Rabies virus cross-reactive murine T cell clones: analysis of helper and delayed-type hypersensitivity function.

Three T cell clones derived from rabies virus-immunized BALB/c mice were analysed for specificity and function. The clones proved to be broadly cross-reactive by responding to different rabies virus isolates (PM, ERA, CVS, HEP) and other representatives of the genus Lyssavirus, like the Duvenhage-6 (DUV6) and Mokola (MOK) viruses. The clones detected three different epitopes: an epitope expressed on the matrix protein (M) shared by PM, HEP, MOK and DUV6 viruses (clone AA8), an epitope expressed on the M-protein shared by PM, ERA, CVS, HEP and MOK viruses (clone 35A) and finally an epitope expressed on the glycoprotein (G-protein) shared by PM, ERA, CVS, HEP and MOK viruses (clone BG2). Antigen recognition of all clones proved to be MHC-restricted and they all displayed the CD4+ CD8- phenotype. Intravenous inoculation of the T cells in syngeneic mice, which had been injected intracutaneously in the ear with HEP virus, resulted in a localized DTH reaction characteristic for TH1 cells. In vitro, the clones were able to provide help to rabies virus-primed B cells, resulting in the production of virus-specific antibodies directed against all the four structural proteins of rabies virus. Further analysis of this antibody response revealed that part of it was directed against antigenic determinants of the G-protein which induce virus neutralizing antibody

Multicompartment dendrimicelles with binary, ternary and quaternary core composition

Hierarchically built-up multicompartment nanoaggregate systems are of interest for, e.g., novel materials and medicine. Here we present a versatile strategy to generate and unambiguously characterize complex coacervate-core micelles by exploiting four different dendrimeric subcomponents as core-units. The resulting mesoscale structures have a hydrodynamic diameter of 50 nm and a core size of 33 nm, and host about thirty 6th generation polyamidoamine (PAMAM) dendrimers. We have used FRET (efficiency of similar to 0.2) between fluorescein and rhodamine moieties immobilized on separate PAMAM dendrimers (G6-F and G6-R, respectively) to prove synchronous encapsulation in the micelle core. Tuning the proximity of the FRET pair molecules either by varying the G6-F : G6-R ratio, or by co-assembling non-functionalized dendrimer (G6-E) in the core, reveals the optimal FRET efficiency to occur at a minimum of 70% loading with G6-F and G6-R. Additional co-encapsulation of 6th generation gold dendrimer-encapsulated nanoparticles (G6-Au) in the micelle core shows a dramatic reduction of the FRET efficiency, which can be restored by chemical etching of the gold nanoparticles from within the micellar core with thiols, leaving the micelle itself intact. This study reveals the controlled co-assembly of up to four different types of subcomponents in one single micellar core and concomitantly shows the wide variety of structures that can be made with a well-defined basic set of subcomponents. It is straightforward to design related strategies, to incorporate inside one micellar core, e.g., even more than 4 different dendrimers, or other classes of (macro)molecules, with different functional groups, other FRET pairs or different encapsulated metal nanoparticles.Imaging- and therapeutic targets in neoplastic and musculoskeletal inflammatory diseas

A KDM4A-PAF1-mediated epigenomic network is essential for acute myeloid leukemia cell self-renewal and survival

Epigenomic dysregulation is a common pathological feature in human hematological malignancies. H3K9me3 emerges as an important epigenomic marker in acute myeloid leukemia (AML). Its associated methyltransferases, such as SETDB1, suppress AML leukemogenesis, whilst H3K9me3 demethylases KDM4C is required for mixed-lineage leukemia rearranged AML. However, the specific role and molecular mechanism of action of another member of the KDM4 family, KDM4A has not previously been clearly defined. In this study, we delineated and functionally validated the epigenomic network regulated by KDM4A. We show that selective loss of KDM4A is sufficient to induce apoptosis in a broad spectrum of human AML cells. This detrimental phenotype results from a global accumulation of H3K9me3 and H3K27me3 at KDM4A targeted genomic loci thereby causing downregulation of a KDM4A-PAF1 controlled transcriptional program essential for leukemogenesis, distinct from that of KDM4C. From this regulatory network, we further extracted a KDM4A-9 gene signature enriched with leukemia stem cell activity; the KDM4A-9 score alone or in combination with the known LSC17 score, effectively stratifies high-risk AML patients. Together, these results establish the essential and unique role of KDM4A for AML self-renewal and survival, supporting further investigation of KDM4A and its targets as a potential therapeutic vulnerability in AML

Mechanistic basis of the Cu(OAc)2 catalyzed azide-ynamine (3 + 2) cycloaddition reaction

R.P.B. and G.A.B. thank GSK and the Engineering and Physical Sciences Research Council (EPSRC) for an industrial CASE studentship (EP/P51066X/1). G.A.B., F.P., and A.J.B.W. thank the Leverhulme Trust (RP-2020-380). A.T.S. and G.A.B. thank the Biotechnology and Biological Research Council (BBSRC) for its support (BB/V017586/1; BB/T000627/1). A.J.B.W. and M.J.A. thank the EPSRC for its support (EP/R025754/1). A.J.B.W. thanks the Leverhulme Trust for a Research Fellowship (RF-2022-014).The Cu-catalyzed azide–alkyne cycloaddition (CuAAC) reaction is used as a ligation tool throughout chemical and biological sciences. Despite the pervasiveness of CuAAC, there is a need to develop more efficient methods to form 1,4-triazole ligated products with low loadings of Cu. In this paper, we disclose a mechanistic model for the ynamine-azide (3 + 2) cycloadditions catalyzed by copper(II) acetate. Using multinuclear nuclear magnetic resonance spectroscopy, electron paramagnetic resonance spectroscopy, and high-performance liquid chromatography analyses, a dual catalytic cycle is identified. First, the formation of a diyne species via Glaser–Hay coupling of a terminal ynamine forms a Cu(I) species competent to catalyze an ynamine-azide (3 + 2) cycloaddition. Second, the benzimidazole unit of the ynamine structure has multiple roles: assisting C–H activation, Cu coordination, and the formation of a postreaction resting state Cu complex after completion of the (3 + 2) cycloaddition. Finally, reactivation of the Cu resting state complex is shown by the addition of isotopically labeled ynamine and azide substrates to form a labeled 1,4-triazole product. This work provides a mechanistic basis for the use of mixed valency binuclear catalytic Cu species in conjunction with Cu-coordinating alkynes to afford superior reactivity in CuAAC reactions. Additionally, these data show how the CuAAC reaction kinetics can be modulated by changes to the alkyne substrate, which then has a predictable effect on the reaction mechanism.Peer reviewe

Hepatitis C virus in hemophilia:Health-related quality of life after successful treatment in the sixth Hemophilia in the Netherlands study

INTRODUCTION: Persons with hemophilia and hepatitis C virus (HCV) infection have a lower health‐related quality of life (HRQoL) than those never HCV infected. However, it is unknown whether HRQoL after HCV eradication is comparable to individuals never HCV infected. We aimed to compare HRQoL between HCV‐cured and never chronically HCV‐infected persons with hemophilia. METHODS: All persons with hemophilia in the Netherlands were invited for a nationwide study conducted in 2018–2019. For the current analysis, participants born before 1992 with data on HRQoL and HCV status were included. HCV status was collected from medical records. HRQoL was measured by RAND‐36 questionnaire, with a minimally important difference set at 4.0 points. Multivariable linear regression was used to adjust for age, hemophilia severity, HIV status, and self‐reported joint impairment. RESULTS: In total, 486 persons were eligible; 180 were HCV cured and 306 never chronically HCV infected. Compared with those never HCV infected, HCV‐cured individuals were older (57 vs. 53 years), more often had severe hemophilia (67% vs. 21%), and reported more impaired joints (median 3 vs. 0). Compared with those never HCV infected, adjusted RAND‐36 domain scores of HCV‐cured individuals cured were lower on all RAND‐36 domains except Pain, ranging from a difference of 4.5 (95% CI, −8.8 to −0.3) for Physical functioning to 11.3 (95% CI, −19.4 to −3.1) for Role limitations due to physical problems. CONCLUSION: Despite effective HCV treatment, HRQoL of HCV‐cured persons with hemophilia is still lower than HRQoL of those never chronically HCV‐infected on all RAND‐36 domains. This implies that careful psychosocial follow‐up and support are indicated

Socioeconomic participation of persons with hemophilia:Results from the sixth hemophilia in the Netherlands study

Background and objectives Treatment availability and comprehensive care have resulted in improved clinical outcomes for persons with hemophilia. Recent data on socioeconomic participation in the Netherlands are lacking. This study assessed participation in education, in the labor market, and social participation for persons with hemophilia compared with the general male population. Methods Dutch adults and children (5-75 years) of all hemophilia severities (n = 1009) participated in a questionnaire study that included sociodemographic, occupational, and educational variables. Clinical characteristics were extracted from electronic medical records. General population data were extracted from Statistics Netherlands. Social participation was assessed with the PROMIS Ability to Participate in Social Roles and Activities short form, with a minimal important difference set at 1.0. Results Data from 906 adults and children were analyzed. Participation in education of 20 to 24 year olds was 68% (general male population: 53%). Educational attainment was higher compared with Dutch males, especially for severe hemophilia. Absenteeism from school was more common than in the general population. The employment-to-population ratio and occupational disability were worse for severe hemophilia than in the general population (64.3% vs. 73.2% and 14.7% vs. 4.8%, respectively), but similar for nonsevere hemophilia. Unemployment was 5.4% (general male population: 3.4%). Absenteeism from work was less common (38% vs. 45.2%). Mean PROMIS score was similar to or higher than in the general population (54.2; SD 8.9 vs. 50; SD 10). Conclusion Socioeconomic participation of persons with nonsevere hemophilia was similar to the general male population. Some participation outcomes for persons with severe hemophilia were reduced

Maternal and neonatal bleeding complications in relation to peripartum management in hemophilia carriers:A systematic review

Currently, there is no consensus on the optimal management to prevent postpartum hemorrhage (PPH) in hemophilia carriers. We aimed to evaluate peripartum management strategies in relation to maternal and neonatal bleeding outcomes by performing an extensive database search up to August 2020. Seventeen case-reports/series and 11 cohort studies were identified of overall 'poor' quality describing 502 deliveries. The PPH incidence in the individual patient data was 63%; 44% for those women receiving prophylaxis to correct coagulation and 77% for those without (OR 0.23, CI 0.09-0.58) and in cohort data 20.3% (26.8% (11/41) vs. 19.4% (55/284) (OR: 1.53, 95% CI: 0.72-3.24), respectively. Peripartum management strategies mostly consisted of clotting factor concentrates, rarely of desmopressin or plasma. Tranexamic acid appears promising in preventing secondary PPH, but was not used consistently. Neonatal bleeding was described in 6 affected male neonates, mostly after instrumental delivery or emergency CS, but insufficient information was provided to reliably investigate neonatal outcome in relation to management. The high PPH risk seems apparent, at most mildly attenuated by prophylactic treatment. Prospective cohort studies are needed to determine the optimal perinatal management in hemophilia.Thrombosis and Hemostasi

'Be active, eat right', evaluation of an overweight prevention protocol among 5-year-old children: design of a cluster randomised controlled trial

BACKGROUND: The prevalence of overweight and obesity in children has at least doubled in the past 25 years with a major impact on health. In 2005 a prevention protocol was developed applicable within Youth Health Care. This study aims to assess the effects of this protocol on prevalence of overweight and health behaviour among children. METHODS AND DESIGN: A cluster randomised controlled trial is conducted among 5-year-old children included by 44 Youth Health Care teams randomised within 9 Municipal Health Services. The teams are randomly allocated to the intervention or control group. The teams measure the weight and height of all children. When a child in the intervention group is detected with overweight according to the international age and gender specific cut-off points of BMI, the prevention protocol is applied. According to this protocol parents of overweight children are invited for up to three counselling sessions during which they receive personal advice about a healthy lifestyle, and are motivated for and assisted in behavioural change.The primary outcome measures are Body Mass Index and waist circumference of the children. Parents will complete questionnaires to assess secondary outcome measures: levels of overweight inducing/reducing behaviours (i.e. being physically active, having breakfast, drinking sweet beverages and watching television/playing computer games), parenting styles, parenting practices, and attitudes of parents regarding these behaviours, health-related quality of life of the children, and possible negative side effects of the prevention protocol. Data will be collected at baseline (when the children are aged 5 years), and after 12 and 24 months of follow-up. Additionally, a process and a cost-effectiveness evaluation will be conducted. DISCUSSION: In this study called 'Be active, eat right' we evaluate an overweight prevention protocol for use in the setting of Youth Health Care. It is hypothesized that the use of this protocol will result in a healthier lifestyle of the children and an improved BMI and waist circumference. TRIAL REGISTRATION: Current Controlled Trials ISRCTN04965410

Liver-related complications before and after successful treatment of chronic hepatitis C virus infection in people with inherited bleeding disorders

Introduction: With availability of direct-acting antivirals (DAA), most persons with inherited bleeding disorders are currently cured of hepatitis C virus (HCV) infection. The risk of liver-related complications following HCV cure has not been reported for this population. Aim: Reporting liver-related complications during long-term chronic HCV infection and following sustained virological response (SVR) in this population. Methods: Retrospective follow-up of a prospective single-centre cohort of HCV antibody-positive persons with inherited bleeding disorders. Primary endpoint was liver-related complications [hepatocellular carcinoma (HCC), decompensated cirrhosis, bleeding gastroesophageal varices]. Liver-related complications were reported separately during chronic HCV and following SVR, stratified for interferon-based and DAA-based SVR. Results: In total 309/381 (81%) HCV antibody-positive individuals developed chronic HCV infection. Median follow-up was 44 years [interquartile range (IQR): 34–50]. Liver-related complications occurred in 36/309 (12%) of individuals with chronic HCV infection after median 31 years of chronic infection. Of 199 individuals with SVR, 97 were cured with interferon-based regimens and 102 with DAA after median infection durations of 29 and 45 years, respectively. At end of follow-up, respectively, 21% and 42% had advanced fibrosis or cirrhosis. Post-SVR, seven (4%) individuals had a liver-related complication, mainly HCC (n = 4). Incidence of liver-related complications per 100 patient-years post-SVR follow-up was.2 for interferon-cured and 1.0 for DAA-cured individuals (p =.01). Conclusion: Successful HCV treatment does not eliminate the risk of liver-related complications in persons with inherited bleeding disorders. Due to higher baseline risk, incidence was higher after DAA than interferon-based SVR. We advise continuing HCC surveillance post-SVR in all with advanced fibrosis or cirrhosis

Genetic biomarkers for intravenous immunoglobulin response in chronic inflammatory demyelinating polyradiculoneuropathy

Background and purpose Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a clinical and electrophysiological heterogeneous immune-mediated polyneuropathy. Intravenous immunoglobulin (IVIg), corticosteroids, and plasma exchange are proven effective treatments for CIDP. The clinical response to IVIg is variable between patients and currently unexplained. Finding biomarkers related to treatment response can help to understand the diversity of CIDP and personalise treatment choice.Methods We investigated whether genetic variation between patients may explain some of these differences in treatment response. Based on previous publications, we selected six candidate genes that might affect immune and axonal functions, IVIg metabolism, and treatment response in CIDP. Genetic variants were assessed in 172 CIDP patients treated with at least one course of IVIg (2 g/kg). A response to IVIg was defined by >= 1 grade improvement on the modified Rankin Scale. Blood samples were tested for variations in CNTN2, PRF1, FCGRT, FCGR2B, GJB1, and SH2D2A genes.Results In univariate analysis, patients with the FCGR2B promoter variant 2B.4/2B.1 responded more often to IVIg than patients with the 2B.1/2B.1 variant (odds ratio [OR] = 6.9, 95% confidence interval [CI] = 1.6-30; p = 0.003). Patients with the p.(Ala91Val) variant of PRF1 were less often IVIg responsive (OR = 0.34, 95% CI = 0.13-0.91; p = 0.038). In multivariate analysis, both PRF1 and FCGR2B showed discriminative ability to predict the chance of IVIg response (area under the curve = 0.67).Conclusions Variations in PRF1 and the promoter region of FCGR2B are associated with the response to IVIg in CIDP. These findings, which require validation, are a first step towards the understanding of the heterogeneity in the treatment response in CIDP.Genetics of disease, diagnosis and treatmen