Dual-probe assay for rapid detection of drug-resistant Mycobacterium tuberculosis by real-time PCR

Mutations in particular nucleotides of genes coding for drug targets or drug-converting enzymes lead to drug resistance in Mycobacterium tuberculosis. For rapid detection of drug-resistant M. tuberculosis in clinical specimens, a simple and applicable method is needed. Eight TaqMan minor groove binder (MGB) probes, which discriminate one-base mismatches, were designed (dual-probe assay with four reaction tubes). The target of six MGB probes was the rpoB gene, which is involved in rifampin resistance; five probes were designed to detect for mutation sites within an 81-bp hot spot of the rpoB gene, and one probe was designed as a tuberculosis (TB) control outside the rpoB gene hot-spot. We also designed probes to examine codon 315 of katG and codon 306 of embB for mutations associated with resistance to isoniazid and ethambutol, respectively. Our system was M. tuberculosis complex specific, because neither nontuberculous mycobacteria nor bacteria other than mycobacteria reacted with the system. Detection limits in direct and preamplified analyses were 250 and 10 fg of genomic DNA, respectively. The system could detect mutations of the rpoB, katG, and embB genes in DNAs extracted from 45 laboratory strains and from sputum samples of 27 patients with pulmonary TB. This system was much faster (3 h from DNA preparation) than conventional drug susceptibility testing (3 weeks). Results from the dual-MGB-probe assay were consistent with DNA sequencing. Because the dual-probe assay system is simple, rapid, and accurate, it can be applied to detect drug-resistant M. tuberculosis in clinical laboratories.     

Activation of SiO2-supported zirconocene catalysts by common trialkylaluminiums

A modified SiO₂ was prepared by reacting SiO₂ with Cl₂Si(CH₃)₂ in toluene, on which methylaluminoxane (MAO) was supported to obtain a catalyst precursor. The mixture of the precursor and Cp₂ZrCl₂ (Cp: cyclopentadienyl) gave polyethylene in a high yield even by using common trialkylaluminiums as cocatalyst. Surprisingly, the MAO-free catalyst system composed of the modified SiO₂ and Cp₂ZrCl₂ was also found to be activated by common trialkylaluminiums.     

Study of the biological activities of toxic shock syndrome toxin-1: II. Induction of the proliferative response and the interleukin 2 production by T cells from human peripheral blood mononuclear cells stimulated with the toxin

Toxic shock syndrome toxin-1 (TSST-1) is an exotoxin produced by Staphylococcus aureus isolated from patients with toxic shock syndrome. We investigated the proliferative response of human lymphocytes and their interleukin 2 (IL-2) production after stimulation with TSST-1 in vitro. Human cord blood mononuclear cells (HCBM) and human peripheral blood mononuclear cells (HPBM) could proliferate with TSST-1 stimulation. T cell-depleted HPBM showed only a marginal response to this toxin. A IL-2-like factor with a molecular weight of 15-18 kD was obtained from the supernatants of TSST-1-stimulated HPBM cultures. The factor was absorbed by CTLL-2 cells but not by T cell-depleted murine spleen cells, indicating that it is IL-2. HPBM are very sensitive to TSST-1: a low concentration of TSST-1 (0.01 ng/ml in 36 h stimulation) and a short period of stimulation (8 h at 10 ng/ml of the toxin) were fully effective for HPBM to produce substantial amounts of IL-2. Removal of T cells abrogated the TSST-1-induced IL-2 production by HPBM. Reconstituted cell cultures of nylon wool column-passed T cells and macrophages produced IL-2 by TSST-1 stimulation and, furthermore, the accessory activity of the macrophages could be partially replaced by a macrophage-derived factor containing interleukin 1. These findings indicate that T cells require macrophages or IL-1 for TSST-1-induced production of IL-2. The roles of lymphokines, including IL-2, in the development of this illness are discussed.     

Purification and characterization of Staphylococcus aureus FRI 1169 and 587 toxic shock syndrome exotoxins.

An exotoxin was purified from a toxic shock toxin (TST)-producing Staphylococcus aureus strain, FRI 1169, and another exotoxin was purified from a pyrogenic exotoxin C (PEC)-producing S. aureus strain, 587. Both strains had been isolated from toxic-shock syndrome patients. The two exotoxins were purified by the same method of ion-exchange chromatography, chromatofocusing, and gel filtration. After purification, those exotoxins gave a line of identity against an anti-TST serum and also were immunologically similar to TST in a double-diffusion test. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, each exotoxin gave a single band with a relative mobility identical to that of the other. Their molecular weights (24,000), isoelectric points (7.0), amino acid compositions, and NH2-terminal amino acid sequences (the first four residues) were identical. Both produced fever and enhanced host susceptibility to lethal endotoxin shock in rabbits, comparable with PEC. These findings show that the two exotoxins are the same protein, which is assumed to be TST. When injected into rabbits, the culture supernatant of strain 587 showed biological activity like that described above, whereas the culture supernatant neutralized with anti-TST immunoglobulin did not. This showed that PEC-producing strain 587 does not produce any toxin with these biological activities in rabbits except TST.     

Dose- and time-dependent responses for micronucleus induction by X-rays and fast neutrons in gill cells of medaka (Oryzias latipes)

Medaka fish (Oryzias latipes) were exposed to various doses of X-rays or fast neutrons, and the frequency of micronucleated cells (MNCs) was measured in gills sampled at 12- or 24-hr intervals from 12 to 96 hr after exposure. The resulting time course of MNC frequency was biphasic, with a clear peak 24 hr after exposure, irrespective of the kind of radiation applied and the dose used. The half-life of MNCs induced in the gill tissues by the two exposures fluctuated around 28 hr, with no significant dose-dependent trend for either X-ray- or neutron-exposed fish. As assayed 24 hr after exposure, the MNC frequency increased linearly over the control level with increasing doses of both X-rays and fast neutrons. The relative biological effectiveness (RBE) of fast neutrons to X-rays for MNC induction was estimated to be 4.3 +/- 0.6. This value is close to the RBE value of 5.1 +/- 0.3 reported for fast neutron induction of somatic crossing-over mutations in Drosophila melanogaster that arise from recombination repair of DNA double-strand breaks. These results and other data support our conclusion that the medaka gill cell micronucleus assay is a reliable short-term test for detecting potential inducers of DNA double-strand breaks.     

TWO CASES OF ACQUIRED TOXOPLASMIC LYMPHADENITIS. Light and Electron Microscopic and Immunohistochemical Studies

We report two cases of acquired toxoplasmic lymphadenitis, one with toxoplasmic cysts and the organisms of Toxoplasma gondii and the other with the organisms only. These cysts and organisms were observed in paraffin-embedded sections, touch smears and ultrathin sections for electron microscopy. Touch smears were especially valuable for the quick and accurate diagnosis of toxoplasmic lymphadenitis. We also studied immature sinus histiocytosis (ISH) in these cases. The predominant cells of ISH were confirmed to be B lymphocytes immunohistochemically, the majority being positive for polyclonal surface IgM. ISH was observed in the perifollicular and paracortical areas surrounding post-capillary venules (PCV), whereas the sinuses were only partially involved.