Generation of functional and mature dendritic cells from cord blood and bone marrow CD34+ cells by two-step culture combined with calcium ionophore treatment

The object of this study is to explore a culture method to generate a large number of functional and mature dendritic cells (DC) from human CD34+ hematopoietic progenitor cells. In the present study, we used a two-step method combined with calcium ionophore to induce DC from cord blood (CB) or normal human bone marrow (BM) CD34+ progenitor cells. The two-step method consists of 10 days of first step culture for the expansion and proliferation of CD34+ hematopoietic progenitor cells in the presence of SCF, IL-3, IL-6, G-CSF, and 7--11 days of second step culture for the induction of DC in the presence of GM-CSF, IL-4 and TNF-alpha. By the two-step culture, total nucleated cells were increased 208+/-66 (+/-SD, n=13), or 94+/-29 (n=5)-fold in the culture of CB or BM cells, respectively, compared with the number of CD34+ cells at the time of starting culture. Out of the total nucleated cells, 23 +/-10.4% of cells in CB cell culture and 25 +/-5% of cells in the BM cell culture acquired DC characteristic phenotypes, which were marked expressions of CD1a, HLA-DR, co-stimulatory molecules such as CD80, CD40, and adhesion molecule such as CD58. In allogeneic mixed leukocyte reaction (MLR), two-step cultured cells showed potent allo-stimulatory capacity. With this two-step culture, the absolute number of CD1a+ cells that co-expressed HLA-DR, CD80, CD40 and CD58 was enhanced approximately 3 times in CB cell culture and 1.9 times in BM cell culture, compared with the commonly used one-step culture method for the generation of DC from CD34+ cells using SCF, GM-CSF and TNF-alpha. However, on these DC generated in the two-step culture, the expressions of co-stimulatory molecule CD86 and mature DC marker CD83 were not sufficient. By the treatment of two-step cultured cells with calcium ionophore agent (A23187), the expression of co-stimulatory molecules such as CD86 and CD80 (especially CD86) was up-regulated. Besides, the expression of mature DC marker CD83 was remarkably induced by treatment with A23187 for a short duration (24 h). Consistent with the up-regulation of surface molecules CD86, CD80 and CD83, the two-step cultured cells treated with A23187 also showed a stronger allo-stimulatory capacity compared with the cells without A23187 treatment. In conclusion, the present study demonstrated that the two-step culture method effectively improved the yield of CD1a+ DC generated from CD34+ cells, and the phenotypes and functions of these CD1a+ DC could be enhanced efficiently by treatment with a calcium ionophore agent.     

The inhibitory effects of isoflavonoids and resveratrol on oxdized low-density lipoprotein uptake in macrophage cell line J774.1

The interaction between oxidized low-density lipoprotein (LDL) and macrophages are known to be important in the development of arteriosclerosis. Macrophages take up oxidized LDL, becoming foam cells, which contribute to the thickening of the blood vessel wall. The antioxidant properties of polyphenols found in vegetables and other foods have been shown to have a protective effect against arteriosclerosis. To elucidate the effect of flavonoids from fruits, vegetables and cereals on oxidized LDL uptake in macrophages, the inhibitory activity of various flavonoids on DiI-ac-LDL uptake reaction in mouse macrophage cell line J774.1 was measured. We found significant uptake of DiI-ac-LDL, but not DiI-LDL, into the J774.1 cells. In addition, polyinosin and dextran sulphate inhibited uptake, but apigenin, kaempferol, and naringenin, did not. Isoflavones and resveratrol significantly inhibit uptake of DiI-ac-LDL into macrophages, and have a protective effect against arteriosclerosis.     

Twenty-six-Week carcinogenicity study of chloroform in CB6F1 rasH2-transgenic mice

The carcinogenic potential of chloroform was evaluated in a short-term carcinogenicity testing system using CB6F1 rasH2-Tg (rasH2-Tg) mice. Chloroform was administered to rasH2-Tg males at doses of 28, 90, or 140 mg/kg and rasH2-Tg females at 24, 90, or 240 mg/kg by oral gavage for 26 weeks. Wild-type (non-Tg) male and female mice received doses of 140 mg/kg and 240 mg/kg, respectively. N-methyl-N-nitrosourea was administered to rasH2-Tg mice by single intraperitoneal injection (75 mg/kg) as a positive control. In both the rasH2-Tg and non-Tg mice, there was no significant increase in the incidence of neoplastic lesions by chloroform treatment. The incidence of hepatocellular foci in the rasH2- and non-Tg females receiving 240 mg/kg was increased. Forestomach tumors and malignant tumors occurred in most of the rasH2-mice in the positive control group. Swelling or vacuolation of hepatocytes, a toxic change induced by chloroform, occurred in both the rasH2-Tg and non-Tg mice. It is concluded that chloroform, a putative human noncarcinogen, did not show evidence of carcinogenic potential in the present study using rasH2-Tg mice. This study suggests that the rasH2-Tg mouse model may not be appropriate for detecting nongenotoxic carcinogens. However, the sensitivity of rasH2-Tg mice to nongenotoxic carcinogens should be assessed with consideration of the results from the other ILSI-HESI project studies.     

Preparation and physicochemical properties of compression-molded keratin films

The S-sulfo keratin was extracted from wool and was then spray-dried to give S-sulfo keratin powder. Differential scanning calorimetry analysis showed that the glass transition temperature of S-sulfo keratins became lowered with the increase of moisture content, while perfectly dried S-sulfo keratin powder did not give thermal transition in the temperature range 30-130 degrees C. The compression molding of the S-sulfo keratin powder supplemented with one-tenth weight of water afforded a plastic-like transparent proteinous film above the glass transition temperature. The film obtained from the powder without water addition or compression molded below glass transition temperature partly remained powdery. The film compression molded at 120 degrees C gave the maximum ultimate strength and Young's modulus, 27.8 +/- 2.9 and 1218 +/- 80 MPa, respectively. Obtained film was insoluble and slightly swelled in water, but, in the presence of reducing agent, the film significantly swelled at pH 7.0 and even dissolved at pH 9.0, suggesting the relevance of abundant disulfide linkage. The film supported the mammalian cell adhesion and proliferation, demonstrating the biocompatibility of S-sulfo keratin films.     

Relationships between oral allergy syndrome and sensitization to pollen antigen, especially to mugwort]

BACKGROUND: In order to know the relationships between mugwort pollinosis and oral allergy syndrome (OAS), an etiological study was performed at Muroran City, where mugwort is the most frequent cause of pollinosis. METHODS: Allergic rhinitis patients positive to serum IgE to birch, mugwort or grass pollen visited to the outpatient-clinic of Otorhinolaryngology of Muroran City General Hospital from 1998 to 2002, were studied by a questionnaire concerning a past-history of OAS. RESULTS: The prevalence of OAS was significantly higher in patients positive to serum IgE antibody specific to birch pollen or mugwort pollen than those negative to each pollen-specific antibody (birch; 54.5 vs 23.5%, p<0.0001, mugwort; 41.0 vs 21.5%, p<0.01). The main causative foods were fruits of rose family in patients with only birch pollen-specific IgE antibody, and were those other than rose family, such as kiwi, melon, orange, celery and onion in those with only mugwort pollen-specific IgE antibody. The patients group with high Lumiward score to mugwort pollen tended to include severe OAS cases. CONCLUSION: A close relationship was suggested between mugwort pollen sensitization and OAS.     

Pathogenesis of pancreatic perilobular necrosis in patients with liver disease.

We often see perilobular necrosis of the pancreas in patients with liver disease at autopsy. This study was undertaken to determine the frequency and the mechanism of development of pancreatic perilobular necrosis in patients with liver disease. Pancreatic perilobular necrosis was seen in 21 per cent of 261 autopsied patients: in 41 per cent of 73 autopsied patients with liver disease and in 13 per cent of 188 autopsied patients without liver disease. Moreover, splanchnic congestion was present in 90 per cent of 30 pancreatic perilobular necrosis patients with liver disease. These data indicate that patients with liver disease develop perilobular necrosis of the pancreas more often than patients without liver disease, and that the high frequency may be a sequela of splanchnic congestion; that is, congestion of the pancreas and endotoxaemia due to congestion of the gut.     

Development of a diagnostic test for Entamoeba histolytica using idiotype expression in human

The protozoan parasite Entamoeba histolytica is the etiological agent of human amebiasis. The pathology of the disease starts with the cytolysis of the host target cells by amoebae. It is initiated by the adhesion of trophozoites to the host cells, through surface lectin via specific receptors. These adherence lectins have been demonstrated to be highly conserved, and can be recognised by serum antibodies from patients with invasive amebiasis.Some of these molecules have been used as antigens in serologic studies, which has been very helpful in the diagnosis of invasive intestinal amebiasis. However, false-positive serologic reactivity can occur using E. histolytica extracts and purified antigens. Additional problems are because the extracts display a great enzymatic activity. Several diagnostic methods, using different molecules and techniques, have been described. However, the problem still remains since these tests are not capable of differentiating between amoebic liver abscess (ALA) and intestinal amebiasis.Here, the research has been addressed to the 66-kDa antigen, which is a part of the outer membrane proteins from the E. histolytica strain HM1-IMSS trophozoites. First of all, we characterized the 66-kDa antigen in order to prove the relevance. We found that the 66-kDa antigen is a part of the plasma membranes and is distributed rather homogeneously on the cell surface of trophozoites. Apparently, the 66-kDa antigen is a glycoprotein. Using a monoclonal antibody (MAb), we found 25% of inhibition in the erythrophagocytosis by the trophozoites.Starting form one monoclonal antibody, we prepared an anti-idiotype (anti-Id) antibody reagent, with the purpose of searching for the different expressions of the idiotype between the sera from ALA and the intestinal amebiasis patients. Moreover, we produced the antibody Ab3 that is capable of recognising the 66-kDa antigen; it means that the Ab2 displays the internal image of the antigen. We found that 91.6% of the serum from ALA patients displayed the expression of the Id. In contrast, 15.7% of the E. histolytica asymtomatic cyst carriers displayed the Id expression, 6.6% of the patients with another parasite infection, and 11% of the negative controls (serum from umbilical cords of newborn babies). Our results showed that the expression of the Id could be differentiated among the AHA patients from the other groups with a 91.6% sensibility and 88.3% specificity.     

The pathogenesis of hyaline globules in liver cells after partial hepatectomy in rats

To clarify the pathogenesis of hyaline globules in liver cells after partial hepatectomy, the present study was undertaken in rats. When partial hepatectomy was performed by the surgical procedure of Higgins and Anderson, the inferior vena cava pressure was raised, and many hyaline globules, which are very similar to those caused by condensation of the contents of vacuoles in liver cells after temporary pressure elevation of the inferior vena cava, were induced in liver cells. On the other hand, when the median lobe and the left lateral lobe were removed carefully one at a time to avoid operative narrowing of the inferior vena cava, the inferior vena cava pressure was not elevated, and only very few hyaline globules were formed. This suggests that hyaline globules in liver cells following partial hepatectomy in rats may be caused by elevation of inferior vena cava pressure due to narrowing of the inferior vena cava by surgical procedure.     

A study of endotoxin-associated hepatotoxicity on proliferating hepatocytes.

It is thought that regeneration of the liver provides a state of preparedness for the Shwartzman reaction and contributes to the development of endotoxin-associated massive hepatic necrosis following partial hepatectomy. Therefore we examined endotoxin hepatotoxicity in rats with hepatic regeneration after 35% hepatectomy and in rats with liver cell proliferation induced by lead nitrate. Biochemical and histopathological studies showed no enhanced endotoxin hepatotoxicity in either partially hepatectomized rats or in rats with lead nitrate-induced liver cell proliferation. These results indicate that the development of endotoxin-associated hepatic damage after partial hepatectomy may not relate to regeneration and proliferation of the liver.