Resistance to immunity is associated with the selection of cancer cells with superior capacities to survive inflammatory reactions. Here, we tailored an ex vivo immune selection model for acute ...myeloid leukemia (AML) and isolated the residual subpopulations as “immune‐experienced” AML (ieAML) cells. We confirmed that upon surviving the immune reactions, the malignant blasts frequently decelerated proliferation, displayed features of myeloid differentiation and activation, and lost immunogenicity. Transcriptomic analyses revealed a limited number of commonly altered pathways and differentially expressed genes in all ieAML cells derived from distinct parental cell lines. Molecular signatures predominantly associated with interferon and inflammatory cytokine signaling were enriched in the AML cells resisting the T‐cell‐mediated immune reactions. Moreover, the expression and nuclear localization of the transcription factors c‐MYB and KLF6 were noted as the putative markers for immune resistance and identified in subpopulations of AML blasts in the patients’ bone marrow aspirates. The immune modulatory capacities of ieAML cells lasted for a restricted period when the immune selection pressure was omitted. In conclusion, myeloid leukemia cells harbor subpopulations that can adapt to the harsh conditions established by immune reactions, and a previous “immune experience” is marked with IFN signature and may pave the way for susceptibility to immune intervention therapies.
In an ex vivo immune selection model for acute myeloid leukemia (AML), residual subpopulations were isolated as — immune‐experienced — AML (ieAML) cells. Immunological properties and molecular signatures of ieAML cells were investigated. Our data note the heterogeneity in leukemia as a factor increasing the possibility of immune escape and secondary immune resistance.
Immune checkpoint inhibitor (ICI) immunotherapy relies on the restoration of T‐cell functions. The ICI receptors are not only found on exhausted T cells but also upregulated upon activation and reach ...high levels on effector T cells. In an ex vivo model, this study explored the consequences of PD‐1 and cytotoxic T‐lymphocyte antigen (CTLA‐4) blockade applied during specific time frames of T‐cell stimulation that coincide with distinct functional phases in type 1 helper T (Th1) cells. When applied at an early stimulation stage, the checkpoint blockade interfered with the upregulation of multiple inhibitory receptors such as PD‐1, LAG3, TIM‐3 and CTLA‐4. Moreover, extension of the blockade period restricted the hyporesponsiveness in T cells. Alternatively, a short‐term ICI treatment was advantageous when applied at late time frames of Th1 cell stimulation. Here, a transition phase from effector to exhausted state, which coincided with the late time frames of Th1 stimulation, was clearly determined together with the transcriptomics data demonstrating the initiation of significant alterations in metabolic pathways, genetic information processes, effector and exhaustion specific pathways. Applied in this transition phase, PD‐1 and/or CTLA‐4 blockade downregulated the inhibitory receptors which were already present on the effector Th1 cells, potentially through endocytic pathways. Therefore, the efficacy of ICI therapy was modulated by the functional status of T cells and can be improved by modifying the timing and duration of PD‐1 and CTLA‐4 blockade. In conclusion, the ICI therapy not only supports the reactivation of T cells but can also constrain de novo exhaustion.
Our ex vivo model explored the consequences of PD‐1 and CTLA‐4 blockade applied during specific time frames of T‐cell stimulation that coincide with distinct functional phases in Type 1 helper T (Th1) cells. Prolonged targeting of PD‐1 and CTLA‐4 receptors on the effector Th1 cells can diminish the acquisition of a hyporesponsive state. Alternatively, a short‐term immune checkpoint inhibitor immunotherapy was found to be efficient for the by‐pass of exhaustion and restoration of effector functions on Th1 cells.
Retinal dystrophies are a common health problem worldwide that are currently incurable due to the inability of retinal cells to regenerate. Inherited retinal diseases (IRDs) are a diverse group of ...disorders characterized by progressive vision loss caused by photoreceptor cell dysfunction. The eye has always been an attractive organ for the development of novel therapies due to its independent access to the systemic pathway. Moreover, anti-sense oligonucleotides (ASOs), which facilitate manipulation of unwanted mRNAs via degradation or splicing, are undergoing rapid development and have been clinically deployed for the treatment of several diseases. The primary aim of this study was to establish a reliable in vitro model utilizing induced photoreceptor-like cells (PRCs) for assessing the efficacy and safety of ASOs targeting the BEST1 gene. Despite advances in gene therapy, effective treatments for a broad range of IRDs remain limited. An additional aim was to develop an in vitro model for evaluating RNA-based therapeutics, specifically ASOs, for the treatment in IRDs.
Firstly, a cell culture model was established by induction of PRCs from dermal fibroblasts via direct programming. The induced PRCs were characterized at both the transcriptomic and protein level. Then, a common single nucleotide polymorphism (SNP) was identified in the BEST1 gene (rs1800007) for targeting with ASOs. ASOs were designed using the GapmeR strategy to target multiple alleles of this SNP, which is potentially suitable for a large proportion of the population. The efficacy and possible off-target effects of these ASOs were also analyzed in the induced PRC model.
The findings show that the selected ASOs achieved allele-specific mRNA degradation with virtually no off-target effects on the global transcriptome profile, indicating their potential as safe and effective therapeutic agents. The presented in vitro model is a valuable platform for testing personalized IRD treatments and should inspire further research on RNA-based therapeutics. To the best of our knowledge this study is the first to test RNA-based therapeutics involving the use of ASOs in an induced PRC model. Based on the present findings, it will be possible to establish an ex vivo disease model using dermal fibroblast samples from affected individuals. In other words, the disease model and the ASOs that were successfully designed in this study can serve as a useful platform for the testing of personalized treatments for IRDs.
•Allele-specific BEST1 mRNA degradation is possible with unique ASO design.•RNA-Seq analysis showed no significant off-target effects.•ASO designed for common SNP could be used for targeting different mutations.•This study is an example for the first step of personalized medicine approach.
Deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessive autoinflammatory disorder associated with
mutations. We aimed to investigate the characteristics and ADA2 enzyme activities of ...patients with DADA2 compared to non-DADA2 patients.
This is a descriptive study of 24 patients with DADA2 who were admitted to the Adult and Pediatric Rheumatology, Pediatric Haematology, and Pediatric Immunology Departments of Hacettepe University. All
exons were screened by Sanger sequencing. Serum ADA2 enzyme activity was measured by modified spectrophotometric method.
Twenty-four patients with DADA2 were included: 14 with polyarteritis nodosa (PAN)-like phenotype (Group 1); 9 with Diamond-Blackfan anemia (DBA)-like features, and 1 with immunodeficiency (Group 2). Fourteen PAN-like DADA2 patients did not have the typical thrombocytosis seen in classic PAN. Inflammatory attacks were evident only in Group 1 patients. Serum ADA2 activity was low in all patients with DADA2 except one, who was tested after hematopoietic stem cell transplantation. There was no significant difference in ADA2 activities between PAN-like and DBA-like patients. In DADA2 patients with one
mutation, serum ADA2 activities were as low as those of patients with homozygote DADA2. ADA2 activities were normal in non-DADA2 patients.
mutations were affecting the dimerization domain in Group 1 patients and the catalytic domain in Group 2 patients.
We suggest assessing ADA2 activity along with genetic analysis because there are patients with one
mutation and absent enzyme activity. Our data suggest a possible genotype-phenotype correlation in which dimerization domain mutations are associated with PAN-like phenotype, and catalytic domain mutations are associated with hematological manifestations.
Metabolic diseases or injuries damage bone structure and self-renewal capacity. Trace elements and hydroxyapatite crystals are important in the development of biomaterials to support the renewal of ...bone extracellular matrix. In this study, it was assumed that the boron-loaded nanometer-sized hydroxyapatite composite supports the construction of extracellular matrix by controlled boron release in order to prevent its toxic effect. In this context, boron release from nanometer-sized hydroxyapatite was calculated by ICP-MS as in large proportion within 1 h and continuing release was provided at a constant low dose. The effect of the boron-containing nanometer-sized hydroxyapatite composite on the proliferation of SaOS-2 osteoblasts and human bone marrow-derived mesenchymal stem cells was evaluated by WST-1 and compared with the effects of nano-hydroxyapatite and boric acid. Boron increased proliferation of mesenchymal stem cells at high doses and exhibited different effects on osteoblastic cell proliferation. Boron-containing nano-hydroxyapatite composites increased osteogenic differentiation of mesenchymal stem cells by increasing alkaline phosphatase activity, when compared to nano-hydroxyapatite composite and boric acid. The molecular mechanism of effective dose of boron-containing hydroxyapatite has been assessed by transcriptomic analysis and shown to affect genes involved in Wnt, TGF-β, and response to stress signaling pathways when compared to nano-hydroxyapatite composite and boric acid. Finally, a safe osteoconductive dose range of boron-containing nano-hydroxyapatite composites for local repair of bone injuries and the molecular effect profile in the effective dose should be determined by further studies to validation of the regenerative therapeutic effect window.
Abstract
Objective
Autoinflammatory diseases (AIDs) are characterized by recurrent sterile systemic inflammation attacks. More than half of the patients remain genetically undiagnosed with ...next-generation sequencing panels for common AIDs. In this study, we aimed to define phenotype-genotype correlations in a cohort of unclassified AID patients via whole exome sequencing (WES).
Methods
Patients with features of AIDs were included in this study followed in the Department of Pediatric Rheumatology at Hacettepe University. They were first screened for MEFV with Sanger sequencing and then WES performed for the patients with clinically insignificant results. Pre-analysis of WES data was done by considering the 13 most common AID-related genes. Further bioinformatic analysis was performed if the patient remained genetically undiagnosed.
Results
The median age at disease onset was 1.2 years (range 0.2–16) and at the time of study recruitment was 14 years (range 3.5–17). In our cohort, WES provided a definite or probable disease-causing variant in 4 of 11 patients (36%). Heterozygous mutations for two of these genes were previously associated with neurological defects (ADAM17, TBK1), also homozygous ADAM17 mutations were observed in one family with neonatal inflammatory skin and bowel disease. Besides, two genes (LIG4, RAG1) were associated with immunodeficiency although the patients had presented with inflammatory features. Finally, for one patient, we associated a strong candidate gene (NLRC3) with autoinflammatory features.
Conclusion
WES strategy is cost-effective and provides substantial results for a selected group of undefined AID patients. Our results will contribute to the spectrum of unclassified AIDs.
Idiopathic nephrotic syndrome (INS) is a genetically heterogeneous group of disorders characterized by proteinuria, hypoalbuminemia, and edema. Because it typically results in end-stage kidney ...disease, the steroid-resistant subtype (SRNS) of INS is especially important when it occurs in children. The present study included 29 affected and 22 normal individuals from 17 SRNS families; genome-wide analysis was performed with Affymetrix 250K SNP arrays followed by homozygosity mapping. A large homozygous stretch on chromosomal region 12p12 was identified in one consanguineous family with two affected siblings. Direct sequencing of protein tyrosine phosphatase receptor type O (PTPRO; also known as glomerular epithelial protein-1 GLEPP1) showed homozygous c.2627+1G>T donor splice-site mutation. This mutation causes skipping of the evolutionarily conserved exon 16 (p.Glu854_Trp876del) at the RNA level. Immunohistochemistry with GLEPP1 antibody showed a similar staining pattern in the podocytes of the diseased and control kidney tissues. We used a highly polymorphic intragenic DNA marker—D12S1303—to search for homozygosity in 120 Turkish and 13 non-Turkish individuals in the PodoNet registry. This analysis yielded 17 candidate families, and a distinct homozygous c.2745+1G>A donor splice-site mutation in PTPRO was further identified via DNA sequencing in a second Turkish family. This mutation causes skipping of exon 19, and this introduces a premature stop codon at the very beginning of exon 20 (p.Asn888Lysfs∗3) and causes degradation of mRNA via nonsense-mediated decay. Immunohistochemical analysis showed complete absence of immunoreactive PTPRO. Ultrastructural alterations, such as diffuse foot process fusion and extensive microvillus transformation of podocytes, were observed via electron microscopy in both families. The present study introduces mutations in PTPRO as another cause of autosomal-recessive nephrotic syndrome.
Abstract
Current knowledge about the molecular properties of the crustacean ion channels is rather limited even if crustaceans have been widely used as a model in neuroscience. We cloned for the ...first time two different potassium channel genes from the freshwater crayfish Astacus leptodactylus (Eschscholtz, 1823), one of the genes functionally expressed in the Xenopus oocytes. The open-reading frames of the genes were 1,203 and 3,447 bp, respectively. The nucleic acid sequence of the genes and associated proteins were similar to those of a typical potassium channel. BLAST analyses indicated that one of the cloned genes had a substantial similarity to an inward-rectifier potassium channel whereas the other gene was similar to a high-conductance-KCa type potassium channel reported in related species. Transmembrane topology and three-dimensional structure of the coded proteins were calculated and functional regions of the channel proteins responsible for ion selectivity, voltage sensing, gating, and calcium binding were identified. One of the cloned channel genes has been expressed in the Xenopus oocytes. Analysis of the expressed potassium currents confirmed that the cloned gene was coding a typical Kir-type potassium channel with ATP sensitivity.
Abstract
Ion channels gated selectively by mechanical stimulus are the key elements of mechanosensation. Several genes have been associated with putative mechanosensitive ion channels or ...mechanosensitive channel complexes. Transmembrane channel (TMC)-like protein is one of those candidate proteins that have been explored in mammals and several invertebrates. The presence and possible function of TMC related genes has not been investigated yet in crustaceans. In the present work an mRNA coding TMC-like protein was firstly cloned in Astacus leptodactylus (Eschscholtz, 1823) (Decapoda: Astacidea: Astacidae) and expressed in HEK293T cells. Three-dimensional structural calculations of the protein predicted a channel. Functional studies, however, indicated that the mechanosensitivity of the transfected HEK293T cells is similar to that in the control cells. It was concluded that a TMC-like protein is present in the crayfish but future studies are necessary to define its function.
Background and objective
Primary ciliary dyskinesia (PCD) is a rare and genetically heterogeneous disease and the severity of the disease related with genetic analysis has been described in some ...previous studies. The main aim of our study was to describe the clinical characteristics and laboratory findings of patients with genetically diagnosed PCD and to investigate the correlation between clinical, radiologic, and laboratory findings and genetic analyses of these patients.
Method
This is a cohort study in which we analyzed the clinical characteristics, laboratory findings, and genetic results of 46 patients with genetically diagnosed PCD through whole‐exome sequencing at our single center from a total of 265 patients with PCD within a 5‐year period.
Results
Genetic analysis revealed pathogenic variants in DNAH5 (n = 12 individuals, 12 families), CCDC40 (n = 9 individuals, six families), RSPH4A (n = 5 individuals, three families), DNAH11 (n = 4 individuals, four families), HYDIN (n = 5 individuals, five families), CCNO (n = 4 individuals, four families), DNAI1 (n = 2 individuals, one family), ARMC4 (n = 2 individuals, two families), TTC25 (n = 1), DNAH1 (n = 1), and CCDC39 (n = 1) genes. Although not statistically significant, the age at diagnosis was lower (median: 3 years; range, 6 months‐4 years) in patients with CCNO pathogenic variants due to the early reporting of symptoms, and the median body mass index (BMI) and BMI z scores were lower in patients at 18.7 and 16 kg/m2, and −0.78 and −1.2 with CCDC40 and CCNO pathogenic variants, respectively. The median forced expiratory flow in 1 second (FEV1%), forced vital capacity (FVC%), and forced expiratory flow (FEF)25‐75% were 53%, 64%, and 28%, respectively; these parameters were also lower in the CCDC40 group than in the other groups. There was no significant correlation between the genetic results and symptoms, radiologic findings, and microbiologic data of patients with PCD.
Conclusion
In PCD, there was significant heterogeneity of lung disease, patients who had pathogenic variants in CCNO presented earlier, and those with CCDC40 and CCNO had worse lung disease, and poorer nutritional status compared with the other subgroups. We hope that whole genotype‐phenotype and clinical relationships will be identified in PCD.
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