Characterization of recombinant Streptococcus mitis-derived human platelet aggregation factor.

We previously purified Streptococcus mitis-derived human platelet aggregation factor (Sm-hPAF) from the culture supernatant of S. mitis strain Nm-65, isolated from the tooth surface of a patient with Kawasaki disease. Here we produced recombinant Sm-hPAF protein (rSm-hPAF) in Escherichia coli, to determine whether rSm-hPAF conserves its platelet aggregation activity. rSm-hPAF precursor (665 amino acids) shows up to 36-56% identity with the family of cholesterol-dependent cytolysins (CDCs), and rSm-hPAF displayed potent hemolytic activity toward mammalian erythrocytes, including human erythrocytes with platelet aggregation activity. The 162-amino acid amino-terminal domain of rSm-hPAF was found in no other CDCs except lectinolysin; this domain is homologous to a portion of pneumococcal fucolectin-related protein. Interestingly, suilysin (SLY) and pneumolysin (PLY) of CDCs also exhibit substantial human platelet aggregation activity, similar to rSm-hPAF, and the platelet aggregation by rSm-hPAF, SLY, and PLY was morphologically confirmed using light and electron microscopy.

[Evaluation of the GonoGen II kit for rapid identification of Neisseria gonorrhoeae using monoclonal antibody directed at gonococcal outer membrane protein 1].

The Gonogen II test for rapid identification of Neisseria gonorrhoeae (Gonococcus, GC) was evaluated. The test is based on a colorimetric reaction with monoclonal antibody to GC outer membrane protein 1. Of the 50 clinical isolates of GC, 49 isolates tested positive and only one strain tested negative. Other Neisseria. spp, H. influenzae, H. parainfluenzae, E. corrodens, M. catarrhalis, and A. baumannii showed negative test results. Non-Neisseriae. spp, such as S. aureus. P. aeruginosa, E. faecalis, and E. coli also showed negative test results. No cross-reactivity was found between GC and other Neisseriae. spp or non-Neisseriae. spp. In a mixed suspension of GC and all of non-Neisseriae. spp as mentioned above, the GonoGen II test was positive. The specificity and sensitivity of the test for the identification of GC were 98% and 100%. The minimum limit of detection of GC was > or = 1 x 10(5) cfu/mL. Decision making based on the test result is possible within 10 minutes. These findings also suggest that the test does not require pure GC. The GonoGen II test appears to be a reliable, quick and easy-to-use assay, and also to not require viable GC. Thus GonoGen II is shown to be a very useful test for the identification of GC.

Studies of recombinant streptococcal pyrogenic exotoxin B/cysteine protease (rSPE B/SCP) in the skin of guinea pigs & the release of histamine from cultured mast cells & basophilic leukocytes.

BACKGROUND & OBJECTIVES: Streptococcal pyrogenic exotoxin B/streptococcal cysteine protease (SPE B/SCP) is considered to be one of the virulence factors of Streptococcus pyogenes (S. pyogenes) which causes serious diseases such as severe invasive infections and streptococcal toxic shock syndrome (STSS). There are no reports on the histamine releasing activity of SPE B/SCP from mast cells, although several biological activities have been studied. It is not clear whether SPE B/SCP have the superantigenic activity. We studied whether SPE B/SCP plays as a pathogenic factor in streptococcal infections and STSS through a histamine releasing activity. METHODS: Human mast cells and basophils were generated from CD34 positive cells isolated from cord blood and cultured in the presence of rIL-6, stem cell factor and/or rIL-3. The capacity of increasing capillary permeability of recombinant SPE B/SCP (rSPE B/SCP) was studied by using the skin of guinea pigs. Mitogenic activity to human T-cells of rSPE B/SCP was studied by incorporation of (3)Hthymidine. The levels of histamine in the plasma of patients with STSS and controls were measured by ELISA kit. RESULTS: rSPE B/SCP induced increased capillary permeability in the skin of guinea pigs, but both SPE A and SPE C did not exhibit such activity. Histamine was released from cultured human mast cells stimulated with rSPE B/SCP. The rSPE B/SCP did not exhibit mitogenic activity to human T-cells. Three of the 7 patients with STSS showed higher levels of plasma histamine than those of normal subjects. INTERPRETATION & CONCLUSION: The results suggested that increased capillary permeability and histamine release from mast cells induced by rSPE B/SCP might be involved in STSS and/or streptococcal infection of skin and mucous membrane.

Moderate hypothermia delays proinflammatory cytokine production of human peripheral blood mononuclear cells.

OBJECTIVE: To clarify the influence of moderate hypothermia on the production of proinflammatory cytokines. DESIGN: Controlled in vitro study. SETTING: Research laboratory. SUBJECTS: Peripheral blood mononuclear cells from healthy adult human subjects. INTERVENTIONS: Stimulation with 1 microg/mL lipopolysaccharide at 33 degrees C and 37 degrees C. MEASUREMENTS: Concentrations of released tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 were measured chronologically by enzyme immunoassay. The number of mRNA copies of these cytokines was determined by competitive reverse transcriptase-polymerase chain reaction analysis, and nuclear factor-kappaB activations were assessed by electrophoretic mobility shift assay. MAIN RESULTS: Significant reduction of the released-tumor necrosis factor-alpha concentration was observed 1 and 2 hrs after the stimulation with lipopolysaccharide at 33 degrees C compared with 37 degrees C. The peak release of interleukin-1beta at 33 degrees C was delayed 12 hrs later than that at 37 degrees C. A delayed peak in the release of interleukin-6 also was observed at 33 degrees C. The peaks of cytokines were confirmed at the mRNA expression level by competitive reverse transcriptase-polymerase chain reaction analysis at both temperatures. The peak of the tumor necrosis factor-alpha mRNA expression level was observed at 1 hr after the stimulation at 37 degrees C and 2 hrs after the stimulation at 33 degrees C. In the interleukin-1beta mRNA expression, at 37 degrees C the first peak appeared 1 hr and the second 6 hrs after the stimulation. In contrast, at 33 degrees C, the first peak appeared 2 hrs and the second 12 hrs after the stimulation. Whereas interleukin-6 mRNA expression at 37 degrees C peaked 6 hrs after the stimulation, no definite peak was observed at 33 degrees C and the expression level was approximately half of that at 37 degrees C. The maximum intensity of nuclear factor-kappaB activation at 33 degrees C was delayed by 1.5 hrs compared with that at 37 degrees C. CONCLUSIONS: Moderate hypothermia delays the induction of proinflammatory cytokines in human peripheral blood mononuclear cells.

Cysteine protease activity and histamine release from the human mast cell line HMC-1 stimulated by recombinant streptococcal pyrogenic exotoxin B/streptococcal cysteine protease.

We constructed the expression vector pSK-SCP containing the streptococcal exotoxin B gene (spe b) which expressed protease activity. We showed that the recombinant streptococcal pyogenic exotoxin B/streptococcal cysteine protease (rSPE B/SCP) was secreted into the culture supernatant of the transformant and retained its SCP activity, which was equivalent to or greater than that of the naturally occurring molecule. The secreted rSPE B/SCP induced histamine release and degranulation of the human mast cell line HMC-1. This study may contribute to the understanding of the pathogenic role of SPE B/SCP in streptococcal infection and streptococcal toxic shock syndrome.