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.

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.

The extent of clonal structure in different lymphoid organs.

To gain insight into the clonal organization of lymphoid organs, we studied the distribution in situ of donor-derived cells in near-physiological chimeras. We introduced RT7b fetal liver cells into nonirradiated congenic RT7a neonatal rats. The chimerism 6-20 wk after injection ranged from 0.3 to 20%. The numbers of cell clones simultaneously contributing to cell generation in a particular histological feature were deduced from the variance in donor cell distribution. In bone marrow and thymus, donor-derived lymphoid cells were found scattered among host cells, indicating a high mobility of cells. In bone marrow, donor cells were evenly distributed over the entire marrow, even at low chimerism. This indicates that leukopoiesis is maintained by the proliferation of many clones. In the thymus, the various lobules showed different quantities of donor-derived lymphoid cells. Mathematical analysis of these differences indicated that 17-18 cell division cycles occur in the cortex. In spleen, the distribution of donor-derived cells over the germinal centers indicated that 5 d after antigenic stimulation, germinal centers develop oligoclonally. The main conclusions of this work are that (a) bone marrow and thymus are highly polyclonal; (b) 17-18 divisions occur between prothymocyte and mature T cell; and (c) lymphoid cells disperse rapidly while proliferating and differentiating.

LAMA tumor in the rat as an experimental model for pre-B-cell leukemia.

A late pre-B-cell leukemia model in the rat, the LAMA tumor, is described. A mouse monoclonal antibody (HIS30) was developed against LAMA cells. HIS30 reacts with a membrane antigen in tumor tissue, whereas its reactivity with normal tissues is limited to the zona glomerulosa of the adrenal cortex and to the adrenal medulla. HIS30 was used for both the immunohistological detection of tumor cells in tissue sections and the immunolocalization of tumor cells in vivo. To enable in vitro studies with the LAMA model, an in vitro growing cell line (LAMA-K1) was established from the LAMA tumor. LAMA-K1 is immunophenotypically similar to the original tumor. Two tumor transplantation models were characterized. In the first model LAMA was implanted s.c., and local tumor growth occurred at the injection site, which was then followed by lymphatogenic and subsequently hematogenic tumor spread. In the second model i.v. transplantation caused direct hematogenic tumor dissemination. In both models early dissemination was especially prominent to the bone marrow, spleen, and liver. Later in the disease most visceral organs became involved, and partial paralysis of the animal was observed in the end stage of the disease. In combination with HIS30, the LAMA pre-B-cell tumor offers a model for both the investigation of in vivo transplanted tumor cells and for the in vivo detection of tumor cells by HIS30 in LAMA tumor-bearing rats.

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.

In vivo distribution and characterization of two novel mononuclear phagocyte differentiation antigens in mice.

Flow cytometry, immunohistology, and SDS-PAGE Western blot analysis were used to characterize two novel anti-mouse mononuclear phagocyte differentiation antigens defined by monoclonal antibodies. One antibody, Monts-4, recognized an 80-100 kd cell-surface protein expressed on resident macrophages in the peritoneum and stained macrophages in the splenic white pulp and marginal zone, liver, lymph node paracortical regions, Peyers patches, and cortex of the thymus. Monts-4 did not stain blood monocytes or monocyte-derived inflammatory macrophages in the peritoneal cavity. Macrophages that were Monts-4 positive became Monts-4 negative within 24-72 h after culturing in vitro. Monoclonal antibody SK39 recognized a 180 kd cell-surface molecule expressed on inflamed peritoneal monocytes/macrophages and stained macrophages in the splenic red pulp, cortex of the thymus, subcapsule and medullary regions of lymph nodes, and in sites of acute and chronic inflammation. No differences were seen in the expression of these antigens in the immunodeficient SCID versus normal BALB/c mouse. A comparison of the distribution and molecular weights of the Monts-4 and SK39 antigens with other described mononuclear phagocyte-specific antigens, including F4/80 and those defined by the M1/70, ERTR9, MOMA1, MOMA2, and SER4 monoclonal antibodies, shows these to be novel mononuclear phagocyte-specific differentiation antigens.

Immunohistochemical antigen demonstration in plastic-embedded lymphoid tissue.

We describe a method for post-embedding immunohistochemical demonstration of a wide range of antigens in glycol methacrylate-embedded tissue. Rat spleen and thymus tissues were fixed by immersion in fixatives containing different concentrations of paraformaldehyde, washed in sucrose phosphate buffer, dehydrated in acetone, infiltrated in a glycol methacrylate mixture in which the commonly used softener 2-butoxyethanol was replaced by butaandiol monoacrylate, and embedded. Trypsin was used to re-expose the masked antigenicity. Excellent results were obtained with a panel of monoclonal antibodies (MoAbs) directed against T-cells, B-cells, Ia-positive cells, macrophages, follicular dendritic cells, and leucocyte common antigen-bearing cells. The method described combines exact localization of antigens with optimal tissue morphology.

Dendritic immune complex trapping cells in the spleen of the snake, Python reticulatus.

The spleen of the snake Python reticulatus, was investigated as to its general histology as well as the presence of immune complex trapping cells both at the light and electron microscopical level. Histological examination revealed that the spleen of this reptile was encapsulated and contained some trabeculae. In the splenic parenchyma two different regions could be distinguished: viz. red and white pulp. The white pulp appeared to be arranged around "central arterioles" and their smaller branches extending towards the periphery of the white pulp. The red pulp was composed of blood sinusoids and cell cords. Electron microscopy revealed at least three types of non-lymphoid cells in the white pulp of the spleen of python: reticulum cells, forming the framework; some macrophages and dendritic cells predominantly located in the periphery of the white pulp. Of these types of non-lymphoid cells, only dendritic cells were able to trap and to retain intravenously injected horseradish peroxidase (HRP)-rabbit-anti-HRP immune complexes on their cell surface as determined by enzymehistochemistry at the light and electron microscopical level. These dendritic cells were frequently found in association with collagen fibres and did not engulf large quantities of carbon particles. These data suggest that dendritic cells in the spleen of the python might be the phylogenetic precursors of the mammalian follicular dendritic cells.

B cells specific for bromelain-treated erythrocytes are not derived from adult rat bone marrow.

As part of an evolutionary layered hematopoietic system, the B lymphocyte compartment consists of different lineages of B lymphocytes, which evolve sequentially during ontogeny. In mice, there is ample evidence for the existence of at least two lineages, a layer of B-1 cells (Ly-1 B cells) and the evolutionary more advanced layer consisting of conventional B cells. In a previous study we were unable to detect B-1 cells in the rat as determined by phenotypic markers. Here we studied the possible existence of putative B-1 cells in the rat based on some functional and developmental characteristics as have been described for mouse B-1 cells. We show that B cells secreting antibodies that recognize bromelain-treated mouse red blood cells (BrMRBC) can be identified in rat spleen, whereas these cells (in contrast to DNP-specific B cells) are virtually absent in lethally X-irradiated and bone marrow (BM) reconstituted animals. The number of anti-rMRBC-secreting B cells could not be restored to control levels by reconstitution with fetal liver cells or by cotransfer of 10(7) cells from peritoneal cavity, lymph node or Peyer's patches or up to 2 x 10(8) splenocytes. Although our findings thus suggest that B-1 cells (or B-1 like cells) may be present in rats, formal proof for the existence of such a lineage in rats awaits definition of these cells at the progenitor level.

The architecture of the spleen of the Red-eared Slider, Chrysemys scripta elegans (Reptilia, Testudines).

The morphology of the spleen of the Red-eared Slider, Chrysemys scripta elegans, has been described. The outer surface of the spleen is covered by a thin fibromuscular capsule, from which a number of trabeculae extend into the parenchyma. The parenchyma of the spleen shows a definite demarcation into a red and white pulp. The white pulp is composed of two lymphoid compartments: lymphoid tissue surrounds both central arterioles and ellipsoids, forming the periarteriolar and periellipsoidal lymphocyte sheath (PALS and PELS, respectively). The ellipsoids consist of a thick layer of reticular tissue, enclosing a capillary with cuboidal endothelium. In contrast with the PALS, reticular fibers are rarely found in the PELS. Even after severe stimulation with paratyphoid vaccine, germinal centers are not found in the spleen of C.s. elegans. The red pulp of the spleen is composed of a system of venous sinuses and cords.

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.