Long-term outcomes of patients with 10 or more colorectal liver metastases.

BACKGROUND: Although the number of colorectal liver metastases (CLM) is decreasingly considered as a contraindication to surgery, patients with 10 CLM or more are often denied liver surgery. This study aimed to evaluate the outcome after liver surgery and to identify prognostic factors of survival in such patients. METHODS: The study population consisted of a multicentre cohort of patients with CLM (N=12 406) operated on, with intention to resect, from January 2005-June 2013 and whose data were prospectively collected in the LiverMetSurvey registry. RESULTS: Overall, the group ⩾10 CLM (N=529, 4.3%) experienced a 5-year overall survival (OS) of 30%. A macroscopically complete (R0/R1) resection (72.8% of patients) was associated with a 3- and 5-year OS of 61% and 39% vs 29% and 5% for R2/no resection patients (P<0.0001). At multivariate analysis, R0/R1 resection emerged as the strongest favourable factor of OS (HR 0.35 (0.26-0.48)). Other independent favourable factors were as follows: maximal tumour size <40 mm (HR 0.67 (0.49-0.92)); age <60 years (HR 0.66 (0.50-0.88)); preoperative MRI (HR 0.65 (0.47-0.89)); and adjuvant chemotherapy (HR 0.73 (0.55-0.98)). The model showed that 5-year OS rates of 30% was possible provided R0/R1 resection associated with at least an additional favourable factor. CONCLUSIONS: Liver resection might provide long-term survival in patients with ⩾10 CLM staged with preoperative MRI, provided R0/R1 resection followed by adjuvant therapy. A validation of these results in another cohort is needed.

Soluble CD40-ligand (sCD40L, sCD154) plays an immunosuppressive role via regulatory T cell expansion in HIV infection.

CD40/CD40-ligand (CD40L) signalling is a key stimulatory pathway which triggers the tryptophan (Trp) catabolizing enzyme IDO in dendritic cells and is immunosuppressive in cancer. We reported IDO-induced Trp catabolism results in a T helper type 17 (Th17)/regulatory T cell (Treg ) imbalance, and favours microbial translocation in HIV chronic infection. Here we assessed the link between sCD40L, Tregs and IDO activity in HIV-infected patients with different clinical outcomes. Plasmatic sCD40L and inflammatory cytokines were assessed in anti-retroviral therapy (ART)-naive, ART-successfully treated (ST), elite controllers (EC) and healthy subjects (HS). Plasma levels of Trp and its metabolite Kynurenine (Kyn) were measured by isotope dilution tandem mass spectrometry and sCD14 was assessed by enzyme-linked immunosorbent assay (ELISA). IDO-mRNA expression was quantified by reverse transcription-polymerase chain reaction (RT-PCR). The in-vitro functional assay of sCD40L on Treg induction and T cell activation were assessed on peripheral blood mononuclear cells (PBMCs) from HS. sCD40L levels in ART-naive subjects were significantly higher compared to ST and HS, whereas EC showed only a minor increase. In ART-naive alone, sCD40L was correlated with T cell activation, IDO-mRNA expression and CD4 T cell depletion but not with viral load. sCD40L was correlated positively with IDO enzymatic activity (Kyn/Trp ratio), Treg frequency, plasma sCD14 and inflammatory soluble factors in all HIV-infected patients. In-vitro functional sCD40L stimulation induced Treg expansion and favoured Treg differentiation by reducing central memory and increasing terminal effector Treg proportion. sCD40L also increased T cell activation measured by co-expression of CD38/human leucocyte antigen D-related (HLA-DR). These results indicate that elevated sCD40L induces immunosuppression in HIV infection by mediating IDO-induced Trp catabolism and Treg expansion.

Systematic review and meta-analysis of liver resection for metastatic melanoma.

BACKGROUND: The multidisciplinary management of metastatic melanoma now occasionally includes major hepatic resection. The objective of this work was to conduct a systematic review of the literature on liver resection for metastatic melanoma. METHODS: MEDLINE, Embase, the Cochrane Library and Scopus were searched (1990 to December 2012). Studies with at least ten patients undergoing liver resection for metastatic melanoma were included. Data on the outcomes of overall survival (OS) and/or disease-free survival (DFS) were abstracted and synthesized. Hazard ratios (HRs) were derived from survival curves and subjected to meta-analysis using random-effects models. RESULTS: Twenty-two studies involving 579 patients (13 per cent weighted resection rate) who underwent liver resection were included. Study quality was poor to moderate. Median follow-up ranged from 9 to 59 months. Median DFS ranged from 8 to 23 months, and median OS ranged from 14 to 41 months (R0, 22-66 months, R2, 10-16 months; R0 versus R1/R2: HR 0.52, 95 per cent confidence interval (c.i.) 0.37 to 0.73). The OS rate was 56-100 per cent at 1 year, 34-53 per cent at 3 years and 11-36 per cent at 5 years. Median OS with non-operative management ranged from 4 to 12 months. Comparison of OS with resection and non-operative management favoured resection (HR 0.32, 95 per cent c.i. 0.22 to 0.46). CONCLUSION: Radical resection of liver metastases from melanoma appears to improve overall survival compared with non-operative management or incomplete resection, but this observation requires future confirmation as selection bias may have confounded the results.

Carcinoma of the gallbladder: patterns of presentation, prognostic factors and survival rate. An 11-year single centre experience.

BACKGROUND: This report examines the patterns of presentation, prognostic factors and survival rate of all patients with gallbladder cancer (GBC) evaluated at our tertiary academic hospital over an 11-year period. METHODS: A retrospective review of a prospectively collected database of all patients with GBC presenting between January 1998 and December 2008 was performed. RESULTS: 102 GBC-patients were included: 69 women and 33 men (median age: 65,5 years). Forty-five patients presented with incidental gallbladder cancer (IGC) and 57 with nonincidental cancer (NIGC). Curative surgery rate was 84.4% for IGC and 29.8% for NIGC (p < 0.001). Five-year actuarial survival rate was 63.2% for patients with curative intent surgery and 0% for patients with palliative approach. Patients with IGC had a longer survival rate compared to patients with NIGC (median: 25.8 vs. 4.4 months, p < 0.0001). For patients with radical resection (42 patients), there was no difference between IGC and NIGC. The incidence of liver involvement was respectively 0%, 20.8%, 58.3%, 100% for pT1, pT2, pT3 and pT4 tumors. Univariate analysis showed that survival rate was significantly affected by perineural invasion, T, N and M-stage, R0 resection, liver involvement, CA-19.9. In multivariate analysis, liver involvement was the only independent factor. CONCLUSIONS: Majority of patients with a potentially curable disease had IGC. Almost 80% of patients with NIGC presented with unresectable disease. For patients who underwent resection with curative intent, actuarial 5-year survival was 63.2%. Liver involvement was the only independent prognostic factor. All patients with IGC and a pT2 or more advanced T stage should undergo a second radical resection.

Double-blind, vehicle-controlled randomized twelve-month neurodevelopmental toxicity study of common aluminum salts in the rat.

This good laboratory practice (GLP) study of aluminum salts in Sprague-Dawley rats was conducted according to double-blind, vehicle-controlled randomized design by exposing offspring to aluminum citrate in-utero, through lactation, and then in drinking water post-weaning. Three dose levels were used: 30, 100, 300 mg Al/kg bw/day, in addition to control groups that received either water or a sodium citrate solution (27.2 g/L). Endpoints were assessed in both female and male pups: behavioral (motor activity, T-maze, auditory startle, the Functional Observational Battery (FOB) with domains targeting autonomic function, activity, neuromuscular function, sensimotor function, and physiological function), cognitive function (Morris swim maze), brain weight, clinical chemistry, hematology, tissue/blood levels of aluminum and neuropathology. The most notable treatment-related effect observed in the offspring was renal pathology, most prominently in the male pups. Higher mortality and significant morbidity were observed in the male pups in the high Al-citrate dose group; leading to euthanization of this group at day 89. There was evidence for dose-response relationships between neuromuscular measurements-hind-limb and fore-limb grip strength-and Al-treatment in both males and females, although some of the effects may be secondary to body weight changes. No consistent treatment-related effects were observed in ambulatory counts (motor activity) in the different cohorts. No significant effects were observed for the auditory startle response, T-maze tests (pre-weaning day 23 cohort) or the Morris water maze test (day 120 cohort). None of the lesions seen on histopathological examination of brain tissues of the day 364 group was reported as treatment-related and, as these were also seen in the control group, were likely due to aging. In conclusion, these results indicate that concentrations of aluminum in the drinking water that are required to produce minimally detectable neurobiological effects in the rat are about 10,000 times higher than what is typically found in potable drinking water.

[Outpatient laparoscopic cholecystectomy: a one year experience on unselected patients]. / Cholécystectomie coelioscopique ambulatoire: expérience d'un an sur des patients non sélectionnés.

UNLABELLED: This study reviews our experience with outpatient laparoscopic cholecystectomy (CCA) to evaluate the benefits of this approach to routine clinical practice. PATIENTS AND METHODS: Of 217 consecutive patients undergoing laparoscopic cholecystectomy over a one-year period (2002-2003) at our university medical center, 151 were selected for same day surgery and discharge according to the following selection criteria: non-urgent surgery, no major co-morbidities, domicile within one hour of the hospital. Patients were typically discharged the afternoon of their surgery if their clinical condition was stable. RESULTS: Of 151 planned outpatient CCA's, 122 (81%) were discharged on the day of surgery. Of these, 16 had a post-operative complication and three required readmission; no patient required reoperation. Univariate analysis revealed three factors predictive of failure of the outpatient strategy: age >65 (p=0.015), operative duration (p<0.0001), and surgical start time after 11 am (p<0.0001). CONCLUSIONS: Outpatient laparoscopic cholecystectomy can be routinely accomplished in unselected patients in an academic center. The low rate of in-patient admission is acceptable. The out-patient strategy for laparascopic cholecystectomy allows for a reduction in waiting time at our institution.

Peripherin-2: an intracellular analogy to viral fusion proteins.

The C-terminus of the intracellular retinal rod outer segment disk protein peripherin-2 binds to membranes, adopts a helical conformation, and promotes membrane fusion, which suggests an analogy to the structure and function of viral envelope fusion proteins. Nuclear magnetic resonance (NMR) data and fluorescence data show that a 63-residue polypeptide comprising the C-terminus of bovine peripherin-2 (R284-G346) binds to the membrane mimetic, dodecylphosphocholine micelles. High-resolution NMR studies reveal that although this C-terminal fragment is unstructured in solution, the same fragment adopts helical structure when bound to the micelles. The C-terminus may be a member of the class of intrinsically unstructured protein domains. Using methods developed for the G-protein coupled receptor rhodopsin, a model for the structure of the transmembrane domain of peripherin-2 was constructed. Previously published data showed that both peripherin-2 and viral fusion proteins are transmembrane proteins that promote membrane fusion and have a fusion peptide sequence within the protein that independently promotes membrane fusion. Furthermore, the fusion-active sequence of peripherin-2 exhibits a sequence motif that matches the viral fusion peptide of influenza hemagglutinin (HA). These observations collectively suggest that the mechanism of intracellular membrane fusion induced by peripherin-2 and the mechanism of enveloped viral fusion may have features in common.

The Wnt pathway regulator DKK1 is preferentially expressed in hormone-resistant breast tumours and in some common cancer types.

In addition to new tumour antigens, new prognostic and diagnostic markers are needed for common cancers. In this study, we report the expression of Dickkopf-1 (DKK1) in multiple common cancers. This constitutes a comprehensive analysis of the DKK1 expression profile. Dickkopf-1 expression was evaluated by classical and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbant assay for protein determination, in cancer lines and clinical specimens of several cancer origins. For breast cancer, expression was correlated with clinicopathological parameters. Dickkopf-1 expression was confirmed in several cancer cell lines derived from breast and other common cancers. Dickkopf-1 protein secretion was documented in breast, prostate and lung cancer lines, but was negligible in melanoma. Analysis of DKK1 expression in human cancer specimens revealed DKK1 expression in breast (21 out of 73), lung (11 out of 23) and kidney cancers (six out of 20). Interestingly, DKK1 was preferentially expressed in oestrogen and progesterone receptor-negative tumours (ER(-)/PR(-); P=0.005) and in tumours from women with a family history of breast cancer (P=0.024). Importantly, DKK1 protein production was confirmed in multiple breast cancer specimens that were positive by RT-PCR. This work establishes DKK1 as a potential prognostic and diagnostic marker for cohorts of breast cancer patients with poor prognosis. Dickkopf-1 may also become a relevant candidate target for immunotherapy of different cancers.

Treatment of post liver transplantation bile duct stricture with self-expandable metallic stent.

OBJECTIVE: The aim of this study is to report our experience using self-expandable covered metallic stents (Wallstent) to treat different types of biliary strictures after orthotopic liver transplantation (OLT). PATIENTS AND METHODS: Between January 1999 and July 2004, 222 OLTs were performed with choledocho-choledochostomy (CC) bile duct reconstruction. An anastomotic biliary stricture was diagnosed and treated by endoscopic retrograde cholangiopancreatography (ERCP) or percutaneous procedures in 100 patients (45%). The group of 21 patients (mean age 57.0+/-5.6 years) that were eventually treated with a biliary Wallstent was studied retrospectively. RESULTS: Significant persistent proximal or anastomotic strictures were diagnosed in 4 and 17 patients, respectively. A Wallstent was inserted by ERCP or through a percutaneous route in 18 and 3 patients, respectively. The mean interval between diagnosis and Wallstent insertion was 179.7+/-292.8 (0-1113) days. The mean total number of procedures required per patient was 7.4+/-5.5. The mean stent primary patency duration was 10.8+/-7.8 (0.9-25.1) months with a 24-month primary patency rate of 26% at a mean follow-up time of 37.8+/-17.2 months. A hepatico-jejunostomy was performed in five patients (24%). Two patients (10%) underwent retransplantation for diffuse ischemic cholangitis or chronic rejection. The overall complication rate was 4%. CONCLUSION: Treatment of post-transplant biliary stenosis using a Wallstent is a valuable option for delaying or avoiding surgery in up to 70% of patients. Proximal stenosis can be treated in the same manner in selected patients with major comorbidities.

Phase II study of troxacitabine in chemotherapy-naive patients with advanced cancer of the pancreas: gastrointestinal tumors.

BACKGROUND: Troxacitabine (Troxatyl) is a novel L-enantiomer nucleoside analog with activity in pancreatic cancer xenograft models. PATIENTS AND METHODS: Troxacitabine 1.5 mg/m(2) was administered by 30-min infusions daily x5 every 4 weeks to 54 patients with advanced pancreatic cancer. Patients were evaluated for objective tumor response, time to tumor progression (TTP), changes in tumor marker CA 19-9, survival, safety, pain, analgesic consumption, Karnofsky performance status and weight change. RESULTS: Median TTP was 3.5 months (95% CI 2.0-3.8), median survival 5.6 months (95% CI 4.9-7.4), and the 1 year survival rate 19%. Best responses were stable disease in 24 patients with eight patients having stable disease for at least 6 months (15%). A 50% or greater decrease in CA 19-9 was seen in seven of 44 assessed patients (16%). Grade 3 and 4 neutropenia were observed in 37% and 30% of patients with one episode of febrile neutropenia. The most common drug-related non-hematological toxic effects reported were cutaneous, with 22% and 6% of patients reporting grade 2 and 3 skin rash, respectively and 4% grade 2 hand-foot syndrome. CONCLUSION: Troxacitabine administered by a bolus daily x5 monthly regimen has modest activity in advanced pancreatic adenocarcinoma.

Anti-apoptotic effect of insulin on normal hepatocytes in vitro and in vivo.

Liver growth factors are known to be anti-apoptotic for hepatocytes. The potential of insulin, a liver co-mitogen, has not been thoroughly evaluated. We studied the anti-apoptotic role of insulin on primary cultures of rat hepatocytes exposed to transforming growth factor-beta (TGF-beta) as the apoptotic agent and in the left portal vein ligation model (LVPL) of liver atrophy. Results show that insulin decreases apoptosis of TGF-beta-treated hepatocyte cultures by 43% (P < 0.002) and the alanine amino transferase (ALT) release by 49% (P < 0.001). Left lobes of LPVL animals displayed a significant increase in the levels of TGF-beta mRNA. In LPVL rats receiving continuous infusion of insulin in the left lobes, the weight of the atrophic lobes was higher over a 7-day period in comparison to control animals. This was associated with lower levels of serum ALT and with a five-fold decrease in the apoptotic index in the left lobes (P < 0.0001). Induction of Akt phosphorylation and increased expression of Bcl-xl were observed in the left lobes of insulin-treated animals. In conclusion, these results show that insulin is anti-apoptotic for normal hepatocytes both in vitro and in vivo and that the action of insulin is associated with increased Bcl-xl expression and Akt activation.

Immunization against endogenous retroviral tumor-associated antigens.

Endogenous retroviral gene products have been found in some human tumors, and therefore, may serve as antigens for immunotherapy approaches. The murine colorectal carcinoma CT26 and melanoma B16 have recently been found to express the endogenous retroviral gene products gp70 and p15E, respectively, that can serve as antigens recognized by T cells. To date, though, there has been no demonstration of tumor treatment using an endogenous retroviral protein. In this study, we demonstrate that mice immunized with recombinant vaccinia encoding the gp70 H2-L(d)-restricted minimal determinant were protected from CT26 tumor challenge. Splenocytes from mice immunized with vaccinia gp70 specifically secreted IFN-gamma in response to gp70 peptide-pulsed stimulators. Although this strategy could protect against subsequent tumor challenge, it was ineffective against established tumors. Therefore, to investigate the treatment of established CT26 or B16 lung metastases, mice were treated with cultured dendritic cells (DCs) pulsed with gp70 or p15E peptide. Significant inhibition of established lung metastases required immunization with peptide-pulsed DCs pretreated with CD40 ligand that has been demonstrated to increase the T-cell stimulatory activity of DCs. The ability to immunize against endogenous retroviral tumor antigens may have relevance in the induction of antitumor immunity for some human cancers.

Retrovirally transduced human dendritic cells can generate T cells recognizing multiple MHC class I and class II epitopes from the melanoma antigen glycoprotein 100.

Involvement of tumor-Ag specific CD4(+) and CD8(+) T cells could be critical in the generation of an effective immunotherapy for cancer. In an attempt to optimize the T cell response against defined tumor Ags, we previously developed a method allowing transgene expression in human dendritic cells (DCs) using retroviral vectors. One advantage of using gene-modified DCs is the potential ability to generate CD8(+) T cells against multiple class I-restricted epitopes within the Ag, thereby eliciting a broad antitumor immune response. To test this, we generated tumor-reactive CD8(+) T cells with DCs transduced with the melanoma Ag gp100, for which a number of HLA-A2-restricted epitopes have been described. Using gp100-transduced DCs, we were indeed able to raise T cells recognizing three distinct HLA-A2 epitopes within the Ag, gp100(154-162), gp100(209-217), and gp100(280-288). We next tested the ability of transduced DCs to raise class II-restricted CD4(+) T cells. Interestingly, stimulation with gp100-transduced DCs resulted in the generation of CD4(+) T cells specific for a novel HLA-DRbeta1*0701-restricted epitope of gp100. The minimal determinant of this epitope was defined as gp100(174-190) (TGRAMLGTHTMEVTVYH). These observations suggest that retrovirally transduced DCs have the capacity to present multiple MHC class I- and class II-restricted peptides derived from a tumor Ag, thereby eliciting a robust immune response against that Ag.

HIV-1 induces phenotypic and functional perturbations of B cells in chronically infected individuals.

A number of perturbations of B cells has been described in the setting of HIV infection; however, most remain poorly understood. To directly address the effect of HIV replication on B cell function, we investigated the capacity of B cells isolated from HIV-infected patients to respond to a variety of stimuli before and after reduction of viremia by effective antiretroviral therapy. B cells taken from patients with high levels of plasma viremia were defective in their proliferative responses to various stimuli. Viremia was also associated with the appearance of a subpopulation of B cells that expressed reduced levels of CD21. After fractionation into CD21(high)- and CD21(low)-expressing B cells, the CD21(low) fraction showed dramatically reduced proliferation in response to B cell stimuli and enhanced secretion of immunoglobulins when compared with the CD21(high) fraction. Electron microscopic analysis of each fraction revealed cells with plasmacytoid features in the CD21(low) B cell population but not in the CD21(high) fraction. These results indicate that HIV viremia induces the appearance of a subset of B cells whose function is impaired and which may be responsible for the hypergammaglobulinemia associated with HIV disease.

Functional community ambulation requirements in incomplete spinal cord injured subjects.

STUDY DESIGN: A group of people with incomplete spinal cord injuries (SCI) were evaluated and compared with able-bodied individuals during several walking conditions. OBJECTIVES: To evaluate the functional community ambulation and estimated energy expenditure in persons with incomplete SCI and able-bodied individuals. METHODS: A list of criteria was used to evaluate functional community ambulation among participants. Physiological variables, such as the heart rate, oxygen uptake and the lactate concentration, were also measured. RESULTS: Three of nine incomplete SCI subjects and all able-bodied subjects were able to meet all the criteria measured. The required velocity to safely cross an intersection was the criterion that the incomplete SCI group had the most difficulty reaching. The able-bodied subjects had a comfortable walking velocity twice that of the incomplete SCI subjects' preferred velocity. When walking at the same velocity (incomplete SCI subjects' preferred velocity), the incomplete SCI subjects had a rate of oxygen uptake 26% greater than the healthy subjects and were 200% less efficient. The lactate concentration also proved to be a useful tool when evaluating the incomplete SCI subjects' walking efficiency. The incomplete SCI subjects lactate concentration increased after walking at their preferred velocity, meaning that the anaerobic pathways were used to meet energy demands. CONCLUSION: Rehabilitation centers should adapt their evaluation forms and increase their criteria requirements to more suitable criteria that are found in the SCI patient's community. The physiological cost should also be taken into consideration when evaluating the SCI patient's functional ambulation.

Simian virus 40 large tumor antigen modulates the Raf signaling pathway.

The large tumor antigen of simian virus 40 (SVLT) is a potent oncogene. Although inactivation of the p53 and pRb tumor suppressors has been causally linked to the transforming properties of SVLT, its exact mechanism of action remains undefined. Previous data indicated that Ras is activated in SVLT-expressing cells. In this report we show that SVLT also increases Raf kinase activity in both insect and mammalian cells, thus identifying the Raf kinase as an additional target of SVLT. Our results further show that SVLT was still able to activate Raf in cells where Ras levels had been drastically reduced through expression of an antisense construct, indicating that SVLT may activate Raf at least partly by a mechanism that is independent of its stimulatory effect on Ras.

Expression of CYP27A, a gene encoding a vitamin D-25 hydroxylase in human liver and kidney.

Vitamin D3 (D3) is not active but must be hydroxylated at C-25 in liver before acquiring its hormonal potential in the kidney. The sterol-27 hydroxylase (gene symbol: CYP27A) catalyses the oxidation of sterol side chain in bile acid synthesis but the enzyme is also known as a D3-25 hydroxylase. The study examined the expression of the gene encoding CYP27A in adult and fetal human livers and kidneys. Thirty-nine adults (18 men and 21 women; mean age 58 years in men and 57 years in women) and three normal fetuses gestational age 17-19 weeks were studied. Tissue CYP27A mRNA and serum 25OHD concentrations were measured. Normal specimens: CYP27A transcript was found to be higher in adult than in fetal livers but its expression was similar in adult and fetal kidneys. In fetuses, no difference was observed between CYP27A levels in livers and kidneys. In adult livers CYP27A levels were higher in women than in men. Hepatic CYP27A mRNA and serum 25OHD concentrations were both found to be higher in summer than in winter. Multiple linear regression analyses indicate that the season of the year and the serum 25OHD concentrations (but not 1,25(OH)2D concentrations) are the best predictors of CYP27A mRNA abundance in normal adult livers. In situ hybridization illustrates a clear label in hepatocytes which increases in intensity in the perivenous region of the hepatic acinus. Pathological specimens: In one man with an hepatic carcinoma there was a very large increase in CYP27A (> 1000 fold) compared to the level found in the normal liver. In that patient, serum 25OHD concentrations were found to be high considering the level of CYP27A mRNA in the normal hepatic area suggesting that the neoplastic tissue contributed to the C-25 hydroxylation of vitamin D. Specimens obtained from two patients suffering from focal hepatic hyperplasia indicate that in one case the level of CYP27A mRNA was twice as high in the pathological than in the normal area while in the other its levels were similar in both areas. No difference in the CYP27A transcript was observed between specimens obtained from normal areas and those obtained form either an hepatic adenoma or from two intrahepatic colonic metastases. CYP27A is present not only in the human adult liver but also in the adult kidney, and in the fetal liver and kidney. Our findings illustrate that CYP27A can be significantly upregulated in certain pathological situations such as in hepatic carcinoma and that the neoplastic tissue could contribute to the circulating concentration of 25OHD.

Human dendritic cells require multiple activation signals for the efficient generation of tumor antigen-specific T lymphocytes.

Dendritic cells (DC) are specialized cells of the immune system responsible for the initiation and regulation of both cellular and humoral responses. DC function is highly dependent on their level of maturation. In this study, we postulated that full DC maturation would require a combination of activating signals. When cultured monocyte-derived DC received stimulation with CD40 ligand (CD40L) and lipopolysaccharide (LPS) together, the IL-12 secretion increased 5-60-fold and the IL-10 secretion increased 5-15-fold when compared with either stimulation alone. In addition, poly I.C, a double-stranded RNA analog that mimics viral infection, also synergized with CD40L to stimulate DC to secrete high levels of IL-12 and IL-10. Flow cytometry revealed an up-regulation in the expression of CD80, CD86 and CD83 following activation with a soluble trimeric form of CD40L (CD40Ls) or LPS. However, no further up-regulation was observed when both CD40Ls and LPS were used together compared with a single stimulatory signal, suggesting that there was no correlation between the expression of these markers and the level of IL-12/IL-10 secretion. Finally, specific cytotoxic T lymphocytes (CTL) were generated using DC pulsed with a modified HLA-A2-restricted peptide epitope derived from the melanoma antigen MART-1. DC activated with a combination of CD40Ls and LPS were more efficient in eliciting MART-specific reactivity compared to DC activated with CD40Ls or LPS alone. These results demonstrate that multiple maturational signals have a positive impact on the ability of DC to secrete IL-12 and IL-10 and more importantly, to generate antigen-specific T lymphocytes.