Toxicity of Shiga toxin 1 in the central nervous system of rabbits.

The action of Shiga toxin (Stx) on the central nervous system was examined in rabbits. Intravenous Stx1 was 44 times more lethal than Stx2 and acted more rapidly than Stx2. However, Stx1 accumulated more slowly in the cerebrospinal fluid than did Stx2. Magnetic resonance imaging demonstrated a predominance of Stx1-dependent lesions in the spinal cord. Pretreatment of the animals with anti-Stx1 antiserum intravenously completely protected against both development of brain lesions and mortality.

Effects of Shiga toxin 2 on lethality, fetuses, delivery, and puerperal behavior in pregnant mice.

Shiga toxin 2 (Stx2) is produced by enterohemorrhagic Escherichia coli (EHEC) and is known as the major virulence factor of EHEC. The aim of this study was to evaluate the effects of Stx2 on (i) maternal lethality, (ii) fetuses, (iii) delivery period, and (iv) maternal behavior after delivery. Timed pregnant ICR mice were injected intravenously with Stx2 on day 5 of pregnancy (early stage) or on day 15 (late stage). In early-stage experiments, the number of normal fetuses of mice injected with Stx2 was significantly lower than that of control mice. In late-stage experiments, mothers injected with Stx2 delivered normal numbers of neonates, but could not take care of them. The lethal doses of Stx2 were not different for pregnant and nonpregnant female mice at either stage. We conclude that Stx2 is toxic to the fetus in early pregnancy and affects maternal puerperal behavior in late pregnancy.

Reconstitution of active recombinant Shiga toxin (Stx)1 from recombinant Stx1-A and Stx1-B subunits independently produced by E. coli clones.

Escherichia coli clones expressing recombinant Shiga toxin (Stx)1-A and recombinant Stx1-B subunits, were established. Culture supernatants of these clones were examined for inhibitory activity on in vitro protein synthesis using luciferase as a reporter enzyme. Culture supernatant of the clone expressing Stx1-A, but not Stx1-B, showed the inhibitory activity. Neither recombinant Stx1-A nor Stx1-B showed Vero cell cytotoxicity. For reconstitution of biologically active toxin, the culture supernatants of the Stx1-A clone and the Stx1-B clone were mixed. The reconstituted recombinant Stx1 showed both Vero cell cytotoxicity and inhibition of in vitro protein synthesis.

Brainstem mechanisms of autonomic dysfunction in encephalopathy-associated Shiga toxin 2 intoxication.

Acute encephalopathy is the major determinant of death in an early stage of Shiga toxin (Stx)-producing Escherichia coli infection. Rapid progress toward refractory hypotension and dysfunction of breathing implies autonomic center dysfunction of patients. To clarify whether autonomic dysfunction becomes an ultimate cause of death in Shiga toxemia, we injected purified Stx2 (20 microg/kg) intravenously into rabbits, and monitored changes in cardiovascular and respiratory function together with renal sympathetic nerve activity (RSNA) in the conscious state. After an approximately 24-hour silent (lag) period, all rabbits given Stx2 developed hemorrhagic diarrhea (25.7 +/- 1.1 hours) and limb paralysis (31.2 +/- 1.3 hours). This limb paralysis was observed initially in the hind legs, and then it gradually extended to the forelegs. After 23.2 +/- 2.3 hours, RSNA increased gradually, and arterial blood pressure was maintained within normal limits together with an increase in the maximum gain of baroreflex response. Severe hypotension developed within 34.8 +/- 2.2 hours, without any increase in heart rate; RSNA significantly increased by 39.5 +/- 0.9 hours. In the final stage, RSNA decreased concurrently with decreases in arterial blood pressure, heart rate, and baroreflex response, suggesting dysfunction of the baroreflex control system. Thereafter, all rabbits died within 47.8 +/- 1.2 hours after the intravenous Stx2 injection. Magnetic resonance imagings of the central nervous system (T2-weighted images) showed high-intensity areas in the dorsal two-thirds of the cervical spinal cord and brainstem 48 hours after Stx2 administration. These results show that the cause of death is circulatory failure caused by impairment of the cardiovascular center in the medulla. We believe that this animal model helps to clarify the mechanism of rapid progress to death of patients with Shiga toxin-producing E. coli infection.

Polyclonal expansion of TCRBV2- and TCRBV6-bearing T cells in patients with Kawasaki disease.

We examined T-cell receptor (TCR) usage, cytokine production and antibody responses to superantigens in patients with Kawasaki disease (KD) to facilitate a better understanding of the immunopathogenesis of KD. The mean percentage of VB2- or VB6. 5-bearing T cells in peripheral blood mononuclear cells (PBMC) of patients with acute-phase KD was significantly higher than that of patients in the convalescent phase of KD or in healthy donors. Expansion of VB2- or VB6.5-bearing T cells was polyclonal because DNA sequences in the complementarity determining region 3 of VB2- and VB6.5-positive cDNA clones were all different from each other. The plasma levels of interleukin (IL)-1beta, IL-2, IL-6, IL-8, IL-10, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and granulocyte colony-stimulating factor (G-CSF) were elevated in the acute phase of KD. We previously reported that streptococcal pyrogenic exotoxin C (SPEC) was a potent stimulator of VB2- and VB6.5-positive T cells and, furthermore, serum levels of anti-SPEC antibodies were significantly higher in patients with acute and convalescent KD than in age-matched controls. The results of the present study, together with those of our previous report, suggest that SPEC induces activation and polyclonal expansion of VB2- and VB6.5-positive T cells, and that SPEC-induced activation of T cells may lead to the pathogenesis of KD.

Neurotoxicity of intrathecal Shiga toxin 2 and protection by intrathecal injection of anti-Shiga toxin 2 antiserum in rabbits.

The initial brain lesions in rabbits given intravenous Shiga toxin 2 (Stx2) were noted at 24 h in an area around the third ventricle (Fujii et al., Infect Immun 1996, 64: 5053-60). This result implied that Stx2 is present in the cerebrospinal fluid (CSF) despite the fact that the toxin was administered intravenously. We measured Stx2 activity in CSF by using a Vero cell cytotoxicity assay at various times after an intravenous injection of Stx2. Stx2 was detected from 2 h after the injection, and its concentration in CSF remained at a high level for a further 6 h. Fifty percent lethal doses (LD 50) of Stx2 were measured in rabbits after intravenous and intrathecal Stx2 injections; The LD 50 after an intrathecal injection of Stx2 was 0. 36 microg/kg, which was 9.2-fold lower than that of an intravenous injection of Stx2 (3.4 microg/kg). Magnetic resonance images obtained after an intrathecal Stx2 injection (5 microg/kg) were compared with those obtained after an intravenous Stx2 injection (5 microg/kg). At 48 h, the cerebellar lesions had spread from the area in contact with the CSF on a T2-weighted image, which suggests that the intrathecal Stx2 may invade the cerebellum directly. We then examined whether anti-Stx2 antiserum injected intrathecally protects rabbits against brain damage. Eighty percent of the rabbits infected with Stx2 at 5 microg/kg died within 8 days from brain damage. Rabbit anti-Stx2 sera (with titres of x16 and x64 by the Ouchterlony precipitation method) were administered into the CSF space through the cisterna magna. All the rabbits ( n=10) survived when they were given an intrathecal injection of rabbit anti-Stx2 antiserum 2 h before the intravenous injection of Stx2. Our results suggest that a leakage of Stx2 into the CSF from the choroid plexus causes brain damage, and that an intrathecal injection of anti-Stx2 antiserum could be a therapy for acute encephalopathy caused by Stx2-producing Escherichia coli.

Serologic evidence that streptococcal superantigens are not involved in the pathogenesis of Kawasaki disease.

Kawasaki disease (KD) is an acute multisystem vasculitis of unknown etiology and is associated with marked activation of T cells and monocyte macrophages, leading to the assumption that superantigens are involved in its pathogenesis. To determine if an association exists between streptococcal superantigens and KD, we examined serum antibody responses to superantigens in sera from 50 paired acute and convalescent KD patients using purified recombinant streptococcal superantigens, such as SPEA, SPEC, SSA and MF. We found a very low frequency of detection of anti-superantigen antibodies by ELISA and no marked IgG seroconversion to each superantigen, indicating the absence of a serological relationship between toxin-producing streptococcal infection and the onset of KD.

Definition of the mitogenic factor (MF) as a novel streptococcal superantigen that is different from streptococcal pyrogenic exotoxins A, B, and C.

Human T cell activation by recombinant mitogenic factor (rMF) was investigated in comparison with that by recombinant streptococcal pyrogenic exotoxins (rSPE) A, B, and C. Recombinant MF, rSPEA, and rSPEC were mitogenic for peripheral blood mononuclear cells (PBMC), whereas rSPEB was not. Recombinant MF required only HLA-DR for the stimulation of PBMC, as determined using monoclonal antibodies (mAb) to HLA class II molecules and the mouse L cells transfected with HLA class II molecules. Recombinant SPEA and rSPEC required HLA-DR or HLA-DQ molecule. Recombinant MF selectively stimulated V beta 2, V beta 7, V beta 8, V beta 18 and V beta 21-bearing T cells, whereas rSPEA and rSPEC activated V beta 2 and V beta 6-bearing T cells as evaluated by the quantitative T cell receptor (TCR) analytical method. No clonality was observed in the nucleotide sequences of complementarity determining region 3 of TCR V beta in T cells responding to rMF. The profiles of cytokine production by PBMC in response to rMF, rSPEA, and rSPEC were quite similar. In summary, these results demonstrate that both HLA class II molecules and the TCR V beta required for rMF-mediated T cell activation are distinct from those required for rSPEA or rSPEC-mediated activation. Therefore, the MF is a novel streptococcal super-antigen which is different from SPEA, SPEB, and SPEC.

The gene encoding a new mitogenic factor in a Streptococcus pyogenes strain is distributed only in group A streptococci.

We recently cloned a gene encoding a new mitogenic factor (MF) from Streptococcus pyogenes NY-5. In the present study, we determined the distribution of this MF gene (mf) by PCR based upon its sequence. Of 371 streptococcal group A strains isolated from clinical specimens, 370 (99.7%) were positive for mf. The strain that was negative for the MF gene was also negative for the streptolysin O gene (slo). Some streptococcal strains belonging to groups C and G were negative for mf but positive for slo. Group B strains were negative for both. Furthermore, we examined the presence of mf in 54 strains belonging to 28 families and found mf only in group A streptococci. These results indicate that mf is distributed specifically in group A streptococci and the presence of mf in clinical samples strongly suggests infection with group A streptococci.

Cloning and sequencing the streptolysin O genes of group C and group G streptococci.

On the basis of the known streptolysin O (SLO) genomic sequence of Streptococcus pyogenes group A, we identified the SLO genes in some strains of group C and group G streptococci by the polymerase chain reaction procedure (PCR). The entire open reading frame region of these genes was cloned and analyzed. Their nucleotide sequence data showed that the defined SLO genes in group C and group G are almost identical to that of group A.

Cloning, characterization and overexpression of a Streptococcus pyogenes gene encoding a new type of mitogenic factor.

A new type of mitogenic factor, termed MF, has been found in the culture supernatant of Streptococcus pyogenes and its N-terminal amino acid sequence has been determined. On the basis of this sequence, an S. pyogenes gene encoding MF was cloned and its nucleotide sequence was determined. The MF gene includes a long, open reading frame with 813 nucleotides capable of encoding the MF precursor protein with 271 amino acids. Removal of the putative 43 residues as a signal peptide results in the mature MF protein with 228 amino acids. The molecular mass of the mature MF is calculated as 25,363 which is consistent with the previously determined value of 25,370 for MF secreted from S. pyogenes. Neither nucleotide nor amino acid sequence homology was found between the mature MF and other streptococcal pyrogenic exotoxins, such as SPE A, SPE B and SPE C. The mature MF was recombinantly overexpressed as a fusion protein with glutathione S-transferase in Escherichia coli. The recombinant protein showed mitogenic activity in rabbit peripheral blood lymphocytes and immunoreactivity with the rabbit antiserum raised against the secreted MF from S. pyogenes. These data indicate that a unique gene encoding MF was cloned from S. pyogenes.

Two-dimensional echocardiographic assessment of papillary muscle contractility in patients with prior myocardial infarction.

OBJECTIVES: This study was performed to assess the length and contractile performance of human left ventricular papillary muscles and to determine the relation between papillary muscle dysfunction and mitral regurgitation. BACKGROUND: Assessment of human papillary muscle contractility remains a clinical challenge. METHODS: Two-dimensional echocardiographic examinations were performed in 16 normal subjects and 31 patients with prior myocardial infarction. Apical echocardiograms were used to obtain long-axis views of the anterior and posterior papillary muscles. The end-systolic and end-diastolic lengths of the papillary muscles were measured and fractional shortening was calculated. RESULTS: Fractional shortening in normal subjects was 27 +/- 8% for the anterior papillary muscle and 30 +/- 8% for the posterior papillary muscle. In patients with prior myocardial infarction, a significant decrease in fractional shortening was observed in proportion to the severity of left ventricular wall motion abnormalities at the site of papillary muscle implantation. Moderate or severe mitral regurgitation was significantly more frequent in patients with combined anterior and posterior papillary muscle dysfunction than in those with isolated anterior or posterior dysfunction or with normal function of both papillary muscles (p < 0.05). CONCLUSIONS: Two-dimensional echocardiography is useful for demonstrating abnormal contractility of human left ventricular papillary muscles. Papillary muscle contractility should be analyzed in each case to elucidate the mechanism of mitral regurgitation in patients with papillary muscle dysfunction.

A new type of mitogenic factor produced by Streptococcus pyogenes.

A new type of mitogenic factor (protein) was purified from the culture supernatant of a strain of Streptococcus pyogenes by SP-Sephadex C-25 column chromatography, preparative isoelectric focusing and reversed-phase high-performance liquid chromatography. The purified factor, showing marked mitogenic activity in rabbit peripheral blood lymphocytes, gave a single-band staining for protein on SDS-PAGE. The molecular weight of the purified mitogenic factor was determined to be 25,370, which was different from those calculated from reported amino acid sequences deduced from 4 different nucleotide sequences of 3 kinds of streptococcal pyrogenic exotoxins (two SPEAs, SPEB and SPEC). The amino acid sequence of the N-terminal region of the purified mitogenic factor was determined to be Gln-Thr-Gln-Val-Ser-Asn-Asp-Val-Val-Leu-Asn-Asp-Gly-Ala-Ser-Lys-Tyr-Leu- Asn-Glu - Ala-, which was also different from the reported N-terminal sequences deduced from the 4 different nucleotide sequences. These data indicate that this mitogenic factor is distinct from the already described streptococcal pyrogenic exotoxins.

[Pulmonary venous flow patterns in normal subjects and cardiac patients: a transesophageal echocardiographic study].

The aim of this study is, first, to analyze pulmonary venous flow velocity (PVFV) pattern in normal subjects and second, to compare it with the various diseased state. PVFV was recorded in eleven normal volunteers, five patients with lone atrial fibrillation, twenty eight patients with valvular heart diseases and six patients with cardiomyopathy using transesophageal color Doppler echocardiography by placing the sample volume at the junction of the left superior pulmonary vein and left atrium. PVFV in normal subjects demonstrated distinct four waveforms: due to atrial systole (AS wave) and diastole (AD wave), and due to ventricular systole (VS wave) and diastole (VD wave). PVFV changed with respiration in normal subjects. The peak velocity of VD wave was increased with inspiration (p less than 0.001). The ratio of velocity VS/VD was increased during expiration (p less than 0.01). The ratio of area AD + VS/VD was significantly decreased with inspiration (p less than 0.01). We feel that this is the normal variation in pulmonary venous return during respiration , influenced by changes in the venous return on the right side of the heart. In all patients with atrial fibrillation, AS and AD waves were disappeared. The negative deflection occasionally observed was due to mitral valve closure. In patients with mitral stenosis, the peak velocity of VD wave was significantly decreased compared to that of normal subjects, but it was not significantly different between the patients with mitral valve replacement and normal subjects. The peak velocity of VD wave was also correlated with pressure half time among the patients with mitral stenosis, mitral valve replacement and mitral commissurotomy. On the other hand, it was significantly increased in patients with mitral regurgitation and returned to normal level after the operation. The peak velocity of VS wave was correlated with the left atrial dimension among the patients with mitral valve diseases except these with mitral regurgitation. In patients with mitral regurgitation, the peak velocity was decreased compared to that of normal subjects and reversed flow was seen in half of the patients. Also, it was decreased in patients with dilated cardiomyopathy and increased in patients with hypertrophic cardiomyopathy. In conclusion, PVFV is influenced not only by changes of venous return with respiration but also by the left atrial size, presence or absence of MS or MR, left atrial or left ventricular systolic and diastolic function in the various diseased states.

[Bacteriological investigation on an outbreak of acute enteritis associated with verotoxin-producing Escherichia coli O111:H-].

An outbreak of acute enteritis in children aged one to thirty-three months occurred from June 10th to 23rd, 1986, at a private orphanage in Matsuyama City. Twenty-two out of 23 children suffered from diarrhea. Nine of the 22 children excreted bloody stool. Fever and vomiting were observed with a few patients. One of them, a 33-month-old girl, developed hemolytic uremic syndrome, and died twelve days after the admission. Escherichia coli O111:H- was isolated from fecal specimens of 7 out of 15 patients. The culture filtrate of the isolate caused fluid accumulation in ligated ileal loops in a rabbit, and was lethal to mice. It was found that all isolates produced two kinds of Vero toxins (VT1 and VT2, or shiga-like toxin I and II). The amount of VT2 produced in vitro was about 10 times more than that of VT1.

[Continuous wave Doppler echocardiographic evaluations of the severity of mitral regurgitation].

To ascertain the usefulness of continuous wave Doppler echocardiography in evaluating the severity of mitral regurgitation (MR), 29 patients with MR and 10 normal subjects were examined. The patients were categorized in three groups according to the angiographic evidence of severity of MR. To analyze the flow velocity patterns of MR, the time to peak velocity index (time from onset of MR signal to peak flow velocity/duration of MR signal), the A/B ratio (the ratio of the first and second half of the systolic MR signal area), systolic peak velocity, and diastolic peak velocity were measured using continuous wave Doppler echocardiograms. The velocity patterns of MR differed significantly among the three groups. With severer MR, the flow velocity pattern showed an earlier appearance of the peak in systole, a steeper decrease in systole and a greater increase in early diastole. The time to peak velocity index was 55 +/- 7% (mean +/- SD) in mild MR, 42 +/- 6% in moderate MR and 35 +/- 5% in severe MR. This index shortened significantly in accord with the severity of MR (mild vs moderate MR: p less than 0.001, moderate vs severe MR: p less than 0.05). The A/B ratio was 1.06 +/- 0.12 in mild MR, 1.23 +/- 0.10 in moderate MR and 1.41 +/- 0.07 in severe MR. This ratio increased significantly with the severity of MR (mild vs moderate MR: p less than 0.01, moderate vs severe MR: p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and some properties of a Vero toxin from a human strain of Escherichia coli that is immunologically related to Shiga-like toxin II (VT2).

A cytotoxin to Vero cells (Vero toxin), which was immunologically related to Shiga-like toxin II (SLT-II) (or VT2), was purified from a stain of Escherichia coli isolated from a patient with hemolytic uremic syndrome. The toxin was active on Vero cells but much less active on HeLa cells, a property similar to that of the recently identified SLT-II variant from E. coli strains that caused edema disease of swine. Thus the toxin purified in this report was tentatively named Shiga-like toxin II variant (Vero toxin 2 variant). The purification procedures consisted of ammonium sulfate fractionation, DEAE-Sepharose CL-6B column chromatography, chromatofocusing column chromatography, and repeated high performance liquid chromatography (HPLC) on TSK-gel G-2000SW column and on TSK-gel DEAE-5PW columns. About 90 micrograms of purified toxin was obtained from 451 of the culture supernatant with a yield of about 16%. The purified toxin consisted of A and B subunits of molecular sizes similar to those of SLT-II (VT2). The isoelectric point of the purified toxin was 6.1, which was different from that of SLT-II (VT2) (pI = 4.1). In an Ouchterlony double gel diffusion test, purified toxin and SLT-II (VT2) formed precipitin lines with spur formation against anti-purified toxin and anti-SLT-II (anti-VT2), respectively. The purified toxin was cytotoxic to Vero cells, about 6 pg of the toxin killing 50% of the Vero cells, and showed lethal toxicity to mice when injected intraperitoneally, the LD50 being about 2.7 ng per mouse.

Identity of molecular structure of Shiga-like toxin I (VT1) from Escherichia coli O157:H7 with that of Shiga toxin.

The primary structures of the A and B subunits of Shiga toxin and of Shiga-like toxin I (VT1), isolated from the culture supernatants of Shigella dysenteriae 1 and Escherichia coli O157:H7, respectively, were analyzed by Edman degradation of intact proteins and peptides in their digests with trypsin or Achromobacter protease I and also by fast atom bombardment mass spectrometry of the digests. The results indicated that the A and B subunits of Shiga toxin and Shiga-like toxin I have the same primary structures. The identity of their primary structures was confirmed by determining the nucleotide sequence of the gene encoding Shiga-like toxin I cloned from a Shiga-like toxin I converting phage. This nucleotide sequence was different from that reported by Jackson et al. (Microbial Pathogenesis 1987; 2: 147-153), by Calderwood et al. (Proc Natl Acad Sci USA 1987; 84: 4364-8) and by Grandis et al. (J Bacteriol 1987; 169: 4313-9) in one base at position 231, which was found to be adenine instead of thymine, which they reported. The amino acid residue at position 45 from the N-terminus of the A subunit of Shiga-like toxin I deduced from the nucleotide sequence determined in this study is threonine, which corresponds with that found by amino acid sequencing, whereas from previous reports by other investigators it is serine. Edman degradation of the intact A subunit of Shiga toxin indicated that the A subunit was nicked between Ala253 and Ser254 to form A1 and A2 fragments linked by a disulfide bond.