Proinflammatory cytokine responses induced by influenza A (H5N1) viruses in primary human alveolar and bronchial epithelial cells

Background

Fatal human respiratory disease associated with influenza A subtype H5N1 has been documented in Hong Kong, and more recently in Vietnam, Thailand and Cambodia. We previously demonstrated that patients with H5N1 disease had unusually high serum levels of IP-10 (interferon-gamma-inducible protein-10). Furthermore, when compared with human influenza virus subtype H1N1, the H5N1 viruses in 1997 (A/Hong Kong/483/97) (H5N1/97) were more potent inducers of pro-inflammatory cytokines (e.g. tumor necrosis factor-a) and chemokines (e.g. IP-10) from primary human macrophages in vitro, which suggests that cytokines dysregulation may play a role in pathogenesis of H5N1 disease. Since respiratory epithelial cells are the primary target cell for replication of influenza viruses, it is pertinent to investigate the cytokine induction profile of H5N1 viruses in these cells.

Methods

We used quantitative RT-PCR and ELISA to compare the profile of cytokine and chemokine gene expression induced by H5N1 viruses A/HK/483/97 (H5N1/97), A/Vietnam/1194/04 and A/Vietnam/3046/04 (both H5N1/04) with that of human H1N1 virus in human primary alveolar and bronchial epithelial cells in vitro.

Results

We demonstrated that in comparison to human H1N1 viruses, H5N1/97 and H5N1/04 viruses were more potent inducers of IP-10, interferon beta, RANTES (regulated on activation, normal T cell expressed and secreted) and interleukin 6 (IL-6) in primary human alveolar and bronchial epithelial cells in vitro. Recent H5N1 viruses from Vietnam (H5N1/04) appeared to be even more potent at inducing IP-10 than H5N1/97 virus.

Conclusion

The H5N1/97 and H5N1/04 subtype influenza A viruses are more potent inducers of proinflammatory cytokines and chemokines in primary human respiratory epithelial cells than subtype H1N1 virus. We suggest that this hyper-induction of cytokines may be relevant to the pathogenesis of human H5N1 disease.     

The role of site-specific N-glycosylation in secretion of soluble forms of rabies virus glycoprotein

Rabies virus glycoprotein is important in the biology and pathogenesis of neurotropic rabies virus infection. This transmembrane glycoprotein is the only viral protein on the surface of virus particles, is the viral attachment protein that facilitates virus uptake by the infected cell, and is the target of the host humoral immune response to infection. The extracellular domain of this glycoprotein has N- glycosylation sequons at Asn37, Asn247, and Asn319. Appropriate glycosylation of these sequons is important in the expression of the glycoprotein. Soluble forms of rabies virus glycoprotein were constructed by insertion of a stop codon just external to the transmembrane domain. Using site-directed mutagenesis and expression in transfected eukaryotic cells, it was possible to compare the effects of site-specific glycosylation on the cell-surface expression and secretion of transmembrane and soluble forms, respectively, of the same glycoprotein. These studies yielded the surprising finding that although any of the three sequons permitted cell surface expression of full-length rabies virus glycoprotein, only the N-glycan at Asn319 permitted secretion of soluble rabies virus glycoprotein. Despite its biological and medical importance, it has not yet been possible to determine the crystal structure of the full-length transmembrane form of rabies virus glycoprotein which contains heterogeneous oligosaccharides. The current studies demonstrate that a soluble form of rabies virus glycoprotein containing only one sequon at Asn319 is efficiently secreted in the presence of the N-glycan processing inhibitor 1-deoxymannojirimycin. Thus, it is possible to purify a conformationally relevant form of rabies virus glycoprotein that contains only one N-glycan with a substantial reduction in its microheterogeneity. This form of the glycoprotein may be particularly useful for future studies aimed at elucidating the three-dimensional structure of this important glycoprotein.      

A new quantitative air-lift sampler for collecting macroinvertebrates on stony bottoms in deep rivers

SUMMARY. 1. As three previous comparative studies of deep-water samplers for benthic macroinvertebrates in rivers highlighted the need for a quantitative sampler for use on stony substrata, a new air-lift sampler was developed. It can be operated from a small boat by two people, weighs 13.5–20.0 kg, depending on the length of riser used, and extends the maximum range of substrata that may be sampled quantitatively from 16–32 mm to 128—256 mm. The sampling area is isolated by forcing a collecting cylinder into the substratum, and rapid evacuation of the contents is assisted by a vibrator.
2. All the major specifications of the sampler were determined experimentally in a large tank using three sizes of substrata and plastic pellets to represent invertebrates. The sampler performed accurately to a depth of at least 8 cm on substrata ranging from gravel (2–4 mm) to large stones (32–36 mm long).
3. The performance of the sampler was compared with that of a Ponar grab and Pearson el at. air-lift sampler at two sites on a large river and also with a Naturalist's dredge and a diver-operated Hess-Waters sampler at one of the sites where there were large stones up to 280 mm long. In terms of both mean taxa per sample and mean numbers m⁻², samples taken using the new air-lift sampler provided estimates comparable to or belter than those obtained with the other samplers.     

A comparative study of seven grabs used for sampling benthic macroinvertebrates in rivers

SUMMARY. After considering the large number of grabs described in the literature, seven grabs of weight < 25 kg were chosen for manual operation from a small boat: Van-Veen grab, weighted and unweighted Ponar grabs, Friedinger version of the Petersen grab, Dietz-La Fond mud-snapper, pole-operated Birge-Ekman grab and pole-operated Allan grab. Random samples (number of sampling units n= 10) were taken in a large tank with a known number of 2-mm cylindrical plastic pellets amongst stones of uniform size. Separate experiments were performed with four sizes of stones (model ranges: 2–4 mm, 8–16 mm, 16–32 mm, 32–64 mm). Stratified random samples (n= 10) were taken in rivers and the modal particle sizes at four sites were 0.004–0.06 mm, 0.5–2 mm, 16–64 mm and 64–128 mm. All grabs usually took a representative sample of the substratum at each site with no strong bias towards a particular particle size. The general performance of the Friedinger, Dietz-La Fond and Allan grabs was poor, except on a muddy bottom, with frequent failure to operate, small samples of substratum and a mean depth of penetration < 3 cm in all substrata except mud for the Dietz-La Fond and Allan grabs. The Van-Veen and Birge-Ekman grabs sampled to a mean depth < 3 cm in mud and fine gravel (2–4 mm), but the Birge-Ekman jammed frequently in fine gravel. Both Ponar grabs operated well and sampled to a mean depth ≥ 5 cm in mud and fine gravel, > 3 cm when small stones (8–16 mm) were present and 2 cm (weighted Ponar only) when larger stones (> 16 mm) were present in a gravel bottom. The mean depth was <0.8 cm for all grabs when larger stones (>16 mm) were predominant on the bottom. In the tank experiments with pellets, the efficiencies for the total catches of the Friedinger, Dietz-La Fond and Allan grabs were low with values <45% for fine gravel (2–4 mm), < 22% for small stones (8–16 mm) and <5% for a substratum of larger stones (>16 mm). If 50% is the minimum acceptable efficiency, then the Ponar, Van-Veen and Birge-Ekman grabs were adequate for fine gravel, only the two Ponar grabs were adequate for small stones and no grabs were adequate for sampling a substratum of larger stones (>16 mm). In field trials, the relative abundances of major taxa were similar for most grabs at each site; Friedinger and Dietz-La Fond grabs were the major exceptions. In terms of both mean number of taxa and mean number of invertebrates m^?2 the Ponar, Birge-Ekman and Allan grabs performed well on the predominantly muddy substratum at site 1, but only the weighted Ponar grab performed adequately on the predominantly gravel bottom with some large stones (>16 mm) at site 2. All grabs performed badly when larger stones (>16 mm) were predominant on the bottom (sites 3, 4). The relationship between the variances and means of the samples taken with each grab followed a power law for the catches of pellets in tank experiments, and for major taxa and total numbers at each site in field trials. Values of exponents in the power law lay within the range 1.14–2.34. The coefficient of variation was also frequently related to the sample mean and was an unreliable statistic for comparing the precision of grabs.     

Loss of alveolar membrane diffusing capacity and pulmonary capillary blood volume in pulmonary arterial hypertension

Background

Reduced gas transfer in patients with pulmonary arterial hypertension (PAH) is traditionally attributed to remodeling and progressive loss of pulmonary arterial vasculature that results in decreased capillary blood volume available for gas exchange.

Methods

We tested this hypothesis by determination of lung diffusing capacity (DL) and its components, the alveolar capillary membrane diffusing capacity (D_m) and lung capillary blood volume (V_c) in 28 individuals with PAH in comparison to 41 healthy individuals, and in 19 PAH patients over time. Using single breath simultaneous measure of diffusion of carbon monoxide (DL_CO) and nitric oxide (DL_NO), DL and D_m were respectively determined, and V_c calculated. D_m and V_c were evaluated over time in relation to standard clinical indicators of disease severity, including brain natriuretic peptide (BNP), 6-minute walk distance (6MWD) and right ventricular systolic pressure (RVSP) by echocardiography.

Results

Both DL_CO and DL_NO were reduced in PAH as compared to controls and the lower DL in PAH was due to loss of both D_m and V_c (all p < 0.01). While DL_CO of PAH patients did not change over time, DL_NO decreased by 24 ml/min/mmHg/year (p = 0.01). Consequently, D_m decreased and V_c tended to increase over time, which led to deterioration of the D_m/V_c ratio, a measure of alveolar-capillary membrane functional efficiency without changes in clinical markers.

Conclusions

The findings indicate that lower than normal gas transfer in PAH is due to loss of both D_m and V_c, but that deterioration of D_m/V_c over time is related to worsening membrane diffusion.