The Protein S Erlangen Mutation PROS1c.1904T>C (F635S) Suppresses Secretion.

BACKGROUND: The recently identified PROS1 mutation Protein S Erlangen c.1904T>C, resulting in amino acid exchange F635S, is associated with severe quantitative protein S (PS) deficiency and clinical thrombosis. It was hypothesized that this deficiency is due to a secretion defect [1]. This report aims to further elucidate the potential secretion defect of PS Erlangen. METHODS: Coding sequences (CDS) of wild type (WT) PROS1 (encoding PS) and mutated PROS1c.1904T>C (encoding PSF635S) were cloned in front of the CDS of green fluorescent protein (GFP), and the respective plasmids were introduced into HEK293T cells. PROS1-GFP and PROS1c.1904T>C-GFP expressing HEK293T cell lines were analyzed by confocal laser scanning microscopy and western blot for cellular proteins and proteins secreted to the growth medium. RESULTS: Western blot analysis revealed a significantly reduced secretion of PSF635S compared to WT PS. This observation was confirmed by the detection of mutant PSF635S-GFP fusion exclusively in the endoplasmic reticulum (ER), while PS-GFP passed through the entire secretory pathway, as indicated by the localization within both the ER and Golgi apparatus. CONCLUSIONS: The Protein S Erlangen mutation results in type I PS deficiency caused by a secretion defect.

Validation of a Parvovirus B19 NAT Assay for Screening of Umbilical Cord Blood for Allogenic Hematopoietic Stem Cell Donation.

Introduction: Parvovirus B19 transmitted by umbilical cord blood (UCB) products may cause severe disease in allogenic hematopoietic stem cell transplant recipients. Thus, commercially available nucleic acid test (NAT) assays for highly sensitive detection of parvovirus B19 DNA validated for the specimen cord blood plasma (CBP) are required to avoid parvovirus B19 transmission by umbilical hematopoietic stem cell preparations. Methods: The multiplex cobas DPX NAT assay was validated for detection of parvovirus B19 DNA in CBP derived from citrate anticoagulated UCB units which have been processed by the Rubinstein method. In total, 363 retained CBP samples pretested negative for parvovirus B19 DNA were prepared for analyzing sensitivity, specificity, and interference of that NAT assay. The 3rd WHO International Standard for parvovirus B19 DNA was used for determining the 95% limit of detection (LOD95) by probit analysis. Results: The validation of the parvovirus B19 NAT assay for CBP demonstrated high sensitivity, specificity, intra- and inter-assay precision. Dilution series and replicate analyses showed a high linearity of the assay with a coefficient of determination above 0.99 and revealed a LOD95 of 17 International Units (IU)/mL (95% confidence interval, 14-44 IU/mL) for parvovirus B19 DNA in CBP samples. Conclusion: The validation of a commercially available parvovirus B19 NAT assay for the specimen CBP demonstrated a high assay performance fulfilling German guidelines and international regulations.

SERPINC1 c.1247dupC: a novel SERPINC1 gene mutation associated with familial thrombosis results in a secretion defect and quantitative antithrombin deficiency.

BACKGROUND: Antithrombin (AT) is an important anticoagulant in hemostasis. We describe here the characterization of a novel AT mutation associated with clinically relevant thrombosis. A pair of sisters with confirmed type I AT protein deficiency was genetically analyzed on suspicion of an inherited SERPINC1 mutation. A frameshift mutation, c.1247dupC, was identified and the effect of this mutation was examined on the cellular and molecular level. METHODS: Plasmids for the expression of wild-type (WT) and mutated SERPINC1 coding sequence (CDS) fused to green fluorescent protein (GFP) or hemagglutinin (HA) tag were transfected into HEK293T cells. Subcellular localization and secretion of the respective fusion proteins were analyzed by confocal laser scanning microscopy and Western blot. RESULTS: The c.1247dupC mutation results in a frameshift in the CDS of the SERPINC1 gene and a subsequently altered amino acid sequence (p.Ser417LysfsTer48). This alteration affects the C-terminus of the AT antigen and results in impaired secretion as confirmed by GFP- and HA-tagged mutant AT analyzed in HEK293T cells. CONCLUSION: The p.Ser417LysfsTer48 mutation leads to impaired secretion, thus resulting in a quantitative AT deficiency. This is in line with the type I AT deficiency observed in the patients.

Virus-reactive T cells expanded in aplastic anemia eliminate hematopoietic progenitor cells by molecular mimicry.

ABSTRACT: Acquired aplastic anemia is a bone marrow failure syndrome characterized by hypocellular bone marrow and peripheral blood pancytopenia. Frequent clinical responses to calcineurin inhibition and antithymocyte globulin strongly suggest critical roles for hematopoietic stem/progenitor cell-reactive T-cell clones in disease pathophysiology; however, their exact contribution and antigen specificities remain unclear. We determined differentiation states and targets of dominant T-cell clones along with their potential to eliminate hematopoietic progenitor cells in the bone marrow of 15 patients with acquired aplastic anemia. Single-cell sequencing and immunophenotyping revealed oligoclonal expansion and effector differentiation of CD8+ T-cell compartments. We reexpressed 28 dominant T-cell receptors (TCRs) of 9 patients in reporter cell lines to determine reactivity with (1) in vitro-expanded CD34+ bone marrow, (2) CD34- bone marrow, or (3) peptide pools covering immunodominant epitopes of highly prevalent viruses. Besides 5 cytomegalovirus-reactive TCRs, we identified 3 TCRs that recognized antigen presented on hematopoietic progenitor cells. T cells transduced with these TCRs eliminated hematopoietic progenitor cells of the respective patients in vitro. One progenitor cell-reactive TCR (11A5) also recognized an epitope of the Epstein-Barr virus-derived latent membrane protein 1 (LMP1) presented on HLA-A∗02:01. We identified 2 LMP1-related mimotopes within the human proteome as activating targets of TCR 11A5, providing proof of concept that molecular mimicry of viral and self-epitopes can drive T cell-mediated elimination of hematopoietic progenitor cells in aplastic anemia.

HLA-C*04:09N is expressed at the cell surface and triggers peptide-specific T-cell activation.

The null allele HLA-C*04:09N differs from HLA-C*04:01 in a frameshift mutation within its cytoplasmic domain, resulting in translation of 32 additional amino acids that are assumed to prevent cell surface expression. However, we recently identified a multiple myeloma-reactive T-cell receptor (TCR) that appeared to recognize antigen presented on HLA-C*04:09N and encouraged us to ask whether HLA-C*04:09N, albeit not easily detectable at the cell surface, can present antigen sufficient for T-cell activation. We generated two HLA-class I-deficient cell lines, re-expressed HLAC* 04:09N, detected HLA expression by flow cytometry, and tested for T-cell activation using a cytomegalovirus peptide- specific HLA-C*04:01-restricted TCR. In both cell lines, HLA-C*04:09N expression was detectable at the cell surface and could be enhanced by IFN-γ exposure. Recombinant HLA-C*04:09N expression was sufficient for T-cell activation in vitro, which could be blocked by an HLA-class I-specific antibody, suggesting HLA-TCR interaction at the cell surface. Peripheral blood mononuclear cells isolated from an individual who physiologically expressed HLA-C*04:09N triggered peptide-specific T-cell activation, confirming our results with cells with natural HLA expression levels. In conclusion, we present peptide-specific HLA-C*04:09N-restricted T-cell activation and suggest consideration of this allele in the appropriate clinical context, such as allogeneic stem cell transplantation, or in the setting of cellular therapy.

Human T cells loaded with superparamagnetic iron oxide nanoparticles retain antigen-specific TCR functionality.

Background: Immunotherapy of cancer is an emerging field with the potential to improve long-term survival. Thus far, adoptive transfer of tumor-specific T cells represents an effective treatment option for tumors of the hematological system such as lymphoma, leukemia or myeloma. However, in solid tumors, treatment efficacy is low owing to the immunosuppressive microenvironment, on-target/off-tumor toxicity, limited extravasation out of the blood vessel, or ineffective trafficking of T cells into the tumor region. Superparamagnetic iron oxide nanoparticles (SPIONs) can make cells magnetically controllable for the site-specific enrichment. Methods: In this study, we investigated the influence of SPION-loading on primary human T cells for the magnetically targeted adoptive T cell therapy. For this, we analyzed cellular mechanics and the T cell response after stimulation via an exogenous T cell receptor (TCR) specific for the melanoma antigen MelanA or the endogenous TCR specific for the cytomegalovirus antigen pp65 and compared them to T cells that had not received SPIONs. Results: SPION-loading of human T cells showed no influence on cellular mechanics, therefore retaining their ability to deform to external pressure. Additionally, SPION-loading did not impair the T cell proliferation, expression of activation markers, cytokine secretion, and tumor cell killing after antigen-specific activation mediated by the TCR. Conclusion: In summary, we demonstrated that SPION-loading of T cells did not affect cellular mechanics or the functionality of the endogenous or an exogenous TCR, which allows future approaches using SPIONs for the magnetically enrichment of T cells in solid tumors.

An Exploratory Study Using Next-Generation Sequencing to Identify Prothrombotic Variants in Patients with Cerebral Vein Thrombosis.

Prothrombotic hereditary risk factors for cerebral vein thrombosis (CVT) are of clinical interest to better understand the underlying pathophysiology and stratify patients for the risk of recurrence. This study explores prothrombotic risk factors in CVT patients. An initial screening in patients of the outpatient clinic of the Department of Transfusion Medicine and Hemostaseology of the University Hospital Erlangen, Germany, revealed 183 patients with a history of CVT. An initial screening identified a number of common prothrombic risk factors, including Factor V Leiden (rs6025) and Prothrombin G20210A (rs1799963). All patients without relevant findings (58 individuals) were invited to participate in a subsequent genetic analysis of 55 relevant genes using next-generation sequencing (NGS). Three intron variants (ADAMTS13: rs28446901, FN1: rs56380797, rs35343655) were identified to occur with a significantly higher frequency in the CVT patient cohort compared to the general European population. Furthermore, the combined prevalence of at least two of four potentially prothrombic variants (FGA (rs6050), F13A1 (rs5985), ITGB3 (rs5918), and PROCR (rs867186)) was significantly higher in the CVT subjects. The possible impact of the identified variants on CVT is discussed.

Deep characterization of human γδ T cell subsets defines shared and lineage-specific traits.

Under non-pathological conditions, human γδ T cells represent a small fraction of CD3+ T cells in peripheral blood (1-10%). They constitute a unique subset of T lymphocytes that recognize stress ligands or non-peptide antigens through MHC-independent presentation. Major human γδ T cell subsets, Vδ1 and Vδ2, expand in response to microbial infection or malignancy, but possess distinct tissue localization, antigen recognition, and effector responses. We hypothesized that differences at the gene, phenotypic, and functional level would provide evidence that γδ T cell subpopulations belong to distinct lineages. Comparisons between each subset and the identification of the molecular determinants that underpin their differences has been hampered by experimental challenges in obtaining sufficient numbers of purified cells. By utilizing a stringent FACS-based isolation method, we compared highly purified human Vδ1 and Vδ2 cells in terms of phenotype, gene expression profile, and functional responses. We found distinct genetic and phenotypic signatures that define functional differences in γδ T cell populations. Differences in TCR components, repertoire, and responses to calcium-dependent pathways suggest that Vδ1 and Vδ2 T cells are different lineages. These findings will facilitate further investigation into the ligand specificity and unique role of Vδ1 and Vδ2 cells in early immune responses.

Medium supplementation with human, but not fetal calf serum facilitates endocytosis of PLGA nanoparticles by human primary B-lymphocytes via complement opsonization.

The "biological identity" of nanoparticles (NPs) is governed by a shell consisting of various biomolecules that is formed upon exposure to biological media, the so-called biomolecule corona. Consequently, supplementation of cell culture media with e.g. different sera is likely to affect interactions between cells and NPs ex-vivo, especially endocytosis. We aimed to investigate the differential impact of human and fetal-bovine serum on the endocytosis of poly (lactic-co-glycolic acid) NPs by human peripheral blood mononuclear cells via flow cytometry. Furthermore, we employed different methods to inhibit endocytosis, providing mechanistic insights. The resulting biomolecule corona was characterized via denaturing gel electrophoresis. We found profound differences between human and fetal bovine serum regarding the endocytosis of fluorescently labeled PLGA nanoparticles by different classes of human leukocytes. Uptake by B-lymphocytes was particularly sensitive. We further present evidence, that these effects are mediated by a biomolecule corona. We demonstrate to our knowledge for the first time that the complement is an important contributor to the endocytosis of non-surface-engineered PLGA-nanoparticles prepared via emulsion solvent evaporation by human immune cells. Our data demonstrates that results obtained with xenogeneic culture supplements such as fetal bovine serum may have to be interpreted with caution.

Characterization of the impact of immune checkpoint inhibitors on platelet activation and aggregation.

Immune checkpoint inhibitors (ICIs) are effective oncological drugs which block cellular check-point receptors typically targeted by tumor immune evasion strategies. Despite their benefits, clinicians have reported treatment-associated thromboembolism during ICI therapy in recent years. Though several theories on this ICI-associated pathogenesis exist, the direct effects of ICIs on platelets remains unknown. We therefore investigated the potential direct and indirect effect of PD-1, PD-L1 and CTLA-4-targeting ICIs on platelet functionality in multifaceted in vitro experiments. Interestingly, we could not observe a clear effect of ICI on platelet aggregation and primary hemostasis in whole blood and platelet concentrate-based assays. Furthermore, the presence of ICIs in toll-like receptor stimulation had no significant impact on platelet surface marker expression. In a second approach, we investigated the indirect immunological impact of ICIs on platelet activation by exposing platelets to supernatants from ICI- and Staphylococcal enterotoxin B-exposed PBMCs. Whereas ICIs affected IL-2 levels in supernatants, we could not detect clear differences in the secretion of pro-thrombogenic factors and platelet responses. The obtained data suggest that the direct influence of ICIs on platelet activation or the influence of altered T cell function on platelet activation cannot be considered a major factor in the development of thrombotic events.

Autologous regulatory T-cell transfer in refractory ulcerative colitis with concomitant primary sclerosing cholangitis.

OBJECTIVE: Ulcerative colitis (UC) is a chronic, debilitating immune-mediated disease driven by disturbed mucosal homeostasis, with an excess of intestinal effector T cells and an insufficient expansion of mucosal regulatory T cells (Tregs). We here report on the successful adoptive transfer of autologous, ex vivo expanded Tregs in a patient with refractory UC and associated primary sclerosing cholangitis (PSC), for which effective therapy is currently not available. DESIGN: The patient received a single infusion of 1×106 autologous, ex vivo expanded, polyclonal Tregs per kilogram of body weight, and the clinical, biochemical, endoscopic and histological responses were assessed 4 and 12 weeks after adoptive Treg transfer. RESULTS: The patient showed clinical, biochemical, endoscopic and histological signs of response until week 12 after adoptive Treg transfer, which was associated with an enrichment of intestinal CD3+/FoxP3+ and CD3+/IL-10+ T cells and increased mucosal transforming growth factor beta and amphiregulin levels. Moreover, there was marked improvement of PSC with reduction of liver enzymes. This pronounced effect lasted for 4 weeks before values started to increase again. CONCLUSION: These findings suggest that adoptive Treg therapy might be effective in refractory UC and might open new avenues for clinical trials in PSC. TRIAL REGISTRATION NUMBER: NCT04691232.

Notch1 Induces Defective Epithelial Surfactant Processing and Pulmonary Fibrosis.

Rationale: Although type II alveolar epithelial cells (AEC2s) are chronically injured in idiopathic pulmonary fibrosis (IPF), they contribute to epithelial regeneration in IPF. Objectives: We hypothesized that Notch signaling may contribute to AEC2 proliferation, dedifferentiation characterized by loss of surfactant processing machinery, and lung fibrosis in IPF. Methods: We applied microarray analysis, kinome profiling, flow cytometry, immunofluorescence analysis, western blotting, quantitative PCR, and proliferation and surface activity analysis to study epithelial differentiation, proliferation, and matrix deposition in vitro (AEC2 lines, primary murine/human AEC2s), ex vivo (human IPF-derived precision-cut lung slices), and in vivo (bleomycin and pepstatin application, Notch1 [Notch receptor 1] intracellular domain overexpression). Measurements and Main Results: We document here extensive SP-B and -C (surfactant protein-B and -C) processing defects in IPF AEC2s, due to loss of Napsin A, resulting in increased intra-alveolar surface tension and alveolar collapse and induction of endoplasmic reticulum stress in AEC2s. In vivo pharmacological inhibition of Napsin A results in the development of AEC2 injury and overt lung fibrosis. We also demonstrate that Notch1 signaling is already activated early in IPF and determines AEC2 fate by inhibiting differentiation (reduced lamellar body compartment, reduced capacity to process hydrophobic SP) and by causing increased epithelial proliferation and development of lung fibrosis, putatively via altered JAK (Janus kinase)/Stat (signal transducer and activator of transcription) signaling in AEC2s. Conversely, inhibition of Notch signaling in IPF-derived precision-cut lung slices improved the surfactant processing capacity of AEC2s and reversed fibrosis. Conclusions: Notch1 is a central regulator of AEC2 fate in IPF. It induces alveolar epithelial proliferation and loss of Napsin A and of surfactant proprotein processing, and it contributes to fibroproliferation.

Biomimetic Magnetic Particles for the Removal of Gram-Positive Bacteria and Lipoteichoic Acid.

Gram+ bacteria are very common in clinical medicine and responsible for a large number of infectious diseases. For example, Gram+ bacteria play a major role in causing bloodstream infections and sepsis. Therefore, the detection of Gram+ bacteria is of great importance for the diagnosis and treatment of infectious diseases. Furthermore, these bacteria are often present in biofilms that cover implants. Recent research work has mainly focused on the biologic activity and removal of Gram-negative bacteria or bacterial components such as lipopolysaccharides (LPS). In contrast, the effects of lipoteichoic acid (LTA) have been less well studied so the relevance of their removal from body fluids is possibly underestimated. To address this topic, we evaluated superparamagnetic iron oxide particles (SPION) carrying different peptides derived from the innate immune receptor (GP-340) for their ability to bind and remove Gram+ bacteria and LTA from different media. Our results show that, beyond S. aureus, effective agglutinating and removing of S. pneumoniae was possible. Furthermore, we were able to show for the first time that this was possible with LTA alone and that the magnetic removal of bacteria was also efficient under flow conditions. We also found that this method was able to capture Stapyhylococcus aureus from platelet concentrates, which can help to enhance the sensitivity of microbiological diagnostics, quality control measures, and blood product safety.

Immune Phenotypes and Target Antigens of Clonally Expanded Bone Marrow T Cells in Treatment-Naïve Multiple Myeloma.

Multiple myeloma is a hematologic malignancy of monoclonal plasma cells that accumulate in the bone marrow. Despite their clinical and pathophysiologic relevance, the roles of bone marrow-infiltrating T cells in treatment-naïve patients are incompletely understood. We investigated whether clonally expanded T cells (i) were detectable in multiple myeloma bone marrow, (ii) showed characteristic immune phenotypes, and (iii) whether dominant clones recognized antigens selectively presented on multiple myeloma cells. Single-cell index sorting and T-cell receptor (TCR) αß sequencing of bone marrow T cells from 13 treatment-naïve patients showed dominant clonal expansion within CD8+ cytolytic effector compartments, and only a minority of expanded T-cell clones expressed the classic immune-checkpoint molecules PD-1, CTLA-4, or TIM-3. To identify their molecular targets, TCRs of 68 dominant bone marrow clones from five selected patients were reexpressed and incubated with multiple myeloma and non-multiple myeloma cells from corresponding patients. Only 1 of 68 TCRs recognized antigen presented on multiple myeloma cells. This TCR was HLA-C-restricted, self-peptide-specific and could be activated by multiple myeloma cells of multiple patients. The remaining dominant T-cell clones did not recognize multiple myeloma cells and were, in part, specific for antigens associated with chronic viral infections. In conclusion, we showed that dominant bone marrow T-cell clones in treatment-naïve patients rarely recognize antigens presented on multiple myeloma cells and exhibit low expression of classic immune-checkpoint molecules. Our data provide experimental context for experiences from clinical immune-checkpoint inhibition trials and will inform future T cell-dependent therapeutic strategies.

Correction: Sommer et al. Decreased Thymic Output Contributes to Immune Defects in Septic Patients. J. Clin. Med. 2020, 9, 2695.

In the original article, there were errors in Figure 1D (panel CD8-Control) [...].

Emergency Use of Convalescent Plasma: Perception of the Regulatory Framework from a Clinical Perspective.

The pandemic spread of an infectious disease poses a plethora of challenges to society, clinicians, health care providers and regulating authorities. In order to mount a rapid response and to provide hope in a potentially catastrophic situation as the current COVID-19 pandemic, emergency plans, regulations and funding strategies have to be developed on regional, national and international levels. The speed needed to establish rapid response programs is challenged by the dynamics of the spread of the disease, the concurrent and competing development of different and potentially more effective treatment options, and not the least by regulatory uncertainty. Convalescent plasma, that is plasma collected from patients who have recovered from COVID-19 infections, has emerged as one of the first potential treatment options in the absence of drugs or vaccines with proven efficacy against SARS-CoV-2. The societal aspects of convalescent plasma and the public awareness gave an additional boost to the rapid employment of convalescent plasma donation platforms immediately after the SARS-CoV-2 outbreak. At the same time, uncertainty remains as to the efficacy of convalescent plasma. With evidence mostly limited to empirical reports, convalescent plasma has been used for decades for the prophylaxis and treatment of various infectious diseases. Clinical trials have addressed different infectious agents, stages of disease, target groups of patients and yielded sometimes inconclusive results. The aim of this short review is to delineate the regulatory background for the emergency use of convalescent plasma in the USA, in the European Union and in Germany, and the transition to the setting of clinical trials. In addition, we describe observations made in the process of collecting COVID-19 convalescent plasma (herein referred to as CCP), and formulate proposals to further improve the framework for rapid responses in future emergency situations.

Differential LysoTracker Uptake Defines Two Populations of Distal Epithelial Cells in Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal degenerative lung disease of unknown etiology. Although in its final stages it implicates, in a reactive manner, all lung cell types, the initial damage involves the alveolar epithelial compartment, in particular the alveolar epithelial type 2 cells (AEC2s). AEC2s serve dual progenitor and surfactant secreting functions, both of which are deeply impacted in IPF. Thus, we hypothesize that the size of the surfactant processing compartment, as measured by LysoTracker incorporation, allows the identification of different epithelial states in the IPF lung. Flow cytometry analysis of epithelial LysoTracker incorporation delineates two populations (Lysohigh and Lysolow) of AEC2s that behave in a compensatory manner during bleomycin injury and in the donor/IPF lung. Employing flow cytometry and transcriptomic analysis of cells isolated from donor and IPF lungs, we demonstrate that the Lysohigh population expresses all classical AEC2 markers and is drastically diminished in IPF. The Lysolow population, which is increased in proportion in IPF, co-expressed AEC2 and basal cell markers, resembling the phenotype of the previously identified intermediate AEC2 population in the IPF lung. In that regard, we provide an in-depth flow-cytometry characterization of LysoTracker uptake, HTII-280, proSP-C, mature SP-B, NGFR, KRT5, and CD24 expression in human lung epithelial cells. Combining functional analysis with extracellular and intracellular marker expression and transcriptomic analysis, we advance the current understanding of epithelial cell behavior and fate in lung fibrosis.

Scavenging of bacteria or bacterial products by magnetic particles functionalized with a broad-spectrum pathogen recognition receptor motif offers diagnostic and therapeutic applications.

Sepsis is a dysregulated host response of severe bloodstream infections, and given its frequency of occurrence and high mortality rate, therapeutic improvements are imperative. A reliable biomimetic strategy for the targeting and separation of bacterial pathogens in bloodstream infections involves the use of the broad-spectrum binding motif of human GP-340, a pattern-recognition receptor of the scavenger receptor cysteine rich (SRCR) superfamily that is expressed on epithelial surfaces but not found in blood. Here we show that these peptides, when conjugated to superparamagnetic iron oxide nanoparticles (SPIONs), can separate various bacterial endotoxins and intact microbes (E. coli, S. aureus, P. aeruginosa and S. marcescens) with high efficiency, especially at low and thus clinically relevant concentrations. This is accompanied by a subsequent strong depletion in cytokine release (TNF, IL-6, IL-1ß, Il-10 and IFN-γ), which could have a direct therapeutic impact since escalating immune responses complicates severe bloodstream infections and sepsis courses. SPIONs are coated with aminoalkylsilane and capture peptides are orthogonally ligated to this surface. The particles behave fully cyto- and hemocompatible and do not interfere with host structures. Thus, this approach additionally aims to dramatically reduce diagnostic times for patients with suspected bloodstream infections and accelerate targeted antibiotic therapy. STATEMENT OF SIGNIFICANCE: Sepsis is often associated with excessive release of cytokines. This aspect and slow diagnostic procedures are the major therapeutic obstacles. The use of magnetic particles conjugated with small peptides derived from the binding motif of a broad-spectrum mucosal pathogen recognition protein GP-340 provides a highly efficient scavenging platform. These peptides are not found in blood and therefore are not subject to inhibitory mechanisms like in other concepts (mannose binding lectine, aptamers, antibodies). In this work, data are shown on the broad bacterial binding spectrum, highly efficient toxin depletion, which directly reduces the release of cytokines. Host cells are not affected and antibiotics not adsorbed. The particle bound microbes can be recultured without restriction and thus be used directly for diagnostics.

Protein S Erlangen: a novel PROS1 gene mutation associated with quantitative protein S deficiency.

The members of a Caucasian family were genetically analyzed on suspicion of hereditary protein S deficiency. A novel mutation, c.1904T>C, associated with severe quantitative protein S deficiency was found. The novel PROS1 mutation was identified by sequencing of the PROS1 gene coding sequence. The identified c.1904T>C point mutation results in p.Phe635Ser amino acid exchange, which is located in the Laminin G-like 2 domain of protein S. Computational analysis indicates that this amino acid exchange affects the correct folding of the protein S antigen. Furthermore, this mutation is located in a region of the Laminin G-like 2 domain where changes in the amino acid sequence often result in decreased secretion. We postulate that the novel p.Phe635Ser mutation might lead to an incorrect folding, and thus, to a strongly impaired secretion of this protein S variant. We named this novel variant protein.