Elektrophysiologische Charakterisierung und pharmakologische Blockade von Listeriolysin O-induzierten Membranporen

Listeriolysin O (LLO), ein porenbildendes Toxin aus der Gruppe der Cholesterolabhängigen Zytolysine (CDTX), ist ein essentieller Pathogenitätsfaktor des humanpathogenen Bakteriums Listeria monocytogenes. In dieser Arbeit konnte elektrophysiologisch direkt nachgewiesen werden, dass Dithiotreitol (DTT) und saure (5,9) pH-Werte die porenbildende Aktivität von LLO um das 3-5fache steigern. Offene LLO-Poren schliessen in der Whole-Cell Messkonfiguration gleich häufig schnell oder langsam. Nominell Ca2+-freie Intrazellulärlösung führte zu einer signifikanten Abnahme langsamer Porenschlüsse, zugunsten schneller Porenschlüsse. In den Outside-Out Patches sind beide Formen des Porenschlusses hochsignifikant seltener, wobei langsame Porenschlüsse signifikant seltener sind als statistisch im Vergleich zu den schnellen Porenschlüssen zu erwarten ist. Dies passt gut zu der Hypothese, dass es sich bei den langsamen Porenschlüssen um Endozytose-Prozesse handelt. In Inside-Out Patches konnte direkt gezeigt werden, dass LLO in der zytoplasmatischen Seite der Zellmembran Poren induzieren kann, was bei der interzellulären Ausbreitung von L. monocytogenes von entscheidender Bedeutung ist. Im Rahmen eines Screenings auf Poren-blockierende Substanzen konnte gezeigt werden, dass die LLO-Poren durch trivalente Ionen, wie Lanthanoide und Aluminium vollständig blockiert wurden. Es handelte sich hierbei um einen vom Ionenradius abhängigen, reversiblen und membranpotentialabhängigen Block. Die zweiwertigen Metall- Ionen, wie Cd2+, Ni2+, Ru2+ und Zn2+ blockierten die LLO-induzierten Porenströme nur teilweise. Eosin Y war der einzige nicht-metallische Blocker der LLO-Poren. LLO-induzierte Ca2+-Oszillationen wurden durch Gd3+ vollständig blockiert. Im Gegensatz dazu konnten Inhibitoren der intrazellulären Ca2+-Freisetzung oder von Ca2+-Kanälen die Ca2+-Oszillationen nicht verändern. Dies zeigt, dass der Anstieg der intrazellulären Ca2+-Konzentration bei den LLO-induzierten Ca2+-Oszillationen allein durch die LLO-Poren bedingt ist. Die Blockade der plasmamembranständigen Ca2+-ATPasen (PMCA) durch Gd3+ führt zur Hemmung Ca2+-Sequestration bei LLO -induzierten Ca2+-Oszillationen. Das unterstreicht die entscheidende Rolle der PMCA bei den LLO-induzierten Ca2+-Oszillationen.Listeriolysin O is a pore-forming toxin belonging to the familiy of cholesteroldependent cytolysins and is the major pathogenicity factor of L. monocytogenes. By electrophysiological measurement studies, it could be directly shown that dithiotreitol (DTT) and acidic (5.9) pH raise LLOs pore-forming activity 3 to 5 fold. In whole-cell measurements LLO-pores close equally often in a quick or slow way. Ca2+-free intracellular medium leads to a significant decrease in slow closings in favour of fast closings. In outside-out patches both types of pore closings were significantly decreased compared to whole-cell measurements. Compared to the quick closings the slow closings were furthermore significantly reduced. This observation favours our hypothesis that slow closings are caused by endocytosis. As clearly shown in the above study, pore formation in inside-out patches proves that LLO can form pores in the cytoplasmatic leaflet of the cell membrane which underlines its importance in cell-to-cell spreading. On screening several substances for pore blocking abilities, we found several of them that could block the LLO-pores. The trivalent ions like lanthanoids and aluminium blocked the LLO-pores completely. This block induced by lanthanoid was fully reversible and voltage dependent. The IC50 of lanthanoids was dependent on its ionic radius. Divalent ions like cadmium, nickel, zinc and ruthenium only partially blocked the current evoked by LLO-pores. Eosin Y could be identified as a non-metal pore blocking agent. LLO induced Ca2+-oscillations were completely blocked by Gd3+ whereas blocking the intracellular Ca2+-release or Ca2+-channels did not lead to any changes. This clearly shows that the Ca2+-influx exclusively goes through the LLO-pores. Gd3+ also blocks plasma membrane Ca2+-ATPases thereby inhibiting Ca2+- sequestration in LLO-induced Ca2+-oscillations. This implies the importance of PMCAs in LLO-induced Ca2+-oscillations

SARS-CoV-2 vaccination in cardiothoracic organ transplant recipients: effective strategies wanted

(...

Advances in Lymphoma Molecular Diagnostics

Lymphomas encompass a diverse group of malignant lymphoid neoplasms. Over recent years much scientific effort has been undertaken to identify and understand molecular changes in lymphomas, resulting in a wide range of genetic alterations that have been reported across all types of lymphomas. As many of these changes are now incorporated into the World Health Organization’s defined criteria for the diagnostic evaluation of patients with lymphoid neoplasms, their accurate identification is crucial. Even if many alterations are not routinely evaluated in daily clinical practice, they may still have implications in risk stratification, treatment, prognosis or disease monitoring. Moreover, some alterations can be used for targeted treatment. Therefore, these advances in lymphoma molecular diagnostics in some cases have led to changes in treatment algorithms. Here, we give an overview of and discuss advances in molecular techniques in current clinical practice, as well as highlight some of them in a clinical context

Prophylactic Peripheral Blood Stem Cell Collection in Patients with Extensive Bone-Marrow Infiltration of Neuroendocrine Tumours Prior to Peptide Receptor Radionuclide Therapy with 177Lu-DOTATATE

Peptide receptor radionuclide therapy (PRRT) of metastatic neuroendocrine tumors (NET) can be successfully repeated but may eventually be dose-limited. Since 177Lu-DOTATATE dose limitation may come from hematological rather than renal function, hematological peripheral blood stem cell backup might be desirable. Here, we report our initial experience of peripheral blood stem-cell collection (PBSC) in patients with treatment-related cytopenia and therefore high risk of bone-marrow failure. Five patients with diffuse bone-marrow infiltration of NET and relevant myelosuppression (≥grade 2) received PBSC before one PRRT cycle with 177Lu-DOTATATE (7.6 ± 0.8 GBq/cycle). Standard stem-cell mobilization with Granulocyte-colony stimulating factor (G-CSF) was applied, and successful PBSC was defined as a collection of >2 × 106/kg CD34+ cells. In case of initial failure, Plerixafor was administered in addition to G-CSF prior to apheresis. PBSC was successfully performed in all patients with no adverse events. Median cumulative activity was 44.8 GBq (range, 21.3–62.4). Three patients had been previously treated with PRRT, two of which needed the addition of Plerixafor for stem-cell mobilization. Only one of five patients required autologous peripheral blood stem-cell transplantation during the median follow up time of 28 months. PBSC collection seems to be feasible in NET with bone-marrow involvement and might be worth considering as a backup strategy prior to PRRT, in order to overcome dose-limiting bone-marrow toxicity

Vitamin D Enhances Immune Effector Pathways of NK Cells Thus Providing a Mechanistic Explanation for the Increased Effectiveness of Therapeutic Monoclonal Antibodies

Patients with diffuse large cell lymphoma who have an adequate vitamin D supply derive significantly more benefit from immuno-chemotherapy with rituximab than patients with vitamin D deficiency; this is especially true for female patients. We have already been able to show that vitamin D increases the antibody-dependent cytotoxicity (ADCC) of NK cells in a sex-dependent manner, but it is unclear how vitamin D makes NK cells more efficient. Methods: Healthy individuals with vitamin D deficiency were supplemented with vitamin D to sufficient levels. NK cells were isolated from blood samples before and after vitamin D saturation. For transcriptome analysis, we used the Affymetrix Gene-Chip 2.0™. Gene expression analysis as well as supervised and unsupervised pathway analysis were performed. Results: Among others the “NK cell-associated cytotoxicity pathway” increased after vitamin D substitution. Five IFN-α subtypes (2, 4, 6, 7 and 10) and IFN-κ were more highly expressed and are mainly responsible in these pathways. In contrast, the pathway “interferon-gamma response”, as well as other sets in cytokine production and chemotaxis showed a reduction. Toll-like receptor genes (TLR-8, TLR-7, TLR-2) were downregulated and, therefore, are responsible for the decline of these pathways. The same could be shown for the “ubiquitin-ligase” pathway. Conclusions: Increased expression of several IFN-α subtypes may explain the increased ADCC of NK cells in vitamin D-replenished and otherwise healthy subjects. Other regulators of interferon production and ADCC are compensatory upregulated in compensation, such as Toll-like receptors and those of the ubiquitin ligase, and normalize after vitamin D substitution

VEGFR2 and VEGFA polymorphisms are not associated with an inferior prognosis in Caucasian patients with aggressive B-cell lymphoma

Purpose Previous published data showed an impact of single‐nucleotide polymorphisms in the VEGF A and VEGFR2 genes on the survival of patients with various malignancies, among others diffuse large B‐cell lymphoma (DLBCL). Patients and Methods We investigated the role of four VEGF‐A and two VEGFR‐2 gene polymorphisms on the outcome of 273 patients with diffuse large B‐cell lymphoma who were treated with R‐CHOP within a prospective, randomized trial of the German High‐Grade Non‐Hodgkin Lymphoma Study Group (DSHNHL). The genomic DNA samples were analyzed using commercial DNA Probes (Applied Biosystems, USA) to detect single‐nucleotide polymorphisms in the VEGF A rs699947, rs1570360, rs2010963, rs3025039 and rs1870377, and rs2305948 in the VEGFR2 receptor. Hundred healthy blood donors served as a control. Results There was no difference between the SNP allele frequencies in lymphoma patients compared to the control group for all investigated SNPs. None of the investigated SNPs was significantly associated with EFS or OS. After adjusting for the International Prognostic Index risk factors in a multivariate analysis, these results could be confirmed. Conclusion Single‐nucleotide polymorphisms of the VEGF and VEGFR2 were not associated with a worse outcome in Caucasian patients with DLBCL

Application and clinical impact of the RESIST-4 O.K.N.V. rapid diagnostic test for carbapenemase detection in blood cultures and clinical samples

Invasive infections caused by carbapenemase-producing bacteria are associated with excess mortality. We applied a rapid diagnostic test (RDT) on clinical samples with an elevated likelihood of carbapenemase-producing bacteria and documented its impact on antibiotic treatment decisions. Among 38 patients, twelve tested positive for infections caused by carbapenemase-producing bacteria (31.6%), mainly in blood cultures. KPC (n = 10) was more frequent than OXA-48 (n = 2). RDT-based carbapenemase detection led to a treatment modification to ceftazidime/avibactam-containing regimens in all patients before detailed antibiotic testing results became available. Eleven patients (92%) survived the acute infection, whereas one patient with a ceftazidime/avibactam- and colistin-resistant OXA-48-positive isolate died

Outcomes of haploidentical stem cell transplantation for chronic lymphocytic leukemia: a retrospective study on behalf of the chronic malignancies working party of the EBMT

Allogeneic hematopoietic stem cell transplantation (HCT) may result in long-term disease control in high-risk chronic lymphocytic leukemia (CLL). Recently, haploidentical HCT is gaining interest because of better outcomes with post-transplantation cyclophosphamide (PTCY). We analyzed patients with CLL who received an allogeneic HCT with a haploidentical donor and whose data were available in the EBMT registry. In total 117 patients (74% males) were included; 38% received PTCY as GVHD prophylaxis. For the whole study cohort OS at 2 and 5 yrs was 48 and 38%, respectively. PFS at 2 and 5 yrs was 38 and 31%, respectively. Cumulative incidence (CI) of NRM in the whole group at 2 and 5 years were 40 and 44%, respectively. CI of relapse at 2 and 5 yrs were 22 and 26%, respectively. All outcomes were not statistically different in patients who received PTCY compared to other types of GVHD prophylaxis. In conclusion, results of haploidentical HCT in CLL seem almost identical to those with HLA-matched donors. Thereby, haploidentical HCT is an appropriate alternative in high risk CLL patients with a transplant indication but no available HLA-matched donor. Despite the use of PTCY, the CI of relapse seems not higher than observed after HLA-matched HCT

Characterization of an HLA-restricted and human cytomegalovirus-specific antibody repertoire with therapeutic potential

With an infection rate of 60–90%, the human cytomegalovirus (HCMV) is very common among adults but normally causes no symptoms. When T cell-mediated immunity is compromised, HCMV reactivation can lead to increased morbidity and mortality. HCMV antigens are processed and presented as peptides on the cell surface via HLA I complexes to the T cell receptor (TCR) of T cells. The generation of antibodies against HCMV peptides presented on HLA complexes (TCR-like antibodies) has been described, but is without therapeutic applications to date due to the polygenic and polymorphic nature of HLA genes. We set out to obtain antibodies specific for HLA/HCMV-peptides, covering the majority of HLA alleles present in European populations. Using phage display technology, we selected 10 Fabs, able to bind to HCMV-peptides presented in the 6 different HLA class I alleles A*0101, A*0201, A*2402, B*0702, B*0801 and B*3501. We demonstrate specific binding of all selected Fabs to HLA-typed lymphoblastoid cell lines (EBV-transformed B cells) and lymphocytes loaded with HCMV-peptides. After infection with HCMV, 4/10 tetramerized Fabs restricted to the alleles HLA-A*0101, HLA-A*0201 and HLA-B*0702 showed binding to infected primary fibroblasts. When linked to the pseudomonas exotoxin A, these Fab antibodies induce highly specific cytotoxicity in HLA matched cell lines loaded with HCMV peptides. TCR-like antibody repertoires therefore represent a promising new treatment modality for viral infections and may also have applications in the treatment of cancers

The addition of rituximab to chemotherapy improves overall survival in mantle cell lymphoma—a pooled trials analysis

Mantle cell lymphoma (MCL) is a distinct subtype of B-cell lymphoma and commonly used induction immunochemotherapies include the anti-CD20 antibody rituximab. However, efficacy data for rituximab regarding overall survival (OS) in first line MCL therapy remain conflicting. We report long-term outcomes of a pooled trials analysis comparing Cyclophosphamide, Doxorubicine, Vincristine, Prednisone (CHOP) to R-CHOP in MCL to confirm efficacy on failure free survival (FFS) and OS in relevant subgroups. Untreated, adult MCL patients of two prospective trials assigned to CHOP or R-CHOP were included. Primary endpoints were FFS and OS, secondary endpoints included duration of response (DOR), secondary malignancies and OS after relapse. Between 1996 and 2003, 385 MCL patients were assigned to CHOP (201) or R-CHOP (184). After a median follow-up of 13.4 years, the addition of Rituximab significantly improved FFS (1.36 vs. 2.07 years, HR 0.62 (0.50–0.77)), OS (4.84 vs. 5.81 years, HR 0.78 (0.61–0.99)) and DOR (1.48 vs. 2.08 years, HR 0.67 (0.53–0.86)). Furthermore, Rituximab improved survival across different MCL risk groups. In a post-hoc analysis of OS after relapse comparing patients receiving chemotherapy with / without rituximab, rituximab maintained efficacy with a median OS of 3.10 vs. 2.11 years (HR 0.70, 0.54–0.91). The rate of secondary malignancies was 0.5 and 3.9% for hematological and 7 and 8% for non-hematological malignancies for CHOP and R-CHOP patients, respectively. We present mature results of a pooled MCL cohort, demonstrating prolonged FFS, OS and DOR for the combined immuno-chemotherapy, confirming the standard of care in first line treatment