Human B-cell Research Articles

Anti-Myeloma Effects of the Selective JAK1 Inhibitor INCB052793 Using a Platform Study Utilizing the Lagκ-1A Multiple Myeloma Xenograft Model

Introduction: We have previously shown that severe combined immune deficiency (SCID)mice bearing the human multiple myeloma (MM) LAGk-1A xenograft develop significantly smaller tumors when treated with the JAK1 inhibitor INCB052793 (INCB; Incyte, Wilmington, DE) alone. Additionally, mice receiving the doublets of this agent and dexamethasone (DEX), lenalidomide (LEN) or pomalidomide (POM) demonstrated enhanced anti-MM effects on LAGk-1A growth in vivo that was superior to the doublets of DEX with LEN or POM. Mice receiving the triple combination of INCB + DEX with LEN or POM demonstrated the most pronounced anti-MM effect on tumor growth in vivo compared to all other combinations tested. Herein, we evaluated the response and toxicity of animals treated in combination or sequentially with INCB as well as with LEN plus DEX. For example, is there enhanced anti-MM activity when INCB + LEN are administered first, and DEX is added after disease progression? Or is there enhanced anti-MM activity when INCB + LEN is administered first and then substituted with DEX? This same treatment strategy was evaluated with INCB + DEX administered first, followed by the various permutations of LEN. Additionally, given that in the clinical setting DEX plus LEN is commonly administered to patients with MM, we evaluated these combinations with the addition of INCB or substitution of one of these drugs with INCB. Similarly, we evaluated the anti-MM effects of mice progressing from DEX therapy and added in INCB, substituted DEX with INCB, continued DEX and added in INCB + LEN.Materials and Methods: For the in vivo studies, each SCID mouse was implanted into the hind limb with a piece of the human MM tumor LAGk-1A. All animal studies were approved by the Institutional Animal Care and Use Committee. Fourteen days post-implantation mice were randomized into various treatment groups, and tumor size was measured on a weekly basis following treatment with various drugs following progression from their initial treatment regimen. Progression is defined as an increase in human B-cell maturation antigen (BCMA) equal to or above 25% when compared to the prior assessment (plasma BCMA levels were assessed on a weekly basis). After the mouse meets the criteria for disease progression, the mouse was placed into their respective treatment group.Results: When each individual mouse progressed, their dosing regimen was changed according to the treatment group to which they were randomized. We observed pronounced anti-MM effects when DEX was administered, followed by additions, or substitutions, of INCB and/or LEN. No significant anti-MM effects were observed when comparing the vehicle control group to mice treated in the LEN, INCB or DEX + LEN cohorts. However, mice treated with the doublets INCB + DEX or INCB + LEN, or triplet of INCB + LEN + DEX showed smaller tumors compared to the vehicle control group, (P= 0.0420; P= 0.0388; P= 0.0079, respectively). Furthermore, mice treated with the triplet, INCB + LEN + DEX, had markedly smaller tumors when compared to those treated with LEN (P= 0.0005), DEX + LEN(P= 0.0244), or INCB + DEX(P= 0.0357).Conclusions: These in vivo studies should provide important data in support of the optimal clinical sequencing of INCB, as they clearly show that the sequencing of drug combinations involving the novel JAK1 inhibitor INCB052793 + anti-MM agents have a profound impact on the anti-MM effects of this new treatment regimen, and provide further support for the clinical evaluation of these drug combinations to treat relapsed/refractory MM patients. DisclosuresBerenson:Incyte: Consultancy, Research Funding; OncoTracker, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

A Novel Antibody-Cell Conjugation Method to Enhance Cellular Therapies for the Treatment of Hematological Malignancies

IntroductionCellular therapies including γδ T cells, NK-92 cells, and cytokine-induced killer (CIK) cells have shown modest anti-tumor activity against multiple hematological and solid malignancies in preclinical and phase I/II clinical trials. We investigate whether the antitumor activity of these cellular therapies can be augmented by using a novel method that rapidly affixes tumor-targeting antibodies directly to cell surface proteins.MethodsMonoclonal antibodies including rituximab and daratumumab were conjugated to the cell surface proteins using a three step approach. First, we conjugate single-stranded DNAs (ssDNAs) to the therapeutic monoclonal antibodies, as previously described (ref below). Next, we conjugate complementary ssDNAs to surface proteins of cells being used for cellular therapy. Finally, the modified antibodies are attached to the modified cells via hybridization of the complementary DNA strands.ResultsWe demonstrate that multiple anti-neoplastic monoclonal antibodies including rituximab and daratumumab can be conjugated to the surface proteins on a variety of cell including γδ T cells, NK-92 cells, CIK cells, and primary human T cells. Then we evaluated the killing activity of these conjugated cells in vitro, in vivo, and ex vivo . In vitro, Rituximab-armed γδ T cells and NK-92 cells demonstrated increased cytokine production (IFN-γ and granzyme B) and enhanced cytotoxic killing against of multiple human B-cell non-Hodgkin lymphoma (NHL) and multiple myeloma cell lines. Similar enhanced anti-tumor activity was seen with rituximab-armed or daratumumab-armed CIK cells, as well as increased CD107a expression (a marker of cytotoxicity activity) by the antibody-armed CIK cells. Rituximab-armed primary T cells also show enhanced cytotoxic killing against Raji cells, although not as pronounced as autologous rituximab-armed CIK cells. We next evaluated the therapeutic potential of antibody-armed cell therapies in vivo in a disseminated xenograft mouse model using SCID-beige mice. Rituximab-armed γδ T cells exhibited potent anti-lymphoma activity against Raji cells, which lead to improved overall survival; whereas unarmed γδ T cells alone or with rituximab co-infusion demonstrated limited anti-lymphoma activity. We also evaluated the therapeutic activity of antibody-armed cell therapies against a primary lymphoma target ex vivo. Rituximab-arm CIK cells demonstrated enhanced cytolytic activity against primary follicular lymphoma freshly collected from a patient via fine needle aspiration biopsy.DiscussionWe have demonstrated a novel approach to create tumor-specific cellular immunotherapies without the need for genetic engineering. The antibody to cell conjugation method is straight forward. The modified antibodies can be made and stored until needed. It takes approximately 1 hour to attaching these modified antibodies to the cell surface and is performed in standard aqueous buffers, such as phosphate-buffered saline (PBS). This method is also flexible being able to conjugate multiple different antibodies to the surface proteins of a variety of different cell types. We also see enhanced cell killing across multiple hematological targets using multiple cell populations including γδ T cells, NK-92 cells, CIK cells, and primary human T cells. Overall, our data demonstrate existing monoclonal antibodies can be used to “antibody program” cell therapies for the treatment of hematological malignancies.RefHsiao et al Langmuir 2009,25(12), 6985-6991 DisclosuresHsiao:Acepodia, Inc: Employment.

Combined Targeting of the Proteasome and Bcl-2 Sensitizes Human B-Cell Lymphoma and Multiple Myeloma to Cyclophosphamide in In Vitro and In Vivo Models of These Diseases.

The balance of apoptotic proteins is a critical mechanism used by the cell to maintain the mitochondrial transmembrane potential, which plays a critical role in governing apoptosis. Bortezomib (B) is a proteasome inhibitor that selectively inhibits the 26S proteasome, leading to an increase in many intracellular proteins, including proteins that lead to inhibition of NF-kB, as well as proapoptotic proteins like bik and Bax. Oblimersen (Obl) is a 18 mer oligonucleotide results in decreased transcription of the antiapoptotic protein bcl-2. We hypothesized that the mitochondrial membrane potential could be influenced by the co-administration of these agents to favor aopotosis. We report results of pre-clinical in vitro and in vivo experiments using these agents in combination in various lymphoma (NHL) & myeloma (MM) models. NHL & MM cell lines were exposed to Obl in the presence of cationic lipid and incubated for 48 hours. DNA synthesis & cell growth arrest was quantified by 3H-thymidine incorporation assays at 72 hrs, & revealed a concentration dependent cell killing. Induction of apoptosis in the presence or absence of different concentrations of B was detected by multiparameter flow cytometric analysis. Data reveals addition of bortezomib, even at relatively low concentrations (10 nM), additively improved the effects of Obl in all the cell lines studied. A SCID beige model of aggressive large B-cell lymphoma was used by injecting 1x10e7 cells (SKI-DLCL-1 or RL, a cell line over expressing bcl-2 through the 14:18 translocation) as a subcutaneous flank tumor. In over 6 in vivo experiments, the combination of B (0.5 mg/kg days 3 & 7) + Obl (3 mg/kg days 1,3,5, &7) consistently resulted in about 50% inhibition of growth of both SKI-DLCL-1 & RL compared to that in the control. Surprisingly, results were evident even using a low dose schedule of bortezomib (about one quarter of the MTD), suggesting the possibility of a small sensitizing dose of this drug. In all experiments, growth delay seen with the doublet exceeded the results noted with any singlet (B or Obl alone). Integration of cyclophosphamide at a dose of 50 mg/kg produced marked tumor shrinkage that appeared synergistic in several circumstances. Single agent CTX produced excellent growth delay (similar to any doublet), but in all these cases, tumor re-growth was evident by 21–25 days post tumor transplant. CTX dose & schedule was also important. Same day ‘lump and dump' of all drugs failed to effect cured, while CTX preceding B consistently produced best results, with cures found only in this triplet cohort. Results suggest that optimal scheduling of agents requires a ‘lead in' exposure of Obl, followed by CTX preceding all doses of bortezomib. These studies support the efficacy of this regimen.

Targeting TfR1 with the ch128.1/IgG1 Antibody Inhibits EBV-driven Lymphomagenesis in Immunosuppressed Mice Bearing EBV+ Human Primary B-cells.

Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with the development of hematopoietic cancers of B-lymphocyte origin, including AIDS-related non-Hodgkin lymphoma (AIDS-NHL). Primary infection of B-cells with EBV results in their polyclonal activation and immortalization. The transferrin receptor 1 (TfR1), also known as CD71, is important for iron uptake and regulation of cellular proliferation. TfR1 is highly expressed in proliferating cells, including activated lymphocytes and malignant cells. We developed a mouse/human chimeric antibody targeting TfR1 (ch128.1/IgG1) that has previously shown significant antitumor activity in immunosuppressed mouse models bearing human malignant B-cells, including multiple myeloma and AIDS-NHL cells. In this article, we examined the effect of targeting TfR1 to inhibit EBV-driven activation and growth of human B-cells in vivo using an immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl /SzJ [NOD/SCID gamma (NSG)] mouse model. Mice were implanted with T-cell-depleted, human peripheral blood mononuclear cells (PBMCs), either without EBV (EBV-), or exposed to EBV in vitro (EBV+), intravenously via the tail vein. Mice implanted with EBV+ cells and treated with an IgG1 control antibody (400 μg/mouse) developed lymphoma-like growths of human B-cell origin that were EBV+, whereas mice implanted with EBV+ cells and treated with ch128.1/IgG1 (400 μg/mouse) showed increased survival and significantly reduced inflammation and B-cell activation. These results indicate that ch128.1/IgG1 is effective at preventing the growth of EBV+ human B-cell tumors in vivo, thus, indicating that there is significant potential for agents targeting TfR1 as therapeutic strategies to prevent the development of EBV-associated B-cell malignancies. SIGNIFICANCE: An anti-TfR1 antibody, ch128.1/IgG1, effectively inhibits the activation, growth, and immortalization of EBV+ human B-cells in vivo, as well as the development of these cells into lymphoma-like tumors in immunodeficient mice.

Immunoinformatics and molecular dynamics approaches: Next generation vaccine design against West Nile virus.

West Nile Virus (WNV) is a life threatening flavivirus that causes significant morbidity and mortality worldwide. No preventive therapeutics including vaccines against WNV are available for human use. In this study, immunoinformatics approach was performed to design a multi epitope-based subunit vaccine against this deadly pathogen. Human (HLA) and Mice (H-2) allele specific potential T-cell and B-cell epitopes were shortlisted through a stringent procedure. Molecular docking showed selected epitopes that have stronger binding affinity with human TLR-4. Molecular dynamics simulation confirmed the stable nature of the docked complex. Furthermore, in silico cloning analysis ensures efficient expression of desired gene in the microbial system. Interestingly, previous studies showed that two of our selected epitopes have strong immune response against WNV. Therefore, selected epitopes could be strong vaccine candidates to prevent WNV infections in human. However, further in vitro and in vivo investigations could be strengthening the validation of the vaccine candidate against WNV.

Functional Characterization of Endogenously Expressed Human RYR1 Variants.

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia susceptibility, core myopathies and centronuclear myopathy. Because of the diverse phenotypes linked to RYR1 mutations it is fundamental to characterize their functional effects to classify variants carried by patients for future therapeutic interventions and identify non-pathogenic variants. Many laboratories have been interested in developing methods to functionally characterize RYR1 mutations expressed in patients' cells. This approach has numerous advantages, including: mutations are endogenously expressed, RyR1 is not over-expressed, use of heterologous RyR1 expressing cells is avoided. However, since patients may present mutations in different genes aside RYR1, it is important to compare results from biological material from individuals harboring the same mutation, with different genetic backgrounds. The present manuscript describes methods developed to study the functional effects of endogenously expressed RYR1 variants in: (a) Epstein Barr virus immortalized human B-lymphocytes and (b) satellite cells derived from muscle biopsies and differentiated into myotubes. Changes in the intracellular calcium concentration triggered by the addition of a pharmacological RyR1 activators are then monitored. The selected cell type is loaded with a ratiometric fluorescent calcium indicator and intracellular [Ca2+] changes are monitored either at the single cell level by fluorescence microscopy or in cell populations using a spectrofluorometer. The resting [Ca2+], agonist dose response curves are then compared between cells from healthy controls and patients harboring RYR1 variants leading to insight into the functional effect of a given variant.

Intrinsic 5-lipoxygenase activity regulates migration and adherence of mantle cell lymphoma cells

Human B-lymphocytes express 5-lipoxygenase (5-LOX) and 5-LOX activating protein (FLAP) and can convert arachidonic acid to leukotriene B4. Mantle cell lymphoma (MCL) cells contain similar amounts of 5-LOX as human neutrophils but the function and mechanism of activation of 5-LOX in MCL cells, and in normal B-lymphocytes, are unclear. Here we show that the intrinsic 5-LOX pathway in the MCL cell line JeKo-1 has an essential role in migration and adherence of the cells, which are important pathophysiological characteristics of B-cell lymphoma. Incubation of JeKo-1 with the FLAP inhibitor GSK2190915 or the 5-LOX inhibitor zileuton, at a concentration below 1 μM, prior to stimulation with the chemotactic agent CXCL12, led to a significant reduction of migration. CRISPR/Cas9 mediated deletion of ALOX5 gene in JeKo-1 cells also led to a significantly decreased migration of the cells. Furthermore, 5-LOX and FLAP inhibitors markedly decreased the adherence of JeKo-1 cells to stromal cells. In comparison, these drugs had a similar effect on adherence of JeKo-1 cells as the Bruton tyrosine kinase inhibitor ibrutinib, which has a proven anti-tumour effect. These results indicate that inhibition of 5-LOX may be a novel treatment for MCL and certain other B-cell lymphomas.

Polymorphism of P66 in Borrelia Burgdorferi Strains in China

ObjectiveTo study the polymorphism in P66 and its human B-cell epitopes of Borrelia burgdorferi strains in China. MethodsPolymerase chain reaction (PCR) and sequencing were used to obtain the P66 sequences of 59 Chinese B. burgdorferi. Then the sequences were analyzed by MEGA 5.10 software and compared with the human B-cell epitope sequences from the Immune Epitope Database (IEDB) based on the reference strain of each genotype. ResultsResults showed that genetic and amino acid diversity presented in the 66 kD protein of all 59 Chinese strains, especially in Borrelia garinii (B.g) and Borrelia afzelii (B.a) strains. B.g strains were divided into three subclusters and two scattered strains JC1-7 and JC2-2 according to the amino acid sequences of P66. The P66 sequences of 15 Xinjiang strains represented by XI91-12 in the B.g subcluster 1, changed from CAA to TAA codon at 508aa position, resulting in early termination. Bases A and C were inserted at sequence position 1 523 bp of strains FP1, LB20, LB21, and SZ21 in the B.a genotype, which resulted to early termination at position 511 aa. G base was inserted at 438 bp of LIP94-11 strain, which led to early termination at position 172 aa. ConclusionIn P66 of 59 Chinese strains, polymorphisms were widely distributed. More importantly, the P66 amino acid sequences of B.g strains had a certain regional character. One of the characteristics of Xinjiang B.g isolates might be the variation at the 508aa location in 15 Xinjiang B.g strains, which may be related to the strains’ pathogenicity in this area.

Single-cell BCR and transcriptome analysis after influenza infection reveals spatiotemporal dynamics of antigen-specific B cells.

SummaryB cell responses are critical for antiviral immunity. However, a comprehensive picture of antigen-specific B cell differentiation, clonal proliferation, and dynamics in different organs after infection is lacking. Here, by combining single-cell RNA and B cell receptor (BCR) sequencing of antigen-specific cells in lymph nodes, spleen, and lungs after influenza infection in mice, we identify several germinal center (GC) B cell subpopulations and organ-specific differences that persist over the course of the response. We discover transcriptional differences between memory cells in lungs and lymphoid organs and organ-restricted clonal expansion. Remarkably, we find significant clonal overlap between GC-derived memory and plasma cells. By combining BCR-mutational analyses with monoclonal antibody (mAb) expression and affinity measurements, we find that memory B cells are highly diverse and can be selected from both low- and high-affinity precursors. By linking antigen recognition with transcriptional programming, clonal proliferation, and differentiation, these finding provide important advances in our understanding of antiviral immunity.

Biology of Germinal Center B Cells Relating to Lymphomagenesis.

The germinal center (GC) reaction is a key feature of adaptive humoral immunity. GCs represent the site where mature B cells refine their B-cell receptor (BCR) and are selected based on the newly acquired affinity for the antigen. In the GC, B cells undergo multiple cycles of proliferation, BCR remodeling by immunoglobulin somatic hypermutation (SHM), and affinity-based selection before emerging as effector memory B cells or antibody-secreting plasma cells. At least 2 histologically and functionally distinct compartments are identified in the GC: the dark zone (DZ) and the light zone (LZ). The proliferative burst and immunoglobulin remodeling by SHM occur prevalently in the DZ compartment. In the LZ, GC B cells undergo an affinity-based selection process that requires the interaction with the antigen and accessory cells. GC B cells are also targeted by class switch recombination, an additional mechanism of immunoglobulin remodeling that ensures the expression of diverse isotype classes. These processes are regulated by a complex network of transcription factors, epigenetic modifiers, and signaling pathways that act in concert with mechanisms of intra-GC B-cell trafficking. The same mechanisms underlying the unique ability of GC B cells to generate high affinity antibodies and ensure immunological memory are hijacked during lymphomagenesis and become powerful weapons for malignant transformation. This review will summarize the main processes and transcriptional networks that drive GC B-cell development and are relevant for human B-cell lymphomagenesis.

Intoxication of Cypermethrin on Binding Site of Human Oxyhaemoglobin

Pesticide exposure can occur by several routes in the same individual due to multiple uses, and there is likely to be additive toxicity in human. Cypermethrin a synthetic pyrethroid can produce a variety of acute health conditions and also carries several ecological risks. Hemoglobin is the protein that transports oxygen in the blood. Any change in heamoglobin may direct or indirect effect the human body. In this work, we report computational prediction of binding of cypermethrin pesticides on human oxyhaemoglobin [using Molegro Virtual Docker (MVD)] and evaluating the computational prediction on binding of human oxyhaemoglobin with cypermethrin pesticide and evaluate the Mole Dock Score, RMSD, affinity, interacting residues of receptor human oxyhaemoglobin, number of H-bond interaction, docking score and intracting interaction of residues. The energy bound conformation with lower value of selected ligand shows hydrogen bonding and electrostatic interactions. The binding affinity of cypermethrin pesticides is found to be -35.7414 KJ/mol, and bind with two hydrogen bonds with tyrosine 35 and serine 133. Frequent alterations in the expression of amino acid due to pesticide interaction with oxyhemoglobin may lead to produce carcinogenic cells in human beings.

Minimalistic In Vitro Culture to Drive Human Naive B Cell Differentiation into Antibody-Secreting Cells.

High-affinity antibody-secreting cells (ASC) arise from terminal differentiation of B-cells after coordinated interactions with T follicular helper (Tfh) cells in germinal centers (GC). Elucidation of cues promoting human naive B-cells to progress into ASCs is challenging, as this process is notoriously difficult to induce in vitro while maintaining enough cell numbers to investigate the differentiation route(s). Here, we describe a minimalistic in vitro culture system that supports efficient differentiation of human naive B-cells into antibody-secreting cells. Upon initial stimulations, the interplay between level of CD40 costimulation and the Tfh cell-associated cytokines IL-21 and IL-4 determined the magnitude of B-cell expansion, immunoglobulin class-switching and expression of ASC regulator PRDM1. In contrast, the B-cell-specific transcriptional program was maintained, and efficient ASC formation was hampered. Renewed CD40 costimulation and Tfh cytokines exposure induced rapid secondary STAT3 signaling and extensive ASC differentiation, accompanied by repression of B-cell identity factors PAX5, BACH2 and IRF8 and further induction of PRDM1. Our work shows that, like in vivo, renewed CD40L costimulation also induces efficient terminal ASC differentiation after initial B-cell expansion in vitro. This culture system for efficient differentiation of human naive B-cells into ASCs, while also maintaining high cell numbers, may form an important tool in dissecting human naive B-cell differentiation, thereby enabling identification of novel transcriptional regulators and biomarkers for desired and detrimental antibody formation in humans.

Novel violet fluorescent nanoparticles for immunophenotyping using flow cytometry

Abstract A novel series of fluorescent nanoparticles with violet excitation have been developed for use with flow cytometry antibodies. These exceptionally bright, photostable dyes also have narrow emission spectra, making them ideal for inclusion in high parameter multicolor panels. For this study, StarBright Violets 440, 515, 610, 670, 710 and 790 were conjugated to human B-cell, T-cell, and Treg marker monoclonal antibodies and used to immunophenotype human peripheral blood mononuclear cells (PBMCs) in an 8-color panel. Tests with this panel were conducted in different staining buffers and compared to a panel with dyes having similar spectra and the same clonality. The new StarBright multicolor panel showed better resolution of Treg populations and reliable identification of the different T- and B-cell populations across different stain buffers and conditions, which makes the StarBright Violet nanoparticles an excellent addition to the field of high parameter flow cytometry.

Universal Detection of Mia Antigen and Frequencies of Glycophorin Hybrids among Blood Donors in Taiwan by Human Monoclonal Antibodies against Mia (MNS7), Mur (MNS10), and MUT (MNS35) Antigens.

Glycophorin hybrids such as GP.Mur are common in Southeast Asians. In Taiwan, clinically significant alloantibodies to the GP.Mur phenotype are the most important issue in blood banks. A large-scale screening of glycophorin hybrids in the Taiwanese population is urgently needed to ensure transfusion safety. Four clones of human hybridomas that secrete anti-Mia, anti-MUT, and anti-Mur were established by fusing human B-lymphocytes and myeloma cells (JMS-3). The specificity of each monoclonal antibody (MoAb) was characterized. Three MoAbs were applied on an Automated Pretransfusion Blood Testing Analyzer (PK7300/PK7400) for donor screening. Genotyping was performed to determine the detailed subgrouping of glycophorin hybrids. Four MoAbs are IgM antibodies. Anti-Mia (377T) binds to 46DXHKRDTYA54, 48HKRDTYAAHT57 peptides, and anti-Mia (367T) binds to 43QTNDXHKRD51 peptides (X indicates T, M, or K). Anti-Mur is reactive with 49KRDTYPAHTA58 peptides. Anti-MUT is reactive with 47KHKRDTYA54. A total of 78,327 donors were screened using three MoAbs, and 3690 (4.71%) were GP.Mur, 20 (0.025%) were GP.Hut, and 18 (0.022%) were GP.Vw. When the Mia antigen was introduced as routine screening, the frequency of Mi(a+) among blood donors in Taiwan was 4.66% (67,348/1,444,541). Mia antigen was implemented as a routine blood testing, and the results were labeled on all red blood cell (RBC) units.

Mechanistic understanding of the combined immunodeficiency in complete human CARD11 deficiency

Germline pathogenic variants impairing the caspase recruitment domain family member 11 (CARD11)-B cell chronic lymphocytic leukemia/lymphoma 10 (BCL10)-MALT1 paracaspase (MALT1) (CBM) complex are associated with diverse human diseases including combined immunodeficiency (CID), atopy, and lymphoproliferation. However, the impact of CARD11 deficiency on human B-cell development, signaling, and function is incompletely understood. This study sought to determine the cellular, immunological, and biochemical basis of disease for 2 unrelated patients who presented with profound CID associated with viral and fungal respiratory infections, interstitial lung disease, and severe colitis. Patients underwent next-generation sequencing, immunophenotyping by flow cytometry, signaling assays by immunoblot, and transcriptome profiling by RNA-sequencing. Both patients carried identical novel pathogenic biallelic loss-of-function variants in CARD11 (c.2509C>T; p.Arg837∗) leading to undetectable protein expression. This variant prevented CBM complex formation, severely impairing the activation of nuclear factor-κB, c-Jun N-terminal kinase, and MALT1 paracaspase activity in B and T cells. This functional defect resulted in a developmental block in B cells at the naive and type 1 transitional B-cell stage and impaired circulating T follicular helper cell (cTFH) development, which was associated with impaired antibody responses and absent germinal center structures on lymph node histology. Transcriptomics indicated that CARD11-dependent signaling is essential for immune signaling pathways involved in the development of these cells. Both patients underwent hematopoietic stem cell transplantations, which led to functional normalization. Complete human CARD11 deficiency causes profound CID by impairing naive/type 1 B-cell and cTFH cell development and abolishing activation of MALT1 paracaspase, NF-κB, and JNK activity. Hematopoietic stem cell transplantation functionally restores impaired signaling pathways.

Tumor-targeted nanoparticles improve the therapeutic index of BCL2 and MCL1 dual inhibition

Cancer and normal cells use multiple antiapoptotic BCL2 proteins to prevent cell death. Therapeutic targeting of multiple BCL2 family proteins enhances tumor killing but is also associated with increased systemic toxicity. Here, we demonstrate that the dual targeting of MCL1 and BCL2 proteins using the small molecules S63845 and venetoclax induces durable remissions in mice that harbor human diffuse large B-cell lymphoma (DLBCL) tumors but is accompanied by hematologic toxicity and weight loss. To mitigate these toxicities, we encapsulated S63845 or venetoclax into nanoparticles that target P-selectin, which is enriched in tumor endothelial cells. In vivo and ex vivo imaging demonstrated preferential targeting of the nanoparticles to lymphoma tumors over vital organs. Mass spectrometry analyses after administration of nanoparticle drugs confirmed tumor enrichment of the drug while reducing plasma levels. Furthermore, nanoparticle encapsulation allowed 3.5- to 6.5-fold reduction in drug dose, induced sustained remissions, and minimized toxicity. Our results support the development of nanoparticles to deliver BH3 mimetic combinations in lymphoma and in general for toxic drugs in cancer therapy.

Dynamic Intracellular Metabolic Cell Signaling Profiles During Ag-Dependent B-Cell Differentiation.

Human B-cell differentiation has been extensively investigated on genomic and transcriptomic grounds; however, no studies have accomplished so far detailed analysis of antigen-dependent maturation-associated human B-cell populations from a proteomic perspective. Here, we investigate for the first time the quantitative proteomic profiles of B-cells undergoing antigen-dependent maturation using a label-free LC-MS/MS approach applied on 5 purified B-cell subpopulations (naive, centroblasts, centrocytes, memory and plasma B-cells) from human tonsils (data are available via ProteomeXchange with identifier PXD006191). Our results revealed that the actual differences among these B-cell subpopulations are a combination of expression of a few maturation stage-specific proteins within each B-cell subset and maturation-associated changes in relative protein expression levels, which are related with metabolic regulation. The considerable overlap of the proteome of the 5 studied B-cell subsets strengthens the key role of the regulation of the stoichiometry of molecules associated with metabolic regulation and programming, among other signaling cascades (such as antigen recognition and presentation and cell survival) crucial for the transition between each B-cell maturation stage.

Evidence for additive and synergistic action of mammalian enhancers during cell fate determination.

Enhancer activity drives cell differentiation and cell fate determination, but it remains unclear how enhancers cooperate during these processes. Here we investigate enhancer cooperation during transdifferentiation of human leukemia B-cells to macrophages. Putative enhancers are established by binding of the pioneer factor C/EBPα followed by chromatin opening and enhancer RNA (eRNA) synthesis from H3K4-monomethylated regions. Using eRNA synthesis as a proxy for enhancer activity, we find that most putative enhancers cooperate in an additive way to regulate transcription of assigned target genes. However, transcription from 136 target genes depends exponentially on the summed activity of its putative paired enhancers, indicating that these enhancers cooperate synergistically. The target genes are cell type-specific, suggesting that enhancer synergy can contribute to cell fate determination. Enhancer synergy appears to depend on cell type-specific transcription factors, and such interacting enhancers are not predicted from occupancy or accessibility data that are used to detect superenhancers.

AP-64, Encoded by C5orf46, Exhibits Antimicrobial Activity against Gram-Negative Bacteria.

Antimicrobial peptides (AMPs), which are evolutionarily conserved components of the innate immune response, contribute to the first line of defense against microbes in the skin and at mucosal surfaces. Here, we report the identification of a human peptide, encoded by the chromosome 5 open reading frame 46 (C5orf46) gene, as a type of AMP, which we termed antimicrobial peptide with 64 amino acid residues (AP-64). AP-64 is an anionic amphiphilic peptide lacking cysteines (MW = 7.2, PI = 4.54). AP-64 exhibited significant antibacterial activity against Gram-negative bacteria, including Escherichia coli DH5α, Escherichia coli O157:H7, Vibrio cholerae, and Pseudomonas aeruginosa. Moreover, AP-64 was efficient in combating Escherichia coli O157:H7 infections in a mouse model and exhibited cytotoxic effects against human T-cell lymphoma Jurkat and B-cell lymphoma Raji cells. We also observed that Gm94, encoded by mouse C5orf46 homologous gene, closely resembles AP-64 in its antibacterial properties. Compared with other human AMPs, AP-64 has distinct characteristics, including a longer sequence length, absence of cysteine residues, a highly anionic character, and cell toxicity. Together, this study identified that AP-64 is an AMP worthy of further investigation.

MOSQUITO‐NET: A deep learning based CADx system for malaria diagnosis along with model interpretation using GradCam and class activation maps

AbstractMalaria is considered one of the deadliest diseases in today's world, which causes thousands of deaths per year. The parasites responsible for malaria are scientifically known as Plasmodium, which infects the red blood cells in human beings. Diagnosis of malaria requires identification and manual counting of parasitized cells in microscopic blood smears by medical practitioners. Its diagnostic accuracy is primarily affected by extensive scale screening due to the unavailability of resources. State of the art Computer‐Aided Diagnostic techniques based on deep learning algorithms such as CNNs, which perform an end to end feature extraction and classification, have widely contributed to various image recognition tasks. In this paper, we evaluate the performance of Mosquito‐Net, a custom made convnet to classify the infected and uninfected blood smears for malaria diagnosis. The CADx system can be deployed on IoT and mobile devices due to its fewer parameters and computation power, making it wildly preferable for diagnosis in remote and rural areas that lack medical facilities. Statistical analysis demonstrates that the proposed model achieves greater accuracy than the previous SOTA architectures for malaria diagnosis despite being 10 times lighter in parameters and inference time. Mosquito‐Net achieves an AUC of 99.009% and an F‐1 score of 96.7% on the validation set.