Augumentation of splenic antitumor immunity by local immunotherapy in gastric cancer patients

We previously reported that the antitumor effect of OK-432, a streptococcal preparation, was markedly augmented when this agent was injected into tumors together with fibrinogen. In order to elucidate the effect of this treatment on the spleen, we assessed splenic function in gastric cancer patients receiving preoperative local immunotherapy with OK-432 and fibrinogen. Immunohistochemical studies of the spleen at 7 days after intratumoral injection therapy revealed numerous macrophages phagocytizing OK-432 in the splenic sinuses. Phenotypic analysis of splenocytes by flow cytometry revealed an increase in the CD4/CD8 ratio and in the expression of HLA-DR, CD25, and Leu M3 by splenic T cells of the patients treated with OK-432 plus fibrinogen when compared to patients treated with OK-432 alone or untreated patients. Splenic T cells from patients treated with OK-432 plus fibrinogen showed significantly higher cytotoxicity against Daudi and K562 cells than T cells from control patients (p<0.05), and culture of these splenic T cells with recombinant IL-2 induced the expansion of lymphokine-activated killer cells. These results demonstrate that local immunotherapy with a mixture of OK-432 and fibrinogen effectively augumented splenic antitumor immunity in gastric cancer patients.     

Involvement of the 3’ Untranslated Region in Encapsidation of the Hepatitis C Virus

Although information regarding morphogenesis of the hepatitis C virus (HCV) is accumulating, the mechanism(s) by which the HCV genome encapsidated remains unknown. In the present study, in cell cultures producing HCV, the molecular ratios of 3’ end- to 5’ end-regions of the viral RNA population in the culture medium were markedly higher than those in the cells, and the ratio was highest in the virion-rich fraction. The interaction of the 3’ untranslated region (UTR) with Core in vitro was stronger than that of the interaction of other stable RNA structure elements across the HCV genome. A foreign gene flanked by the 3’ UTR was encapsidated by supplying both viral NS3-NS5B proteins and Core-NS2 in trans. Mutations within the conserved stem-loops of the 3’ UTR were observed to dramatically diminish packaging efficiency, suggesting that the conserved apical motifs of the 3´ X region are important for HCV genome packaging. This study provides evidence of selective packaging of the HCV genome into viral particles and identified that the 3’ UTR acts as a cis-acting element for encapsidation.     

Studies on the Recombination Genes of Bacteriophage T4: Suppression of uvsX and uvsY Mutations by uvsW Mutations

Genes uvsW, uvsX and uvsY are dispensable for T4 growth but are implicated in recombination and in the repair of damaged DNA. We found that large-plaque mutants arose efficiently from small-plaque uvsX and uvsY mutants at 42 degrees and were pseudorevertants containing a new mutation in uvsW. Using reconstructed double mutants, we confirmed that a mutation in uvsW partially increases the burst size and UV resistance of uvsX and uvsY mutants. At 41 degrees the uvsW mutation completely restores the arrest in DNA synthesis caused by mutations in genes uvsX, uvsY and 46, but at 30 degrees it only partially restores DNA synthesis in a gene 46 mutant and does not restore DNA synthesis in uvsX and uvsY mutants. Restored DNA synthesis at 41 degrees was paralleled by the overproduction of single-stranded DNA and gene 32 protein. Based on these findings, we propose that the uvsW gene regulates the production of single-stranded DNA and we discuss the phenotype of uvsW mutants and their suppression of some uvsX and uvsY phenotypes. Infection of restrictive cells with am uvsW mutants revealed a defect in the synthesis of a protein of molecular weight 53,000 daltons, suggesting that this protein is the uvsW gene product.     

Impact of Cytotoxic-T-Lymphocyte Memory Induction without Virus-Specific CD4+ T-Cell Help on Control of a Simian Immunodeficiency Virus Challenge in Rhesus Macaques

Despite many efforts to develop AIDS vaccines eliciting virus-specific T-cell responses, whether induction of these memory T cells by vaccination before human immunodeficiency virus (HIV) exposure can actually contribute to effective T-cell responses postinfection remains unclear. In particular, induction of HIV-specific memory CD4⁺ T cells may increase the target cell pool for HIV infection because the virus preferentially infects HIV-specific CD4⁺ T cells. However, virus-specific CD4⁺ helper T-cell responses are thought to be important for functional CD8⁺ cytotoxic-T-lymphocyte (CTL) induction in HIV infection, and it has remained unknown whether HIV-specific memory CD8⁺ T cells induced by vaccination without HIV-specific CD4⁺ T-cell help can exert effective responses after virus exposure. Here we show the impact of CD8⁺ T-cell memory induction without virus-specific CD4⁺ T-cell help on the control of a simian immunodeficiency virus (SIV) challenge in rhesus macaques. We developed a prophylactic vaccine by using a Sendai virus (SeV) vector expressing a single SIV Gag_241-249 CTL epitope fused with enhanced green fluorescent protein (EGFP). Vaccination resulted in induction of SeV-EGFP-specific CD4⁺ T-cell and Gag_241-249-specific CD8⁺ T-cell responses. After a SIV challenge, the vaccinees showed dominant Gag_241-249-specific CD8⁺ T-cell responses with higher effector memory frequencies in the acute phase and exhibited significantly reduced viral loads. These results demonstrate that virus-specific memory CD8⁺ T cells induced by vaccination without virus-specific CD4⁺ T-cell help could indeed facilitate SIV control after virus exposure, indicating the benefit of prophylactic vaccination eliciting virus-specific CTL memory with non-virus-specific CD4⁺ T-cell responses for HIV control.Virus-specific T-cell responses are crucial for controlling human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication (3, 4, 12, 20, 28, 36, 37). Therefore, a great deal of effort has been exerted to develop AIDS vaccines eliciting virus-specific T-cell responses (23, 27, 30, 47), but whether this approach actually results in HIV control remains unclear (1, 6). It is important to determine which T-cell responses need to be induced by prophylactic vaccination for HIV control after virus exposure.Because HIV preferentially infects HIV-specific CD4⁺ T cells (5), induction of HIV-specific memory CD4⁺ T cells by vaccination may increase the target cell pool for HIV infection and could enhance viral replication (42). However, CD4⁺ helper T-cell responses are important for functional CD8⁺ cytotoxic-T-lymphocyte (CTL) induction (11, 40, 43, 46), and it has remained unknown whether HIV-specific memory CD8⁺ T cells induced by vaccination with non-virus-specific CD4⁺ T-cell help (but without HIV-specific CD4⁺ T-cell help) can exert effective responses after virus exposure. Indeed, the real impact of prophylactic induction of CTL memory itself on HIV replication has not been well documented thus far.We previously developed a prophylactic AIDS vaccine consisting of DNA priming followed by boosting with a recombinant Sendai virus (SeV) vector expressing SIVmac239 Gag (26). Evaluation of this vaccine''s efficacy against a SIVmac239 challenge in Burmese rhesus macaques showed that some vaccinees contained SIV replication whereas unvaccinated animals developed AIDS (15, 27). In particular, vaccination consistently resulted in control of SIV replication in those animals possessing the major histocompatibility complex class I (MHC-I) haplotype 90-120-Ia. Gag_206-216 (IINEEAADWDL) and Gag_241-249 (SSVDEQIQW) epitope-specific CD8⁺ T-cell responses were shown to be involved in SIV control in these vaccinated macaques (14, 16).In the present study, focusing on CD8⁺ T-cell responses directed against one of these epitopes, we have evaluated the efficacy of a vaccine expressing the Gag_241-249 epitope fused with enhanced green fluorescent protein (EGFP) against a SIVmac239 challenge in 90-120-Ia-positive rhesus macaques. The animals exhibited this single-epitope-specific CD8⁺ T-cell response and SeV-EGFP-specific CD4⁺ T-cell responses after vaccination and showed rapid, dominant induction of potent secondary Gag_241-249-specific CD8⁺ T-cell responses after a SIV challenge. Plasma viral loads in these vaccinees were significantly reduced compared to those of naive controls. These results indicate that induction of CD8⁺ T-cell memory without virus-specific CD4⁺ T-cell help by prophylactic vaccination can result in effective CD8⁺ T-cell responses after virus exposure.     

S19 ribosomal protein dimer augments metal-induced apoptosis in a mouse fibroblastic cell line by ligation of the C5a receptor

To analyze the role of S19 ribosomal protein (RP S19) in apoptosis, murine NIH3T3 were transfected with either hemagglutinin peptide-tagged (HA) wild-type human RP S19 or a mutant (Gln137Asn) that is resistant to transglutaminase-catalyzed cross-linked-dimerization. Transfection with the mutant HA-RP S19 inhibited manganese (II) (Mn II)-induced apoptosis whereas the wild-type HA-RP S19 augmented apoptosis and a mock transfection had no effect. Release of the wild-type HA-RP S19 dimer but not the mutant HA-RP S19 was observed during the apoptosis. The reduced rate of apoptosis of the cells transfected with the mutant HA-RP S19 was overcome by addition of extracellular wild-type RP S19 dimer. The apoptosis rates in cells transfected with either form of human HA-RP S19 and in mock transfectants were reduced to about 40% by the presence of anti-RP S19 antibody in the culture medium. Immunofluorescence staining and fluorescence-activated cell sorting (FACS) analysis showed that the cell surface expression of the receptor for cross-linked RP S19 dimer, C5a receptor, increased during apoptosis, concomitant with phosphatidylserine exposure. The expression of the C5a receptor gene also increased twofold. Apoptosis rates in the transfected and control cell lines were also reduced by the presence of an anti-mouse C5a receptor monoclonal antibody or of a peptide C5a receptor antagonist. These results indicated the presence of an RP S19 dimer- and C5a receptor-mediated autocrine-type augmentation mechanism during Mn II-induced apoptosis in the mouse fibroblastic cell line. In contrast to the RP S19 dimer, C5a actually inhibited apoptosis, suggesting that signaling through the C5a receptor varies depending on the ligand bound.     

Relationship between cerebral activity and movement frequency of maximal finger tapping

To examine the cerebral activity of the motor cortex during maximum movement, we measured regional cerebral blood flow (rCBF) in twelve normal volunteers, using near infrared spectroscopy (NIRS). Repetitive tapping of the right index finger was performed at 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, and 4.5 Hz, and during maximum effort (ME). The relative increase rate of rCBF during movement beginning with a resting condition was calculated for each movement condition. The left primary sensorimotor cortex showed significant activation during ME compared to the other frequencies. The rapid increase of rCBF was seen immediately after the initiation of finger tapping at all the tested frequencies but showed no increase following that. However, the rCBF during ME continued to increase until the end of the task.Change of the integrated electromyogram (iEMG) for the frequency and change of rCBF for the frequency at all the tested frequencies showed similar tendencies.     

Molecular monitoring of the developmental bacterial community in the gastrointestinal tract of Japanese infants

The dynamics of the developmental bacterial community in the Japanese neonatal gastrointestinal tract were examined by monitoring 16S ribosomal RNA gene (rDNA) diversity in fecal samples by PCR and denaturing gradient gel electrophoresis (DGGE). The results showed a certain pattern common in infants without antibiotic treatment, in which aerobes, e.g., Pseudomonas, appeared first and were then immediately replaced by facultative anaerobe, Enterococcus, Streptococcus, and Enterobacteriaceae through the first month, and finally strictly anaerobic Bifidobactrerium appeared.     

Function of cloned T4 recombination genes, uvsX and uvsY, in cells of Escherichia coli

Summary Genes uvsX and uvsY of bacteriophage T4 both control genetic recombination and repair of damaged DNA, and their mutant phenotypes bear a striking resemblance to each other. It has been shown recently that the uvsX gene product is analogous to the recA gene product of Escherichia coli (Yonesaki et al. 1985; Yonesaki and Minagawa 1985; Formosa and Alberts 1986), but the function of the uvsY gene is unknown. To obtain further insight into the function of these genes we introduced plasmid-borne copies of the two genes separately or together into E. coli. The uvsX gene rendered recA ^- cells more resistant to UV and raised the recombination frequency of phage and E. coli, but hampered induction of the prophage and the SOS function of E. coli. The uvsY gene had no detectable function when introduced alone into E. coli but significantly enhanced the function of the uvsX gene when the two plasmid-borne genes were introduced together.     

Identification and recombinant analysis of botrocetin-2, a snake venom cofactor for von Willebrand factor-induced platelet agglutination +

Botrocetin is a heterodimer snake venom protein that induces von Willebrand factor (VWF)- and platelet glycoprotein Ib (GPIb)-dependent platelet agglutination in vitro. We have cloned cDNAs for a botrocetin-2 from a cDNA library of the venom gland of Bothrops jararaca having a high similarity with botrocetin subunits. Recombinant botrocetin-2, expressed in 293T cells, showed cofactor activity comparable to natural botrocetin. In a single subunit expression experiment, a dimer of the β subunit was obtained, and it showed reduced, but apparent, platelet agglutination activity. Ala scanning mutagenesis showed that substitutions at Asp62, Asp70, Arg115, or Lys117 in the β subunit reduced platelet agglutination activity. The 3D homology modeling of botrocetin-2 complexed with the VWF A1 domain and GPIbα indicated that Asp62, Arg115, and Lys117 of the β subunit are located near Arg218 and Asp222 of GPIbα, respectively, and that Aspβ70 is in proximity to Gln1391 of the A1 domain. Our results indicate that these charged amino acid residues in the β subunit have a preferential role in the activity of botrocetin-2. Since it has been time-consuming and difficult to obtain homogeneous botrocetin from natural venom, recombinant botrocetin-2 has potential benefits for clinical and basic investigations into hemostasis and thrombosis as a standard reagent.