Phagocytosis represents a central element of the host response to microbial invasion. We describe a flow cytometric method for measuring the kinetics of phagocytosis of two bacteria by human polymorphonuclear leukocytes (PMNs). Over a 60-min period, isolated human PMNs were exposed to Staphylococcus aureus (rapidly phagocytosed) and Klebsiella pneumoniae (slowly phagocytosed). This method distinguished adherent from ingested bacteria by quenching fluorescein isothiocyanate-labeled extracellular bacteria with ethidium bromide. This further allowed the exclusion of dead, highly permeable, and subsequently bright-red fluorescent PMNs. Our experiments with two different bacteria, various PMN-to-bacteria ratios (1 : 1, 1 : 10, 1 : 100), and different individuals proved that 1) flow cytometric analysis is accurate and useful for characterizing phagocytosis, 2) adherent bacteria can be distinguished from ingested bacteria after quenching with ethidium bromide, and that 3) phagocytosis kinetics of two bacteria with different onsets of phagocytosis can be determined by flow cytometry and the assessment of a score that quantifies phagocytosis.

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Using a series of oligonucleotides synthesized on the basis of conserved nucleotide motifs in heat-shock genes, the groESL heat-shock operon from a Vibrio cholerae TSI-4 strain has been cloned and sequenced, revealing that the presence of two open reading frames (ORFs) of 291 nucleotides and 1, 632 nucleotides separated by 54 nucleotides. The first ORF encoded a polypeptide of 97 amino acids, GroES homologue, and the second ORF encoded a polypeptide of 544 amino acids, GroEL homologue. A comparison of the deduced amino acid sequences revealed that the primary structures of the V. cholerae GroES and GroEL proteins showed significant homology with those of the GroES and GroEL proteins of other bacteria. Complementation experiments were performed using Escherichia coli groE mutants which have the temperature-sensitive growth phenotype. The results showed that the groES and groEL from V. cholerae were expressed in E. coli, and groE mutants harboring V. cholerae groESL genes regained growth ability at high temperature. The evolutionary analysis indicates a closer relationship between V. cholerae chaperonins and those of the Haemophilus and Yersinia species.

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Attempts were made to detect Mycoplasma fermentans in saliva sampled from 201 subjects (108 males and 93 females) aged from 4 months to 59 years by a polymerase chain reaction-based assay. M. fermentans was detected in saliva from 110 (54.7%) of 201 subjects, and 10 (28.6%) of 35 subjects aged from 4 months to 3 years. Of ten positive subjects, three were aged from 16 to 23 months and five were from 26 to 31 months. The incidence tended to increase with age up to the teens. The incidence was significantly greater in teenagers than in subjects aged from 7 to 12 years, but there was no significant difference in the incidence between the group of teenagers and each of the groups of subjects older than the teenagers. Thus, it was suggested that M. fermentans colonized the mouth at the age of about 16 months up to the age of 19 years.

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Recent evidence suggests an interaction between immune, enteric neural and fibroblasts in the regulation of intestinal function. Earlier, we have reported that lipopolysaccharide (LPS) induced cell proliferation, collagen synthesis and production of proinflammatory mediators in lamina propria fibroblasts. In this report, we investigated the change in transepithelial resistance (TER) as a marker of epithelial barrier function by lipopolysaccharide (LPS) and its modulation by human small intestinal lamina propria fibroblasts (HSILPF). Epithelial cells incubated with LPS alone did not show any change in the TER at any concentration or prolonged exposure. However, co-cultivation of epithelial cells with lamina propria fibroblasts which had been exposed to LPS resulted in a rapid decrease in TER by 2hr. The decrease in the TER was continued till 8hr followed by returning to the basal level by 24hr. The supernatant of LPS-treated HSILPF was less effective in causing a fall in the TER than HSILPF itself. The fall in TER was accompanied by loosening of tight junctions as depicted by increased penetration of horse radish peroxidase (HRP) across the epithelial cells from the apical to the basal side. Increased incorporation of ³[H]thymidine (tritiated thymidine) in epithelial cells was observed at 48hr in the presence of LPS-treated HSILPF. The decrease in TER during the early time period in epithelial cells was abrogated to 70% by incubating the LPS-treated HSILPF and the conditioned medium of LPS-treated HSILPF with anti-TNFα antibody, and not with antibody to other cytokines like IL1α, IL1β, IL6 and IL8. Overall, these results suggest that TNFα produced by HSILPF in response to LPS as a soluble form cause a decrease in the TER and loosening of tight junctions, and such early changes in the epithelial barrier may contribute to local inflammation in the gut.

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A conjugate vaccine against Salmonella typhi was prepared by covalently binding capsular polysaccharide (Vi) with porin, both isolated from S. typhi. First, Vi and porins were extracted. The Vi was purified from S. typhi Ty2. The purified Vi conformed to the requirements of the World Health Organization. Porins were purified from S. typhi 0901. The Vi was bound to the porins by a heterobifunctional cross-linking reagent, N-succinimidyl-3-(2-pyridyl dithio)-propionate (SPDP). After preparing the Vi-porin conjugate, its protective ability and immunogenicity were studied in mice following systemic immunization. The results showed that the conjugate is 6.5-fold more protective than Vi alone against S. typhi. The mice immunized with conjugate elicited higher anti-Vi antibody (IgG) levels (P<0.01) than the mice immunized with Vi alone. Anti-porin antibodies were also induced by the conjugate. To study the mucosal immune responses, secretory IgA (sIgA) in the intestinal fluid was measured. Conjugate-immunized mice showed the induction of sIgA as compared to Vi alone. The results showed that when Vi is bound to porins, both isolated from same organism, the resultant conjugate induced both systemic and mucosal immune responses and provided better protection against S. typhi than Vi alone.

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Samples of feces and vomit collected from patients during 13 non-bacterial gastroenteritis outbreaks which occurred in Hokkaido between 1995 and 1998 were examined by electron microscopy (EM) and reverse-transcription polymerase chain reaction (RT-PCR) for evidence of infection with human caliciviruses (HuCVs). In 6 food-borne outbreaks, oysters were the probable source of infection, while the origin of HuCVs was not found out for the other 7 outbreaks. One-hundred-eleven of 214 stool, vomit and oyster specimens examined gave positive results by RT-PCR, while HuCVs were detected by EM in 36 of 121 stool specimens examined. We determined the nucleotide sequences of 470-bp or 373-bp PCR products amplified from the RNA polymerase region of the HuCV genomes with primer sets MR3/4 and Yuri22F/R, respectively. The sequences of different strains revealed great heterogenicity, with a range of 60 to 100% homology among strains. In a few cases, a mixed genotype was found in the same patient or same outbreak by means of nested PCR and cloning of PCR products into an appropriate vector. Of the 19 different strains found, 4 strains could be classified as Norwalk virus (genogroup 1) and the other 15 strains as Snow Mountain agent (genogroup 2) based on genotyping with homology analysis. Furthermore, the strains belonging to genogroup 2 could be classified into 4 subgroups with more than 93% homology in amino acids among strains in the subgroup.

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For the purpose of making clear the dynamics of rabies viruses that are prevalent among dogs in Asia, especially Thailand, nucleoprotein (N) genes of isolates derived from Thailand were partially sequenced, and a phylogenetic analysis was performed on the basis of the sequencing data. Firstly, all 27 isolates from Thailand belonged to one group that was distantly related to an isolate from China and was separated into at least six lineages. On the other hand, the isolate from Japan was related to viruses from the Arctic. Secondly, in order to analyze the diversity of the N gene more conveniently, restriction fragment length polymorphism (RFLP) analysis was performed on the N gene of 27 isolates from Thailand. The RFLP analysis could distinguish the lineages of each isolate, and the lineages of additional 34 isolates were deduced by this method. On examination of the geographical distribution of the six lineages, based on the results of phylogenetic and RFLP analyses, it was clear that infection cycles of the rabies virus in Thailand have tended to be maintained endemically.

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Two RESA repeat sequences, (EENVEHDA)₂ and (DDEHVEEPTVA)₂, were chemically linked to a universal T-cell epitope, CS.T3 and polytuftsin, and a natural immunopotentiator, was physically mixed with these conjugates. The immunogens were studied for in vitro antigen-induced T-cell proliferation, and cytokine levels were measured in the culture supernatants. The RESA peptide(s)-CS.T3 conjugate containing polytuftsin showed the highest stimulation index (SI) as compared to the RESA peptide-CS.T3 conjugates or RESA peptides alone. Spleen cells from mice primed with either RESA peptide(s)-CS.T3 conjugate or RESA peptide-CS.T3 conjugate containing polytuftsin, when pulsed in vitro with the respective RESA peptide, showed a higher proliferation index as compared to spleen cells primed and pulsed in vitro with the respective RESA peptides. This observation has an important relevance during natural reinfection for boosting the immune response. The culture supernatants from the cells primed and pulsed in vitro with RESA peptide-CS.T3 conjugate and RESA peptide-CS.T3 conjugate containing polytuftsin showed higher IL-2 and IFN-γ levels as compared to the RESA peptides alone. Very low IL-4 levels were detected with the above formulations. The cytokine profile is suggestive of a CD4⁺ TH1 type of immune response, which is ideal for the killing of intracellular pathogens like the malarial parasite.

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Synthetic peptides representing repeat sequences of ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum have shown poor immunogenicity and protection. In this study, the RESA peptides [(EENVEHDA)₂ and (DDEHVEEPTVA)₂] were chemically linked to a universal T-cell determinant, CS.T3, derived from the CS protein of P. falciparum. Polytuftsin (TKPR)₄₀, a polymer of naturally occurring immunomodulator “tuftsin, ” was physically mixed with these conjugates. These preparations in alum and liposomes were immunized in four inbred strains of mice with different genetic backgrounds to study the humoral response. In the case of liposome-entrapped preparations, a 10μg dose of antigen showed the optimum antibody response. Mice immunized with liposome containing RESA peptide(s)-CS.T3 conjugate along with polytuftsin showed the highest antibody levels in all the strains, whereas the RESA peptide(s) alone, adsorbed on alum or entrapped in liposomes, showed either poor or moderate antibody levels. The antibodies raised against liposome-entrapped preparations in both high-responder strain (SJL/J H-2^s) and low-responder strain (FVB/J H-2^q) showed 2-4-fold lower Kd values as compared to the alum adsorbed preparations, suggestive of high affinity antibodies. All the antigen preparations predominantly induced IgG2a and IgG2b isotype response, suggesting that the T-helper response involved is of the CD4⁺ Th1 type. The in vitro merozoite reinvasion inhibition assay showed 50-92% inhibition with sera raised against different antigen formulations. The highest percentage inhibition was observed with the RESA peptide-CS.T3 conjugate containing polytuftsin in liposomes. Thus, the incorporation of peptide antigens inside liposomes not only reduced the antigen dose by 5-fold but also elicited a high titre with high affinity antibodies and the inhibition of merozoites to RBC in vitro. Therefore, we conclude that the incorporation of these synthetic constructs in liposomes could be a useful strategy for the development of a subunit immunogen against malaria.

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The T-cell receptor (TCR) BV gene of human TCR AV24⁺ double-negative (DN) T cells, a novel subset of natural killer (NK) T cells, was investigated by single-cell sorting and single-cell polymerase chain reaction (PCR) methods. Seven of eleven TCR AV24⁺ DN T-cell clones utilized TCR BV8, three BV9, and one BV6. Six of seven TCR AV24/BV8⁺ DN T-cell clones had identical TCR β and α chains, indicating that they were the same clone. All three TCR AV24/BV9⁺ DN T-cell clones also demonstrated the same amino acids in the CDR3 region. These findings strongly suggest that the usage of TCR β and α chains on TCR AV24⁺ DN T cells is extremely restricted, supporting the notion that these cells recognize highly limited T-cell epitopes on antigens. All TCR AV24⁺ clones expressed the NKR-P1A mRNA, and so were true NK T cells. IL-2 and IL-4 mRNAs were detected in all clones, suggesting that the majority of these cells were Th0-type T cells. Six clones overexpressed Fas-ligand (Fas-L) mRNA and Fas antigen was detected on all clones at the mRNA level. In conclusion, TCR AV24⁺ DN T cells might recognize restricted T-cell epitopes on antigens and function as Th0-type T cells, inducer cells to Th1-or Th2-type T cells (regulatory T cells), and as Fas-L-positive cytolytic T cells.

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The presence of C5b-9 complexes, some complement regulators, and abundant cytokines in atherosclerotic lesions has been reported. However, it is unclear whether these complement-associated proteins are produced by vascular smooth muscle cells (SMCs) and how they are influenced by the cytokines. In the present study, we demonstrated, by the reverse transcription-polymerase chain reaction method, the mRNA expression of complement components (C3, C4, and C5) and membrane regulators (decay-accelerating factor, membrane cofactor protein, Crry, and CD59) in cultured SMCs derived from the rat carotid artery. The expression of C9 mRNA was also induced upon stimulation by interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α) and/or lipopolysaccharide (LPS). Northern blot analysis showed that the mRNA expression of C3, C4, DAF and Crry was up-regulated, but that of CD59 was down-regulated by IFN-γ, TNF-α and/or LPS alon or by synergy. The increase of C3 mRNA by TNF-α or LPS and that of C4 mRNA by IFN-γ was induced in a dose-dependent manner. The results indicate that the arterial SMCs of rat have the ability to produce complement components and regulators, which is affected by cytokines and/or LPS. Since atherosclerosis is characterized by the intimal proliferation of SMCs, the complement system including its regulators may be involved in the pathogenesis of the disease.

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Parabiotic pairs of B6.Ly5.1 and B6.Ly5.2 mice were used to investigate how lymphocytes in various organs and various lymphocyte subsets mixed with partner cells. The origin of partner cells was determined by using anti-Ly5.1mAb in conjunction with immunofluorescence tests. Parabiosis was also produced after the irradiation of B6.Ly5.2 mice at various doses to prepare an immunosuppressive partner. Irrespective of irradiation, lymphocytes and other hematopoietic cells in the bone marrow and lymphocytes in the thymus showed a low mixture of partner cells in comparison with those of all other organs tested. On the other hand, lymphocytes in the blood, spleen, and lymph nodes became a half-and-half mixture of their own cells and partner cells by 14 days after parabiosis. Among lymphocyte subsets, intermediate CD3 cells (i.e., CD3^int cells) and NKT cells (i.e., NK1.1⁺ subset of CD3^intcells) in the liver also showed a low mixture of partner cells. The present results raise the possibility that lymphocytes in the bone marrow and thymus, and extrathymic T cells in the liver might be in situ generated from their own preexisting precursor cells. Another observation was that, after irradiation, partner cells showed accelerated mixture even if they showed a low mixture under non-irradiated conditions. However, only lymphocyte subsets with the same phenotype as those of preexisting cells entered the corresponding sites.

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Feline parvovirus (FPV) was isolated rather frequently from the peripheral blood mononuclear cells (PBMCs) of cats in northern Vietnam by coculturing with MYA-1 cells (an interleukin-2-dependent feline T lymphoblastoid cell line) or Crandell feline kidney (CRFK) cells (a feline renal cell line). Efficiency of virus isolation was higher in MYA-1 cells than in CRFK cells. Interestingly, among the 17 cats from which FPV was isolated, 9 cats were positive for virus neutralizing (VN) antibody against FPV, indicating that FPV infected PBMCs and was not eliminated from PBMCs even in the presence of VN antibodies in the cats.

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