Neuroblastoma patients with high-affinity FCGR2A, -3A and stimulatory KIR 2DS2 treated by long-term infusion of anti-GD2 antibody ch14.18/CHO show higher ADCC levels and improved event-free survival.

Polymorphisms in Fc-gamma-receptor (FCGR) genes as well as killer cell immunoglobulin-like receptor (KIR) and KIR ligand (KIRL) repertoires may influence antitumor effects of monoclonal antibodies (mAb). Here, we systematically analyzed high- and low-affinity FCGR2A and -3A genotypes as well as stimulating and inhibitory KIR/KIRL combinations in 53 neuroblastoma (NB) patients treated by long-term infusion (LTI) of anti-GD2 IgG1 Ab ch14.18/CHO using validated real-time PCR methods. Patients with high-affinity FCGR2A and -3A genotypes showed a higher level of Ab-dependent cell-mediated cytotoxicity (ADCC) on day 8 after the start of ch14.18/CHO and superior event-free survival (EFS) compared to patients with low FCGR genotypes. Similar observations were made for patients with stimulatory KIR/KIRL haplotype B (combination of KIR genes including activating receptor genes) compared to inhibitory haplotype A (a fixed set of genes encoding for inhibitory receptors, except 2DS4) and stronger effects were found in patients when haplotype B and high-affinity FCGRs were combined. Surprisingly, independent analysis of KIRs showed a major role of activating KIR 2DS2 for high ADCC levels and prolongation of EFS. The greatest effect was observed in 2DS2-positive patients that also had high-affinity FCGR2A and -3A genotypes. In summary, the presence of the activating KIR 2DS2 has a major effect on ADCC levels and survival in NB patients treated by LTI of ch14.18/CHO and may therefore be a useful biomarker in combination with FCGR polymorphisms for Ab-based immunotherapies.

Pneumocephalus Following Thoracic Surgery with Posterior Chest Wall Resection.

Pneumocephalus can be seen after head injury with fracture of the skull-base or in cerebral neoplasm, infection, or after intracranial or spinal surgery. We report on a 69-year-old male patient with pneumocephalus after right-sided lobectomy and en bloc resection of the chest wall for non-small-cell lung cancer. Postoperatively, the patient showed a reduced vigilance level with no response to pain stimuli and anisocoria. The CCT scan revealed an extensive pneumocephalus; following which, the patient underwent neurosurgery with laminectomy and ligature of the transected nerve roots. After operation the patient returned to his baseline mental status.

Human adenovirus-specific γ/δ and CD8+ T cells generated by T-cell receptor transfection to treat adenovirus infection after allogeneic stem cell transplantation.

Human adenovirus infection is life threatening after allogeneic haematopoietic stem cell transplantation (HSCT). Immunotherapy with donor-derived adenovirus-specific T cells is promising; however, 20% of all donors lack adenovirus-specific T cells. To overcome this, we transfected α/ß T cells with mRNA encoding a T-cell receptor (TCR) specific for the HLA-A*0101-restricted peptide LTDLGQNLLY from the adenovirus hexon protein. Furthermore, since allo-reactive endogenous TCR of donor T lymphocytes would induce graft-versus-host disease (GvHD) in a mismatched patient, we transferred the TCR into γ/δ T cells, which are not allo-reactive. TCR-transfected γ/δ T cells secreted low quantities of cytokines after antigen-specific stimulation, which were increased dramatically after co-transfection of CD8α-encoding mRNA. In direct comparison with TCR-transfected α/ß T cells, TCR-CD8α-co-transfected γ/δ T cells produced more tumor necrosis factor (TNF), and lysed peptide-loaded target cells as efficiently. Most importantly, TCR-transfected α/ß T cells and TCR-CD8α-co-transfected γ/δ T cells efficiently lysed adenovirus-infected target cells. We show here, for the first time, that not only α/ß T cells but also γ/δ T cells can be equipped with an adenovirus specificity by TCR-RNA electroporation. Thus, our strategy offers a new means for the immunotherapy of adenovirus infection after allogeneic HSCT.

Tasmancin and lysogenic bacteriophages induced from Erwinia tasmaniensis strains.

Mitomycin C treatment of Erwinia tasmaniensis strains from Australia induced prophages and the expression of bacteriocins. The bacteriocin named tasmancin inhibited E. tasmaniensis strains from South Africa and Germany. A gene cluster with a klebicin-related operon and an immunity protein was detected on plasmid pET46 from E. tasmaniensis strain Et1/99. PCR reactions using primers directed to this region produced signals for several strains originating from Australia, but not for strains isolated in South Africa and Germany. The latter isolates lacked plasmid pET46. Bacteriophages were induced from E. tasmaniensis strains Et88 and Et14/99, both isolates from South-Eastern Australia. These phages formed plaques on several other strains from this region, as well as on E. tasmaniensis strains from South Africa and Germany. Sequencing revealed similarity of phages ϕEt88 and ϕEt14, which shared the host range on E. tasmaniensis strains. Bacteriophages and tasmancin may interfere with the viability of several related E. tasmaniensis strains in the environment of carrier strains.

Expression of lysozymes from Erwinia amylovora phages and Erwinia genomes and inhibition by a bacterial protein.

Genes coding for lysozyme-inhibiting proteins (Ivy) were cloned from the chromosomes of the plant pathogens Erwinia amylovora and Erwinia pyrifoliae. The product interfered not only with activity of hen egg white lysozyme, but also with an enzyme from E. amylovora phage ΦEa1h. We have expressed lysozyme genes from the genomes of three Erwinia species in Escherichia coli. The lysozymes expressed from genes of the E. amylovora phages ΦEa104 and ΦEa116, Erwinia chromosomes and Arabidopsis thaliana were not affected by Ivy. The enzyme from bacteriophage ΦEa1h was fused at the N- or C-terminus to other peptides. Compared to the intact lysozyme, a His-tag reduced its lytic activity about 10-fold and larger fusion proteins abolished activity completely. Specific protease cleavage restored lysozyme activity of a GST-fusion. The bacteriophage-encoded lysozymes were more active than the enzymes from bacterial chromosomes. Viral lyz genes were inserted into a broad-host range vector, and transfer to E. amylovora inhibited cell growth. Inserted in the yeast Pichia pastoris, the ΦEa1h-lysozyme was secreted and also inhibited by Ivy. Here we describe expression of unrelated cloned 'silent' lyz genes from Erwinia chromosomes and a novel interference of bacterial Ivy proteins with a viral lysozyme.

Mild hyperthermia enhances human monocyte-derived dendritic cell functions and offers potential for applications in vaccination strategies.

Dendritic cell (DC)-based immunotherapy has been shown to be a promising strategy for anti-cancer therapy. Nevertheless, only a low overall clinical response rate has been observed in vaccinated patients with advanced cancer and therefore methods to improve DC immuno-stimulatory functions are currently under intense investigation. In this respect, we exposed human monocyte-derived DCs to a physiological temperature stress of 40°C for up to 24 h followed by analysis for (i) expression of different heat shock proteins, (ii) survival, (iii) cell surface maturation markers, (iv) cytokine secretion, and (v) migratory capacity. Furthermore, we examined the ability of heat-shocked DCs to prime naïve CD8(+) T cells after loading with MelanA peptide, by transfection with MelanA RNA, or by transduction with MelanA by an adenovirus vector. The results clearly indicate that in comparison to control DCs, which remained at 37°C, heat-treated cells revealed no differences concerning the survival rate or their migratory capacity. However, DCs exposed to thermal stress showed a time-dependent enhanced expression of the immune-chaperone heat shock protein 70A and both an up-regulation of co-stimulatory molecules such as CD80, CD83, and CD86 and of the inflammatory cytokine TNF-α. Moreover, these cells had a markedly improved capacity to prime autologous naïve CD8(+) T cells in vitro in an antigen-specific manner, independent of the method of antigen-loading. Thus, our strategy of heat treatment of DCs offers a promising means to improve DC functions during immune activation which, as a physical method, facilitates straight-forward applications in clinical DC vaccination protocols.

Molecular and physiological properties of bacteriophages from North America and Germany affecting the fire blight pathogen Erwinia amylovora.

For possible control of fire blight affecting apple and pear trees, we characterized Erwinia amylovora phages from North America and Germany. The genome size determined by electron microscopy (EM) was confirmed by sequence data and major coat proteins were identified from gel bands by mass spectroscopy. By their morphology from EM data, φEa1h and φEa100 were assigned to the Podoviridae and φEa104 and φEa116 to the Myoviridae. Host ranges were essentially confined to E. amylovora, strains of the species Erwinia pyrifoliae, E. billingiae and even Pantoea stewartii were partially sensitive. The phages φEa1h and φEa100 were dependent on the amylovoran capsule of E. amylovora, φEa104 and φEa116 were not. The Myoviridae efficiently lysed their hosts and protected apple flowers significantly better than the Podoviridae against E. amylovora and should be preferred in biocontrol experiments. We have also isolated and partially characterized E. amylovora phages from apple orchards in Germany. They belong to the Podoviridae or Myoviridae with a host range similar to the phages isolated in North America. In EM measurements, the genome sizes of the Podoviridae were smaller than the genomes of the Myoviridae from North America and from Germany, which differed from each other in corresponding nucleotide sequences.

A well protected intruder: the effective antimicrobial defense of the invasive ladybird Harmonia axyridis.

The harlequin ladybird Harmonia axyridis (Coleoptera: Coccinellidae) is a polyphagous predatory beetle native to Central and Eastern Asia. Since 2007 it has established all over Central Europe. In order to elucidate which defense strategy is responsible for its high resistance to diseases, we tested hemolymph as well as eleven main components of the headspace of H. axyridis for antimicrobial activity against Gram-positive (Bacillus subtilis, B. thuringiensis ssp. tenebrionis, Micrococcus luteus) and Gram-negative bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae). While three of the volatile compounds weakly reduced the growth of microorganisms, hemolymph of adults and larvae of H. axyridis strongly inhibited the growth of Gram-positive and Gram-negative bacteria as well as yeast. Furthermore, we compared the antimicrobial activity in the hemolymph of H. axyridis and Coccinella septempunctata. Antimicrobial activity in H. axyridis was about a thousand times higher compared to hemolymph from C. septempunctata. In contrast to C. septempunctata, the antimicrobial activity in H. axyridis was present without prior challenge. Minimal inhibitory concentration (MIC) of the hemolymph of H. axyridis was lowest against E. coli and yeast followed by B. subtilis, and was highest against entomopathogenic B. thuringiensis ssp. tenebrionidae. Furthermore, MIC values of the hemolymph obtained from live beetles were significantly lower than from frozen insects. This suggests that the active antimicrobial compound is affected by freezing and subsequent thawing of the beetles. Potential implications of our findings for the competitive advantages of H. axyridis over C. septempunctata are discussed.

Modulation of viability and maturation of human monocyte-derived dendritic cells by oncolytic adenoviruses.

Adenoviral oncolysis is a promising new modality for treatment of cancer based on selective viral replication in tumor cells. However, tumor cell killing by adenoviral oncolysis needs to be improved to achieve therapeutic benefit in the clinic. Towards this end, the activation of anti-tumor immunity by adenoviral oncolysis might constitute a potent mechanism for systemic killing of uninfected tumor cells, thereby effectively complementing direct tumor cell killing by the virus. Knowledge of anti-tumor immune induction by adenoviral oncolysis, however, is lacking mostly due to species-specificity of adenovirus replication, which has hampered studies of human oncolytic adenoviruses in animals. We suggest the analysis of interactions of oncolytic adenoviruses with human immune cells as rational basis for the implementation of adenoviral oncolysis-induced anti-tumor immune activation. The goal of our study was to investigate how oncolytic adenoviruses affect human dendritic cells (DCs), key regulators of innate and adoptive immunity that are widely investigated as tumor vaccines. We report that melanoma-directed oncolytic adenoviruses, like replication-deficient adenoviruses but unlike adenoviruses with unrestricted replication potential, are not toxic to monocyte-derived immature DCs and do not block DC maturation by external stimuli. Of note, this is in contrast to reports for other viruses/viral vectors and represents a prerequisite for anti-tumor immune activation by adenoviral oncolysis. Furthermore, we show that these oncolytic adenoviruses alone do not or only partially induce DC maturation. Thus additional signals are required for optimal immune activation. These could be delivered, for example, by inserting immunoregulatory transgenes into the oncolytic adenovirus genome.

Introduction of functional chimeric E/L-selectin by RNA electroporation to target dendritic cells from blood to lymph nodes.

BACKGROUND: Inefficient migration of dendritic cells (DC) to regional lymph nodes (LN) upon intracutaneous injection is a major obstacle for effective DC vaccination. Intravenous vaccination is unfavorable, because DC cannot migrate directly from the blood into LN. METHODS: To enable human monocyte-derived (mo)DC to enter LN directly from the blood, we manipulated them by RNA electroporation to express a human chimeric E/L-selectin (CD62E/CD62L) protein, which binds to peripheral node addressin expressed on high endothelial venules. RESULTS: Transfection efficiency exceeded 95%, and high E/L-selectin surface expression was detected for >48 h. E/L-selectin RNA-transfected DC displayed an identical mature DC phenotype as mock-transfected DC. Furthermore, E/L-selectin-transfected DC maintained their normal CCR7-mediated migration capacity, and their ability to prime and expand functional cytotoxic T cells recognizing MelanA. Most importantly, E/L-selectin-RNA-transfected DC gained the capability to attach to and roll on sialyl-Lewis(X) in vitro. OUTLOOK: The presented strategy can be readily translated into the clinic, as it involves no stable genetic manipulation or viral transformation, and allows targeting of a large number of LN.

A new way to generate cytolytic tumor-specific T cells: electroporation of RNA coding for a T cell receptor into T lymphocytes.

Effective T cell receptor (TCR) transfer until now required stable retroviral transduction. However, retroviral transduction poses the threat of irreversible genetic manipulation of autologous cells. We, therefore, used optimized RNA transfection for transient manipulation. The transfection efficiency, using EGFP RNA, was >90%. The electroporation of primary T cells, isolated from blood, with TCR-coding RNA resulted in functional cytotoxic T lymphocytes (CTLs) (>60% killing at an effector to target ratio of 20:1) with the same HLA-A2/gp100-specificity as the parental CTL clone. The TCR-transfected T cells specifically recognized peptide-pulsed T2 cells, or dendritic cells electroporated with gp100-coding RNA, in an IFNgamma-secretion assay and retained this ability, even after cryopreservation, over 3 days. Most importantly, we show here for the first time that the electroporated T cells also displayed cytotoxicity, and specifically lysed peptide-loaded T2 cells and HLA-A2+/gp100+ melanoma cells over a period of at least 72 h. Peptide-titration studies showed that the lytic efficiency of the RNA-transfected T cells was similar to that of retrovirally transduced T cells, and approximated that of the parental CTL clone. Functional TCR transfer by RNA electroporation is now possible without the disadvantages of retroviral transduction, and forms a new strategy for the immunotherapy of cancer.

Generation of an optimized polyvalent monocyte-derived dendritic cell vaccine by transfecting defined RNAs after rather than before maturation.

Transfection with RNA is an attractive method of Ag delivery to dendritic cells (DCs), but has not yet been standardized. We describe in this study the methods to efficiently generate an optimized mature monocyte-derived DC vaccine at clinical scale based on the electroporation of several RNAs either into immature DC followed by maturation or, alternatively, directly into mature DCs, which has not been possible so far with such high efficiency. Electroporation of DCs resulted in high yield, high transfection efficiency (>90%), and high migration capacity. Intracellular staining allowed the study of the expression kinetics of Ags encoded by the transfected RNAs (MelanA, MAGE-3, and survivin) and a validation of the vaccine (>/=90% transfection efficiency). Expression of all three Ags peaked 3-4 h after electroporation in DC transfected either before or after maturation, but decreased differently. The DC vaccine can also be cryopreserved and nevertheless retains its viability, stimulatory capacity as well as migratory activity. In addition, we uncover that DC transfected after rather than before maturation appear to be preferable vaccines not only from a production point of view but also because they appear to be immunologically superior for CTL induction in sharp contrast to common belief. DCs transfected after maturation not only more effectively generate and present the Mage-3.A1 and MelanA.A2.1 epitopes to T cell clones, but they even are superior in priming to the standard proteasome-dependent MelanA.A2.1 wild-type prototype tumor epitope, both in terms of T cell expansion and effector function on a per cell basis.