Identification and HLA-Tetramer-Validation of Human CD4+ and CD8+ T Cell Responses against HCMV Proteins IE1 and IE2

Human cytomegalovirus (HCMV) is an important human pathogen. It is a leading cause of congenital infection and a leading infectious threat to recipients of solid organ transplants as well as of allogeneic hematopoietic cell transplants. Moreover, it has recently been suggested that HCMV may promote tumor development. Both CD4⁺ and CD8⁺ T cell responses are important for long-term control of the virus, and adoptive transfer of HCMV-specific T cells has led to protection from reactivation and HCMV disease. Identification of HCMV-specific T cell epitopes has primarily focused on CD8⁺ T cell responses against the pp65 phosphoprotein. In this study, we have focused on CD4⁺ and CD8⁺ T cell responses against the immediate early 1 and 2 proteins (IE1 and IE2). Using overlapping peptides spanning the entire IE1 and IE2 sequences, peripheral blood mononuclear cells from 16 healthy, HLA-typed, donors were screened by ex vivo IFN-γ ELISpot and in vitro intracellular cytokine secretion assays. The specificities of CD4⁺ and CD8⁺ T cell responses were identified and validated by HLA class II and I tetramers, respectively. Eighty-one CD4⁺ and 44 CD8⁺ T cell responses were identified representing at least seven different CD4 epitopes and 14 CD8 epitopes restricted by seven and 11 different HLA class II and I molecules, respectively, in total covering 91 and 98% of the Caucasian population, respectively. Presented in the context of several different HLA class II molecules, two epitope areas in IE1 and IE2 were recognized in about half of the analyzed donors. These data may be used to design a versatile anti-HCMV vaccine and/or immunotherapy strategy.     

Bioinformatics-driven identification and examination of candidate genes for non-alcoholic fatty liver disease

Objective

Candidate genes for non-alcoholic fatty liver disease (NAFLD) identified by a bioinformatics approach were examined for variant associations to quantitative traits of NAFLD-related phenotypes.

Research Design and Methods

By integrating public database text mining, trans-organism protein-protein interaction transferal, and information on liver protein expression a protein-protein interaction network was constructed and from this a smaller isolated interactome was identified. Five genes from this interactome were selected for genetic analysis. Twenty-one tag single-nucleotide polymorphisms (SNPs) which captured all common variation in these genes were genotyped in 10,196 Danes, and analyzed for association with NAFLD-related quantitative traits, type 2 diabetes (T2D), central obesity, and WHO-defined metabolic syndrome (MetS).

Results

273 genes were included in the protein-protein interaction analysis and EHHADH, ECHS1, HADHA, HADHB, and ACADL were selected for further examination. A total of 10 nominal statistical significant associations (P<0.05) to quantitative metabolic traits were identified. Also, the case-control study showed associations between variation in the five genes and T2D, central obesity, and MetS, respectively. Bonferroni adjustments for multiple testing negated all associations.

Conclusions

Using a bioinformatics approach we identified five candidate genes for NAFLD. However, we failed to provide evidence of associations with major effects between SNPs in these five genes and NAFLD-related quantitative traits, T2D, central obesity, and MetS.     

Altered Response to A(H1N1)pnd09 Vaccination in Pregnant Women: A Single Blinded Randomized Controlled Trial

Background

Pregnant women were suspected to be at particular risk when H1N1pnd09 influenza became pandemic in 2009. Our primary objective was to compare the immune responses conferred by MF59®-adjuvanted vaccine (Focetria®) in H1N1pnd09-naïve pregnant and non-pregnant women. The secondary aims were to compare influences of dose and adjuvant on the immune response.

Methods

The study was nested in the Copenhagen Prospective Studies on Asthma in Childhood (COPSAC₂₀₁₀) pregnancy cohort in 2009-2010 and conducted as a single-blinded block-randomised [1∶1∶1] controlled clinical trial in pregnant women after gestational week 20: (1) 7.5 µg H1N1pnd09 antigen with MF59-adjuvant (Pa7.5 µg); (2) 3.75 µg antigen half MF59-adjuvanted (Pa3.75 µg); (3) 15 µg antigen unadjuvanted (P15 µg); and in non-pregnant women receiving (4) 7.5 µg antigen full adjuvanted (NPa7.5 µg). Blood samples were collected at baseline, 3 weeks, 3 and 10 months after vaccination, adverse events were recorded prospectively.

Results

58 pregnant women were allocated to Pa7.5 µg and 149 non-pregnant women were recruited to NPa7.5 µg. The sero-conversion rate was significantly increased in non-pregnant (NPa7.5 µg) compared with pregnant (Pa7.5 µg) women (OR = 2.48 [1.03–5.95], p = 0.04) and geometric mean titers trended towards being higher, but this difference was not statistically significant (ratio 1.27 [0.85–1.93], p = 0.23). The significant titer increase rate showed no difference between pregnant (Pa7.5 µg) and non-pregnant (NPa7.5 µg) groups (OR = 0.49 [0.13–1.85], p = 0.29).

Conclusion

Our study suggests the immune response to the 7.5 µg MF59-adjuvanted Focetria® H1N1pnd09 vaccine in pregnant women may be diminished compared with non-pregnant women.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01012557","term_id":"NCT01012557"}}NCT01012557.     

Mechanisms of Topoisomerase I (TOP1) Gene Copy Number Increase in a Stage III Colorectal Cancer Patient Cohort

Background

Topoisomerase I (Top1) is the target of Top1 inhibitor chemotherapy. The TOP1 gene, located at 20q12-q13.1, is frequently detected at elevated copy numbers in colorectal cancer (CRC). The present study explores the mechanism, frequency and prognostic impact of TOP1 gene aberrations in stage III CRC and how these can be detected by fluorescent in situ hybridization (FISH).

Methods

Nine CRC cell line metaphase spreads were analyzed by FISH with a TOP1 probe in combination with a reference probe covering either the centromeric region of chromosome 20 (CEN-20) or chromosome 2 (CEN-2). Tissue sections from 154 chemonaive stage III CRC patients, previously studied with TOP1/CEN-20, were analyzed with TOP1/CEN-2. Relationships between biomarker status and overall survival (OS), time to recurrence (TTR) in CRC and time to local recurrence (LR; rectal cancer only) were determined.

Results

TOP1 aberrations were observed in four cell line metaphases. In all cell lines CEN-2 was found to reflect chromosomal ploidy levels and therefore the TOP1/CEN-2 probe combination was selected to identify TOP1 gene gains (TOP1/CEN-2≥1.5). One hundred and three patients (68.2%) had TOP1 gain, of which 15 patients (14.6%) harbored an amplification (TOP1/CEN-20≥2.0). TOP1 gene gain did not have any association with clinical endpoints, whereas TOP1 amplification showed a non-significant trend towards longer TTR (multivariate HR: 0.50, p = 0.08). Once amplified cases were segregated from other cases of gene gain, non-amplified gene increases (TOP1/CEN-2≥1.5 and TOP1/CEN-20<2.0) showed a trend towards shorter TTR (univariate HR: 1.57, p = 0.07).

Conclusions

TOP1 gene copy number increase occurs frequently in stage III CRC in a mechanism that often includes CEN-20. Using CEN-2 as a measurement for tumor ploidy levels, we were able to discriminate between different mechanisms of gene gain, which appeared to differ in prognostic impact. TOP1 FISH guidelines have been updated.     

Effect of anti-inflammatory medication on the running-induced rise in patella tendon collagen synthesis in humans

NSAIDs are widely used in the treatment of inflammatory diseases as well as of tendon diseases associated with pain in sports and labor. However, the effect of NSAID intake, and thus blockade of PGE(2) production, on the tendon tissue adaptation is unknown. The purpose of the present study was to elucidate the possible effects of NSAID intake on healthy tendon collagen turnover in relation to a strenuous bout of endurance exercise. Fifteen healthy young men were randomly assigned into two experimental groups, with one group receiving indomethacin (oral 2 × 100 mg Confortid daily for 7 days; NSAID; n = 7) and a placebo group (n = 8). Both groups were exposed to a prolonged bout of running (36 km). The collagen synthesis NH?-terminal propeptide of type I (PINP) and PGE? concentrations were measured before and 72 h following the run in the patella tendon by microdialysis. The peritendinous concentrations of PINP increased significantly in the placebo group as a result of the run, as shown previously. PGE? levels were significantly decreased 72 h after the run compared with basal levels in the subjects treated with NSAID and unchanged in the placebo group. The NSAID intake abolished the adaptive increase in collagen synthesis in the patella tendon found in the placebo group in response to the prolonged exercise (P < 0.05). The present study demonstrates that intake of NSAID decreased interstitial PGE? and abolished the exercise-induced adaptive increase in collagen synthesis in human tendons.     

Human 2'-phosphodiesterase localizes to the mitochondrial matrix with a putative function in mitochondrial RNA turnover

The vertebrate 2-5A system is part of the innate immune system and central to cellular antiviral defense. Upon activation by viral double-stranded RNA, 5'-triphosphorylated, 2'-5'-linked oligoadenylate polyribonucleotides (2-5As) are synthesized by one of several 2'-5'-oligoadenylate synthetases. These unusual oligonucleotides activate RNase L, an unspecific endoribonuclease that mediates viral and cellular RNA breakdown. Subsequently, the 2-5As are removed by a 2'-phosphodiesterase (2'-PDE), an enzyme that apart from breaking 2'-5' bonds also degrades regular, 3'-5'-linked oligoadenylates. Interestingly, 2'-PDE shares both functionally and structurally characteristics with the CCR4-type exonuclease-endonuclease-phosphatase family of deadenylases. Here we show that 2'-PDE locates to the mitochondrial matrix of human cells, and comprise an active 3'-5' exoribonuclease exhibiting a preference for oligo-adenosine RNA like canonical cytoplasmic deadenylases. Furthermore, we document a marked negative association between 2'-PDE and mitochondrial mRNA levels following siRNA-directed knockdown and plasmid-mediated overexpression, respectively. The results indicate that 2'-PDE, apart from playing a role in the cellular immune system, may also function in mitochondrial RNA turnover.     

Rapid determination of Phytophthora infestans sporangia using a surface plasmon resonance immunosensor

Phytophthora infestans is the cause of late blight disease in potato and is an economically important pathogen worldwide. Early disease detection is important to implement disease control measures. In this study a surface plasmon resonance (SPR) immunosensor for detection of P. infestans sporangia is presented. The specificity of an existing mouse monoclonal antibody (phyt/G1470 mAb) against P. infestans was investigated in plate-trapped antigen ELISA and in subtractive inhibition ELISA. No or only limited cross-reactivity was observed against representatives having air-borne spores from Ascomycetes, Deuteromycetes as well as Basidiomycetes. phyt/G1470 mAb was incorporated in a subtractive inhibition SPR assay, consisting of a pre-incubation of mAb and sporangia, a centrifugation step to remove sporangia-bound phyt/G1470 mAb and quantification of remaining phyt/G1470 mAb by SPR. Good intra- and interday assay variability was observed and the assay had a detection limit of 2.2x10(6) sporangia/ml. Analysis time was 75 min, which is superior to existing P. infestans detection methods.     

Detection of fungal spores using a generic surface plasmon resonance immunoassay

This paper describes a biosensor-based method for detection of fungal spores using surface plasmon resonance (SPR). The approach involves the use of a mouse monoclonal antibody (Pst mAb8) and a SPR sensor for label-free detection of urediniospores from the model organism Puccinia striiformis f.sp. tritici (Pst). In the subtractive inhibition assay, urediniospores and Pst mAb8 were mixed, urediniospore-bound Pst mAb8 removed by centrifugation and the remaining Pst mAb8 quantified using the SPR sensor. Assay conditions were optimised and a detection limit of 3.1 x 10(5)urediniospores/ml was achieved. Spiked Pst samples were further examined in a background of a related spore and it was found that Pst detection was possible in this mixture. This study represent the first use of SPR technology for fungal spore detection as well as the first report of a successful biosensor-based detection strategy for Pst.     

Gas in Scattering Media Absorption Spectroscopy (GASMAS) Detected Persistent Vacuum in Apple Tissue After Vacuum Impregnation

The microstructure and the capillary pressure of the pore space are important variables for better understanding of the complex phenomena occurring during vacuum impregnation (VI) of plant tissues. In this study, we used GASMAS (Gas in Scattering Media Absorption Spectroscopy) of oxygen to, non-destructively, measure the dynamics of the internal pressure in apple pieces after restoration of the atmospheric pressure. Apple pieces were impregnated with isotonic sucrose solution (18% w/v) at different reduced pressures (15, 30, 45 kPa (abs.)). After restoration of the atmospheric pressure, the pressure of the remaining pore space gas could remain as low as 50 kPa (abs) and rise slowly toward ambient over a time scale of hours. Both the residual vacuum and the timescale of pressure equilibration with ambient varied with applied vacuum level and apple variety. It is proposed that at least a part of the pore space of apples may be hydrophobic, giving rise to a negative Laplace pressure, and thus the convective flow of impregnating solution is arrested at a mechanical equilibrium where internal pressure is lower than external pressure. Further pressure equilibration can then only be achieved either by gas diffusion in gas phase, or by gradual wetting of the pores.     

A whole-tree chamber system for examining tree-level physiological responses of field-grown trees to environmental variation and climate change

A whole-tree chamber (WTC) system was installed at Flakaliden in northern Sweden to examine the long-term physiological responses of field-grown 40-year-old Norway spruce trees [Picea abies (L.) Karst.] to climate change. The WTCs were designed as large cuvettes to allow the net tree-level CO(2) and water fluxes to be measured on a continuous basis. A total of 12 WTCs were used to impose combinations of atmospheric carbon dioxide concentration, [CO(2)], and air temperature treatments. The air inside the ambient and elevated [CO(2)] WTCs was maintained at 365 and 700 micromol mol(-1), respectively. The air temperature inside the ambient temperature WTCs tracked air temperature outside the WTCs. Elevated temperatures were altered on a monthly time-step and ranged between +2.8 and +5.6 degrees C above ambient temperature. The system allowed continuous, long-term measurement of whole-tree photosynthesis, night-time respiration and transpiration. The performance of the WTCs was assessed using winter and spring data sets. The ability of the WTC system to measure tree-level physiological responses is demonstrated. All WTCs displayed a high level of control over tracking of air temperatures. The set target of 365 micromol mol(-1) in the ambient [CO(2)] chambers was too low to be maintained during winter because of tree dormancy and the high natural increase in [CO(2)] over winter at high latitudes such as the Flakaliden site. Accurate control over [CO(2)] in the ambient [CO(2)] chambers was restored during the spring and the system maintained the elevated [CO(2)] target of 700 micromol mol(-1) for both measurement periods. Air water vapour deficit (VPD) was accurately tracked in ambient temperature WTCs. However, as water vapour pressure in all 12 WTCs was maintained at the level of non-chambered (reference) air, VPD of elevated temperature WTCs was increased.