Molecular typing of the bacterial flora in sputum of cystic fibrosis patients

Despite recent advances in therapy, lower airway infections remain the major cause of morbidity and mortality in cystic fibrosis (CF) patients. Bacterial colonisation of the lower airways in CF is limited to a few bacterial species, commonly Staphylococcus aureus, Pseudomonas aeruginosa and Haemophilus influenzae. Burkholderia cepacia colonisation is much rarer, but it has been thought to be associated with more advanced lung disease and increased mortality. A rapid characterisation of the bacterial flora in sputum of CF patients is of great importance for proper treatment. The aim of this study was to establish bacterial profiles and to identify pathogenic bacteria in respiratory specimens by means of molecular methods including temporal temperature gradient gel electrophoresis (TTGE) and DNA sequencing of PCR amplicons derived from 16S rDNA variable V3 and V6 regions. Sputa of 13 CF patients (7 males/6 females, age 19-59 years) collected at the Stockholm CF centre were analysed. TTGE revealed the presence of complex bacterial profiles in all samples. The V3 and V6 PCR amplicons were cloned and sequenced by real-time DNA Pyrosequencing. DNA from Staphylococcus aureus, Haemophilus influenzae, and Pseudomonas aeruginosa, respectively, was identified together with sequences from normal oral cavity flora. The results were in reasonable agreement with those obtained by conventional bacterial culture, considering that only known CF pathogens are included in routine reports. However, the methodology seems too elaborate to be introduced into daily routine     

Residual effects of prior exercise and recovery on subsequent exercise-induced metabolic responses

Abstract Data on the metabolic responses to repeated endurance exercise sessions are limited. Thus, the aims of this study were to examine (1) the impact of prior exercise on metabolic responses to a subsequent exercise session and (2) the effect of different recovery periods between two daily exercise sessions on metabolic responses to the second bout of exercise. Nine male elite athletes participated in four 25-h trials: one bout of exercise (ONE), two bouts of exercise separated by 3 h of rest and one meal (SHORT), two bouts of exercise separated by 6 h of rest and two meals (LONG), and a trial with no exercise (REST). All exercise bouts consisted of 10 min cycling at 50% followed by 65 min at 75% of maximal O₂ uptake. Compared to no prior exercise (ONE), a previous bout of exercise (SHORT) was followed by higher mean O₂ uptake, heart rate (HR), rectal temperature (T_R), excess post-exercise oxygen consumption and lower respiratory exchange ratio (R) during and after a similar exercise session 3 h later. A longer rest interval between the two exercise bouts (6 h versus 3 h) and an additional meal resulted in a decrease in O₂ uptake, HR, T_R and an increase in R during the second bout of exercise, but no effects on post-exercise metabolism were found. Thus, augmented metabolic stress was observed when strenuous exercise was repeated after only 3 h of recovery, but this was attenuated when a longer recovery period including an additional meal was provided between the exercise sessions.     

A new, simple and rapid method for enumerating human T lymphocytes in full blood: the E. coli (ATCC 11303) rosette test

A new bacterial rosette technique for enumerating T lymphocytes is described. E. coli (strain B; ATCC 11303), fixed in formaldehyde after overnight growth in thioglycolate medium, are mixed with washed whole blood cells (100 μl) and after incubation at 4°C, slides are made, stained and counted. The nature of the lymphocytes forming E. coli rosettes was demonstrated by comparing their cytochemical staining characteristics with those of E rosetted lymphocytes, and by mixed E. coli and E, mouse E rosette and Fc receptor tests, and by mixed E. coli rosette tests and anti-Ig staining. E. coli and E rosette tests in controls and pediatric patients were also compared. The results show that T_μ and T_γ cells rosette with E. coli.     

Evidence of allosteric conformational changes in the antibody constant region upon antigen binding

We have addressed the question of whether antigen binding induces a conformational change in the heavy chain constant (C(H)) domain of antibodies using staphylococcal protein A or streptococcal protein G as probes, since these proteins are known to bind to IgG domains such as C(H)1 and C(H)2-C(H)3 domains. Biosensor assays on interactions between these proteins and mouse IgG specific to (4-hydroxy-3-nitrophenyl)acetyl (NP) or their enzymatic fragments conducted in the presence or absence of the hapten, NP-epsilon-aminocaproic acid (NP-Cap), showed that the binding of IgG to these proteins was inhibited by the binding of NP-Cap. The results of isothermal titration calorimetry also revealed that the association constant for the interaction of protein A with IgG2b decreased by the addition of NP-Cap. These results suggested that antigen binding induced conformational changes in binding sites for protein G or protein A located at C(H)1 and C(H)2-C(H)3 domains, respectively.     

Ambient temperature related sleep changes in rats neonatally treated with capsaicin

Ambient temperature related sleep changes in rats neonatally treated with capsaicin. PHYSIOL BEHAV 00(0) 000-000, 2004. The study was conducted on adult male Wistar rats, neonatally treated with capsaicin to destroy the peripheral warm receptors. The sleep-wakefulness was recorded for 5 h at an ambient temperature (T(amb)) of 18, 24, 30 and 33 degrees C on different days. The rectal temperatures (T(r)) of the rats were studied on exposure to 6 and 37 degrees C for 2 h to assess their thermoregulatory ability. The changes in the behavioral thermoregulation were assessed by noting the thermal preference of rats when they were placed in an environmental chamber with 3 interconnected compartments maintained at 24, 27 and 30 degrees C. Slow wave (SWS) and rapid eye movement (REM) sleep were decreased at 18 degrees C and increased at 30 degrees C, in control rats. There was a decrease in REM sleep and no change in SWS when T(amb) was raised from 30 to 33 degrees C. However, in neonatally capsaicin treated rats, sleep was increased even at 33 degrees C, though there was no significant change in sleep when T(amb) was increased from 18 to 24 degrees C. Capsaicin treated rats showed thermoregulatory deficiency at 37 degrees C but the thermal preference was unaltered in these rats. The results suggest that the central warm receptors can produce alteration in sleep at different T(amb), even in absence of peripheral warm receptors. The behavioral thermoregulation was unaffected in these rats, though their ability to defend the body temperature in warm environment was affected.     

Changes in hypothalamic temperature of rats after daily exposure to heat at a fixed time

Rats were subjected to an ambient temperature (T_a) of 33°C for ca. 5 h during the last half of the dark phase for 5, 14 or 28 consecutive days (heat-exposed rats, HE), while control rats were kept at a constant T_a of 24°C. After the heat exposure schedule, the levels of hypothalamic temperature (T_hy) as an index of body core temperature in the HE were significantly lower than those of the controls for 2–4 h in the last half of the dark phase. The low levels of T_hy persisted during the specific period for 1, 3 and 6 days after the end of the 5-, 14- and 28-day heat exposure schedules, respectively. These results confirm that, in rats subjected to daily heat exposure for ca. 5 h at a fixed time per day, their T_hy falls during the period when the rats were previously exposed to heat, and suggest that the duration of the specific T_hy change observed after completing the heat exposure schedule depends on the length of the heat exposure schedule.     

Vaccination of the Leishmania major susceptible BALB/c mouse. I. The precise selection of peptide determinant influences CD4+ T cell subset expression

BALB/c mice are susceptible to cutaneous lelshmanlasls uponinfection with Leishmania major while C57BL/6 are not. Thereis a major promastigote surface protease (PSP or gp63) whichis available in both native and recomblnant forms, and for whichthe primary amlno acid sequence is known. Immunization withPSP has been shown to offer some protection against challengewith the live organism. Therefore, we attempted to develop apeptide vaccine with PSP peptldes. In the first experiments,recall prollferatlve responses to PSP were measured using aset of 15mer peptldes spanning the entire PSP molecule whichallowed designation of major determinant regions in BALB/c,C57BL/6, and CBA mice. Several of these determinants were promiscuousand shared almost the identical core amlno acid residues inthe different strains. Immunization with major determinant peptldeswas recalled vigorously with L. major soluble antigen as wellas with PSP. The response to peptide was almost entirely T_h1as measured by a localized ELISA assay for single-cell productionof IFN-. A similar assay for IL-5, which overcomes problemsof sensitivity and inhibition by lymphoklnes produced by T_h1cells, Indicates very little production of T_h1 cells even byBALB/c. It was found that if a major responsive peak was examinedby recall with overlapping peptldes, the highest, central peptidegave a mainly Th1 response while the boundary, less efficientpeptldes gave more of a T_h2 response. Possible reasons for thiswere discussed. These results point to the importance of selectingthe exactly appropriate peptide in considering a vacclnogenthat might protect susceptible individuals. Even the choiceof a somewhat immunogenlc peptide within the determinant envelopemight actually exacerbate infection by steering the responsein a T_h2 direction.     

胸部体表导纳变化与胸腔中血管导纳变化的关系

目的：探讨胸部体表导纳变化与胸腔中血管导纳变化的关系。方法：根据导纳与阻抗互为倒数的关系，导出胸部体表导纳变化公式，并用模型实验证明。结果：单根血管在胸部体表引起的导纳变化与血管本身的导纳变化和基础导纳的比值成正比，与血管的长度和胸部体表两检测电极之间的基础导纳成正比，与血管到两检测电极连线的距离成反比；多根血管在胸部体表共同引起的导纳变化等于各血管单独在胸部体表产生的导纳变化的代数和，即在电导纳图测量中导纳变化遵从标量迭加原理。结论：本文结果可为心导纳图的波形重建奠定理论基础。