Changes in peripheral lymphocyte subpopulations after direct moxibustion.

We measured peripheral lymphocyte subpopulations after direct moxibustion using moxa cones the size of a half-rice grain. In humans at 2 hrs after the direct moxibustion, NK cell percentage decreased and CD4/CD8 ratio increased significantly. Although the same trends were observed in the control session, those changes were not statistically significant. In rabbits at 3 and 12 hrs after direct moxibustion, CD4/CD8 ratio significantly increased, and recovered at between 24 and 72 hrs after treatment. Similarly, the CD4/CD8 ratio significantly increased in the control session, but they seemed to have a shorter duration. Although it is not yet clear whether the effects are beneficial, we found that direct moxibustion influences the immune system at least transiently.

Serotypes VI and VIII predominate among group B streptococci isolated from pregnant Japanese women.

Infection by group B streptococcus (GBS) is an important cause of bacterial disease in neonates, pregnant women, and nonpregnant adults. Whereas serotypes Ia, Ib, II, III, and V are most commonly associated with colonization and disease in the United States, strains of other serotypes have been isolated from patients in Japan. By use of an inhibition ELISA, the serotypes of 73 vaginal colonizing GBS strains isolated from healthy pregnant Japanese women were investigated. Twenty-six (35.6%) were type VIII, 18 (24.7%) were type VI, and the remaining 29 were distributed among more traditional serotypes. Strains were also tested by immunoblot for the presence of GBS surface proteins. Fifty-three (72.6%) of the 73 strains expressed one or more laddering GBS proteins. These data show that type VI and VIII GBS strains are common vaginal isolates in pregnant Japanese women and that one or more laddering proteins are present in most GBS strains.

Detection of human serum antibody to encapsulated strains of Staphylococcus aureus by enzyme-linked immunosorbent assay inhibition test.

A specific and rapid enzyme-linked immunosorbent assay (ELISA) inhibition test was employed for detection of immunoglobulins to Staphylococcus aureus (S. aureus) capsular polysaccharide in human serum. Cap-sular polysaccharide antigens obtained from Smith diffuse (capsular type 2), Reynolds (capsular type 5), or Becker (capsular type 8) strains of S. aureus were added to microplates coated with these strains. Seventy-four patients with open fractures (31 serum samples from those with staphylococcal infections, 10 serum samples from those with non-staphylococcal infections, and 33 serum samples from the non-infected group) and 28 serum samples from healthy controls were then added. The plates were incubated at 37 degreesC for 2 h and the ELISA was performed. The ELISA inhibition assay showed remarkable inhibition with the capsular type 2, 5, and 8 polysaccharides in the 33 serum samples from the non-infected group and in the 28 serum samples from the healthy controls, but low inhibition was observed with the 31 sera with staphylococcal infections. Positive immunoglobulin (Ig)G and IgM titers showed marked inhibition with this assay, but IgA titer were not seen in any samples. These results indicate that the quantitation of human serum antibody against S. aureus capsular polysaccharide by the ELISA inhibition assay is useful for the demonstration of protective activities against S. aureus.

Effects of moxibustion on the enhancement of serum antibody in rabbit against Staphylococcus aureus.

The titer and activity of antibody in rabbits immunized with heat-killed vaccine were assessed with and without moxibustion treatment. Enzyme-linked immunosorbent assay (ELISA) was applied for the detection of immunoglobulins to Smith strain of Staphylococcus aureus. Positive IgM titer of more than 0.4 were observed with this assay against the moxibustion group (P < 0.05). The titer of IgG antibody also increased; however, there was no significant difference between the moxibustion group and the control group. The ELISA inhibition test showed significantly higher protective activity of the sera in the moxibustion group at the 9th week after the first immunization (P < 0.05).

Some biochemical properties of the components of Staphylococcus aureus binding to human platelets.

The binding properties of Staphylococcus aureus in relation to human platelets were investigated. Protease digestion (pronase E, proteinase K, trypsin), heat treatment (80 degrees C, 30 min), and sonication for 5 min significantly reduced the binding abilities of the staphylococcal cells to 0% (p < .01), 50 +/- 5% (p < .05), and 38 +/- 9% (p < .05), respectively, while mixed glycosidases did not. Inhibition experiments indicated that protein A and various sugars were ineffective. A binding study using biotinylated cell surface fractions extracted from the whole cells of S. aureus indicated that the proteins having apparent molecular weights of 14400 and 16500 estimated by sodium dodecylsulfate-polyacrylamide gel electrophoresis were involved in the binding between S. aureus and human platelets.

Experimental hematogenous osteomyelitis by Staphylococcus aureus.

To assess bone marrow lodgement of bacteria that produce osteomyelitis, 10(6) colony forming units of 16 nonhemolytic strains of Staphylococcus aureus was injected intravenously into mice. Eleven of 16 strains showed bone marrow lodgement without the death of mice. The M-138 strain induced osteomyelitis in 100% of the mice. Furthermore, the difference of compact colony forming active substance activity between bone marrow lodgement and nonlodgement strains was statistically significant. Compact colony forming active substance, which is an alkali stable polysaccharide located on the cell surface of Staphylococcus aureus strain, caused compact formation of the strains in serum soft agar or fibrinogen soft agar, and it clotted animal plasma. These results suggest that bacterial factors are important for bacterial lodgement at the onset of staphylococcal hematogenous osteomyelitis.

Conversion of biological and immunological properties during a process of decapsulation in a strain of Staphylococcus hyicus.

Repeated subculture at 42 degrees C of Staphylococcus hyicus strain ST67P, which exhibits streaming-type growth in a soft-agar medium, yielded three variants, ST67L, ST67S and ST67C, which had different colonial morphologies; small compact colonies possessing long and short tails and perfect compact colonies. The parent strain and ST67L respectively gave strong and weak positive intensity when stained by rabbit antisera prepared by capsular type I and II strains of Staph. epidermidis conjugated with fluorescein isothiocyanate. Variant ST67L gave a positive result with antiserum prepared by capsular type I strain and no staining was observed with variants ST67S and ST67C against these antisera preparations. Strain ST67C had the lowest virulence although no remarkable difference was shown between the parent strain and variants ST67L and ST67S. The cell volume index of the parent strain was 1.35, 2.43 and 3.71 times larger than those of ST67L, ST67S and ST67C, respectively. The converting activity of rabbit anti-ST67P strain serum absorbed by strain ST67C required four times more of the organisms than strain ST67P, changing the colonial morphology of the strain from diffuse to compact type by the addition of antiserum to soft agar medium. Positive coagulase and false positive clumping factor reaction were shown in variants ST67C, but no remarkable alteration was observed with 19 biochemical properties determined by a conventional identification kit. In ulta-thin sections of the parent strain labelled with rabbit anti-strain ST67P serum conjugated with ferritin, large capsule surrounded by ferritin granules were demonstrated by electron microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

Relation of human serum antibody against Staphylococcus epidermidis cell surface polysaccharide detected by enzyme-linked immunosorbent assay to passive protection in the mouse.

A specific and rapid enzyme-linked immunosorbent assay (ELISA) has been applied for the detection of immunoglobulins to Staphylococcus epidermidis cell surface polysaccharides in human serum. Positive IgG, IgM and IgA titres of more than 1: 6400, 1: 1600 and 1: 400 were observed with this assay against passive protective human serum. However, IgG, IgM and IgA titres of less than 1: 400, 1: 100 and 1: 50 were shown in non-protective serum. When the cross-reactivity of passive protective human serum to homologous and heterologous cell surface polysaccharides was examined by inhibition test with ELISA, remarkable inhibition was shown with homologous cell surface polysaccharide, whereas no inhibition was observed with heterologous substances. According to these results, the quantitation of human serum antibody by the ELISA method against Staph. epidermidis cell surface polysaccharide was found to be significant for the demonstration of passive protective activities against Staph. epidermidis.

Monoclonal IgM antibody protection in mice against infection with an encapsulated strain of Staphylococcus epidermidis.

Passive protective activities of three different classes of monoclonal antibodies in mice against challenge with strain ATCC 31432 (capsular type I) of Staphylococcus epidermidis were examined. Monoclonal IgM antibody passively protected mice against challenge with the homologous strain, whereas monoclonal IgG1 and IgG2b antibodies did not. The protective activity of IgM was absorbed by the cell surface antigen extracted from the homologous strain but not by the antigen from heterologous strains. Rapid reduction of viable cells took place in the peritoneal cavity of mice immunized with monoclonal IgM as early as 6 h after the challenge with the homologous strain. An enzyme-linked immunosorbent inhibition assay showed there was remarkable inhibition with the homologous cell surface antigen but not with heterologous preparations from other strains. Results suggest that in the mouse the major passive protection against the S. epidermidis strain is provided by the IgM antibody to the cell surface antigen.

Platelet aggregation induced by strains of various species of coagulase-negative staphylococci.

Major species of coagulase-negative staphylococci (CNS) were tested for their ability to induce platelet aggregation in rabbit platelet-rich plasma (PRP). Among 11 species of CNS tested, a majority of the strains of 10 species of CNS (S. epidermidis, S. simulans, S. capitis, S. hyicus, S. sciuri, S. cohnii, S. xylosus, S. hominis, S. haemolyticus, S. warneri) caused induction of the platelet aggregation and serotonin release, while S. saprophyticus did not show such activity. The addition of aspirin (10 mM) or quinacrine (1 mM) to PRP resulted in no remarkable effect on the platelet aggregation induced by these strains and it was shown that the platelet aggregation did not require arachidonate pathways. Complement system components were shown to be one of the plasma factors required for platelet aggregation by ten strains of each species of CNS. The bacterial substance participating in the platelet aggregation by ten species of CNS tested was indicated to be heat-stable and trypsin-resistant, while the activity of a strain of S. epidermidis was susceptible to trypsin.

Platelet aggregation by strains of enterococci.

The platelet aggregation capability of whole cells of Enterococcus faecalis, E. faecium and E. avium was tested. The optimum ratios of bacteria to platelets in E. faecalis (strain SMU-37), E. faecium (strain SMU-138) and E. avium (strain SMU-197) were 1.0, 1.2 and 2.0, respectively. During the platelet aggregation induced by the three strains of enterococci, 65-69% of total serotonin was released. The aggregation was totally inhibited by ethylenediaminetetraacetate (10 mM) and apyrase (1 mg/ml), while no effect was shown by aspirin (10 mM), indomethacin (10 mM) and quinacrine (1 mM). By pretreatment of platelet-poor plasma with heat (56 C, 30 min) or zymosan, the reactivities with platelets of each strain of species were markedly diminished. These results suggest that enterococci-induced platelet aggregation was an ion-dependent, cyclooxygenase-insensitive event, and plasma component(s) was (were) required for the reaction.

[Cross protection between an encapsulated strain of Staphylococcus hyicus and an encapsulated strain of Staphylococcus epidermidis].

Strain ST67P of Staphylococcus hyicus was capsular type I (++)/II(+) of S. epidermidis as determined by the method of Ichiman. To mice immunized with heat-killed vaccine of strain ST67P, homologous strain and strain ATCC 31432 (capsular type I), SE-360 (capsular type II) and SE-10 (capsular type III) of S. epidermidis were injected intraperitoneally into mice, then, the viable cell number of the organisms in the peritoneal cavity were enumerated of 30 minutes and 20 hours after the injection. Results showed that the viable cell number of the homologous strain and strain ATCC 31432 was remarkably decreased at 20 hours after the injection, however, the cells were increased with strain SE-360 and SE-10. Passive protective activity of rabbit anti-ATCC 31432 serum was absorbed either with homologous strain or strain ST67P in the mouse, however, protective activity of anti-SE-360 strain serum and anti-SE-10 strain serum was not absorbed with these organisms although the activity was absorbed with homologous organisms. With an ultrathin section preparation of strain ST67P conjugated with ferritin-labelled rabbit anti-homologous strain serum, numerous ferritin granules surrounding the outermost layer of large capsule were electronmicro-scopically demonstrated. In the same organisms treated with ferritin-labelled anti-ATCC31432 strain serum, the all walls were surrounded by a relatively thinner capsule and a number of ferritin granules were located in the outermost layer of the capsule. However, with the organisms treated with ferritin-labelled anti-SE-360 strain serum only a number of ferritin granules were shown on the surface of the cell walls, and neither capsule nor ferritin granules were exhibited in the organisms treated with ferritin-labelled anti-SE-10 strain serum.

Inhibition of platelet aggregation by a whole cell extract from strains of group B streptococcus.

A whole cell extract (HCl-Ext) from strains of group B streptococci (GBS) possessing fibrinogen binding activity prevented the platelet aggregation induced with adenosine 5'-diphosphate (ADP), collagen and thrombin, while aggregation by epinephrine and ristocetin was slightly inhibited and arachidonic acid-induced platelet aggregation was not affected whatsoever. When the HCl-Ext was added after commencement of the aggregation, deaggregation was observed in cases induced by ADP, collagen, and thrombin. By precoating the washed platelets with HCl-Ext, both of ADP- and collagen-induced platelet aggregation were suppressed. The active factor in the HCl-Ext seemed to be undialyzable, trypsin-susceptible, and proteinaceous substance, unlike GBS polysaccharide type antigen.

Protection of mice by a pseudodiffuse strain of Staphylococcus aureus possessing polyvalent capsular type antigen.

Staphylococcus aureus strain MC31 showed pseudodiffuse growth in serum-soft agar and reacted with immune rabbit sera to strains Smith diffuse (capsular type A), NS58D (type B) and NS41D (type C) but not with strain NS68D (type D) in serum-soft agar. Immunisation of mice with 300 micrograms of cell-surface polysaccharide extracted from strain MC31 protected against lethal infection by strain MC31 and the strains of capsular types A, B and C. Immune rabbit serum prepared against strain MC31 passively protected mice against challenge infection with the homologous strain, but approximately 30 times more anti-MC31 serum was required to protect against infection with the strains of capsular types A, B and C. Absorption of the passive protective activity of immune sera raised against the three capsular type strains required at least 10 times the quantity of MC31 cell-surface polysaccharide than the quantity of cell-surface polysaccharide from the homologous capsular strain. Electronmicrographs of strain MC31 treated with ferritin-labelled antisera to the three capsular strains showed only small amounts of ferritin granules around the cell wall.

Induction of resistance with heat-killed unencapsulated strains of Staphylococcus epidermidis against challenge with encapsulated strains of Staphylococcus epidermidis.

Active immunization of mice with high doses of heat-killed unencapsulated strains of Staphylococcus epidermidis, which were grown in brain heart infusion media, protected mice against challenge with encapsulated strains of S. epidermidis. The unencapsulated strains were capable of absorbing the protective antibody in rabbit hyperimmune sera prepared with the encapsulated strains. Also, mice treated with rabbit hyperimmune sera prepared with the unencapsulated strains were protected against challenge with the encapsulated strains. The protective activities of these rabbit hyperimmune sera were assumed to be essentially identical to those of the protective antibody induced by the encapsulated strains.

Cross protection between an encapsulated strain of Staphylococcus hyicus and encapsulated strains of Staphylococcus aureus.

Strain ST67P of Staphylococcus hyicus grew diffusely in regular serum-soft agar. With the addition of rabbit antisera prepared with Staph. aureus strains, Smith, NS58D or NS41D, capsular type A, B or C, respectively, the organisms converted to compact type growth. Mice immunized with heat-killed vaccine of strain ST67P showed significant resistance against either homologous or heterologous strains, Smith, NS58D and NS41D. Passive protective activities in rabbit antisera prepared with strains Smith, NS58D and NS41D were absorbed out with either homologous cell surface polysaccharide fraction or cell surface polysaccharide fraction extracted from strain ST67P. Well-defined large capsules were observed in ultra-thin sections treated with rabbit antiserum prepared with homologous strain conjugated with ferritin. Also, the capsule surrounded by ferritin granules was shown in ultra-thin sections treated with ferritin conjugated with antisera prepared with those heterologous strains although the capsular size was significantly smaller than those observed by homologous antiserum.

Possible common biological and immunological properties for detecting encapsulated strains of Staphylococcus epidermidis.

Twenty strains of capsular type II Staphylococcus epidermidis, determined by the method of Ichiman, were obtained from clinical specimens. Among them, 5, 5, and 10 strains were 4+, 3+, and 2+ in the intensities of their reactions against fluorescent antibody, respectively. Strains exhibiting 4+ and 3+ intensities were mouse virulent and phage nontypable, while 2+ strains were mouse avirulent and phage typable. When three strains randomly selected from each of the mouse-virulent and mouse-avirulent strains were compared in terms of their cell volume indices, all mouse-virulent strains had significantly higher indices (average, 1.86 times) than the mouse-avirulent strains. With intraperitoneal injection of the strains into mice, strains with higher cell volume indices resisted ingestion by peritoneal cells, while strains with low cell volume indices were sensitive to phagocytosis. When the capacity to absorb a definite amount of passive protective activity in rabbit antiserum prepared with capsular type II strains was compared among these strains, 10 to 20 mg of mouse-virulent strains was capable of completely absorbing the passive protective activity, whereas more than 80 mg of the cells was required for similar absorption by mouse-avirulent strains. In ultra-thin sections of three mouse-virulent strains stained with ferritin-conjugated rabbit antiserum, well-defined capsules were detected around cell walls; however, no capsule was seen around the walls of three mouse-avirulent strains.