Neutralization of Chlamydia (C.) psittaci avian strain P-1041 was examined in vitro using monoclonal antibodies (MAbs). Of the 10MAbs used, 6 were found to exhibit neutralizing capability. These include 3 against major outer membrane protein (MOMP), 1 against lipopolysaccharide (LPS) and 2 against other protein molecules [90 kilodalton (kDa) and 90/50kDa]. Most neutralizing MAbs were dependent on complement for efficient neutralization, while a strain-specific MAb (2B5) against the 90kDa protein displayed a different requirement for complement and neutralized the infectivity of the P-1041 at high concentrations without complement. By competitive inhibition enzyme-linked immunosorbent assay (competitive inhibition ELISA), all 3 neutralizing anti-MOMP MAbs were demonstrated to recognize different epitopes found in very close proximity to each other on the outer membrane.

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In Cryptococcus neoformans, enzymic oxidation of various catechols leads to melanin, a proposed virulence factor. A phenol oxidase enzyme of Cryptococcus neoformans var. neoformans produced at 25C has been purified from an ultracentrifugal supernatant of an extract of broken cells. Hydrophobic interaction chromatography followed by anion-exchange column chromatography allowed purification of the phenol oxidase. The molecular weight of the enzyme estimated by gel filtration was about 80, 000 and a dimeric species (Mw=160, 000) was suggested. The isoelectric point of the protein was approximately 4.1. An NH₂-terminal 31 amino acid sequence was determined using phenol oxidase electroblotted onto a PVDF membrane after nondenaturing gel electrophoresis. Upon searching the Peptide Institute (Osaka) data base, no proteins with high degrees of homology were found.

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We have investigated the characteristics and utilities of streptavidin-binding to gram-negative and gram-positive bacteria and Candida spp. The pre-treatment of these microbes with chemical reagents such as CHCl₃, NaOH, and Tween 20 have allowed colorimetric visualization under light microscopy or quantitation on nitrocellulose membranes, using streptavidin/biotinylated alkaline phosphatase conjugates. Analysis of this binding was confirmed by western blot. These binding reactions were due to the specific interaction of streptavidin with biotinylated proteins present in the microbes. Competition assays with free biotin or inhibition by an antibiotin antibody confirmed binding to these proteins. With knowledge of these strongly specific interactions, we attempted to reveal the biotinylated proteins within these microbes using clinical specimens. Using phagocyte-smears from blood, urine, and ascites, these intracellular microbes were easily detected by light microscopy. One of the septic blood samples stained by our technique revealed semi-digested microbial signals despite the absence of a signal with routine staining. This detection system, which combines streptavidin as a probe and biotinylated proteins as a microbial marker, is useful in staining for intracellular bacteria or fungi (e.g., microbial infections in phagocyte-smears).

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To determine whether the avidin-biotin complex enzyme-linked immunosorbent assay (ABC-E) is a potentially useful method for detection of herpes simplex virus type 1 (HSV-1) antibody in saliva, paired serum and saliva samples from 129 healthy individuals aged 18 to 25 years were collected simultaneously and subjected to a neutralization test (NT) for neutralizing antibody and also to an indirect ELISA (IE) and ABC-E for HSV-1 specific IgG detection. Compared with the results of NT, the sensitivities of the IE and ABC-E for serum were both 100% (45/45), and for saliva 82.2% (37/45) and 93.3% (42/45), respectively. The specificity of all these methods was 100% (84/84). With the same ABC-E method, a significant correlation (r=0.66, P<0.001) between the OD-difference (d-OD) values of positive serum and saliva samples was observed. Furthermore, the consistency of ABC-E for salivary antibody detection was confirmed with the paired serum and saliva samples which were collected from four individuals followed up for eight months. It was clear that the ABC-E method for saliva can be used in place of the NT and ABC-E method for serum for seroprevalence studying of HSV-1 infection.

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Although chronic fatigue syndrome (CFS) is known to be the syndrome that begins with an acute flu-like illness that may be due to the exposure to an infectious agent, there has been no convincing evidence on the causative agents. Recently, human T-lymphotropic virus type II (HTLV-II)-like virus has been reported to be associated with the CFS by using HTLV Western blot analysis and polymerase chain reaction. However, some investigators could not detect HTLV-II by indirect immunofluorescence analysis. Lately, CFS patients have been reported in Japan. We detected all 30 tested patients with CFS were seronegative for HTLV-II, HTLV-I and HIV by specific peptide ELISA and Western blot. Further, PCR analysis was negative for HTLV-II and retrovirus was not detected by coculture method with patients' PBMC. Thus, known human retrovirus infections do not cause a CFS in Japan.

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Variations in cytokine production in patients with human immunodeficiency virus (HIV) infection could be involved in the physiopathology and in the progression of the disease. Therefore we studied the level of granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor α (TNFα) produced in patients with HIV infection at stage II (asymptomatic seropositives) and stage IV (AIDS) of the CDC classification, by using an enzyme amplified sensitivity immunoassay. We measured the level of GM-CSF and TNFα in supernatant of phytohemagglutinin-activated peripheral blood mononuclear cells from patients and healthy individuals. In one out of 10 stage II patients and 4 out of 14 stage IV patients, we obtained higher levels of GM-CSF than the mean+2S.D. of controls, but in 3 stage IV patients with very low CD4⁺ T lymphocyte counts (<50/mm^-3) compared to other patients, the GM-CSF values were very low. High levels of TNFα were detected in 3 out of 10 stage II and 6 out of 11 stage IV patients. The high values of TNFα were associated with high values of GM-CSF in stage II and in most of AIDS patients except those with very low CD4⁺ T cell counts, who produced low levels of GM-CSF. Plasma levels of cytokines were evaluated in 10 stage II, 22 stage IV patients and 20 controls. Increased levels of GM-CSF (more than 9pg/ml) were observed in the plasma from 8 out of 10 stage II patients and 17 out of 22 stage IV patients. The tendency that increased levels of GM-CSF were associated with increased levels of TNFα was observed in plasma from stage IV patients. We report a disarray of GM-CSF production in patients with HIV infection that could be involved in clinical manifestations and progression of the disease.

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Porins isolated from Salmonella typhi have been demonstrated to protect against the challenge with this bacteria in mice. The mechanism has not been clarified, but could be associated with activation of both humoral and cellular immunity. In order to evaluate the induction of specific T cell responses, the lymphocytic proliferation to porins isolated from Salmonella typhimurium, Salmonella typhi and Escherichia coli was examined by ³H-thymidine incorporation assay in mice immunized with three different antigens: acetone-killed S. typhimurium, its porins, or outer-membrane proteins (OMPs) isolated from S. typhi. Higher proliferative responses were observed in mice immunized with porins and OMPs compared with those which received the acetone-killed bacteria. Although cross-reactivity was observed between porins, they were not mitogenic. Moreover, porins were able to activate T lymphocytes isolated from mice immunized with S. typhi OMPs. These results suggest that T cell activation, through the release of lymphokines, may play a role in the induction of protective immunity with porins.

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It is known that polymorphonuclear leukocytes (PMNs) emerge first in local inflammatory sites, and then they are followed and scavenged by macrophages. We focused on the effect of PMN on tumor necrosis factor (TNF) release activity of macrophages, which is viewed as a possible indicator of the status of macrophage activation. One day after macrophages were cultured with fresh, intact murine PMNs which were induced with sodium casein, the release of TNF triggered by lipopolysaccharide (LPS) was augmented by low concentrations of PMNs, but suppressed by their high concentrations. When the PMN samples were fractionated into soluble and insoluble fractions, the augmenting and suppressing activity was partitioned; the relatively high concentrations of soluble fraction showed the suppressive effect whereas the insoluble fraction in lower concentrations showed augmentation. The suppressive activity was stable at 100C, but the filtrates of the soluble fraction with membranes having cut-offs of 5, 000 or 10, 000 were not suppressive at all, suggesting the suppression is not due to low molecular compounds. It was also suggested that the suppressive effect for TNF release was not due to contaminating LPS or transforming growth factor-β. Inflammatory processes may thus be positively and negatively controlled by a quantitative factor of initial PMN populations by regulating the TNF release activity of the subsequent macrophages.

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Spore coat protein of Bacillus subtilis was purified by electrophoretic elution procedure. Solubilized coat protein components were separated on SDS-PAGE and the desired protein was recovered from the gel pieces under the optimal condition examined. Two purified polypeptides with molecular weights of about 40kDa were obtained; each of them was in very closed size on SDS-PAGE, both retaining antigenic activity against anti-spore coat protein serum on immunoblot analysis. The N-terminal 23 and 30 amino acid sequences of them were determined, and they were not identical to each other and also not homologous in the sequences of coat proteins previously reported.

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Quantitative microanalysis of bacterial endotoxin was performed using [³H]-labeled L-glycero-D-mannoheptitol (LD-Heptitol) as a marker. Several different amounts of authentic L-glycero-D-mannoheptose (LD-Heptose) were reduced with 20μg of cold NaBH₄ containing 2μg of NaB³H₄ (40Ci/mmol) in 20μl of 1mM NaOH at 4C for 48hr. The product, [1-³H]-labeled LD-Heptitol, has high specific activity, and was purified by HPLC and detected using a liquid-scintillation counter. As little as 50pg of LD-Heptose was detectable, and the radioactivity increased dose-dependently in the 100pg to 80ng range tested. More than 2ng of Salmonella abortus equi endotoxin could be accurately determined by this method. It is possible to detect 50pg of endotoxin by this method, if 100% hot material (NaB³H₄) is used for [³H]-labeling.

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A phylogenetic tree constructed by the unweighted pair group method with arithmetic means (UPGMA) for the VP8* gene from 13 human and two feline rotavirus strains possessing the AU-1 gene 4 allele revealed that these strains could be classified into two clusters which did not correspond with the year of isolation or the host species from which they originated. Nine of 10 human strains and one feline strain isolated in Japan formed one cluster, whereas three human strains from Italy and one feline strain from Australia formed the other. Human strain K8 from Japan was distantly related to the Australo-European clustering.

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