Altered effector function of peripheral cytotoxic cells in COPD

Background

There is mounting evidence that perforin and granzymes are important mediators in the lung destruction seen in COPD. We investigated the characteristics of the three main perforin and granzyme containing peripheral cells, namely CD8⁺ T lymphocytes, natural killer (NK; CD56⁺CD3^-) cells and NKT-like (CD56⁺CD3⁺) cells.

Methods

Peripheral blood mononuclear cells (PBMCs) were isolated and cell numbers and intracellular granzyme B and perforin were analysed by flow cytometry. Immunomagnetically selected CD8+ T lymphocytes, NK (CD56⁺CD3^-) and NKT-like (CD56⁺CD3⁺) cells were used in an LDH release assay to determine cytotoxicity and cytotoxic mechanisms were investigated by blocking perforin and granzyme B with relevant antibodies.

Results

The proportion of peripheral blood NKT-like (CD56⁺CD3⁺) cells in smokers with COPD (COPD subjects) was significantly lower (0.6%) than in healthy smokers (smokers) (2.8%, p < 0.001) and non-smoking healthy participants (HNS) (3.3%, p < 0.001). NK (CD56⁺CD3^-) cells from COPD subjects were significantly less cytotoxic than in smokers (16.8% vs 51.9% specific lysis, p < 0.001) as were NKT-like (CD56⁺CD3⁺) cells (16.7% vs 52.4% specific lysis, p < 0.001). Both cell types had lower proportions expressing both perforin and granzyme B. Blocking the action of perforin and granzyme B reduced the cytotoxic activity of NK (CD56⁺CD3^-) and NKT-like (CD56⁺CD3⁺) cells from smokers and HNS.

Conclusion

In this study, we show that the relative numbers of peripheral blood NK (CD56⁺CD3^-) and NKT-like (CD56⁺CD3⁺) cells in COPD subjects are reduced and that their cytotoxic effector function is defective.     

The structure of interrupted human AC microsatellites

Microsatellite lengths change over evolutionary time through a process of replication slippage. A recently proposed model of this process holds that the expansionary tendencies of slippage mutation are balanced by point mutations breaking longer microsatellites into smaller units and that this process gives rise to the observed frequency distributions of uninterrupted microsatellite lengths. We refer to this as the slippage/point-mutation theory. Here we derive the theory's predictions for interrupted microsatellites comprising regions of perfect repeats, labeled segments, separated by dinucleotide interruptions containing point mutations. These predictions are tested by reference to the frequency distributions of segments of AC microsatellite in the human genome, and several predictions are shown not to be supported by the data, as follows. The estimated slippage rates are relatively low for the first four repeats, and then rise initially linearly with length, in accordance with previous work. However, contrary to expectation and the experimental evidence, the inferred slippage rates decline in segments above 10 repeats. Point mutation rates are also found to be higher within microsatellites than elsewhere. The theory provides an excellent fit to the frequency distribution of peripheral segment lengths but fails to explain why internal segments are shorter. Furthermore, there are fewer microsatellites with many segments than predicted. The frequencies of interrupted microsatellites decline geometrically with microsatellite size measured in number of segments, so that for each additional segment, the number of microsatellites is 33.6% less. Overall we conclude that the detailed structure of interrupted microsatellites cannot be reconciled with the existing slippage/point-mutation theory of microsatellite evolution, and we suggest that microsatellites are stabilized by processes acting on interior rather than on peripheral segments.     

Pulmonary expression of leukemia inhibitory factor induces B cell hyperplasia and confers protection in hyperoxia

Leukemia inhibitory factor (LIF) is produced by a large number of pulmonary cells in response to diverse stimuli. Exaggerated levels of LIF have also been detected in the adult respiratory distress syndrome and other disorders. The biologic effects of LIF in the lung, however, have not been elucidated. To define the respiratory effects of LIF, we generated transgenic mice in which human LIF was selectively targeted to the mature lung. In these mice, transgene activation caused an impressive increase in bronchoalveolar lavage (BAL) cellularity with a significant increase in BAL and tissue B lymphocytes. LIF also conferred protection in 100% O2 where it decreased alveolar-capillary protein leak and enhanced survival. This protective effect was associated with the induction of interleukin (IL)-6 mRNA and protein. LIF transgenic mice with a null mutation in IL-6 were more sensitive to the toxic effects of 100% O2 than LIF-transgenic animals with a wild-type IL-6 locus. These studies demonstrate that LIF induces B cell hyperplasia and confers protection in hyperoxic acute lung injury. They also demonstrate that LIF induces IL-6 and that the protective effects of LIF are mediated, in part, via this inductive event. LIF may be an important regulator of B cell-mediated responses and oxidant injury in the lung.     

Effects of inspiratory flow on diaphragmatic motor output in normal subjects.

Increasing inspiratory flow (V) has been shown to shorten neural inspiratory time (TI(n)) in normal subjects breathing on a mechanical ventilator, but the effect of V on respiratory motor output before inspiratory termination has not previously been studied in humans. While breathing spontaneously on a mechanical ventilator, eight normal subjects were intermittently exposed to 200-ms-duration positive pressure pulses of different amplitudes at the onset of inspiration. Based on the increase in V above control breaths (DeltaV), trials were grouped into small, medium, and large groups (mean DeltaV: 0.51, 1.11, and 1.65 l/s, respectively). We measured TI(n), transdiaphragmatic pressure (Pdi), and electrical activity (electromyogram) of the diaphragm (EMGdi). Transient increases in V caused shortening of TI(n) from 1.34 to 1.10 (not significant), 1.55 to 1.11 (P < 0.005), and 1.58 to 1.17 s (P < 0. 005) in the small, medium, and large DeltaV groups, respectively. EMGdi measured at end TI(n) of the pulse breaths was 131 (P < 0.05), 142, and 155% (P < 0.05) of the EMGdi of the control breaths at an identical time point in the small, medium, and large trials, respectively. The latency of the excitation was 126 +/- 42 (SD) ms, consistent with a reflex effect. Increasing V had two countervailing effects on Pdi: 1) a depressant mechanical effect due primarily to the force-length (11.2 cmH(2)O/l) relation of the diaphragm, and 2) an increase in diaphragm activation. For the eight subjects, mean peak Pdi did not change significantly, but there was significant intersubject variability, reflecting variability in the strength of the excitation reflex. We conclude that increasing inspiratory V causes a graded facilitation of EMGdi, which serves to counteract the negative effect of the force-length relation on Pdi.     

Predicting Epileptic Seizures in Advance

Epilepsy is the second most common neurological disorder, affecting 0.6–0.8% of the world''s population. In this neurological disorder, abnormal activity of the brain causes seizures, the nature of which tend to be sudden. Antiepileptic Drugs (AEDs) are used as long-term therapeutic solutions that control the condition. Of those treated with AEDs, 35% become resistant to medication. The unpredictable nature of seizures poses risks for the individual with epilepsy. It is clearly desirable to find more effective ways of preventing seizures for such patients. The automatic detection of oncoming seizures, before their actual onset, can facilitate timely intervention and hence minimize these risks. In addition, advance prediction of seizures can enrich our understanding of the epileptic brain. In this study, drawing on the body of work behind automatic seizure detection and prediction from digitised Invasive Electroencephalography (EEG) data, a prediction algorithm, ASPPR (Advance Seizure Prediction via Pre-ictal Relabeling), is described. ASPPR facilitates the learning of predictive models targeted at recognizing patterns in EEG activity that are in a specific time window in advance of a seizure. It then exploits advanced machine learning coupled with the design and selection of appropriate features from EEG signals. Results, from evaluating ASPPR independently on 21 different patients, suggest that seizures for many patients can be predicted up to 20 minutes in advance of their onset. Compared to benchmark performance represented by a mean S1-Score (harmonic mean of Sensitivity and Specificity) of 90.6% for predicting seizure onset between 0 and 5 minutes in advance, ASPPR achieves mean S1-Scores of: 96.30% for prediction between 1 and 6 minutes in advance, 96.13% for prediction between 8 and 13 minutes in advance, 94.5% for prediction between 14 and 19 minutes in advance, and 94.2% for prediction between 20 and 25 minutes in advance.     

Differential Activation of Killer Cells in the Circulation and the Lung: A Study of Current Smoking Status and Chronic Obstructive Pulmonary Disease (COPD)

Background

CD8⁺ T-lymphocytes, natural killer T-like cells (NKT-like cells, CD56⁺CD3⁺) and natural killer cells (NK cells, CD56⁺CD3⁻) are the three main classes of human killer cells and they are implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Activation of these cells can initiate immune responses by virtue of their production of inflammatory cytokines and chemokines that cause lung tissue damage, mucus hypersecretion and emphysema. The objective of the current study was to investigate the activation levels of human killer cells in healthy non-smokers, healthy smokers, ex-smokers with COPD and current smokers with COPD, in both peripheral blood and induced sputum.

Methods/Principal Findings

After informed consent, 124 participants were recruited into the study and peripheral blood or induced sputum was taken. The activation states and receptor expression of killer cells were measured by flow cytometry. In peripheral blood, current smokers, regardless of disease state, have the highest proportion of activated CD8⁺ T-lymphocytes, NKT-like cells and NK cells compared with ex-smokers with COPD and healthy non-smokers. Furthermore, CD8⁺ T-lymphocyte and NK cell activation is positively correlated with the number of cigarettes currently smoked. Conversely, in induced sputum, the proportion of activated killer cells was related to disease state rather than current smoking status, with current and ex-smokers with COPD having significantly higher rates of activation than healthy smokers and healthy non-smokers.

Conclusions

A differential effect in systemic and lung activation of killer cells in COPD is evident. Systemic activation appears to be related to current smoking whereas lung activation is related to the presence or absence of COPD, irrespective of current smoking status. These findings suggest that modulating killer cell activation may be a new target for the treatment of COPD.     

Bacterial assay for the rapid assessment of antifouling and fouling release properties of coatings and materials

An assay has been developed to accurately quantify the growth and release behaviour of bacterial biofilms on several test reference materials and coatings, using the marine bacterium Cobetia marina as a model organism. The assay can be used to investigate the inhibition of bacterial growth and release properties of many surfaces when compared to a reference. The method is based upon the staining of attached bacterial cells with the nucleic acid-binding, green fluorescent SYTO 13 stain. A strong linear correlation exists between the fluorescence of the bacterial suspension measured (RFU) using a plate reader and the total bacterial count measured with epifluorescence microscopy. This relationship allows the fluorescent technique to be used for the quantification of bacterial cells attached to surfaces. As the bacteria proliferate on the surface over a period of time, the relative fluorescence unit (RFU) measured using the plate reader also shows an increase with time. This was observed on all three test surfaces (glass, Epikote and Silastic T2) over a period of 4 h of bacterial growth, followed by a release assay, which was carried out by the application of hydrodynamic shear forces using a custom-made rotary device. Different fixed rotor speeds were tested, and based on the release analysis, 12 knots was used to provide standard shear force. The assay developed was then applied for assessing three different antifouling coatings of different surface roughness. The novel assay allows the rapid and sensitive enumeration of attached bacteria directly on the coated surface. This is the first plate reader assay technique that allows estimation of irreversibly attached bacterial cells directly on the coated surface without their removal from the surface or extraction of a stain into solution.     

Dynamics of HIV infection studied with cellular automata and conformon-P systems

Recently a cellular automaton (CA) has been used to model the dynamics of HIV infection, with interesting results. We replicate and further test this model, and we introduce an alternative model based on conformon-P (cP) systems. We find (in common with other recently published comments) that the CA model is very sensitive to initial conditions and produces appropriate qualitative dynamics only for a narrow range of rule probabilities. In contrast, the cP system model is robust to a wide range of conditions and parameters, with more reproducible qualitative agreement to the overall dynamics and to the densities of healthy and infected cells observed in vivo.     

垃圾填埋场中厌氧真菌18SrDNA的PCR扩增及鉴定

采用机械破壁法直接从来自7个不同地区的垃圾填埋场滤液样本中提取真菌DNA,应用真菌通用引物NS1和NS8扩增18SrDNA（约1800bp),多聚酶链式反应（PCR）产物的琼脂糖凝胶电泳结果表明所有的样本均得到了扩增;以PCR产物作为模板,采用厌氧真菌Chytridiomycetes科的专用引物Chyt-719和Chyt-1553进行二次PCR扩增（约857bp),该阳性扩增产物克隆和测序结果首次表明在食草动物瘤胃中存在的厌氧真菌Chytridiomycetes也存在于垃圾填埋场中,且为Neocallimastix属。