When apoptotic cells are not cleared in an efficient and timely manner, they progress to secondary necrosis and lose their membrane integrity. This results in a leakage of immunostimulatory, danger associated molecular patterns (DAMPs), similar to accidental (or primary) necrosis. However, primary necrosis is a sudden event with an inadvertent release of almost unmodified DAMPs. Secondary necrotic cells, in contrast, have gone through various modifications during the process of apoptosis. Recent research revealed that the molecules released from the cytoplasm or exposed on the cell surface differ between primary necrosis, secondary necrosis, and regulated necrosis such as necroptosis. This review gives an overview of these differences and focusses their effects on the immune response. The implications to human physiology and diseases are manifold and will be discussed in the context of cancer, neurodegenerative disorders and autoimmunity.

BackgroundMonoclonal antibodies (mAb), such as trastuzumab are a valuable addition to breast cancer therapy. Data obtained from neoadjuvant settings revealed that antibody-dependent cell-mediated cytotoxicity (ADCC) is a major mechanism of action for the mAb trastuzumab. Conflicting results still call into question whether disease progression, prolonged treatment or concomitant chemotherapy influences ADCC and related immunological phenomena.MethodsWe analyzed the activity of ADCC and antibody-dependent cell-mediated phagocytosis (ADCP) of peripheral blood mononuclear cells (PBMCs) from human epidermal growth factor receptor 2 (HER2/neu) positive breast cancer patients receiving trastuzumab therapy either in an adjuvant (n = 13) or metastatic (n = 15) setting as well as from trastuzumab treatment-naive (t-naive) HER2/neu negative patients (n = 15). PBMCs from healthy volunteers (n = 24) were used as controls. ADCC and ADCP activity was correlated with the expression of antibody binding Fc-gamma receptor (FcγR)I (CD64), FcγRII (CD32) and FcγRIII (CD16) on CD14+ (monocytes) and CD56+ (NK) cells, as well as the expression of CD107a+ (LAMP-1) on CD56+ cells and the total amount of CD4+CD25+FOXP3+ (Treg) cells. In metastatic patients, markers were correlated with progression-free survival (PFS).ResultsADCC activity was significantly down regulated in metastatic, adjuvant and t-naive patient cohorts as compared to healthy controls. Reduced ADCC activity was inversely correlated with the expression of CD107a on CD56+ cells in adjuvant patients. ADCC and ADCP activity of the patient cohorts were similar, regardless of treatment duration or additional chemotherapy. PFS in metastatic patients inversely correlated with the number of peripheral Treg cells.ConclusionThe reduction of ADCC in patients as compared to healthy controls calls for adjuvant strategies, such as immune-enhancing agents, to improve the activity of trastuzumab. However, efficacy of trastuzumab-specific ADCC and ADCP appears not to be affected by treatment duration, disease progression or concomitant chemotherapy. This finding supports the application of trastuzumab at any stage of the disease.

NS1 protein of influenza virus is a virulence factor that counteracts Type I interferon (IFN)- Key words: conditionally-replicating-virus; STAT1; SCIDKnowledge of the pathogenesis of viral diseases and the ability to manipulate specific regions of viral genomes permit the construction of conditionally replicating viruses that are attenuated in normal cells but retain their ability to lyse tumor cells. [1][2][3] We show that the NS1 protein of influenza A virus is a virulence factor that counteracts the interferon (IFN)-mediated antiviral cellular response. 4 As a consequence, an influenza virus that lacks a functional NS1 protein due to an almost total deletion of the NS1 open reading frame (delNS1 virus) fails to replicate in normal cells and is apathogenic for wild-type mice. 5 The delNS1 virus, however, grows to titers similar to wild-type virus in dsRNA-activated kinase (PKR) knockout mice. 6 The conditionally replicating phenotype of the delNS1 virus in PKR-defective systems could be exploited for virally-induced oncolysis in tumors expressing oncogenic ras. 7 This observation is based on the fact that oncogenic ras inhibits PKR activation. 8 For this reason, melanoma cells became permissive for productive delNS1 virus replication upon transfection and expression of oncogenic N-ras. Moreover, delNS1 virus treatment of subcutaneous N-ras-expressing melanomas in SCID mice showed that this virus has tumor-ablative potentials in vivo.The delNS1 virus was also shown to replicate effectively in STAT1 knockout mice. 9 STAT1 exists in 2 isoforms, a 91-kD protein (STAT1␣) and a 84-kD splice variant (STAT1␤). 10 Upon activation by Type I IFN signaling STAT1 forms a heterodimer with STAT2 and becomes an essential part of the IFN-receptor induced transcription complex ISGF3. A reduced expression of STAT1 is associated with IFN resistance. 11 Alterations in the IFN-dependent signal cascades, including changes in STAT1 and Type I IFN-receptor molecules, have been described to occur frequently in malignantly transformed cells. For example, several melanoma and lymphoma cell lines contain no or reduced levels of STAT1. 12,13 In addition, leukemia cell lines were shown to be defective in IFN genes. 14 We hypothesized, therefore, that IFN resistance is a common tumor characteristic, which may allow delNS1 virus-mediated oncolysis.We analyzed the growth of delNS1 virus in IFN-resistant tumor cell lines of various histological origins. We compared the oncolytic effect of the delNS1 virus with a second virus, in which the NS1 was only deleted to the N-terminal 99 amino acids (NS1-99). In mice, the latter virus has intermediate attenuation properties ranging between delNS1 and wild-type viruses. 15 The NS1-99 virus gave us the possibility to investigate whether the attenuation level of NS1-deletion viruses influences the efficiency of oncolysis in vivo. The in vitro growth of the NS1-deletion mutants inversely correlated with the IFN resistance of the assayed cell lines. Both delNS1 and NS1-99 viruses induced a tumor-ablative ef...

BackgroundWe developed a novel intranasal influenza vaccine approach that is based on the construction of replication-deficient vaccine viruses that lack the entire NS1 gene (ΔNS1 virus). We previously showed that these viruses undergo abortive replication in the respiratory tract of animals. The local release of type I interferons and other cytokines and chemokines in the upper respiratory tract may have a “self-adjuvant effect”, in turn increasing vaccine immunogenicity. As a result, ΔNS1 viruses elicit strong B- and T- cell mediated immune responses.Methodology/Principal FindingsWe applied this technology to the development of a pandemic H5N1 vaccine candidate. The vaccine virus was constructed by reverse genetics in Vero cells, as a 5∶3 reassortant, encoding four proteins HA, NA, M1, and M2 of the A/Vietnam/1203/04 virus while the remaining genes were derived from IVR-116. The HA cleavage site was modified in a trypsin dependent manner, serving as the second attenuation factor in addition to the deleted NS1 gene. The vaccine candidate was able to grow in the Vero cells that were cultivated in a serum free medium to titers exceeding 8 log10 TCID50/ml. The vaccine virus was replication deficient in interferon competent cells and did not lead to viral shedding in the vaccinated animals. The studies performed in three animal models confirmed the safety and immunogenicity of the vaccine. Intranasal immunization protected ferrets and mice from being infected with influenza H5 viruses of different clades. In a primate model (Macaca mulatta), one dose of vaccine delivered intranasally was sufficient for the induction of antibodies against homologous A/Vietnam/1203/04 and heterologous A/Indonesia/5/05 H5N1 strains.Conclusion/SignificanceOur findings show that intranasal immunization with the replication deficient H5N1 ΔNS1 vaccine candidate is sufficient to induce a protective immune response against H5N1 viruses. This approach might be attractive as an alternative to conventional influenza vaccines. Clinical evaluation of ΔNS1 pandemic and seasonal influenza vaccine candidates are currently in progress.

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 ␣ with or without addition of IFN-␥. 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-␥ 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.

The cyanobacterium Synechocystis sp. PCC 6803 is transformable at high efficiency and integrates DNA by homologous double recombination. However, several genetic mapping procedures depend on the ability to generate transformants even with very small amounts of added DNA. This study is aimed at optimizing the transformation efficiency at limiting concentrations of exogenous DNA. The transformation efficiency showed little sensitivity to experimental conditions. Transformation with circular plasmid DNA was found to be no more than 30% more efficient than with linearized plasmid DNA. The efficiency of transformation remained essentially the same in the presence of competing DNA, indicating that the capacity of DNA uptake by the cells is not limiting. The incubation time of cells with DNA before plating (0-8 h) affected the transformation efficiency by up to 3-fold. Only minor changes in the efficiency were observed as a function of the presence of a membrane filter on the plate or the presence of TAE or TBE gel buffer residues in the transformation mixture. However, transformability of the host strain of Synechocystis sp. PCC 6803 was increased by two orders of magnitude if the sll1354 gene encoding the exonuclease RecJ was deleted. Therefore, the transformation efficiency of Synechocystis sp. PCC 6803 with exogenous DNA appears to be determined primarily by intracellular processes such as the efficiency of DNA processing and homologous recombination.

The response to neoadjuvant chemotherapy in breast cancer patients is usually assessed by pCR and RCB score. However, the prognostic value of these parameters is still in discussion. We showed recently that an epirubicin/docetaxel therapy is associated with an increase in the cell death marker high‐mobility group box 1 protein (HMGB1) in the circulation. Here, we investigate whether this increase correlates with the long‐term outcome. Thirty‐six early breast cancer patients under neoadjuvant epirubicin/docetaxel combination chemotherapy were included in this study. To determine the immediate effect of this treatment on HMGB1, we collected blood samples before and 24–96 h after the initial dose. This time course was then compared to the 5‐year follow‐up of the patients. HMGB1 levels varied before chemotherapy between 4.1 and 11.3 ng/mL and reacted differently in response to therapy. Some patients showed an increase while others did not show any changes. Therefore, we subdivided the patient collective into two groups: patients with an at least 1.1 ng/mL increase in HMGB1 and patients with smaller changes. The disease‐free survival was longer in the HMGB1 increase group (56.2 months vs. 46.6 months), but this difference did not reach significance. The overall survival (OS) was significantly better in patients with an increase in HMGB1 (log rank P = 0.021). These data suggest that an immediate increase in HMGB1 levels correlates with improved outcome in early breast cancer patients receiving neoadjuvant chemotherapy, and may be a valuable complementary biomarker for early estimation of prognosis.

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