Widespread use of DNA restriction fragment length polymorphism (RFLP) to differentiate strains of Mycobacterium tuberculosis to monitor the transmission of tuberculosis has been hampered by the need to culture this slow-growing organism and by the level of technical sophistication needed for RFLP typing. We have developed a simple method which allows simultaneous detection and typing of M. tuberculosis in clinical specimens and reduces the time between suspicion of the disease and typing from 1 or several months to 1 or 2 days. The method is based on polymorphism of the chromosomal DR locus, which contains a variable number of short direct repeats interspersed with nonrepetitive spacers. The method is referred to as spacer oligotyping or "spoligotyping" because it is based on strain-dependent hybridization patterns of in vitro-amplified DNA with multiple spacer oligonucleotides. Most of the clinical isolates tested showed unique hybridization patterns, whereas outbreak strains shared the same spoligotype. The types obtained from direct examination of clinical samples were identical to those obtained by using DNA from cultured M. tuberculosis. This novel preliminary study shows that the novel method may be a useful tool for rapid disclosure of linked outbreak cases in a community, in hospitals, or in other institutions and for monitoring of transmission of multidrugresistant M. tuberculosis. Unexpectedly, spoligotyping was found to differentiate M. bovis from M. tuberculosis, a distinction which is often difficult to make by traditional methods.
Chronic lung disease (CLD) of prematurity is associated with an initial increase in pulmonary neutrophils followed by pulmonary fibrosis. We determined whether the proinflammatory cytokines, IL-1 beta and IL-6, were increased in the bronchoalveolar lavage fluid obtained from nine infants (median gestation 25 wk, birthweight 820 g) who developed CLD, seven (28 wk, 1110 g) who recovered from the respiratory distress syndrome (RDS), and four (38 wk, 2690 g) control infants. IL-1 beta and IL-6 protein were both increased in the bronchoalveolar lavage fluid from the CLD groups when compared with the RDS and control groups. This difference for both the cytokines was most marked on d 10 of age, when results from infants with and without CLD were compared (IL-1 beta, 4.6 versus 1.1 ng/mL, p < 0.05; and IL-6, 9.5 versus 1.5 ng/mL, p < 0.05). Immunocytochemistry of lavage cells for IL-1 beta, IL-6, and IL-8 protein showed alveolar macrophages to contain all three cytokines, with lesser staining evident in neutrophils, and in epithelial cells occasionally obtained by lavage. The contribution of alveolar macrophages and luminal cells to the increase in IL-6 and IL-1 was determined by performing semiquantitative reverse transcription-polymerase chain reactions on RNA extracted from lavage cells. IL-6 mRNA expression was increased in lavage cells from the CLD infants when compared with the RDS group. However, the expression for IL-1 beta and IL-8 mRNA was similar in both groups. These results suggest that IL-1 beta, IL-6, and IL-8 may contribute to the pathogenesis of CLD, and that, in CLD, IL-6 may be produced by cells within the air spaces.
Interleukin-8 (IL-8), soluble intercellular adhesion molecule-i (sICAM), elastase and neutrophils were assessed in bronchoalveolar lavage fluid from nine infants who developed chronic lung disease (CLD) after respiratory distress syndrome (RDS), seven who had recovered from RDS, and in four control infants. IL-8, sICAM, elastase and neutrophils in bronchoalveolar lavage fluid were increased in the CLD group, the differences being most pronounced at 10 days of age. When babies with and without CLD were compared at 10 days of age, bronchoalveolar lavage fluid from the babies with CLD had significantly increased (114.0 vs 12*7 nglml), sICAM (19.0 vs 1*1 ,ug/ml), elastase (6.9 vs 0*9 ,ug/ml) and neutrophils (1.9 vs 0.4X109/1). In serum the increased concentration of IL-8 observed at birth in the CLD (247 pg/ml) and RDS (192 pg/ml) groups decreased over three weeks to the concentrations observed in the controls (<70 pg/ml). Persistent inflammation could be a major contributory factor in the development of CLD.
New episodes of asthma during the epidemic on 24 and 25 June 1994 were associated with a fall in air temperature and a rise in grass pollen concentration. Non-epidemic asthma was significantly associated with a greater number of environmental changes. This may indicate that the patients with thunderstorm associated asthma were a separate population, sensitive to different environmental stimuli.
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