Chronic lymphocytic leukemia (CLL) is characterized by the clonal expansion of small mature-looking CD19+ CD23+ CD5+ B-cells that accumulate in the blood, bone marrow, and lymphoid organs. To date, no consensus has been reached concerning the normal cellular counterpart of CLL B-cells and several B-cell types have been proposed. CLL B-cells have remarkable phenotypic and gene expression profile homogeneity. In recent years, the molecular and cellular biology of CLL has been enriched by seminal insights that are leading to a better understanding of the natural history of the disease. Immunophenotypic and molecular approaches (including immunoglobulin heavy-chain variable gene mutational status, transcriptional and epigenetic profiling) comparing the normal B-cell subset and CLL B-cells provide some new insights into the normal cellular counterpart. Functional characteristics (including activation requirements and propensity for plasma cell differentiation) of CLL B-cells have now been investigated for 50 years. B-cell subsets differ substantially in terms of their functional features. Analysis of shared functional characteristics may reveal similarities between normal B-cell subsets and CLL B-cells, allowing speculative assignment of a normal cellular counterpart for CLL B-cells. In this review, we summarize current data regarding peripheral B-cell differentiation and human B-cell subsets and suggest possibilities for a normal cellular counterpart based on the functional characteristics of CLL B-cells. However, a definitive normal cellular counterpart cannot be attributed on the basis of the available data. We discuss the functional characteristics required for a cell to be logically considered to be the normal counterpart of CLL B-cells.

The widely used organophosphorus pesticide chlorpyrifos (CPF) is often detected in food. CPF inhibits acetylcholinesterase and can modify muscle contractility and respiratory patterns. We studied the effects of chronic exposure to CPF on respiratory parameters and diaphragm contractility in 21- and 60-days old rats. Pregnant rats were exposed to oral CPF (1 or 5 mg/ kg /day: CPF-1 or CPF-5 groups vs vehicle: controls) from gestation onset up to weaning of the pups that were individually gavaged (CPF or vehicle) thereafter. Two developmental time points were studied: weaning (day 21) and adulthood (day 60). Whole-body plethysmography was used to score breathing patterns and apnea index during sleep. Then, diaphragm strips were dissected for the assessment of contractility and acetylcholinesterase activity. Results showed that the sleep apnea index was higher in CPF-exposed rats than in controls. In adult rats, the expiratory time and tidal volume were higher in CPF-exposed animals than in controls. At both ages, the diaphragm’s amplitude of contraction and fatigability index were higher in the CPF-5 group, due to lower acetylcholinesterase activity. We conclude that chronic exposure to CPF is associated with higher sleep apnea index and diaphragm contractility, and modifies respiratory patterns in sleeping juvenile and adult rats.

Neuroinflammation and iron accumulation are hallmarks of a variety of adult neurodegenerative diseases. In Sanfilippo syndrome (mucopolysaccharidosis type III, MPSIII, a pediatric neurodegenerative disease that shares some features with adult neurodegenerative diseases), the progressive accumulation of heparan sulfate oligosaccharides (HSOs) induces microglia and astrocytes to produce pro-inflammatory cytokines leading to severe neuroinflammation. The objectives of the present study were (1) to measure the local iron concentration and to assess iron metabolism in the brain of a MPSIIIB murine model and (2) to identify the brain cells involved in this accumulation. We found that iron accumulation in MPSIIIB mice primarily affected the cerebral cortex where hepcidin levels were higher than in wild-type mice, and increased with aging. This increase was correlated with low expression of ferroportin 1 (FPN1), and thus brain iron retention. Moreover, we showed in vitro that HSOs are directly responsible for the production of hepcidin and the relative decrease in FPN1 expression when added to cultures of microglia and, to a lesser extent, to cultures of astrocytes. In contrast, no significant differences were observed in neurons. Hepcidin induction results from activation of the TLR4 pathway and STAT3 signaling, and leads to iron retention within microglia. Our results show that microglia have a key role in cerebral hepcidin overexpression and thus in the brain iron accumulation observed in the MPSIIIB model.

Objective— Cardiovascular diseases constitute the leading cause of mortality worldwide. Calcification of the vessel wall is associated with cardiovascular morbidity and mortality in patients having many diseases, including diabetes mellitus, atherosclerosis, and chronic kidney disease. Vascular calcification is actively regulated by inductive and inhibitory mechanisms (including vascular smooth muscle cell adaptation) and results from an active osteogenic process. During the calcification process, extracellular vesicles (also known as matrix vesicles) released by vascular smooth muscle cells interact with type I collagen and then act as nucleating foci for calcium crystallization. Our primary objective was to identify new, natural molecules that inhibit the vascular calcification process. Approach and Results— We have found that oligogalacturonic acids (obtained by the acid hydrolysis of polygalacturonic acid) reduce in vitro inorganic phosphate–induced calcification of vascular smooth muscle cells by 80% and inorganic phosphate–induced calcification of isolated rat aortic rings by 50%. A specific oligogalacturonic acid with a degree of polymerization of 8 (DP8) was found to inhibit the expression of osteogenic markers and, thus, prevent the conversion of vascular smooth muscle cells into osteoblast-like cells. We also evidenced in biochemical and immunofluorescence assays a direct interaction between matrix vesicles and type I collagen via the GFOGER sequence (where single letter amino acid nomenclature is used, O=hydroxyproline) thought to be involved in interactions with several pairs of integrins. Conclusions— DP8 inhibits vascular calcification development mainly by inhibition of osteogenic marker expression but also partly by masking the GFOGER sequence—thereby, preventing matrix vesicles from binding to type I collagen.

Recent research has shown that chronic lymphocytic leukemia (CLL) B-cells display a strong tendency to differentiate into antibody-secreting cells (ASCs) and thus may be amenable to differentiation therapy. However, the effect of this differentiation on factors associated with CLL pathogenesis has not been reported. In the present study, purified CLL B-cells were stimulated to differentiate into ASCs by phorbol myristate acetate or CpG oligodeoxynucleotide, in combination with CD40 ligand and cytokines in a two-step, seven-day culture system. We investigated (i) changes in the immunophenotypic, molecular, functional, morphological features associated with terminal differentiation into ASCs, (ii) the expression of factors involved in CLL pathogenesis, and (iii) the expression of pro- and anti-apoptotic proteins in the differentiated cells. Our results show that differentiated CLL B-cells are able to display the transcriptional program of ASCs. Differentiation leads to depletion of the malignant program and deregulation of the apoptosis/survival balance. Analysis of apoptosis and the cell cycle showed that differentiation is associated with low cell viability and a low rate of cell cycle entry. Our findings shed new light on the potential for differentiation therapy as a part of treatment strategies for CLL.

Prenatal and postnatal exposure to CPF in the rat is associated with weaker contraction of ileal longitudinal smooth muscle via a nitrergic mechanism with increased iNOS expression.

Background Novel, less toxic, cost-effective and safe therapeutic strategies are needed to improve treatment of chronic lymphocytic leukemia (CLL). Ascorbic acid (AA, vitamin C) has shown a potential anti-cancer therapeutic activity in several cancers. However, the anti-cancer effects of ascorbic acid on CLL B-cells have not been extensively studied. We aimed in this study to evaluate the in vitro therapeutic activity using clinically relevant conditions. Methods Primary CLL B-cells and two CLL cell lines were exposed to a dose that is clinically achievable by AA oral administration (250 μM), and cell death and potential mechanisms were assessed. The role of the protective CLL microenvironment was studied. Synergistic interaction between AA and CLL approved drugs (Ibrutinib, Idelalisib and Venetoclax) was also evaluated. Results Ascorbic acid is cytotoxic for CLL B-cells at low dose (250 μM) but spares healthy B-cells. Ascorbic-acid-induced cytotoxicity involved pro-oxidant damage through the generation of reactive oxygen species in the extracellular media and in CLL cells, and induced caspase-dependent apoptosis. We also found that AA treatment overcame the supportive survival effect provided by microenvironment including bone marrow mesenchymal stem cells, T-cell cues (CD40L + IL-4), cytokines and hypoxia. Our data suggest that resistance to AA could be mediated by the expression of the enzyme catalase in some CLL samples and by the glucose metabolite pyruvate. We also demonstrated that AA synergistically potentiates the cytotoxicity of targeted therapies used in or being developed for CLL. Conclusion These preclinical results point to AA as an adjuvant therapy with potential to further improve CLL treatments in combination with targeted therapies.

Aberrant NF-κB activation is a hallmark of most B-cell malignancies. Recurrent inactivating somatic mutations in the NFKBIE gene, which encodes IκBε, an inhibitor of NF-κB-inducible activity, are reported in several B-cell malignancies with highest frequencies in chronic lymphocytic leukemia and primary mediastinal B-cell lymphoma, and account for a fraction of NF-κB pathway activation. The impact of NFKBIE deficiency on B-cell development and function remains, however, largely unknown. Here, we show that Nfkbie-deficient mice exhibit an amplification of marginal zone B cells and an expansion of B1 B-cell subsets. In germinal center (GC)-dependent immune response, Nfkbie deficiency triggers expansion of GC B-cells through increasing cell proliferation in a B-cell autonomous manner. We also show that Nfkbie deficiency results in hyperproliferation of a B1 B-cell subset and leads to increased NF-κB activation in these cells upon Toll-like receptor stimulation. Nfkbie deficiency cooperates with mutant MYD88 signaling and enhances B-cell proliferation in vitro. In aged mice, Nfkbie absence drives the development of an oligoclonal indolent B-cell lymphoproliferative disorders, resembling monoclonal B-cell lymphocytosis. Collectively, these findings shed light on an essential role of IκBε in finely tuning B-cell development and function.