F4-Neuroprostane Effects on Human Sperm.

Swim-up selected human sperm were incubated with 7 ng F4-neuroprostanes (F4-NeuroPs) for 2 and 4 h. Sperm motility and membrane mitochondrial potential (MMP) were evaluated. The percentage of reacted acrosome was assessed by pisum sativum agglutinin (PSA). Chromatin integrity was detected using the acridine orange (AO) assay and localization of the ryanodine receptor was performed by immunofluorescence analysis. Sperm progressive motility (p = 0.02) and the percentage of sperm showing a strong MMP signal (p = 0.012) significantly increased after 2 h F4-NeuroP incubation compared to control samples. The AO assay did not show differences in the percentage of sperm with dsDNA between treated or control samples. Meanwhile, a significantly higher number of sperm with reacted acrosomes was highlighted by PSA localization after 4 h F4-NeuroP incubation. Finally, using an anti-ryanodine antibody, the immunofluorescence signal was differentially distributed at 2 and 4 h: a strong signal was evident in the midpiece and postacrosomal sheath (70% of sperm) at 2 h, whereas a dotted one appeared at 4 h (53% of sperm). A defined concentration of F4-NeuroPs in seminal fluid may induce sperm capacitation via channel ions present in sperm cells, representing an aid during in vitro sperm preparation that may increase the positive outcome of assisted fertilization.

Redox imbalance induced by docetaxel in the neuroblastoma SH-SY5Y cells: a study of docetaxel-induced neuronal damage.

OBJECTIVES: In cancer survivors, chemotherapy-associated adverse neurological effects are described as side effects in non-targeted tissue. We investigated the role of redox-imbalance in neuronal damage by a relative low dose of Docetaxel (DTX). METHODS: The neuroblastoma cells (SH-SY5Y cells) were exposed to DTX at a dose of 1.25 nM for 6 h. Antioxidant defenses (i.e. ascorbic acid, glutathione, and catalase) and lipid oxidation products (i.e. F2-isoprostanes) were evaluated. To investigate cell ultrastructure and tubulin localisation, transmission electron microscopy (TEM) and immunofluorescence techniques were applied. RESULTS: In the SH-SY5Y cells, DTX induced a significant reduction of total glutathione (P < 0.001) and ascorbic acid (P < 0.05), and an increase in both total F2-Isoprostanes (P < 0.05) and catalase activity (P < 0.05), as compared to untreated cells. Additionally, TEM showed a significant increase in cells with apoptotic characteristics. Immunolocalisation of tubulin showed a compromised cytoskeletal organisation. DISCUSSION: The investigated sublethal dose of DTX, to which non-targeted cells may be exposed throughout the duration of chemotherapy treatment, induces a redox imbalance resulting in a specific modulation of the antioxidant response. This study provides new insights into DTX-induced cellular mechanisms useful for evaluating whether the concomitant use of antioxidants associated with chemotherapy mitigates chemotherapy side effects in cancer survivors.

Cytotoxic Effects of Cannabinoids on Human HT-29 Colorectal Adenocarcinoma Cells: Different Mechanisms of THC, CBD, and CB83.

In this study, we investigated the effects of exposition to IC50 dose for 24 h of a new synthetic cannabinoid (CB83) and of phytocannabinoids Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) on HT-29 colorectal carcinoma cells. Cell viability and proliferative activity evaluated using the MTT, lactate dehydrogenase (LDH), and CyQUANT assays showed that cell viability was significantly affected when CB83, THC, and CBD were administered to cells. The results obtained showed that the reduced glutathione/oxidized glutathione ratio was significantly reduced in the cells exposed to CBD and significantly increased in the cells treated with the CB83 when compared to the controls. CBD treatment causes a significant increase in malondialdehyde content. The catalase activity was significantly reduced in HT-29 cells after incubation with CB83, THC, and CBD. The activities of glutathione reductase and glutathione peroxidase were significantly increased in cells exposed to THC and significantly decreased in those treated with CBD. The ascorbic acid content was significantly reduced in cells exposed to CB83, THC, and CBD. The ultrastructural investigation by TEM highlighted a significantly increased percentage of cells apoptotic and necrotic after CB83 exposition. The Annexin V-Propidium Iodide assay showed a significantly increased percentage of cells apoptotic after CB83 exposition and necrotic cells after CBD and THC exposition. Our results proved that only CBD induced oxidative stress in HT-29 colorectal carcinoma cells via CB receptor-independent mechanisms and that CB83 caused a mainly CB2 receptor-mediated antiproliferative effect comparable to 5-Fuorouracil, which is still the mainstay drug in protocols for colorectal cancer.

Resistin in Human Seminal Plasma: Relationship with Lipid Peroxidation, CAT Activity, GSH/GSSG Ratio, and Semen Parameters.

Resistin is an adipokine involved in inflammation and able to induce the expression of other proinflammatory cytokines. It is known that, in human semen, resistin is correlated with inflammatory cytokines and sperm quality. The aim of this prospective study was to explore the potential relationship between resistin, lipid peroxidation (LPO), catalase (CAT) activity, and reduced and oxidized glutathione (GSH/GSSG) ratio in semen samples of infertile patients with leukocytospermia (no. 19), infertile patients with varicocele (no. 17), and fertile men (no. 17). Semen analysis was performed following the WHO guidelines, and sperm apoptosis and necrosis were evaluated with annexin V/propidium iodide assay. Seminal plasma samples were used to determine resistin levels by an immunological method, MDA concentration by a HPLC analysis with UV detection, GSH/GSSG ratio by an enzymatic method, CAT activity by a spectrophotometric method. The results showed that, in both groups of infertile patients, semen parameters were significantly reduced (P < 0.001) and sperm apoptosis and necrosis percentages were increased. Resistin levels were significantly higher in leukocytospermia and varicocele groups (P < 0.001 and P < 0.01, respectively) as well as MDA concentration (P < 0.001) compared to controls. The MDA level was also significantly increased in the leukocytospermia group versus the varicocele group (P < 0.05). The GSH/GSSG ratio was higher in fertile controls than the leukocytospermia group (P < 0.05) and the varicocele group (P < 0.001) and in the leukocytospermia group versus the varicocele group (P < 0.05). Both the leukocytospermia and varicocele groups showed increased values of CAT activities (P < 0.001) than controls. Briefly, the correlation between variables, calculated in the whole patient population, showed that resistin levels positively correlated with MDA levels, CAT activity, sperm apoptosis, and necrosis and negatively with sperm parameters and GSH/GSSG ratio. These results support an active role of resistin in an inflammatory process causing LPO, increase of CAT activity, and decrease of GSH/GSSG ratio in seminal plasma of infertile men vs. fertile controls.

Relationships between Ghrelin and Obestatin with MDA, Proinflammatory Cytokines, GSH/GSSG Ratio, Catalase Activity, and Semen Parameters in Infertile Patients with Leukocytospermia and Varicocele.

Ghrelin and obestatin are involved in many biological functions including reproduction. Growing evidences suggest that both peptides could exert protective and antioxidant activities. In this study, the relationships between ghrelin/obestatin, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), malondialdehyde (MDA), reduced glutathione (GSH), oxidized glutathione (GSSG), expressed as the GSH/GSSG ratio, catalase (CAT), and semen parameters in infertile patients with varicocele or leukocytospermia and controls were investigated. Fifty-six infertile patients (32 with leukocytospermia and 24 with varicocele) and 14 controls participated in this study. Semen analysis was performed following the WHO guidelines. Apoptotic and necrotic sperm were scored by annexin V/propidium iodide assay. Seminal plasma samples were used for the following determinations: ghrelin, obestatin, IL-6, and TNF-α were measured by an immunological method, GSH/GSSG by an enzymatic method, and CAT by spectrophotometric determination. With respect to controls, both the leukocytospermia and varicocele groups showed altered sperm parameters, significantly increased sperm apoptosis (P = 0.009 and P = 0.011, respectively), IL-6 (P = 0.0001 and P = 0.004, respectively), and TNF-α levels (P = 0.0001 and P = 0.002, respectively); both groups had significantly decreased levels of ghrelin (P = 0.0001), obestatin (P = 0.0001 and P = 0.006, respectively), and GSH/GSSG ratio (P = 0.003 and P = 0.0001, respectively). The MDA concentration was significantly increased in the leukocytospermia group vs. controls (P = 0.0001), in the varicocele group vs. controls (P = 0.011), and in the leukocytospermia group vs. the varicocele group (P = 0.008). CAT activity was augmented in both the leukocytospermia and varicocele groups (P = 0.0001)vs. controls. The results indicate that both ghrelin and obestatin may play a protective role in human semen and this effect is probably due to their antioxidant properties.

Myocardial oxidative damage is induced by cardiac Fas-dependent and mitochondria-dependent apoptotic pathways in human cocaine-related overdose.

The aim of this study is to analyse cardiac specimens from human cocaine-related overdose, to verify the hypothesis that cardiac toxicity by acute exposure to high dosage of cocaine could be mediated by unbalanced myocardial oxidative stress, and to evaluate the apoptotic response. To address these issues, biochemical and immunohistological markers of oxidative/nitrosative stress were evaluated. We found that i-NOS, NOX2 and nitrotyrosine expression were significantly higher in the hearts of subjects who had died from high doses of cocaine, compared to the control group. Increase of these markers was associated with a dramatic increase in 8-OHdG, another marker of oxidative stress. A high number of TUNEL-positive apoptotic myocells was observed in the study group compared to the control group. The immunoexpression of TNF-α was significantly higher in the cocaine group compared to the control group. Furthermore, we detected a significantly stronger immunoresponse to anti-SMAC/DIABLO in our study group compared to control cases. Both cardiac Fas-dependent and mitochondria-dependent apoptotic pathways appeared to be activated to a greater extent in the cocaine group than in the control group. Our results highlight the central role of oxidative stress in cocaine toxicity. High levels of NOS can promote the oxidation process and lead to apoptosis.

Phase I trial of thymidylate synthase poly-epitope peptide (TSPP) vaccine in advanced cancer patients.

Thymidylate synthase (TS) poly-epitope peptide (TSPP) is a 27-mer peptide vaccine containing the amino acidic sequences of three epitopes with HLA-A2.1-binding motifs of TS, an enzyme overexpressed in cancer cells, which plays a crucial role in DNA repair and replication. Based on the results of preclinical studies, we designed a phase Ib trial (TSPP/VAC1) to investigate, in a dose escalation setting, the safety and the biological activity of TSPP vaccination alone (arm A) or in combination with GM-CSF and IL-2 (arm B) in cancer patients. Twenty-one pretreated metastatic cancer patients, with a good performance status (ECOG ≤ 1) and no severe organ failure or immunological disease, were enrolled in the study (12 in arm A, nine in arm B) between April 2011 and January 2012, with a median follow-up of 28 months. TSPP resulted safe, and its maximal tolerated dose was not achieved. No grade 4 toxicity was observed. The most common adverse events were grade 2 dermatological reactions to the vaccine injection, cough, rhinitis, fever, poly-arthralgia, gastro-enteric symptoms and, to a lesser extent, moderate hypertension and hypothyroidism. We detected a significant rise in auto-antibodies and TS-epitope-specific CTL precursors. Furthermore, TSPP showed antitumor activity in this group of pretreated patients; indeed, we recorded one partial response and seven disease stabilizations (SD) in arm A, and three SD in arm B. Taken together, our findings provide the framework for the evaluation of the TSPP anti-tumor activity in further disease-oriented clinical trials.

Hepatotoxicity of Teucrium chamaedrys L. decoction: role of difference in the harvesting area and preparation method.

AIM: Two recurrent cases of severe acute liver injury attributed to the use of a wild germander decoction, prepared with some variation in traditional method has been reported. The aim of the present study was to correlate the hepatotoxic effect observed in patients who consumed germander decoction with teucrin A levels. Antioxidant properties were analyzed to assess any possible differences between the decoction used traditionally by the family (without negative consequences) and the decoction taken by the patients. MATERIALS AND METHODS: Different types of germander decoctions were prepared in the laboratory by simulating the same conditions for preparing the decoction by the patients and their family members. The levels of teucrin A, the polyphenols and the antioxidant power were determined. One-way analysis of variance was used to test for differences between the groups. RESULTS AND CONCLUSIONS: The extract consumed by the patients had higher concentration of teucrin A, lower antioxidant activity and lower content of polyphenols compared with the traditional decoction, revealing an inverse relationship between teucrin A content and antioxidant capacity. These case reports emphasize that more information is needed on the safety and quality of these natural products.

Severe hypoglycemia associated with levofloxacin in Type 2 diabetic patients receiving polytherapy: two case reports.

OBJECTIVE: Although hypoglycemia is known to be associated with levofloxacin, patients are continually being hospitalized because of this adverse event. Here we have reported two further cases of severe hypoglycemia and discussed the possibility that the hypoglycemia is the result of interactions between levofloxacin and certain drugs, in order to alert physicians to be aware that geriatric patients, particularly those with Type 2 diabetes and in polytherapy, are at risk of showing this adverse reaction. CASES SUMMARY: A 91-year-old woman with Type 2 diabetes on metformin and glibenclamide, also under treatment with oral antihypertensive drugs, platelet antiaggregant, low-molecular- weight heparin and buprenorphine, was prescribed levofloxacin for a bacterial infection. Liver function and renal function parameters were within normal limits. After repeated administration of levofloxacin, her serum glycemic levels had decreased to 47 mg/dl and the patient was in a coma. After stopping levofloxacin her glycemia level returned to the normal value. A 61 year-old male, affected by tonsillar squamous cell carcinoma, with Type 2 diabetes on metformin and glibenclamide, under treatment with low-molecular-weight heparin for deep venous thrombosis, hydromorphone and undergoing nutritional support, was treated with levofloxacin for a bacterial infection. After 72 h the patient was unresponsive and his blood glucose levels were 38 mg/dl. After discontinuation of levofloxacin administration the patient was treated with glucose infusion and his glycemic values gradually returned to the normal range. DISCUSSION: The causality relationship between the levofloxacin and the hypoglycemia was established using Naranjo's ADR probability scale. These case reports confirm the literature data that serious hypoglycemia may develop due to the use of levofloxacin and appears to occur most frequently in elderly patients with Type 2 diabetes who are receiving oral hypoglycemic agents. We described the possible pharmacokinetic and pharmacodinamic mechanisms of the interaction between levofloxacin and the other drugs. CONCLUSIONS: We hypothesized that the concomitant use of several drugs and particularly levofloxacin in association with oral antidiabetic drugs, opioid analgesics and low-molecular-weight heparin could concur to cause this side effect. The safety and tolerability of this anti-infective agent should be revised urgently.

In vitro antioxidant activity of aged extracts of some Italian Allium species.

Antioxidant activity of fresh Allium sativum L. (garlic) is well known and is mainly due to unstable and irritating organosulphur compounds. Fresh garlic extracted over a prolonged period (up to 20 months) produces odourless aged garlic extract (AGE) containing stable and water soluble organosulphur compounds that prevent oxidative damage by scavenging free radicals. The aim of this study was to investigate the in vitro antioxidant activity of aged (up to 20 months) 15% hydroethanolic extracts of different parts (bulbs, bulblets, flower bulblets, flowers, and leaves) of three Allium spontaneous species which are endemic for Italian flora: Allium neapolitanum Cyr., Allium subhirsutum L., Allium roseum L. and to compare it with the in vitro antioxidant activity of aged 15% hydroethanolic extracts of bulbs and leaves of garlic. The antioxidant potential of aged extracts of all species has been evaluated using two different spectrophotometric assays: 2,2-diphenylpicrylhydrazyl (DPPH) test and the ferric reducing/antioxidant power (FRAP) assay. Furthermore the polyphenol content was determined. The aged extracts obtained from the leaves showed the best antioxidant activity, followed by flowers and then by bulbs in both used tests, while flower bulblets and bulblets exhibited lower results or no activity. The polyphenol content was generally directly correlated with antioxidant/antiradical activity. This study confirms the data obtained in previous researches, the wild-type species of Allium and in particular organs other than bulbs are more active and efficacious than garlic bulb. Surely leaves of these Allium spp. deserve special attention.

Evaluation of antioxidative properties of Allium species growing wild in Italy.

The genus Allium (Alliaceae) is an important dietary source of antioxidant phytochemical products. The antioxidant activity of some Allium species is well known but no information is available on the in vitro antioxidant activities of Italian Allium species growing wild. The aim of this study was to examine the in vitro antioxidant activity of aqueous extracts of different parts belonging to three Allium species growing wild, endemic to Italian flora: Allium neapolitanum Cyr., Allium subhirsutum L. and Allium roseum L., compared with the in vitro antioxidant activity of aqueous extracts of bulbs and leaves of the greatly studied garlic (Allium sativum L.). The antioxidant potential of extracts was evaluated using two different spectrophotometric assays: the DPPH test and FRAP assay. Furthermore the polyphenolic content was determined in all Allium species. The flowers of species growing wild showed the higher antioxidant power. Interesting results were shown even by the leaves, while the antioxidant capacity of the bulbs was lower. A correlation between the phenolic contents and the antioxidant activity was discovered. The differences in antioxidant capacity reflect the variability in the Allium species and the parts of the plant used and that the bulb of Allium sativum did not show higher antioxidant power.

Correlation between cardiac oxidative stress and myocardial pathology due to acute and chronic norepinephrine administration in rats.

BACKGROUND: To investigate the cardiotoxic role of reactive oxygen species (ROS) and of products derived from catecholamines auto-oxidation, we studied: (1) the response of antioxidant cardiac cellular defence systems to oxidative stress induced by norepinephrine (NE) administration, (2) the effect of NE administration on cardiac beta1-adrenergic receptors by means of receptor binding assay, (3) the cellular morphological alterations related to the biologically cross-talk between the NE administration and cytokines [tumor necrosis factor-alpha (TNF-alpha), monocyte chemotactic protein-1 (MCP-1), interleukins IL6, IL8, IL10]. METHODS AND RESULTS: A total of 195 male rats was used in the experiment. All animals underwent electrocardiogram (EKG) before being sacrificed. The results obtained show that NE administration influences the antioxidant cellular defence system significantly increasing glutathione peroxidase (GPx) activity, glutathione reductase (GR) and superoxide dismutase (SOD). The oxidized glutathione (GSH/GSSG) ratio significantly decreases and malondialdehyde (MDA) levels increase showing a state of lipoperoxidation of cardiac tissue. We describe a significant apoptotic process randomly sparse in the damaged myocardium and the effect of ROS on the NE-mediated TNF-alpha, MCP-1, and IL6, IL8, IL10 production. CONCLUSIONS: Our results support the hypothesis that catecholamines may induce oxidative damage through reactive intermediates resulting from their auto-oxidation, irrespective of their interaction with adrenergic receptors, thus representing an important factor in the pathogenesis of catecholamines-induced cardiotoxicity. The rise of the cardioinhibitory cytokines may be interpreted as the adaptive response of jeopardized myocardium with respect to the cardiac dysfunction resulting from NE injection.

In vitro antiproliferative effect of six Salvia species on human tumor cell lines.

This study was designed to examine the in vitro antiproliferative activity of the methanol crude extracts of six Salvia species: Salvia dominica L. leaves, Salvia lanigera Desf. aerial parts, Salvia menthaefolia Ten. roots, Salvia palaestina Benth. aerial parts, Salvia sclarea L. roots and Salvia spinosa L. aerial parts. Extracts were screened for their possible antitumoral activity by MTT test on nine human cancer cell lines: glioblastoma (DBTRG-05MG, T98G, U-87MG), colorectal adenocarcinoma (WiDr and HT-29), prostate adenocarcinoma (MDA Pca2b), choriocarcinoma (JEG-3), endometrium adenocarcinoma (HEC-1A) and B lymphoblast (CIR). IC(50) values were determined for only five extracts and ranged from 90 to 400 microg/mL approximately. Salvia menthaefolia extract exhibited marked antiproliferative activity against all tumor cell lines showing lower IC(50) values, while S. spinosa, S. sclarea and S. dominica extracts showed a degree cytotoxic activity dependent on the cell line type. Finally S. palaestina extract revealed a moderate antiproliferative effect only against three cell lines. Salvia lanigera extract displayed toxic activity at all concentrations tested. The results strengthen the evidence that the genus Salvia could be considered a natural resource of potential antitumor agents.

Chemo-immunotherapy of colorectal carcinoma: preclinical rationale and clinical experience.

Advanced colorectal cancer is a common disease with an high mortality rate. For four decades, pharmacological treatment of the advanced disease was based on the use of 5-fluorouracil alone or in combination with biomodulators such as folinic acid and intereferon alpha. In the last 5 years, response to therapy has been considerably ameliorated thanks to the discovery of new drugs such as oxaliplatin and CPT-11. These agents, in combination with 5-fluorouracil, according to various schedules of treatment, have reached a significant improvement of palliation, response rate and survival. Immunotherapy is an uprising modality of treatment for human cancer including colorectal carcinoma. Its rationale is based on the knowledge that tumour cells are genetically unstable and produce molecular structures which allow their recognition and destruction by the immune-surveillance system. Therefore, humoral as well as cellular compartments of the immune system can be utilized according to a "passive" strategy (e.g. monoclonal antibody administration and adoptive immunotherapy) or an "active" approach, by using different modalities of vaccine therapy. In this context, monoclonal antibodies (mAbs) and cancer vaccines are being tested for the treatment of advanced colorectal cancer. Due to their genetic instability and extraordinary adaptative potential, tumour cells may acquire resistance to the immune effectors and mAbs exactly as they do for cytotoxic drugs. To improve the results of both immunological and chemical modality of cancer treatment, an increasing number of authors is starting to combine chemo and immunotherapy in the attempt to circumvent the limitations of both strategies. This report tries to review the possible rationale of the chemo-immunotherapy combination, illustrating preliminary results of preclinical and clinical studies.

Chemo-immunotherapy of metastatic colorectal carcinoma with gemcitabine plus FOLFOX 4 followed by subcutaneous granulocyte macrophage colony-stimulating factor and interleukin-2 induces strong immunologic and antitumor activity in metastatic colon cancer patients.

PURPOSE: Tumor cell killing by anticancer drugs may be supported by their immuno- and pharmacologic effects. Chemotherapy is in fact able to (A) upregulate tumor-associated antigen expression, including carcinoembryonic antigen (CEA) or other target molecules such as thymidylate synthase (TS); and (B) downregulate tumor cell resistance to the death signals induced by tumor antigen-specific cytotoxic T lymphocytes. This provides the rationale for combining chemo- and immunotherapy. MATERIALS AND METHODS: We describe the results of a translational phase II trial designed to evaluate the toxicity, antitumor activity and immunologic effects of gemcitabine + FOLFOX-4 (oxaliplatin, fluorouracil, and folinic acid) polychemotherapy followed by the subcutaneous administration of granulocyte macrophage colony-stimulating factor and low-dose interleukin-2 in colorectal carcinoma patients. The study involved 29 patients (16 males and 13 females with a mean age of 69 years), 21 of whom had received a previous line of treatment, and 19 had liver involvement. RESULTS: The treatment was well tolerated and induced very high objective response (68.9%) and disease control rates (96.5%), with an average time to progression of 12.5 months. An immunologic study of peripheral blood mononuclear cells (PBMCs) taken from 20 patients showed an enhanced proliferative response to colon carcinoma antigen and a significant reduction in suppressive regulatory T lymphocytes (CD4+CD25T-reg+). A cytofluorimetric study of the PBMCs of five HLA-A(*)02.01+ patients who achieved an objective response showed an increased frequency of cytolytic T lymphocyte precursors specific for known CEA- and TS-derived epitopes. CONCLUSION: The results show that our regimen has strong immunologic and antitumor activity in colorectal cancer patients and deserves to be investigated in phase III trials.

Dendritic cell-mediated cross-presentation of antigens derived from colon carcinoma cells exposed to a highly cytotoxic multidrug regimen with gemcitabine, oxaliplatin, 5-fluorouracil, and leucovorin, elicits a powerful human antigen-specific CTL response with antitumor activity in vitro.

Gemcitabine, oxaliplatin, leucovorin, and 5-fluorouracil (GOLF) is a novel multidrug regimen inducing high levels of necrosis and apoptosis in colon carcinoma cells. This regimen is also able to promote a process of Ag remodeling including up-regulation of immunotherapy targets like carcinoembryonic Ag (CEA), thymidylate synthase (TS). We have conducted a preclinical study aimed to investigate whether these drug-induced modifications would also enhance colon cancer cell immunogenicity. Several CTL lines were thus generated by in vitro stimulating human HLA-A(*)02.01(+) PBMCs, from normal donors and colon cancer patients, with autologous dendritic cells cross-primed with cell lysates of colon cancer cells untreated, irradiated, or previously exposed to different drug treatments including the GOLF regimen. Class I HLA-restricted cytolytic activity of these CTL lines was tested against colon cancer cells and CEA and TS gene transfected target cells. These experiments revealed that CTLs sensitized with GOLF-treated cancer cells were much more effective than those sensitized with the untreated colon carcinoma cells or those exposed to the other treatments. CTL lines sensitized against the GOLF-treated colon cancer cells, also expressed a greater percentage of T-lymphocyte precursors able to recognize TS- and CEA-derived peptides. These results suggest that GOLF regimen is a powerful antitumor and immunomodulating regimen that can make the tumor cells a suitable means to induce an Ag-specific CTL response. These results suggest that a rationale combination of GOLF chemotherapy with cytokine-based immunotherapy could generate a chemotherapy-modulated Ag-specific T-lymphocyte response in cancer patients able to destroy the residual disease survived to the cytotoxic drugs.

Endothelin-1 triggers placental oxidative stress pathways: putative role in preeclampsia.

CONTEXT: Preeclampsia (PE) is a disorder that occurs only during pregnancy. The placenta has a controlling role in this condition. Recent literature suggests that the oxidative stress is a component of PE and plays a main role in the link between decreased placental perfusion and the impaired function of maternal endothelium. OBJECTIVE: Because the human placenta expresses endothelin-1 (ET-1) and its circulating levels are high in pregnancies complicated with PE, the present study investigated the role of ET-1 on placental oxidative stress pathways. DESIGN: Human placental explants, JEG-3, and primary cytotrophoblast cells were cultured with increasing ET-1 concentrations for 6 and 24 h. SETTING: The study was conducted at tertiary clinical care centers in Siena and Padova, Italy. INTERVENTIONS: Human placental explants, JEG-3, and primary cytotrophoblast cells were used to test ET-1 effect. MAIN OUTCOME MEASURE(S): The main outcome measure was ET-1 mRNA and its receptor mRNAs, type A and B, detection by RT-PCR. The common markers of oxidative stress [malondialdehyde (MDA), glutathione (GSH), glutathione disulfide (GSSG), ascorbic acid (AA)] as well as cell proliferation and vitality were measured after stimulation periods. RESULTS: ET-1 inhibits cell proliferation and vitality and triggers oxidative stress in the human placenta by altering the balance between oxidant (increased MDA levels) and antioxidant (decreased GSH, GSSG, and AA) forces in favor of oxidation. CONCLUSIONS: Because MDA damages endothelial cells, whereas GSH, GSSG, and AA protect them, we postulate that ET-1 may be one of the key links between primary placental disorders and the systemic endothelial dysfunction of PE.

Treatment of colon and breast carcinoma cells with 5-fluorouracil enhances expression of carcinoembryonic antigen and susceptibility to HLA-A(*)02.01 restricted, CEA-peptide-specific cytotoxic T cells in vitro.

Cancer vaccines directed against tumor associate antigen (TAA) have produced encouraging results in preclinical models but not in cancer patients. A major limitation of this strategy is the relative degree of tolerance to these antigens and the low and heterogeneous tumor cell expression of TAA and major histocompatibility complex (MHC). Previous studies have shown that 5-fluorouracil (5-FU) can upregulate the expression of membrane-associated carcino-embryonic antigen (CEA), and MHC molecules in colon and breast carcinoma cell lines. We have investigated whether this drug can also enhance their sensitivity to the lytic effects of CEA-peptide specific Cytotoxic T cell lymphocytes (CTL). The CEA peptide-specific CTLs generated in our laboratory from normal HLA-A(*)02.01(+) donor PBMCs, were able to kill HLA-A(*)02.01(+)/CEA(+) breast (MCF-7-T103) and colon (HLA-A(*)02.01 gene-transfected HT-29 and C22.20) carcinoma cells in HLA-A(*)02.01 restricted manner. The treatment of target cells with 5-FU, enhanced their CEA expression and susceptibility to CTL-mediated lysis. Cold competition assays confirmed these results, thus supporting the hypothesis that immune target cell lysis and 5-FU mediated enhancement were dependent on CEA peptide presentation by cancer cells. 5-FU treatment of functionally "mature" CTL after in vitro expansion, did not reduce their cytolytic activity against MT-2 target cells but, when the anti-metabolite was added during the immune-sensitization phase, CTL generation was significantly inhibited. These results provide a rationale for investigating a possible new role of 5-FU as an immuno targeting amplifier agent in breast and colorectal cancer patients immunized with CEA-directed cancer vaccines.