Effects of the Protein Kinase Inhibitor PKC412 on Gene Expression and Link to Physiological Effects in Zebrafish Danio rerio Eleuthero-Embryos

To identify molecular effects of the antineoplastic agent protein kinase C inhibitor 412 (PKC412) (midostaurin), we applied gene expression profiling in zebrafish using whole-genome microarrays. Behavioral, developmental, and physiological effects were investigated in order to analyze for correlations between altered gene expression profiles with effects on development and physiology. Zebrafish blastula-stage embryos were exposed for 6 days postfertilization to nominal levels of 2 and 40 μg/l PKC412. Among the 259 and 511 altered transcripts at both concentrations, respectively, the expressions of genes involved in the circadian rhythm were further investigated. Alteration of swimming behavior was not observed. Pathways of interest affected by PKC412 were angiogenesis, apoptosis, DNA damage response, and response to oxidative stress. Angiogenesis was analyzed in double-transgenic zebrafish embryos Tg(fli1a:EGFP)y1;Tg(gata1:dsRed)sd2; no major defects were induced by PKC412 treatment at both concentrations. Apoptosis occurred in olfactory placodes of embryos exposed to 40 μg/l, and DNA damage was induced at both PKC412 concentrations. However, there were no significant effects on reactive oxygen species formation. This study leads to the conclusion that PKC412-induced alterations of gene transcripts are partly paralleled by physiological effects at high, but not at low PKC412 concentrations expected to be of environmental relevanc

Influence of Chronic Exposure to Treated Sewage Effluent on the Distribution of White Blood Cell Populations in Rainbow Trout (Oncorhynchus mykiss) Spleen

Impairment of immune function in aquatic animals has been proposed as a possible consequence of low-level contamination of surface waters with anthropogenic substances such as through the discharge of wastewater into rivers, lakes, and oceans. The study at hand investigated the effects of chronic (32 weeks) exposure to sewage treatment plant (STP) effluent on the prevalence and distribution of different leucocyte populations in spleen samples of rainbow trout (Oncorhynchus mykiss). To simulate an infection, fish were injected intraperitoneally (ip) with inactivated Aeromonas salmonicida salmonicida, 6 weeks prior to the termination of the experiment. Immunohistological analysis revealed a marked decrease in thrombocyte numbers, an increase of monocytes, altered distribution of B-cells, and higher surface immunoglobulin expression, as well as activation of MHC class II expression in the spleen after exposure to 15% (v/v) effluent. The most prominent finding of the present study, however, was the occurrence of intraplasmatic deposits or inclusions with strong autofluorescence in spleen sections from effluent-exposed trout. In addition to effluent effects, injection of trout with A. salmonicida stimulated infiltration of monocytes, increased staining intensity on thrombocytes, and enhanced MHC class I expression in larger leucocytes surrounding melanomacrophage centres. In general, the current study demonstrates a marked, potentially adverse effect of STP effluent on spleen leucocytes and on the integrity of spleen tissue. The observed response suggests a constant unspecific stimulation of different leucocyte populations and is reminiscent of chronic inflammatio

Germline biallelic mutation affecting the transcription factor Helios causes pleiotropic defects of immunity

Helios, a member of the Ikaros family of transcription factors, is predominantly expressed in developing thymocytes, activated T cells, and regulatory T cells (T-regs). Studies in mice have emphasized its role in maintenance of T-reg immunosuppressive functions by stabilizing Foxp3 expression and silencing the Il2 locus. However, its contribution to human immune homeostasis and the precise mechanisms by which Helios regulates other T cell subsets remain unresolved. Here, we investigated a patient with recurrent respiratory infections and hypogammaglobulinemia and identified a germline homozygous missense mutation in IKZF2 encoding Helios (p.Ile325Val). We found that Helios(I325V) retains DNA binding and dimerization properties but loses interaction with several partners, including epigenetic remodelers. Whereas patient T-regs showed increased IL-2 production, patient conventional T cells had decreased accessibility of the IL2 locus and consequently reduced IL-2 production. Reduced chromatin accessibility was not exclusive to the IL2 locus but involved a variety of genes associated with T cell activation. Single-cell RNA sequencing of peripheral blood mononuclear cells revealed gene expression signatures indicative of a shift toward a proinflammatory, effector-like status in patient CD8(+) T cells. Moreover, patient CD4(+) T cells exhibited a pronounced defect in proliferation with delayed expression of surface checkpoint inhibitors, suggesting an impaired onset of the T cell activation program. Collectively, we identified a previously uncharacterized, germline-encoded inborn error of immunity and uncovered a cell-specific defect in Helios-dependent epigenetic regulation. Binding of Helios with specific partners mediates this regulation, which is ultimately necessary for the transcriptional programs that enable T cell homeostasis in health and disease.Peer reviewe

Identification of germline monoallelic mutations in IKZF2 in patients with immune dysregulation

Helios, encoded by IKZF2, is a member of the Ikaros family of transcription factors with pivotal roles in T-follicular helper, NK- and T-regulatory cell physiology. Somatic IKZF2 mutations are frequently found in lymphoid malignancies. Although germline mutations in IKZF1 and IKZF3 encoding Ikaros and Aiolos have recently been identified in patients with phenotypically similar immunodeficiency syndromes, the effect of germline mutations in IKZF2 on human hematopoiesis and immunity remains enigmatic. We identified germline IKZF2 mutations (one nonsense (p.R291X)- and 4 distinct missense variants) in six patients with systemic lupus erythematosus, immune thrombocytopenia or EBV-associated hemophagocytic lymphohistiocytosis. Patients exhibited hypogammaglobulinemia, decreased number of T-follicular helper and NK cells. Single-cell RNA sequencing of PBMCs from the patient carrying the R291X variant revealed upregulation of proinflammatory genes associated with T-cell receptor activation and T-cell exhaustion. Functional assays revealed the inability of HeliosR291X to homodimerize and bind target DNA as dimers. Moreover, proteomic analysis by proximity-dependent Biotin Identification revealed aberrant interaction of 3/5 Helios mutants with core components of the NuRD complex conveying HELIOS-mediated epigenetic and transcriptional dysregulation.Peer reviewe

Mutations affecting the actin regulator WD repeat–containing protein 1 lead to aberrant lymphoid immunity

Background: The actin-interacting protein WD repeat–containing protein 1 (WDR1) promotes cofilin-dependent actin filament turnover. Biallelic WDR1 mutations have been identified recently in an immunodeficiency/autoinflammatory syndrome with aberrant morphology and function of myeloid cells. Objective: Given the pleiotropic expression of WDR1, here we investigated to what extent it might control the lymphoid arm of the immune system in human subjects. Methods: Histologic and detailed immunologic analyses were performed to elucidate the role of WDR1 in the development and function of B and T lymphocytes. Results: Here we identified novel homozygous and compound heterozygous WDR1 missense mutations in 6 patients belonging to 3 kindreds who presented with respiratory tract infections, skin ulceration, and stomatitis. In addition to defective adhesion and motility of neutrophils and monocytes, WDR1 deficiency was associated with aberrant T-cell activation and B-cell development. T lymphocytes appeared to develop normally in the patients, except for the follicular helper T-cell subset. However, peripheral T cells from the patients accumulated atypical actin structures at the immunologic synapse and displayed reduced calcium flux and mildly impaired proliferation on T-cell receptor stimulation. WDR1 deficiency was associated with even more severe abnormalities of the B-cell compartment, including peripheral B-cell lymphopenia, paucity of B-cell progenitors in the bone marrow, lack of switched memory B cells, reduced clonal diversity, abnormal B-cell spreading, and increased apoptosis on B-cell receptor/Toll-like receptor stimulation. Conclusion: Our study identifies a novel role for WDR1 in adaptive immunity, highlighting WDR1 as a central regulator of actin turnover during formation of the B-cell and T-cell immunologic synapses

Human NF-κB1 Haploinsufficiency and Epstein–Barr Virus-Induced Disease—Molecular Mechanisms and Consequences

Nuclear factor kappa-light-chain-enhancer of activated B cells 1 (NF-κB1)-related human primary immune deficiencies have initially been characterized as defining a subgroup of common variable immunodeficiencies (CVIDs), representing intrinsic B-cell disorders with antibody deficiency and recurrent infections of various kind. Recent evidence indicates that NF-κB1 haploinsufficiency underlies a variable type of combined immunodeficiency (CID) affecting both B and T lymphocyte compartments, with a broadened spectrum of disease manifestations, including Epstein–Barr virus (EBV)-induced lymphoproliferative disease and immediate life-threatening consequences. As part of this review series focused on EBV-related primary immunodeficiencies, we discuss the current clinical and molecular understanding of monoallelic NFKB1 germline mutations with special focus on the emerging context of EBV-associated disease. We outline mechanistic implications of dysfunctional NF-κB1 in B and T cells and discuss the fatal relation of impaired T-cell function with the inability to clear EBV infections. Finally, we compare common and suggested treatment angles in the context of this complex disease

Human NF-κB1 Haploinsufficiency and Epstein–Barr Virus-Induced Disease—Molecular Mechanisms and Consequences

Nuclear factor kappa-light-chain-enhancer of activated B cells 1 (NF-B1)-related human primary immune deficiencies have initially been characterized as defining a subgroup of common variable immunodeficiencies (CVIDs), representing intrinsic B-cell disorders with antibody deficiency and recurrent infections of various kind. Recent evidence indicates that NF-B1 haploinsufficiency underlies a variable type of combined immunodeficiency (CID) affecting both B and T lymphocyte compartments, with a broadened spectrum of disease manifestations, including EpsteinBarr virus (EBV)-induced lymphoproliferative disease and immediate life-threatening consequences. As part of this review series focused on EBV-related primary immunodeficiencies, we discuss the current clinical and molecular understanding of monoallelic NFKB1 germline mutations with special focus on the emerging context of EBV-associated disease. We outline mechanistic implications of dysfunctional NF-B1 in B and T cells and discuss the fatal relation of impaired T-cell function with the inability to clear EBV infections. Finally, we compare common and suggested treatment angles in the context of this complex disease.(VLID)470196

Distribution of intraperitoneally injected diclofenac in brown trout (Salmo trutta f. fario)

The detection of low levels of pharmaceuticals in aquatic environments has lately raised concerns regarding possible adverse effects of these highly active substances on aquatic organisms. The non steroidal anti inflammatory drug diclofenac (DCF) is one of the pharmaceutical substances regularly detected in surface waters and has lately been demonstrated to elicit adverse effects in salmonid species at environmentally relevant concentrations. The aim of the present study was to investigate the distribution of DCF in indigenous brown trout (Salmo trutta f. fario) following intraperitoneal (i.p.) injection of a single dose of 14C labelled DCF. A distribution kinetic over 36 h provides information on possible accumulation of DCF in different organs as well as on DCF detoxification in trout, possibly enabling identification of sites of preferential toxicity. Approximately 57% of the total single DCF dose appeared in the bile 6 h after i.p. application. Subsequently, DCF was observed to undergo enterohepatic cycling with an amount of 14C activity comparable to the 6 h bile values reappearing in bile 36 h after application. Results for 14C activity in intestine and pylori support the observation of enterohepatic cycling with a small peak in intestine at 3 h post i.p. injection and a low peak in intestine and pylori at 6 h post i.p. injection, reflecting presence of the drug substance in bile. The highest activity in intestine was found 24 h post injection coinciding with low levels in bile, followed by a gradual decrease of activity in intestine mirroring the re uptake of DCF into bile. The finding of enterohepatic cycling of DCF in brown trout is suggestive of a prolonged retention of DCF in brown trou

Distribution of intraperitoneally injected diclofenac in brown trout (Salmo trutta f. fario)

The detection of low levels of pharmaceuticals in aquatic environments has lately raised concerns regarding possible adverse effects of these highly active substances on aquatic organisms. The non steroidal anti inflammatory drug diclofenac (DCF) is one of the pharmaceutical substances regularly detected in surface waters and has lately been demonstrated to elicit adverse effects in salmonid species at environmentally relevant concentrations. The aim of the present study was to investigate the distribution of DCF in indigenous brown trout (Salmo trutta f. fario) following intraperitoneal (i.p.) injection of a single dose of 14C labelled DCF. A distribution kinetic over 36 h provides information on possible accumulation of DCF in different organs as well as on DCF detoxification in trout, possibly enabling identification of sites of preferential toxicity. Approximately 57% of the total single DCF dose appeared in the bile 6 h after i.p. application. Subsequently, DCF was observed to undergo enterohepatic cycling with an amount of 14C activity comparable to the 6 h bile values reappearing in bile 36 h after application. Results for 14C activity in intestine and pylori support the observation of enterohepatic cycling with a small peak in intestine at 3 h post i.p. injection and a low peak in intestine and pylori at 6 h post i.p. injection, reflecting presence of the drug substance in bile. The highest activity in intestine was found 24 h post injection coinciding with low levels in bile, followed by a gradual decrease of activity in intestine mirroring the re uptake of DCF into bile. The finding of enterohepatic cycling of DCF in brown trout is suggestive of a prolonged retention of DCF in brown trou

Mutational Analysis of a Conserved Glutamate Reveals Unique Mechanistic and Structural Features of the Phosphatase PRL‑3

Phosphatase of regenerating liver (PRL)-3 (<i>PTP4A3</i>) has gained much attention in cancer research due to its involvement in tumor promoting and metastatic processes. It belongs to the protein tyrosine phosphatase (PTP) superfamily and is thought to follow the catalytic mechanism shared by this family, which aside from the conserved active-site amino acids includes a conserved glutamic acid residue that is usually required for the integrity of the active site in PTPs. We noted that in structures of PRL-3, PRL-1, and PTEN these residues do not clearly align and therefore we sought to investigate if the glutamic acid residue fulfills its usual function in these proteins. Although this residue was essential for PTEN’s catalytic activity, it was nonessential for PRL-1 and PRL-3. Surprisingly, the mutation E50R increased PRL-3 activity against all tested in vitro substrates and also enhanced PRL-3-promoted cell adhesion and migration. We show that the introduction of Arg50 leads to an enhancement of substrate turnover for both PRL-3 and, to a lesser extent, PRL-1, and that the stronger gain in activity correlates with a higher structural flexibility of PRL-3, likely allowing for conformational adaptation during catalysis. Thus, in contrast to its crucial functions in other PTPs, this conserved glutamic acid can be replaced in PRL-3 without impairing the structural integrity. The variant with enhanced activity might serve as a tool to study PRL-3 in the future