Cytotoxic T cells expressing the co-stimulatory receptor NKG2 D are increased in cigarette smoking and COPD

Background

A suggested role for T cells in COPD pathogenesis is based on associations between increased lung cytotoxic T lymphocyte (CD8⁺) numbers and airflow limitation. CD69 is an early T cell activation marker. Natural Killer cell group 2 D (NKG2D) receptors are co-stimulatory molecules induced on CD8⁺ T cells upon activation. The activating function of NKG2 D is triggered by binding to MHC class 1 chain-related (MIC) molecules A and B, expressed on surface of stressed epithelial cells. The aim of this study was to evaluate the expression of MIC A and B in the bronchial epithelium and NKG2 D and CD69 on BAL lymphocytes in subjects with COPD, compared to smokers with normal lung function and healthy never-smokers.

Methods

Bronchoscopy with airway lavages and endobronchial mucosal biopsy sampling was performed in 35 patients with COPD, 21 healthy never-smokers and 16 smokers with normal lung function. Biopsies were immunohistochemically stained and BAL lymphocyte subsets were determined using flow cytometry.

Results

Epithelial CD3⁺ lymphocytes in bronchial biopsies were increased in both smokers with normal lung function and in COPD patients, compared to never-smokers. Epithelial CD8⁺ lymphocyte numbers were higher in the COPD group compared to never-smoking controls. Among gated CD3⁺cells in BAL, the percentage of CD8⁺ NKG2D⁺ cells was enhanced in patients with COPD and smokers with normal lung function, compared to never-smokers. The percentage of CD8⁺ CD69⁺ cells and cell surface expression of CD69 were enhanced in patients with COPD and smokers with normal lung function, compared to never-smokers. No changes in the expression of MIC A or MIC B in the airway epithelium could be detected between the groups, whereas significantly decreased soluble MICB was detected in bronchial wash from smokers with normal lung function, compared to never-smokers.

Conclusions

In COPD, we found increased numbers of cytotoxic T cells in both bronchial epithelium and airway lumen. Further, the proportions of CD69- and NKG2D-expressing cytotoxic T cells in BAL fluid were enhanced in both subjects with COPD and smokers with normal lung function and increased expression of CD69 was found on CD8⁺ cells, indicating the cigarette smoke exposure-induced expansion of activated cytotoxic T cells, which potentially can respond to stressed epithelial cells.     

The spatial and temporal expression of VEGF and its receptors 1 and 2 in post-traumatic bone bridge formation of the growth plate

Injuries to growth plates may initiate the formation of reversible or irreversible bone-bridges, may leading to bone length discrepancy or axis deviation. As vascular invasion is essential for the formation of bone tissue, the aim of our study was to investigate the kinetic expression of Vascular Endothelial Growth Factor (VEGF) and its receptors R1 and R2 and the ingrowth of vessels in the formation of bone bridges in a rat physeal injury model. Quantitative Real-Time Polymerase Chain Reaction was performed for VEGF and its receptors. Samples from the proximal physis of the tibial bone were immunohistochemically evaluated for the expression of VEGF and its R1 and R2 receptors and Laminin. Morphologically, physeal bone bridge formation was validated by means of Magnetic Resonance Imaging. Kinetic expression of VEGF and VEGF-R1 mRNA documented a tendency towards an increase in expression on day 7. Histological analyses showed a hematoma containing bone debris on day 1 which was replaced with bony trabeculae by day 14, forming a bone bridge by day 28 which was preceded and accompanied by angiogenesis and consistent with MRI data. VEGF and VEGF-R2 was expressed on the debris within the hematoma and bone trabeculae from days 1 to 28. VEGF-R1 expression was only noted until day 14. The findings of our study suggest that physeal bone bridge formation is in part triggered by VEGF expression and associated with angiogenesis, which was shown to precede bone bridge formation and may be further stimulated through VEGF-positive bone debris.     

Sequencing of DISC1 pathway genes reveals increased burden of rare missense variants in schizophrenia patients from a northern Swedish population

In recent years, DISC1 has emerged as one of the most credible and best supported candidate genes for schizophrenia and related neuropsychiatric disorders. Furthermore, increasing evidence--both genetic and functional--indicates that many of its protein interaction partners are also involved in the development of these diseases. In this study, we applied a pooled sample 454 sequencing strategy, to explore the contribution of genetic variation in DISC1 and 10 of its interaction partners (ATF5, Grb2, FEZ1, LIS-1, PDE4B, NDE1, NDEL1, TRAF3IP1, YWHAE, and ZNF365) to schizophrenia susceptibility in an isolated northern Swedish population. Mutation burden analysis of the identified variants in a population of 486 SZ patients and 514 control individuals, revealed that non-synonymous rare variants with a MAF<0.01 were significantly more present in patients compared to controls (8.64% versus 4.7%, P?=?0.018), providing further evidence for the involvement of DISC1 and some of its interaction partners in psychiatric disorders. This increased burden of rare missense variants was even more striking in a subgroup of early onset patients (12.9% versus 4.7%, P?=?0.0004), highlighting the importance of studying subgroups of patients and identifying endophenotypes. Upon investigation of the potential functional effects associated with the identified missense variants, we found that ～90% of these variants reside in intrinsically disordered protein regions. The observed increase in mutation burden in patients provides further support for the role of the DISC1 pathway in schizophrenia. Furthermore, this study presents the first evidence supporting the involvement of mutations within intrinsically disordered protein regions in the pathogenesis of psychiatric disorders. As many important biological functions depend directly on the disordered state, alteration of this disorder in key pathways may represent an intriguing new disease mechanism for schizophrenia and related neuropsychiatric diseases. Further research into this unexplored domain will be required to elucidate the role of the identified variants in schizophrenia etiology.     

The N-methyl-d-aspartic acid receptor antagonist memantine ameliorates and delays the development of arthritis by enhancing regulatory T cells

The neuroendocrine impact on rheumatoid arthritis is not yet fully described although numerous neurotransmitters are shown to act as inflammatory modulators. One of these is the excitatory transmitter glutamate (Glu). In this study, the influence of the Glu receptor (GluR)-mediated effects on collagen-induced arthritis (CIA) was investigated. CIA was induced in DBA/1 mice by immunization with chicken collagen type II (CII). Mice were exposed to the following GluR antagonists: group 1, the N-methyl-D-aspartic acid (NMDA) receptor channel blocker memantine; group 2, the metabotropic GluR antagonist AIDA, and group 3, the excitatory amino acid receptor antagonist kynurenic acid (KA). Arthritis was evaluated clinically and histologically and compared to PBS-treated controls. The effects of treatment on T cell populations and the levels of anti-CII and anti-citrullinated peptide antibodies were evaluated. Memantine treatment significantly improved the course of CIA, reducing synovitis (p = 0.007) and the frequency of erosions (p = 0.007). Memantine treatment up-regulated the expression of Foxp3 in spleen CD4+ T cells followed by an increase in CD4+CD25+ regulatory T cells. The other GluR antagonists, AIDA and KA, had no effect on CIA. These results demonstrate that blockade of the NMDA receptor channel with memantine delays and attenuates the development of arthritis, probably by promoting the development of regulatory T lymphocytes.     

Identification of DNA-Synthesizing Bacterial Cells in Coastal North Sea Plankton

We describe a method for microscopic identification of DNA-synthesizing cells in bacterioplankton samples. After incubation with the halogenated thymidine analogue bromodeoxyuridine (BrdU), environmental bacteria were identified by fluorescence in situ hybridization (FISH) with horseradish peroxidase (HRP)-linked oligonucleotide probes. Tyramide signal amplification was used to preserve the FISH staining during the subsequent immunocytochemical detection of BrdU incorporation. DNA-synthesizing cells were visualized by means of an HRP-labeled antibody Fab fragment and a second tyramide signal amplification step. We applied our protocol to samples of prefiltered (pore size, 1.2 μm) North Sea surface water collected during early autumn. After 4 h of incubation, BrdU incorporation was detected in 3% of all bacterial cells. Within 20 h the detectable DNA-synthesizing fraction increased to >14%. During this period, the cell numbers of members of the Roseobacter lineage remained constant, but the fraction of BrdU-incorporating Roseobacter sp. cells doubled, from 24 to 42%. In Alteromonas sp. high BrdU labeling rates after 4 to 8 h were followed by a 10-fold increase in abundance. Rapid BrdU incorporation was also observed in members of the SAR86 lineage. After 4 h of incubation, cells affiliated with this clade constituted 8% of the total bacteria but almost 50% of the visibly DNA-synthesizing bacterial fraction. Thus, this clade might be an important contributor to total bacterioplankton activity in coastal North Sea water during periods of low phytoplankton primary production. The small size and low ribosome content of SAR86 cells are probably not indications of inactivity or dormancy.     

Comparison of Fluorescently Labeled Oligonucleotide and Polynucleotide Probes for the Detection of Pelagic Marine Bacteria and Archaea

We compared the detection of bacteria and archaea in the coastal North Sea and at Monterey Bay, Calif., after fluorescence in situ hybridization (FISH) either with rRNA-targeted oligonucleotide probes monolabeled with the cyanin dye Cy3 (oligoFISH) or with fluorescein-labeled polyribonucleotide probes (polyFISH). During an annual cycle in German Bight surface waters, the percentages of bacteria visualized by polyFISH (annual mean, 77% of total counts) were significantly higher than those detected by oligoFISH (53%). The fraction of total bacteria visualized by oligoFISH declined during winter, whereas cell numbers determined by polyFISH remained constant throughout the year. Depth profiles from Monterey Bay showed large differences in the fraction of bacterial cells visualized by polyFISH and oligoFISH in the deeper water layers irrespective of the season. Image-analyzed microscopy indicated that the superior detection of cells by polyFISH with fluorescein-labeled probes in bacterioplankton samples was less a consequence of higher absolute fluorescence intensities but was rather related to quasi-linear bleaching dynamics and to a higher signal-to-background ratio. The relative abundances of archaea in North Sea and Monterey Bay spring samples as determined by oligoFISH were on average higher than those determined by polyFISH. However, simultaneous hybridizations with oligonucleotide probes for bacteria and archaea suggested that the oligoFISH probe ARCH915 unspecifically stained a population of bacteria. Using either FISH technique, blooms of archaea were observed in North Sea surface waters during the spring and summer months. Marine group II archaea (Euryarchaeota) reached >30% of total picoplankton abundances, as determined by polyFISH. We suggest that studies of pelagic microbial community structure using oligoFISH with monolabeled probes should focus on environments that yield detections ≥70% of total cell counts, e.g., coastal surface waters during spring and summer.