B Lymphocyte signaling established by the CD19/CD22 loop regulates autoimmunity in the tight-skin mouse

Systemic sclerosis (SSc) is characterized by fibrosis and autoimmmunity. Peripheral blood B cells from SSc patients specifically overexpress CD19, a critical cell-surface signal transduction molecule in B cells. CD19 deficiency in B cells also attenuates skin fibrosis in the tight-skin (TSK/+) mouse, a genetic model for SSc. Herein we analyzed two transgenic mouse lines that overexpress CD19. Remarkably, 20% increase of CD19 expression in mice spontaneously induced SSc-specific anti-DNA topoisomerase I (topo I) antibody (Ab) production, which was further augmented by 200% overexpression. In TSK/+ mice overexpressing CD19, skin thickness did not increase, although anti-topo I Ab levels were significantly augmented, indicating that abnormal CD19 signaling influences autoimmunity in TSK/+ mice and also that anti-topo I Ab does not have a pathogenic role. The molecular mechanisms for abnormal CD19 signaling were further assessed. B-cell antigen receptor crosslinking induced exaggerated calcium responses and augmented activation of extracellular signal-regulated kinase in TSK/+ B cells. CD22 function was specifically impaired in TSK/+ B cells. Consistently, CD19, a major target of CD22-negative regulation, was hyperphosphorylated in TSK/+ B cells. These findings indicate that reduced inhibitory signal provided by CD22 results in abnormal activation of signaling pathways including CD19 in TSK/+ mice and also suggest that this disrupted B cell signaling contribute to specific autoantibody production.     

Derivation and morphological characterization of mouse spermatogonial stem cell lines

Spermatogonial stem cells (SSCs), having yet to possess decisive markers, can only be detected retrospectively by transplantation assay. It was reported recently that mouse gonocytes collected from DBA/2 and ICR neonates propagated in vitro. This cultured germ cell, named the germline stem cell (GS cell), produced functional sperm to make progeny when transplanted into recipient mouse testes. Here we show that GS cell lines can be established not only from neonatal testes but also from the testis of adult mice. We also confirmed that GS cells once transplanted into a host testis can be recovered to resume in vitro expansion, indicating that they are convertible mutually with SSCs in adult testes. Confocal laser microscopic examination showed GS cells resemble undifferentiated spermatogonia in the adult testis. This unique cell line could be useful for research in germ cell biology and applicable as a new tool for the genetic engineering of animals.     

Characterization and localization of side population cells in mouse skin

Recently, the detection of side population (SP) cells, which have the ability to strongly efflux Hoechst 33342 fluorescence dye, has attracted attention as a method of stem cell isolation. We identified SP cells from mouse skin using the same method as from bone marrow. This population almost completely disappeared after treatment with the calcium channel blocker verapamil. SP cells were mainly localized in the epidermis, with a few in the dermis. The ratio of SP cells decreased as the mouse became older. Surface marker analysis revealed that the sorted SP cells expressed alpha6-integrin, beta1-integrin, Sca-1, keratin 14, and keratin 19, which are proliferating and progenitor cell markers, at levels higher than in non-SP cells, while they expressed E-cadherin, CD34, and CD71 at lower levels. The expression of breast cancer resistance protein 1 (BCRP1), which participates in dye efflux, was expressed at high levels at both the protein and mRNA level in sorted SP cells. Immunohistochemical analysis showed that BCRP1 was expressed in the basal layers and hair bulge regions of mouse skin. BCRP1 mRNA was found in basal layers and hair follicles of newborn skin by in situ hybridization. These results indicate that the localization of BCRP1-positive cells is compatible with that of keratinocyte stem cells. Based on the close relationship between BCRP1 and the SP cell phenotype, we conclude that keratinocyte stem cells are closely related to the SP- or BCRP1-positive cells.     

Generation of B lymphocytes from a single hemopoietic progenitor cell in vitro

The first stages of the pathway by which lymphocytes differentiate from hemopoietic stem cells were studied at a clonal level. When 211 interleukin 3 (IL-3)-induced blast colonies shown to be capable of differentiating into a variety of hemopoietic cells were individually transferred into wells containing a monolayer of stromal cells, growth in granulocyte, macrophage, megakaryocyte, or mast cell lineages was observed in 192 wells. In seven of these 192 wells, lymphoid cell growth also was seen. The lymphoid cells were proved to be B lymphocytes by phenotype and immunoglobulin gene rearrangement analyses and by demonstration of surface expression of IgM. The clonal origin of myeloid and B lymphocyte lineage cells was further confirmed by the generation of both myeloid and B lymphoid cells in the same well following FACS clone-sorting of IL-3 induced blast cells. These results provide in vitro evidence that cells of B lymphoid and myeloid lineage can originate clonally from single primitive hemopoietic stem cells.     

Liquid photocurable biodegradable copolymers: in vivo degradation of photocured poly(epsilon-caprolactone-co-trimethylene carbonate)

The present investigation was designed to test cellular toxicity of modern dentin adhesives. With the use of the products Ariston Liner, Etch & Prime 3.0, Optibond Solo, Prime & Bond NT, Scotchbond 1, and Syntac Sprint, test specimens were prepared according to the manufacturers' instructions and transferred into a culture medium. Eluates were obtained and pipetted onto fibroblast cultures, incubated, and subsequently stained. The respective cell densities and the numbers of normal, altered, and dead cells were determined and compared with control cell cultures. Statistical analysis of the data showed that all materials caused cytotoxic effects. Scotchbond 1 displayed the highest number of dead cells. The difference was statistically significant compared to Etch" 3.0, Optibond Solo, Prime&Bond NT, and the control. The lowest cell density was found for Scotchbond 1 and Ariston Liner. The difference was also statistically significant in comparison with Etch" 3.0, Optibond Solo, Prime&Bond NT, and the control. To conclude, all tested dentin adhesives caused cytotoxic reactions. Taking the limitations of an in vitro experiment into consideration, Prime&Bond NT, Optibond Solo, and Etch" 3.0 appear to be the most recommendable products, and Scotchbond 1 and Ariston Liner the least.     

Glucose uptake by individual skeletal muscles during running using whole-body positron emission tomography

The purpose of this study was to examine, by positron emission tomography (PET), the distribution of [¹⁸F]fluoro-deoxy-glucose ([¹⁸F]FDG) uptake by human muscles during 35?min of running. Thirteen healthy male subjects were studied, seven of whom participated in the exercise study. Running intensity was kept constant such that the subjects' heart rates were maintained at between 140 and 150?beats per minute. [¹⁸F]FDG [62.9 (14.8)?MBq, mean (SD)] was injected after 15?min of running. PET imaging was started immediately after the running ended. The ratio of [¹⁸F]FDG uptake by muscles in runners to that in control subjects (r-c ratio) varied from three to six for the muscles of the foot and leg below the knee joint. The r-c ratio of the medial head of the gastrocnemius (MG) was higher than that of its lateral head (LG). The r-c ratio of the rectus femoris (RF) was lower than that of the other three muscles of the quadriceps femoris (QF). The r-c ratio of inactive muscles located above the waist was approximately 0.7. These results suggest that, during the moderate running of this study: (1) glucose uptake by muscles of the foot and leg below the knee joint clearly increases, (2) the r-c ratio differs significantly among the skeletal muscles, which act synergistically, and (3) glucose uptake by inactive skeletal muscles decreases.     

Functional variation of HIV-1 Rev Response Element in a longitudinally studied cohort

We showed previously that HIV-1 Rev Response Element (RRE) contains a certain degree of structural variation, and in a set of limited samples, RRE from HIV-1 natural isolates were found to have functional variability. The significance of the RRE heterogeneity is addressed further by analyzing the functional variation of RREs in a longitudinal cohort. While the RRE activity at early time points was not a good predictor of disease outcome, the RRE activity at late time points was correlated with rates of CD4+ count decline. These data suggest that RRE heterogeneity may be important in viral pathogenesis and disease progression.     

Pharmacokinetics of bisoprolol and its effect on dialysis refractory hypertension

The efficacy, safety, and pharmacokinetics of bisoprolol were investigated following oral administration once daily for 12 weeks in hyperreninemic patients with dialysis-refractory hypertension. Mean blood pressure rapidly fell from 132 to 112 mmHg in the 5.0-mg/day (n = 6) and from 142 to 128 mmHg in the 2.5-mg/day patients (n = 5), which were accompanied by a fall in plasma renin activity. On nondialysis days, Cmax and T1/2 were significantly higher in patients than in healthy control subjects. However, Cmax in the 2.5-mg/day patients was almost equal to that in healthy control subjects receiving 5.0 mg/day of bisoprolol. Plasma bisoprolol was dialyzable. During the course of the study, dialysis hypotension and bradycardia occurred in two patients receiving 5.0 mg/day of bisoprolol. In conclusion, a daily dose of 2.5 mg bisoprolol seems to be an adequate and relatively effective dose in our patients with dialysis-refractory hypertension.     

Virus-associated hemophagocytic syndrome: the diagnostic usefulness of immature histiocytes with benign features in the bone marrow

A 2-year-old girl developed fever, hepatosplenomegaly, jaundice, lymphadenopathy and pancytopenia. Bone marrow examination revealed increased immature histiocytes (5.5%) and mature histiocytes with hemophagocytosis. All the abnormalities were normalized in one month without any chemotherapy. It was suggested that the presence of immature histiocytes with benign features, even if their number exceeds that of mature histiocytes, does not favor the diagnosis of malignant histiocytosis.     

A Morphological and Immunohistochemical Study of Lymphoid Germinal Centers in Synovial and Lymph Node Tissues from Rheumatoid Arthritis Patients with Special Reference to Complement Components and Their Receptors

Rheumatoid arthritis (RA) is one of the immune complex (IC) diseases in which lymphoid germinal centers (GCs) are found in the synovial tissue. Simultaneously, patients with RA often show swelling of lymph nodes. The morphology and function of the lymph node GCs in patients with RA is not clear. The aim of this study was to evaluate the differences in morphology and immunoreactions to complement (C) components, their receptors, and lgM-rheumatoid factor (RF) between synovial GCs and lymph-node GCs in RA. Furthermore, the relationship between these immuno-reactive substances and follicular dendritic cells (FDCs) in GCs was investigated. The tissues examined were 41 RA synovial specimens, seven RA lymph nodes with massive lymphadenopathy, and 10 non-RA lymph nodes. The number of synovial GCs was relatively decreased in comparison with lymph-node GCs in RA, and the diameter of each synovial GC was smaller than that of each lymph-node GC. The synovial GCs were edematous and less cellular, and moreover, those from RF-seronegative cases were smaller than those from RF seropositive cases. On the other hand, the lymph-node GCs in RA were larger, more cellular and hyperplastic, but contained more tingible-body macro-phages (TBMs) and neutrophils. In the GCs of both synovial tissues and lymph nodes in RA, early C components (C1q, C4, C3c, C3d), IgM RF, and C3b receptor (C3bR) and C3d receptor (C3dR) were expressed as a lacy network by light microscopy, and were demonstrated on the surfaces of FDCs and lymphocytes, and in the intercellular spaces by electron microscopy. Furthermore, immuno-staining for dendritic reticulum cells (DRC, DAKO DRC1) was observed in a lacy pattern by light microscopy and on the cell surface of FDCs by electron microscopy. In the GCs of non-RA lymph nodes, early C components, C3bR, C3dR, and DRC showed a similar reaction pattern, but IgMRF did not. Consequently, no marked difference in immunoreactions in the GCs, except for the immunoreactions of late C components, was found between synovial tissues and lymph nodes in RA. On the basis of these findings, we discuss the possibility of the presence of a RF-IC.