Bendamustine, lenalidomide and dexamethasone (BRd) has high activity as 2nd -line therapy for relapsed and refractory multiple myeloma - a phase II trial.

The combination of lenalidomide (Revlimid® , R) and dexamethasone (d) is a standard regimen for patients with relapsed/refractory multiple myeloma (rrMM). With this regimen, only a small fraction of patients will achieve high quality responses [≥ very good partial response (VGPR)]. The combination of bendamustine (B), lenalidomide and dexamethasone (BRd) has shown high efficacy in patients with advanced rrMM. However, dose-limiting haematotoxicity restricted its use in extensively pre-treated patient populations. This prospective, multicentre Phase II study evaluated the efficacy and safety of BRd in rrMM patients with one prior line of therapy. Fifty patients were enrolled (median age 68·5 years [range 46-83]) and were treated with B 75 mg/m2  days 1, 2; R 25 mg days 1-21 and d (40/20 mg) days 1, 8, 15 and 22, for 6 28-day induction cycles, followed by 12 cycles with Rd alone. Pegfilgrastim was administered according to protocol-defined criteria. The study aimed to demonstrate a complete response (CR)/VGPR rate of >40% after induction therapy. Of 45 evaluable patients, 23 (51%) achieved a CR/VGPR. Grade 4 neutropenia or thrombocytopenia occurred in 17 (34%) and 8 (16%) of patients, respectively. BRd is a safe and efficacious regimen as a second line treatment for rrMM, leading to high quality responses in a considerable proportion of patients.

Prevention of lethal tumor growth and generation of long-lasting immunity in vivo using CD137L and interleukin-12 gene transfer.

In a wide range of solid tumors, overexpression of CD137L has been shown to induce tumor immunity partly due to the stimulation of CD8+ CTL, which was even increased when immunotherapy with interleukin-12 (IL12) was additionally employed. However, little in known regarding hematologic neoplasias in this respect. Of the 8 animals receiving IL12-secreting tumor cells, 2 died. Animals treated with CD137L-expressing tumor cells and the combination group, all animals survived. Interestingly, re-challenge with wild-type tumor cells was rejected by all animals in the CD137L group and all remaining animals in the IL12 group, while these in the control group died. IL12- and CD137L-transfected plasmocytoma cells prevented tumor growth and induced long-lasting immunity. Our results warrant follow-up for future clinical use in patients with myeloma.

Minimal size MIDGE vectors improve transgene expression in vivo.

Viral and plasmid vectors may cause immunological side-effects resulting from the expression of therapeutically unwanted genes and from CpG motifs contained in their sequence. A new vector type for minimalistic, immunological-defined gene expression (MIDGE) may overcome these problems. MIDGE is a minimal size gene transfer unit consisting of the expression cassette, including promotor, gene and RNA-stabilizing sequences, flanked by two short hairpin oligonucleotide sequences. DNA not encoding the desired gene is reduced to a minimum. To compare transfection efficiencies in vivo hydrodynamics-based, systemic transfection was performed in BALB/c mice with MIDGE vectors and corresponding plasmids. The transfection efficiencies of the MIDGE vectors as measured by luciferase expression were significantly higher in liver (2.5-fold), lung (3.5-fold), kidneys (3.9-fold) and heart (17-fold) as compared to plasmids. The mean numbers of MIDGE vector molecules per cell as measured by quantitative PCR were also significantly higher. These advantages suggest the preferential use of this new vector type for clinical gene therapy studies.

Comparison of rapidly cycled tandem high-dose chemotherapy plus peripheral-blood stem-cell support versus dose-dense conventional chemotherapy for adjuvant treatment of high-risk breast cancer: results of a multicentre phase III trial.

BACKGROUND: Breast cancer with extensive axillary-lymph-node involvement has a poor prognosis after conventional treatment. In trials with historical controls, high-dose chemotherapy produced improved outcomes. We compared an intensive double-cycle high-dose chemotherapy regimen with an accelerated conventionally dosed regimen in high-risk breast cancer in a multicentre trial. METHODS: Patients with at least nine positive nodes were randomly assigned either two courses of accelerated (2-week intervals, with filgrastim support), conventionally dosed epirubicin and cyclophosphamide followed by two courses of high-dose chemotherapy (epirubicin, cyclophosphamide, and thiotepa supported by peripheral-blood progenitors) or four identical cycles of epirubicin and cyclophosphamide followed by three cycles of accelerated cyclophosphamide, methotrexate, and fluorouracil. The primary endpoint was event-free survival. Analyses were done both by intention to treat and per protocol. FINDINGS: 403 patients were enrolled; 201 were assigned high-dose chemotherapy and 202 conventional treatment. The mean number of positive nodes was 17.6, and median follow-up was 48.6 months. 4-year event-free survival (intention-to-treat analysis) was 60% (95% CI 53-67) in the high-dose chemotherapy group and 44% (37-52) in the control group (p=0.00069). The corresponding overall survival was 75% (69-82) versus 70% (64-77; p=0.02). There were no treatment-related deaths. INTERPRETATION: Our finding of significant improvements in both event-free and overall survival for high-dose chemotherapy compared with a dose-dense conventional regimen contrasts with the results of other studies. The discrepancy might be due partly to design differences (tandem, brief induction) between our regimen and those studied in other trials. This approach merits further study.

Total ascitic fluid leukocyte count for reliable exclusion of spontaneous bacterial peritonitis in patients with ascites.

BACKGROUND AND AIMS: The diagnosis of spontaneous bacterial peritonitis (SBP) in patients with ascites is established by definition with a polymorphonuclear (PMN) cell count in the ascitic fluid greater than 0.250 g/l determined via cytological (microscopic) examination. In this study, we correlated the automatically assessed total ascitic nucleated cell count with PMN and determined its predictive value for diagnosis of SBP. METHODS: Six hundred and eleven consecutive paracenteses of 179 patients with ascites of various aetiologies (liver cirrhosis, hepatocellular carcinoma, peritoneal carcinomatosis, and ascites of other aetiology) were studied retrospectively. RESULTS: The most reliable diagnostic cut-off level was determined for differentiation between SBP and non-SBP via receiver operating characteristics analysis. A total ascitic nucleated cell count less than 1.0 g/l is unlikely to represent SBP (negative predictive value, 95.5%). CONCLUSIONS: If ascitic fluid samples with machine-made total ascitic nucleated cell count below 1.0 g/l are not followed by additional laboratory tests, the risk of missing the diagnosis of SBP is low. Applying these criteria we would have classified 51 samples of 611 samples (20 of 179 patients) wrongly using the cut-off value of 1 g/l. On the other hand we would have spared cytologic evaluation in about 63% of paracentesis performed in our hospital. Nevertheless, to insure patient safety, standard laboratory analysis is recommended in circumstances of clinical uncertainty. Thus, patients with first manifestation of ascites should always receive cytologic examination and full diagnostic investigation to exclude other causes of ascites.

Invasive fungal infections in neutropenic enterocolitis: a systematic analysis of pathogens, incidence, treatment and mortality in adult patients.

BACKGROUND: Neutropenic enterocolitis is a life-threatening complication most frequently occurring after intensive chemotherapy in acute leukaemias. Gramnegative bacteria constitute the most important group of causative pathogens. Fungi have also been reported, but their practical relevance remains unclear. The guidelines do not address concrete treatment recommendations for fungal neutropenic enterocolitis. METHODS: Here, we conducted a metaanalysis to answer the questions: What are frequency and mortality of fungal neutropenic enterocolitis? Do frequencies and microbiological distribution of causative fungi support empirical antimycotic therapy? Do reported results of antimycotic therapy in documented fungal neutropenic enterocolitis help with the selection of appropriate drugs? Following a systematic search, we extracted and summarised all detail data from the complete literature. RESULTS: Among 186 articles describing patients with neutropenic enterocolitis, we found 29 reports describing 53 patients with causative fungal pathogens. We found no randomised controlled trial, no good quality cohort study and no good quality case control study on the role of antifungal treatment. The pooled frequency of fungal neutropenic enterocolitis was 6.2% calculated from all 860 reported patients and 3.4% calculated from selected representative studies only. In 94% of the patients, Candida spp. were involved. The pooled mortality rate was 81.8%. Most authors did not report or perform antifungal therapy. CONCLUSION: In patients with neutropenic enterocolitis, fungal pathogens play a relevant, but secondary role compared to bacteria. Evidence concerning therapy is very poor, but epidemiological data from this study may provide helpful clues to select empiric antifungal therapy in neutropenic enterocolitis.

Intratumoral immunization with tumor RNA-pulsed dendritic cells confers antitumor immunity in a C57BL/6 pancreatic murine tumor model.

Pancreatic tumors lack adequate recruitment of immunocompetent cells, especially dendritic cells (DCs). We have shown previously that coculturing of natural killer-like T cells with DCs transfected with pancreatic tumor cell line-derived RNA reverses pancreatic carcinoma cell resistance by directly triggering natural killer-like T lymphocytes in vitro. In the present study, we tested triggering of specific T lymphocytes in vivo by using an immunocompetent mouse strain (C57BL/6). Syngenic, bone marrow-derived DCs were pulsed with tumor RNA derived from the pancreatic cell line PANC02. This cell line is a ductal pancreatic adenocarcinoma and shows high resistance to every known class of clinically active antitumor agent. PANC02 cells were implanted orthotopically via ultrasound guidance and led to pancreatic tumor formation in all of the mice. Thereafter, tumor RNA-pulsed DCs were injected intratumorally. Intratumoral administration of tumor RNA-pulsed DCs induced significantly more potent protective immunity than s.c. or i.v. administration. It was significantly more effective than administration with unpulsed DCs or DCs pulsed with a control tumor RNA derived from a lymphomatous cell line (EL4). The antitumor effect was caused by induction of antigen-specific T lymphocytes as shown by additional in vitro studies. These results favor intratumoral injection of tumor RNA-pulsed DCs for immunotherapy of pancreatic cancer.

Retroviral IFN-alpha gene transfer combined with gemcitabine acts synergistically via cell cycle alteration in human pancreatic carcinoma cells implanted orthotopically in nude mice.

Standard chemotherapy for pancreatic carcinoma is based on the use of gemcitabine. The clinical benefit of interferon-alpha (IFN-alpha) in advanced pancreatic cancer has been shown. However, it has been demonstrated that to be effective, there is a need for a constant amount of IFN-alpha at the site of the tumor. Therefore, we examined transfection of the human pancreatic cancer cell line DAN-G with a retrovirus encoding for IFN-alpha and the effect of IFN-alpha gene expression alone or in combination with gemcitabine on growth inhibition of DAN-G pancreatic cancer cells in vitro and in vivo in orthotopically implanted DAN-G cells in nude mice. DAN-G cells could be efficiently transfected retrovirally by the human IFN-alpha gene and significantly enhanced the levels of IFN-alpha mRNA. In vitro gemcitabine led to an alteration of G1/S phase progression in transduced as well as untransduced cells, whereas IFN-alpha led to a significant decrease in cell viability in the transduced cells via delay in the progression of the S phase but no alteration of G1/S phase progression. In vivo, tumor volume in mice was reduced significantly with gemcitabine combined with IFN-alpha (76% +/- 8.3%) compared with gemcitabine alone (62.9% +/- 7.3%) or IFN-alpha alone (24.4% +/- 5.2%) compared with untreated animals. We conclude that gemcitabine and IFN-alpha concomitantly inhibited tumor cell proliferation significantly.

High-dose chemotherapy followed by autologous stem cell transplantation as first-line therapy in aggressive non-Hodgkin's lymphoma: a meta-analysis.

BACKGROUND AND OBJECTIVES: High-dose chemotherapy followed by autologous stem cell transplantation (HDT/ASCT) has proven to be superior to conventional chemotherapy in patients with chemosensitive relapse of aggressive non-Hodgkin's lymphoma (NHL). Therefore, HDT/ASCT was evaluated as part of first-line therapy. Several trials generated conflicting results. This meta-analysis summarizes the available evidence from all suitable studies. DESIGN AND METHODS: Prospective, randomized trials with HDT/ASCT as first-line therapy of aggressive lymphoma were included in this meta-analysis. The primary outcome was overall mortality. Statistical analysis applied the odds ratio (OR) and a fixed effects model. RESULTS: Eleven trials with 2228 patients were eligible for meta-analysis. Overall mortality was comparable in the HDT/ASCT and in control arms (OR=0.97, 95% CI: 0.69;1.36, p=0.9), with statistically significant heterogeneity between the trials. To resolve this, we tried to identify variables that could explain this heterogeneity. Among a range of methodological, patient- or treatment-related factors, subgroups formed by the proportion of bulky disease in treated patients, the type of therapy prior to HDT/ASCT, the drop-out rate from the HDT/ASCT arm, and the presence of high or high-intermediate risk IPI showed significant benefit for any of the treatment modalities. However, such post-hoc subgroup analysis may be considerably influenced by random or systemic biases. INTERPRETATION AND CONCLUSIONS: Overall, the analysis of published evidence reveals very heterogeneous results and no overall survival benefit. Therefore, HDT/ASCT cannot be recommended as standard first line treatment for patients with aggressive NHL. However, the exploratory analyses presented here may help to design new trials for this treatment modality.

Human cytokine-induced killer cells have enhanced in vitro cytolytic activity via non-viral interleukin-2 gene transfer.

Modulation of the immune system by genetically modified immunological effector cells is of potential therapeutic value in the treatment of malignancies. Interleukin-2 (IL-2) is a crucial cytokine which induces potent antitumor response. Cytokine-induced killer cells (CIK) have been described as highly efficient cytotoxic effector cells capable of lysing tumor cell targets and are capable of recognizing these cells in a non-MHC restricted fashion. Dendritic cells (DC) are the major antigen presenting cells. This study evaluated the antitumor effect of CIK cells which were non-virally transfected with IL-2 and co-cultured with pulsed and unpulsed DC. Human CIK cells generated from peripheral blood were transfected in vitro with plasmid encoding for the human IL-2. Transfection involved a combination of electrical parameters and a specific solution to deliver plasmid directly to the cell nucleus by using the Nucleofector® electroporation system. Nucleofection resulted in the production of IL-2 with a mean of 478.5 pg/106 cells (range of 107.6-1079.3 pg /106 cells/24 h) compared to mock transfected CIK cells (31 pg/106 cells) (P = 0.05). After co-culturing with DC their functional ability was assessed in vitro by a cytotoxicity assay. On comparison with non-transfected CIK cells co-cultured with DCs (36.5 ± 5.3 %), transfected CIK cells co-cultured with DC had a significantly higher lytic activity of 58.5 ± 3.2% (P = 0.03) against Dan G cells, a human pancreatic carcinoma cell line.

Effects of recombinant adenovirus-mediated expression of IL-2 and IL-12 in human B lymphoma cells on co-cultured PBMC.

BACKGROUND: Modulation of the immune system by genetically modified lymphoma cell vaccines is of potential therapeutic value in the treatment of B cell lymphoma. However, the anti-tumor effect of any single immunogene transfer has so far been limited. Combination treatment of recombinant IL-2 and IL-12 has been reported to be synergistic for inducing anti-tumor responses in solid tumors but the potential of IL-2/IL-12 gene modified B cell lymphoma cells has not been explored yet. METHODS: Using three different human B cell lymphoma cell lines and primary samples from patients with B cell neoplasms, expression levels of the coxsackie B-adenovirus receptor (CAR) and alpha (v) integrins were analyzed by fluorescence-activated cell sorter (FACS). Adenoviral transduction efficiencies were determined by GFP expression analysis and IL-2 and IL-12 cytokine production was quantified by enzyme-linked immunosorbent (ELISA) assays. Proliferative activities of peripheral blood mononuclear cells (PBMC) stimulated with either cytokine derived from supernatants of transduced lymphoma cells were measured by cell proliferation (MTT) assays. An EuTDA cytotoxicity assay was used to compare cytotoxic activities of IL-2 and/or IL-12 stimulated PBMC against unmodified lymphoma cells. RESULTS: We found that B cell lymphoma cell lines could be transduced with much higher efficiency than primary tumor samples, which appeared to correlate with the expression of CAR. Adenoviral-expressed IL-2 and IL-12 similarly led to dose-dependent increases in proliferation rates of PBMC obtained from healthy donors. IL-2 and/or IL-12 transduced lymphoma cells were co-cultured with PBMC, which were assayed for their cytolytic activity against unmodified lymphoma cells. We found that IL-2 stimulated PBMC elicited a significant anti-tumor effect but not the combined effect of IL-2/IL-12 or IL-12 alone. CONCLUSION: This study demonstrates that the generation of recombinant adenovirus modified lymphoma cell vaccines based on lymphoma cell lines expressing IL-2 and IL-12 cytokine genes is technically feasible, induces increases in proliferation rates and cytotoxic activity of co-cultured PBMC, and warrants further development for the treatment of lymphoma patients in the future.

Sequential high-dose chemotherapy with autologous stem cell support in patients with limited-stage small cell lung cancer.

To determine the feasibility and safety of multiple sequential courses of high-dose chemotherapy with peripheral blood progenitor cells (PBSCs) followed by thoracic irradiation (54 Gy) and prophylactic cranial irradiation (PCI 30 Gy) in patients with limited disease (LD) small cell lung cancer (SCLC), six patients received one cycle of conventional-dose chemotherapy followed by four cycles of high-dose chemotherapy with stem cell support. Neither significant adverse side-effects nor treatment mortality were observed. CR and almost CR (> 90%) was obtained in five out of six patients while one patient had a partial response (PR). The median survival was 21.3 months (range 8-34). However, the benefit of the high CR rate and prolonged survival did not result in cure even in these favorable circumstances, because all the patients died of recurrence in the cerebral nervous system. According to our data we stopped this chemotherapy regime because it was not able to prevent cerebral disease.

Primary small cell carcinoma of the esophagus: patient data metaanalysis and review of the literature.

We analysed the typical features of primary small cell carcinoma of the esophagus (SCCE) with emphasis on occurrence, behaviour, outcome and treatment options. This metaanalysis was aimed at collecting and analyzing information from international studies about handling this disease. This seems necessary due to the rarity of this disease. Studies were acquired from electronic databases and reference lists. We finally analysed 313 patient cases from the literature with oesophageal SCC. A data extraction was accomplished referring to 13 evaluable features that are described in the "methods", whereof 7 were analyzed with univariate and multivariate tests. Three hundred thirteen cases were analyzed, 109 patients (35%) had limited stage (LS), whereas 167 (54%) had extensive stage (ES). There is no information about the remaining 35 patients concerning the stage. Univariate and multivariate analysis showed only age (<50 years vs. >50 years, HR 1.024; 95% CI 1.000-1.041, P<0.0001) and disease stage (LS vs. ES, HR 4.884; 95% CI 2.572-9.27, P<0.0001) as significant prognostic factors. There also was a statistically significant difference in survival between those patients who received therapy compared to those who only received best supportive care (11.6 months vs. 0.8 months, HR 0.093, CI 95% 0.053-0.16, P<0.001). In this first multivariate analysis for SCCE we show that SCCE is an aggressive type of tumour with a shorter survival rate compared to its counterpart from the lung. It is demonstrated that only disease stage (limited vs. extensive stage), age (<50 years vs. >50 years) and therapy are independent significant predictors of prognosis.

Increase of in vivo antitumoral activity by CD40L (CD154) gene transfer into pancreatic tumor cell-dendritic cell hybrids.

OBJECTIVES: Fusion of dendritic cells (DC) with tumor cells is an approach in immunotherapy combining antigenicity and capacity of antigen presentation to activate T cells for the induction of tumor-specific cytotoxic immunity. Although there have been reports of clinical benefit, response rates have been limited and further improvements are warranted. METHODS: We used murine DC and a novel protocol for an effective fusion of those cells with the murine pancreatic cell line Panc02. RESULTS: We observed 2 events: only moderate in vitro and in vivo cytotoxicity of tumor cell/DC hybrids and a down-regulation of costimulatory molecules on fused cells. Therefore, we transfected tumor cell/DC hybrids with an adenovirus expressing CD154 to improve DC activation and generating antitumor immune response without the need of CD4 T cells. High CD154 expression could be obtained by transfection of DC and Panc02 cells prior fusion. Furthermore, vaccination with CD154-transfected tumor cell/DC hybrid led to a significantly increased induction of cytotoxic T cells in vitro and to an improved antitumoral effect in an orthotopic in vivo mouse model. CONCLUSIONS: CD154-transfected tumor cell/DC hybrids are a promising approach to increase the efficiency of antitumoral response.

Pancreas carcinoma antigen fused to invariant chain elicits T-cell response and tumor growth inhibition.

OBJECTIVES: The major histocompatibility complex class II chaperone invariant chain (Ii) is widely used as a carrier for inserted antigenic sequences and their introduction into the class II processing pathway. The tumor-associated antigen core 2beta 1,6 N-acetylglucosaminyltransferase (C2GnT), a glycosyltransferase present in human pancreatic tumor cells, is not expressed by normal pancreatic tissues. METHODS: A set of expression vectors was engineered where the class II binding region of Ii was replaced by C2GnT-derived sequences. We investigated in vitro whether dendritic cells transfected with Ii-C2GnT constructs were capable to stimulate proliferation of CD4 T cells. We also tested whether vaccination with Ii-C2GnT would protect mice from tumor development. RESULTS: Invariant chain-C2GnT fusion proteins bind to human DR1, DR3, DR4 and to mouse I-A molecules. Our results demonstrate that the plasmid DNA encoding the C2GnT epitope embedded in Ii induces tumor-specific T-cell responses. Mice immunized with the Ii constructs showed reduced growth of Panc02 pancreatic tumor cells. CONCLUSIONS: Therefore, Ii clipped with the tumor-associated antigen C2GnT shows promise for the treatment of pancreatic cancer.

Real-time high-resolution compound imaging allows percutaneous initiation and surveillance in an orthotopic murine pancreatic cancer model.

OBJECTIVES: Orthotopic tumor models are regarded as being suitable for preclinical research on pancreatic cancer. The anatomic localization of the tumor in the retroperitoneum, however, provides little possibility for monitoring tumor growth. METHODS: To assess time-related changes in orthotopic tumor volume, we applied transabdominal high-resolution compound imaging to the murine pancreas. A 15-MHz ultrasound probe was used to determine the feasibility of real-time transabdominal high-resolution ultrasonography to initiate tumor growth by inoculation of pancreatic tumor cells into the pancreas and monitor tumor growth, as well as use as a tool for assessing response to chemotherapy. RESULTS: High-resolution ultrasound allows for precise tumor inoculation in the pancreas. Sonographic-evaluated tumor weight was found to be closely related to actual tumor weight (R = 0.97) measured during necropsy. CONCLUSIONS: High-resolution real-time compound imaging substitutes killing of mice during longitudinal studies and can be used for minimizing animal consumption because each mouse can be followed in an experimental group rather than having to resort to euthanasia for tissue harvesting.

Pegfilgrastim as hematopoietic support for dose-dense chemoimmunotherapy with R-CHOP-14 as first-line therapy in elderly patients with diffuse large B cell lymphoma.

GOALS OF WORK: Recently, 6 cycles of R-CHOP-14 have been recommended as the reference standard regimen for the treatment of elderly patients with diffuse large B-cell lymphoma (DLBCL). Pegfilgrastim has been shown to facilitate dose-dense chemotherapy schedules with a single administration per chemotherapy cycle. The aims of this study were to evaluate the use of pegfilgrastim in combination with the R-CHOP-14 regimen in a homogenous group of previously untreated elderly patients with DLBCL and to assess the pharmacokinetics of pegfilgrastim within this patient population. MATERIALS AND METHODS: Ten patients with DLBCL between 60 and 80 years of age received a single subcutaneous injection of 6 mg pegfilgrastim 24 h after administration of R-CHOP chemoimmunotherapy, which was repeated for 6 to 8 cycles in two-weekly intervals. A total of 348 blood samples were collected. Pegfilgrastim plasma levels and absolute neutrophil counts were measured every other day during the first treatment cycle and twice weekly during all consecutive cycles. MAIN RESULTS: Sixty-three of 72 cycles (87.5%) could be delivered on time and at the planned dose. Median absolute neutrophil nadir was 0.32 g/l on day 9. Grade 3/4 granulocytopenia occurred in all patients. Febrile neutropenia occurred in two patients. Plasma levels of pegfilgrastim remained elevated during the neutropenic phase. At the start of hematologic recovery, plasma concentrations of pegfilgrastim decreased rapidly to baseline levels. Median pegfilgrastim trough plasma level was 0.43 ng/ml on the day preceding the next application. CONCLUSIONS: A single fixed dose of 6 mg of pegfilgrastim given once per cycle of R-CHOP-14 is effective in supporting neutrophil recovery to allow two-weekly drug administration in previously untreated elderly patients with DLBCL. However, close monitoring for infectious complications is mandatory in this patient population.

Transfection of human monocyte-derived dendritic cells with CpG oligonucleotides.

Monocyte-derived dendritic cells (mDC), the most frequently applied DC subset in clinical studies, which can be obtained easily from peripheral blood monocytes after incubation with GM-CSF and IL-4, have not been clearly demonstrated to be activated by CpG oligodeoxynucleotides (ODN). The development of novel molecular strategies - such as the use of CpG-ODN - to increase immunological functions and thus improve the therapeutic efficiency of mDC vaccines in the treatment of malignant diseases is highly desirable. CpG-ODN need to be internalized into specific intracellular compartments to be active. Therefore, we applied electroporation and lipofection and compared these techniques with incubation to overcome possible defects in localization. Conditions of CpG-ODN transfection of these cells were optimized using fluorescein-marked ODN 2216. We were able to achieve high transfection efficiencies with various methods of delivery. However, we did not observe increased expression of maturation-associated and functionally relevant surface antigens (CD14, HLA-DR, CD40, CD83, CD80 and CD86), significant secretion of IL-12 and IFN-alpha in culture supernatant, or enhanced antitumour activation of cytokine-induced killer cells. In conclusion, our results show that non-viral transfection of CpG-ODN is not sufficient to overcome resistance of mDC to CpG activation.

Neutropenic enterocolitis in adults: systematic analysis of evidence quality.

OBJECTIVE: Neutropenic enterocolitis is a life-threatening complication occurring most frequently after intensive chemotherapy in acute leukaemias. The literature is heterogeneous and a systematic review is lacking. METHODS: Following a systematic search we categorised all relevant reports according to their quality and extracted evidence to answer the questions: Which diagnostic criteria are appropriate? What is the incidence of neutropenic enterocolitis? Are there good quality studies supporting specific interventions: Which empiric antimicrobial therapy is recommendable? Is neutropenic enterocolitis without surgical emergency complications an indication for bowel resection? RESULTS: We found and analysed 145 articles of these reports: 64 were reports of single cases, 30 papers reported of two or three cases, 13 were narrative reviews, 34 were retrospective case series of more than three cases and four were prospective diagnostic studies. There were no prospective trials or case control studies on the therapy of neutropenic enterocolitis. There was no consensus on diagnostic criteria. We discuss the difficulty to define diagnostic criteria without having a disease definition. Histology is mostly not available in the living patients. We suggest applying a combination of clinical and radiological criteria: fever, abdominal pain and any bowel wall thickening >4 mm detected by ultrasonography (US) or computed tomography. We calculated a pooled incidence rate from 21 studies of 5.3% (266/5058; 95% CI: 4.7%-5.9%) in patients hospitalised for haematological malignancies, for high-dose chemotherapy in solid tumours or for aplastic anaemia. CONCLUSIONS: This systematic review provides diagnostic criteria for neutropenic enterocolitis, presents a quantitative synthesis on its incidence and discusses its treatment recommendations. Prospective studies are clearly warranted.