0001). Moreover, we found that CRCs with a high density of pCXCR4+ tumor-infiltrating immune cells (TICs) also showed a favorable prognosis in a test and validation group (p = 0.054 and p = 0.004). Univariate Cox regression analysis for TICs revealed that a high density of pCXCR4+ TICs was a favorable prognostic marker for overall survival (HR = 0.97,95%CI = 0.96-1.00; p = 0.01). In multivariate Cox regression survival analyses a high expression of pCXCR4 in tumor cells lost its association with a better overall survival (HR = 0.99; 95%CI = 0.99-1.00, p = 0.098). CONCLUSION: Our results show that high densities of CXCR4 and pCXCR4 positive TICs are favorable prognostic factors in CRC.

[Expression of cancer testis (CT) antigens in pediatric and adolescent melanomas]. / Expression von Cancer-Testis(CT)-Antigenen in Melanomen des Kindes- und Jugendalters.

BACKGROUND: One of the main problems in the diagnostics of pediatric melanomas is the differentiation from benign dermal lesions typical for this age group, such as Spitz nevus. The biological behavior of pediatric melanomas differs considerably from that of melanomas in adults. MATERIAL AND METHODS: Cancer testis (CT) antigens are named after their typical expression pattern since they are present in various types of malignant tumors but in normal adult tissues are solely expressed in testicular germ cells. Because of this tumor-associated expression pattern, CT antigens are regarded as potential targets for vaccine-based immunotherapy of cancer and might be used as diagnostic tools in surgical pathology. In adults, melanoma is among the tumors showing a high incidence of CT antigen expression; however, while there is ample knowledge about adult melanomas, little is known about the presence of CT antigens in pediatric melanomas. Consequently, the expression of CT antigens MAGE-A1, MAGE-A4, CT7/MAGE-C1, NY-ESO-1, and GAGE was analyzed in a series of pediatric melanomas. The study was restricted to cases of metastatic disease and/or fatal outcome. A total of 12 cases were available and immunohistochemically analyzed with monoclonal antibodies (mAb). RESULTS: The expression of CT antigens was generally low and present in only 4 of 12 cases. This is in stark contrast to the expression of these antigens in adult melanomas. Moreover, the extent of expression was very limited with most cases showing only a focal CT antigen expression and only marked in very small tumor areas (<5%). CONCLUSION: Despite the low case numbers this study indicates that CT antigens are most likely not useful as diagnostic markers in pediatric melanomas or as targets for vaccine-based immunotherapy. It supports the notion that pediatric melanomas show a different biological behavior than their adult counterparts.

Cancer-testis antigen expression in triple-negative breast cancer.

BACKGROUND: cancer-testis (CT) antigens, frequently expressed in human germline cells but not in somatic tissues, may become aberrantly reexpressed in different cancer types. The aim of this study was to investigate the expression of CT antigens in breast cancer. PATIENTS AND METHODS: a total of 100 selected invasive breast cancers, including 50 estrogen receptor (ER) positive/HER2 negative and 50 triple negative (TN), were examined for NY-ESO-1 and MAGE-A expression by immunohistochemistry. RESULTS: a significantly higher expression of MAGE-A and NY-ESO-1 was detected in TN breast cancers compared with ER-positive tumors (P = 0.04). MAGE-A expression was detected in 13 (26%) TN cancers compared with 5 (10%) ER-positive tumors (P = 0.07). NY-ESO-1 expression was confirmed in nine (18%) TN tumor samples compared with two (4%) ER-positive tumors. CONCLUSIONS: MAGE-A and NY-ESO-1 CT antigens are expressed in a substantial proportion of TN breast cancers. Because of the limited therapeutic options for this group of patients, CT antigen-based vaccines might prove to be useful for patients with this phenotype of breast cancer.

Prognostic impact of the expression of putative cancer stem cell markers CD133, CD166, CD44s, EpCAM, and ALDH1 in colorectal cancer.

BACKGROUND: The aim of this study was to elucidate the prognostic impact of putative cancer stem cell markers CD133, CD166, CD44s, EpCAM, and aldehyde dehydrogenase-1 (ALDH1) in colorectal cancer. METHODS: A tissue microarray of 1420 primary colorectal cancers and 57 normal mucosa samples was immunostained for CD133, CD166, CD44s, EpCAM, and ALDH1 in addition to 101 corresponding whole tissue sections. Invasive potential of three colorectal cancer cell lines was tested. RESULTS: Differences between normal tissue and cancer were observed for all markers (P<0.001). Loss of membranous CD166 and CD44s were linked to higher pT (P=0.002, P=0.014), pN (P=0.004, P=0.002), an infiltrating growth pattern (P<0.001, P=0.002), and worse survival (P=0.015, P=0.019) in univariate analysis only. Loss of membranous EpCAM expression was also linked to higher pN (P=0.023) and infiltrating growth pattern (P=0.005). The CD44s, CD166, and EpCAM expression were lost towards the invasive front. The CD44-/CD166- cells from three colorectal cancer cell lines exhibited significantly higher invasive potential in vitro than their positive counterparts. CONCLUSIONS: Loss, rather than overexpression, of membranous CD44s, CD166, and EpCAM is linked to tumour progression. This supports the notion that the membranous evaluation of these proteins assessed by immunohistochemistry may be representative of their cell adhesion rather than their intra-cellular functions.

CD8+ lymphocytes/ tumour-budding index: an independent prognostic factor representing a 'pro-/anti-tumour' approach to tumour host interaction in colorectal cancer.

BACKGROUND: The tumour-host interaction at the invasive front of colorectal cancer, including the epithelial-mesenchymal transition and its hallmark 'tumour budding', is an important area of investigation in terms of prognosis. The aim of this study was to determine the prognostic impact of a 'pro-/anti-tumour' approach defined by an established 'pro-tumour' (tumour budding) and host-related 'anti-tumour' factor of the adaptive immunological microenvironment (CD8+ lymphocytes). METHODS: Double immunostaining for CK22/CD8 on whole tissue sections (n=279; Cohort 1) and immunohistochemistry for CD8+ using tissue microarrays (n=191; Cohort 2) was carried out. Tumour buds, CD8+ and CD8+ T-lymphocytes : tumour buds indices were evaluated per high-power field. RESULTS: In Cohort 1, a low-CD8+/ buds index was associated with lymph node metastasis (P<0.001), vascular invasion (P=0.009), worse survival in univariate (P<0.001) and multivariable (P<0.001) analysis, and furthermore in lymph node-negative patients (P=0.002). In Cohort 2, the CD8+/ buds index was associated with T stage (P<0.001), N stage (P=0.041), vascular invasion (P=0.005) and survival in patients with TNM stage II (P=0.019), stage III (P=0.004), and adjuvantly untreated (P=0.009) and treated patients (P<0.001). CONCLUSION: The CD8+ lymphocyte : tumour-budding index is an independent prognostic factor in colorectal cancer and a promising approach for a future prognostic score for patients with this disease.

Interleukin-6 affects cell death escaping mechanisms acting on Bax-Ku70-Clusterin interactions in human colon cancer progression.

Activation of pro-survival pathways and apoptotic cell death escape are considered hallmarks of oncogenic cell transformation. Tissue microenvironment strongly influences tumorigenesis, redirecting some pathways versus a persisting pro-survival state. Here, we report evidence on the role of interleukin 6 (IL-6) in affecting pro-survival pathways in colon cancer progression, modulating the expression and the molecular interactions among the pro-apoptotic factor Bax, the DNA repair proteins Ku70/86 and Clusterin isoforms. In human colorectal carcinomas (n = 50) at different stages of disease, we found an increased IL-6 production, the loss of Ku86 and Clusterin 50-55 kDa pro-apoptotic isoform. Conversely, we observed the overexpression of Bax and the 40 kDa prosurvival sClusterin (sCLU) isoform. Bax co-localized with Ku70 that was found atypically expressed in the cytoplasm of advanced stage colon cancers (Dukes'C-D; n = 22). IL-6 treatment of a colon cancer cell line, Caco-2, modulated the expression of genes involved in tumor invasion and apoptosis, as observed by microarrays. In particular, IL-6 downmodulated Bax expression at mRNA level. Concomitantly, IL-6 exposure influenced Bax also at protein level acting on the Bax-Ku70-sCLU physical interactions in the cytoplasm, by affecting the Ku70 acetylation and phosphorylation state, thus leading to the inhibition of Bax pro-apoptotic activity. In addition, we found that IL-6 treatment induced a significant downregulation of Ku86 and a strong increase of sCLU, confirming tumor biopsies data. In contrast Somatostatin treatment of Caco-2 cells was able to restore apoptosis, demonstrating that Ku70-Bax-CLU interactions could be dynamically modulated. Hence, IL-6 could favor tumor expansion, promoting cell survival and apoptosis escape throughout the different stages of tumor evolution. Uncovering the molecular mechanisms of action of these factors may offer strategies for selectively manipulate the cancer cells sensitivity to therapy.

Human bone marrow mesenchymal stem cells and chondrocytes promote and/or suppress the in vitro proliferation of lymphocytes stimulated by interleukins 2, 7 and 15.

OBJECTIVES: To investigate whether human bone marrow-derived mesenchymal stem cells (BM-MSCs) and articular chondrocytes (ACs) affect the in vitro proliferation of T lymphocytes and peripheral blood mononuclear cells (PBMCs) driven by the homeostatic interleukin (IL)2, IL7 and IL15 cytokines binding to the common cytokine receptor gamma-chain (gamma(c)) in the absence of T cell receptor (TCR) triggering. METHODS: PBMCs, total T cells and T cell subsets (CD4+ and CD8+) were stimulated with IL2, IL7 or IL15 and exposed to cultured BM-MSCs and ACs at varying cell:cell ratio either in contact or in transwell conditions. Lymphocyte proliferation was measured by (3)H-thymidine uptake or by flow cytometry of carboxyfluorescein succinimidyl ester (CFSE)-labelled lymphocytes. RESULTS: MSCs and ACs enhanced and inhibited lymphocyte proliferation depending on the extent of lymphocyte baseline proliferation and on the MSC/AC to lymphocyte ratio. Enhancement was significant on poorly proliferating lymphocytes and mostly at lower MSC/AC to lymphocyte ratio. Suppression occurred only on actively proliferating lymphocytes and at high MSC/AC to lymphocyte ratio. Neither enhancement nor inhibition required cell-cell contact. CONCLUSIONS: There is a dichotomous effect of MSCs/ACs on lymphocytes proliferating in response to the homeostatic IL2, IL7 and IL15 cytokines likely to be encountered in homeostatic and autoimmune inflammatory conditions. The effect is determined by baseline lymphocyte proliferation, cell:cell ratio and is dependent on soluble factor(s). This should be taken into account when planning cellular therapy for autoimmune disease (AD) using stromal-derived cells such as MSCs.

[Evaluation of occupational exposure to biological agents, endotoxins and PNOC in a sewage treatment plant]. / Valutazione dell'esposizione professionale ad agenti biologici, endotossine e PNOC in un impianto di depurazione.

The aim of this work has been to determine the occupational exposure to the biological agents and airborne dust in a sewage treatment plant in south Italy. The air samplings were performed in a sewage treatment plant in Calabria, in two different seasons (spring and summer) at 5 sites associated with various phases of sewage treatment process. In addition we have estimated the concentration of airborne endotoxins and PNOC (Particles Not Otherwise Classified) by using personal samplers. The results showed a significant variation in exposure to bioaerosols, endotoxins and PNOC depending on the sampling season: the PNOC concentration increase as much as the endotoxins concentration in spring and decrease in summer

Multiple mechanisms underlie defective recognition of melanoma cells cultured in three-dimensional architectures by antigen-specific cytotoxic T lymphocytes.

Cancer cells' growth in three-dimensional (3D) architectures promotes resistance to drugs, cytokines, or irradiation. We investigated effects of 3D culture as compared to monolayers (2D) on melanoma cells' recognition by tumour-associated antigen (TAA)-specific HLA-A(*)0201-restricted cytotoxic T-lymphocytes (CTL). Culture of HBL, D10 (both HLA-A(*)0201+, TAA+) and NA8 (HLA-A(*)0201+, TAA-) melanoma cells on polyHEMA-coated plates, resulted in generation of 3D multicellular tumour spheroids (MCTS). Interferon-gamma (IFN-gamma) production by HLA-A(*)0201-restricted Melan-A/MART-1(27-35) or gp 100(280-288)-specific CTL clones served as immunorecognition marker. Co-culture with melanoma MCTS, resulted in defective TAA recognition by CTL as compared to 2D as witnessed by decreased IFN-gamma production and decreased Fas Ligand, perforin and granzyme B gene expression. A multiplicity of mechanisms were potentially involved. First, MCTS per se limit CTL capacity of recognising HLA class I restricted antigens by reducing exposed cell surfaces. Second, expression of melanoma differentiation antigens is downregulated in MCTS. Third, expression of HLA class I molecules can be downregulated in melanoma MCTS, possibly due to decreased interferon-regulating factor-1 gene expression. Fourth, lactic acid production is increased in MCTS, as compared to 2D. These data suggest that melanoma cells growing in 3D, even in the absence of immune selection, feature characteristics capable of dramatically inhibiting TAA recognition by specific CTL.

Bone marrow mesenchymal stromal cells (BM-MSCs) from healthy donors and auto-immune disease patients reduce the proliferation of autologous- and allogeneic-stimulated lymphocytes in vitro.

OBJECTIVES: To investigate the ability of bone marrow (BM)-derived mesenchymal stromal cells (BM-MSCs) in suppressing the proliferation of stimulated lymphocytes across a range of conditions including autologous BM-MSCs derived from autoimmune disease (AD) patients. METHODS: In vitro cultures of BM-MSCs from healthy donors and AD patients were established and characterized by their differentiation potential into adipocytes and osteoblasts, and their fibroblast-colony-forming unit (CFU-F) ability and phenotype by flow cytometry. BM-MSCs (irradiated and non-irradiated) from healthy and AD patients were tested for their ability to suppress the in vitro proliferation of autologous and allogeneic peripheral blood mononuclear cells (PBMC) (from healthy donors and patients suffering from various ADs) stimulated with anti-CD3epsilon antibody alone or in combination with anti-CD28 antibody. The anti-proliferative effect of the BM-MSCs from healthy donors was tested also on transformed B-cell lines as a model of non-antigen-stimulated lymphocytes. RESULTS: BM-MSCs from healthy donors and AD patients reduced the proliferation of autologous and allogeneic PBMCs by up to 90% in a cell dose-dependent fashion. The immunosuppression was independent of the proliferation of the BM-MSCs and was also effective on already proliferating cells. It was independent also of the clinical activity of AD. An MSC dose-dependent pattern of suppression of proliferation was observed also with transformed B-cell lines, similar to that observed with proliferating PBMC. CONCLUSIONS: The BM-MSCs exhibit extensive anti-proliferative properties against lymphocytes under different conditions. This property might offer a form of immunomodulatory cellular therapy for AD patients if further confirmed in animal models.

[Mount Reventino greenstone: assessment of tremolite fibre dispersion in the workplace]. / Le pietre verdi del Reventino: valutazione della dispersione di fibre di tremolite nelle attività lavorative.

BACKGROUND: Mount Reventino, a massif located in the Calabria Region of Italy, has several ophiolite outcrops of greenstone. These deposits are an important economical resource in the surrounding area. Some rock layers contain tremolite, a type of asbestos fibre. OBJECTIVES: The aim of this paper was to analyze the chemical and physical structure of the outcrops of Mount Reventino, and to assess and reduce the risk to workers associated with exposure to airborne fibres. METHODS: Personal and environmental samples were collected and analysed by Scanning Electron Microscopy equipped with energy-dispersive X-ray analysis. RESULTS: The analysis of samples showed a difference in mineralogical features not only between the quarries under study, but also between the two opposite sides of the mountain. Exploitation of the quarries produces a fibre dispersion that is higher than the natural emission. Occupational exposure to asbestos fibres during greenstone transformation was confirmed by by the results of analysis of the collected samples. CONCLUSIONS: This study made it possible to identify working activities with highest exposure to asbestos and establish the correct procedures to abate fibre dispersion, in order to reduce the correlated risk. Environmental samples collected in the urban area surrounding the quarries showed that the asbestos fibre concentrations were very low, however, further studies are needed in order to confirm these findings.

Expression of MAGE-A cancer/testis antigens in esophageal squamous cell carcinomas.

BACKGROUND: Although the diagnosis and therapy of esophageal cancer have improved over the past decade, the prognosis remains dismal. Since MAGE-A cancer/testis antigens (CTA) are potential targets for immunotherapy, this study was aimed at evaluating their expression in these patients and its prognostic value. MATERIALS AND METHODS: Using 57B monoclonal antibody, MAGE-A CTA expression was analyzed in paraffin-embedded tumor specimens of 98 patients with esophageal squamous cell carcinoma or adenocarcinomas who had undergone surgical resection. For all patients, a postoperative follow-up of at least 4 years was available. The expression was quantified using a scoring system considering intensity and homogeneity of the immunostaining. The prognostic relevance of MAGE-A expression was analyzed in univariate analyses as well as Cox proportional hazard regression analysis. RESULTS: 57B positivity could be detected in 38 tumors (38.8%). Positive staining was observed in five out of 32 adenocarcinomas (15.2%) and in 33 out of 66 (50%) squamous cell carcinomas. MAGE-A expression did not correlate with the TNM classification, grading or age of the patients. Both univariate (p=0.88) and multivariate analyses (p = 0.82) revealed that MAGE-A expression lacked prognostic significance in esophageal carcinomas. CONCLUSION: MAGE-A was expressed in half of the squamous cell carcinomas of the esophagus, but rarely in adenocarcinomas. Although its immunodetection was insufficient for prognostic evaluation, the high expression rate suggests MAGE-A as a potential target for immunotherapy in the first group with the ability for pretherapeutic testing.

Follow up study on the immune response to low frequency electromagnetic fields in men and women working in a museum.

Seven women and eight men, exposed to low frequency (50 Hz) electromagnetic fields (EMFs) in a museum for 20 hours a week, were investigated in the years 1999 and 2005. During the first study, the mean EMF exposure in the working place was 1.7 microT and 1.1 microT, respectively. In the first investigation, the EMF-exposed men showed reduced blood NK lymphocytes in relation to controls, while EMF-exposed women presented reduced PHA-stimulated IFN-gamma release from peripheral blood mononuclear cells (PBMC). In the year 2005, blood cytotoxic activity, state and trait anxiety (STAI I and II, respectively) and occupational stress were also investigated. The scores of STAI I and II of the control women were slightly higher than those of the control men. EMF-exposed men showed higher occupational stress but normal immune parameters. EMF-exposed women showed, in relation to controls, lower PHA-stimulated IFN-gamma release from PBMC and reduced blood cytotoxic activity/CD45+-CD16+-56+ NK lymphocytes (but not per ml of blood). One of the women exposed to EMF, who worked a night shift, showed marked lymphopenia with very low NK lymphocytes and reduced IFN-gamma release; these immune parameters returned to normal following a change of work site. This study suggests that low frequency EMFs affect the immune functions of women more than those of men. Moreover, the determination of immune parameters seems to be a useful marker of the health effects of exposure to EMFs.

The immune response of women with prolonged exposure to electromagnetic fields produced by radiotelevision broadcasting stations.

Twelve women, five of them housewives, exposed in their residences to electromagnetic fields (EMFs)emitted by radio-television broadcasting stations for a mean period of 13 years, were investigated. The EMFs in the balconies of the homes were (mean + S.D.) 4.3 + 1.4 V/m in the year 2000 and 3.7 + 1.3 V/m in 2005, while the exposure in the nearby area was <2.0 V/m. The EMF exposed women showed in 2000 reduced blood NK lymphocytes as well as PHA stimulated PBMC proliferation and IL-2 and IFN-gamma release. In the year 2005, the EMF exposed women and 48 control women with similar ages(mean 43 years), smoking habits, atopy and social level were investigated. State (temporary) and trait(tendency of the personality) anxiety were determined by STAI I and II, respectively. Blood cytotoxic activity and lymphocyte subsets were also determined. The ratio STAI I/STAI II of the EMF exposed group was lower than that of the control group. The blood cytotoxic activity of the exposed women was lower (p<0.01), percent of B CD45+-CD19+ lymphocytes higher and percent of CD45+-CD3+-CD8+ cells lower (p<0.05). Moreover, cytotoxic activity/CD45+-CD16+-56+ NK lymphocytes of the controls was negatively correlated with STAI I and STAI II (p<0.001). In conclusion, this study demonstrates reduced blood cytotoxic activity and increased trait anxiety in relation to state anxiety in EMF exposed women. An effect of EMFs on immune functions, in part mediated by nervous mechanisms, may be hypothesized. However, the influence of lifestyle may not be excluded.

[Biological risk in the operating room: microbiological monitoring of the environment and analysis of the associated variables]. / Rischio biologico in sala operatoria: monitoraggio micro- biologico ambientale ed analisi delle variabili associate.

The authors present an environmental microbiological monitoring programme carried out over a period of 15 months in 16 operating theatres performing specific types of surgery. The levels of microbial contamination of the air and of four of the most representative surfaces of the clean area were determined at 3 different times for each theatre, both before and during surgery. For the air assessment, the results obtained with three different samplers, Sed-3 Unit, SAS and RCS, were compared. The results were on the whole acceptable, but some poor conditions were detected during the theatres in use, especially in general surgery theatres; in some of these the floors showed levels of contamination consistently exceeding the reference limits. As the monitoring programme proceeded, the microbiological quality of the air and of the surfaces in the theatres notably improved. The three air samplers showed different conditions expressed with units of measure not always readily comparable. For active samplers, the bacterial load determined by RCS, although less variable, were always higher (even 2-3 fold) than those obtained with the SAS. Passive sampling takes longer but determines the real risk of infection for the patients; contemporary determination of the fall-out and the CFU/m3 helps to identify the occupational risks. Since the limit values established by the ISPESL guidelines for the operating theatres have been defined only for active samplers, there is urgent need for more exhaustive national guidelines to define similar values also for passive sampling. The Authors conclude stressing the importance of promoting continuing information-education programmes to heighten the awareness of all those involved in operating theatre activities.

[Preventive measures suggested to protect health workers from the risk due to a potential exposure to SARS agents]. / Misure di prevenzione e protezione per la potenziale esposizione all'agente biologico responsabile della SARS in ambiente ospedaliero.

The Authors describe the personal and environmental preventive measures suggested to protect health workers from the risks due to a potential exposure to SARS agents. The Authors stress the need that workers are allowed to wear individual protective disposable complying with technical regulations in order to be assured the best protection.

Encapsulation into sterically stabilised liposomes enhances the immunogenicity of melanoma-associated Melan-A/MART-1 epitopes.

Tumour-associated antigens (TAA)-specific vaccination requires highly immunogenic reagents capable of inducing cytotoxic T cells (CTL). Soluble peptides are currently used in clinical applications despite an acknowledged poor immunogenicity. Encapsulation into liposomes has been suggested to improve the immunogenicity of discrete antigen formulations. We comparatively evaluated the capacity of HLA-A2.1 restricted Melan-A/MART-1 epitopes in soluble form (S) or following inclusion into sterically stabilised liposomes (SSL) to be recognised by specific CTL, to stimulate their proliferation and to induce them in healthy donors' peripheral blood mononuclear cells (PBMC), as well as in melanoma-derived tumour-infiltrating lymphocytes (TIL). HLA-A2.1(+), Melan-A/MART-1-NA-8 melanoma cells served as targets of specific CTL in 51Cr release assays upon pulsing by untreated or human plasma-treated soluble or SSL-encapsulated Melan-A/MART-1 27-35 (M27-35) or 26-35 (M26-35) epitopes. These reagents were also used to stimulate CTL proliferation, measured as 3H-thymidine incorporation, in the presence of immature dendritic cells (iDC), as antigen-presenting cells (APC). Induction of specific CTL upon stimulation with soluble or SSL-encapsulated peptides was attempted in healthy donors' PBMC or melanoma-derived TIL, and monitored by 51Cr release assays and tetramer staining. Na-8 cells pulsing with SSL M27-35 resulted in a five-fold more effective killing by specific CTL as compared with equal amounts of S M27-35. Encapsulation into SSL also provided a partial (50%) protection of M27-35 from plasma hydrolysis. No specific advantages regarding M26-35 were detectable in these assays. However, at low epitope concentrations (

Pesticides re-entry dermal exposure of workers in greenhouses.

This research has the aim to evaluate the risk of pesticide dermal exposure for workers in greenhouses. We considered the following crops: tomato, cucumber and strawberry, largely spread in Bracciano lake district. The pesticides monitored were: tetradifon on strawberry: metalaxyl, azoxystrobin and fenarimol on cucumber; acrinathrin, azoxystrobin and chlorpyrifos ethyl on tomato. The dermal exposure was evaluated by Dislodgeable Foliar Residue (DFR) measurements employing transfer coefficients got from literature. For risk evaluation, we have compared the dermal exposures with Acceptable Operator Exposure Levels (AOEL). The re-entry time were obtained intercepting the dose decay curves with AOEL values. The re-entry times result higher than two days in the cases of chlorpyrifos on tomato (re-entry time: 3 days), azoxystrobin on tomato (4 days), and tetradifon on strawberry (8 days). The need of measuring specific transfer coefficients is pointed out.

Phase I/II clinical trial of a nonreplicative vaccinia virus expressing multiple HLA-A0201-restricted tumor-associated epitopes and costimulatory molecules in metastatic melanoma patients.

We performed a phase I/II clinical trial in metastatic melanoma patients with an ultraviolet (UV)-inactivated nonreplicating recombinant vaccinia virus enabling the expression, from a single construct, of endoplasmic reticulum-targeted HLA-A0201-restricted Melan-A/MART-1(27-35), gp100(280-288), and tyrosinase(1-9) epitopes, together with CD80 and CD86 costimulatory proteins. Corresponding soluble peptides were used to boost responses and granulocyte-macrophage colony-stimulating factor was used as systemic adjuvant. Safety and immunogenicity, as monitored with in vitro-restimulated peripheral blood mononuclear cells by cytotoxic T lymphocyte precursor (CTLp) frequency analysis and tetramer staining, were specifically addressed. Of 20 patients entering the protocol, 2 had to withdraw because of rapidly progressing disease. Immune responses were evaluated in 18 patients (stage III, n = 5; stage IV, n = 13) and increases in specific CTLp frequencies were observed in 15. In 16 patients responsiveness against all 3 antigens could be analyzed: 7 (43%), including all stage III cases, showed evidence of induction of CTLs specific for the three epitopes, and 2 (12%) and 4 (25%), respectively, showed reactivity against two or one tumor-associated antigen. In three stage IV patients no specific CTL reactivity could be induced. Increases in CTLp frequency were detected mostly after viral vaccine injections. However, in a majority of patients final CTLp levels were comparable to initial levels. Tetramer characterization of Melan-A/MART-1(27-35)-specific CTLs during the protocol also suggested preferential expansion after recombinant virus administration. Vector-specific humoral responses, frequently undetectable in stage IV patients, did not appear to prevent tumor-associated antigen-specific CTL induction. Aside from a single occurrence of transient grade 3 leukopenia, no major clinical toxicity was reported. Seventeen of 18 patients completed the 3-month trial (one patient died before the last delayed-type hypersensitivity test). Three displayed regression of individual metastases, seven had stable disease, and progressive disease was observed in seven patients. This is the first report on the administration of a UV-inactivated recombinant vaccinia virus coexpressing five transgenes in cancer patients. The results described here, in terms of safety and immunogenicity, support the use of this reagent in active specific immunotherapy.