ß-Adrenergic signaling induces Notch-mediated salivary gland progenitor cell control.

ß-Adrenergic signaling blockade is a mainstay of hypertension management. One percent of patients taking ß-blockers develop reduced salivary gland (SG) function. Here we investigate the role of SG progenitor cells in ß-blocker-induced hyposalivation, using human SG organoid cultures (SGOs). Compared with control SGs, initial low SG progenitor cell yield from patients taking ß-blockers was observed. When passaged, these SGOs recovered self-renewal and upregulated Notch pathway expression. Notch signaling was downregulated in situ in ß-adrenergic receptor-expressing luminal intercalated duct (ID) cells of patients taking ß-blockers. Control SGOs treated with ß-adrenergic agonist isoproterenol demonstrated increased proportion of luminal ID SGO cells with active Notch signaling. Control SGOs exposed to isoproterenol differentiated into more mature SGOs (mSGOs) expressing markers of acinar cells. We propose that ß-blocker-induced Notch signaling reduction in luminal ID cells hampers their ability to proliferate and differentiate into acinar cells, inducing a persistent hyposalivation in some patients taking ß-blocking medication.

Abatacept treatment reduces disease activity in early primary Sjögren's syndrome (open-label proof of concept ASAP study).

OBJECTIVE: To assess the efficacy and safety of abatacept in patients with early and active primary Sjögren's syndrome (pSS). METHODS: All 15 patients (12 women, three men) included in the open-label Active Sjögren Abatacept Pilot study met the revised American-European Consensus Group criteria for pSS and were biological disease-modifying antirheumatic drug-naive. Patients were treated with eight intravenous abatacept infusions on days 1, 15 and 29 and every 4 weeks thereafter. Follow-up was conducted at 4, 12, 24 (on treatment), 36 and 48 weeks (off treatment). Disease activity was assessed with European League Against Rheumatism (EULAR) Sjögren's Syndrome Disease Activity Index (ESSDAI) and EULAR Sjögren's Syndrome Patient Reported Index (ESSPRI). Several other functional, laboratory and subjective variables were analysed. Generalised estimating equations were used to analyse parameters over time. RESULTS: ESSDAI, ESSPRI, rheumatoid factor and IgG levels decreased significantly during abatacept treatment and increased post-treatment. Salivary and lacrimal gland function did not change during treatment. Fatigue and health-related quality of life (HR-QoL) improved significantly during treatment. No serious side effects or infections were seen. CONCLUSIONS: In this open-label study, abatacept treatment is effective, safe and well tolerated, and results in improved disease activity, laboratory parameters, fatigue and HR-QoL in patients with early and active pSS. TRIAL REGISTRATION NUMBER: 2009-015558-40.

Blood cell dynamics during hibernation in the European Ground Squirrel.

Hibernation is a unique natural model to study large and specific modulation in numbers of leukocytes and thrombocytes, with potential relevance for medical application. Hibernating animals cycle through cold (torpor) and warm (arousal) phases. Previous research demonstrated clearance of leukocytes and thrombocytes from the circulation during torpor, but did not provide information regarding the timing during torpor or the subtype of leukocytes affected. To study the influence of torpor-bout duration on clearance of circulating cells, we measured blood cell dynamics in the European Ground Squirrel. Numbers of leukocytes and thrombocytes decreased within 24h of torpor by 90% and remained unchanged during the remainder of the torpor-bout. Differential counts demonstrated that granulocytes, lymphocytes and monocytes are all affected by torpor. Although a decreased production might explain the reduced number of thrombocytes, granulocytes and monocytes, this cannot explain the observed lymphopenia since lymphocytes have a much lower turnover rate than thrombocytes, granulocytes and monocytes. In conclusion, although underlying biochemical signaling pathways need to be unraveled, our data show that the leukocyte count drops dramatically after entrance into torpor and that euthermic cell counts are restored within 1.5h after onset of arousal, even before body temperature is fully normalized.

Bacterial population analysis of human colon and terminal ileum biopsies with 16S rRNA-based fluorescent probes: commensal bacteria live in suspension and have no direct contact with epithelial cells.

BACKGROUND: The commensal intestinal microflora has important metabolic and perhaps also immune modulatory functions. Evidence has accumulated that the microflora plays a role in the pathogenesis of inflammatory bowel disease. Therefore, there is a growing interest in the intestinal microflora and its interaction with the host. Presumably, this interaction takes place at the mucus layer. In this study, we investigated the microflora that is present at the mucus layer and addressed the following questions. Does a specific mucus-adherent microflora exist? Is there direct contact between commensal bacteria and epithelial cells? METHODS: Snap-frozen biopsies were taken of 5 colon regions and of the terminal ileum in 9 subjects with a normal colon. Fecal samples were also collected. Bacteria were detected in cryosections with fluorescent in situ hybridization (FISH) with 16S ribosomal (r)RNA-targeted probes for all bacteria and specific probes for the major representatives of anaerobic microflora (bifidobacteria, Bacteroides, clostridia, atopobia) and aerobic microflora (Enterobacteriaceae, enterococci, streptococci, lactobacilli). RESULTS: With this sensitive technique, bacteria were only observed at the luminal side of the intestinal mucus layer. Very few microcolonies were present at the mucus layer, and the composition of the bacterial microflora present in the feces was similar to that at the mucus layer of the terminal ileum and colon regions. CONCLUSIONS: We did not observe direct contact between bacteria and epithelial cells. The equal distribution of bacterial species suggests that intestinal commensal bacteria live in suspension in the lumen and that there is no specific mucus-adherent microflora.

Slow recovery of follicular B cells and marginal zone B cells after chemotherapy: implications for humoral immunity.

Although most chemotherapeutic agents are known to cause primarily reduction or suppression of immune responses, surprisingly little is known about the influence of cytostatic agents on lymphoid tissue compartments such as the splenic marginal zone. The marginal zone plays an important role in the defence against encapsulated bacteria, which are potential candidates for postchemotherapeutic infections. We studied the effect of three different cytostatic agents (cisplatin, methotrexate, and cyclophosphamide) on B cell subpopulations in a rat model. Rats received a single dose of a single cytostatic agent and were sacrificed at different time points after treatment. Bone marrow, blood, mesenteric lymph nodes and spleens were analysed by flow-cytometry and immunohistochemistry. All three cytostatic agents showed severe bone marrow depression. CP and MTX showed only mild reduction of cell populations in the spleen. CyPh showed a severe reduction of recirculating follicular B (RF-B) cells and marginal zone B (MZ-B) cells. At day 24 most populations were already recovered, but RF-B cells and MZ-B cells were still reduced. The reduction of the marginal zone and late recovery may imply that, beside the overall increased infection risk due to neutropenia, patients treated with chemotherapy are at risk for developing infections from encapsulated bacteria for a considerable period of time after treatment, extending beyond the period of bone marrow depression.

Immunoglobulin VH-gene usage of autoantibodies in mercuric chloride-induced membranous glomerulopathy in the rat.

Brown-Norway (BN) and Dorus Zadel Black (DZB) rats develop a T-cell-dependent membranous glomerulopathy (MGP) with high proteinuria and antiglomerular basement membrane (GBM) autoreactive antibodies (Abs), upon exposure to mercuric chloride (HgCl2). Laminin is an important autoantigenic target of the anti-GBM Abs, absorbing approximately 30% of the anti-GBM reactivity. Although many anti-GBM Abs have undergone isotype switching, it is currently unclear whether affinity maturation occurs during the HgCl2-induced autoimmune response. To address this question we analysed the rearranged immunoglobulin heavy chain variable-region genes (VHDJH regions) of 15 mAbs that were previously obtained from HgCl2-treated rats. Seven of these mAbs exhibit reactivity towards laminin. Our study showed that the VH-gene usage of antilaminin mAbs is largely restricted to the PC7183 VH-gene family (six out of seven). In addition, we demonstrated that at least three out of six laminin reactive and five out of six non-laminin-binding mAbs are encoded by germline VH genes (a total of eight out of 12 mAbs). Of the eight mAbs that are encoded by germline VH genes, seven are of a non-immunoglobulin M (IgM) isotype, indicating that isotype switching has occurred in these mAbs in the absence of somatic mutations. The mutations observed in the VH genes of the four remaining mAbs do not provide strong evidence for antigenic selection. The data support the notion that B cells in this model of MGP are not subjected to affinity maturation and probably result from polyclonal B-cell activation.

CD27 expression in the human splenic marginal zone: the infant marginal zone is populated by naive B cells.

The splenic marginal zone of adult humans contains B cells, of which most express CD27, an antigen only recently identified as a marker for somatically mutated memory B cells. We investigated whether and to which extent the developing marginal zone in infants and children is populated by either memory (CD27+) or naive (CD27-) B cells. Frozen sections of 32 spleens of infants and children ranging in age from 6 days to 15 years and 6 adult spleens were investigated. The expression of CD27 in combination with monoclonal antibodies against CD3, CD21, IgM, IgD and ASM-1 was analyzed by immunohistochemistry. The marginal zone was already present at 4 months after birth but CD21 expression was observed first after 2 years. CD27-positive marginal zone B cells were observed firstly 2 years after birth and increased in number to adult levels at the age of 5 years. We demonstrated that the MZ of infants and young children is populated by naive B cells, which are replaced by memory B cells in a time frame of 2 to 5 years. Before the age of 2 years, although present, memory B cells appear to be unable to colonize the marginal zone. Because of the absence of memory B cells in the marginal zone, the immune system of a child is not capable to initiate a rapid secondary humoral immune response comparable to the adult immune response.

Most marginal zone B cells in rat express germline encoded Ig VH genes and are ligand selected.

The present study was performed to analyze whether marginal zone B (MZ-B) cells in nondeliberately immunized adult rats are selected on basis of the specificity of their B cell receptor, and to determine to what extent memory B cells contribute to the MZ-B cell subset. To this end, the Ig PC7183 V(H) gene repertoire was studied among V(H)DJ(H)-mu transcripts expressed in four sequential stages of B cell development, of two individual untreated adult rats. B cell subsets, i.e., pro/pre-B cells and newly formed B (NF-B) cells from bone marrow, and recirculating follicular B cells and MZ-B cells from spleen were sorted by flow cytometry. In addition, from one these rats, cells were microdissected from follicular and MZ areas of the spleen and productive PC7183 V(H) gene rearrangements were analyzed for the presence of somatic mutations. Sequence analysis reveals that most MZ-B cells in the adult rat, either defined by flow cytometry or by their anatomical location in the spleen, express germline encoded V(H) genes (naive MZ-B cells) and a minor fraction (about 20%) of the MZ-B cells carry somatic mutations (memory MZ-B cells). In addition, we show that naive MZ-B cells are a selected population of cells, both based on PC7183 V(H) gene repertoire and on the length of the Ig heavy (H) chain complementarity-determining region 3 (H-CDR3) region, i.e., PC7183 V(H)DJ(H)-mu transcripts of MZ-B cells carry significantly shorter H-CDR3 regions than other B cell subsets.

The origin of marginal zone B cells in the rat.

The marginal zone is a unique compartment that is only found in the spleen. Rat marginal zone B cells (MZ-B) can be distinguished from other B cells, e.g. recirculating follicular B cells (RF-B), by several phenotypic characteristics. Typically MZ-B cells are surface (s)IgMhi, sIgDlo and CD45R(B220)lo, whereas RF-B cells are sIgMlo, sIgDhi and CD45Rhi. In addition, MZ-B cells stain strongly with HIS57, a newly developed monoclonal antibody. The developmental pathway and origin of MZ-B cells are not exactly known. However, previous studies indicate that recirculating (i. e. thoracic duct) B cells can give rise to MZ-B cells. Here the origin of (naive) MZ-B cells was studied using adriamycin (doxorubicin)-induced B cell depletion. Using three-color flow cytometry and immunohistology we show that 2 days after a single i.v. injection of the anti-tumor drug adriamycin only RF-B cells can be detected, while all other B cell subpopulations are depleted, including all bone marrow precursor B cells. By studying the sequential reappearance of various B cell subsets and their precursors after adriamycin administration we show that MZ-B cells and the splenic marginal zone can be detected at a time point at which newly generated B cells (immature B cells) are not yet present. Given the observation that only RF-B cells were present at this time, we conclude that RF-B cells are the immediate MZ-B precursor cells.

Presence of germline and full-length IgA RNA transcripts among peritoneal B-1 cells.

Next to conventional B cells (or B-2 cells), peritoneal B-1 cells have been shown to contribute significantly to the production of IgA-secreting plasma cells in the gut. Evidence for this was mainly based on studies comprising manipulated animals, including lethally X-irradiated and transgenic mice. To examine the ability of peritoneal B-1 cells from untreated mice to switch actively to IgA in vivo, we performed RT-PCR analysis on FACS-sorted peritoneal B-cell subsets from untreated BALB/c mice in order to examine the presence of germline C alpha mRNA and mature C alpha mRNA transcripts. Germline C alpha and mature C alpha transcripts were readily detectable in peritoneal B-1 cells (defined as IgMbright/IgDdull), but not, or very little, in peritoneal B-2 cells (defined as IgMdull/IgDbright). Moreover, by subdividing the B-1-cell population in CD5+ B-1a cells and CD5- B-1b cells, it was shown that in vivo expression of germline C alpha and mature C alpha transcripts was largely restricted to the B-1b-cell lineage. These results indicate that peritoneal B-1 cells indeed are capable to switch to IgA under normal physiological conditions and hereby further support the view that B-1 cells contribute significantly to the mucosal IgA response, albeit this function appears to be restricted to the B-1b-cell subset.

Monoclonal gammopathies in aging mu, kappa-transgenic mice: involvement of the B-1 cell lineage.

Monoclonal gammopathies (MG) are monoclonal proliferative disorders of B cells at the differentiation stage of Ig production. They can be detected in the serum, either as transient or as persistent homogenous immunoglobulin (H-Ig) components. The exact phenotype, localization, and cell lineage origin of the precursor cells of MG are unknown, but may be crucial for both the correct diagnosis and for timely efficient treatment of the malignant forms. We used for the first time transgenic (Tg) mice (Sp6; mu/kappa) to study the origin of MG. In the mu, kappa Tg mice a small proportion of B cells can still produce endogenous IgM. These cells are of B-1 cell origin. The MG in Tg mice showed a later onset and a lower frequency than those in littermate control mice, mainly due to a four times lower frequency of benign monoclonal gammopathy. The 10% of B-1 cells that were able to produce endogenous Ig led to the development of MG in a frequency that was half the number of MG found in normal littermates. None of the MG in Tg mice produced an Ig of the Tg origin and therefore it can be concluded that they originated from B-1 cells.