No abstract

SCI-25 The B-cell receptor (BCR) is a flexible and variable environmental sensor with no fixed ligand. The central component is surface Ig (sIg), which appears to function at several levels, mediating a “tonic” signal essential for survival and also binding to antigens via its variable (V) regions. Expression of sIg and reliance on BCR signals generally persist in neoplastic B cells. Indolent tumors, including chronic lymphocytic leukemia (CLL), allow insight into the pathogenetic role of the BCR prior to therapy as well as revealing key proteins within these pathways for drug targeting. CLL is heterogeneous, arising at two points of differentiation, generating two major subsets, one with unmutated Ig V genes (U-CLL), another with mutated Ig V genes (M-CLL). U-CLL appears to develop from naïve B cells of the natural antibody repertoire aimed against common pathogens. The clinical behavior of the two subsets differs, with U-CLL being of poorer prognosis. Evidence for antigen drive on both subsets comes from detecting “endocytosis in vivo,” whereby sIgM expression and signal capacity in blood cells are variably downmodulated, but can recover in vitro. Mysteriously, sIgD of the same presumed antigen specificity shows no evidence for endocytic downmodulation in vivo. CLL cells apparently engage antigens via sIgM in tissue sites, leading to proliferation and downmodulation, with reexpression gradually occurring during transit through the blood. Expression of CXCR4 closely follows that of sIgM, and clonal analysis reveals subpopulations of potentially dangerous cells with high sIgM/CXCR4 primed for tissue-based proliferative stimulation. In contrast to normal B cells, this is an iterative process exposing the proliferating CLL cells to further genetic changes. Overall higher sIgM levels and increased signal capacity in U-CLL likely account for more aggressive clinical behavior. BCR-induced membrane-proximal events include LYN-mediated phosphorylation of Iga/b followed by recruitment of the tyrosine kinase Syk. Signal propagation then involves Btk and PLCg2. LYN-dependent phosphorylation of CD19 also recruits the p85 subunit of PI3K, a known survival mechanism in CLL. Downstream events include upregulation of MYC proto-oncoprotein expression and induction of MYC-regulated target genes such as cyclin D2, with both proteins detected in proliferation centers. Pathways to increased cell survival include induction of the antiapoptotic MCL1 protein and inactivation of the proapoptotic activity of BIM(EL/L) via enhanced phosphorylation. The ability to phosphorylate BIM(EL) was highly correlated with mutational status and with requirement for treatment. While these events delineate BCR-activated pathways, they provide only the skeleton. sIgM signaling is highly dependent on the polymeric nature of the antigen, with responses to solid-phase stimulus producing a higher and more prolonged signal than the soluble form. Clearly, CLL cells have to integrate BCR signals with those from other receptors for the multitude of microenvironmental factors. This is a two-way process, since BCR signals operate “inside-out” by modulating the expression of molecules involved in migration and adhesion. The fact that the glycan composition of sIgM is also modulated to a mannosylated form, potentially able to bind to mannose-binding lectins, could contribute to the latter. Clinical effects of Syk, Btk and PI3Kd inhibitors, known to affect BCR signaling and potentially other pathways, are both explicable and exciting. Disclosures: No relevant conflicts of interest to declare.

B-cell receptor and microenvironment-derived signals promote accumulation of chronic lymphocytic leukemia (CLL) cells through increased proliferation and/or decreased apoptosis. In this study, we investigated the regulation of BIM, a proapoptotic BCL2-related protein, which is tightly regulated by phosphorylation. Surface IgM stimulation increased phosphorylation of 2 BIM isoforms, BIMEL and BIML, in a subset of CLL samples. In contrast, in normal B cells, anti-IgM triggered selective phosphorylation of BIMEL only. In CLL, anti-IgM–induced BIM phosphorylation correlated with unmutated IGHV gene status and with progressive disease. Strikingly, it was also associated with progressive disease within the mutated IGHV gene subset. BIM phosphorylation was dependent on MEK1/2 kinase activity, and we identified BIMEL serine 69, previously linked to pro-survival responses, as the major site of phosphorylation in CLL and in Ramos cells. BIMEL/BIML phosphorylation was associated with release of the pro-survival protein MCL1. Coculture of CLL cells with HK cells, a model of the CLL microenvironment, promoted CLL cell survival and was associated with MEK1/2 activation and BIMEL phosphorylation. Hence, BIM phosphorylation appears to play a key role in apoptosis regulation in CLL cells, potentially coordinating antigen and microenvironment-derived survival signals. Antigen-mediated effects on BIM may be an important determinant of clinical behavior.

The hypothalamic supraoptic nucleus (SON) is a core osmoregulatory control centre that deciphers information about the metabolic state of the organism and orchestrates appropriate homeostatic (endocrine) and allostatic (behavioural) responses. We have used RNA sequencing to describe the polyadenylated transcriptome of the SON of the male Wistar Han rat. These data have been mined to generate comprehensive catalogues of functional classes of genes (enzymes, transcription factors, endogenous peptides, G protein coupled receptors, transporters, catalytic receptors, channels and other pharmacological targets) expressed in this nucleus in the euhydrated state, and that together form the basal substrate for its physiological interactions. We have gone on to show that fluid deprivation for 3 days (dehydration) results in changes in the expression levels of 2247 RNA transcripts, which have similarly been functionally catalogued, and further mined to describe enriched gene categories and putative regulatory networks (Regulons) that may have physiological importance in SON function related plasticity. We hope that the revelation of these genes, pathways and networks, most of which have no characterised roles in the SON, will encourage the neuroendocrine community to pursue new investigations into the new ‘known‐unknowns’ reported in the present study.

The use of Next Generation Sequencing (NGS) techniques has generated a wide variety of blood microbiome data. Due to the large variation in bacterial DNA profiles between studies and the likely high concentrations of cell-free bacterial DNA in the blood, it is still not clear how such microbiome data relates to viable microbiota. For these reasons much remains to be understood about the true nature of any possible healthy blood microbiota and of bacteraemic events associated with disease. The gut, reproductive tracts, skin, and oral cavity are all likely sources of blood-borne bacteria. Oral bacteria, especially those associated with periodontal diseases, are also commonly associated with cardiovascular diseases such as infective endocarditis, and also have been linked to rheumatoid arthritis and Alzheimer’s disease. Periodontal treatment, dental probing, and toothbrushing have been shown to cause transient bacteraemia and oral bacteria from the phyla Firmicutes (e.g. Streptococci) and Bacteroidetes (e.g. Porphyromonas) are found in cardiovascular lesions (CVD). Many studies of blood bacterial DNA content however, find Proteobacteria DNA to be the dominant microbiome component, suggesting a gut origin. Most studies of this type use total DNA extracted from either whole blood or blood fractions, such as buffy coat. Here, using a method that purifies DNA from intact bacterial cells only, we examined blood donated by those with active, severe periodontitis and periodontally healthy controls and show that 43–52% of bacterial species in blood are classified as oral. Firmicutes, consisting largely of members of the Streptococcus mitis group and Staphylococcus epidermidis, were predominant at 63.5% of all bacterial sequences detected in periodontal health and, little changed at 66.7% in periodontitis. Compared to studies using total DNA Proteobacteria were found here at relatively low levels in blood at 13.3% in periodontitis and 17.6% in health. This study reveals significant phylogenetic differences in blood bacterial population profiles when comparing periodontal health to periodontal disease cohorts.

No abstract

Transcription factor cAMP responsive element‐binding protein 3 like 1 (Creb3l1) is a non‐classical endoplasmic reticulum stress molecule that is emerging as an important component for cellular homeostasis, particularly within cell types with high peptide secretory capabilities. We have previously shown that Creb3l1 serves an important role in body fluid homeostasis through its transcriptional control of the gene coding for antidiuretic hormone arginine vasopressin in the neuropeptide‐rich magnocellular neurones of the supraoptic nucleus. In response to osmotic stimuli such as dehydration, vasopressin magnocellular neurones undergo remarkable transcriptome changes, including increased Creb3l1 expression, to ensure that the supply of vasopressin meets demand. To determine where else Creb3l1 fits into the secretory cell supply chain, we performed RNA‐sequencing of Creb3l1 knockdown anterior pituitary mouse corticotroph cell line AtT20. The target chosen for further investigation was Pcsk1, which encodes proprotein convertase enzyme 1 (PC1/3). PC1/3 is crucial for processing of neuropeptides and peptide hormones such as pro‐opiomelanocortin (POMC), proinsulin, proglucagon, vasopressin and oxytocin. Viral manipulations in supraoptic nuclei by over‐expression of Creb3l1 increased Pcsk1, whereas Creb3l1 knockdown decreased Pcsk1 expression. In vitro promoter activity and binding studies showed that Creb3l1 was a transcription factor of the Pcsk1 gene binding directly to a G‐box motif in the promoter. In the dehydrated rat anterior pituitary, Creb3l1 and Pcsk1 expression decreased in parallel compared to control, supporting our findings from manipulations in AtT20 cells and the supraoptic nucleus. No relationship was observed between Creb3l1 and Pcsk1 expression in the neurointermediate lobe of the pituitary, indicating a different mechanism of PC1/3 synthesis by these POMC‐synthesising cells. Therefore, Creb3l1, by regulating the expression of Pcsk1, does not control the processing of POMC peptides in the intermediate lobe.

Elderly people exhibit a diminished capacity to cope with osmotic challenges such as dehydration. We have undertaken a detailed molecular analysis of arginine vasopressin (AVP) biosynthetic processes in the supraoptic nucleus (SON) of the hypothalamus and secretory activity in the posterior pituitary of adult (3 months) and aged (18 months) rats, to provide a comprehensive analysis of age-associated changes to the AVP system. By matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis, we identified differences in pituitary peptides, including AVP, in adult and aged rats under both basal and dehydrated states. In the SON, increased Avp gene transcription, coincided with reduced Avp promoter methylation in aged rats. Based on transcriptome data, we have previously characterized a number of novel dehydration-induced regulatory factors involved in the response of the SON to osmotic cues. We found that some of these increase in expression with age, while dehydration-induced expression of these genes in the SON was attenuated in aged rats. In summary, we show that aging alters the rat AVP system at the genome, transcriptome, and peptidome levels. These alterations however did not affect circulating levels of AVP in basal or dehydrated states.