Single-cell transcriptomics reveal different maturation stages and sublineage commitment of human thymic invariant natural killer T cells.

Invariant natural killer T cells are a rare, heterogeneous T-cell subset with cytotoxic and immunomodulatory properties. During thymic development, murine invariant natural killer T cells go through different maturation stages differentiating into distinct sublineages, namely, invariant natural killer T1, 2, and 17 cells. Recent reports indicate that invariant natural killer T2 cells display immature properties and give rise to other subsets, whereas invariant natural killer T1 cells seem to be terminally differentiated. Whether human invariant natural killer T cells follow a similar differentiation model is still unknown. To define the maturation stages and assess the sublineage commitment of human invariant natural killer T cells during thymic development, in this study, we performed single-cell RNA sequencing analysis on human Vα24+Vß11+ invariant natural killer T cells isolated from thymocytes. We show that these invariant natural killer T cells displayed heterogeneity, and our unsupervised analysis identified 5 clusters representing different maturation stages, from an immature profile with high expression of genes important for invariant natural killer T cell development and proliferation to a mature, fully differentiated profile with high levels of cytotoxic effector molecules. Evaluation of expression of sublineage-defining gene sets revealed mainly cells with an invariant natural killer T2 signature in the most immature cluster, whereas the more differentiated ones displayed an invariant natural killer T1 signature. Combined analysis with a publicly available single-cell RNA sequencing data set of human invariant natural killer T cells from peripheral blood suggested that the 2 main subsets exist both in thymus and in the periphery, while a third more immature one was restricted to the thymus. Our data point to the existence of different maturation stages of human thymic invariant natural killer T cells and provide evidence for sublineage commitment of invariant natural killer T cells in the human thymus.

Personalized aerosolised bacteriophage treatment of a chronic lung infection due to multidrug-resistant Pseudomonas aeruginosa.

Bacteriophage therapy has been suggested as an alternative or complementary strategy for the treatment of multidrug resistant (MDR) bacterial infections. Here, we report the favourable clinical evolution of a 41-year-old male patient with a Kartagener syndrome complicated by a life-threatening chronic MDR Pseudomonas aeruginosa infection, who is treated successfully with iterative aerosolized phage treatments specifically directed against the patient's isolate. We follow the longitudinal evolution of both phage and bacterial loads during and after phage administration in respiratory samples. Phage titres in consecutive sputum samples indicate in patient phage replication. Phenotypic analysis and whole genome sequencing of sequential bacterial isolates reveals a clonal, but phenotypically diverse population of hypermutator strains. The MDR phenotype in the collected isolates is multifactorial and mainly due to spontaneous chromosomal mutations. All isolates recovered after phage treatment remain phage susceptible. These results demonstrate that clinically significant improvement is achievable by personalised phage therapy even in the absence of complete eradication of P. aeruginosa lung colonization.

Platform combining statistical modeling and patient-derived organoids to facilitate personalized treatment of colorectal carcinoma.

BACKGROUND: We propose a new approach for designing personalized treatment for colorectal cancer (CRC) patients, by combining ex vivo organoid efficacy testing with mathematical modeling of the results. METHODS: The validated phenotypic approach called Therapeutically Guided Multidrug Optimization (TGMO) was used to identify four low-dose synergistic optimized drug combinations (ODC) in 3D human CRC models of cells that are either sensitive or resistant to first-line CRC chemotherapy (FOLFOXIRI). Our findings were obtained using second order linear regression and adaptive lasso. RESULTS: The activity of all ODCs was validated on patient-derived organoids (PDO) from cases with either primary or metastatic CRC. The CRC material was molecularly characterized using whole-exome sequencing and RNAseq. In PDO from patients with liver metastases (stage IV) identified as CMS4/CRIS-A, our ODCs consisting of regorafenib [1 mM], vemurafenib [11 mM], palbociclib [1 mM] and lapatinib [0.5 mM] inhibited cell viability up to 88%, which significantly outperforms FOLFOXIRI administered at clinical doses. Furthermore, we identified patient-specific TGMO-based ODCs that outperform the efficacy of the current chemotherapy standard of care, FOLFOXIRI. CONCLUSIONS: Our approach allows the optimization of patient-tailored synergistic multi-drug combinations within a clinically relevant timeframe.

FOLFOXIRI Resistance Induction and Characterization in Human Colorectal Cancer Cells.

FOLFOXIRI, i.e., the combination of folinic acid, 5-fluorouracil, oxaliplatin, and irinotecan, is a first-line treatment for colorectal carcinoma (CRC), yet non-personalized and aggressive. In this study, to mimic the clinical situation of patients diagnosed with advanced CRC and exposed to a chronic treatment with FOLFOXIRI, we have generated the CRC cell clones chronically treated with FOLFOXIRI. A significant loss in sensitivity to FOLFOXIRI was obtained in all four cell lines, compared to their treatment-naïve calls, as shown in 2D cultures and heterotypic 3D co-cultures. Acquired drug resistance induction was observed through morphometric changes in terms of the organization of the actin filament. Bulk RNA sequencing revealed important upregulation of glucose transporter family 5 (GLUT5) in SW620 resistant cell line, while in the LS174T-resistant cell line, a significant downregulation of protein tyrosine phosphatase receptor S (PTPRS) and oxoglutarate dehydrogenase-like gene (OGDHL). This acquired resistance to FOLFOXIRI was overcome with optimized low-dose synergistic drug combinations (ODCs) acting via the Ras-Raf-MEK-ERK pathway. The ODCs inhibited the cell metabolic activity in SW620 and LS174T 3Dcc, respectively by up to 82%.

Hairy cell leukemia: a specific 17-gene expression signature points to new targets for therapy.

BACKGROUND: Hairy cell leukemia (HCL) is a rare chronic B cell malignancy, characterized by infiltration of bone marrow, blood and spleen by typical "hairy cells" that bear the BRAFV600E mutation. However, in addition to the intrinsic activation of the MAP kinase pathway as a consequence of the BRAFV600E mutation, the potential participation of other signaling pathways to the pathophysiology of the disease remains unclear as the precise origin of the malignant hairy B cells. MATERIALS AND METHODS: Using mRNA gene expression profiling based on the Nanostring technology and the analysis of 290 genes with crucial roles in B cell lymphomas, we defined a 17 gene expression signature specific for HCL. RESULTS: Separate analysis of samples from classical and variant forms of hairy cell leukemia showed almost similar mRNA expression profiles apart from overexpression in vHCL of the immune checkpoints CD274 and PDCD1LG2 and underexpression of FAS. Our results point to a post-germinal memory B cell origin and in some samples to the activation of the non-canonical NF-κB pathway. CONCLUSIONS: This study provides a better understanding of the pathogenesis of HCL and describes new and potential targets for treatment approaches and guidance for studies in the molecular mechanisms of HCL.

Comparison of Two Experimental Mouse Dry Eye Models through Inflammatory Gene Set Enrichment Analysis Based on a Multiplexed Transcriptomic Approach.

The goal of this study was to explore the specific signaling pathways related to inflammation in two experimental mouse dry eye (EDE) models. Female C57BL/6 mice housed for 10 days in a controlled desiccative environment were either treated with scopolamine (EDE-1; n = 18) or subjected to extraorbital lacrimal gland excision bilaterally (EDE-2; n = 10). Non-induced mice (n = 20) served as healthy controls. A corneal fluorescein staining (CFS) scoring was used at baseline through to day (D) 10 to evaluate epitheliopathy. At D10, corneas and conjunctivas were collected for multiplexed transcriptomic analysis with the NanoString® mouse inflammatory CodeSet. Both EDE-1 and EDE-2 mice presented a change in corneal integrity, with a significant increase in CFS scores at D10. More gene transcripts were identified in EDE-2 compared with EDE-1 (116 vs. 96, respectively), and only a few were common to both models, 13 for the cornea and 6 for the conjunctiva. The gene functional annotation analysis revealed that the same inflammatory pathways were involved in both models. Comparative profiling of gene expression in the two EDE models leads to the identification of various targets and signaling pathways, which can be extrapolated to and confirmed in human disease.

Drug Repurposing to Identify a Synergistic High-Order Drug Combination to Treat Sunitinib-Resistant Renal Cell Carcinoma.

Repurposed drugs have been evaluated for the management of clear cell renal cell carcinoma (ccRCC), but only a few have influenced the overall survival of patients with advanced disease. To combine repurposed non-oncology with oncological drugs, we applied our validated phenotypic method, which consisted of a reduced experimental part and data modeling. A synergistic optimized multidrug combination (ODC) was identified to significantly reduce the energy levels in cancer remaining inactive in non-cancerous cells. The ODC consisted of Rapta-C, erlotinib, metformin and parthenolide and low doses. Molecular and functional analysis of ODC revealed a loss of adhesiveness and induction of apoptosis. Gene-expression network analysis displayed significant alterations in the cellular metabolism, confirmed by LC-MS based metabolomic analysis, highlighting significant changes in the lipid classes. We used heterotypic in vitro 3D co-cultures and ex vivo organoids to validate the activity of the ODC, maintaining an efficacy of over 70%. Our results show that repurposed drugs can be combined to target cancer cells selectively with prominent activity. The strong impact on cell adherence and metabolism indicates a favorable mechanism of action of the ODC to treat ccRCC.

Molecular and Functional Analysis of Sunitinib-Resistance Induction in Human Renal Cell Carcinoma Cells.

Resistance in clear cell renal cell carcinoma (ccRCC) against sunitinib is a multifaceted process encompassing numerous molecular aberrations. This induces clinical complications, reducing the treatment success. Understanding these aberrations helps us to select an adapted treatment strategy that surpasses resistance mechanisms, reverting the treatment insensitivity. In this regard, we investigated the dominant mechanisms of resistance to sunitinib and validated an optimized multidrug combination to overcome this resistance. Human ccRCC cells were exposed to single or chronic treatment with sunitinib to obtain three resistant clones. Upon manifestation of sunitinib resistance, morphometric changes in the cells were observed. At the molecular level, the production of cell membrane and extracellular matrix components, chemotaxis, and cell cycle progression were dysregulated. Molecules enforcing the cell cycle progression, i.e., cyclin A, B1, and E, were upregulated. Mass spectrometry analysis revealed the intra- and extracellular presence of N-desethyl sunitinib, the active metabolite. Lysosomal sequestration of sunitinib was confirmed. After treatment with a synergistic optimized drug combination, the cell metabolic activity in Caki-1-sunitinib-resistant cells and 3D heterotypic co-cultures was reduced by >80%, remaining inactive in non-cancerous cells. These results demonstrate geno- and phenotypic changes in response to sunitinib treatment upon resistance induction. Mimicking resistance in the laboratory served as a platform to study drug responses.

Blood virosphere in febrile Tanzanian children.

Viral infections are the leading cause of childhood acute febrile illnesses motivating consultation in sub-Saharan Africa. The majority of causal viruses are never identified in low-resource clinical settings as such testing is either not part of routine screening or available diagnostic tools have limited ability to detect new/unexpected viral variants. An in-depth exploration of the blood virome is therefore necessary to clarify the potential viral origin of fever in children. Metagenomic next-generation sequencing is a powerful tool for such broad investigations, allowing the detection of RNA and DNA viral genomes. Here, we describe the blood virome of 816 febrile children (<5 years) presenting at outpatient departments in Dar es Salaam over one-year. We show that half of the patients (394/816) had at least one detected virus recognized as causes of human infection/disease (13.8% enteroviruses (enterovirus A, B, C, and rhinovirus A and C), 12% rotaviruses, 11% human herpesvirus type 6). Additionally, we report the detection of a large number of viruses (related to arthropod, vertebrate or mammalian viral species) not yet known to cause human infection/disease, highlighting those who should be on the radar, deserve specific attention in the febrile paediatric population and, more broadly, for surveillance of emerging pathogens.Trial registration: ClinicalTrials.gov identifier: NCT02225769.

Conjunctival transcriptome analysis reveals the overexpression of multiple pattern recognition receptors in vernal keratoconjunctivitis.

BACKGROUND: Vernal keratoconjunctivitis (VKC) is a chronic, potentially blinding ocular allergic disease affecting children with uncertain pathogenic mechanisms. OBJECTIVE: To identify differences in gene expression between VKC and normal subjects (CT) and to evaluate the expression of pattern recognition receptors (PRRs). METHODS: Conjunctival cells were collected by impression cytology device from 25 VKC patients and 10 CT. Isolated RNA was assayed with the NanoString human immunology codeset to evaluate the expression levels of immunology-related genes. RESULTS: Of the 579 genes, 398 were detected and 58 were significantly differently expressed in VKC compared to CT. The number of significantly differentially expressed genes (DEG) in the 3 different phenotypes vs CT were 149 in tarsal, 17 in limbal and 68 in the mixed form of VKC. The list of the most overexpressed genes included several chemokines (CCL24, CCL18, CCL22, CXCL1), proinflammatory cytokines (IL-1ß, IL-6, IL-8, TGFß-1) and genes related to Th2- and Th17-signaling families. Toll like receptors (TLR)4 and TLR8, Dectin-1/CLEC7A, mincle/CLEC4E, MCR1, NOD2 and NLRP3 and several of their pathway-related genes were significantly overexpressed in VKC. The number of DEG increased with the disease severity either in IgE+ or IgE- patients. Immunohistochemistry analysis of VKC conjunctival tissues confirmed an increased expression of these molecules at protein level. CONCLUSIONS: The increased expression of several chemotactic factors and co-stimulatory signals required for T-cell activation, confirms that VKC is mostly cell-mediated with local eosinophilia. The multiple expression of PRRs suggests a role of host-pathogens interaction in VKC development.

Optimized low-dose combinatorial drug treatment boosts selectivity and efficacy of colorectal carcinoma treatment.

The current standard of care for colorectal cancer (CRC) is a combination of chemotherapeutics, often supplemented with targeted biological drugs. An urgent need exists for improved drug efficacy and minimized side effects, especially at late-stage disease. We employed the phenotypically driven therapeutically guided multidrug optimization (TGMO) technology to identify optimized drug combinations (ODCs) in CRC. We identified low-dose synergistic and selective ODCs for a panel of six human CRC cell lines also active in heterotypic 3D co-culture models. Transcriptome sequencing and phosphoproteome analyses showed that the mechanisms of action of these ODCs converged toward MAP kinase signaling and cell cycle inhibition. Two cell-specific ODCs were translated to in vivo mouse models. The ODCs reduced tumor growth by ~80%, outperforming standard chemotherapy (FOLFOX). No toxicity was observed for the ODCs, while significant side effects were induced in the group treated with FOLFOX therapy. Identified ODCs demonstrated significantly enhanced bioavailability of the individual components. Finally, ODCs were also active in primary cells from CRC patient tumor tissues. Taken together, we show that the TGMO technology efficiently identifies selective and potent low-dose drug combinations, optimized regardless of tumor mutation status, outperforming conventional chemotherapy.

Contribution of exome sequencing to the identification of genes involved in the response to clopidogrel in cardiovascular patients.

BACKGROUND: On-clopidogrel platelet reactivity (PR) is associated with the risk of thrombotic or bleeding event in selected populations of high-risk patients. PR is a highly heritable phenotype and a few variants of cytochrome genes, essentially CYP2C19, are associated with PR but only explain 5% to 12% of the variability. OBJECTIVE: The aim of this study is to delineate genetic determinants of on-clopidogrel PR using high-throughput sequencing. METHODS: We performed a whole exome sequencing of 96 low- and matched high-PR patients in a discovery cohort. Exomes from genes with variants significantly associated with PR were sequenced in 96 low- and matched high-PR patients from an independent replication cohort. RESULTS: We identified 585 variants in 417 genes with an adjusted P value < .05. In the replication cohort, all top variants including CYP2C8, CYP2C18, and CYP2C19 from the discovery population were found again. An original network analysis identified several candidate genes of potential interest such as a regulator of PI3K, a key actor in the downstream signaling pathway of the P2Y12 receptor. CONCLUSION: This study emphasizes the role of CYP-related genes as major regulators of clopidogrel response, including the poorly investigated CYP2C8 and CYP2C18.

Viral Sequences Detection by High-Throughput Sequencing in Cerebrospinal Fluid of Individuals with and without Central Nervous System Disease.

: Meningitis, encephalitis, and myelitis are various forms of acute central nervous system (CNS) inflammation, which can coexist and lead to serious sequelae. Known aetiologies include infections and immune-mediated processes. Despite advances in clinical microbiology over the past decades, the cause of acute CNS inflammation remains unknown in approximately 50% of cases. High-throughput sequencing was performed to search for viral sequences in cerebrospinal fluid (CSF) samples collected from 26 patients considered to have acute CNS inflammation of unknown origin, and 10 patients with defined causes of CNS diseases. In order to better grasp the clinical significance of viral sequence data obtained in CSF, 30 patients without CNS disease who had a lumbar puncture performed during elective spinal anaesthesia were also analysed. One case of human astrovirus (HAstV)-MLB2-related meningitis and disseminated infection was identified. No other viral sequences that can easily be linked to CNS inflammation were detected. Viral sequences obtained in all patient groups are discussed. While some of them reflect harmless viral infections, others result from reagent or sample contamination, as well as index hopping. Altogether, this study highlights the potential of high-throughput sequencing in identifying previously unknown viral neuropathogens, as well as the interpretation issues related to its application in clinical microbiology.

Detection of dicistroviruses RNA in blood of febrile Tanzanian children.

Fever is the leading cause of paediatric outpatient consultations in Sub-Saharan Africa. Although most are suspected to be of viral origin, a putative causative pathogen is not identified in over a quarter of these febrile episodes. Using a de novo assembly sequencing approach, we report the detection (15.4%) of dicistroviruses (DicV) RNA in sera collected from 692 febrile Tanzanian children. In contrast, DicV RNA was only detected in 1/77 (1.3%) plasma samples from febrile Tanzanian adults, suggesting that children could represent the primary susceptible population. Estimated viral load by specific quantitative real-time RT-PCR assay ranged from < 1.32E3 to 1.44E7 viral RNA copies/mL serum. Three DicV full-length genomes were obtained, and a phylogenetic analyse on the capsid region showed the presence of two clusters representing tentative novel genus. Although DicV-positive cases were detected throughout the year, a significantly higher positivity rate was observed during the rainy season. This study reveals that novel DicV RNA is frequently detected in the blood of Tanzanian children, paving the way for further investigations to determine if DicV possibly represent a new agent in humans.

Modulation of Inflammation-Related Genes in the Cornea of a Mouse Model of Dry Eye upon Treatment with Cyclosporine Eye Drops.

Purpose/Aim: Inflammation is recognized as playing an etiological role in dry eye disease. This study aimed to assess the efficacy of various topical cyclosporine A (CsA) formulations on cornea inflammatory markers in a mouse model of dry eye. MATERIAL AND METHODS: Six- to 7-week-old mice treated with scopolamine were housed in a controlled environment room to induce dry eye. Following dry eye confirmation by corneal fluorescein staining (CFS), the mice were treated three times a day with: 0.05%CsA (Restasis, Allergan), 0.1%CsA (Ikervis, Santen), 1%CsA oil solution, and 0.5% loteprednol etabonate (LE, Lotemax, Baush+Lomb), or left untreated. Aqueous tear production and CFS scores were assessed during the treatment period, and corneas were collected to measure the expression profile of a selection of inflammatory genes. RESULTS: After 7 days of treatment, the CFS scores were reduced by 21%, 31%, and 44% with 0.05%CsA, 0.1%CsA, and 1%CsA eye drops, respectively. By contrast, 0.5% LE did not decrease corneal fluorescein staining at day 10. A statistically significant dose-dependent CFS reduction was observed only between the 0.05% and 1%CsA formulations. The gene expression profiles indicated that 12, 18, 17 genes were downregulated by 0.05%CsA, 0.1%CsA, 1%CsA, respectively. Among them, the genes significantly downregulated were: IL1A, IL1R1, and TLR4 with 0.05%CsA; H2-Eb1, IL1A, IL1B, IL1RN, IL6, TGFB2, TGFB3, TLR2, TLR3, and TLR4 with 0.1%CsA; IL1B, IL6, TGFB3, and TLR4 with 1%CsA. TGFB1 and TGFBR1 were the only genes upregulated in all groups, but only TGFB1 upregulation reached significance. IL6RA was significantly upregulated by 0.05%CsA. CONCLUSIONS: This study indicates that the three CsA formulations effectively modulated TLR4, TGFß1, IL1, and IL6 pathways to reduce corneal epithelium lesions in a mouse model of severe dry eye. The study also suggests that the different anti-inflammatory eye drops modulated inflammatory genes in a slightly different manner.

Conjunctival Inflammatory Gene Expression Profiling in Dry Eye Disease: Correlations With HLA-DRA and HLA-DRB1.

Purpose: In several multicenter clinical trials, HLA-DR was found to be a potential biomarker of dry eye disease (DED)'s severity and prognosis. Given the fact that HLA-DR receptor is a heterodimer consisting in an alpha and a beta chain, we intended to investigate the correlation of inflammatory targets with the corresponding transcripts, HLA-DRA and HLA-DRB1, to characterize specific targets closely related to HLA-DR expressed in conjunctival cells from patients suffering from DED of various etiologies. Methods: A prospective study was conducted in 88 patients with different forms of DED. Ocular symptom scores, ocular-staining grades, tear breakup time (TBUT) and Schirmer test were evaluated. Superficial conjunctival cells were collected by impression cytology and total RNAs were extracted for analyses using the new NanoString® nCounter technology based on an inflammatory human code set containing 249 inflammatory genes. Results: Two hundred transcripts were reliably detected in conjunctival specimens at various levels ranging from 1 to 222,546 RNA copies. Overall, from the 88 samples, 21 target genes showed a highly significant correlation (R > 0.8) with HLA-DRA and HLA-DRB1, HLA-DRA and B1 presenting the highest correlation (R = 0.9). These selected targets belonged to eight family groups, namely interferon and interferon-stimulated genes, tumor necrosis factor superfamily and related factors, Toll-like receptors and related factors, complement system factors, chemokines/cytokines, the RIPK enzyme family, and transduction signals such as the STAT and MAPK families. Conclusions: We have identified a profile of 21 transcripts correlated with HLA-DR expression, suggesting closely regulated signaling pathways and possible direct or indirect interactions between them. The NanoString® nCounter technology in conjunctival imprints could constitute a reliable tool in the future for wider screening of inflammatory biomarkers in DED, usable in very small samples. Broader combinations of biomarkers associated with HLA-DR could be analyzed to develop new diagnostic approaches, identify tighter pathophysiological gene signatures and personalize DED therapies more efficiently.

Transcriptomic approach for assessment of the impact on microalga and macrophyte of in-situ exposure in river sites contaminated by chlor-alkali plant effluents.

Water quality degradation is a worldwide problem, but risk evaluation of chronic pollution in-situ is still a challenge. The present study aimed to evaluate the potential of transcriptomic analyses in representative aquatic primary producers to assess the impact of environmental pollution in-situ: the microalga Chlamydomonas reinhardtii and the macrophyte Elodea nuttallii were exposed 2 h in the Babeni Reservoir of the Olt River impacted by chlor-alkali plant effluent release resulting in increased concentrations of Hg and NaCl in receiving water. The response at the transcriptomic level was strong, resulting in up to 5485, and 8700 dysregulated genes (DG) for the microalga and for the macrophyte exposed in the most contaminated site, respectively. Transcriptomic response was congruent with the concentrations of Hg and NaCl in the water of the impacted reservoir. Genes involved in development, energy metabolism, lipid metabolism, nutrition, and RedOx homeostasis were dysregulated during in-situ exposure of both organisms. In addition, genes involved in the cell motility of C. reinhardtii and development of the cell wall of E. nuttallii were affected. DG were in line with adverse outcome pathways and transcriptomic studies reported after exposure to high concentrations of Hg and NaCl under controlled conditions in the laboratory. Transcriptomic response provided a sensitive measurement of the exposure as well as hints on the tolerance mechanisms of environmental pollution, and is thus promising as an early-warning tool to assess water quality degradation.