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.     

Differentiation of human CD8(+) T cells from a memory to memory/effector phenotype

Previous studies of perforin expression and cytokine production in subsets of peripheral human CD45RA(-)CD8(+) T cells with different CD28/CD27 phenotypes showed that CD28(+)CD45RA(-)CD8(+) and CD27(+)CD45RA(-)CD8(+) T cells have characteristics of memory T cells, whereas CD28(-)CD45RA(-)CD8(+) and CD27(-)CD45RA(-)CD8(+) T cells have characteristics of both memory and effector T cells. However, the differentiation pathway from memory CD8(+) T cells into memory/effector CD8(+) T cells has not been completely clarified. We investigated this differentiation pathway using EBV- and human CMV (HCMV)-specific CD8(+) T cells. Three subsets of CD45RA(-)CD8(+) T cells were observed in both total CD8(+) T cells and EBV- or HCMV-specific CD8(+) T cells: CD27(+)CD28(+), CD27(+)CD28(-), and CD27(-)CD28(-). A significant number of the CD27(-)CD28(+) subset was observed in total CD8 T cells. However, this subset was barely detectable in EBV- or HCMV-specific CD8(+) T cells. Analysis of perforin expression and cytotoxic activity in the first three subsets suggested the following differentiation pathway: CD27(+)CD28(+)CD45RA(-)-->CD27(+)CD28(-)CD45RA(-)-->CD27(-)CD28(-)CD45RA(-). This was supported by the observation that the frequency of CCR5(+) cells and CCR7(+) cells decreased during this sequence. Analysis of CCR5 and CCR7 expression in the CD27(+)CD28(+) memory cell subset demonstrated the presence of three CCR5/CCR7 populations: CCR5(-)CCR7(+), CCR5(+)CCR7(+), and CCR5(+)CCR7(-). These findings suggested the following differentiation pathway: CD27(+)CD28(+)CD45RA(-) (CCR5(-)CCR7(+)-->CCR5(+)CCR7(+)-->CCR5(+)CCR7(-))-->CD27(+)CD28(-)CD45RA(-)-->CD27(-)CD28(-)CD45RA(-). The presence of a CD27(-)CD28(+) subset with a CCR5(+)CCR7(-) phenotype implies a specialized role for this subset in the differentiation of CD8(+) T cells.     

Genomic affinities in Arachis section Arachis (Fabaceae): molecular and cytogenetic evidence

Section Arachis is the largest of nine sections in the genus Arachis and includes domesticated peanut, A. hypogaea L. Most species are diploids (x=10) with two tetraploids and a few aneuploids. Three genome types have been recognized in this section (A, B and D), but the genomes are not well characterized and relationships of several newly described species are uncertain. To clarify genomic relationships in section Arachis, cytogenetic information and molecular data from amplified fragment length polymorphism (AFLP) and the trnT-F plastid region were used to provide an additional insight into genome composition and species relationships. Cytogenetic information supports earlier observations on genome types of A. cruziana, A. herzogii, A. kempff-mercadoi and A. kuhlmannii but was inconclusive about the genome composition of A. benensis, A. hoehnei, A. ipaensis, A. palustris, A. praecox and A. williamsii. An AFLP dendrogram resolved species into four major clusters and showed A. hypogaea grouping closely with A. ipaensis and A. williamsii. Sequence data of the trnT-F region provided genome-specific information and showed for the first time that the B and D genomes are more closely related to each other than to the A genome. Integration of information from cytogenetics and biparentally and maternally inherited genomic regions show promise in understanding genome types and relationships in Arachis.     

Different distribution patterns of the two mannose 6-phosphate receptors in rat liver.

Two mannose 6-phosphate receptors, cation-dependent and -independent receptors (CDMPR and CIMPR), play an important role in the intracellular transport of lysosomal enzymes. To investigate functional differences between the two in vivo, their distribution was examined in the rat liver using immunohistochemical techniques. Positive signals corresponding to CIMPR were detected intensely in hepatocytes and weakly in sinusoidal Kupffer cells and interstitial cells in Glisson's capsule. In the liver acinus, hepatocytes in the perivenous region showed a more intense immunoreactivity than those in the periportal region. On the other hand, positive staining of CDMPR was detected at a high level in Kupffer cells, epithelial cells of interlobular bile ducts, and fibroblast-like cells, but the corresponding signal was rather weak in hepatocytes. In situ hybridization analysis also revealed a high level of expression of CIMPR mRNAs in hepatocytes and of CDMPR mRNA in Kupffer cells. By double immunostaining, OX6-positive antigen-presenting cells in Glisson's capsule were co-labeled with the CDMPR signal but were only faintly stained with anti-CIMPR. These different distribution patterns of the two MPRs suggest distinct functional properties of each receptor in liver tissue.     

Investigation of the protective effect of ellagic acid for preventing kidney injury in rats exposed to nicotine during the fetal period

We investigated the possible protective effects of ellagic acid on rat kidneys exposed to nicotine during the fetal period. Twenty pregnant female rats were divided randomly into four groups: control (C), nicotine (N), ellagic acid (EA) and nicotine + ellagic acid (N + EA). Nicotine and ellagic acid treatments were continued throughout the pregnancies and for 15 days after delivery. On day 15, all neonatal pups were sacrificed and their kidneys were removed for biochemical and histopathological examination. The nicotine treatment significantly decreased body weight, total glutathione (GSH), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities, and increased malondialdehyde (MDA) and nitric oxide (NO) levels in the N group compared to controls. EA treatment ameliorated decreased body weight, GSH, GSH-Px and SOD activities, and increased MDA and NO levels in group N + EA compared to group N (p < 0.05). Nicotine caused kidney damage as shown by incomplete development of glomeruli and Bowman's capsules. Nicotine also caused greater apoptosis in group N compared to group C. Ellagic acid treatment produced histological kidney structure that was closer to normal and it exerted an anti-apoptotic effect in the N + EA group compared to the N group. EA played a protective role against nicotine-induced nephrotoxicity and oxidative stress in rats owing to its antioxidant, radical scavenging and anti-apoptotic effects.     

YM-09151-2 but Not l-Sulpiride Induces Transient Dopamine Release in Rat Striatum via a Tetrodotoxin-Insensitive Mechanism

Abstract: The effects of the selective dopamine D₂ receptor antagonists YM-09151-2 and l -sulpiride on the in vivo release of dopamine (DA), l -3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in rat striatum were investigated. The drugs were injected into the striatum through a microinjection needle attached to a dialysis probe. YM-09151-2 (0.1 or 1.0 μg/0.5 μl) injected into the striatum produced a dramatic rapid-onset transient increase in striatal DA release in a dose-dependent manner. However, the DA increase induced by l -sulpiride (15 or 75 ng/0.5 μl) was small and of slower onset. An increase of DOPAC levels by YM-09151-2 was biphasic: The first peak occurred at 40 min, followed by a delayed-onset gradual increase. Slower-onset gradual increases were also found in DOPAC levels after l -sulpiride injection and in HVA levels after injections of both YM-09151-2 and l -sulpiride. The infusion of tetrodotoxin (TTX; 2 μM) revealed two different types of DA release mechanisms: The rapid-onset transient DA release induced by YM-09151-2 was TTX insensitive, whereas the slower-onset DA release induced by l -sulpiride was TTX sensitive. Moreover, the rapid-onset transient DA release was Ca²⁺ independent and was not affected by pre-treatment with l -sulpiride or nomifensine. Therefore, it is concluded that YM-09151-2 injected into the striatum produced a transient striatal DA release that is independent of D₂ receptors and the action potential.     

Demonstration of high-affinity interleukin-2 receptors on B-chronic lymphocytic leukemia cells: functional and structural characterization

Functional and structural characteristics of interleukin 2 (IL-2) receptors on B-cell chronic lymphocytic leukemia (B-CLL) cells were analyzed by a proliferation assay, IL-2 binding assay and cross-linking study. In the 3H-thymidine incorporation assay, purified B-CLL cells from four out of sixteen cases, in which the percentage of Tac antigen (Tac Ag) positive cells in peripheral blood lymphocytes ranged from 0 to 48.8%, responded to IL-2 (100 U/ml) after both 3- and 6-day incubation. No relationship was found between the responsiveness to IL-2 and the percentage of Tac Ag positive cells. In the radiolabeled IL-2 binding assay, however, B-CLL cells from all seven cases examined, including three cases with mitogenic response to IL-2 and four cases without mitogenic response, were shown to have both high- and low-affinity receptors. The number of high- and low-affinity receptors per cell ranged from 29-186 and from 420 to 1,800, respectively. Furthermore, with the affinity cross-linking method p55 (Tac Ag) and p70/75 were found even in cases without mitogenic response in their B-CLL cells. In conclusion, the B-CLL cells so far examined possessed high-affinity IL-2 receptors consisting of p55 and p70/75; nevertheless, this was not sufficient to respond to the mitogenic signal of IL-2.