Differential Responses of Human Regulatory T Cells (Treg) and Effector T Cells to Rapamycin

Background: The immunosuppressive drug rapamycin (RAPA) promotes the expansion of CD4+ CD25highFoxp3+ regulatory\ud T cells via mechanisms that remain unknown. Here, we studied expansion, IL-2R-c chain signaling, survival pathways and resistance to apoptosis in human Treg responding to RAPA.\ud Methodology/Principal Findings: CD4+CD25+ and CD4+CD25neg T cells were isolated from PBMC of normal controls (n = 21)\ud using AutoMACS. These T cell subsets were cultured in the presence of anti-CD3/CD28 antibodies and 1000 IU/mL IL-2 for 3 to 6 weeks. RAPA (1–100 nM) was added to half of the cultures. After harvest, the cell phenotype, signaling via the PI3K/ mTOR and STAT pathways, expression of survival proteins and Annexin V binding were determined and compared to values obtained with freshly-separated CD4+CD25high and CD4+CD25neg T cells. Suppressor function was tested in co-cultures with autologous CFSE-labeled CD4+CD25neg or CD8+CD25neg T-cell responders. The frequency and suppressor activity of Treg were increased after culture of CD4+CD25+ T cells in the presence of 1–100 nM RAPA (p,0.001). RAPA-expanded Treg were largely CD4+CD25highFoxp3+ cells and were resistant to apoptosis, while CD4+CD25neg T cells were sensitive. Only Treg upregulated anti-apoptotic and down-regulated pro-apoptotic proteins. Treg expressed higher levels of the PTEN protein than CD4+CD25neg cells. Activated Treg6RAPA preferentially phosphorylated STAT5 and STAT3 and did not utilize the PI3K/ mTOR pathway.\ud Conclusions/Significance: RAPA favors Treg expansion and survival by differentially regulating signaling, proliferation and sensitivity to apoptosis of human effector T cells and Treg after TCR/IL-2 activation

Salvage surgery for head and neck squamous cell carcinoma

n/

Reduction of Radiation Dosage in Visualization of Paranasal Sinuses in Daily Routine

Background. Preoperative imaging of the nose and paranasal sinus is standard in otorhinolaryngology. Previous studies on phantoms demonstrated the potential for dose reduction of cone beam computed tomography (CBCT) by varying the application parameters. Methodology. Based on previous studies, the standard protocol of paranasal sinus imaging by CBCT was altered. One hundred and fifty examinations using the old protocol (01/2010–01/2011, high dosage) and 150 examinations using the new protocol (09/2012–09/2013, low dosage) were evaluated and compared for the visibility of 17 anatomical structures, the Lund-Mackay Score, and technical parameters. Results. Alteration of the protocol resulted in a significant reduction in dosage (6.64 mGy versus 2.88 mGy). Both groups showed the same amount of pathology (Lund-Mackay Score: 4.95±3.79 versus 5.26±5.77; p=0.558). There was a significant better visibility of the anatomical structures (all visible = 1, nothing visible = 4) (results: 1.25 versus 1.17; p=0.001) in the low-dosage group. Conclusion. Despite a significant reduction in the applied dosage, reliable visualization of the bony anatomy of the anterior skull base is possible by CBCT. This demonstrates the need for the discussion of the required clinical imaging quality

Tumor-Derived Microvesicles Induce, Expand and Up-Regulate Biological Activities of Human Regulatory T Cells (Treg)

Background: Tumor-derived microvesicles (TMV) or exosomes are present in body fluids of patients with cancer and might be involved in tumor progression. The frequency and suppressor functions of peripheral blood CD4 + CD25 high FOXP3 + Treg are higher in patients with cancer than normal controls. The hypothesis is tested that TMV contribute to induction/ expansion/and activation of human Treg. Methodology/Principal Findings: TMV isolated from supernatants of tumor cells but not normal cells induced the generation and enhanced expansion of human Treg. TMV also mediated conversion of CD4 + CD25 neg T cells into CD4 + CD25 high FOXP3 + Treg. Upon co-incubation with TMV, Treg showed an increased FasL, IL-10, TGF-b1, CTLA-4, granzyme B and perforin expression (p,0.05) and mediated stronger suppression of responder cell (RC) proliferation (p,0.01). Purified Treg were resistant to TMV-mediated apoptosis relative to other T cells. TMV also increased phospho-SMAD2/3 and phospho-STAT3 expression in Treg. Neutralizing Abs specific for TGF-b1 and/or IL-10 significantly inhibited TMV ability to expand Treg. Conclusions/Significance: This study suggests that TMV have immunoregulatory properties. They induce Treg, promote Treg expansion, up-regulate Treg suppressor function and enhance Treg resistance to apoptosis. Interactions of TMV wit

Evaluation eines Tacrolimus-beschichteten Stents zur Prävention der Restenosierung im diabetischen Tiermodell

In spite of improved technical methods, restenosis following stent implantation remains a central problem in interventional cardiology, especially in diabetic patients. Eminent for the development of restenosis are the proliferation of smooth muscle cells, the production of extracelluar matrix and inflammatory processes. Recently, a number of studies using drug-eluting stents have been performed, since the local and sustained application of antiproliferativ drugs can achieve a decrease in restenosis rates. Tacrolimus, a Rapamycin-derivative, owns, as an immunsuppressiv drug, antiinflammatoric as well as antiprolifative features. Aim of this study was to evaluate a Tacrolimus-eluting stent regarding restenosis in a diabetic animal model. Tacrolimus-eluting stents (TES) with a dosage of 173µg and Bare Metal Stents (BMS) were implanted biiliacal in alloxan-treated diabetic rabbits as well as non-diabetic rabbits. At the RWTH Aachen, an appropriate diabetic rabbit model was successfully established. The animals were partly sacrified after two weeks, and partly after 8 ± 1 weeks. 12 TES and 14 BMS could be analysed in the diabetic and 13 TES and 14 BMS in the non-diabetic group. Histomorphometric and immunhistochemical analyses and gene expression studies were performed. The procentual restenosis was lower with the BMS than with the TES (22 ± 6% vs. 31 ± 10%, p=0,05). The average rate of neointimal formation tended to be lower in the BMS group in comparism to the TES group (0,88 ± 0,32mm² vs. 1,13 ± 0,31mm², p=0,11). The average number of macrophages in the neointima was significantly decreased in the TES as compared to the BMS group (16,81± 5,55 vs. 24,31 ± 5,88, p=0,01). The relative VCAM 1 - expression was also decreased by the TES (1,9x 10-3 ± 3 *10-4 vs. 8x10-4 ± 6 *10-4 , p=0,05). Consequently, the inflammatory reaction was successfully suppressed by the TES, but the neointimal proliferation could not been positively influenced by this stent. Therfore, clinical evaluation of the TES in its current form, seems not justified yet in diabetic patients. Tacrolimus is offering a promising onset in the prevention of restenosis after stent implantation, which should be investigated further more

Breast Cancer Cell-Derived Adenosine Enhances Generation and Suppressor Function of Human Adaptive Regulatory T Cells

Introduction: Adaptive regulatory T cells (Tr1) are induced in the periphery by environmental stimuli. CD73 expression and adenosine (ADO) production by tumor cells may influence Tr1 generation and their immunosuppressive activity. Material and Methods: Tr1 were generated in co-cultures of CD4+CD25neg T cells, autologous immature dendritic cells (iDC), and irradiated ADO-producing CD73+ or non-producing CD73neg breast cancer (BrCa) cell lines (TU). The expression of ectonucleotidases and other surface markers on Tr1 was determined by flow cytometry. Tr1-mediated suppression of proliferation was evaluated in CFSE-based assays. Luciferase-based ATP detection assays and mass spectrometry were used to measure ATP hydrolysis and ADO levels. Cytokine levels were measured by ELISA or Luminex. CD73 expression on tumor cells or T cells in TU tissues was assessed by immunofluorescence. Results: CD73+ TU induced higher numbers of Tr1 cells (p < 0.01) than CD73neg TU. Tr1TU73+ hydrolyzed more exogenous ATP, produced more ADO, and mediated higher suppression than Tr1TU73neg (p < 0.05 for all). ARL67156, an ectonucleotidase inhibitor, and ZM241385, A2A receptor antagonist, reduced suppression of proliferation mediated by Tr1TU73+ cells (p < 0.01). Basal-like primary BrCa cells expressed higher levels of ectonucleotidases and induced more Tr1 than less aggressive primary luminal-like BrCa. Conclusion: BrCa producing ADO (CD73+ TU) favor the induction of Tr1, which expresses CD39 and CD73, hydrolyzes ATP to ADO, and effectively suppresses anti-tumor immunity

Transoral surgery for oropharyngeal tumors using the Medrobotics(®) Flex(®) System - a case report.

Transoral resection of pharyngeal tumors with acceptable oncological and functional results can be challenging due to their location in a narrow anatomic space

TMV promote differentiation of human Treg in culture.

<p>(<b><u>A</u></b>) Purified CD3⁺CD4⁺ T cells were labeled with CFSE and cultured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011469#s4" target="_blank">Materials and Methods</a> ± TMV or DC-derived MV (5 µg/mL). On days 3, 5 and 8, the frequency of CD4⁺CD25⁺FOXP3⁺ Treg among proliferating T cells was determined by flow cytometry. The data (means ± SD) represent three independent experiments (*p<0.01). (<b><u>B</u></b>) Proliferating CD3⁺CD4⁺ T cells (squares) were tested for co-expression of CD25 in a representative co-culture ± TMV. A higher proportion of proliferating CD4⁺ T cells expressed CD25 in the co-culture with TMV than without TMV. (<b><u>C</u></b>) The proliferating CD4⁺CD25⁺ T cells in the co-cultures with TMV were evaluated for the frequency of FOXP3⁺ T cells upon gating on the CD4⁺CD25^high subset (see box). Over 90% of these cells also expressed intracellular FOXP3. Data are representative for one out of 6 cultures tested.</p

TMV promote expansion of human Treg in culture.

<p>The fold expansion of fresh (<i>left panel</i>) or rapamycin-expanded (<i>right panel</i>) CD4⁺CD25^high T cells to which TMV or DC-derived MV were added on day 0. Cells were stimulated with OKT3, anti-CD28 Abs and IL-2 (500 IU/mL) and cultured for 14–21 d. The data are means ± SD of six independent co-cultures. Asterisks indicate significant differences (p<0.05) between the cultures ± TMV.</p