Aberrant B1 cell migration into the thymus results in activation of CD4 T cells through its potent antigen-presenting activity in the development of murine lupus

B1 cells have different origin and function from conventional B (B2) cells and are considered to be involved in autoantibody production in the development of autoimmune disease. We found that B1 cells preferentially accumulated in the target organs including thymus in aged BWF1 mice, a murine model for systemic lupus erythematosus, and that B lymphocyte chemoattractant (BLC/CXCL13) expression was increased in the thymus before the onset of lupus nephritis, while stromal cell-derived factor-1 (SDF-1/CXCL12) and secondary lymphoid tissue chemokine (SLC/CCL21) expression remained unchanged. Adhesion molecules such as peripheral node addressin (PNAd), ICAM-1, and VCAM-1 were also expressed on endothelial cells in the enlarged thymic perivascular space (PVS) in aged BWF1 mice. BLC protein and PNAd were co-localized on these high-endothelial-venules-like vessels in enlarged PVS. B1 cells expressed higher level of costimulatory molecules and showed a potent antigen-presenting activity in allogeneic mixed lymphocyte reaction comparable to splenic dendritic cells. Interestingly, B1 cells stimulated proliferation of autologous thymic CD4 T cells in the presence of IL-2. These results indicate that aberrant B1 cell trafficking into the thymus due to ectopic high expression of BLC may result in an activation of self-reactive T cells in the development of murine lupus.     

Clinical heterogeneity of neonatal intrahepatic cholestasis caused by citrin deficiency: case reports from 16 patients

A deficiency of citrin, which is encoded by the SLC25A13 gene, causes both adult-onset type II citrullinemia (CTLN2) and neonatal intrahepatic cholestasis (NICCD). We analyzed 16 patients with NICCD to clarify the clinical features of the disease. Severe intrahepatic cholestasis with fatty liver was the most common symptom, but the accompanying clinical features were variable, namely; suspected cases of neonatal hepatitis or biliary atresia, positive results from newborn screening, tyrosinemia, failure to thrive, hemolytic anemia, bleeding tendencies and ketotic hypoglycemia. Laboratory data showed elevated serum bile acid levels, hypoproteinemia, low levels of vitamin K-dependent coagulation factors, and hypergalactosemia. Hypercitrullinemia was detected in 11 out of 15 patients examined. Most of the patients were given a lactose-free and/or medium chain triglycerides-enriched formula and lipid-soluble vitamins. The prognosis of the 16 patients is going fairy well at present, but we should observe these patients carefully to see if they manifest any symptom of CTLN2 in the future.     

Multinucleated cells formed on calcified dentine from mouse bone marrow cells treated with 1α,25-dihydroxyvitamin D3 have ruffled borders and resorb dentine

Osteoclast-like multinucleated cells were formed from mouse bone marrow mononuclear cells, and their morphology on coverslips and on calcified dentine slices was compared by means of transmission electron microscopy. Addition of 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] to bone marrow cells cultured on coverslips greatly stimulated the formation of multinucleated cells within 8 days. These multinucleated cells had the cytological features of osteoclasts (abundant pleomorphic mitochondria, a large number of vacuoles and lysosomes, many stacks of Golgi membranes, and an extensive canalicular system), but they developed neither ruffled borders nor clear zones. The multinucleated cells appeared to result from direct fusion of mononuclear progenitor cells, whose structural features were similar to those of multinucleated cells. Like isolated osteoclasts, both multinucleated cells and their precursors exhibited an intense reaction for tartrate-resistant acid phosphatase (TRACP) in the cisterns of endoplasmic reticulum and lysosomes. Multinucleated cells formed from alveolar macrophages in the presence of 1α,25(OH)₂D₃ were totally negative for TRACP reaction. When marrow cells were cultured on dentine slices in the presence of 1α,25(OH)₂D₃, some of the multinucleated cells were located in the shallow resorption lacunae of dentine surfaces, and they developed the characteristic ruffled borders and clear zones. The narrow extracellular spaces of the ruffled borders, the adjacent pale endocytotic vacuoles, and the dark lysosomes located in the perinuclear cytoplasm of the multinucleated cells contained numerous apatite crystals delete in resorption lacunae. These results indicate that (1) the multinucleated cells formed on coverslips from mouse marrow cells treated with 1α,25(OH)₂D₃ exhibit non-functional basic features of osteoclast morphology, and (2) differentiation of the multinucleated cells into functional osteoclasts requires some components of calcified dentine.     

Synaptic responses of guinea pig and rat central amygdala neurons in vitro.

1. To investigate postsynaptic potentials (PSPs), we made intracellular recordings from neurons of the amygdaloid central nucleus in slices from the guinea pig and rat brains maintained in vitro. The results from guinea pigs and rats were very similar. 2. In the presence of bicuculline (20 microM), focal electrical stimulation of the amygdaloid basal nucleus with low intensities elicited short-latency excitatory PSPs (EPSPs) followed by long-latency EPSPs. The short-latency EPSP was selectively blocked by 6-cyano-7-nitroquinoxaline-2,3-dion (CNQX; 10-20 microM). The long-latency EPSP was preferentially abolished by D,L-2-amino-5-phosphonovaleric acid (D,L-APV; 40 microM) and was augmented by removal of extracellular Mg2+. The compound EPSP reversed at -4 mV, which was close to -1 mV, the reversal potential for pressure-ejected glutamate (Glu). 3. When the intensity of the focal stimulation was increased in the presence of bicuculline (20 microM), CNQX (20 microM), and D,L-APV (50 microM), a second EPSP with a short latency and a prolonged duration could be evoked in approximately 65% of the neurons. The EPSPs were reversibly blocked by d-tubocurarine (50 microM) or hexamethonium (200 microM) but were unaffected by atropine (1 microM) or a 5-hydroxytryptamine type 3 receptor antagonist, ICS-205930 (5-10 microM). In these neurons, acetylcholine (ACh; 1-3 mM) caused a depolarization, associated with a decreased input resistance. 4. In the presence of CNQX (20 microM) and D,L-APV (50 microM), single focal stimulation of the dorsolateral subdivision in the central nucleus with low intensities elicited a depolarizing inhibitory PSP (IPSP). The IPSP was reversibly abolished by bicuculline (20-40 microM). The reversal potential (-63 mV) for the IPSP was similar to the reversal potential (-61 mV) for the response to gamma-aminobutyric acid (GABA) applied by pressure ejection. 5. In the presence of bicuculline (20-40 microM) and CNQX (20 microM), a repetitive focal stimulus with high intensities delivered to the dorsolateral subdivision produced a hyperpolarizing PSP followed by a slow depolarization in most neurons. Of putative inhibitory amino acid transmitters, glycine (Gly; 3 mM) produced only a hyperpolarization, associated with a decrease in input resistance. Strychnine (1-2 microM) reversibly blocked both the Gly hyperpolarization and the synaptically evoked hyperpolarization. The reversal potential of -81 mV for the hyperpolarizing PSP was close to -82 mV for the Gly hyperpolarization. The reversal potential for the Gly response was shifted to less negative values by increasing the external K+ concentration or decreasing the extracellular Cl- concentration.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cutaneous basophil hypersensitivity and inhibited macrophage migration in guinea-pigs with schistosomiasis

In guinea-pigs infected with schistosomes, delayed cutaneous reactions rich in basophils (CBH) were found to characterize skin test responses to schistosome egg antigens. In addition, strong contact hypersensitivity-like skin eruptions with large basophil infiltrates resulted from skin penetration challenge by live cercariae (larvae) in these animals. Oedema and diminished basophil granule staining were noted around schistosomula which had penetrated the skin of sensitized animals. CBH responses to egg antigens and to live cercarial challenges were also noted after immunization with a single injection of dead cercariae.

Using peritoneal exudates from guinea-pigs immunized with dead cercariae or infected with schistosomes, direct macrophage migration inhibition with schistosome antigens was found only in animals with infections. Thus, CBH correlated with intradermal exposure to schistosome cercarial antigens, while MMI correlated with live infections. It is suggested that cutaneous basophil responses may play a role in protection from re-infection with schistosomes, and that dead cercarial vaccines might stimulate this beneficial response, without immunizing for potentially harmful granulomatous hypersensitivity.

     

Bone bonding behavior of three kinds of apatite containing glass ceramics

We have produced three kinds of apatite-containing glass ceramics of the same chemical composition: MgO (4.6), CaO (44.9), SiO2 (34.2), P2O5 (16.3), CaF2 (0.5) (in weight ratio). They contain different crystal combinations and have different mechanical properties. The first glass ceramic (A-GC) was prepared by heating a glass plate to 870 degrees C. It contains only oxy- and fluoroapatite (35 wt%). The second glass ceramic (A-W-GC), and the third (A-W-CP-GC), were prepared by heating glass powder compacts to 1050 degrees C and 1200 degrees C, respectively. A-W-GC contains oxyapatite and fluoroapatite (Ca10(PO4)6(O,F2] (35 wt%) and beta-wollastonite (40 wt%). A-W-CP-GC contains oxyapatite and fluoroapatite (20 wt%), beta-wollastonite (CaO X SiO2) (55 wt%), and beta-whitlockite (3CaO X P2O5) (15 wt%). The bending strengths of A-GC, A-W-GC, and A-W-CP-GC were 88MPa, 178MPa, and 213MPa, respectively, in air. Rectangular ceramic plates (15mm X 10mm X 2mm) were implanted into a rabbit tibia. Ten and 25 weeks after implantation, the segment of tibia with implant was excised for examination. The segment was held by a special jig and the traction breaking load (failure load) was measured by an autograph. A-GC showed a lower load than A-W-GC and A-W-CP-GC. The loads for A-W-GC and A-W-CP-GC were almost equal. The failure loads did not change significantly between 10 and 25 weeks for any of the materials. The interface was examined by Giemsa surface staining, contact micro-radiography, and SEM-EPMA. Giemsa surface staining and CMR revealed direct bonding between the materials and the bone for all the three materials. SEM-EPMA showed that Si and Mg content decreased, Ca content did not change, and P content increased at the reaction zone between all three glass ceramics and bone. This was observed at 10 weeks, as well as at 25 weeks, after implantation. The reaction zone was narrowest with A-GC, wider with A-W-GC, and widest with A-W-CP-GC.     

Pathogenic mechanism of mouse brain damage caused by oral infection with Shiga toxin-producing Escherichia coli O157:H7

In a previous study, we showed that infection with Shiga toxin (Stx)-producing Escherichia coli O157:H7 (strain Sm(r)N-9) caused neurologic symptoms in malnourished mice with positive immunoreactions of Stx2 in brain tissues. The present study explores the mechanism of how Stx injures the vascular endothelium to enter the central nervous system in mice. Oral infection with strain Sm(r)N-9 elicited a tumor necrosis factor alpha (TNF-alpha) response in the blood as early as 2 days after infection, while Stx was first detected at 3 days postinfection. In the brain, TNF-alpha was detected at day 3, and its quantity was increased over the next 3 days. Frozen sections of the brains from moribound mice contained high numbers of apoptotic cells. Glycolipids recognized by an anti-Gb3 monoclonal antibody were extracted from the brain, and purified Stx2 was able to bind to the glycolipids. In human umbilical vascular endothelial cells (HUVEC) cultured with fluorescein-labeled Stx2 (100 ng/ml), TNF-alpha (20 U/ml) significantly facilitated the intracellular compartmentalization of fluorescence during 24 h of incubation, suggesting the enhanced intracellular processing of Stx2. Consequently, higher levels of apoptosis in HUVEC were found at 48 h. Short-term exposure of HUVEC to Stx2 abrogated their apoptotic response to subsequent incubation with TNF-alpha alone or TNF-alpha and Stx2. In contrast, primary exposure of HUVEC to TNF-alpha followed by exposure to Stx2 alone or TNF-alpha and Stx2 induced apoptosis at the same level as obtained after 48-h incubation with these two agents. These results suggest that the rapid production of circulating TNF-alpha after infection induces a state of competence in vascular endothelial cells to undergo apoptosis, which would be finally achieved by subsequent elevation of Stx in the blood. In this synergistic action, target cells must be first exposed to TNF-alpha. Such cell injury may be a prerequisite to brain damage after infection with Stx-producing E. coli O157:H7.     

Use of the genomic subtractive hybridization technique to develop a real-time PCR assay for quantitative detection of Prevotella spp. in oral biofilm samples

Genomic subtractive hybridization was used to design Prevotella nigrescens-specific primers and TaqMan probes. Based on this technique, a TaqMan real-time PCR assay was developed for quantifying four oral black-pigmented Prevotella species. The combination of real-time PCR and genomic subtractive hybridization is useful for preparing species-specific primer-probe sets for closely related species.     

Bilateral ovarian hemangiomas associated with diffuse hemangioendotheliomatosis: a case report

A patient who had disseminated vascular tumors involving the bilateral ovaries, bilateral lungs and pleura, pericardium, and mediastinum is reported. The tumors were histologically of the capillary, and partly the cavernous, type of hemangioma. However, endothelial cell growth was prominent in some areas, especially in the lung, and the histology of the lung tumor resembled epithelioid hemangioendothelioma or intravascular bronchiolo-alveolar tumor (IVBAT). In the endocardium of the right atrium, an endothelial tumorous projection was observed, and there were tiny foci of tumor cell nests in the abdominal venous wall. Small lymphangiomas were also found in the subcapsular region of the spleen. These findings suggest that there had been an abnormal proliferation of systemic endothelial cells and that tumors of endothelial cell origin with diverse histological patterns developed with this condition as a background. The autopsy finding of fibrin thrombi in multiple organs as well as laboratory data including thrombocytopenia suggest that this case belongs to the "Kasabach-Merritt syndrome."