Immunomagnetic exclusion of E-cadherin-positive hepatoblasts in fetal mouse liver cell cultures impairs morphogenesis and gene expression of sinusoidal endothelial cells

Previous studies have shown that various cell-cell interactions between hepatoblasts and nonparenchymal cells, including sinusoidal endothelial cells and stellate cells, are indispensable for the development of fetal murine hepatic architecture. The present study was undertaken to determine the effects of hepatoblasts on the sinusoidal structural formation using a culture system of fetal mouse livers. Primitive sinusoidal structures extensively developed in fetal livers, and were composed of LYVE-1- and PECAM-1-positive endothelial cells, desmin-positive stellate cells and F4/80-positive macrophages. When fetal liver cells at 12.5 days of gestation were cultured in vitro, hepatoblasts spread on glass slides and gave rise to hepatocytes on day 5. Desmin-positive stellate cells also spread on the glass slides. PECAM-1-positive endothelial cells became slender and developed into anastomosing capillary networks. When fetal liver cells were cultured without hepatoblasts, which were excluded by an immunomagnetic method using anti-E-cadherin antibodies, endothelial cells had impaired growth and capillary formation. These results demonstrated that capillary formation of endothelial cells was induced by the presence of hepatoblasts. VEGF and the conditioned medium containing humoral factors produced by hepatoblasts/hepatocytes did not induce capillary formation of endothelial cells in cultures of nonparenchymal cells, although they significantly increased the number of endothelial cells on the glass slides. The presence of hepatoblasts also significantly stimulated expression of CD32b mRNA, which is a sinusoidal endothelial marker. Hepatoblasts may work as a positive stimulator of sinusoid morphogenesis and maturation in liver development, in which a signal other than VEGF may play a decisive role, together with VEGF.     

The role of tumor necrosis factor (TNF)-α in the antitumor effect of intrapleural injection of Lactobacillus casei strain Shirota in mice

The involvement of several cytokines in the antitumor effect induced by intrapleural (i.pl.) injection of heat-killed cells of Lactobacillus casei strain Shirota (LC 9018) in mice was investigated. Injection of LC 9018 i.pl. into Meth A fibrosarcoma (Meth A)-bearing mice not only significantly prolonged the survival of the mice, but also effectively inhibited the accumulation of malignant pleural fluid in the thoracic cavity. In the thoracic cavity of tumor-bearing mice treated with LC 9018, we observed large amounts of several cytokines including interleukin (IL)-1β, interferon (IFN)-γ, IL-12 and tumor necrosis factor (TNF)-α. Both anti-IFN-γ and anti-IL-12 monoclonal antibody (mAb) treatments partially diminished the antitumor activity of LC 9018 in vivo, while the treatment of anti-IL-1β mAb did not influence the survival of the mice. However, anti-TNF-α mAb treatment completely abolished the antitumor effect of LC 9018 in vivo, suggesting that in this model LC 9018 has a survival-prolonging effect involving certain cytokines. Moreover, i.pl. injection of mouse recombinant TNF-α into Meth A-bearing mice pretreated with anti-TNF-α mAb partially restored the survival-enhancing effect of LC 9018. These results led us to conclude that TNF-α induced by i.pl. injection of LC 9018 plays an important role in the antitumor effect of LC 9018 in vivo. Received: 22 February 1999     

Diminution of experimental autoimmune uveoretinitis (EAU) in mice depleted of NK cells

To evaluate the potential role of NK1.1 (CD161c) cells in autoimmune uveoretinitis, we treated experimental autoimmune uveoretinitis (EAU)-susceptible mice with anti-CD161c antibodies (PK136) to deplete natural killer (NK) cells. Injection of anti-CD161c antibodies deleted NK cells from the peripheral blood of EAU-susceptible mice. The T cell proliferative response against the ocular autoantigen K2 was not suppressed in mice treated with anti-CD161c antibody when compared with T cells from control mice. Although mice treated with anti-CD161c developed EAU, the clinical severity on days 17 and 19 after induction of EAU was significantly mild in anti-CD161c-treated mice compared with control mice. In addition, the histopathological severity of EAU was significantly milder in mice treated with anti-CD161c antibodies than controls 21 days after induction of EAU. Our results indicate that the severity of EAU is augmented by NK1.1(+) NK cells.     

Vaccination of mice with MUC1 cDNA suppresses the development of lung metastases

C57BL/6 mice were immunized intradermally with various doses of purified pCEP4 plasmid DNA containing full-length MUC1 cDNA (22 tandem repeats). Mice immunized with MUC1 DNA three times at weekly intervals had serum antibodies to a synthetic peptide corresponding to the tandem repeats of MUC1. The antibody titer correlated with the plasmid DNA dose. After the third immunization mice were injected intravenously with 5×10⁵ B16-F10 melanoma cells that had been stably transfected with MUC1 cDNA (F10-MUC1-C8 clone cells). The number of lung metastatic nodules three weeks after inoculation of F10-MUC1-C8 cells was significantly lower in mice immunized with MUC1 plasmid DNA than in mice immunized with the vector DNA alone. Thus, the suppression of lung metastasis was antigen-specific. In vivo depletion of lymphocyte subpopulations by specific antibodies revealed that natural killer cells are the major effector cells responsible for the suppression of lung metastasis. CD4⁺ cells and CD8⁺ cells apparently played some roles too. This revised version was published online in July 2006 with corrections to the Cover Date.     

Different Ca2+ dynamics between isolated hippocampal pyramidal cells and dentate granule cells

The hippocampal formation contains a variety of neuronal types. The principal neurons are granule cells in the dentate gyrus and pyramidal cells in Ammon's horn. These two neuron types show distinct cell morphology and display a different vulnerability to ischemic injury or various neurotoxins. In order to illustrate the difference in the pathophysiological properties of these neurons, we established a method for separately culturing granule cells and pyramidal cells. They were prepared from the dentate gyrus and Ammon's horn of 3-day-old Wistar rat pups and maintained for 7–9 days in culture. After transient exposure to N-methyl-D-aspartate or glutamate, both the cultured neuron populations displayed somatic Ca²⁺ transients with similar amplitudes, but the subsequent recovery to baseline was about twice as fast in granule cells than in pyramidal cells. Similar results were obtained for K⁺ depolarization-induced Ca²⁺ elevation, suggesting that the relatively rapid Ca²⁺ clearance in granule cells is independent of Ca²⁺ influx pathways. The present study provides the first evidence for a difference in Ca²⁺ dynamics and homeostasis between granule and pyramidal cells and may represent a cellular basis for the differential vulnerability of hippocampal neurons.     

Association analysis of toll-like receptor 4 polymorphisms in Japanese primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is characterized by portal inflammation and immune-mediated destruction of intrahepatic bile ducts that often result in liver failure. Toll-like receptor (TLR) 4 recognizes lipopolysaccharides of Gram-negative bacteria. Infectious agents have been suspected to play a crucial role in PBC pathogenesis since TLR4 expression was found in bile duct epithelial cells and periportal hepatocytes in liver tissues of PBC. To assess the potential contribution of TLR4 SNPs to the development of this disease, we genotyped five SNPs in TLR4 in 261 PBC patients and 359 controls using a TaqMan assay. No significant positive associations with either PBC susceptibility or progression were uncovered. These results indicate that TLR4 polymorphisms do not play a prominent role in the development of PBC in Japanese patients.     

Comprehensive molecular analysis of Japanese patients with pediatric-onset MODY-type diabetes mellitus

Yorifuji T, Fujimaru R, Hosokawa Y, Tamagawa N, Shiozaki M, Aizu K, Jinno K, Maruo Y, Nagasaka H, Tajima T, Kobayashi K, Urakami T. Comprehensive molecular analysis of Japanese patients with pediatric‐onset MODY‐type diabetes mellitus. Background: In Asians, mutations in the known maturity‐onset diabetes of the young (MODY) genes have been identified in only <15% of patients. These results were obtained mostly through studies on adult patients. Objective: To investigate the molecular basis of Japanese patients with pediatric‐onset MODY‐type diabetes. Subjects: Eighty Japanese patients with pediatric‐onset MODY‐type diabetes. Methods: Mitochondrial 3243A>G mutation was first tested by the polymerase chain reaction restriction fragment length polymorphism analysis for maternally inherited families. Then, all coding exons and exon–intron boundaries of the HNF1A, HNF1B, GCK, and HNF4A genes were amplified from genomic DNA and directly sequenced. Multiplex ligation‐dependent probe amplification analysis was also performed to detect whole‐exon deletions. Results: After excluding one patient with a mitochondrial 3243A>G, mutations were identified in 38 (48.1%) patients; 18 had GCK mutations, 11 had HNF1A mutations, 3 had HNF4A mutations, and 6 had HNF1B mutations. In patients aged <8 yr, mutations were detected mostly in GCK at a higher frequency (63.6%). In patients >9 yr of age, mutations were identified less frequently (45.1%), with HNF1A mutations being the most frequent. A large fraction of mutation‐negative patients showed elevated homeostasis model assessment (HOMA) insulin‐resistance and normal HOMA‐β indices. Most of the HNF1B mutations were large deletions, and, interestingly, renal cysts were undetectable in two patients with whole‐gene deletion of HNF1B. Conclusion: In Japanese patients with pediatric‐onset MODY‐type diabetes, mutations in known genes were identified at a much higher frequency than previously reported for adult Asians. A fraction of mutation‐negative patients presented with insulin‐resistance and normal insulin‐secretory capacities resembling early‐onset type 2 diabetes.     

Relevance of inflammatory cell infiltrates for complete atrioventricular block in experimental murine myocarditis.

BACKGROUND: There are few systemic pathologic studies on myocarditis. This study aimed to clarify the pathologic characteristics of murine myocarditis. METHODS: We recorded serial electrocardiograms in experimental viral myocarditis in mice and then examined their cardiac pathology. After taking baseline electrocardiograms, we inoculated the mice intraperitoneally with the encephalomyocarditis virus. Electrocardiograms were serially recorded until 220 days after the virus inoculation. RESULTS: Serial electrocardiograms revealed ectopic beats, low voltage of the QRS complex, and the appearance of complete atrioventricular (AV) block. Corresponding myocardial lesions were found in the hearts of mice with these ectopic beats. Mononuclear cell infiltrations into the His bundle were most frequently found in mice with complete AV block. CONCLUSIONS: Inflammatory change with cellular infiltrations was the most common pathologic finding in mice with complete AV block. In clinical settings, anti-inflammatory therapy might be recommended for patients with myocarditis complicated with conduction disturbances.