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01 juni 2023, Item does not contain fulltext, BACKGROUND: Novel mRNA-based vaccines have been used to protect against SARS-CoV-2, especially in vulnerable populations who also receive an annual influenza vaccination. The TACTIC study investigated potential immune interference between the mRNA COVID-19 booster vaccine and the quadrivalent influenza vaccine, and determined if concurrent administration would have effects on safety or immunogenicity. METHODS: TACTIC was a single-blind, placebo-controlled randomized clinical trial conducted at the Radboud University Medical Centre, the Netherlands. Individuals ≥60 years, fully vaccinated against COVID-19 were eligible for participation and randomized into one of four study groups: 1) 0.5 ml influenza vaccination Vaxigrip Tetra followed by 0.3 ml BNT162b2 COVID-19 booster vaccination 21 days later, (2) COVID-19 booster vaccination followed by influenza vaccination, (3) influenza vaccination concurrent with the COVID-19 booster vaccination, and (4) COVID-19 booster vaccination only (reference group). Primary outcome was the geometric mean concentration (GMC) of IgG against the spike (S)-protein of the SARS-CoV-2 virus, 21 days after booster vaccination. We performed a non-inferiority analysis of concurrent administration compared to booster vaccines alone with a predefined non-inferiority margin of -0.3 on the log10-scale. FINDINGS: 154 individuals participated from October, 4, 2021, until November, 5, 2021. Anti-S IgG GMCs for the co-administration and reference group were 1684 BAU/ml and 2435 BAU/ml, respectively. Concurrent vaccination did not meet the criteria for non-inferiority (estimate -0.1791, 95% CI -0.3680 to -0.009831) and antibodies showed significantly lower neutralization capacity compared to the reference group. Reported side-effects were mild and did not differ between study groups. INTERPRETATION: Concurrent administration of both vaccines is safe, but the quantitative and functional antibody responses were marginally lower compared to booster vaccin

01 juli 2023, Item does not contain fulltext

Contains fulltext : 294983.pdf (Publisher’s version ) (Open Access), An increasing number of patients develop an atherothrombotic myocardial infarction (MI) in the absence of standard modifiable risk factors (SMuRFs). Monocytes and macrophages regulate the development of atherosclerosis, and monocytes can adopt a long-term hyperinflammatory phenotype by epigenetic reprogramming, which can contribute to atherogenesis (called "trained immunity"). We assessed circulating monocyte phenotype and function and specific histone marks associated with trained immunity in SMuRFless patients with MI and matched healthy controls. Even in the absence of systemic inflammation, monocytes from SMuRFless patients with MI had an increased overall cytokine production capacity, with the strongest difference for LPS-induced interleukin-10 production, which was associated with an enrichment of the permissive histone marker H3K4me3 at the promoter region. Considering the lack of intervenable risk factors in these patients, trained immunity could be a promising target for future therapy.

Item does not contain fulltext, Revertant mosaicism is an infrequently observed phenomenon caused by spontaneous correction of a pathogenic allele. We have observed such reversions caused by mitotic recombination of mutant TERC (telomerase RNA component) alleles in six patients from four families affected by dyskeratosis congenita (DC). DC is a multisystem disorder characterized by mucocutaneous abnormalities, dystrophic nails, bone-marrow failure, lung fibrosis, liver cirrhosis, and cancer. We identified a 4 nt deletion in TERC in a family with an autosomal-dominant form of DC. In two affected brothers without bone-marrow failure, sequence analysis revealed pronounced overrepresentation of the wild-type allele in blood cells, whereas no such skewing was observed in the other tissues tested. These observations suggest that this mosaic pattern might have resulted from somatic reversion of the mutated allele to the normal allele in blood-forming cells. SNP-microarray analysis on blood DNA from the two brothers indeed showed independent events of acquired segmental isodisomy of chromosome 3q, including TERC, indicating that the reversions must have resulted from mitotic recombination events. Subsequently, after developing a highly sensitive method of detecting mosaic homozygosity, we have found four additional cases with a mosaic-reversion pattern in blood cells; these four cases are part of a cohort of 17 individuals with germline TERC mutations. This shows that revertant mosaicism is a recurrent event in DC. This finding has important implications for improving diagnostic testing and understanding the variable phenotype of DC.

Contains fulltext : 88486.pdf (publisher's version ) (Closed access), Resistance to glucocorticoids (GCs) is a major clinical problem in the treatment of acute lymphoblastic leukemia (ALL), but the underlying mechanisms are not well understood. Although mutations in the glucocorticoid receptor (GR) gene can give rise to therapy resistance in vitro, acquired somatic mutations in the GR are rarely encountered in patients. Here we report that the protein encoded by the BTG1 gene, which is frequently deleted in (pediatric) ALL, is a key determinant of GC responsiveness. Using RNA interference, we show that loss of BTG1 expression causes GC resistance both by decimating GR expression and by controlling GR-mediated transcription. Conversely, reexpression of BTG1 restores GC sensitivity by potentiating GC-induced GR expression, a phenomenon known as GR autoinduction. In addition, the arginine methyltransferase PRMT1, a BTG1-binding partner and transcriptional coactivator, is recruited to the GR gene promoter in a BTG1-dependent manner. These results implicate the BTG1/PRMT1 complex in GR-mediated gene expression and reveal that deregulation of a nuclear receptor coactivator complex can give rise to GC resistance. Further characterization of this complex as part of the GR regulatory circuitry could offer novel opportunities for improving the efficacy of GC-based therapies in ALL and other hematologic malignancies.

Contains fulltext : 47884.pdf (publisher's version ) (Closed access), Protein ubiquitination plays important roles in a variety of basic cellular processes. Proteins are ubiquitinated by E2-E3 ubiquitin ligase complexes. Depending on the type of ubiquitin chain conjugated, proteins are either targeted for degradation by the proteasome or their activity is specifically altered. We describe a novel conserved nuclear protein, Triad1 (2 RING [really interesting new gene] fingers and DRIL [double RING finger linked] 1), which is strongly induced during myeloid differentiation. Triad1 contains a TRIAD motif that harbors 2 RING finger structures. Triad1 binds the E2 ubiquitin-conjugating enzyme UbcH7 as well as ubiquitinated proteins and supports the formation of ubiquitin chains that are recognized by the proteasome. The biologic function of Triad1 in myelopoiesis was studied by performing granulocyte-macrophage colony-forming unit (CFU-GM) assays using retrovirally transduced primary murine bone marrow cells. Triad1 severely inhibited myeloid colony formation. In contrast, 2 Triad1 RING finger point mutants that failed to bind UbcH7 did not affect colony formation. Moreover, proteasome inhibition counteracted the inhibition of colony formation exerted by wild-type Triad1. In liquid cultures, Triad1 did not influence differentiation but strongly inhibited proliferation resulting in a G0/G1 accumulation. We conclude that proteasomal degradation of proteins that are ubiquitinated by Triad1 affects the clonogenic growth of primary myeloid progenitor cells.

Item does not contain fulltext, OBJECTIVE: Quantitative assessment of gene expression in stem cells is essential for understanding the molecular events underlying normal and malignant hematopoiesis. The aim of the present study was to develop a method for precise quantitation of gene expression in small subsets of highly purified CD34(+)CD38(-) stem cell populations. MATERIALS AND METHODS: Real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to quantitate housekeeping and drug resistance gene expression in cDNA obtained from 300 CD34(+)CD38(-) cells without cDNA amplification or nested PCR techniques. RESULTS: Validation experiments in cell lines showed efficient, representative and reproducible gene amplification using 300-cell real-time quantitative RT-PCR. Sensitivity was confirmed in dilutional experiments and by detection of the low-copy gene PBGD. GAPDH was found to be a useful reference gene in normal and leukemic CD34(+)CD38(-) cells. In contrast, 18S rRNA content varied 100-fold to 1000-fold in these populations. Moreover, expression of 18S rRNA was significantly lower in leukemic CD34(+)CD38(+) cells compared to normal CD34(+)CD38(+) cells (p = 0.002). Expression of MDR-1 (18-fold, p < 0.0005), MRP-1 (3.8-fold, p < 0.05), and LRP (1.8-fold, NS) was higher in normal CD34(+)CD38(-) compared to CD34(+)CD38(+) cells. CONCLUSIONS: Real-time quantitative RT-PCR is a valuable tool for precise quantitation of gene expression in small subsets of hematopoietic cells. Using this method, we showed the inappropriateness of 18S as a reference gene in these progenitors and the down-regulation of drug-resistance-related genes early in hematopoiesis.

Item does not contain fulltext, This report describes the preparation of an immunotoxin-combination, consisting of an anti-CD3 and anti-CD7 monoclonal antibody (MoAb) both conjugated to the A-chain of plant toxin ricin, for the experimental treatment of graft-versus-host disease. MoAbs and toxin were conjugated by conventional biochemical and chromatographic techniques. Raw materials, intermediate and final products were evaluated in accordance with the relevant 'points to consider' of the FDA. Yields, purity and sterility of the two final products were all satisfactory. Preservation of MoAb-affinity and toxin-activity were confirmed in biological assays. The LD50, 25-45 mg immunotoxin-combination/kg mouse, equalled that of similar immunotoxins already in clinical use. Because in vitro cross-reactivity screening revealed an unexpected binding of the CD3-MoAb to the esophagus epithelium, human doses of immunotoxin-combination were administered to two cynomolgus monkeys. Clinically relevant serum concentrations were obtained without irreversible toxicities occurring. The T(1/2) varied between approximately 6 and 9 h and the C(max) ranged from 1.8 to 3.9 microg/ml. The main side effect was a transient rise of serum creatine kinase. Importantly, neither damage nor binding of the CD3-immunotoxin to the monkey esophagus epithelium could be demonstrated. It was concluded that sufficient material of proper quality and with an acceptable toxicity profile was produced, warranting the evaluation in a clinical pilot-study.

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