Stability of a Recombinant Adenoviral Vector: Optimization of Conditions for Storage, Transport and Delivery

Recombinant viral vectors have been developed for use as therapeutic agents and for the introduction of exogenous genes into living cells. However, little is known about the viability and stability of such recombinant viruses during storage, transport and delivery under various conditions. We describe here an analysis of the stability of an adenoviral vector in crude solutions of cell lysates during freezing and thawing and during storage at various temperatures in the presence and in the absence of glycerol. For example, the titer of adenoviruses in crude lysates of infected cells was reduced only ten-fold or three-fold after two hundred rounds of freezing and thawing or after incubation at 28°C for 14 days, respectively. Our observations indicate that recombinant adenoviral vector was more stable than expected both during freezing and thawing and during storage at low temperatures. Our results confirm the importance of appropriate conditions for the delivery and transport of recombinant adenoviral vectors.     

Association of a haplotype (196Phe/532Ser) in the interleukin-1-receptor-associated kinase (IRAK1) gene with low radial bone mineral density in two independent populations.

Osteoporosis, a multifactorial common disease, is believed to result from the interplay of multiple environmental and genetic determinants, including factors that regulate bone mineral density. Interleukin-1 (IL-1) is one of the most potent bone-resorbing factors, and interleukin-1-associated kinase 1 (IRAK1) is an essential effector of the IL-1 receptor signaling cascade. In genetic studies of two independent populations of postmenopausal women (cohort A: 220 individuals and cohort T: 126 individuals) from separated geographical regions of Japan, we found that radial bone mineral density levels had similar associations with IRAK1 genotypes in both populations. Two amino acid-substituting variations in the gene, encoding Phe196Ser and Ser532Leu, were in complete linkage disequilibrium (D' = 1.0000, r2 = 1.0000, chi2 = 192.000, p = 1.2 x 10(-43)), and we found two exclusive haplotypes (196F/532S, frequency 0.74; 196S/532L, frequency 0.26) of the IRAK1 gene among our test subjects. In both populations, a significant association with decreased radial bone mineral density was identified with haplotype 196F/532S (in cohort A: r = 0.21, p = 0.0017; in cohort T: r = 0.23, p = 0.011). Radial bone mineral density was lowest among 196F/532S homozygotes, highest among 196S/532L homozygotes, and intermediate among heterozygotes. Accelerated bone loss also correlated with the 196F/532S haplotype in a 5-year follow-up. These results suggest that variation of IRAK1 may be an important determinant of postmenopausal osteoporosis, in part through the mechanism of accelerated postmenopausal bone loss.     

Suppression of glutathione S-transferase placental form-positive foci development in rat hepatocarcinogenesis by Chlorella pyrenoidosa

The modifying effects of dietary administration of dried Chlorella pyrenoidosa powder (C. pyrenoidosa) on the development of glutathione S-transferase placental form-positive foci (GST-P-positive foci), which are putative preneoplastic lesions, in male F344 rats were investigated using a medium-term liver bioassay system. In rats given 10% C. pyrenoidosa in a basal diet, the number and area of GST-P-positive foci in the rat livers, which diethylnitrosamine (DEN) initiated and 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline (MeIQx) promoted, were significantly decreased compared with those fed a basal diet not containing C. pyrenoidosa. The inhibition percentage of the number and area of GST-P-positive foci > or =0.2 mm in diameter was 67.6 and 74.2%, respectively (p<0.01). Furthermore, C. pyrenoidosa significantly decreased the number of GST-P-positive foci induced by MeIQx alone. The inhibition percentage of the number of GST-P-positive foci <0.2 mm in diameter was 52% (p<0.01). These results suggest that C. pyrenoidosa has chemopreventive effects against hepatocarcinogenesis in rats. C. pyrenoidosa appears to be a promising chemopreventive agent for human liver neoplasia and carcinogenesis induced by heterocyclic amines such as MeIQx.     

A FAMILY OF GARDNER'S SYNDROME WITH REPORT OF AN AUTOPSY CASE

A family with Gardner's syndrome including one autopsy case is reported. A 22 year-old man had multiple polyposis of the colon and the small intestine, epidermal cysts, soft part tumor of the abdominal skin and osteomas in the calvarlum and rib, and died of a carcinoma in the transverse colon and embolism of the brain. Histologically, all polyps were diagnosed as adenomatous polyp. The greater their diameter, the higher the tendency of atypism was observed. However, the polyps around the cancer were few, small, and with low atypism, and we could not obtain evidence showing direct transformation of adenomatous polyps into carcinoma. His two elder brothers had multiple polyposis of the colon, epidermal cysts and osteomas in the calvarlum or mandibular bone, but are still alive.     

BMP signals inhibit proliferation and in vivo tumor growth of androgen-insensitive prostate carcinoma cells

Prostate cancer is one of the most common cancers in men. Several lines of evidence have suggested that bone morphogenetic protein (BMP) signals play important roles in the generation and progression of prostate cancers. In the present study, we show that BMP-7 inhibits the proliferation of androgen-insensitive PC-3 and DU-145 prostate cancer cells in a medium containing 1% fetal bovine serum, observed as decreased incorporation of [(3)H]thymidine and decreased cell number. Cell cycle analysis by flow cytometry showed an increased fraction of cells in the G1 phase and subsequent decrease in both S and G2/M phase after BMP-7 stimulation. BMP-7 caused an upregulation of the cyclin-dependent kinase inhibitor (CDKI) p21(CIP1/WAF1), and decreased the activity of Cdk2, leading to hypophosphorylation of Rb proteins. Furthermore, in order to evaluate the impact of BMP signals on prostate tumor growth, we generated the PC-3 cell lines expressing a constitutively active BMP type I receptor (constitutively active (c.a.) activin receptor-like kinase (ALK)-6) in a tetracycline (Tet)-regulated manner. Tet/doxycycline-regulated expression of c.a.ALK-6 resulted in the inhibition of in vitro cell proliferation and reduction of the size of tumors derived from the PC-3 cells subcutaneously injected into immune-deficient mice. Collectively, these findings suggest that BMP signals inhibit growth and proliferation of prostate tumor cells through induction of CDKI. Furthermore, this is the first report of a role for BMP signaling in reducing growth kinetics of androgen-insensitive prostate tumors.     

Expression of the interleukin-21 gene in murine colon carcinoma cells generates systemic immunity in the inoculated hosts

Interleukin-21 (IL-21) is a novel cytokine that can induce proliferation of activated T cells and maturation of natural killer (NK) cells. We therefore examined whether expression of the IL-21 gene in tumor cells could generate antitumor responses. Murine colon carcinoma Colon 26 cells that were transduced with the mouse IL-21 gene (Colon 26/IL-21) were rejected in syngeneic mice and the mice subsequently acquired protective immunity. The growth of Colon 26/IL-21 tumors developed in nude mice was retarded compared with that of parent tumors, and this growth suppression was not observed in nude mice that were treated with anti-asialo GM(1) antibody. Spleen cells from the mice that had rejected Colon 26/IL-21 cells showed cytotoxic activity to Colon 26 but not to irrelevant tumor cells, and produced larger amounts of interferon-gamma upon stimulation with irradiated Colon 26 cells. Spleen cells from Colon 26/IL-21-tumor- but not parent-tumor-bearing mice had lytic activity to YAC-1 cells. These data suggest that expression of IL-21 in tumors induces T- and NK-cell-dependent antitumor effects.     

Epidemic study of pet allergy in Wakayama Prefecture

In Japan, the number of households who have pets has gradually increased, together with the number of people who have pet allergies. Many reports exist on pollen and mite allergy, but few on pet allergy. We conducted an epidemic study in 531 first-year junior high school students in Wakayama prefecture in 1999. Questionnaires covered allergy and measurement of total IgE antibody using CAP system (Pharmacia Co. Ltd.) and specific IgE antibody using MAST26 system (Hitachi chemical Co. Ltd.). Of 306 students having pets, 11 were allergic to dogs, 11 to cats, 8 to both, and 1 to rabbits. Clinical symptoms were various. No differences in symptoms were observed among allergens. Serum total IgE tended to increase in students who suffered from pet allergies. Positive rates of specific IgE antibodies were high in mites and Japanese cedar pollen (36.7% for mites and 37.0% for Japanese cedar pollen), and also in dogs and cats (15.4% for dogs and 18.2% for cats). Specific dog and cat IgE antibodies increased significantly (p = 0.033 for dog and for p < 0.0001 cat), but no significant correlation was found between the positive specific IgE antibody and history of pet keeping.     

ULTRASTRUCTURAL STUDY OF LYMPHOCYTIC INTERACTION WITH HEPATOCYTES AND ENDOTHELIAL CELLS IN ACUTE NON-A, NON-B HEPATITIS

In the liver biopsy specimens of all six patients with acute non-A, non-B hepatitis, the lymphocytic interaction with hepatocytes and sinusoidal endothelial cells was observed by electron microscopic study. Lymphocytes were in a close contact with damaged hepatocytes and interrupted endothelial cells, and the microvilli on the surface of these damaged hepatocytes were degenerated and lost. These findings pointed out the possibility that the lymphocyte may play one of the important roles in hepatocytic damage and endothelial cell damage in acute non-A, non-B hepatitis.