Migrating Platelets Are Mechano-scavengers that Collect and Bundle Bacteria.

Blood platelets are critical for hemostasis and thrombosis and play diverse roles during immune responses. Despite these versatile tasks in mammalian biology, their skills on a cellular level are deemed limited, mainly consisting in rolling, adhesion, and aggregate formation. Here, we identify an unappreciated asset of platelets and show that adherent platelets use adhesion receptors to mechanically probe the adhesive substrate in their local microenvironment. When actomyosin-dependent traction forces overcome substrate resistance, platelets migrate and pile up the adhesive substrate together with any bound particulate material. They use this ability to act as cellular scavengers, scanning the vascular surface for potential invaders and collecting deposited bacteria. Microbe collection by migrating platelets boosts the activity of professional phagocytes, exacerbating inflammatory tissue injury in sepsis. This assigns platelets a central role in innate immune responses and identifies them as potential targets to dampen inflammatory tissue damage in clinical scenarios of severe systemic infection.

Lymphocyte Circadian Clocks Control Lymph Node Trafficking and Adaptive Immune Responses.

Lymphocytes circulate through lymph nodes (LN) in search for antigen in what is believed to be a continuous process. Here, we show that lymphocyte migration through lymph nodes and lymph occurred in a non-continuous, circadian manner. Lymphocyte homing to lymph nodes peaked at night onset, with cells leaving the tissue during the day. This resulted in strong oscillations in lymphocyte cellularity in lymph nodes and efferent lymphatic fluid. Using lineage-specific genetic ablation of circadian clock function, we demonstrated this to be dependent on rhythmic expression of promigratory factors on lymphocytes. Dendritic cell numbers peaked in phase with lymphocytes, with diurnal oscillations being present in disease severity after immunization to induce experimental autoimmune encephalomyelitis (EAE). These rhythms were abolished by genetic disruption of T cell clocks, demonstrating a circadian regulation of lymphocyte migration through lymph nodes with time-of-day of immunization being critical for adaptive immune responses weeks later.

Helicobacter pylori binds human Annexins via Lipopolysaccharide to interfere with Toll-like Receptor 4 signaling.

Helicobacter pylori colonizes half of the global population and causes gastritis, peptic ulcer disease or gastric cancer. In this study, we were interested in human annexin (ANX), which comprises a protein family with diverse and partly unknown physiological functions, but with a potential role in microbial infections and possible involvement in gastric cancer. We demonstrate here for the first time that H. pylori is able to specifically bind ANXs. Binding studies with purified H. pylori LPS and specific H. pylori LPS mutant strains indicated binding of ANXA5 to lipid A, which was dependent on the lipid A phosphorylation status. Remarkably, ANXA5 binding almost completely inhibited LPS-mediated Toll-like receptor 4- (TLR4) signaling in a TLR4-specific reporter cell line. Furthermore, the interaction is relevant for gastric colonization, as a mouse-adapted H. pylori increased its ANXA5 binding capacity after gastric passage and its ANXA5 incubation in vitro interfered with TLR4 signaling. Moreover, both ANXA2 and ANXA5 levels were upregulated in H. pylori-infected human gastric tissue, and H. pylori can be found in close association with ANXs in the human stomach. Furthermore, an inhibitory effect of ANXA5 binding for CagA translocation could be confirmed. Taken together, our results highlight an adaptive ability of H. pylori to interact with the host cell factor ANX potentially dampening innate immune recognition.

Widespread amyloidogenicity potential of multiple myeloma patient-derived immunoglobulin light chains.

BACKGROUND: In a range of human disorders such as multiple myeloma (MM), immunoglobulin light chains (IgLCs) can be produced at very high concentrations. This can lead to pathological aggregation and deposition of IgLCs in different tissues, which in turn leads to severe and potentially fatal organ damage. However, IgLCs can also be highly soluble and non-toxic. It is generally thought that the cause for this differential solubility behaviour is solely found within the IgLC amino acid sequences, and a variety of individual sequence-related biophysical properties (e.g. thermal stability, dimerisation) have been proposed in different studies as major determinants of the aggregation in vivo. Here, we investigate biophysical properties underlying IgLC amyloidogenicity. RESULTS: We introduce a novel and systematic workflow, Thermodynamic and Aggregation Fingerprinting (ThAgg-Fip), for detailed biophysical characterisation, and apply it to nine different MM patient-derived IgLCs. Our set of pathogenic IgLCs spans the entire range of values in those parameters previously proposed to define in vivo amyloidogenicity; however, none actually forms amyloid in patients. Even more surprisingly, we were able to show that all our IgLCs are able to form amyloid fibrils readily in vitro under the influence of proteolytic cleavage by co-purified cathepsins. CONCLUSIONS: We show that (I) in vivo aggregation behaviour is unlikely to be mechanistically linked to any single biophysical or biochemical parameter and (II) amyloidogenic potential is widespread in IgLC sequences and is not confined to those sequences that form amyloid fibrils in patients. Our findings suggest that protein sequence, environmental conditions and presence and action of proteases all determine the ability of light chains to form amyloid fibrils in patients.

Azacitidine, lenalidomide and donor lymphocyte infusions for relapse of myelodysplastic syndrome, acute myeloid leukemia and chronic myelomonocytic leukemia after allogeneic transplant: the Azalena-Trial.

Azacitidine (Aza) combined with donor lymphocyte infusions (DLI) is an established treatment for relapse of myeloid malignancies after allogeneic transplantation. Based on its immunomodulatory and anti-leukemic properties we considered Lenalidomide (Lena) to act synergistically with Aza/DLI to improve outcome. We, therefore, prospectively investigated tolerability and efficacy of this combination as first salvage therapy for adults with post-transplant relapse of acute myeloid leukemia, myelodysplastic syndromes and chronic myelomonocytic leukemia. Patients were scheduled for eight cycles Aza (75 mg/m2 day 1-7), Lena (2.5 or 5 mg, days 1-21) and up to three DLI with increasing T-cell dosages (0.5×106-1.5×107 cells/kg). Primary endpoint was safety, while secondary endpoints included response, graft-versus-host disease (GvHD) and overall survival (OS). Fifty patients with molecular (52%) or hematological (48%) relapse of myelodysplastic syndromes (n=24), acute myeloid leukemia (n=23) or chronic myelomonocytic leukemia (n=3) received a median of seven (range, 1-8) cycles including 14 patients with 2.5 mg and 36 with 5 mg Lena daily dosage. Concomitantly, 34 patients (68%) received at least one DLI. Overall response rate was 56% and 25 patients (50%) achieved complete remission being durable in 80%. Median OS was 21 months and 1-year OS rate 65% with no impact of type of or time to relapse and Lena dosages. Treatment was well tolerated indicated by febrile neutropenia being the only grade ≥3 non-hematologic adverse event in >10% of patients and modest acute (grade 2-4 24%) and chronic (moderate/severe 28%) GvHD incidences. In summary, Lena can be safely added to Aza/DLI without excess of GvHD and toxicity. Its significant anti-leukemic activity suggests that this combination is a novel salvage option for post-transplant relapse (clinicaltrials gov. Identifier: NCT02472691).

Kinetics of CagA type IV secretion by Helicobacter pylori and the requirement for substrate unfolding.

Type IV secretion of effector proteins is an important principle for interaction of human pathogens with their target cells. The corresponding secretion systems may transport a multitude of effector proteins that have to be deployed in the respective spatiotemporal context, or only a single translocated protein, as in the case of the CagA effector protein produced by the human gastric pathogen Helicobacter pylori. For a more detailed analysis of the kinetics and mode of action of CagA type IV secretion by H. pylori, we describe here, a novel, highly sensitive split luciferase-based translocation reporter which can be easily adapted to different end-point or real-time measurements. Using this reporter, we showed that H. pylori cells are able to rapidly inject a limited amount of their CagA supply into cultured gastric epithelial cells. We have further employed the reporter system to address the question whether CagA has to be unfolded prior to translocation by the type IV secretion system. We showed that protein domains co-translocated with CagA as protein fusions are more readily tolerated as substrates than in other secretion systems, but also provide evidence that unfolding of effector proteins is a prerequisite for their transport.

Substrate usage determines carbon flux via the citrate cycle in Helicobacter pylori.

Helicobacter pylori displays a worldwide infection rate of about 50%. The Gram-negative bacterium is the main reason for gastric cancer and other severe diseases. Despite considerable knowledge about the metabolic inventory of H. pylori, carbon fluxes through the citrate cycle (TCA cycle) remained enigmatic. In this study, different 13 C-labeled substrates were supplied as carbon sources to H. pylori during microaerophilic growth in a complex medium. After growth, 13 C-excess and 13 C-distribution were determined in multiple metabolites using GC-MS analysis. [U-13 C6 ]Glucose was efficiently converted into glyceraldehyde but only less into TCA cycle-related metabolites. In contrast, [U-13 C5 ]glutamate, [U-13 C4 ]succinate, and [U-13 C4 ]aspartate were incorporated at high levels into intermediates of the TCA cycle. The comparative analysis of the 13 C-distributions indicated an adaptive TCA cycle fully operating in the closed oxidative direction with rapid equilibrium fluxes between oxaloacetate-succinate and α-ketoglutarate-citrate. 13 C-Profiles of the four-carbon intermediates in the TCA cycle, especially of malate, together with the observation of an isocitrate lyase activity by in vitro assays, suggested carbon fluxes via a glyoxylate bypass. In conjunction with the lack of enzymes for anaplerotic CO2 fixation, the glyoxylate bypass could be relevant to fill up the TCA cycle with carbon atoms derived from acetyl-CoA.

The Helicobacter pylori adhesin protein HopQ exploits the dimer interface of human CEACAMs to facilitate translocation of the oncoprotein CagA.

Helicobacter pylori infects half of the world's population, and strains that encode the cag type IV secretion system for injection of the oncoprotein CagA into host gastric epithelial cells are associated with elevated levels of cancer. CagA translocation into host cells is dependent on interactions between the H. pylori adhesin protein HopQ and human CEACAMs. Here, we present high-resolution structures of several HopQ-CEACAM complexes and CEACAMs in their monomeric and dimeric forms establishing that HopQ uses a coupled folding and binding mechanism to engage the canonical CEACAM dimerization interface for CEACAM recognition. By combining mutagenesis with biophysical and functional analyses, we show that the modes of CEACAM recognition by HopQ and CEACAMs themselves are starkly different. Our data describe precise molecular mechanisms by which microbes exploit host CEACAMs for infection and enable future development of novel oncoprotein translocation inhibitors and H. pylori-specific antimicrobial agents.

Acute myeloid leukemia-induced functional inhibition of healthy CD34+ hematopoietic stem and progenitor cells.

Acute myeloid leukemia (AML) is characterized by an expansion of leukemic cells and a simultaneous reduction of normal hematopoietic precursors in the bone marrow (BM) resulting in hematopoietic insufficiency, but the underlying mechanisms are poorly understood in humans. Assuming that leukemic cells functionally inhibit healthy CD34+ hematopoietic stem and progenitor cells (HSPC) via humoral factors, we exposed healthy BM-derived CD34+ HSPC to cell-free supernatants derived from AML cell lines as well as from 24 newly diagnosed AML patients. Exposure to AML-derived supernatants significantly inhibited proliferation, cell cycling, colony formation, and differentiation of healthy CD34+ HSPC. RNA sequencing of healthy CD34+ HSPC after exposure to leukemic conditions revealed a specific signature of genes related to proliferation, cell-cycle regulation, and differentiation, thereby reflecting their functional inhibition on a molecular level. Experiments with paired patient samples showed that these inhibitory effects are markedly related to the immunomagnetically enriched CD34+ leukemic cell population. Using PCR, ELISA, and RNA sequencing, we detected overexpression of TGFß1 in leukemic cells on the transcriptional and protein level and, correspondingly, a molecular signature related to TGFß1 signaling in healthy CD34+ HSPC. This inhibitory effect of TGFß1 on healthy hematopoiesis was functionally corrobated and could be pharmacologically reverted by SD208, an inhibitor of TGFß receptor 1 signaling. Overall, these data indicate that leukemic cells induce functional inhibition of healthy CD34+ HSPC, at least in part, through TGFß1, suggesting that blockage of this pathway may improve hematopoiesis in AML.

18F-FDG-PET/CT in relapsed multiple myeloma: Are prognostic thresholds different from first-line therapy?

PURPOSE: While 18F-FDG PET/CT yields valuable prognostic information for patients in first-line therapy of multiple myeloma (MM), its prognostic relevance in relapse is not established. Available studies of relapsed MM describe prognostic thresholds for frequently used PET/CT parameters that are significantly higher than those identified in the first-line setting. The purpose of this study was to evaluate the prognostic role of PET/CT in relapsed MM, based on parameters used in the first-line setting. METHODS: Our retrospective study included 36 patients with MM who had received autologous or allogeneic stem cell transplantation, suffered at least one relapse, and underwent FDG-PET/CT at relapse. Number of focal bone lesions (FL), maximal standardised uptake value (SUVmax), and presence of PET-positive extramedullary lesions (EMD) were analysed. RESULTS: For the number of FLs, the prognostic value was demonstrated with a cut-off of > 3 (median OS 3.8 months vs. not reached, p = 0.003). Median OS of patients with SUVmax ≤ 4 was not reached, while it was 3.9 months in patients with SUVmax > 4 (p = 0.014). Presence of EMD was a significant prognostic parameter too, with median OS of 3.6 months versus not reached (p = 0.004). The above-mentioned parameters showed prognostic significance for PFS as well. Combination of higher ISS stage and PET/CT parameters identified patients with particularly short OS (3.7 months vs. not reached, p < 0.001) and PFS (3.6 vs. 11.7 months p < 0.001). CONCLUSION: The PET/CT parameters SUVmax > 4, nFL > 3, and presence of EMD identify patients with poor prognosis not only in the first-line setting but also in relapsed MM.

Myelodysplastic syndrome patients display alterations in their immune status reflected by increased PD-L1-expressing stem cells and highly dynamic exhausted T-cell frequencies.

Little data are available for the expression of immune checkpoint (IC) molecules within myelodysplastic syndrome (MDS). Here, we report increased PD-L1+ CD34+ CD38- and PD-L1+ CD34+ CD38+ stem cell frequencies within MDS patients compared to stem cell recipients in remission. Additionally, we observed exceedingly similar PD1+ and Tim-3+ T-cell frequencies between acute myeloid leukaemia (AML) and MDS samples that were elevated compared to patients in remission. Furthermore, we found highly dynamic Tim-3+ and PD1+ T-cell frequencies within serial samples of relapsing MDS with excess blasts (MDS-EB II) patients, correlating with further disease markers. These findings support the idea of a potential successful implementation of IC inhibitor treatment in suitable MDS patients.

De Novo Sequencing of Antibody Light Chain Proteoforms from Patients with Multiple Myeloma.

In multiple myeloma diseases, monoclonal immunoglobulin light chains (LCs) are abundantly produced, with, as a consequence in some cases, the formation of deposits affecting various organs, such as the kidney, while in other cases remaining soluble up to concentrations of several g·L-1 in plasma. The exact factors crucial for the solubility of LCs are poorly understood, but it can be hypothesized that their amino acid sequence plays an important role. Determining the precise sequences of patient-derived LCs is therefore highly desirable. We establish here a novel de novo sequencing workflow for patient-derived LCs, based on the combination of bottom-up and top-down proteomics without database search. PEAKS is used for the de novo sequencing of peptides that are further assembled into full length LC sequences using ALPS. Top-down proteomics provides the molecular masses of proteoforms and allows the exact determination of the amino acid sequence including all posttranslational modifications. This pipeline is then used for the complete de novo sequencing of LCs extracted from the urine of 10 patients with multiple myeloma. We show that for the bottom-up part, digestions with trypsin and Nepenthes digestive fluid are sufficient to produce overlapping peptides able to generate the best sequence candidates. Top-down proteomics is absolutely required to achieve 100% final sequence coverage and characterize clinical samples containing several LCs. Our work highlights an unexpected range of modifications.

Influence of platelet count at diagnosis and during the course of disease on prognosis in MDS patients.

Thrombocytopenia at diagnosis and platelet drop within the first 6 months have an adverse effect on prognosis of MDS patients. We therefore were interested in the association and impact on prognosis of morphologic findings of megakaryocytes and platelets with platelet count at diagnosis, bleeding complications, and the drop of platelets during the course of disease. This retrospective analysis was based on 334 MDS patients from the Duesseldorf MDS registry that were followed up for blood counts, bleeding, transfusion dependency, and AML evolution and correlated with morphology of the megakaryocytes and platelets. Thrombocytopenia was found more frequently in higher risk MDS and was associated with hypocellularity of the megakaryocytes in the bone marrow. Signs of bleeding were present at diagnosis in 14% and occurred during the disease in 48% of all MDS patients. Death due to bleeding was ranked third behind infections and AML. A decrement of platelets during the first 6 months was associated with an inferior overall survival of 21 vs. 49 months and with a higher cumulative 2-year AML rate of 22.2% vs. 8.3% (p = 0.001). In a multivariate analysis, besides bone marrow blasts and karyotype, decreasing platelets were also associated with an inferior outcome. Signs of bleeding are present in a relevant number of MDS patients and account for significant morbidity and mortality in MDS. We could demonstrate the prognostic importance of decreasing platelets during the course of disease in all MDS patients, identifying patients at higher risk for death or AML progression.

Treatment of myeloid malignancies relapsing after allogeneic hematopoietic stem cell transplantation with venetoclax and hypomethylating agents-a retrospective multicenter analysis on behalf of the German Cooperative Transplant Study Group.

Treatment of relapse after allogeneic hematopoietic stem cell transplantation (alloHSCT) remains a great challenge. Aiming to evaluate the combination of venetoclax and hypomethylating agents (HMAClax) for the treatment of relapse of myeloid malignancies after alloHSCT, we retrospectively collected data from 32 patients treated at 11 German centers. Venetoclax was applied with azacitidine (n = 13) or decitabine (n = 19); 11 patients received DLI in addition. HMAClax was the first salvage therapy in 8 patients. The median number of cycles per patient was 2 (1-19). All but 1 patient had grade 3/4 neutropenia. Hospital admission for grade 3/4 infections was necessary in 23 patients (72%); 5 of these were fatal. In 30 evaluable patients, overall response rate (ORR) was 47% (14/30, 3 CR MRDneg, 5 CR, 2 CRi, 1 MLFS, 3 PR). ORR was 86% in first salvage patients versus 35% in later salvage patients (p = 0.03). In 6 patients with molecular relapse (MR), ORR was 67% versus 42% in patients with hematological relapse (HR) (n = 24, p = n.s.). After a median follow-up of 8.4 months, 25 patients (78%) had died and 7 were alive. Estimated median overall survival was 3.7 months. Median survival of patients with HMAClax for first versus later salvage therapy was 5.7 and 3.4 months (p = n.s.) and for patients with MR (not reached) compared to HR (3.4 months, p = 0.024). This retrospective case series shows that venetoclax is utilized in various different combinations, schedules, and doses. Toxicity is substantial and patients who receive venetoclax/HMA combinations for MR or as first salvage therapy derive the greatest benefit.

Prognostic impact of pretransplant measurable residual disease assessed by peripheral blood WT1-mRNA expression in patients with AML and MDS.

OBJECTIVE: As peripheral blood (PB) Wilm's Tumor 1 (WT1)-mRNA expression is established as MRD-marker during conventional AML chemotherapy, impact of pretransplant WT1 expression remains unclear. Therefore, we aimed to assess prognostic impact of pretransplant WT1 expression on post-transplant outcome in patients with AML/MDS. METHODS: In 64 AML/MDS patients, pretransplant WT1 expression was retrospectively analyzed using a standardized assay offering high sensitivity, specificity, and a validated cut-off. Patients were divided into three groups determined by pretransplant remission and WT1 expression. Post-transplant outcome of these groups was compared regarding cumulative incidence of relapse (CIR), relapse-free (RFS), and overall survival (OS). RESULTS: Pretransplant forty-six patients (72%) showed hematologic remission, including 21 (46%) MRD-negative and 25 (54%) MRD-positive patients indicated by WT1 expression, while 18 refractory patients (28%) showed active disease. Two-year estimates of post-transplant CIR, RFS, and OS were similar in MRD-positive (61%, 37%, 54%) and refractory patients (70%, 26%, 56%), but significantly inferior compared with MRD-negative patients (10%, 89%, 90%). After multivariable adjustment, pretransplant MRD negativity measured by WT1 expression retained its prognostic impact on CIR (P = .008), RFS (P = .005), and OS (P = .049). CONCLUSIONS: PB WT1 expression represents a useful method to estimate pretransplant MRD, which is highly predictable for post-transplant outcome and may help improving peri-transplant management in AML/MDS patients.

fuPCR as diagnostic method for the detection of rare fungal pathogens, such as Trichosporon, Cryptococcus and Fusarium.

Fungal respiratory tract colonization is a common finding in patients with hematologic neoplasms due to immunosuppression inherent in the diseases and exacerbated by therapy. This greatly increases the risk of fungal infections of the lungs, which is associated with significant mortality. Therefore, reliable diagnostic methods with rapidly available results are needed to administer adequate antifungal therapy. We have established an improved method for fungal DNA extraction and amplification that allows simultaneous detection of fungal families based on a set of multiplexed real-time PCR reactions (fuPCR). We analyzed respiratory rinses and blood of 94 patients with hematological systemic diseases by fuPCR and compared it with the results of culture and serological diagnostic methods. 40 healthy subjects served as controls. Regarding Candida species, the highest prevalence resulted from microbiological culture of respiratory rinses and from detection of antibodies in blood serum in patients (61 and 47%, respectively) and in the control group (29 and 51%, respectively). Detection of other pathogenic yeasts, such as Cryptococcus and Trichosporon, and molds, such as Fusarium, was only possible in patients by fuPCR from both respiratory rinses and whole blood and serum. These fungal species were found statistically significantly more frequent in respiratory rinses collected from patients after myeloablative therapy for stem cell transplantation compared to samples collected before treatment (P < 0.05i). The results show that fuPCR is a valuable complement to culturing and its inclusion in routine mycological diagnostics might be helpful for early detection of pathophysiologically relevant respiratory colonization for patients with hematologic neoplasms.

We validated a set of PCR reactions (fuPCR) for use in routine diagnostic. In contrast to culture and serological methods, only by fuPCR pathogenic yeasts (Cryptococcus and Trichosporon) and molds (Aspergillus and Fusarium) were detected in respiratory rinses and blood of hematological patients.

Integrin but not CEACAM receptors are dispensable for Helicobacter pylori CagA translocation.

Translocation of the Helicobacter pylori (Hp) cytotoxin-associated gene A (CagA) effector protein via the cag-Type IV Secretion System (cag-T4SS) into host cells is a hallmark of infection with Hp and a major risk factor for severe gastric diseases, including gastric cancer. To mediate the injection of CagA, Hp uses a membrane-embedded syringe-like molecular apparatus extended by an external pilus-like rod structure that binds host cell surface integrin heterodimers. It is still largely unclear how the interaction of the cag-T4SS finally mediates translocation of the CagA protein into the cell cytoplasm. Recently certain carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), acting as receptor for the Hp outer membrane adhesin HopQ, have been identified to be involved in the process of CagA host cell injection. Here, we applied the CRISPR/Cas9-knockout technology to generate defined human gastric AGS and KatoIII integrin knockout cell lines. Although confocal laser scanning microscopy revealed a co-localization of Hp and ß1 integrin heterodimers on gastric epithelial cells, Hp infection studies using the quantitative and highly sensitive Hp ß-lactamase reporter system clearly show that neither ß1 integrin heterodimers (α1ß1, α2ß1 or α5ß1), nor any other αß integrin heterodimers on the cell surface are essential for CagA translocation. In contrast, deletion of the HopQ adhesin in Hp, or the simultaneous knockout of the receptors CEACAM1, CEACAM5 and CEACAM6 in KatoIII cells abolished CagA injection nearly completely, although bacterial binding was only reduced to 50%. These data provide genetic evidence that the cag-T4SS-mediated interaction of Hp with cell surface integrins on human gastric epithelial cells is not essential for CagA translocation, but interaction of Hp with CEACAM receptors is facilitating CagA translocation by the cag-T4SS of this important microbe.

Introduction of a bead beating step improves fungal DNA extraction from selected patient specimens.

In immunocompromised patients a colonisation with fungi carries the risk to develop serious invasive fungal infection. An early detection is therefore important, but not optimal hitherto. Fortunately, molecular genetic methods have increased the sensitivity of fungal detection and limited the time, until results are available. However, their success depends on an efficient extraction of genomic DNA from the fungal cell in the given diagnostic specimen. To improve the routine DNA preparation method for yeasts and moulds, the impact of bead beating on fungal DNA release was evaluated. PBS, blood and respiratory rinse were spiked with Candida glabrata or Aspergillus fumigatus. DNA was extracted by mechanical bead beating in addition to the different steps of the DNA preparation protocol, which comprised liquid nitrogen treatment, proteinase K digestion and DNA isolation using the EZ1 DNA Tissue Kit and Workstation. In every method variant tested, treatment with liquid nitrogen did not improve the DNA release. Bead beating once followed by proteinase K digestion and EZ1-work-up led to the highest DNA release from fungus, spiked in PBS, and increased the extracted DNA amount of C. glabrata about 100-fold and of A. fumigatus about 10-fold in relation to sole EZ1-work-up. In fungus-spiked respiratory rinse and blood, highest increase in DNA release was measured after triple bead beating with simultaneous proteinase K digestion. Fungal DNA release of C. glabrata increased for >100-fold in respiratory rinse and for >1000-fold in blood and of A. fumigatus for >10-fold in respiratory rinse and about 5- to 10-fold in blood. The data of this study clearly demonstrate that preparation of fungal DNA from human specimens is optimized by introduction of a bead beating step to the conventional DNA-preparation method without the necessity of a liquid nitrogen step.

Surveillance of Myelodysplastic Syndrome via Migration Analyses of Blood Neutrophils: A Potential Prognostic Tool.

Autonomous migration is a central characteristic of immune cells, and changes in this function have been correlated to the progression and severity of diseases. Hence, the identification of pathologically altered leukocyte migration patterns might be a promising approach for disease surveillance and prognostic scoring. However, because of the lack of standardized and robust assays, migration patterns have not been clinically exploited so far. In this study, we introduce an easy-to-use and cross-laboratory, standardized two-dimensional migration assay for neutrophil granulocytes from peripheral blood. By combining time-lapse video microscopy and automated cell tracking, we calculated the average migration of neutrophils from 111 individual participants of the German Heinz Nixdorf Recall MultiGeneration study under steady-state, formyl-methionyl-leucyl-phenylalanine-, CXCL1-, and CXCL8-stimulated conditions. Comparable values were obtained in an independent laboratory from a cohort in Belgium, demonstrating the robustness and transferability of the assay. In a double-blinded retrospective clinical analysis, we found that neutrophil migration strongly correlated with the Revised International Prognostic Scoring System scoring and risk category of myelodysplastic syndrome (MDS) patients. In fact, patients suffering from high-risk subtypes MDS with excess blasts I or II displayed highly significantly reduced neutrophil migration. Hence, the determination of neutrophil migration patterns might represent a useful tool in the surveillance of MDS. Taken together, we suggest that standardized migration assays of neutrophils and other leukocyte subtypes might be broadly applicable as prognostic and surveillance tools for MDS and potentially for other diseases.

The Aggregation Conditions Define Whether EGCG is an Inhibitor or Enhancer of α-Synuclein Amyloid Fibril Formation.

The amyloid fibril formation by α -synuclein is a hallmark of various neurodegenerative disorders, most notably Parkinson's disease. Epigallocatechin gallate (EGCG) has been reported to be an efficient inhibitor of amyloid formation by numerous proteins, among them α -synuclein. Here, we show that this applies only to a small region of the relevant parameter space, in particular to solution conditions where EGCG readily oxidizes, and we find that the oxidation product is a much more potent inhibitor compared to the unmodified EGCG. In addition to its inhibitory effects, EGCG and its oxidation products can under some conditions even accelerate α -synuclein amyloid fibril formation through facilitating its heterogeneous primary nucleation. Furthermore, we show through quantitative seeding experiments that, contrary to previous reports, EGCG is not able to re-model α -synuclein amyloid fibrils into seeding-incompetent structures. Taken together, our results paint a complex picture of EGCG as a compound that can under some conditions inhibit the amyloid fibril formation of α -synuclein, but the inhibitory action is not robust against various physiologically relevant changes in experimental conditions. Our results are important for the development of strategies to identify and characterize promising amyloid inhibitors.