Severe adult hemophagocytic lymphohistiocytosis (HLHa) correlates with HLH-related gene variants.

BACKGROUND: The contribution of genetic factors to the severity of adult hemophagocytic lymphohistiocytosis (HLHa) remains unclear. OBJECTIVE: We sought to assess a potential link between HLHa outcomes and HLH-related gene variants. METHODS: Clinical characteristics of 130 HLHa patients (age ≥ 18 years and HScore ≥ 169) and genotype of 8 HLH-related genes (LYST, PRF1, UNC13-D, STX11, STXBP2, RAB27A, XIAP, and SAP) were collected. A total of 34 variants found in only 6 genes were selected on the basis of their frequency and criteria predicted to impair protein function. Severity was defined by refractory disease to HLH treatment, death, or transfer to an intensive care unit. RESULTS: HLHa-associated diseases (ADs) were neoplasia (n = 49 [37.7%]), autoimmune/inflammatory disease (n = 33 [25.4%]), or idiopathic when no AD was identified (n = 48 [36.9%]). Infectious events occurred in 76 (58.5%) patients and were equally distributed in all ADs. Severe and refractory HLHa were observed in 80 (61.5%) and 64 (49.2%) patients, respectively. HScore, age, sex ratio, AD, and infectious events showed no significant association with HLHa severity. Variants were identified in 71 alleles and were present in 56 (43.1%) patients. They were distributed as follows: 44 (34.4%), 9 (6.9%), and 3 (2.3%) patients carrying 1, 2, and 3 variant alleles, respectively. In a logistic regression model, only the number of variants was significantly associated with HLHa severity (1 vs 0: 3.86 [1.73-9.14], P = .0008; 2-3 vs 0: 29.4 [3.62-3810], P = .0002) and refractoriness (1 vs 0: 2.47 [1.17-5.34], P = .018; 2-3 vs 0: 13.2 [2.91-126.8], P = .0003). CONCLUSIONS: HLH-related gene variants may be key components to the severity and refractoriness of HLHa.

Human RIPK1 deficiency causes combined immunodeficiency and inflammatory bowel diseases.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is a critical regulator of cell death and inflammation, but its relevance for human disease pathogenesis remains elusive. Studies of monogenic disorders might provide critical insights into disease mechanisms and therapeutic targeting of RIPK1 for common diseases. Here, we report on eight patients from six unrelated pedigrees with biallelic loss-of-function mutations in RIPK1 presenting with primary immunodeficiency and/or intestinal inflammation. Mutations in RIPK1 were associated with reduced NF-κB activity, defective differentiation of T and B cells, increased inflammasome activity, and impaired response to TNFR1-mediated cell death in intestinal epithelial cells. The characterization of RIPK1-deficient patients highlights the essential role of RIPK1 in controlling human immune and intestinal homeostasis, and might have critical implications for therapies targeting RIPK1.

SYK expression endows human ZAP70-deficient CD8 T cells with residual TCR signaling.

Autosomal recessive human ZAP70 deficiency is a rare cause of combined immunodeficiency (CID) characterized by defective CD4 T cells and profound CD8 T cell lymphopenia. Herein, we report two novel patients that extend the molecular genetics, the clinical and functional phenotypes associated with the ZAP70 deficiency. The patients presented as infant-onset CID with severe infections caused by varicella zoster virus and live vaccines. Retrospective TCR excision circle newborn screening was normal in both patients. One patient carried a novel non-sense mutation (p.A495fsX75); the other a previously described misense mutation (p.A507V). In contrast to CD4 T cells, the majority of the few CD8 T cells showed expression of the ZAP70-related tyrosine kinase SYK that correlated with residual TCR signaling including calcium flux and degranulation. Our findings highlight the differential requirements of ZAP70 and SYK during thymic development, peripheral homeostasis as well as effector functions of CD4 and CD8 T cells.

The F423Y mutation in the pfmdr2 gene and mutations N51I, C59R, and S108N in the pfdhfr gene are independently associated with pyrimethamine resistance in Plasmodium falciparum isolates.

Screening for in vitro susceptibility to pyrimethamine and sequencing of the pfmdr2 and pfdhfr genes were performed in 140 Plasmodium falciparum isolates. The risk of in vitro resistance to pyrimethamine was analyzed with a logistic regression model. The mutation F423Y in pfmdr2 (odds ratio [OR] = 2.12 [confidence interval {CI}, 1.02 to 4.59]; P = 0.0489) and the mutation N51I, C59R, or S108N in pfdhfr (OR = 42.34 [CI, 5.52 to 324.61]; P = 0.0003) were independently associated with in vitro resistance to pyrimethamine.

Ena/VASP proteins at the crossroads of actin nucleation pathways in dendritic cell migration.

As sentinels of our immune system dendritic cells (DCs) rely on efficient cell migration for patrolling peripheral tissues and delivering sampled antigens to secondary lymphoid organs for the activation of T-cells. Dynamic actin polymerization is key to their macropinocytic and migratory properties. Both major actin nucleation machineries, formins and the Arp2/3 complex, are critical for different aspects of DC functionality, by driving the generation of linear and branched actin filaments, respectively. However, the importance of a third group of actin nucleators, the Ena/VASP family, has not been addressed yet. Here, we show that the two family members Evl and VASP are expressed in murine DCs and that their loss negatively affects DC macropinocytosis, spreading, and migration. Our interactome analysis reveals Ena/VASP proteins to be ideally positioned for orchestrating the different actin nucleation pathways by binding to the formin mDia1 as well as to the WAVE regulatory complex, a stimulator of Arp2/3. In fact, Evl/VASP deficient murine DCs are more vulnerable to inhibition of Arp2/3 demonstrating that Ena/VASP proteins contribute to the robustness and efficiency of DC migration.

Single-cell-resolved dynamics of chromatin architecture delineate cell and regulatory states in zebrafish embryos.

DNA accessibility of cis-regulatory elements (CREs) dictates transcriptional activity and drives cell differentiation during development. While many genes regulating embryonic development have been identified, the underlying CRE dynamics controlling their expression remain largely uncharacterized. To address this, we produced a multimodal resource and genomic regulatory map for the zebrafish community, which integrates single-cell combinatorial indexing assay for transposase-accessible chromatin with high-throughput sequencing (sci-ATAC-seq) with bulk histone PTMs and Hi-C data to achieve a genome-wide classification of the regulatory architecture determining transcriptional activity in the 24-h post-fertilization (hpf) embryo. We characterized the genome-wide chromatin architecture at bulk and single-cell resolution, applying sci-ATAC-seq on whole 24-hpf stage zebrafish embryos, generating accessibility profiles for ∼23,000 single nuclei. We developed a genome segmentation method, ScregSeg (single-cell regulatory landscape segmentation), for defining regulatory programs, and candidate CREs, specific to one or more cell types. We integrated the ScregSeg output with bulk measurements for histone post-translational modifications and 3D genome organization and identified new regulatory principles between chromatin modalities prevalent during zebrafish development. Sci-ATAC-seq profiling of npas4l/cloche mutant embryos identified novel cellular roles for this hematovascular transcriptional master regulator and suggests an intricate mechanism regulating its expression. Our work defines regulatory architecture and principles in the zebrafish embryo and establishes a resource of cell-type-specific genome-wide regulatory annotations and candidate CREs, providing a valuable open resource for genomics, developmental, molecular, and computational biology.

Synergy of mefloquine activity with atorvastatin, but not chloroquine and monodesethylamodiaquine, and association with the pfmdr1 gene.

OBJECTIVES: The aim of the study was to assess the in vitro potentiating effects of atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, in combination with mefloquine, chloroquine or monodesethylamodiaquine against Plasmodium falciparum and to evaluate whether the effects of atorvastatin could be associated with mutations or gene copy number in multidrug resistance (MDR)-like protein genes. METHODS: The susceptibilities of 21 parasite strains to combinations of atorvastatin with mefloquine, chloroquine or monodesethylamodiaquine were assessed using the in vitro isotopic microtest. Genotypes and gene copy number were assessed for pfmdr1, pfmdr2 and pfmrp genes. RESULTS: Atorvastatin demonstrated synergistic effects in combination with mefloquine. The mefloquine IC(50) (50% inhibitory concentration) was reduced by 7%, 24% and 37% in the presence of atorvastatin at concentrations of 0.1, 0.5 and 1.0 microM, respectively. The synergistic effect of atorvastatin on the response to mefloquine was significantly associated with pfmdr1 copy number. The concentration of atorvastatin that could reduce the IC(50) of mefloquine by 50% was 2.4 +/- 1.3 microM for the 12 strains that contained one copy of pfmdr1 and 5.8 +/- 2.1 microM for the 9 strains that contained two copies or more. The synergistic effect of atorvastatin in combination with mefloquine was found to be significantly unrelated to mutations in pfmdr1, pfmdr2 or pfmrp genes. CONCLUSIONS: The synergy of the effect of mefloquine at concentrations relevant to its achievable plasma concentrations in patients taking 80 mg of atorvastatin daily suggests that atorvastatin will be a good candidate in combination with mefloquine for malaria treatment.

Atorvastatin as a potential anti-malarial drug: in vitro synergy in combinational therapy with quinine against Plasmodium falciparum.

BACKGROUND: Quinine (QN) remains the first line anti-malarial drug for the treatment of complicated malaria in Europe and Africa. The emergence of QN resistance has been documented. QN resistance is not yet a significant problem, but there is an urgent need to discover partners for use in combination with QN. The aim of the study was to assess the in vitro potentiating effects of atorvastatin (AVA), a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, in combination with QN against Plasmodium falciparum and to evaluate whether the effects of AVA could be associated with gene copy number or mutations in genes involved in QN resistance, such as pfcrt, pfmdr1, pfmrp and pfnhe. METHODS: The susceptibilities to combination of AVA with QN were assessed against 21 parasite strains using the in vitro isotopic microtest. Genotypes and gene copy number were assessed for pfcrt, pfmdr1, pfmdr2, pfmrp genes. In addition, the number of DNNND, DDNHNDNHNN repeats in pfnhe-1 ms4760 and the ms4760 profile were determined for each strains of P. falciparum. RESULTS: AVA demonstrated synergistic effects in combination with QN against 21 P. falciparum strains. The QN IC50 was reduced by 5% (0% to 15%; 95%CI: 1%-8%), 10% (3% to 23%; 95%CI: 7%-14%) and 22% (14% to 40%; 95%CI: 19%-25%) in presence of AVA at concentrations of 0.1, 0.5 and 1.0 microM, respectively. These reductions were all significant (p < 0.009). The reduction in the QN IC50 in presence of AVA was not significantly correlated with the QN IC50 (r = 0.22, P = 0.3288) or the AVA IC50 (r = 0.03, P = 0.8946). The synergistic effect of AVA in combination with QN was not significantly associated with polymorphisms in the pfcrt, pfmdr1, pfmrp, and pfnhe-1 genes that could be involved in QN resistance. The synergistic effect of AVA on QN responses was not significantly associated with pfmdr1 copy number (P = 0.0428). CONCLUSION: The synergistic effect of AVA in combination with QN was found to be unrelated to mutations occurring in transport protein genes involved in QN drug resistance. The different mechanisms of drug uptake and/or mode of action for AVA compared to the other anti-malarial drugs, as well as the AVA-mediated synergy of the anti-malarial effect of QN, suggests that AVA will be a good candidate for combinatorial malaria treatment. All of these observations support calls for both an in vivo evaluation with pharmacokinetic component and clinical trials of AVA as an anti-malarial therapy.

Tetratricopeptide repeat domain 7A is a nuclear factor that modulates transcription and chromatin structure.

A loss-of-function mutation in tetratricopeptide repeat domain 7A (TTC7A) is a recently identified cause of human intestinal and immune disorders. However, clues to related underlying molecular dysfunctions remain elusive. It is now shown based on the study of TTC7A-deficient and wild-type cells that TTC7A is an essential nuclear protein. It binds to chromatin, preferentially at actively transcribed regions. Its depletion results in broad range of epigenomic changes at proximal and distal transcriptional regulatory elements and in altered control of the transcriptional program. Loss of WT_TTC7A induces general decrease in chromatin compaction, unbalanced cellular distribution of histones, higher nucleosome accessibility to nuclease digestion along with genome instability, and reduced cell viability. Our observations characterize for the first time unreported functions for TTC7A in the nucleus that exert a critical role in chromatin organization and gene regulation to safeguard healthy immune and intestinal status.

TTC7A mutations disrupt intestinal epithelial apicobasal polarity.

Multiple intestinal atresia (MIA) is a rare cause of bowel obstruction that is sometimes associated with a combined immunodeficiency (CID), leading to increased susceptibility to infections. The factors underlying this rare disease are poorly understood. We characterized the immunological and intestinal features of 6 unrelated MIA-CID patients. All patients displayed a profound, generalized lymphocytopenia, with few lymphocytes present in the lymph nodes. The thymus was hypoplastic and exhibited an abnormal distribution of epithelial cells. Patients also had profound disruption of the epithelial barrier along the entire gastrointestinal tract. Using linkage analysis and whole-exome sequencing, we identified 10 mutations in tetratricopeptide repeat domain­7A (TTC7A), all of which potentially abrogate TTC7A expression. Intestinal organoid cultures from patient biopsies displayed an inversion of apicobasal polarity of the epithelial cells that was normalized by pharmacological inhibition of Rho kinase. Our data indicate that TTC7A deficiency results in increased Rho kinase activity, which disrupts polarity, growth, and differentiation of intestinal epithelial cells, and which impairs immune cell homeostasis, thereby promoting MIA-CID development.