ALBERT

Description: Background: In overhead sports like volleyball, the onset of a rotator cuff tendinopathy due to functional overload is a common observation. An angiofibroblastic etiopathogenesis has been hypothesized, whereby a greater anaerobic metabolism occurs in critical zones of the tendon with a lower degree of vascularization; this would induce collagen and extracellular matrix degradation, that could then trigger a compensatory neovascularization response.We performed a clinical observational study of 80 elite volleyball players, monitoring the perfusion values of the rotator cuff tendons by oximetry. Results: No statistically significant differences were found between the oximetry data and age, sex or years of sports activity, nor when comparing the right and left arm or the dominant and non-dominant arm. A statistically significant difference was found for the dominant arm values in relation to the competitive role, higher values being obtained in outside hitters (62.7%) middle hitters (53.7%), opposite hitters (55.5%) and libero players (54.4%) (p 0.05). Conclusions: The different tendon vascularization values found in players with different roles in the team may be attributed to a response to the specific biomechanical demands posed by the different overhead throwing roles.

Description: The coasts of the Mediterranean Sea are dynamic habitats in which human activities have been conducted for centuries and which feature micro-tidal environments with about 0.40 m of range. For this reason, human settlements are still concentrated along a narrow coastline strip, where any change in the sea level and coastal dynamics may impact anthropic activities. In the frame of the RITMARE and the Copernicus Projects, we analyzed light detection and ranging (LiDAR) and Copernicus Earth Observation data to provide estimates of potential marine submersion for 2100 for 16 small-sized coastal plains located in the Italian peninsula and four Mediterranean countries (France, Spain, Tunisia, Cyprus) all characterized by different geological, tectonic and morphological features. The objective of this multidisciplinary study is to provide the first maps of sea-level rise scenarios for 2100 for the IPCC RCP 8.5 and Rahmstorf (2007) projections for the above affected coastal zones, which are the locations of touristic resorts, railways, airports and heritage sites. On the basis of our model (eustatic projection for 2100, glaciohydrostasy values and tectonic vertical movement), we provide 16 high-definition submersion maps. We estimated a potential loss of land for the above areas of between about 148 km2 (IPCC-RCP8.5 scenario) and 192 km2 (Rahmstorf scenario), along a coastline length of about 400 km.

Description: Despite much investigation into T regulatory cells (Tregs), little is known about the mechanism controlling their recruitment and function. Since mesenchymal cells (MSCs) exert an immune regulatory function and suppress T cell proliferation, this in vitro study investigated their role in Treg recruitment and function. hMSCs and different T cell populations (CD3+, CD3+/CD45RA+, CD3+/CD45RO+, CD4+/CD25+, CD4+/CD25+/CD45RO+, CD4+/CD25+/CD45RA+) from healthy donors were co-cultured for up to 15 days. Harvested lymphocytes were analysed by flow-cytometry; FoxP3 and CD127 expression were measured by real time PCR; regulatory activity was assessed. Within CD3+ fraction, percentages of CD4+/CD25bright and CD4+/CD25bright/Foxp3+ cells were higher in the presence of hMSCs (42% vs 34% for the CD4+/CD25bright and 20.5 vs 3.5 for the CD4+/CD25bright/Foxp3+). Within CD3+/CD45RA+ and CD3+/CD45RO+ fractions, the T reg starting fraction of the naïve population rose from 0.05% ± 0.01 CD4/CD25 positive cells to 0.2% ± 0.14 and the T reg starting fraction of the memory population rose from 0.3% ± 0.05 CD4/CD25 positive cells to 1.5% ± 0.9. Within CD4+/CD25+, CD4+/CD25+/CD45RA+, CD4+/CD25+/CD45RO+, FoxP3 expression did not change in CD4+/CD25+ cells. It increased 5.38±2.3 fold in CD4+/CD25+/CD45RA+ and 7.98±1.9 fold in CD4+/CD25+/CD45RO+ cells. CD127 expression decreased by -582±29.2 fold in CD4+/CD25+ cells, by -216±17.5 fold in CD4+/CD25+/CD45RA+ and by -71.95±12.6 fold in CD4+/CD25+/CD45RO+ cells. Cytofluorimetric analysis showed CD4+/CD25+/FoxP3+ was up-regulated to 14%±4 and CD127 down-regulated to 4.4%±1.5 vs respectively 0.2%±0.28 and 21.9%±3.5 in controls without MSCs (CD4+/CD25+ alone). FoxP3 up-regulation was more marked in CD4+/CD25bright cells (51%±10 vs 0.1%±0.7 in controls). Sorted Tregs exert potent immunosuppressive activity on CD4+/CD25- cell populations. Inhibitory activity was lost within 15 days when sorted T regs were cultured without hMSC. On day +15 proliferation using CD4+/CD25+ cells as suppressor cells was 1.7±0.8 vs 45.2±12 in controls; proliferation using CD4/CD25+/CD45RA+ as suppressor cells was 5.4±3.1, vs 21.3±11.2 in controls, and proliferation with CD4+/CD25+/RO+ as suppressor cells was 2.3±2 vs 33.5±8.7 in controls. We demonstrate MSC recruit Tregs from a fraction of CD3+ and from immunoselected CD3+/CD45RA+ and CD3+/CD45RO+ fractions. After culture with MSCs both immunoselected fractions registered increases in the CD4+/CD25bright/FoxP3 subset and CD127 expression was down regulated. When purified Treg populations (CD4/CD25+, CD4/CD25+/CD45RA+ and CD4/CD25+/CD45RO+) were used as fraction for hMSC co-cultures, they maintain FoxP3 expression and CD127 expression is down-regulated. Treg suppressive capacity was maintained in Treg populations that were layered on MSC for up to 15 days while control Tregs lost all suppressive activity after 5 days culture. In conclusion, our study demonstrates that MSCs recruit, regulate and maintain T regulatory phenotype and function over time.

Description: We previously demonstrated that when human bone marrow-derived mesenchymal cells (hMSCs) were co-cultured with CD4+/CD25+ T regulatory cells (Tregs) they maintained the T regulatory phenotype and function over time. Here we studied the TGF-beta/Smad signalling and mitogen-activated protein kinase (MAPK) pathways after Tregs/hMSCs co-culture. After 7 days co-culture of highly purified, immuno-selected Tregs and hMSCs from healthy donors, TGF-beta and IL-10 concentrations were dosed and mRNA of a panel of genes (NFAT, Smad4, Smad7, AP-1, Foxp3 and CD127) was quantified on harvested lymphocytes. Western blotting was performed by incubation with antiSMAD2, anti-pSMAD2, anti-ERK1/2 and anti-pERK1/2 polyclonal antibodies. After immuno-magnetic cell separation the final Treg fraction showed a mean purity of 93.6%±1. The CD4+/CD25+bright constituted 29%±7 of Tregs, FoxP3 cells constituted 51.9%±15.1 and CD127+ cells 19%±11.5. CD4+/CD25+ cells inhibited CD4+/CD25− cells (mean inhibition percentage: 52.1±29.6% (ratio 1:1). Tregs produced no TGF-beta and only a small quantity of IL-10 (8.67±4 pg/ml). hMSCs produced high quantity of TGF-beta (229.3 ± 54.8 pg/ml) associated with little IL-10 (2.7 ± 1.2 pg/ml). After co-culture, the TGF-beta concentration was 91.5±48.5 pg/ml while the IL-10 concentration was no different to baseline. To identify TGF-beta signaling pathways, we focused on Smad2, a central element in the cascade. In Tregs the strongly expressed pSmad2 signal after selection rapidly decreased after 7 days culture. When Tregs were co-cultured in presence of hMSCs the phosphorylated form of Smad2 did not change, indicating TGF-beta signaling was up-regulated. After co-culture mRNA quantification showed hMSCs down-regulated expression of SMAD7 (−8.9± 4.4 times vs Tregs without hMSCs), a negative regulator of TGF-beta signaling. After co-culture with hMSCs non-regulatory CD4+/CD25− control cells did not show any differences in SMAD7 expression. To study the MAPK kinase pathways, harvested Tregs were assayed for ERK1/2 kinase activity by determining their phosphorylated forms. pERK1/2 was almost absent in selected Tregs; it rapidly increased after 7 days in vitro culture without hMSCs but remained low after co-culture with hMSCs, indicating impaired ERK1/2 activation. AP-1 activation was also reduced (AP-1 mRNA −32±29 times less than controls). In conclusion, co-cultures of hMSCs and Tregs have the potential to set up a positive feedback loop that heightens responsiveness to TFG-beta in Treg cells. In fact, hMSCs produce TGF-beta which is consumed in co-culture with Tregs; TGF-beta1 signaling in Tregs is maintained through pSMAD2 up-regulation and SMAD7 down-regulation; hMSCs also inhibit ERK phosphorylation which consequently down-regulates AP-1 mRNA. Thus Tregs signalling is maintained through culture on a layer of hMSCs. Manipulation of Tregs signaling through culture on a layer of hMSCs may represent a novel strategy for maintenance of Treg phenotype and function.

Description: A single activating mutation in Janus Kinase (JAK)-2 is detected in 65%–97% patients with polycythemia vera (PV), in 23%–57% with essential thrombocytemia and in 35%–57% with idiopathic myelofibrosis. Recent studies have demonstrated the heterozygote population contains clonal and polyclonal granulopoiesis; clonality correlates with the Jak2V617F allelic ratio. We developed a microchip diagnostic platform (NMW NanoChip Molecular Biology Workstation, manufactured by Nanogen Inc, San Diego, CA) to identify the JAK-2 mutation in granulocyte-derived DNA and determine the allelic ratio. The system enables electronic deposition of biotinylated amplicons to selected pads. Twenty-three DNA samples from patients with PV were analysed by direct sequencing, AS-PCR and microelectronic chip. Sequencing results: a fragment of 345 genomic DNA was amplified by PCR under standard condition. 21/23 samples showed mutated JAK-2, 18/21 presented wild-type and mutated alleles; 3/21 had only the mutated allele. 21/23 samples showed mutated JAK-2. Microelectronic chip results: 21/23 samples showed mutated JAK-2, with a frequency of 91%. All samples were genotyped: at the first analysis 3 were identified as homozygotes; 18 were identified as heterozygotes. The allelic ratio in the 3 homozygotes shows residual wild-type allele due, in our view, to residual polyclonal granulopoiesis which the microchip detects. The allelic ratio of the 18 designated heterozygotes showed different T/G percentages, suggesting different disease stages. Combining microchip results with clonality assays could distinguish true heterozygotes. Overall genotyping was performed in 100% of positive cases. Sensitivity assay (on serial dilution of mutated HEL vs unmutated K562 cell lines) showed the electronic microchip detects the JAK-2 mutation in at least 1% of a total cell mixture. Our system is a fast, reliable and sensitive method alternative to DNA sequencing, AS-PCR or dHPLC. It overcomes the relative low sensitivity of direct sequencing and at the same time provides genotyping data even when sequencing fails or results are hard to interpret. This electronic microchip system is as sensitive as dHPLC but does not require an extra sequencing step for genotyping. Specific advantages over AS-PCR include automated calculation of the allelic ratio and consequently the genotype without any reduction in sensitivity.

Description: CD4(+)CD25(+)FoxP3(+) T regulatory (T reg) cells constitute 1–2% of peripheral blood cells in adults and function prevalently as immunosuppressors. 70% of T reg are memory/effector cells with a CD45RO+ phenotype. The other 30% have a naive T cell phenotype (CD45RA+). Both sub-populations exert a regulatory function and when sorted and/or purified each inhibits a mixed lymphocyte culture. In the present study, we investigated the effects of human recombinant IL-7 (rIL-7), human mesenchymal cells (hMSC) and hMSC engineered with the IL-7 gene (hMSC/IL-7) on regulatory T cells expressing a memory (CD4/CD25/CD45RO) or naive (CD4/CD25/CD45RA) phenotype. T cells from healthy donors were enriched by immnuselection to provide populations of CD45RA+ cells (95 % ± 2.9) and CD45RO+ cells (97 % ± 0.25). Enriched naive and memory cells were cultured in presence rIL-7 (100 ng/ml), hMSC (ratio 5:1) or hMSC/IL-7 (ratio 5:1). After 7 days’ culture we examined the CD4/CD25+ cells within the naive and memory populations. In the naive T cell population the T reg starting fraction of 0.05 % ± 0.01 of CD4/CD25 positive cells, did not change in the presence of rIL-7 while it rose to 0.2 % ± 0.14 in presence of human MSC and more interestingly reached 1.67 % ± 0.6 in presence of IL-7 engineered human MSC. In the memory T cell population the T reg starting fraction of 0.3 % ± 0.05 of CD4/CD25 positive cells, did not change in presence of rIL-7 while it rose 1.5 % ± 0.9 in the presence of human MSC and more interestingly reached 11.55 % ± 7.5 in the presence of human IL-7 engineered-MSC. We analyzed CD127 expression within different groups. In the naive T reg starting fraction 3 % ± 1.2 expressed CD127 which was down-regulated to 0.96 % ± 0.5 in the presence of rIL-7, to 0.29 % ± 0.2 with human MSC and to 0.37 % ± 0.02 with human IL-7engineered-MSC. Memory T reg cells expressed CD127 in 15% ± 1.2 of the starting fraction which was down-regulated to 1.2 % ± 0.45 in the presence of rIL-7, to 1.32 % ± 0.34 with hMSC and to 4.01% ± 0.74 in presence of hMSC/IL-7. FoxP3 expression was measured by real time quantitative PCR in sort-purified subsets of peripheral blood, identified by staining with a combination of CD4, CD25, CD45RA or CD45RO. In naive T reg FoxP3 expression was increased 1.15 fold in the presence of hMSC and 2.7 fold in presence of hMSC/IL-7. In memory T reg FoxP3 expression was increased 1.14 fold in the presence of hMSC and 2.67 fold in presence of hMSC/IL-7. In conclusion naive T reg cells are IL-7 independent and up-regulated by human MSC. Engineering human MSC with the IL-7 gene enhanced up-regulation. Memory T reg cells are also IL-7 independent and are up-regulated by human MSC. Engineering human MSC with the IL-7 gene markedly increased up-regulation. The different regulatory systems may underlie different functions within the T reg sub-populations.

Description: As Chronic Lymphocytic Leukemia (CLL) is associated with several defects in the T cell compartment, the impact of tumour burden on the autologous immune system was studied. Gene expression profiles (using Applied Biosystem Human Genome Microarray) identified 237 genes with significantly increased expression and 221 genes with significantly decreased expression (p