Measures of infection prevention and incidence of SARS-CoV-2 infections in cancer patients undergoing radiotherapy in Germany, Austria and Switzerland.

PURPOSE: COVID-19 infection has manifested as a major threat to both patients and healthcare providers around the world. Radiation oncology institutions (ROI) deliver a major component of cancer treatment, with protocols that might span over several weeks, with the result of increasing susceptibility to COVID-19 infection and presenting with a more severe clinical course when compared with the general population. The aim of this manuscript is to investigate the impact of ROI protocols and performance on daily practice in the high-risk cancer patients during this pandemic. METHODS: We addressed the incidence of positive COVID-19 cases in both patients and health care workers (HCW), in addition to the protective measures adopted in ROIs in Germany, Austria and Switzerland using a specific questionnaire. RESULTS: The results of the questionnaire showed that a noteworthy number of ROIs were able to complete treatment in SARS-CoV­2 positive cancer patients, with only a short interruption. The ROIs reported a significant decrease in patient volume that was not impacted by the circumambient disease incidence, the type of ROI or the occurrence of positive cases. Of the ROIs 16.5% also reported infected HCWs. About half of the ROIs (50.5%) adopted a screening program for patients whereas only 23.3% also screened their HCWs. The range of protective measures included the creation of working groups, instituting home office work and protection with face masks. Regarding the therapeutic options offered, curative procedures were performed with either unchanged or moderately decreased schedules, whereas palliative or benign radiotherapy procedures were more often shortened. Most ROIs postponed or cancelled radiation treatment for benign indications (88.1%). The occurrence of SARS-CoV­2 infections did not affect the treatment options for curative procedures. Non-university-based ROIs seemed to be more willing to change their treatment options for curative and palliative cases than university-based ROIs. CONCLUSION: Most ROIs reported a deep impact of SARS-CoV­2 infections on their work routine. Modification and prioritization of treatment regimens and the application of protective measures preserved a well-functioning radiation oncology service and patient care.

Cellphone-based devices for bioanalytical sciences.

During the last decade, there has been a rapidly growing trend toward the use of cellphone-based devices (CBDs) in bioanalytical sciences. For example, they have been used for digital microscopy, cytometry, read-out of immunoassays and lateral flow tests, electrochemical and surface plasmon resonance based bio-sensing, colorimetric detection and healthcare monitoring, among others. Cellphone can be considered as one of the most prospective devices for the development of next-generation point-of-care (POC) diagnostics platforms, enabling mobile healthcare delivery and personalized medicine. With more than 6.5 billion cellphone subscribers worldwide and approximately 1.6 billion new devices being sold each year, cellphone technology is also creating new business and research opportunities. Many cellphone-based devices, such as those targeted for diabetic management, weight management, monitoring of blood pressure and pulse rate, have already become commercially-available in recent years. In addition to such monitoring platforms, several other CBDs are also being introduced, targeting e.g., microscopic imaging and sensing applications for medical diagnostics using novel computational algorithms and components already embedded on cellphones. This report aims to review these recent developments in CBDs for bioanalytical sciences along with some of the challenges involved and the future opportunities.

Disulfiram Inhibits Opsonin-Independent Phagocytosis and Migration of Human Long-Lived In Vitro Cultured Phagocytes from Multiple Inflammatory Diseases.

Disulfiram (DSF), an anti-alcoholism medicine, exerts treatment effects in patients suffering from persistent Borreliosis and also exhibits anti-cancer effects through its copper chelating derivatives and induction of oxidative stress in mitochondria. Since chronic/persistent borreliosis is characterized by increased amounts of pro-inflammatory macrophages, this study investigated opsonin-independent phagocytosis, migration, and surface marker expression of in vivo activated and in vitro cultured human monocyte-derived phagocytes (macrophages and dendritic cells) with and without DSF treatment. Phagocytosis of non-opsonized Dynabeads® M-450 and migration of macrophages and dendritic cells were monitored using live cell analyzer Juli™ Br for 24 h, imaging every 3.5 min. To simultaneously monitor phagocyte function, results were analyzed by a newly developed software based on the differential phase contrast images of cells before and after ingestion of Dynabeads. DSF decreased the phagocytic capacities exhibited by in vitro enriched and long-lived phagocytes. Although no chemotactic gradient was applied to the test system, vigorous spontaneous migration was observed. We therefore set up an algorithm to monitor and quantify both phagocytosis and migration simultaneously. DSF not only reduced phagocytosis in a majority of these long-lived phagocytes but also impaired their migration. Despite these selective effects by DSF, we found that DSF reduced the expression densities of surface antigens CD45 and CD14 in all of our long-lived phagocytes. In cells with a high metabolic activity and high mitochondrial contents, DSF led to cell death corresponding to mitochondrial oxidative stress, whereas metabolically inactive phagocytes survived our DSF treatment protocol. In conclusion, DSF affects the viability of metabolically active phagocytes by inducing mitochondrial stress and secondly attenuates phagocytosis and migration in some long-lived phagocytes.

Major endothelial damage markers identified from hemadsorption filters derived from treated patients with septic shock - endoplasmic reticulum stress and bikunin may play a role.

Introduction: In septic patients the damage of the endothelial barrier is decisive leading to circulatory septic shock with disseminated vascular coagulation, edema and multiorgan failure. Hemadsorption therapy leads to rapid resolution of clinical symptoms. We propose that the isolation of proteins adsorbed to hemadsorption devices contributes to the identification of mediators responsible for endothelial barrier dysfunction. Material and methods: Plasma materials enriched to hemadsorption filters (CytoSorb®) after therapy of patients in septic shock were fractionated and functionally characterized for their effect on cell integrity, viability, proliferation and ROS formation by human endothelial cells. Fractions were further studied for their contents of oxidized nucleic acids as well as peptides and proteins by mass spectrometry. Results: Individual fractions exhibited a strong effect on endothelial cell viability, the endothelial layer morphology, and ROS formation. Fractions with high amounts of DNA and oxidized DNA correlated with ROS formation in the target endothelium. In addition, defined proteins such as defensins (HNP-1), SAA1, CXCL7, and the peptide bikunin were linked to the strongest additive effects in endothelial damage. Conclusion: Our results indicate that hemadsorption is efficient to transiently remove strong endothelial damage mediators from the blood of patients with septic shock, which explains a rapid clinical improvement of inflammation and endothelial function. The current work indicates that a combination of stressors leads to the most detrimental effects. Oxidized ssDNA, likely derived from mitochondria, SAA1, the chemokine CXCL7 and the human neutrophil peptide alpha-defensin 1 (HNP-1) were unique for their significant negative effect on endothelial cell viability. However, the strongest damage effect occurred, when, bikunin - cleaved off from alpha-1-microglobulin was present in high relative amounts (>65%) of protein contents in the most active fraction. Thus, a relevant combination of stressors appears to be removed by hemadsorption therapy which results in fulminant and rapid, though only transient, clinical restitution.

Loss-of-function/gain-of-function polymorphisms of the ATP sensitive P2X7R influence sepsis, septic shock, pneumonia, and survival outcomes.

Introduction: Extracellular ATP (eATP) released from damaged cells activates the P2X7 receptor (P2X7R) ion channel on the surface of surrounding cells, resulting in calcium influx, potassium efflux and inflammasome activation. Inherited changes in the P2X7R gene (P2RX7) influence eATP induced responses. Single nucleotide polymorphisms (SNPs) of P2RX7 influence both function and signaling of the receptor, that in addition to ion flux includes pathogen control and immunity. Methods: Subjects (n = 105) were admitted to the ICU at the University Hospital Ulm, Germany between June 2018 and August 2019. Of these, subjects with a diagnosis of sepsis (n = 75), were also diagnosed with septic shock (n = 24), and/or pneumonia (n = 42). Subjects with pneumonia (n = 43) included those without sepsis (n = 1), sepsis without shock (n = 29) and pneumonia with septic shock (n = 13). Out of the 75 sepsis/septic shock patients, 33 patients were not diagnosed with pneumonia. Controls (n = 30) were recruited to the study from trauma patients and surgical patients without sepsis, septic shock, or pneumonia. SNP frequencies were determined for 16 P2RX7 SNPs known to affect P2X7R function, and association studies were performed between frequencies of these SNPs in sepsis, septic shock, and pneumonia compared to controls. Results: The loss-of-function (LOF) SNP rs17525809 (T253C) was found more frequently in patients with septic shock, and non-septic trauma patients when compared to sepsis. The LOF SNP rs2230911 (C1096G) was found to be more frequent in patients with sepsis and septic shock than in non-septic trauma patients. The frequencies of these SNPs were even higher in sepsis and septic patients with pneumonia. The current study also confirmed a previous study by our group that showed a five SNP combination that included the GOF SNPs rs208294 (C489T) and rs2230912 (Q460R) that was designated #21211 was associated with increased odds of survival in severe sepsis. Discussion: The results found an association between expression of LOF P2RX7 SNPs and presentation to the ICU with sepsis, and septic shock compared to control ICU patients. Furthermore, frequencies of LOF SNPs were found to be higher in sepsis patients with pneumonia compared to those without pneumonia. In addition, a five SNP GOF combination was associated with increased odds of survival in severe sepsis. These results suggest that P2RX7 is required to control infection in pneumonia and that inheritance of LOF variants increases the risk of sepsis when associated with pneumonia. This study confirms that P2RX7 genotyping in pneumonia may identify patients at risk of developing sepsis. The study also identifies P2X7R as a target in sepsis associated with an excessive immune response in subjects with GOF SNP combinations.

Inflammation-associated autophagy-related programmed necrotic death of human neutrophils characterized by organelle fusion events.

The most common form of neutrophil death, under both physiological and inflammatory conditions, is apoptosis. In this study, we report a novel form of programmed necrotic cell death, associated with cytoplasmic organelle fusion events, that occurs in neutrophils exposed to GM-CSF and other inflammatory cytokines upon ligation of CD44. Strikingly, this type of neutrophil death requires PI3K activation, a signaling event usually involved in cellular survival pathways. In the death pathway reported in this study, PI3K is required for the generation of reactive oxygen species, which somehow trigger the generation of large cytoplasmic vacuoles, generated by the fusion of CD44-containing endosomes with autophagosomes and secondary, but not primary, granules. Neutrophils demonstrating vacuolization undergo rapid cell death that depends on receptor-interacting protein 1 kinase activity and papain family protease(s), but not caspases, that are most likely activated and released, respectively, during or as a consequence of organelle fusion. Vacuolized neutrophils are present in infectious and autoimmune diseases under in vivo conditions. Moreover, isolated neutrophils from such patients are highly sensitive toward CD44-mediated PI3K activation, reactive oxygen species production, and cell death, suggesting that the newly described autophagy-related form of programmed neutrophil necrosis plays an important role in inflammatory responses.

Old and New Biomarkers for Infection, Inflammation, and Autoimmunity in Treatment-Resistant Affective and Schizophrenic Spectrum Disorders.

Affective (AF) and Schizophrenic (SZ) Spectrum disorders manifest with risk factors, involving inflammatory processes linked to infections and autoimmunity. This study searched for novel biomarkers in cerebrospinal fluid (CSF) and peripheral blood. A total of 29 AF and 39 SZ patients with treatment-resistant disease were included. In CSF, the chemokine IL-8 was significantly elevated in AF and SZ patients. IL-8 promotes chemotaxis by neutrophils and may originate from different tissues. S100B, a glia-derived brain damage marker, was higher in CSF from AF than SZ patients. Among the plasma-derived biomarkers, ferritin was elevated in AF and SZ. Soluble CD25, indicating Treg dysfunction, was higher in SZ than in AF patients. Interferon-γ, implying virus-specific immune activation, was positive in selective AF patients, only. Both groups showed elevated expression of immunosuppressive CD33 on monocytes, but higher amounts of CD123+ plasmacytoid dendritic cells were restricted to SZ. In conclusion, chemotactic IL-8 indicates neuronal stress and inflammation in the CSF of both groups. Novel plasma-derived biomarkers such as sCD25 and monocytic CD33 distinguish SZ from AF with an autoimmune phenotype.

Monocyte subtype expression patterns in septic patients with diabetes are distinct from patterns observed in obese patients.

Background: Sepsis causes a high rate of mortality and long-term morbidity, associated with an imbalance of innate immunity against infections and inflammation. Obesity and diabetes increase the risk for disease severity. Monocyte dysfunction plays a major role and justify further investigations. Objective: To investigate the distribution and inflammatory phenotypes in circulating monocyte subsets in patients manifesting with sepsis including septic shock with and without obesity and diabetes. Methods: A total of 235 blood samples were tested from critically ill adult patients registered at the intensive care unit (ICU). The cohorts were divided into non-diabetic groups with or without obesity and diabetic groups with or without obesity, suffering from sepsis or septic shock. We determined frequencies of total monocytes and of monocyte subsets in the circulation and density expression levels of functional markers, including CD14, CD16, HLA-DR, CD33, CD163, CD206, and arginase-1 by flow cytometric analysis. Results: When progressing to septic shock in non-diabetic and diabetic patients, the percentages of total monocytes among the leukocyte population and of CD33+ and CD14+ monocytes among the monocyte population were consistently down-regulated compared to non-sepsis in non-diabetic and diabetic patients, respectively. Non-diabetic sepsis patients further presented with decreased CD33 and up-regulated CD163 expression density, which was absent in diabetic patients. We subsequently addressed obesity-related changes of monocytes in non-diabetic and diabetic septic patients. Obese septic patients with diabetes were unique in displaying increased monocytic CD16 and CD163 expression. However, obese septic patients without diabetes solely presented with lower amounts of non-classical monocytes. Body mass index (BMI) dependent changes were restricted to diabetic septic patients, with a significantly higher diminution of the classical monocyte subset and concomitantly increased CD16 expression densities. Conclusion: Distribution and phenotypes of monocyte subsets were differentially modulated in critically ill patients with and without metabolic disease when progressing to sepsis or septic shock. Only diabetic septic patients displayed decline of classical monocytes and increase of CD16 expression densities. Therefore, diabetes but not obesity appears to promote the inflammatory phenotype of circulating monocytes in critically ill patients.

Prognostic markers for the clinical course in the blood of patients with SARS-CoV-2 infection.

BACKGROUND: The presentation of peptides and the subsequent immune response depend on the MHC characteristics and influence the specificity of the immune response. Several studies have found an association between HLA variants and differential COVID-19 outcomes and have shown that HLA genotypes are associated with differential immune responses against SARS-CoV-2, particularly in severely ill patients. Information, whether HLA haplotypes are associated with the severity or length of the disease in moderately diseased individuals is absent. METHODS: Next-generation sequencing-based HLA typing was performed in 303 female and 231 male non-hospitalized North Rhine Westphalian patients infected with SARS-CoV2 during the first and second wave. For HLA-Class I, we obtained results from 528 patients, and for HLA-Class II from 531. In those patients, who became ill between March 2020 and January 2021, the 22 most common HLA-Class I (HLA-A, -B, -C) or HLA-Class II (HLA -DRB1/3/4, -DQA1, -DQB1) haplotypes were determined. The identified HLA haplotypes as well as the presence of a CCR5Δ32 mutation and number of O and A blood group alleles were associated to disease severity and duration of the disease. RESULTS: The influence of the HLA haplotypes on disease severity and duration was more pronounced than the influence of age, sex, or ABO blood group. These associations were sex dependent. The presence of mutated CCR5 resulted in a longer recovery period in males. CONCLUSION: The existence of certain HLA haplotypes is associated with more severe disease.

A Thrombomodulin Promoter Gene Polymorphism, rs2239562, Influences Both Susceptibility to and Outcome of Sepsis.

Objective: Disseminated intravascular coagulation plays a key role in the pathophysiology of sepsis. Thrombomodulin is essential in the protein C system of coagulation cascade, and functional polymorphisms influence the human thrombomodulin gene (THBD). Therefore, we conducted a multicenter study to evaluate the influence of such polymorphisms on the pathophysiology of sepsis. Methods: A collaborative case-control study in the intensive care unit (ICU) of each of five tertiary emergency centers. The study included 259 patients (of whom 125 displayed severe sepsis), who were admitted to the ICU of Chiba University Hospital, Chiba, Japan between October 2001 and September 2008 (discovery cohort) and 793 patients (of whom 271 patients displayed severe sepsis), who were admitted to the five ICUs between October 2008 and September 2012 (multicenter validation cohort). To assess the susceptibility to severe sepsis, we further selected 222 critically ill patients from the validation cohort matched for age, gender, morbidity, and severity with the patients with severe sepsis, but without any evidence of sepsis. Results: We examined whether the eight THBD single nucleotide polymorphisms (SNPs) were associated with susceptibility to and/or mortality of sepsis. Higher mortality on severe sepsis in the discovery and combined cohorts was significantly associated with the CC genotype in a THBD promoter SNP (-1920*C/G; rs2239562) [odds ratio [OR] 2.709 (1.067-6.877), P = 0.033 and OR 1.768 (1.060-2.949), P = 0.028]. Furthermore, rs2239562 SNP was associated with susceptibility to severe sepsis [OR 1.593 (1.086-2.338), P = 0.017]. Conclusions: The data demonstrate that rs2239562, the THBD promoter SNP influences both the outcome and susceptibility to severe sepsis.

Association of HLA genotypes, AB0 blood type and chemokine receptor 5 mutant CD195 with the clinical course of COVID-19.

BACKGROUND: COVID-19, the pandemic disease caused by infection with SARS-CoV-2, may take highly variable clinical courses, ranging from symptom-free and pauci-symptomatic to fatal disease. The goal of the current study was to assess the association of COVID-19 clinical courses controlled by patients' adaptive immune responses without progression to severe disease with patients' Human Leukocyte Antigen (HLA) genetics, AB0 blood group antigens, and the presence or absence of near-loss-of-function delta 32 deletion mutant of the C-C chemokine receptor type 5 (CCR5). PATIENT AND METHODS: An exploratory observational study including 157 adult COVID-19 convalescent patients was performed with a median follow-up of 250 days. The impact of different HLA genotypes, AB0 blood group antigens, and the CCR5 mutant CD195 were investigated for their role in the clinical course of COVID-19. In addition, this study addressed levels of severity and morbidity of COVID-19. The association of the immunogenetic background parameters were further related to patients' humoral antiviral immune response patterns by longitudinal observation. RESULTS: Univariate HLA analyses identified putatively protective HLA alleles (HLA class II DRB1*01:01 and HLA class I B*35:01, with a trend for DRB1*03:01). They were associated with reduced durations of disease instead decreased (rather than increased) total anti-S IgG levels. They had a higher virus neutralizing capacity compared to non-carriers. Conversely, analyses also identified HLA alleles (HLA class II DQB1*03:02 und HLA class I B*15:01) not associated with such benefit in the patient cohort of this study. Hierarchical testing by Cox regression analyses confirmed the significance of the protective effect of the HLA alleles identified (when assessed in composite) in terms of disease duration, whereas AB0 blood group antigen heterozygosity was found to be significantly associated with disease severity (rather than duration) in our cohort. A suggestive association of a heterozygous CCR5 delta 32 mutation status with prolonged disease duration was implied by univariate analyses but could not be confirmed by hierarchical multivariate testing. CONCLUSION: The current study shows that the presence of HLA class II DRB1*01:01 and HLA class I B*35:01 is of even stronger association with reduced disease duration in mild and moderate COVID-19 than age or any other potential risk factor assessed. Prospective studies in larger patient populations also including novel SARS-CoV-2 variants will be required to assess the impact of HLA genetics on the capacity of mounting protective vaccination responses in the future.

Informed consent and informed intervention: SARS-CoV-2 vaccinations not just call for disclosure of newly emerging safety data but also for hypothesis generation and testing.

BACKGROUND: COVID-19 infection is a major threat to patients and health care providers around the world. One solution is the vaccination against SARS-CoV-2. METHODS: We performed a comprehensive query of the latest publications on the prevention of viral infections including the recent vaccination program and its side effects. RESULTS: The situation is evolving rapidly and there is no reasonable alternative to population-scale vaccination programs as currently enrolled. CONCLUSION: Therefore, regulatory authorities should consider supplementing their conventional mandate of post-approval pharmacovigilance, which is based on the collection, assessment, and regulatory response to emerging safety findings.

Pyrosequencing-based strategy for a successful SNP detection in two hypervariable regions: HV-I/HV-II of the human mitochondrial displacement loop.

Forensic analysis of mitochondrial displacement loop (D-loop) sequences using Sanger sequencing or SNP detection by minisequencing is well established. Pyrosequencing has become an important alternative because it enables high-throughput analysis and the quantification of individual mitochondrial DNAs (mtDNAs) in samples originating from more than one individual. DNA typing of the mitochondrial D-loop region is usually the method of choice if STR analysis fails because of trace amounts of DNA and/or extensive degradation. The main aim of the present work was to optimize the efficiency of pyrosequencing. To do this, 31 SNPs within the hypervariable regions I and II of the D-loop of human mtDNA were simultaneously analyzed. As a novel approach, we applied two sets of amplification primers for the multiplexing assay. These went in combination with four sequencing primers for pyrosequencing. This method was compared with conventional sequencing of mtDNA from blood and biological trace materials.

Effective Treatment of Glioblastoma Multiforme With Oncolytic Virotherapy: A Case-Series.

Glioblastoma multiforme (GBM) remains an incurable condition, associated with a median survival time of 15 months with best standard of care and 5-year survival rate of <10%. We report on four GBM patients on combination treatment regimens that included oncolytic virus (OV) immunotherapy, who achieved clinical and radiological responses with long-term survival, thus far, of up to 14 years, and good quality of life. We discuss the radiological findings that provide new insights into this treatment, the scientific rationale of this innovative and promising therapy, and considerations for future research.

Hemophagocytic lymphohistiocytosis and Kawasaki disease: combined manifestation and differential diagnosis.

Both hemophagocytic lymphohistiocytosis (HLH) and Kawasaki disease (KD) are diagnosed in patients with prolonged resistant fever by using a scoring system. Concurrent manifestation of both conditions has been reported previously. We describe an infant of 7 weeks whose condition fulfilled the criteria of HLH, but who, after clinical response to treatment, suddenly died from a myocardial infarction at 11 weeks. Post-mortem examination revealed a previously unknown coronary arteritis typical for KD. Since it is difficult to distinguish between KD and HLH, both diseases should be considered in young children with overlapping symptoms. Repeated echocardiograms may be helpful in these cases.

Cytokine Expression Patterns and Single Nucleotide Polymorphisms (SNPs) in Patients with Chronic Borreliosis.

(1) Background: Genetically based hyperinflammation may play a role in pathogen defense. We here questioned whether alterations in circulating monocytes/macrophages, inflammatory biomarkers and a functional SNP (single nucleotide polymorphisms) of the Interleukin-6 (IL-6) promotor might play a role in patients with persistent, and treatment resistant borreliosis. (2) Methods: Leukocyte subpopulations were studied by flow cytometry; plasma cytokines were determined by a chemiluminescence based ELISA (Immulite®), and genotypes of the IL-6 promotor SNP rs1800795 were determined by pyrosequencing. (3) Results: In a cohort of n = 107 Lyme borreliosis patients, who concomitantly manifested either malignant diseases (group 1), autoimmune disorders (group 2), neurological diseases (group 3), or morbidities caused by multiple other infectious complications (group 4), we found decreased numbers of anti-inflammatory CD163-positive macrophages, elevated concentrations of inflammatory cytokines, and an imbalance of IL-6 promotor SNP rs1800795 genotypes. The most prominently upregulated cytokines were IL-1ß, and IL-8. (4) Conclusions: Increased pro-inflammatory phenotypes identified by monocyte/macrophage subtypes and concomitantly increased cytokines appear to be valid to monitor disease activity in patients with persistent Lyme borreliosis. Patterns may vary by additional co-morbidities. In patients with autoimmune diseases, increased frequencies of a heterozygous IL-6 promotor SNP rs1800795 were identified. This functional SNP may guide chronic inflammation, impacting other cytokines to trigger trigger chronicity and therapeutic resistance in Lyme borreliosis.

Bioassay for Endothelial Damage Mediators Retrieved by Hemoadsorption.

Hemoadsorption devices are used to treat septic shock by adsorbing inflammatory cytokines and as yet incompletely defined danger and pathogen associated molecular patterns. In an ideal case, hemoadsorption results in immediate recovery of microvascular endothelial cells' (mEC) function and rapid recovery from catecholamine-dependency and septic shock. We here tested a single device, which consists of polystyrene-divinylbenzene core particles of 450 µm diameter with a high affinity for hydrophobic compounds. The current study aimed at the proof of concept that endothelial-specific damage mediators are adsorbed and can be recovered from hemoadsorption devices. Because of excellent clinical experience, we tested protein fractions released from a hemoadsorber in a novel endothelial bioassay. Video-based, long-term imaging of mEC proliferation and cell death were evaluated and combined with apoptosis and ATP measurements. Out of a total of 39 fractions recovered from column fractionation, we identified 3 fractions that caused i) inhibition of mEC proliferation, ii) increased cell death and iii) induction of apoptosis in mEC. When adding these 3 fractions to mEC, their ATP contents were reduced. These fractions contained proteins of approximately 15 kDa, and high amounts of nucleic acid, which was at least in part oxidized. The efficacy for endothelial cell damage prevention by hemoadsorption can be addressed by a novel endothelial bioassay and long-term video observation procedures. Protein fractionation of the hemoadsorption devices used is feasible to study and define endothelial damage ligands on a molecular level. The results suggest a significant effect by circulating nucleic acids - bound to an as yet undefined protein, which may constitute a major danger-associated molecular pattern (DAMP) in the exacerbation of inflammation when patients experience septic shock. Hemoadsorption devices may thus limit endothelial damage, through the binding of nucleic acid-bearing aggregates and thus contribute to improved endothelial barrier function.

A direct immunoassay assessment of streptavidin- and N-hydroxysuccinimide-modified biochips in validation of serological TNFalpha responses in hemophagocytic lymphohistiocytosis.

The authors report 2 biochip platforms on gold manufactured by either nanoscale biotinylated self-assembled architectures to streptavidin surface or proteins containing free NH(2) groups to N-hydroxysuccinimide (NHS)-activated surfaces and investigated the potential application of tumor necrosis factor-alpha (TNFalpha) serodiagnosis of hemophagocytic lymphohistiocytosis (HLH). Interactions of TNFalpha antigen and TNFalpha antibody on the biochips were optimized using an indirect immunofluorescence method. Variation coefficients were 1.87% to 4.56% on the streptavidin biochip and 5.03% to 8.64% on the NHS biochip. The correlation coefficients (r) in TNFalpha and TNFalpha antibody assays in HLH patients between the 2 biochip formats were 0.9623 and 0.9386 and the concordance frequencies were 92.2% and 96.1%, respectively. To detect plasma TNFalpha-receptor complexes (TNFR1 and R2) in HLH, a biochip assay strategy was developed. Plasma levels of TNFalpha, TNFalpha antibody, and TNFalpha-receptor complexes (TNFR1 and R2) were detected in plasmas from 42 HLH cases using streptavidin biochips. Frequencies of the biomarkers in the plasmas were 40.5% (17/42) for TNFalpha, 30.9% (13/42) for TNFalpha antibody, 28.6% (12/42) for TNFalpha-receptor 1 complex, and 26.1% (11/42) for TNFalpha-receptor 2 complex, respectively. The streptavidin biochip format was more sensitive than the NHS surface and was demonstrated to be a valuable tool to identify individual biomarker molecules and molecular complexes in sera and cell lysates and to track therapeutic progress of patients.

Suspension microarrays for the identification of the response patterns in hyperinflammatory diseases.

Miniaturized and parallelized sandwich immunoassays allow the simultaneous analysis of a variety of parameters in a single experiment. Bead-based protein array systems or suspension microarrays are well-established multiplex sandwich immunoassay formats. To study inflammatory diseases, protein arrays can be used to analyze changes in plasma protein levels, such as cytokines, chemokines, soluble receptors, and matrix metalloproteinases. Using the bead-based Luminex system, multiplexed sandwich immunoassays have been developed to analyze the plasma concentrations of soluble receptors: sTNF-RI, sTNF-RII, sIL-2R, sgp130, sFas, sRAGE, sE-selectin, sICAM-1, sVCAM-1, sMIF-1 and sFasL. This newly established 11-plex soluble receptors assay demonstrated acceptable intra-assay and inter-assay precision, appropriate accuracy, and no crossreactivity between analytes. Using this assay, 100 plasma samples derived from 36 critically ill intensive care unit (ICU) patients with trauma or sepsis were analyzed for their soluble receptor plasma concentrations. Results obtained allowed grouping of patients' samples into a trauma and a sepsis group. Four candidate molecules: sFas, sICAM-1, sTNF-RI, and sTNF-RII had higher concentrations in patients with sepsis than in those with trauma, contributing the highest discriminatory values to define the nature of the inflammatory disease originating from pathogen-involved (sepsis) or pathogen-independent inflammation.

MiR-744-5p inducing cell death by directly targeting HNRNPC and NFIX in ovarian cancer cells.

MicroRNAs (miRNAs) play an important role in the regulation of gene expression. The binding to target messenger RNAs (mRNAs) results in mRNA cleavage or inhibition of the translational machinery leading to decreased protein levels. Various signalling pathways, including apoptosis are modulated by miRNAs. Here, we investigated the role of miR-744-5p in apoptosis signalling in ovarian cancer cell lines. MiR-744-5p expression was reduced in the cancer cell lines independent of the host gene MAP2K4. Overexpression of miR-744-5p activated the intrinsic apoptotic pathway in SKOV3, OVCAR3 and Cisplatin resistant (A2780-cis) and non-resistant A2780 cells leading to cell death. Notably, miR-744-5p overexpression together with Carboplatin treatment led to at least additive pro-apoptotic effects. Investigation of the apoptotic signalling pathways mediated by miR-744-5p revealed that its elevated expression directly downregulated mRNA and protein expression of nuclear factor I X (NFIX) and heterogeneous nuclear ribonucleoprotein C (HNRNPC). HNRNPC caused diminished miR-21 expression and AKT phosphorylation, while NFIX decreased Bcl2 levels, leading to the detected pro-apoptotic effects. Finally, Kaplan-Meier-Plots showed a prolonged median disease-free survival in ovarian serous cystadenocarcinoma patients with high miR-744 expression.