The Cannabinoid Receptor Type 2 as Mediator of Mesenchymal Stromal Cell Immunosuppressive Properties

Mesenchymal stromal cells are non-hematopoietic, multipotent progenitor cells producing cytokines, chemokines, and extracellular matrix proteins that support hematopoietic stem cell survival and engraftment, influence immune effector cell development, maturation, and function, and inhibit alloreactive T-cell responses. The immunosuppressive properties of human mesenchymal stromal cells have attracted much attention from immunologists, stem cell biologists and clinicians.Recently, the presence of the endocannabinoid system in hematopoietic and neural stem cells has been demonstrated. Endocannabinoids, mainly acting through the cannabinoid receptor subtype 2, are able to modulate cytokine release and to act as immunosuppressant when added to activated T lymphocytes.In the present study, we have investigated, through a multidisciplinary approach, the involvement of the endocannabinoids in migration, viability and cytokine release of human mesenchymal stromal cells.We show, for the first time, that cultures of human mesenchymal stromal cells express all of the components of the endocannabinoid system, suggesting a potential role for the cannabinoid CB2 receptor as a mediator of anti-inflammatory properties of human mesenchymal stromal cells, as well as of their survival pathways and their capability to home and migrate towards endocannabinoid sources.     

Influence of aperiodic summer droughts on leaf litter breakdown and macroinvertebrate assemblages: testing the <Emphasis Type="Italic">drying memory</Emphasis> in a Central Apennines River (Aterno River,Italy)

Management of multiple exploited stocks of anadromous salmonids in large catchments requires understanding of movement and catchment use by the migrating fish and of their harvesting. The spawning migration of sea trout (Salmo trutta) and Atlantic salmon (Salmo salar) was studied in the River Tweed, UK, using acoustic telemetry to complement exploitation rate data and to quantify catchment penetration. Salmon (n = 79) and sea trout (n = 65) were tagged in the tidal-influenced Tweed in summer–autumn. No tagged salmon left the river before spawning, but 3% (2010) and 8% (2011) of pre-spawning sea trout dropped out. Combined tag regurgitation/fish mortality in salmon was 12.5%, while trout mortality was 6% (2010) and 0% (2011). The estimated spawning positions of salmon and sea trout differed; tagged salmon were mostly in the main channel while trout occurred mostly in the upper Tweed and tributaries. Early fish migrated upstream slower than later fish, but sea trout moved through the lower-middle river more quickly than salmon, partly supporting the hypothesis that the lower exploitation rate in autumn of trout (1 vs 3.3% for salmon) there is generated by differences in migration behaviour.     

CCDC65 Mutation Causes Primary Ciliary Dyskinesia with Normal Ultrastructure and Hyperkinetic Cilia

Background

Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by impaired ciliary function, leading to chronic sinopulmonary disease. The genetic causes of PCD are still evolving, while the diagnosis is often dependent on finding a ciliary ultrastructural abnormality and immotile cilia. Here we report a novel gene associated with PCD but without ciliary ultrastructural abnormalities evident by transmission electron microscopy, but with dyskinetic cilia beating.

Methods

Genetic linkage analysis was performed in a family with a PCD subject. Gene expression was studied in Chlamydomonas reinhardtii and human airway epithelial cells, using RNA assays and immunostaining. The phenotypic effects of candidate gene mutations were determined in primary culture human tracheobronchial epithelial cells transduced with gene targeted shRNA sequences. Video-microscopy was used to evaluate cilia motion.

Results

A single novel mutation in CCDC65, which created a termination codon at position 293, was identified in a subject with typical clinical features of PCD. CCDC65, an orthologue of the Chlamydomonas nexin-dynein regulatory complex protein DRC2, was localized to the cilia of normal nasal epithelial cells but was absent in those from the proband. CCDC65 expression was up-regulated during ciliogenesis in cultured airway epithelial cells, as was DRC2 in C. reinhardtii following deflagellation. Nasal epithelial cells from the affected individual and CCDC65-specific shRNA transduced normal airway epithelial cells had stiff and dyskinetic cilia beating patterns compared to control cells. Moreover, Gas8, a nexin-dynein regulatory complex component previously identified to associate with CCDC65, was absent in airway cells from the PCD subject and CCDC65-silenced cells.

Conclusion

Mutation in CCDC65, a nexin-dynein regulatory complex member, resulted in a frameshift mutation and PCD. The affected individual had altered cilia beating patterns, and no detectable ultrastructural defects of the ciliary axoneme, emphasizing the role of the nexin-dynein regulatory complex and the limitations of certain methods for PCD diagnosis.     

Population Genomic Analyses Based on 1 Million SNPs in Commercial Egg Layers

Identifying signatures of selection can provide valuable insight about the genes or genomic regions that are or have been under selective pressure, which can lead to a better understanding of genotype-phenotype relationships. A common strategy for selection signature detection is to compare samples from several populations and search for genomic regions with outstanding genetic differentiation. Wright''s fixation index, F_ST, is a useful index for evaluation of genetic differentiation between populations. The aim of this study was to detect selective signatures between different chicken groups based on SNP-wise F_ST calculation. A total of 96 individuals of three commercial layer breeds and 14 non-commercial fancy breeds were genotyped with three different 600K SNP-chips. After filtering a total of 1 million SNPs were available for F_ST calculation. Averages of F_ST values were calculated for overlapping windows. Comparisons of these were then conducted between commercial egg layers and non-commercial fancy breeds, as well as between white egg layers and brown egg layers. Comparing non-commercial and commercial breeds resulted in the detection of 630 selective signatures, while 656 selective signatures were detected in the comparison between the commercial egg-layer breeds. Annotation of selection signature regions revealed various genes corresponding to productions traits, for which layer breeds were selected. Among them were NCOA1, SREBF2 and RALGAPA1 associated with reproductive traits, broodiness and egg production. Furthermore, several of the detected genes were associated with growth and carcass traits, including POMC, PRKAB2, SPP1, IGF2, CAPN1, TGFb2 and IGFBP2. Our approach demonstrates that including different populations with a specific breeding history can provide a unique opportunity for a better understanding of farm animal selection.     

Diastereoselective functionalisation of Baylis–Hillman adducts: a convenient approach to α-methyl-α-amino acids

The N-tosyl carbamates 4a–e, easily prepared starting from the Baylis–Hillman adducts 3a–e, underwent cyclization carried out with I₂/NIS in the presence of NaH, to give the corresponding 2-oxo-1,3-oxazolidines 5a–e in good yield and total stereoselection when the substituent at C-5 is Ar. After the removal of tosyl group, followed by the cleavage of the heterocyclic ring, the α-methyl-α-amino acids 8a,b and 10 were obtained in good yield as hydrochlorides.     

A Scale-Corrected Comparison of Linkage Disequilibrium Levels between Genic and Non-Genic Regions

The understanding of non-random association between loci, termed linkage disequilibrium (LD), plays a central role in genomic research. Since causal mutations are generally not included in genomic marker data, LD between those and available markers is essential for capturing the effects of causal loci on localizing genes responsible for traits. Thus, the interpretation of association studies requires a detailed knowledge of LD patterns. It is well known that most LD measures depend on minor allele frequencies (MAF) of the considered loci and the magnitude of LD is influenced by the physical distances between loci. In the present study, a procedure to compare the LD structure between genomic regions comprising several markers each is suggested. The approach accounts for different scaling factors, namely the distribution of MAF, the distribution of pair-wise differences in MAF, and the physical extent of compared regions, reflected by the distribution of pair-wise physical distances. In the first step, genomic regions are matched based on similarity in these scaling factors. In the second step, chromosome- and genome-wide significance tests for differences in medians of LD measures in each pair are performed. The proposed framework was applied to test the hypothesis that the average LD is different in genic and non-genic regions. This was tested with a genome-wide approach with data sets for humans (Homo sapiens), a highly selected chicken line (Gallus gallus domesticus) and the model plant Arabidopsis thaliana. In all three data sets we found a significantly higher level of LD in genic regions compared to non-genic regions. About 31% more LD was detected genome-wide in genic compared to non-genic regions in Arabidopsis thaliana, followed by 13.6% in human and 6% chicken. Chromosome-wide comparison discovered significant differences on all 5 chromosomes in Arabidopsis thaliana and on one third of the human and of the chicken chromosomes.     

Low 2-dimensional CD4 T cell receptor affinity for myelin sets in motion delayed response kinetics

T cells recognizing self-peptides that mediate autoimmune disease and those that are responsible for efficacious immunity against pathogens may differ in affinity for antigen due to central and peripheral tolerance mechanisms. Here we utilize prototypical self-reactive (myelin) and viral-specific (LCMV) T cells from T cell receptor (TCR) transgenic mice (2D2 and SMARTA, respectively) to explore affinity differences. The T cells responsive to virus possessed >10,000 fold higher 2D affinity as compared to the self-reactive T cells. Despite their dramatically lower affinity for their cognate ligand, 2D2 T cells respond with complete, albeit delayed, activation (proliferation and cytokine production). SMARTA activation occurs rapidly, achieving peak phosphorylation of p38 (1 minute), Erk (30 minutes), and Jun (3 hours) as well as CD69 and CD25 upregulation (3 and 6 hours, respectively), with a corresponding early initiation of proliferation. 2D2 stimulation with MOG results in altered signaling--no phospho-Erk or phospho-p38 accumulation, significantly delayed activation kinetics of Jun (12 hours), and delayed but sustained SHP-1 activity--as well as delayed CD69 and CD25 expression (12-24 hours), and slow initiation of proliferation. This delay was not intrinsic to the 2D2 T cells, as a more potent antigen with >100-fold increased 2D affinity restored rapid response kinetics in line with those identified for the viral antigen. Taken together, these data demonstrate that time can offset low TCR affinity to attain full activation and suggest a mechanism by which low affinity T cells participate in autoimmune disease.     

A novel germline mutation in Peroxisome Proliferator-Activated Receptor γ gene associated with large intestine polyp formation and dyslipidemia

We report a novel PPARG germline mutation in a patient affected by colorectal cancer that replaces serine 289 with cysteine in the mature protein (S289C). The mutant has impaired transactivation potential and acts as dominant negative to the wild type receptor. In addition, it no longer restrains cell proliferation both in vitro and in vivo. Interestingly, the S289C mutant poorly activates target genes and interferes with the inflammatory pathway in tumor tissues and proximal normal mucosa. Consistently, only mutation carriers exhibit colonic lesions that can evolve to dysplastic polyps. The proband presented also dyslipidemia, hypertension and overweight, not associated to type 2 diabetes; of note, family members tested positive for the mutation and display only a dyslipidemic profile at variable penetrance with other biochemical parameters in the normal range. Finally, superimposing the mutation to the crystal structure of the ligand binding domain, the new Cys289 becomes so closely positioned to Cys285 to form an S–S bridge. This would reduce the depth of the ligand binding pocket and impede agonist positioning, explaining the biological effects and subcellular distribution of the mutant protein. This is the first PPARG germline mutation associated with dyslipidemia and colonic polyp formation that can progress to full-blown adenocarcinoma.