An HLA-linked immune suppression gene in man.

Genetic control of immune response in man was investigated with the system of antigen-specific T cell proliferation in vitro against streptococcal cell wall (SCW) antigen. Family analysis by Morton's maximum likelihood scoring method revealed that the low response to SCW antigen was controlled by a single dominant gene. Furthermore, this gene was shown to be closely linked to HLA (lod score was 3,209 at theta = 0). This is the first description of the HLA-linked immune suppression gene in man. The possible mechanism for this gene action was discussed.

Use of a dual priming oligonucleotide system to detect multiple sexually transmitted pathogens in clinical specimens.

AIMS: To evaluate a new dual priming oligonucleotide (DPO)-based multiplex polymerase chain reaction (PCR) assay for detection of six sexually transmitted pathogens, including Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum and Trichomonas vaginalis. METHODS AND RESULTS: Using 130 clinical specimens, the results obtained by the multiplex PCR, previously established in-house PCR and COBAS Amplicor PCR assays were compared. The specimens frequently contained multiple pathogens (34/130 specimens). The multiplex PCR assay had an overall sensitivity of 96% and specificity of 100% compared to the in-house PCR assay at >20 microg ml(-1) of DNA concentrations in samples and there was no cross-reaction with nonpathogenic Neisseria species that cause the majority of false-positive results with the COBAS Amplicor PCR assay. CONCLUSIONS: The DPO-based multiplex PCR assay detected the six sexually transmitted pathogens in clinical specimens with a high sensitivity and specificity, although its sensitivity was dependent on the DNA content of the samples. SIGNIFICANCE AND IMPACT OF THE STUDY: It is the first report about the new DPO-based technique to detect multiple sexually transmitted pathogens in a single assay, which has considerable potential to diagnose the infections accurately and rapidly.

Immunological studies of post-streptococcal sequelae. Evidence for presence of streptococcal antigens in circulating immune complexes.

Since elevated levels of circulating complexes have been noted to occur in the sera of patients with post-streptococcal sequelae, the possibility that these complexes contained streptococcal antigens within the complex was investigated. Sera from these patients were precipitated with polyethylene glycol to extract a fraction rich in these complexes, which was then injected into rabbits. The rabbit sera were then reacted with both cellular and extracellular fractions obtained from streptococcal strains associated with either acute post-streptococcal nephritis (APSGN) or acute rheumatic fever (ARF) by using immunoelectrophoresis and ELISA techniques. The data demonstrate that both ARF and APSGN complexes contain streptococcal antigens. However, APSGN complexes react uniquely to certain extracellular antigens present in those strains associated with nephritis, while ARF complexes react specifically to certain streptococcal extracellular antigens excreted by strains associated with rheumatic fever. Neither of the two groups of complexes appear to contain streptococcal antigens related to any cellular antigens derived from the group A streptococcus. Additionally, a rabbit serum immunized with streptococcal extracellular products reacted directly with complexes isolated from nephritis patients. Removal of the gamma globulin by absorption with an anti-human Fc serum resulted in the concomitant loss of reactivity with the anti-streptococcal serum, strongly suggesting an intimate association of the streptococcal antigen with these complexes. The presence of streptococcal antigens within the circulating immune complex of patients with APSGN coupled with their specific presence in those strains associated with post-streptococcal glomerulonephritis argues strongly for a causal role of these antigens in the disease process.

Superantigenic exotoxin production by isolates of Staphylococcus aureus from the Kawasaki syndrome patients and age-matched control children.

Nineteen strains of Staphylococcus aureus were isolated from the throat or the tooth surfaces of 19 cases amongst 127 patients with Kawasaki syndrome (KS) during the acute phases and 11 S. aureus isolates were obtained from five of 17 diseased controls and six healthy controls. The production of exotoxins, particularly superantigenic toxic shock syndrome toxin-1 (TSST-1), coagulase serotype, pigment production, haemolytic activity and tryptophan auxotrophy of these isolates were compared. Among 10 KS S. aureus strains isolated in 1990-1991, five (50%) secreted TSST-1, a higher frequency than two (18%) of 11 control isolates. In contrast, none of the nine KS strains collected in 1984 produced TSST-1. Four of five TSST-1-secreting KS strains produced white or white to golden pigmentation, whereas the two control strains capable of TSST-1 production formed golden colonies. There were no noticeable differences between S. aureus strains from KS patients and control children in the production of staphylococcal exotoxins A-E, coagulase serotype, haemolysis of sheep erythrocytes and tryptophan auxotrophy. The pathological or aetiological role of a new TSST-1-secreting S. aureus clone in patients with KS was not confirmed.

Biologically active extracellular products of oral viridans streptococci and the aetiology of Kawasaki disease.

A bacteriological study of isolates from the oral cavity of patients with Kawasaki disease (KD), age-matched non-KD patients and healthy children, showed that over half the KD and control isolates had gram-positive, catalase-negative cocci. About 50% of these organisms were identified as viridans streptococci by means of an API Strep 20 kit. Further identification by fluorometric DNA-DNA hybridisation demonstrated that the predominant species were S. oralis and S. mitis, each of which accounted for 25% of the isolates of viridans streptococci; 40% of viridans strains were unidentifiable; and S. sanguis and S. parasanguis were minor components. Studies in vivo showed that insertion of culture supernates of most of the viridans streptococci increased capillary permeability and induced redness with swelling and occasional bleeding in rabbit skin. One-third of S. mitis strains and one-fifth of the unidentified strains caused aggregation of human blood platelets, whereas S. oralis and other strains had no such effect. The distribution of extracellular lipoteichoic acids and glucan produced in the presence of sucrose was also examined. There were no significant differences in the recovery rate of viridans streptococci forming these biologically active extracellular products between KD and control groups.

Detection of nephritis strain-associated streptokinase by monoclonal antibodies.

Monoclonal antibodies (MAbs) N-59 and RU-1 were produced by immunisation of mice with streptokinase secreted by Streptococcus group A, type 12, strain A374 isolated from a patient with post-streptococcal glomerulonephritis (PSGN) and were characterised by Western blot analysis. MAb N-59 recognised antigenic determinants shared by both nephritis strain-associated streptokinase (NSA-SKase) and streptokinase of Streptococcus group C (C-SKase); MAb RU-1 reacted only with NSA-SKase. All nephritis-associated group A streptococcal strains tested reacted with MAb N-59; 87.5% of these strains reacted with MAb RU-1. MAb N-59 reacted with SKase produced by group G streptococcal strains isolated from patients with PSGN, and MAb RU-1 recognised SKase in two out of three of these strains.

Purification and partial characterization of a novel human platelet aggregation factor in the extracellular products of Streptococcus mitis, strain Nm-65.

A human blood platelet aggregation factor was purified from the extracellular products (ECP) of Streptococcus mitis, strain Nm-65 by sequential chromatography on DEAE-Sepharose CL-6B, hydroxyapatite and Superdex 75 columns. The purified factor (S. mitis-derived human platelet aggregation factor, Sm-hPAF) gave a single band with a molecular weight of 66 kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Sm-hPAF showed a peak absorption at 278 nm and an isoelectric point of around 8.5. Chemical analyses revealed that Sm-hPAF contained no sugars and that its first 15 amino-terminal amino acid residues were H-DEQGNRPVETENIAR. Platelet aggregation activity of Sm-hPAF was abolished by heating at 45 degrees C for 10 min. Platelet aggregation by Sm-hPAF was accompanied by a release of prostaglandin E2 (PGE2) in a dose-dependent manner. The platelet aggregation was not inhibited by either prostaglandin E1 (PGE1) or Gly-Arg-Gly-Asp-Ser (GRGDS), that inhibit the platelet aggregation induced by collagen. Twenty (77%) platelet rich-plasma (PRP) specimens derived from 26 healthy volunteers were aggregated by Sm-hPAF, but the remaining 6 (23%) were not reactive. A preliminary study suggested the presence of an inhibitory factor against Sm-hPAF in the plasma from a non-reactive donor.

Efficacy of gentian violet in the eradication of methicillin-resistant Staphylococcus aureus from skin lesions.

The efficacy of gentian violet (Gv) in eradicating methicillin-resistant Staphylococcus aureus (MRSA) in decubitus ulcers was investigated. Decubitus ulcers (a total of 18 cases) were scrubbed with Gv aqueous solution 0.1% and ointment containing Gv 0.1% was applied daily. MRSA was not detected in these lesions for 3-34 days (average, 10.5 +/- 2.5 days) after the application of Gv ointment. Before this trial, all patients were treated with povidone-iodine and antibiotics; however, those treatments were not effective in eradicating MRSA from skin lesions. Skin irritation and other systemic side effects caused by Gv were not observed. Our data suggest that Gv is a useful agent for treatment of the decubitus ulcers infected with MRSA.

Induction of lymphocytes cytotoxic to oral epithelial cells by Streptococcus mitis superantigen.

The preparation of a superantigenic fraction F-2 from the culture supernatant of Streptococcus mitis 108, a fresh isolate from human tooth surfaces, was reported previously. Now, to determine the possible pathogenic role of the superantigen in oral mucosal diseases, we examined the cytotoxic effects of human peripheral blood T-cells activated with F-2 on human oral epithelial cells. T-cells activated with F-2 were cytotoxic to the human squamous carcinoma HO-1-N-1 cells derived from the oral mucosa, similar to those activated with Staphylococcus aureus enterotoxin B (SEB). This cytotoxic effect was increased in a dose-dependent manner by the addition of the respective stimulant, F-2 or SEB, to the cytotoxic assay system. F-2 endowed mainly CD8+ T-cells with cytotoxic activity. Pretreatment with human interferon gamma increased the sensitivity of the HO-1-N-1 cells to the cytotoxic effects of F-2-activated T-cells. The F-2-activated T-cells were also cytotoxic to human keratinocytes derived from gingiva. There was no correlation between the degree of cytotoxicity and the levels of tumor necrosis factor alpha in co-cultures of F-2-activated T-cells and HO-1-N-1 cells. A double-chamber plate experiment revealed no cytotoxic effects when the F-2-activated T-cells were separated from the HO-1-N-1 cells. Supernatants of the co-cultures of target and effector cells were not cytotoxic to HO-1-N-1 cells. These findings suggest that the cytotoxic effects of the F-2-activated T-cells on HO-1-N-1 cells were mediated not by soluble factors but by the direct interaction between the activated T-cells and the target cells. The cytotoxicity of the F-2-activated T-cells against HO-1-N-1 cells was markedly inhibited by monoclonal antibodies (MAbs) against CD11a and CD54, but was only slightly inhibited by MAbs against human leukocyte antigen (HLA)-DR and CD2. Thus, the interaction between lymphocyte-function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) was crucial for the F-2-dependent T-cell-mediated cytotoxicity against oral epithelial cells, while HLA-DR and CD2 molecules are not necessarily involved in the cytotoxicity observed.

Effects of "isolating hemodialysis" on prevention of methicillin-resistant Staphylococcus aureus cross-infection in a hemodialysis unit.

AIM: To evaluate the effects of a contact isolation program against methicillin-resistant Staphylococcus aureus (MRSA) cross-infection among patients in a hemodialysis unit. CLINICAL SETTING AND METHODS: In all patients maintaining hemodialysis therapy were tested for MRSA infection and who had MRSA infection, not only inpatients but also outpatients were separated into a designated area (isolating hemodialysis). Clinically isolated MRSA strains were clonotyped with coagulase typing, staphylococcal enterotoxin typing and restriction enzyme analysis of plasmid DNA. RESULTS: The frequency of patients with MRSA infection was 4.5% before starting this protocol and was reduced to 2.9% two and a half years later. At this time, MRSA was isolated from the 8 patients. These 8 clinical strains were differentiated into 6 clonotypes and 3 strains showed the same patterns. Two of 3 were isolated from inpatients and the other was from a patient with community onset MRSA colitis. In this case, most MRSA infections were independent under prophylaxis control and cross-infection was observed only once between hospitalized patients who stayed in a same ward. CONCLUSION: This "isolating hemodialysis" should be useful to prevent cross-infection among patients in end-stage renal disease in a dialysis unit.

Clonotypes of Staphylococcus aureus isolated from continuous ambulatory peritoneal dialysis patients: what is the vector between nares and infection site?

Staphylococcus aureus is frequently isolated from patients with infections related to continuous ambulatory peritoneal dialysis (CAPD). In many cases, the organism is also isolated simultaneously from the anterior nares. To clarify the transmission trail of S. aureus, we used DNA analysis to identify clonotypes of clinical strains. The nares and exit sites of 32 CAPD patients were swabbed, and PD fluid samples were taken for pathogen culture. Genome DNA of S. aureus was digested with restriction enzyme Sma I for pulsed-field gel electrophoresis. We also asked the patients how they usually performed the PD procedure. S. aureus was isolated from 4 patients, including 3 who hosted two strains isolated separately from different sites. The DNA patterns of the strains isolated from these latter 3 patients were identical. However, the clonotypes from all 4 patients were different. Most of the patients did not wash their hands and wear masks while exchanging PD bags and caring for their exit sites. After the patients were disinfected and re-educated in proper procedures, S. aureus was not detected in any of them. These data suggest that no outbreak occurred in our hospital and that the vectors of endogenous infection were the patients themselves, probably their hands. A bacteriological study presents an efficient opportunity to re-educate patients in PD procedure.

[Synergism on the bactericidal effect of gentian violet (Gv) and acrinol (Ac) against Pseudomonas aeruginosa].

The MBCs of Ac against P. aeruginosa (7 strains) isolated from infected skin lesions of patients were more than 6400 micrograms/ml, and those of Gv were more than 1600 micrograms/ml. When either Ac or Gv was used independently, these dyes did not have the bactericidal effect of P. aeruginosa. When Gv was used in combination with Ac, predominantly synergism on the bactericidal effect of Ac and Gv against P. aeruginosa was observed. The MBCs of an Ac-Gv cocktail were between 100 micrograms/ml and 225 micrograms/ml. We have previously reported that Gv possessed significantly a bactericidal effect to MRSA isolated from clinical specimens. Therefore, these results suggested that a combination treatment by an Ac-Gv cocktail may be one of the useful drugs for the MRSA and P. aeruginosa mixed infection on the skin lesions which is frequently observed clinically.

[Report of surveillance on streptococcal toxic shock syndrome in Japan and presentation of the criteria].

A survey was made on the situation of Group A Streptococcal toxic shock syndrome (STSS) based on questionnaires. The survey was divided into two parts. The first survey was done by sending out an outline of the STSS inquiring if any STSS cases were observed by mail to university hospitals, residence training hospital and other major hospitals totaling 2512 institutes. The second survey was subsequently done to the institutes that had STSS cases asking for the clinical course, data and sampling of the bacteria. The diagnosis of STSS was confirmed based on the diagnostic criteria induced by the working group of the United States. We have found 97 cases of STSS which 48.5% had fatal outcomes. There was no significant sex difference in the onset or the mortality rate. It occurred more in the older population, and occurred through out Japan but was not found to be epidemic. The first case was backed in 1978 and it began to increase since 1993, reaching its peak in 1994 and now decreasing in number. Most of the isolated Group A streptococcus were of type M1 and M3. We have modified the United States diagnostic criteria creating a new Japanese criteria, which includes the symptoms of the central nervous system in the term MOF. The aim for the Japanese criteria is to search for the etiology of the disease. The Japanese criteria requires that the disease progresses rapidly and that the patient be free from any conditions that might suppress the immunal system.

Efficacy of acellular pertussis vaccine in Japan--comparison of two areas, one area where immunization started at 6 months and the other area at over 2 years of age--Regional Surveillance Committee on Tuberculosis and Infectious Diseases of Osaka.

In the Osaka area, a very satisfactory surveillance system of infectious diseases has been achieved with the establishment of a weekly facsimile network, and computer aided graphics and feedback system. A mathematical formula has been devised for calculating the number of reported cases in exactly 100,000 of the population using the constant reported number of cases of exanthema subitum every week. With this method, we compared the incidence of pertussis patients in two areas, one where acellular pertussis vaccine is given to children after 6 months of age and the other where it is given at more than 2 years of age. The former area has the one fifth the incidence of pertussis patients of the latter.

The adjuvant activity of pyrene in diesel exhaust on IgE antibody production in mice.

In this communication, it is shown that pyrene has an adjuvant activity on IgE antibody production when mice are immunized by an intraperitoneal injection of ovalbumin (OA) or Japanese cedar pollen allergen (JCPA) with pyrene. The effects of pyrene on IgE antibody production in mice were investigated to clarify the relation between pollen allergy and the adjuvanticity of the chemical compounds contained in diesel-exhaust particulates (DEP). In the first experiment, three groups of mice were immunized intraperitoneally six times at 2-week intervals with 1 microgram of OA alone, 1 microgram of OA plus 1 mg of pyrene, and 1 microgram of OA plus 1 mg of DEP, respectively. The IgE antibody responses to OA in mice immunized with OA plus pyrene or OA plus DEP were extremely enhanced as compared with those in mice immunized with OA alone, and the highest responses were observed in mice immunized with OA plus DEP. In the second experiment, mice were immunized with 10 micrograms of JCPA alone or 10 micrograms of JCPA plus 5 mg of pyrene in the same way. The IgE antibody responses to JCPA in mice immunized with JCPA plus pyrene were higher than those in mice immunized with JCPA alone. The intraperitoneal macrophages of the mice also clearly stimulated in vitro by pyrene on chemiluminescence assay. These results suggest that pyrene contained in DEP acts as an adjuvant in IgE antibody production when mice are immunized with antigens.

[Adhesion ultrastructures of mononuclear cells in experimentally-induced silicotic granuloma].

Experimental silicosis was induced by intratracheal infusions of 1 ml saline containing 50 mg standard silica (less than 5 microns diameter) in Sprague-Dawley rats. The lung tissues were observed histologically and ultrastructurally from half an hour up to 4 months. Macrophages, neutrophils, desquamated cells and their debris piled up around the alveolar ducts where the central cores of silicotic granuloma appeared. The granuloma became apparent by day 4 after the infusion and were covered by type II alveolar epithelial cells and bronchiolar cuboidal epithelial cells. Macrophages, fibroblasts and epithelial cells began to react to the antibody against proliferating cell nuclear antigen (PCNA) indicating self-replication on day 1. Macrophages in the granuloma made a close interdigitation with adjacent macrophages, and they gradually formed subplasmalemmal linear densities (SPLD) as paired forms between adjacent plasma membranes, and unpaired forms facing the interstitial matrix. SPLD were composed of linear densities with actin-like microfilaments along the leaflets of plasma membrane and were associated with extracellular dense bands which resembled a limited length of basement membrane. Interdigitation and SPLD structures were quite rare on day 1, but the number of macrophages with both structures increasingly appeared. The frequency of SPLD in macrophages also increased on a time course of granuloma maturation up to 4 months. Thus SPLD, which were originally found in the mononuclear phagocytes including macrophages, epithelioid cells and multi-nucleated giant cells, particularly in immune granuloma of man, also played a basic role in immobilizing macrophages in lesions of silica-induced granulomas.

[Effect of gentian violet on the elimination of methicillin-resistant Staphylococcus aureus (MRSA) existing in the decubitus region].

Methicillin-resistant Staphylococcus aureus (MRSA) is frequently isolated from skin lesions, such as in the decubitus region. There is a possibility that MRSA through these lesions can spread widely in a hospital. However, local treatment with most antibiotics and antiseptics (povidone-iodine) is not effective to eradicate MRSA from the infected decubitus. We have recently demonstrated that gentian violet (Gv) possessed a bactericidal effect against MRSA isolated from clinical specimens in vitro. This examination evaluated whether or not a topical ointment containing 0.1% Gv is effective to eradicate MRSA which existed in decubitus regions. Decubitus (14 clinical cases, ages 59-87 years) infected with MRSA were treated with 0.1% Gv-ointment once or twice daily after bathing in 0.1% Gv aqueous solution. Although all patients were treated with povidone-iodine and 9 out of 14 patients were given either local or systemic administration of antibiotics, those treatments were not effective to eradicate MRSA from decubituses. However, MRSA was not detectable in all cases within 34 days (average: 10.8 days +/- 2.7) after treatment with 0.1% Gv-ointment. The eradication of MRSA from decubitus areas tended to be delayed, depending upon the size and depth of decubituses (Grade III and IV) and complications such as diabetes mellitus. Skin irritability was not observed in any patients. These results suggest that 0.1% Gv-ointment is a useful material for the treatment of the MRSA-local wound infection. Treatment with Gv-ointment to MRSA-infected decubitus may exhibit a protective effect with regard to infection with MRSA in hospital.

[Clinical and fundamental studies of streptococcal toxic shock syndrome].

Streptococcal toxic shock syndrome(STSS) is known to progress rapidly into septic shock and multiple organ failure with frequently necrotizing fasciitis, and high mortality (approximately 40%). The diagnosis of STSS is confirmed based on the diagnosis criteria induced by the working group of the United State. Several extracellular products such as SPE A, B, and C having pyrogenic and superantigenic activity as well as SPE F, SPE G, H, J, SME Z, SME Z2, SSA are likely to involved in the pathogenesis of STSS. Recent studies have demonstrated an important role of certain cytokines, such as TNF-alpha, in laboratory animals. The exact role of such products in the pathogenesis of STSS, however, is currently unknown. The role of several virulence factors of group A streptococci in the pathogenesis of STSS was discussed in this review.