Pilot trial of autologous dendritic cells loaded with tumor lysate(s) from allogeneic tumor cell lines in patients with metastatic medullary thyroid carcinoma.

Immunotherapy with autologous dendritic cells (DCs) loaded with tumor lysate(s) from allogeneic tumor cell lines is a novel strategy to induce immune responses in cancer patients. We report on a pilot trial of autologous DCs pulsed with tumor cell lysate derived from allogeneic medullary thyroid carcinoma (MTC) cell lines in patients with metastatic MTC. The purpose of this study was to assess the safety, resulting immune responses and clinical activity of the DCs. DCs were injected into a groin lymph node at 3-week intervals. Monitoring included serial calcitonin tumor marker measurements, radiological imaging and immunological in vitro tests (T-cell interferon-gamma detection assay, T-cell cytotoxicity assay). Ten patients (median age 47 years, range 29-77) were enrolled. DC vaccinations were well-tolerated and safe. After a median follow-up of 11 months, (range 7-26), 3 (30%) of 10 patients had stable disease, while 7 (70%) of the patients progressed during treatment. In 2 patients with stable disease, calcitonin decreased below treatment levels, paralleled by a T-cell-mediated immune response. Notably, treatment with DCs pulsed with a combination of different tumor cell lysates was followed by a calcitonin decrease in 4 patients who had previously experienced a calcitonin increase during monotherapy with DCs pulsed with a single lysate. Allogeneic tumor cell lysate-based DC immunotherapy is well-tolerated and safe. Combined treatment with different tumor cell lysate-pulsed DCs increases the likelihood of a calcitonin tumor marker response and should therefore be preferred over monotherapy with DCs pulsed with a single lysate.

Low telomerase activity: possible role in the progression of human medullary thyroid carcinoma.

Maintenance of telomere length has been reported to be an absolute requirement for unlimited growth of human tumour cells and in about 85% of cases, this is achieved by reactivation of telomerase, the enzyme that elongates telomeres. Only in rare cases, like in human medullary thyroid carcinomas (MTC), telomerase activity (TA) is low or undetectable; however, this does not limit tumours to become clinically significant. Here, we report that very low TA (below 5% of HEK293) observed in MTC cell strains derived from different patients, although not sufficient for immortalising the cells, is necessary for prolonging their replicative life span. Telomere erosion led to induction of a crisis period after long-term in vitro cultivation, which was reached earlier when treating the cells with MST-312, a telomerase inhibitor at non-toxic concentrations. Crisis was bypassed either by ectopic hTERT introduction or by infrequent spontaneous immortalisation, the latter of which was always associated with telomerase reactivation and changes of the cellular phenotype. While confirming the high importance of telomerase for tumour development, these data draw attention to the relevance of low TA: although insufficient for telomere stabilisation, it allows MTC cells to reach more population doublings, increasing both cell numbers as well as the risk of accumulating mutations and thus might support the development of clinically significant MTC.

Endorectal advancement flaps in the treatment of high anal fistula of cryptoglandular origin: full-thickness vs. mucosal-rectum flaps.

PURPOSE: The treatment of high anal fistula using endorectal advancement flaps represents an important technique to attain cure of fistulation and preserve anal continence. The creation of the advancement flap may comprise the rectal mucosa only or involve the full transection of the rectal wall. A comparison between full-thickness flaps and mucosal (partial-thickness) flaps was made to analyze the defining elements of successful fistula treatment: recurrence rates and anal continence. METHODS: A retrospective review of 54 consecutive patients with high anal fistula of cryptoglandular origin was undertaken. Patient risk was categorized according to previous anal surgery. Continence was assessed according to the Vaizey score. Recurrence rates were recorded in a long-term, complete follow-up. RESULTS: Thirty-four patients underwent surgery using a partial-thickness flap; in 20 patients the full-thickness flap was used. There were no major intraoperative or postoperative complications. Continence scores revealed significant incontinence in 11.1 percent of all patients. Full transection of the rectal wall for flap creation did not pose a threat to continence. Twenty-four percent of all patients suffered from a recurrence. Patients with four or more previous anal surgeries were at highest risk for failure. A single patient in the full-thickness flap group (5 percent) as opposed to 12 patients (35.3 percent) in the partial-thickness group suffered from recurrence. CONCLUSION: The comparison of partial-thickness to full-thickness endorectal advancement flaps suggests an improvement of recurrence rates without higher incontinence rates when a full mobilization of the rectal wall is performed.

Heat shock treatment of tumor lysate-pulsed dendritic cells enhances their capacity to elicit antitumor T cell responses against medullary thyroid carcinoma.

BACKGROUND: In vitro and in vivo studies have shown that dendritic cells (DCs) can stimulate antitumor T cell responses against medullary thyroid carcinoma (MTC). However, despite promising results in selected cases, the clinical efficacy of DC immunotherapy in patients with MTC has been limited. Recently, it has been demonstrated in mice that heat shock enhances the capacity of bone-marrow-derived DCs to stimulate antigen-specific T cells. The aim of our investigations was to evaluate whether heat shock also increases the capacity of human monocyte-derived DCs to stimulate antitumor T cell responses against MTC tumor cells. METHODS: DCs from six patients with metastatic MTC were pulsed with tumor lysate derived from allogeneic MTC tumor cells and were heat shocked for 12 h at 40 C or kept at 37 C. Thereafter, the DCs were matured and cocultured with T cells. Finally, the cytotoxic activity of T cells against MTC tumor cells was measured in vitro. RESULTS: In all patient samples, cytotoxic T cell responses against MTC tumor cells could be induced. Notably, heat-shocked DCs were more potent stimulators of cytotoxic T cell responses than control DCs, with T cells stimulated with heat-shocked DCs displaying a significantly increased cytotoxic activity against MTC tumor cells as compared with T cells stimulated with control DCs. In none of the experiments was a cytotoxic T cell response against unrelated pancreatic tumor cells (PANC-1) observed, using both control and heat-shocked DCs. CONCLUSIONS: Our study shows that heat-shocking DCs may be a valuable strategy to increase the immunostimulatory capacity of DCs used for immunotherapy of MTC.

Inosine 5'-monophosphate dehydrogenase inhibition by mycophenolic acid impairs maturation and function of dendritic cells.

BACKGROUND: The mechanism of action of mycophenolic acid (MPA) has been described as a blockade of inosine 5'-monophosphate dehydrogenase (IMPDH) and is thought to selectively influence T- and B-lymphocytes due to their strong dependency on guanine nucleotides synthesized via the de novo purine synthesis pathway. Recent evidence suggests MPA to affect antigen-presenting cells. METHODS: Using CD14+ derived human dendritic cells (DC) we have investigated the effects of MPA on differentiation, maturation and function and studied intracellular nucleotide content and IMPDH activity. RESULTS: GTP content and IMPDH activities of DC were strongly and dose-dependently decreased when MPA was present during the entire culture period or was added after the fifth (immature DC) or the seventh (mature DC) day of culture. Concurrent to low GTP levels, a dose-dependent reduction in the expression of CD80, CD86, CD40, CD54 and CD83 was seen which was accompanied by a decreased capacity of DC to stimulate T-cells. Our data for the first time shows a direct effect of MPA on the maturation and function of human CD14+ derived DC, indicates a role of IMPDH and a dependency on the de novo purine synthesis pathway.

Phase II trial of capecitabine plus nab-paclitaxel in patients with metastatic pancreatic adenocarcinoma.

BACKGROUND: Combination chemotherapy regimens including fluoropyrimidines as well as albumin-bound paclitaxel have shown promising results in patients with metastatic pancreatic adenocarcinoma (mPC). Based on the recently described excellent therapeutic index of capecitabine plus nab-paclitaxel in metastatic breast cancer, the present phase II trial was initiated. METHODS: Patients with previously untreated mPC were treated with capecitabine (825 mg/m(2) orally bid on days 1-15) and nab-paclitaxel (125 mg/m(2) intravenously on days 1 and 8) every 3 weeks. In patients without clinically relevant adverse reactions after the 1st treatment course (≤ grade 2 toxicities according to NCI-CTC vs. 4.0, exuding alopecia and fatigue of any degree) and adequate bone marrow function, the nab-paclitaxel dose was escalated to 100 mg/m(2) on days 1, 8 and 15 of each cycle; this intra-individual dose escalation was maintained during subsequent treatment courses if tolerated. The primary endpoint was objective response rate (ORR) according to RECIST criteria, assessed by an independent radiological review committee with evaluation performed every 2 months. RESULTS: Between 12/2013 and 01/2015, 30 patients were entered in this monocentric academic phase II trial. All patients had an ECOG performance status of 0-1, 80% had liver metastases and 23% had biliary stents in place at time of study initiation. Median CA19-9 was 1,004 U/mL (0.9-100.000 U/mL). In all patients except 2, a dose escalation of nab-paclitaxel after the 1st treatment course could be accomplished. The most common grade 3 adverse events (AEs) included transient sensory neuropathy (23%), (afebrile) neutropenia (17%), hand-foot-syndrome (13%) and phototoxic skin reaction (10%). Among 29 RECIST-response assessable patients, the ORR was 41.4% and stable disease (SD) was noted in 34.5%, resulting in a disease control rate (DCR) of 76%. After a median follow-up duration of 10.3 months (range, 1.9-19.0 months), 13/30 patients (43.3%) are presently being alive. CONCLUSIONS: The combination of capecitabine + nab-paclitaxel at these doses and scheduling was well tolerated and showed substantial antitumor efficacy.

Targeting vaccinia to solid tumors with local hyperthermia.

We have previously demonstrated that mutant vaccinia viruses target tumors in vivo after systemic delivery, and they have potential as vectors for tumor-directed gene therapy. We hypothesized that hyperthermia may augment vaccinia delivery to tumors after systemic injection, as hyperthermia increases the permeability of the endothelial vasculature to nanoparticles. In our in vitro experiments, we have shown that hyperthermia does not alter tumor cells' susceptibility to the intrinsic cytopathogenicity of the vaccinia virus compared with normothermic controls. Hyperthermia also does not change the viral infectivity or the level of viral marker gene expression when compared with normothermia. In an in vitro model of endothelial cell monolayer permeability, we have demonstrated that hyperthermia increases the permeability of the monolayer to vaccinia virus and that this phenomenon is completely reversible. In vivo we have demonstrated that the tumors that were treated with systemic vaccinia under conditions of hyperthermia (41.5 degrees C for 30 min) had significantly higher levels of vaccinia marker gene activity (>100-fold) than those treated under normothermic conditions (p < 0.05) and that this effect was specific to tumor. We also demonstrated that mice with 1 cm subcutaneous tumors treated with a systemically delivered, conditionally replicating vaccinia under conditions of hyperthermia had complete tumor regression in 50% and significantly improved antitumor response, compared with normothermic viral-treated controls (mean tumor volume of 110 mm(3) vs 3169 mm(3), 13 days after treatment) and compared with hyperthermic, nonvirally treated control animals (p < 0.0001). Regional hyperthermia improves vaccinia targeting to tumors, and thereby enhances the antitumor response.

Medullary thyroid carcinoma: autologous tumor cell lines for dendritic cell vaccination.

BACKGROUND: Medullary thyroid carcinoma (MTC) is a calcitonin-producing tumor of the parafollicular C-cells, accounting for 5-10% of all thyroid tumors. To date, the only effective treatment is the early and total surgical removal of all neoplastic tissue. As the prognosis of patients with advanced MTC, unresectable or distant metastases is poor, and chemotherapy or irradiation is of no significant value, alternative strategies have been sought. MATERIALS AND METHODS: A promising treatment approach for human MTC, that has already been introduced at our facility, is based on vaccination with autologous dendritic cells (DCs). Strong evidence that vaccination with autologous tumor lysate-pulsed DCs induces a specific immune response in vivo has been provided. However, the therapeutic success of this approach is sometimes critically limited by the small amount of tumor material available, especially from patients operated at an early tumor stage. Thus, it would be to the best advantage to have sufficient amounts of autologous tumor cells available for DC pulsing. RESULTS: A method to generate viable autologous tumor cell cultures from a variety of MTC tissue samples, even when the sample size is small, has been successfully established. These cell lines maintain their neuroendocrine phenotype. In addition, it can be shown that these cells also display the biological features of neuroendocrine tumor cells at the molecular level. CONCLUSION: The unlimited availability of these MTC cell lines makes it possible to specify cancerogenesis of MTC. In addition, the availability of sufficient amounts of tumor lysate from these cell lines offers the advantage of prolonged immunotherapy. Finally, these cell lines could be elegantly used as read-out system to monitor the in vivo immune response during immunotherapy with DC cell-based vaccination in patients suffering from MTC.

Dendritic cell vaccination in medullary thyroid carcinoma.

PURPOSE: Prognosis and treatment effectiveness for medullary thyroid carcinoma (MTC) are strictly related to tumor stage. Palliative treatment options show no significant benefit. A promising treatment approach for human cancer is based on the vaccination of autologous dendritic cells (DCs). EXPERIMENTAL DESIGN: The objective of this study was to evaluate the effectiveness of DC vaccines in MTC patients. Therefore, we generated autologous tumor lysate-pulsed DCs from 10 patients suffering from advanced MTC for repeated vaccination. Mature DCs were derived from peripheral blood monocytes by using CD14 magnetic bead selection and subsequent culture in the presence of granulocyte macrophage colony-stimulating factor, interleukin 4, and tumor necrosis factor alpha with or without addition of IFN-gamma. DCs were loaded with tumor lysate and further injected into a groin lymph node. Toxicity, tumor marker profile, immune response, and clinical response were determined. RESULTS: Vaccination was well tolerated and induced a positive immunological response in all of the tested patients as evaluated by in vivo delayed-type hypersensitivity reactivity or in vitro intracytoplasmic IFN-gamma detection assay. Three patients had a partial response, 1 patient presented a minor response, and 2 patients showed stable disease. The remaining 4 patients had progressive disease. CONCLUSIONS: These data provide strong evidence that vaccination with tumor-lysate pulsed DCs results in the induction of a specific immune response in patients suffering from MTC. Objective clinical responses could be observed even for far-advanced disease. Therefore, we suggest that MTC is particularly suited for DC-based immunotherapy.

Immunopathologic features of allergic contact dermatitis in humans: participation of plasmacytoid dendritic cells in the pathogenesis of the disease?

Contrary to our abundant knowledge about the sensitization phase of human contact hypersensitivity, little is known about the cell types orchestrating the effector phase. In order to address this issue, we phenotypically analyzed biopsies from 72 h epicutaneous patch test reactions (n=10) and normal human skin (n=5) for the presence of various leukocyte differentiation antigens. The inflammatory infiltrate was dominated by CD3+/CD4+ T cells with approximately 30% of the cells coexpressing CD25 and CTLA-4, a phenotype consistent with either activated effector or regulatory T cells. In our search for professional antigen-presenting cells, we were surprised to find not only sizeable numbers of CD1a+ dendritic cells and CD1c+ dendritic cells, but also of CD123+, CD45RA+, BDCA-2+, CLA+, and CD62L+ plasmacytoid dendritic cells. Although virtually absent in normal human skin, these cells were detectable already 6 h after hapten challenge and were often found in close proximity to CD56+ natural killer cells, indicative of a functional interaction between these cell types. The detailed knowledge of the cellular composition of the inflammatory infiltrate in allergic contact dermatitis and its kinetics should form the basis for the investigation of the immunologic and molecular events operative in the perpetuation and resolution of the eczematous response.

Augmentation of endothelial cell monolayer permeability by hyperthermia but not tumor necrosis factor: evidence for disruption of vascular integrity via VE-cadherin down-regulation.

Following isolated limb perfusion (ILP) with hyperthermia (H T), TNF and melphalan, there is immediate tumor softening secondary to augmentation of capillary leak in the tumor neovasculature. TNF can induce vascular permeability but is always used with HT during ILP and the contribution of the latter on permeability is not known. This study characterizes the effects of HT and TNF on vascular permeability in vitro. Permeability across confluent human umbilical vein endothelial cells exposed to HT (40 degrees C) with or without 0.1-1000 ng/ml TNF was assessed by quantitating flux of albumin bound Evan's Blue dye from the upper to lower chamber. Immunofluorescent staining for VE-cadherin and F-actin was performed after human umbilical vein endothelial cells (hUVECs) were exposed to these conditions. HT induced a significant and reversible increase in permeability compared to untreated hUVECs (p<0.001) whereas barrier function was not altered by TNF. Untreated hUVECs had uniform cell surface staining for VE-cadherin, the primary endothelial intercellular adhesion molecule, with colocalization of F-actin cytoskeletal elements. HT resulted in a marked decrease in VE-cadherin staining and contraction of F-actin at sites of endothelial cell-cell separation. These data demonstrate that under conditions relevant to those used in ILP, HT but not TNF contributes to a rapid and reversible change in endothelial cell permeability in association with a down regulation of VE-cadherin. These data support the use of HT in isolation perfusion and demonstrate a novel mechanism for alterations in microvascular permeability by HT.

Heat shock protein expression induced by percutaneous radiofrequency ablation of hepatocellular carcinoma in vivo.

Heat shock proteins (HSPs) are known to be of crucial importance in host-tumor interactions. The goal of this study was to determine whether there was an increase in HSP expression in a patient suffering from unresectable hepatocellular carcinoma treated with percutaneous radiofrequency (RF) ablation. Immediately before RF ablation, a computed tomography (CT)-guided core needle biopsy (diameter, 18 gauge) was obtained from the tumor. The RF ablation was then performed using a saline-perfused RF ablation system (diameter of RF electrode, 15 gauge; ablation time, 10 min). Twenty-four hours after tumor ablation, core needle biopsy was repeated, and biopsy specimens were obtained from the residual tumor margin visible on contrast-enhanced CT. For both procedures, no side effects or clinically relevant complications were observed. The specimens were mapped by immunohistochemistry, determining the cellular expression of HSP 70 and HSP 90. After RF ablation, in the cytoplasm and at the tumor cell surface, an 8-fold increase in HSP 70 and a 1.2-fold increase in HSP 90 was observed, respectively. In the cell nucleus, the HSP 90 expression before RF ablation was 10%, and decreased to 0% after RF ablation. We have demonstrated that, following RF ablation, cellular expression of HSP 70 and HSP 90 in hepatocellular carcinoma is increased. We further suggest that routing of HSP 90 from the nucleus to the cell surface occurred after RF ablation. This may be of relevance in further therapeutic anti-tumor strategies.

In vivo induction of dendritic cell-mediated cytotoxicity against allogeneic pancreatic carcinoma cells.

The objective of this study was to assess the toxicity and immunological response induced by autologous dendritic cells (DCs) pulsed with allogeneic tumor lysate in a pancreatic cancer patient. The lack of available tumor peptides in pancreatic cancer strongly supports the idea to use allogeneic tumor cells as a source of antigens. The patient suffering from a stage IV pancreatic cancer received 1-2x10(7) autologous monocyte-derived DCs in three-week intervals injected into a groin lymph node. Monocytes from peripheral blood were isolated by magnetic bead selection. For the first ten vaccinations DCs were loaded with autologous tumor cell lysate obtained during surgical exploration. After consumption of the autologous lysate, equal numbers of DCs were pulsed with lysate of the tumor cell line AsPc-1 and BxPc-3 for a further five vaccinations. Peripheral mononuclear cells (PMNCs) were harvested after the seventh and compared with PMNCs obtained after the fourteenth vaccination for immunological response. Delayed type hypersensitivity reactivity to DCs pulsed with autologous and allogeneic tumor lysate was also assessed. The patient received a total of fifteen vaccinations. There was no toxicity or evidence of autoimmunity observed. Delayed type hypersensitivity was found to be positive for the autologous as well as the allogeneic tumor lysate pulsed DCs. in vitro cytotoxicity assays demonstrated a dramatic increase of the PMNC killing capacity against the pancreatic cancer cell lines AsPc-1 and BxPc-3 after the fourteenth compared to the seventh vaccination. CT scans revealed a stable disease for six months. The administration of autologous DCs pulsed with allogeneic tumor lysate is non-toxic and suitable for inducing an immunological anti-tumor response. Even though this study was confined to a single patient, the data might open a door for novel immunotherapeutical strategies.

Hyperthermia improves cellular immune response to human hepatocellular carcinoma subsequent to co-culture with tumor lysate pulsed dendritic cells.

Dendritic cells play a major role in cellular immunity. The crucial steps of antigen presentation and processing by DCs may be limiting factors for adoptive cellular immunotherapy. Here, we investigated whether hyperthermia of human hepatocellular carcinoma (HCC) cells induces enhanced cytotoxic cellular immune response. Peripheral blood mononuclear cell (PBMC)-derived DCs were pulsed with tumor cell lysate of the human HCC cell line HepG2, which had been heat shocked prior to incubation for 5 h. Subsequent to TNFalpha-induced maturation DCs were co-cultured with autologous CD4+ and/or CD8+ cells, and T cell mediated cytolysis of HepG2 cells was assessed. We observed enhanced CD4+/8+ cellular cytotoxicity against HepG2 cells subsequent to co-culture with the heat shocked tumor lysate pulsed DCs as compared to pulsing DCs with lysate of non-heat shocked tumor cells. The improved cellular immune response can be related to enhanced expression of HSP 70 and 90 in HepG2 cells upon hyperthermia.

Gastrointestinal autonomic nerve tumors: a surgical point of view.

AIM: Gastrointestinal autonomic nerve tumors are uncommon stromal tumors of the intestinal tract. Their histological appearance is similar to that of other gastrointestinal stromal tumors. We report two cases and performed an analysis of the literature by comparing our findings with the available case reports in the medical literature. METHODS: Two patients were admitted with abdominal tumor masses. One occurred in the stomach with large multiple liver metastases and the second originated in Meckel's diverticulum. The latter site has never been reported previously. Both patients underwent surgery. In one patient gastrectomy, right liver resection and colon transversum resection were performed to achieve aggressive tumor debulking. In the other patient the tumor bearing diverticulum was removed. RESULTS: Postoperative recovery of both patients was uneventful. Histological examination, immunohistochemical analysis and electron microscopy revealed the diagnosis of a gastrointestinal autonomic nerve tumor. The patient with the tumor in Meckel's diverticulum died 6 mo after surgery because of pneumonia. The patient with liver metastases have been alive 13 years after initial tumor diagnosis and 7 years after surgery with no evidence of tumor progression. In light of our results, we performed a thorough comparison with available literature reports. CONCLUSION: Radical surgical resection of gastrointestinal autonomic nerve tumors seems to be the only available curative approach to date, and long term survival is possible even in large metastasized tumors.

[Primary immune thrombocytopenia in adults: diagnostics and treatment consensus statement of the Austrian Society of Hematology and Oncology (ÖGHO)]. / Primäre Immunthrombozytopenie des Erwachsenen - Diagnostik und Therapie, Konsensus-Statement der Österreichischen Gesellschaft für Hämatologie und Onkologie (ÖGHO).

Immune Thrombocytopenia (ITP) is a rare and - in most patients - mild disease, but might be associated with severe or even life-threatening bleeding complications. The treatment of ITP has partly changed in recent years, due to new therapeutic options. International guidelines changed accordingly. This consensus statement by the Austrian Society of Hematology and Oncology (OEGHO) is not a new evaluation of the current evidence, but rather tries to discuss the available international guidelines and adapt them to the situation in Austria. The subject is primary ITP in adults only. Classification, epidemiology, clinical presentation and diagnostics of ITP, and especially the management of this disease, are discussed in detail. This includes current aspects of first, second, and third line therapies, splenectomy with its indications and contraindications, and the use of new therapeutic options like thrombopoetin receptor agonists (TRA).

Cryopreservation of monocytes is superior to cryopreservation of immature or semi-mature dendritic cells for dendritic cell-based immunotherapy.

Cryopreservation of immature or mature dendritic cells (DC) has been proposed as a suitable method to gain large numbers of DC for immunotherapeutic trials against cancer. However, clinical studies using cryopreserved DC have demonstrated only limited success so far. The aim of this study was to investigate whether cryopreservation of monocytes elicits more potent DC and whether these DC are comparable to freshly generated DC preparations. Monocytes, either separated immunomagnetically or by means of leukapheresis and elutriation, were differentiated into DC and cryopreserved at various developmental stages. DC preparations were analyzed regarding recovery, viability, phenotype, and functional properties. In contrast to DC frozen at their immature or semi-mature state, generation of DC from cryopreserved monocytes elicited viability values comparable with freshly generated DC. Furthermore, using frozen monocytes for DC differentiation revealed improved expression of DC surface markers and interleukin-12p70 secretion as compared with DC generated from frozen immature or frozen semi-mature DC. Impaired phenotypical appearance of the latter DC variants was further substantiated by functional analysis. T-cells cocultured with these DC showed decreased expression of interferon-gamma and granzyme B, and lowered proliferation when compared with T-cells cocultured with DC generated from frozen monocytes or DC generated from freshly isolated monocytes. Induction of regulatory T-cell populations was negligible among all investigated DC preparations. These findings may further improve DC-based immunotherapeutical protocols. Cryopreservation of unchallenged monocytes enables targeted therapy by loading DC with varying antigenic compositions in case of tumor escape during treatment.

Allogeneic tumor lysate can serve as both antigen source and protein supplementation for dendritic cell culture.

BACKGROUND: Recent preclinical and clinical evidence suggests the use of allogeneic tumor as a source of antigen for DC-based immunotherapy against cancer. We hypothesized that addition of allogeneic tumor lysate to monocyte-derived DC culture could serve a dual purpose: (1) antigen source and (2) protein supplementation of DC culture media. Protein supplementation whether of known origin (human serum/plasma, fetal bovine serum, human serum albumin) or undeclared origin ("serum-free" media) is a source of variability and bias. We addressed the question whether protein supplementation can be omitted in the presence of allogeneic tumor lysate. MATERIALS AND METHODS: Human DC cultured in the presence of lysate from medullary thyroid carcinoma (MTC) cell line SHER-I (TuLy-DC) and DC pulsed with the same lysate but cultured in the presence of FBS (FBS-DC) were assessed for morphology, phenotype, maturation and functional properties. RESULTS: In comparison of FBS-DC/TuLy-DC no significant differences in morphology, phenotype and maturation could be detected. Both culture conditions produced CD1a(high), CD14(low) DC with high expression of costimulatory molecules and CD83 upon stimulation. TuLy-DC gave significantly better yields and produced more IL12p70. DC showed high (allo)stimulatory capacity toward T-cells. TuLy-DC induced more intracellular IFNgamma in CD8+T-cells of vaccinated MTC patients. Both types of DC induced killing of SHER-I after short in vitro restimulation. Tumor lysate from SHER-I can substitute for further protein supplementation in DC culture. Allogeneic tumor lysates should be taken into consideration as both source of antigen and protein supplementation in monocyte-derived DC culture.

Prognostic value of T-cell receptor gamma rearrangement in peripheral blood or bone marrow of patients with peripheral T-cell lymphomas.

Peripheral T-cell lymphomas (PTCL) have a variable outcome. We have investigated the prognostic value of molecular staging in non-anaplastic PTCL. T-cell receptor gamma rearrangements were routinely determined in peripheral blood (n = 40) and bone marrow (n = 38) of patients with PTCL (75% unspecified) by conventional PCR at diagnosis. Tissue controls for PCR included 24 tumour biopsies. Twenty-four patients (60%) had a PCR-detectable clonal TCR gamma rearrangement in PB or BM. These TCR gamma PCR positive patients had significantly more stage IV disease (14 patients of 15 patients; P = 0.001), elevated LDH (14 of 18 patients; P = 0.04), higher IPI (16 of 21 patients; P = 0.03), more anemia (15 of 19 patients; P = 0.02) and lower platelet counts (seven of seven patients; P = 0.02). Clinical outcome of this clonal group was characterised by lower complete remission rates (37.5% vs. 62.5%), and overall response rates (58.3% vs. 87.5%; P < 0.05) as well as a significantly shorter median overall survival (12.8 vs. 30.0 months; P = 0.006). Patients with clinical stages I - III but molecular stage IV had an equally poor overall survival when compared with patients with clinical stage IV (15.8 vs. 13.9 months). In contrast, patients with CS I - III in the absence of a TCR gamma rearrangement in PB or BM had a favourable outcome with an estimated overall survival of 70% at 3 and 5 years. Molecular staging in PB and BM by TCR gamma PCR at diagnosis may serve as a useful prognostic tool in PTCL.

Vitamin D3 down-regulates monocyte TLR expression and triggers hyporesponsiveness to pathogen-associated molecular patterns.

Toll-like receptors (TLR) represent an ancient front-line defence system that enables the host organism to sense the presence of microbial components within minutes. As inducers of inflammation, TLR act as important triggers of distinct entities such as sepsis or autoimmune disease exacerbation. We report here that vitamin D3 [1alpha,25-dihydroxycholecalciferol, 1,25(OH)(2)D3] suppresses the expression of TLR2 and TLR4 protein and mRNA in human monocytes in a time- and dose-dependent fashion. Despite 1,25(OH)(2)D3-induced up-regulation of CD14, challenge of human monocytes with either LPS or lipoteichoic acid resulted in impaired TNF-alpha and procoagulatory tissue factor (CD142) production, emphasizing the critical role of TLR in the induction of inflammation. Moreover, reduced TLR levels in 1,25(OH)(2)D3-treated phagocytes were accompanied by impaired NF-kappaB/RelA translocation to the nucleus and by reduced p38 and p42/44 (extracellular signal-regulated kinase 1/2) phosphorylation upon TLR-ligand engagement. Both TLR down-regulation and CD14 up-regulation were substantially inhibited by the vitamin D receptor (VDR) antagonist ZK 159222, indicating that the immunomodulatory effect of 1,25(OH)(2)D3 on innate immunity receptors requires VDR transcription factor activation. Our data provide strong evidence that 1,25(OH)(2)D3 primes monocytes to respond less effectively to bacterial cell wall components in a VDR-dependent mechanism, most likely due to decreased levels of TLR2 and TLR4.