Retroviral transfer of HSV1-TK gene into human lung cancer cell line

We used a recombinant retrovirus as one of the potential vectors for human gene therapy to transfer a drug sensitivity gene into human lung cancer cells. The gene encoding the thymidine kinase (TK) of herpes simplex virus type 1 (HSV1) was used as the drug sensitivity gene. The antiherpes drugs acyclovir (ACV) and ganciclovir (GCV) were chosen to test the HSV1-TK activity transferred into the human lung cancer cell lines. The rationale for this approach was that ACV and GCV are nucleoside analogs specifically converted by HSV1-TK to a toxic form capable of inhibiting DNA synthesis or disrupting cellular DNA replication. The results obtained from our experiments demonstrate that the retroviral vector-mediated HSV1-TK gene transfer leads to ACV- and GCV-dependent cytotoxicity in human lung cancer cell lines, including both small-cell carcinoma and nonsmall-cell carcinoma. Although the gene transfer of HSV1-TK gene into tumor cells would be one model for gene therapy to control lung cancer, further investigations are necessary for the proper choice of the therapeutic gene and vector targeting such as tumor cell specific delivery of the gene or tumor cell specific expression of the transduced gene.Abbreviations ACV Acyclovir - GCV Ganciclovir - HSV1 Herpes simplex virus type 1 - LTR Long terminal repeat - TK Thymidine kinase     

Molecular characterization and heterogeneity of feline immunodeficiency virus isolates

Summary We have molecularly cloned the complete genomic DNA of TM2 strain of feline immunodeficiency virus (FIV) isolated in Japan and compared its nucleotide and the deduced amino acid sequence with those of previously described U.S. isolates, FIV Petaluma and FIV PPR. The infectious molecular clone of FIV TM2 is different from FIV Petaluma in host cell range; the clone can not infect Crandell feline kidney cells which were permissive for FIV Petaluma. The amino acid sequence homologies, ingag, pol, andenv genes between FIV TM2 and Petaluma were 90%, 87%, and 81%, respectively. On the other hand, comparative analysis of each gene between FIV Petaluma and PPR showed 96, 95, and 85%, respectively. These results suggested that the genomic diversity was present among FIV strains isolated from geographically distant areas. Interestingly,tat- andrev-like short open reading frames contained in-frame stop codons in the FIV Petaluma but not in the FIV TM2.     

Distribution of Antigens Detected with MB1, MB2 and MB3 on Non-hematopoietic Human Organs and Various Tumors

We have examined the distribution of antigens detected by MB1, MB2 and MB3 on non-hematopoietic normal human tissues and various types of benign and malignant tumors. MB1 and MB2 reacted with various organs, such as the epithelium of various glands, smooth muscle cells, vascular endothelial cells, and peripheral nerve tissue. The distributions of these two antibodies were essentially identical. Reactivity with MB3 was confined to the ductal eDithelium of salivary glands, the pancreas, and sweat glands, and the cortex of the adrenal gland. lmmunoblotting analysis demonstrated that MB1 and MB2 reacted with a few bands of an extract of myometrial cytoskeletal fraction and salivary gland cytosol fraction, whereas MB3 failed to show any bands on these materials. The reactivities of MB1 and MB2 with various neoplasms were similar to those in normal organs, with slight variations of staining pattern and preponderance in well differentiated tumors. Exceptionally, carcinoid tumor and small round cell tumors, such as small cell carcinoma or neuroblastoma, were not reactive with MB1 and MB2. MB3 reacted with several cases of well differentiated benign and malignant epithelial tumors in various organs, and exceptional cases of malignant schwannoma and glioma. These results indicate that the antigens detected by MB1 and MB2 are distributed broadly on non-hematopoietic normal organs, whereas those detected by MB3 are confined to exceptional cases of epithelial and non-epithelial tumors. Thus, although the use of MB1, MB2 and MB3 is of little value for differential diagnosis of various tumors, these three antibodies may be useful for determining of the origin of some tumor types. Acta Pathol Jpn 42: 339–346, 1992.     

Preparation of blood-compatible hollow fibers from a polymer alloy composed of polysulfone and 2-methacryloyloxyethyl phosphorylcholine polymer

Blood-compatible hollow fibers were successfully prepared from a polymer alloy composed of polysulfone (PSf) and the 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer. To improve the hydrophilicity, fouling-resistance characteristics, and blood compatibility of the PSf hollow fiber in a hemodialyzer, an MPC polymer that can be blended with PSf was synthesized in order to prepare the polymer alloy (PSf/MPC polymer). The contents of the MPC polymer blended in the PSf were 7 and 15 wt%. The PSf/MPC polymer hollow fiber could be prepared by both wet and dry-wet processing methods. The hollow fiber took an asymmetric structure, that is, the hollow-fiber membrane had a dense skin layer on the porous sponge-like structure. The mechanical strength was higher than that of conventional PSf hollow fibers for hemodialysis. The surface characterization of the PSf/MPC polymer hollow fiber by x-ray photoelectron spectroscopy revealed that the MPC units were concentrated at the surface. The permeability for solutes through the PSf/MPC polymer hollow fibers was measured for 4 h. The permeabilities of both a low-molecular-weight compound and protein were greater than those of the PSf hollow fibers. The amount of adsorbed protein was lower on the PSf/MPC polymer hollow fiber when compared to that of the PSf hollow fiber. Moreover, platelet adhesion was also effectively inhibited on the PSf/MPC polymer hollow fiber. Based on these results, the addition of the MPC polymer to the PSf is a very useful method to improve the functions and blood compatibility of the hollow fiber.     

High incidence of pre-excitation syndrome in Japanese families with Leber's hereditary optic neuropathy

Cardiac conduction abnormalities have been reported in families with Leber's hereditary optic neuropathy (LHON). The pre-excitation syndrome, Wolff-Parkinson-White syndrome or Lown-Ganong-Levine syndrome, is reportedly common in Finns with LHON, being seen in 14 (9%) of the 163 individuals with mitochondrial DNA (mtDNA) mutations. While this syndrome is thought to be rare in other ethnic groups with LHON, the present study of 35 Japanese LHON families confirmed that it is also relatively common among Japanese families, being seen in 5 (8%) of the 63 individuals with mtDNA mutations. It remains to be determined whether the high incidence of the pre-excitation syndrome in Finnish and Japanese LHON families is due to a particular genetic composition of ethnic groups such as in Finland and in Japan, or only to a reporting bias.     

Augmentation of antibody responses of mice to inhaled protein antigens by simultaneously inhaled bacterial lipopolysaccharides

Serum antibody responses of mice to repeatedly inhaled protein antigens such as bovine serum albumin and ovalbumin, plus or minus bacterial lipopolysaccharide (LPS) in the form of an aerosol were studied. Results showed that the levels of responses to inhaled protein antigens varied, depending on the mouse strain-antigen combination and that LPS inhaled simultaneously with the antigens definitely augmented the responses which were not otherwise very high. LPS extracted from Klebsiella O3 (LPS-K) but not LPS from Escherichia coli O55 (LPS-E), which was inhaled at the time of initial inhalation of antigen, significantly intensified the priming for the secondary antibody response to the antigen subsequently inhaled. Both LPS-K and LPS-E, however, definitely acted to augment the response when they were inhaled repeatedly together with the antigen. Oral administration of antigen or antigen plus LPS-K did not induce any detectable antibody response in our experiment, ruling out the possibility that the antigen and LPS stimulated the immune system via alimentary canal rather than via lung. Tissue distribution of the radioactivity soon after inhalation of 131I-labeled antigen and decay speed of the radioactivity were not significantly changed by LPS-K inhaled simultaneously. This suggested that the augmentation of responses was not mediated by the action of LPS to modulate the air-blood barrier against the entry of antigen via lung. All the results prove for the first time that inhaled LPS displays a definite adjuvant action on antibody responses to inhaled antigens.     

The appearance and role of gamma delta T cells in the peritoneal cavity and liver during primary infection with Listeria monocytogenes in rats.

We have previously reported that gamma delta T cells play important roles in protection during the early stage of infection with Listeria monocytogenes in mice. To generalize the protective roles of gamma delta T cells in listerial infection to different species, we examined the appearance of gamma delta T cells during infection with L. monocytogenes in Fisher F344 rats. The numbers of bacteria in the peritoneal cavity and liver increased to a maximum level on day 3 and then decreased to an undetectable level by day 10 after an intraperitoneal infection with a sublethal dose (1 x 10(8)) of viable L. monocytogenes in rats. CD3+ alpha beta- T cells in the peritoneal cavity and liver began to increase on day 3, reached a maximum level on day 6, and thereafter decreased gradually by day 10 after infection. Northern blot analysis confirmed that the CD3+ alpha beta- T cells expressed TCR delta and gamma gene messages. In vivo treatment with anti-TCR alpha beta mAb, which suppressed most of the alpha beta T cells in the periphery and impaired resistance during the late stage of listerial infection, did not affect the host defense by day 6 after infection. A significantly increased number of gamma delta T cells was detected in the peritoneal cavity of the TCR alpha beta-suppressed rats on day 6 after infection. These results suggest that the early appearing gamma delta T cells may contribute to the host defense at a relatively early stage during listeriosis in rats.     

Genetic and stimulatory cell type requirements for inducing class I major histocompatibility complex alloantigen-specific in vivo cytotoxic T cell immunity

Current interpretation based on analytical in vitro works that actions of Ia antigens and accessory cells such as macrophages and dendritic cells are crucial for inducing cytotoxic T cell responses to class I major histocompatibility complex (MHC) alloantigens has been challenged by experiments performed in a newly developed system handling in vivo cytotoxic T cell immunity. We first characterized the transplantation immunity for second-set rejection of ascitic tumor allografts as principally induced by allogeneic stimulator cells via direct pathway, and as exclusively mediated by class I MHC alloantigen-specific in vivo cytotoxic T cell activity. By comparison of activities of limiting effective doses (10(4)-10(5) cells per mouse) of various stimulator cells in this defined system, we could demonstrate that genetic disparity at the D region of H-2 to the recipient is just enough for inducing the immunity, and presence of allogeneic or syngeneic Ia antigens in addition to H-2D alloantigens on stimulator cells does not give any premium effect. Further study revealed that allogeneic peritoneal cells rich in macrophages or glass-adherent spleen cells enriched for dendritic cells are not stronger stimulators than allogeneic adherent cell-depleted spleen cells and semi-allogeneic thymocytes. These results fit with the alternative concept that the physiological pathway inducing in vivo cytotoxic T cell immunity for graft rejection entirely depends on class I MHC antigens on live lymphocytes as self-supported stimulators, and does not crucially involve additional stimulator activities of Ia antigens and special accessory cell types, which must be in vivo concerned with induction of other types of transplantation immunity.