Healthy Control Donors Research Articles

Siglec-15 Is a Novel Immunomodulatory Protein and Therapeutic Target in Acute Lymphoblastic Leukemia

Immunotherapies have recently shown efficacy in treatment of aggressive, refractory pediatric B cell acute lymphoblastic leukemia (B-ALL), which remains one of the leading causes of cancer-related death in children. The immune evasion mechanisms of B-ALL are still being explored to discover new therapeutic targets and improve patient outcomes.Recent reports have implicated a role for the molecule Siglec-15 (Sig15) in regulating immune response in solid tumor-infiltrating macrophages. Our lab has found higher expression of SIGLEC15 at the RNA level in primary pediatric B-ALL as compared to healthy donor controls, as well as at the RNA and protein levels across a panel of B-ALL, T cell acute lymphoblastic leukemia (T-ALL), and diffuse large B cell lymphoma (DLBCL) cell lines compared to healthy donor PBMCs. Higher expression of SIGLEC15 in pediatric B-ALL samples from the TARGET database correlates with markers of PKC and NFκB activation known to drive B-ALL leukemogenesis, which we have demonstrated to regulate Sig15 RNA and protein expression in vitro.Knockout of Siglec15 expression in a BCR-ABL1 + murine model of B-ALL engrafted in immunocompetent and Rag1 -/- immunodeficient recipients resulted in leukemia clearance in immunocompetent, but not immunodeficient, recipients and 100% survival (Figure A, p=0.01 Sig15 KO into WT vs. Rag1 -/-). Further study indicates that Siglec15 expression on these leukemia cells suppresses T cell effector and memory population expansion at 7 days post-engraftment (Figure B) and correlates with higher levels of IL-10 and lower levels of CCL17 present in the bone marrow, representing a more immunosuppressive bone marrow milieu. These data suggest a prominent role for Sig15 in the suppression of adaptive immune response to B-ALL as well as other hematological malignancies.We have also reported for the first time the release of a soluble form of Sig15 (sSig15), which we have demonstrated to circulate at higher levels in the plasma of pediatric B-ALL patients compared to healthy donors (Figure C, ****P≤0.0001). Detection of this sSig15 negatively correlated with circulating levels of IL-12 and IL-1α/β (Figure D, depicting correlations of cytokines using Pearson's r), suggesting sSig15 levels correspond to a systemically immunosuppressive phenotype. Flow cytometry of fresh pediatric B ALL cells demonstrates expression of surface Sig15 in a subset of cases. Thus, Sig15 has the capacity to promote immunosuppressive effects at both marrow-localized and systemic levels.Together, these results suggest Siglec-15 is a novel, potent immunosuppressive molecule active in leukemia progression that may be targeted therapeutically to activate T lymphocytes against leukemia cells. [Display omitted] DisclosuresAbukharma: NextCure Inc.: Current Employment. Liu: NextCure: Current Employment, Current holder of stock options in a privately-held company.

Inhibitory effects of tolerogenic probiotics on migratory potential of lupus patient-derived DCs.

Objective(s):The present in vitro study aimed to evaluate whether Lactobacillus delbrueckii and Lactobacillus rhamnosus treatments can induce regulatory phenotype, together with modulating the expression of chemokine receptors (CRs) in dendritic cells (DCs). The CRs of DCs have been found to be involved in the pathogenesis of Systemic lupus erythematosus (SLE) through directing recruitment and migration of immune cells.Materials and Methods: In brief, monocytes of patients with SLE and healthy donors were isolated and differentiated to regulatory or inflammatory mature DCs through treatment with L. delbrueckii, L. rhamnosus, mixed probiotics, and LPS. Results:FACScan analysis showed that the expression of CRs including CXCR3, CCR5, CCR4, and CCR3, was significantly reduced in all probiotic-treated groups of SLE and healthy (control) donors when compared with the LPS treated group. Conclusion:The results demonstrated that tolerogenic probiotics could prevent or decrease the expression of inflammatory CRs on the surface of tolerogenic DCs during the maturation process.

Neutrophil Extracellular Trap Formation Potential Correlates with Lung Disease Severity in COVID-19 Patients.

Severe lung inflammation is common in life-threatening coronavirus disease 2019 (COVID-19). This study tested the hypothesis that polymorphonuclear (PMN, neutrophil) phenotype early in the course of disease progression would predict peak lung disease severity in patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It is increasingly evident that PMN activation contributes to tissue injury resulting from extracellular reactive oxygen species generation, granule exocytosis with release of proteases, neutrophil extracellular trap (NET) formation, and release of cytokines. The current study focuses on PMN activation in response to SARS-CoV-2 infection, specifically, the association between NETs and lung disease. This is a prospective cohort study at an academic medical center with patients enrolled within 4 days of admission at 3 tertiary hospitals: Clements University Hospital, Parkland Memorial Hospital, and Children’s Health in Dallas, TX. Patients were categorized as having minimal or moderate to severe lung disease based on peak respiratory support. Healthy donor controls matched for age, sex, race, and ethnicity were also enrolled. Neutrophils from COVID-19 patients displayed greater IL-8 expression, elastase release, and NET formation as compared with neutrophils from healthy donors. Importantly, neutrophils from COVID-19 patients had enhanced NET formation in the absence of any additional stimulus, not seen in PMN from healthy donors. Moreover, PMA-elicited NET formation by circulating PMN correlated with severity of lung disease. We speculate that neutrophil immuno-phenotyping can be used to predict lung disease severity in COVID-19 patients.SUPPLEMENTARY INFORMATIONThe online version contains supplementary material available at 10.1007/s10753-021-01585-x.

Quantitative LC\u2013MS/MS uncovers the regulatory role of autophagy in immune thrombocytopenia

BackgroundImmune thrombocytopenia (ITP) is an autoimmune haemorrhagic disease whose pathogenesis is associated with bone marrow megakaryocyte maturation disorder and destruction of the haematopoietic stem cell microenvironment.MethodsIn this study, we report the qualitative and quantitative profiles of the ITP proteome. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) was conducted to elucidate the protein profiles of clinical bone marrow mononuclear cell (BMMC) samples from ITP patients and healthy donors (controls). Gene Ontology (GO) and Kyoto Encyclopaedia Genes and Genome (KEGG) pathway analyses were performed to annotate the differentially expressed proteins. A protein–protein interaction (PPI) network was constructed with the BLAST online database. Target proteins associated with autophagy were quantitatively identified by parallel reaction monitoring (PRM) analysis.ResultsOur approaches showed that the differentially expressed autophagy-related proteins, namely, HSPA8, PARK7, YWHAH, ITGB3 and CSF1R, were changed the most. The protein expression of CSF1R in ITP patients was higher than that in controls, while other autophagy-related proteins were expressed at lower levels in ITP patients than in controls.ConclusionBioinformatics analysis indicated that disruption of the autophagy pathway is a potential pathological mechanism of ITP. These results can provide a new direction for exploring the molecular mechanism of ITP.

Complement Activation Is a Prominent Feature of MOGAD.

Myelin oligodendrocyte glycoprotein (MOG)-antibody (Ab)-associated diseases (MOGADs) account for a substantial proportion of pediatric and adult patients who present with acquired demyelinating disorders. Its pathogenesis and optimal therapy are incompletely understood. We profiled systemic complement activation in adult and pediatric patients with MOGAD compared with patients with relapse-onset multiple sclerosis, patients with neuromyelitis optica spectrum disorder, and pediatric control and adult healthy donors. Proteins indicative of systemic classical and alternative complement activation were substantially increased in patients with MOGAD compared to control groups. Elevated levels were detected in both adult and pediatric cases and across all clinical syndromes. Complement inhibition should be explored for its therapeutic merit in patients with MOGAD. ANN NEUROL 2021;90:976-982.

NATURAL KILLER CELLS PHENOTYPE CORRELATES WITH TYROSINE KINASE INHIBITORS TREATMENT RESPONSE IN CHRONIC MYELOID LEUKEMIA PATIENTS

Despite well-known Natural Killer (NK) cells antitumor activity, its potential in regulating Chronic Myeloid Leukemia (CML) is not fully stablished. Tyrosine kinase inhibitors (TKI) treatment, besides reducing BCR-ABL1 transcripts, seems to result in immunomodulation that partly restore the immune dysregulation of the disease. Numerical, functional and maturation defects might be associated with CML risk stratification, molecular response profile or even with resistance to treatment with TKI. Therefore, we aimed to immunophenotypically characterize frequency, subtypes and maturation profile of NK cells in CML patients, by Multiparametric Flow Cytometry (MFC), during monitoring of TKI treatment. 11 Peripheral Blood samples from patients with CML (Clinical Hospital of Ribeirão Preto - HCRP-USP) and 10 healthy donors (controls) were analyzed. Clinical, laboratory and ELN2017 risk stratification were assessed. NK cells were defined as CD19-CD3-CD56+ and: CD56brightCD16- (secretory), CD56dimCD16+ (cytotoxic), CD11b-CD27- (tolerant); CD27+CD11b- (immature secretor), CD27+CD11b+ (secretory) and CD11b+CD27- (cytotoxic). Based on molecular response levels assessed by BCR-ABL1 RT-qPCR, patients were classified in G1 (without loss of molecular response to the 1 st line of TKI; n = 3), G2 (loss of molecular response to 1 st line of TKI; n = 3) and G3 (loss of molecular response to 2 nd generation TKI; n = 5). Mann Whitney or Kruskal-Wallis tests (p < 0.05) were applied using SPSS (v.20). A higher frequency of lymphocytes in CML patients was observed when compared to controls (p = 0.0242), but no difference was found in the frequency of NK cells. G1 and G2 showed a higher frequency of NK cells than G3 (p = 0,7774). Even though no statistical significance was found, CD27-CD11b- and CD27+CD11b- NK cells showed a higher frequency, while CD27+CD11b+ NK cells showed a lower frequency in CML when compared to controls. Frequency of CD27+CD11b- NK cells was higher in G2 and G3 when compared to G1. G3 showed an immature profile with higher percentages of CD27-CD11b- and CD27+CD11b- NK cells when compared to controls, while the more mature CD27+CD11b+ NK cells showed a lower frequency in CML. Furthermore, a lower frequency of CD56brightCD16- (p = 0.0002) and CD56dimCD16+ (p = 0.0448) NK cells was observed in CML, as wells as the frequency of CD27-CD11b+ cytotoxic NK cells, consistent to what we found in G3 and supporting our hypothesis. We observed that patients with loss of molecular response in treatment with 1 st and 2 nd TKI generation presented an immature non-effective NK cell profile, with deficient cytotoxicity. This scenario might result in impaired tumor surveillance of CML, meaning that phenotypic changes in NK cells may directly affect risk stratification and treatment response in CML. However due to the small number of patients and the lack of a functional assay, it is not possible to confirm that the cytotoxic cell may actually be deficient. Our findings and further functional studies can contribute to a better understanding of the physiopathology, prognosis and monitoring of CML, response to TKI therapy, as well as help to define therapeutic strategies based on immunotherapy mediated by NK cells.

Peripheral blood mononuclear cells - Can they provide a clue to the pathogenesis of Graves' Orbitopathy?

Graves' disease (GD) is an autoimmune disorder affecting primarily the thyroid gland. The most common extrathyroidal manifestation of GD is known as Graves' orbitopathy (GO). Bone marrow-derived fibrocytes represent a subset of monocytes in peripheral blood mononuclear cells (PBMCs), infiltrate the orbital tissues, and contribute to the pathogenesis of GO. Hence objectives of the study included whether the concentration of fibrocytes in peripheral blood was higher in GO, whether TSHR m RNA expression and TSHR surface expression in peripheral blood were higher in GO in comparison to Graves' Disease (GD) and Control subjects. The percentage of circulating fibrocytes (FC) along with TSHR on its cell surface (CD 34+, CD 45+, CXCR4+, Collagen 1+, TSHR+) were assessed by flow cytometry of 50 patients with GD and GO and 15 healthy donors (Control). TSHR mRNA expression was measured by q RT PCR. The concentration of circulating fibrocytes was significantly higher in GO compared to GD and control [GO 17% vs GD 3% vs control 0.7% (p < 0.05)]. Moreover, these fibrocytes express a significantly higher level of TSHR in GO. This was corroborated by the measure of TSH mRNA; in GD it was 2.3-fold higher and in GO it was 3.9 fold higher than in control, in GO this transcript level was 1.7fold higher than GD (p < 0.05). TSHR+ fibrocytes were significantly positively correlated with CAS (p = 0.004) and negatively correlated with age (p = 0.01) and duration of disease (p = 0.01) in GO. This study sheds further light on the pathogenesis of GO.

A nanoenzyme linked immunochromatographic sensor for rapid and quantitative detection of SARS-CoV-2 nucleocapsid protein in human blood

The establishment of a simple, low-cost, high-sensitive and rapid immunoassay for detecting SARS-CoV-2 antigen in human blood is an effective mean of discovering early SARS-CoV-2 infection and controlling the pandemic of COVID-19. Herein, a smartphone based nanozyme linked immunochromatographic sensor (NLICS) for the detection of SARS-CoV-2 nucleocapsid protein (NP) has been developed on demand. The system is integrated by disposable immunochromatography assay (ICA) and optical sensor devices. Immunoreaction and enzyme-catalyzed substrate color reaction were carried out on the chromatographic strip in a device, of which the light signal was read by a photometer through a biosensor channel, and the data was synchronously transmitted via the Bluetooth to the app in-stored smartphone for reporting the result. With a limit of detection (LOD) of 0.026 ng/mL NP, NLICS had the linear detection range (LDR) between 0.05 and 1.6 ng/mL NP, which was more sensitive than conventional ICA. NLICS took 25 min for reporting results. For detection of NP antigen in clinical serum samples from 21 COVID-19 patients and 80 healthy blood donor controls, NLICS and commercial enzyme linked immunosorbent assay (ELISA) had 76.2% or 47.6% positivity, and 100% specificity, respectively (P = 0.057), while a good correlation coefficient (r = 0.99) for quantification of NP between two assays was obtained. In conclusion, the NLICS was a rapid, simple, cheap, sensitive and specific immunochromatographic sensing assay for early diagnosis of SARS-CoV-2 infection.

Clinical and prognostic significance of the soluble form of the VISTA immunity control point in patients with primary bone tumors

The data of a comparative enzyme-linked immunosorbent assay of the content of the soluble form of the immunity checkpoint VISTA in the blood serum of 30 healthy donors (control group), 79 patients with primary malignant (osteosarcoma - 30, chondrosarcoma - 31, chordoma - 14) and 14 borderline (giant cell tumor) bone neoplasms are presented. In the general group of patients with malignant neoplasms of bones, the median sVISTA content in blood serum is statistically significant lower than in the control (p = 0.040). In patients with bone tumors and healthy donors over 18 years of age, there was a decrease with age in serum sVISTA levels. There were no significant differences in sVISTA concentration between patients with osteosarcoma, chondrosarcoma and healthy donors. Only in patients with chordoma were sVISTA levels statistically significant lower than in controls (p = 0.013). In the groups of patients with chondrosarcoma and osteosarcoma of the bone, there were no significant associations between the serum sVISTA content and the main clinical and morphological characteristics of the disease. In patients with osteosarcoma, no relationship was found between sVISTA levels and overall survival rates, while in patients with bone chondrosarcoma, there was a tendency towards a favorable prognosis with a high content of the marker in the blood serum.

Circulating microRNAs from the mouse tibia fracture model reflect the signature from patients with complex regional pain syndrome.

Complex regional pain syndrome (CRPS) often results from an initial trauma that later produces a disproportionate amount of pain. The mechanisms underlying CRPS have been studied using a tibia fracture model (TFM) in rodents because this model closely mimics symptoms and has several molecular correlates observed in patients with CRPS. Here, we determined whether the TFM has alterations in circulating microRNAs (miRNAs) and cytokines transported by small extracellular vesicles (sEVs) that faithfully model previously reported miRNA alterations from patients with CRPS. We isolated and characterized serum-derived sEVs from mice 3 weeks after fracture when symptoms such as pain hypersensitivity develop. Whole-transcriptome profiling was used to determine sEV miRNAs, and Bio-Plex Pro Mouse Cytokine 23-plex assay was used to measure cytokines. Differentially expressed miRNAs from TFM were compared with previously reported circulating miRNA alterations from patients with CRPS. Although sEV cytokine levels were unchanged, there were significant changes in sEV miRNA profiles. Differentially expressed miRNAs from TFM sEVs significantly overlapped with those previously reported in patients with CRPS. Of the 57 sEV miRNAs dysregulated in the TFM, 30 were previously reported in patients with CRPS compared with healthy control donors both in sEVs and 23 in whole blood. These findings enhance the validity of TFM as a model for CRPS and suggest that specific miRNA dysregulation may be a shared feature of CRPS and the TFM. These dysregulated miRNAs could help identify mechanistic targets or serve as biomarker candidates for both diagnosis and treatment responses in clinical trials.

Molecular basis of immunoglobulin heavy constant G4 gene (IGHG4)-related low serum IgG4 subclasses in Down syndrome.

Objectives:To investigate the molecular mechanism of low serum IgG4 at the level of the immunoglobulin heavy constant G4 gene (IGHG4). Patients with Down syndrome (DS) are more likely to exhibit immunological abnormalities that predispose them to infection. Among other anomalies, individuals with DS have altered serum concentrations of some subclasses of immunoglobulin G (IgG), particularly the IgG4 subclasses.Methods:In this prospective study, quantitative real-time polymerase chain reaction (qPCR) was carried out from December 2017 to June 2019 in the University Hospital of Saint-Etienne, Saint-Etienne, France to measure the number of IGHG4 copies and to compare those outcomes with a reference gene (36B4). An IGHG4/36B4 ratio was considered normal when between 0.8 and 1.2. Forty-four DS patients, comprising 23 DS patients carrying severe low serum IgG4 and 21 DS patients with normal serum IgG4 (level >0.1 g/L). The patient group was compared with 38 healthy donors (controls) without DS.Results:The heavy chain gene IGHG4 heterozygous deletion was found in 16 (69.57%) DS patients with low serum IgG4 versus in 2 (9.52%) DS with normal serum IgG4 (p=0.0001). In the control group, deletion was found in 5.26% (2/38) of the sample.Conclusion:The heavy chain gene IGHG4 haploinsufficiency is highly correlated with low serum IgG4 in our population with DS, but other relevant factors must be assessed in future work.

The first human induced pluripotent stem cell line of Kashin–Beck disease reveals involvement of heparan sulfate proteoglycan biosynthesis and PPAR pathway

Kashin-Beck disease (KBD) is an endemic osteochondropathy. Due to a lack of suitable animal or cellular disease models, the research progress on KBD has been limited. Our goal was to establish the first disease-specific human induced pluripotent stem cell (hiPSC) cellular disease model of KBD, and to explore its etiology and pathogenesis exploiting transcriptome sequencing. HiPSCs were reprogrammed from dermal fibroblasts of two KBD and one healthy control donor via integration-free vectors. Subsequently, hiPSCs were differentiated into chondrocytes through three-week culture. Gene expression profiles in KBD, normal primary chondrocytes, and hiPSC-derived chondrocytes were defined by RNA sequencing. A Venn diagram was constructed to show the number of shared differentially expressed genes (DEGs) between KBD and normal. Gene oncology and Kyoto Encyclopedia of Genes and Genomes annotations were performed, and six DEGs were further validated in other individuals by RT-qPCR. KBD cellular disease models were successfully established by generation of hiPSC lines. Seventeen consistent and significant DEGs present in all compared groups (KBD and normal) were identified. RT-qPCR validation gave consistent results with the sequencing data. Glycosaminoglycan biosynthesis-heparan sulfate/heparin; PPAR signaling pathway; and cell adhesion molecules (CAMs) were identified to be significantly altered in KBD. Differentiated chondrocytes derived from KBD-origin hiPSCs provide the first cellular disease model for etiological studies of KBD. This study also provides new sights into the pathogenesis and etiology of KBD and is likely to inform the development of targeted therapeutics for its treatment.

Kidney organoids generated from erythroid progenitors cells of patients with autosomal dominant polycystic kidney disease.

BackgroundKidney organoids have been broadly obtained from commercially available induced pluripotent stem cells (iPSCs); however, it has been a great challenge to efficiently produce renal organoid models from patients with autosomal dominant polycystic kidney disease (ADPKD) that recapitulate both embryogenesis and the mechanisms of cystogenesis.MethodsBlood erythroid progenitors (EPs) from two ADPKD patients and one healthy donor (HC) was used as a comparative control to normalize the many technical steps for reprogramming EPs and for the organoids generation. EPs were reprogrammed by an episomal vector into iPSCs, which were differentiated into renal tubular organoids and then stimulated by forskolin to induce cysts formation.ResultsiPSCs derived from EPs exhibited all characteristics of pluripotency and were able to differentiate into all three germ layers. 3D tubular organoids were generated from single cells after 28 days in Matrigel. HC and ADPKD organoids did not spontaneously form cysts, but upon forskolin stimulation, cysts-like structures were observed in the ADPKD organoids but not in the HC-derived organoids.ConclusionThe findings of this study showed that kidney organoids were successfully generated from the blood EP cells of ADPKD patients and a healthy control donor. This approach should contribute as a powerful tool for embryonic kidney development model, which is able to recapitulate the very early pathophysiological mechanisms involved in cytogenesis.

Plasma-Derived Exosomal hsa-miR-4488 and hsa-miR-1228-5p: Novel Biomarkers for Dermatomyositis-Associated Interstitial Lung Disease with Anti-Melanoma Differentiation-Associated Protein 5 Antibody-Positive Subset.

The present study is aimed at profiling circulating exosome-derived microRNAs (miRNAs/miRs) from patients with dermatomyositis (DM), in particular those complicated with interstitial lung disease (ILD) with anti-melanoma differentiation-associated protein 5 (MDA5) antibody-positive. Fifteen participants were enrolled, including five patients with DM complicated with ILDs prior to treatment with circulating anti-MDA5 antibody-positive status [DM-ILD-MDA5 Ab(+)], five DM patients without ILDs who were negative for 16 detectable myositis-specific antibodies [DM-nonILD-MSA16(-)], and five age- and gender-matched healthy donor controls (HCs). The characteristics of the exosomes extracted by Ribo™ Exosome Isolation Reagent were identified using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and flow cytometry. Differentially expressed miRNAs, determined by next-generation deep sequencing, were identified through the criteria of ∣log2 fold change | ≥1 and P < 0.01. A total of 38 miRNAs were significantly upregulated in exosomes from patients with DM-ILD-MDA5 Ab(+) compared to those from HC, while 21 miRNAs were significantly downregulated. Compared to exosomes derived from patients with DM-nonILD-MSA16(-), 51 miRNAs were significantly upregulated and 33 miRNAs were significantly downregulated from patients with DM-ILD-MDA5 Ab(+). A total of 73 exosomal miRNAs were significantly differentially expressed between DM-nonILD-MSA16(-) and HC. In particular, two miRNAs, Homo sapiens- (hsa-) miR-4488 and hsa-miR-1228-5p, were common differentially expressed miRNAs among three comparisons. GO and KEGG analyses suggested that several pathways may contribute the pathogenesis of DM-ILD-MDA5 Ab(+) and DM-nonILD-MSA16(-), while PPI network analysis of hsa-miR-4488 and hsa-miR-1228-5p indicated that their predicted target genes, DExD-box helicase 39B and MDM2, may be involved in the mechanisms of DM-ILD-MDA5 Ab(+).

Expression of NF-kB Regulators (BCL-3, NFKB1, NFKB2) in Metastatic Breast Cancer Patients

Background/aim: BCL-3, upregulated in several cancers, functions as a crucial player not only in cell cycle and apoptosis, but also in metastasis. The study aimed to analyse the expression profile of BCL-3 and its interacting partners in metastatic breast cancer. Materials and methods: mRNA expression levels were evaluated in blood samples of metastatic breast cancer patients (n=55) and in healthy control donors (n=50) by RT qPCR. SPSS 26 program was used for statistical analysis. Results: Expression levels of BCL-3 mRNA was downregulated in metastatic breast cancers patients compared to healthy control group (p= 0.0004). Subsequently, expression levels of the NFKB1 (p= 0.0005), NFKB2 (p=0.0001), CYLD (p= 0.00042) and TP53 (p= 0.0287) were significantly lower in metastatic breast patients compared to healthy controls. CCND1 and CDH2 gene expressions were significantly upregulated in metastatic group (p=0.0009; and p= 0.0030, respectively). GSK3B, SMAD3 and TGFB1 expressions were not significantly different between groups. Conclusion: This study indicates significant expression difference between patients and controls. The NF-κB regulators might induce the metastatic potential through interaction with the other molecules in the microenvironment, nevertheless, it is needed further research whether the importance of BCL-3 as a biomarker for metastatic breast cancer.

Serum Neutrophil Gelatinase Associated Lipocalin in Patients with Toxic Nephropathies. Prospective Study

Background. Drug induced kidney disorder (DIKD) is a frequent adverse event which contributes to morbidity and even incapacitation. Toxic nephropathy also is one of the pathological syndromes or complications in acute alcohol poisoning (AAP). Recent years experience shows the insensitivity of serum creatinine to the early stages of kidney damage. NGAL has been shown as a preferred marker of acute kidney damage in a variety of clinical settings. However, no similar studies of serum NGAL have been performed in patients with toxic nephropathy. The presence of markers discovers the possibility of earlier detection, timely treatment and prevention of disease progression to chronic kidney disease (CKD), which can improve the patients prognosis. Aims to study serum NGAL levels in patients with AAP and DIKD with clinically diagnosed nephropathy and without signs of kidney damage. The effect of glomerular filtration rate (GFR) on serum NGAL levels was studied. Materials. This prospective cross-sectional study was conducted on the basis of the biochemical laboratory of the Karaganda Medical University in conjunction with the toxicological department of the Regional Medical Center (from January 2018 to October 2019). The study included 89 patients with AAP and 50 patients with DIKD. 25 healthy donors (control group) and 25 patients with CKD served as comparison groups. Serum NGAL levels were measured using a commercially available ELISA kit. Results. We detected the increased serum NGAL level in both groups with drug-induced nephropathy and nephropathy, caused by AAP compared with the control group values (p 0.01). However, there were no significant differences in NGAL level, depending on the type of toxic nephropathy. After ranking the GFR, it was revealed that GFR did not affect the serum NGAL level (F = 2.21; p = 0.12) and its increase was observed both at reduced and increased GFR relative to the control group values (p 0.05). Conclusions. The results of our study showed a multiple increase in the concentration of NGAL in serum not only in patients with toxic nephropathy, but also in patients with increased GFR, even in the absence of clinical and laboratory signs of impaired renal function.

Inflammatory epithelial cytokines after in vitro respiratory syncytial viral infection are associated with reduced lung function.

Respiratory syncytial virus (RSV) infections in early life predispose children with cystic fibrosis (CF) to more severe lung function decline in later life. The mechanisms explaining the associations between RSV and progression of CF lung disease are not clear.In this study, a human bronchial epithelial cell line and primary human nasal epithelial cells (PNECs) from individuals with CF and healthy control donors were infected with RSV. Real-time PCR, plaque assay, cytokine detection, immunofluorescence and Western blot analyses were performed.RSV is replicated to a higher degree in CF epithelial cells as compared to control cells; however, no defects in innate immune pathways were identified in CF cells. Rather, primary p.Phe508del cystic fibrosis transmembrane conductance regulator PNECs produced more cytokines after RSV infection than control cells. Moreover, interleukin-8 and tumour necrosis factor-α production post RSV negatively correlated with lung function (% predicted forced expiratory volume in 1 s) in the individuals who donated the cells.These data suggest that CF epithelium has a dysfunctional response to RSV allowing for enhanced viral replication and an exaggerated inflammatory response that ultimately may predispose to greater airway inflammation and reduced lung function.

A Single-Cell Approach to Unraveling Abnormal Megakaryocyte Differentiation and Function in Myelofibrosis

Increased numbers of immature/low ploidy MKs are a hallmark feature of myelofibrosis (MF). MF MKs produce pathological amounts of pro-fibrotic factors including TGFβ causing a severe bone marrow (BM) fibrosis. Isolating MKs in MF is difficult as the fibrosis prevents BM aspiration. Also, the cellular hierarchy of normal megakaryopoiesis remains unclear, complicating any in-depth study of abnormal MK development in MF. Using a single-cell approach, we recently identified CD41 as a marker of a novel, rare population of MK-primed progenitors within peripheral blood (PB) immunophenotypic megakaryocyte-erythroid progenitors (MEP) of mobilized, healthy donor controls (Psaila et al, 2016). However, our analysis did not include MF patients and we did not analyze earlier hematopoietic stem/progenitor cells (HSPCs), which may also include MK-biased subsets that might be disrupted in MF. To examine the abnormalities in MF hematopoiesis more comprehensively and without bias from pre-selected surface markers/gene expression assays, we performed single-cell RNA-sequencing on ~50,000 individual CD34+ Lineage- PB cells of healthy mobilized donors (n=2, 18,500 cells) and 4 JAK2V617F+MF patients (n=4, 32,500 cells, 10X Genomics platform). The data were analyzed using Seurat (Sajita et al, 2015) to perform dimensionality reduction and unsupervised cell clustering and to identify differentially expressed genes. Individual cells from control and MF samples were grouped into distinct ‘clusters’ of cells with similar transcriptional profiles. Identities of the different clusters were determined by unsupervised gene ontology enrichment analyses and manual inspection of top differentially expressed genes for known lineage-specific identifiers.We observed striking differences in the cellular architecture of HSPCs in the controls and patients with MF (Fig 1A). Increased expression of MK-associated genes (incl. VWF, ITGA2B /CD41) was observed throughout HSPC populations in MF (Fig 1B). Similarly, we observed prominent clusters of CD34+ progenitors expressing PF4, a more mature MK marker, and high levels of TGFβ only in MF samples and not in controls (Fig 1B). Analyses to determine the molecular basis for the bias towards MK lineage specification in MF are now underway.As our single cell analysis showed increased expression of CD41 across a number of different HSPCs in MF patients, we compared the frequencies of CD41+, MK-primed populations in PB samples of 11 controls and 15 patients with JAK2V617F+ MF, both within more differentiated CD38+ MEP/CMP as well as primitive, CD38-neg HSPC. A selective expansion of MK-primed populations in MF was observed, with a >5-fold increase in both MK-primed MEP as well as MK-primed HSC/MPP populations, suggesting an expansion of the MK lineage and biased commitment from the apex of the HSPC hierarchy.To study more differentiated MKs, we next carried out RNA-sequencing of immature (CD41+42-) and mature (CD41+42+) MKs cultured in vitro from CD34+ HSPC from controls and patients with JAK2V617F+MF and moderate-severe BM fibrosis. The mRNA signature of immature CD42- MKs was highly distinct for MF patients vs. controls, whereas mature CD42+ MKs were not significantly different. IPA analysis identified tissue fibrosis as the most highly dysregulated pathway. The expression of genes encoding extracellular matrix proteins e.g. elastin were also markedly increased in JAK2V617F+ MKs, suggesting that MKs may themselves produce extracellular matrix factors as well as stimulating their deposition by fibroblasts. Increased expression of hepatocyte growth factor (HGF) was also observed in MF MKs. Treatment of fibroblasts in vitro with HGF or PF4 was found to inhibit TGFβ-mediated collagen deposition (P's<0.01), suggesting a complex interplay of both stimulatory (e.g. TGFβ) and inhibitory (HGF and PF4) regulators of fibrosis in MF MKs.In summary, we demonstrate that a single-cell approach enables a much finer dissection of HSPCs compared to “bulk” assays and allows proper comparison of normal and malignant cellular differentiation pathways, as well as the ability to uncover rare cell types. Our data constitute a rich and unique dataset, illustrating how transcriptomic analysis of HSPCs in MF can identify novel regulators of fibrosis with potential for therapeutic development. [Display omitted] DisclosuresMilojkovic:Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Incyte: Honoraria, Speakers Bureau. Mead:BMS: Honoraria; Pfizer: Honoraria; Novartis: Honoraria, Research Funding, Speakers Bureau.

Whole Genome Sequencing to Detect Small Deletions in Ribosomal Protein Genes in Diamond Blackfan Anemia Patients

Diamond Blackfan Anemia (DBA) is characterized by profound anemia due to red cell aplasia (Vlachos, Blanc and Lipton 2014; Clinton and Gazda 2016). Mutations in ribosomal protein (RP) genes involved in the biogenesis of the small and large ribosomal subunits are detected in approximately 65% of patients and display an autosomal dominant pattern of inheritance. In addition, rare recessive mutations in the GATA1, TSR2, CECR1, and EPO genes have also been associated with a DBA phenotype. The molecular diagnosis of DBA follows a standard genotyping pipeline that employs targeted sequencing, detection of large deletions and whole exome sequencing. However, approximately 35% of patients with a clinical diagnosis of DBA have no detectable mutation.We first hypothesized that mutations in other genes associated with ribosomal biogenesis and/or translation were responsible for DBA in the patients without a molecular diagnosis, and that these mutations would be inherited in an autosomal recessive pattern. To test this, we conducted whole exome sequencing for 12 patients that had a clinical diagnosis of DBA but were negative for previously characterized RP mutations, large deletions, as well as for both parents and a sibling (affected or unaffected). In our 12 families we detected compound heterozygous point mutations in 20 non-ribosomal protein genes. Unfortunately, none of these variants could be convincingly validated in functional assays or by finding an unrelated family with a mutation in the same gene. Similar to previous studies, we conclude that non-RP mutations associated with DBA are rare and that undetected mutations in RP genes were responsible for DBA in the majority of the undiagnosed patients.One limitation in the current genotyping pipelines is that they are unable to detect deletions smaller than 15kb and small insertions or deletions in the exons. Therefore we hypothesized that small deletions in RP genes may be present in DBA patients without a molecular diagnosis. To test this hypothesis we performed whole genome sequencing on five patients that had a clinical diagnosis of DBA but had been shown to be negative for the known RP gene and GATA1 mutations, large RP gene deletions and did not have RP mutations detected by whole exome sequencing. To identify deletions in whole genome sequencing data we used two variant callers, DELLY (Rausch et al, 2012) and CNVkit (Talevich et al, 2016). DELLY analyzes the sequence from the ends of the sequenced DNA fragments and determines whether the ends are the appropriate distance apart compared to reference genome. When DELLY identifies sequences that are not at the correct distance, it analyzes single reads to identify putative deletions. In contrast, CNVkit analyzes the number of reads at each location in the genome and identifies significant changes in sequencing read depth between exons and surrounding DNA in order to detect genomic variants (increased number = duplication, decreased number = deletion).All the variants we discovered in our 5 patients (~42000/patient) were screened against copy number variants found in the general population using the dbVar database. Only novel deletions that overlap with RP genes were considered. Nine ribosomal protein genes were found to carry deletions in at least 1 patient (Table 1). DELLY detected a 419bp deletion overlapping the 3' end of the RPL36AL gene while CNVkit predicted deletions in the RPS27L (1867bp), RPS24 (6666bp), RPS17 (4533bp), RPS13 (3200bp), RPS6 (1067bp), RPL5 (1334bp) and RPS5 (3466bp) genes for one patient each. A deletion in the RPL7, predicted by CNVkit, was shared by 3 of the 5 patients and varied in length between 1867bp and 3200bp.To validate the presence of these deletions, we designed primers that span each predicted deletion. For example we performed PCR on DNA from healthy donor controls and the patient predicted to have the RPL36AL deletion. In the patient DNA we demonstrated both the wildtype band (~850bp) and a band of ~350bp, is consistent with a deletion of 500bp, which is within the confidence limits of the detection software.In conclusion, we identified deletions in five RP genes in five DBA patients that had no molecular diagnosis after targeted sequencing, analysis of SNP arrays and whole exome sequencing. These small deletions are amenable to primer-directed PCR analysis to validate the causative variant in uncharacterized DBA patients. [Display omitted] DisclosuresLipton:BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Preclinical Evaluation of MEDI2228, a BCMA-Targeting Pyrrolobenzodiazepine-Linked Antibody Drug Conjugate for the Treatment of Multiple Myeloma

Despite remarkable progress in treatment over the past two decades, multiple myeloma (MM) remains an incurable disease. B-cell maturation antigen (BCMA, TNFRSF17) is a suitable therapeutic target for the treatment of MM due to its restricted expression on normal plasma cells and universal expression in myeloma cells. Targeting BCMA with an antibody-drug conjugate (ADC) is an attractive therapeutic option, combining the specificity of a monoclonal antibody with a potent cytotoxic drug to preferentially eliminate antigen-positive cells for the treatment of cancer.Here we describe MEDI2228, a novel ADC that targets BCMA. MEDI2228 is composed of a fully human antibody site-specifically conjugated to a DNA cross-linking pyrrolobenzodiazepine (PBD) dimer via a protease-cleavable linker. Flow cytometry studies show that MEDI2228 is rapidly internalized and trafficked to lysosomes. Upon release, the warhead binds to the minor-groove and cross-links DNA, leading to DNA damage and apoptotic cell death.The anti-tumor activity of MEDI2228 was assessed in vitro in 10 MM and plasma cell leukemia (PCL) cell lines encompassing several cytogenetic subtypes. MEDI2228 was highly active in 8 of 10 MM cell lines tested (IC50 range 6 to 210 ng/mL) including cell lines with high (~19,000 receptors/cell) or low (~930 receptors/cell) expression of BCMA. In addition, MEDI2228 is active in the presence of bone marrow stromal cells, which have been shown to play a role in chemotherapy resistance, and in cell models resistant to Lenalidomide. In vivo antitumor activity was evaluated in 4 separate MM and PCL subcutaneous xenograft models and 2 PCL disseminated xenograft models, where a single injection of MEDI2228 induced tumor regression at doses as low as 0.1 mg/kg.A soluble form of BCMA (sBCMA) has been detected in the serum of MM patients (Sanchez et. al. Br J Haematol 2012) and is comprised of the entire extracellular domain of the molecule (Laurent et. al. Nat Commun 2015). Therefore, sBCMA could diminish the effects of antibody-based therapies. We found that levels of sBCMA are elevated in the sera of MM patients compared with healthy donor controls, with levels ranging from 23 ng/mL to 728 ng/mL. The functional features of sBCMA and recombinant monomeric human BCMA are similar (Laurent et. al. Nat Commun 2015). Therefore, to mitigate the potential effects of sBCMA on the efficacy of our drug, the antibody component of MEDI2228 was selected because it possessed weak binding to recombinant monomeric human BCMA and strong binding to membrane-bound BCMA. The in vitro cytotoxicity of MEDI2228 in the presence of clinically-relevant levels of sBCMA was evaluated using NCI-H929 and MM.1S cells. MEDI2228 was cytotoxic to both MM.1S and NCI-H929 in vitro, killing an average of 95% of tumor cells in the presence of sBCMA at levels up to 720 ng/mL with little impact on IC50. ADCs developed from antibodies that possessed a similar affinity between monomeric BCMA and membrane-bound BCMA exhibited a sBCMA-dose dependent drop in potency, with a 20-fold shift in IC50 in the presence of 720 ng/mL sBCMA.MM is a clonally heterogeneous disease, and previously it has been reported that a small population of CD19+CD138- clonogenic cells can be identified in the bone marrow (BM) of MM patients (Matsui et. al. Blood 2004). We used flow cytometric sorting of CD19 and CD138 populations and outgrowth in methocult to confirm that CD19+CD138- cells are the clonogenic cells in MM BM samples. Furthermore, we found that this population contains the same light chain restriction, genetic translocation, and aberrant phenotype as the MM plasma cells. These cells express BCMA and can be killed by MEDI2228 in methocult outgrowth assays.These data demonstrate that MEDI2228 exhibits potent antitumor activity in preclinical models of MM. Importantly, in vitro experiments suggest that this activity is maintained in the presence of sBCMA. MEDI2228, bearing a potent PBD payload, may effectively target both the bulk myeloma plasma cells as well as the more quiescent, CD19+CD138- clonogenic cells, which may offer an opportunity for more durable clinical response in this genetically heterogeneous disease. DisclosuresKinneer:MedImmune: Employment. Meekin:MedImmune: Employment. Varkey:MedImmune: Employment. Xiao:MedImmune: Employment; Bristol-Myers Squibb: Employment. Zhong:MedImmune: Employment. Breen:MedImmune: Employment. Hurt:MedImmune: Employment. Thomas:MedImmune: Employment. Flynn:MedImmune: Employment. Hynes:MedImmune: Employment. Bezabeh:Salubris Biotherapeutics Inc: Employment; MedImmune: Employment. Chen:MedImmune: Employment. Wetzel:MedImmune: Employment. Chen:MedImmune: Employment. Anderson:Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: scientific founder; C4 Therapeutics: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Herbst:MedImmune: Employment. Tice:MedImmune: Employment.