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Background Sickle cell disease (SCD) is increasingly recognized as a red blood cell disorder modulated by abnormally increased inflammation. We have previously shown that in patients with SCD not on a disease-modifying therapy (hydroxyurea or chronic transfusions), natural killer (NK) cell numbers are increased. In the current study, we further investigated the NK cell function to determine if there was evidence of increased activation and cytotoxicity. Procedure We conducted a cross-sectional study of 44 patients with HbSS/HbSβ0 thalassemia at steady state (hydroxyurea = 13, chronic transfusion = 11, no disease-modifying therapy = 20) and 23 healthy controls. Using a fresh blood sample, NK immunophenotyping was performed as follows: NK cells (CD3- CD56+ lymphocytes) were evaluated for makers associated with activation (NKG2D, NKp30, NKp44, and CD69) and maturity (CD57, killer immunoglobulin-like receptors (KIR), and CD56dim). Degranulation and cytotoxicity assays were performed to evaluate NK cell function. Results Patients with SCD who were not on disease-modifying therapy had a higher number of NK cells with an immunophenotype associated with increased cytotoxicity (NKG2D+ , NKp30+ , CD56dim+ , and KIR+ NK cells) compared with healthy controls and patients on hydroxyurea. NK cells from SCD patients not on disease-modifying therapy demonstrated significantly increased cytotoxicity (measured by assaying NK cell killing of the K562 cell line) compared with healthy controls (P = 0.005). Notably, NK cell cytotoxicity against K562 cells in the hydroxyurea or chronic transfusion patients was not significantly different from that in healthy controls. Conclusion SCD is associated with increased NK cell function as well as increased NK cell numbers, which appears to be normalized with disease-modifying therapy.

Background aims Human parainfluenza virus-3 (HPIV) is a common cause of respiratory infection in immunocompromised patients and currently has no effective therapies. Virus-specific T-cell therapy has been successful for the treatment or prevention of viral infections in immunocompromised patients but requires determination of T-cell antigens on targeted viruses. Methods HPIV3-specific T cells were expanded from peripheral blood of healthy donors using a rapid generation protocol targeting four HPIV3 proteins. Immunophenotyping was performed by flow cytometry. Viral specificity was determined by interferon (IFN)-γ ELISpot, intracellular cytokine staining and cytokine measurements from culture supernatants by Luminex assay. Cytotoxic activity was tested by 51 Cr release and CD107a mobilization assays. Virus-specific T cells targeting six viruses were then produced by rapid protocol, and the phenotype of HPIV3-specific T cells was determined by immunomagnetic sorting for IFN-γ-producing cells. Results HPIV3-specific T cells were expanded from 13 healthy donors. HPIV3-specific T cells showed a CD4 + predominance (mean CD4:CD8 ratio 2.89) and demonstrated specificity for multiple HPIV3 antigens. The expanded T cells were polyfunctional based on cytokine production but only had a minor cytotoxic component. T cells targeting six viruses in a single product similarly showed HPIV3 specificity, with a predominant effector memory phenotype (CD3 + /CD45RA – /CCR7 – ) in responder cells. Discussion HPIV3-specific T cells can be produced using a rapid ex vivo protocol from healthy donors and are predominantly CD4 + T cells with Th1 activity. HPIV3 epitopes can also be successfully targeted alongside multiple other viral epitopes in production of six-virus T cells, without loss of HPIV3 specificity. These products may be clinically beneficial to combat HPIV3 infections by adoptive T-cell therapy in immune-compromised patients.

In order to optimize the performance of the suture for tissue closure, it is essential to develop strategies for devising new and improved techniques that can visualize and compare various suturing techniques. This paper describes an experimental and numerical investigation on the performance of sutured tissue. In the experiments, two pieces of glutaraldehyde cross-linked bovine pericardium were sutured together through simple running suture and tensioned to study the performance of the sutured tissue. During testing, the tension load and the total displacement of the specimen were recorded. The strain field of the specimen was simultaneously captured using two high speed cameras and post processed using its associated image processing software. In addition, nonlinear hyperelastic material models for Shelhigh patch and cryopreserved human aorta were derived through least-square fitting into the tensile testing data. Three dimensional finite element models were developed to replicate the behavior of wound closure. The effect of tissue material mismatch, and stiffness of the suture thread on the mechanical behavior of sutured tissue was examined. The stain distributions obtained from simulation agrees with the captured surface strain map from experiments. A relative softer suture thread could reduce the peak stress concentrations at the knotting location. The mechanical performance of sutured tissue depends on the level of mismatch in material stiffness between the native tissue and the replacement material

3042Background: Patients with solid tumors refractory to standard therapies have poor prognoses, and most salvage therapies are toxic and ineffective. Antigen-specific T cell therapies offer a prom...

As an in vivo model for studying human MHC (HLA) class I-restricted CTL responses to viral infection, we established a series of HLA Tg mice expressing HLA-A2, -B7 or -B27 human/mouse hybrid genes on a background deficient for H2 class I (Tg HLA(hyb)/H2 class I DKO). To determine whether CTL recognition of influenza A (flu) infection in Tg HLA-A2(hyb)/H2 DKO mice is similar to HLA-A2+ humans, we compared the HLA-A2-restricted Tg mouse and human CD8+ T-cell responses to an immunodominant flu epitope (wild-type [WT] M1 58-66), as well as a variant of this peptide (var. M1 58-66). Similar to HLA-A2+ humans, our results show WT M1 58-66 is likely the dominant CTL epitope recognized in infected Tg HLA-A2(hyb)/H2 DKO mice. Var. M1 58-66 was also recognized by WT peptide-reactive T cells from both HLA-A2+ humans and Tg mice, although slightly less efficiently than WT M1 58-66 in both cases. Reduced variant recognition was shown to be associated with reduced peptide/A2 binding, as well as a more limited repertoire of utilized TCR Vbeta chains. The similar pattern of recognition and cross reaction observed here for the WT and variant M1 58-66 epitopes with HLA-A2 by human and Tg HLA mouse CTLs indicates that A2-dependent events of Ag processing, presentation and recognition are well-conserved between species. These findings demonstrate that this Tg HLA-A2/H2 DKO model will aid identification and development of epitopes as vaccines for numerous viral and tumor antigens for the HLA-A2 supertype.

Although mice transgenic (Tg) for human MHC (HLA) class I alleles could provide an important model for characterizing HLA-restricted viral and tumor Ag CTL epitopes, the extent to which Tg mouse T cells become HLA restricted in the presence of endogenous H2 class I and recognize the same peptides as in HLA allele-matched humans is not clear. We previously described Tg mice carrying the HLA-B27, HLA-B7, or HLA-A2 alleles expressed as fully native (HLAnat) (with human β2-microglobulin) and as hybrid human/mouse (HLAhyb) molecules on the H2b background. To eliminate the influence of H2b class I, each HLA Tg strain was bred with a H2-Kb/H2-Db-double knockout (DKO) strain to generate mice in which the only classical class I expression was the human molecule. Expression of each HLAhyb molecule and HLA-B27nat/human β2-microglobulin led to peripheral CD8+ T cell levels comparable with that for mice expressing a single H2-Kb or H2-Db gene. Influenza A infection of Tg HLA-B27hyb/DKO generated a strong CD8+ T cell response directed at the same peptide (flu nucleoprotein NP383–391) recognized by CTLs from flu-infected B27+ humans. As HLA-B7/flu epitopes were not known from human studies, we used flu-infected Tg HLA-B7hyb/DKO mice to examine the CTL response to candidate peptides identified based on the B7 binding motif. We have identified flu NP418–426 as a major HLA-B7-restricted flu CTL epitope. In summary, the HLA class I Tg/H2-K/H2-D DKO mouse model described in this study provides a sensitive and specific approach for identifying and characterizing HLA-restricted CTL epitopes for a variety of human disease-associated Ags.

In this work, the decellularized porcine small intestinal submucosa extracellular matrix (SIS-ECM), obtained from the commercial product under the trade name of CorMatrix, were tested in uniaxial tension. Preconditioning under cyclic loading of 2 N was conducted to stabilize the mechanical response of the tissue. The influence of rehydration time on the mechanical properties of the tissue was evaluated. Results suggested that the stiffness of SIS-ECM decreased with longer rehydration time. Considering the application of CorMatrix in pericardial closure, the native pericardium samples were also tested. The comparison indicated that the native pericardium is softer than rehydrated CorMatrix. This work can facilitate the surgeons to better choose the appropriate rehydration time when conducting the extracardiac implantations, such as pericardial reconstruction, pericardial closure, etc.Copyright © 2013 by ASME

Uncontrolled systemic-to-pulmonary shunt results in decreased systemic flow during extracorporeal life support (ECLS). Ligation of systemic-to-pulmonary shunts during ECLS is associated with poor outcome and is not always readily achieved. In ex vivo preparations, alveolar hypoxia results in pulmonary vasoconstriction despite normoxic pulmonary perfusate. We hypothesized that anoxic ventilation would result in reduced pulmonary shunting and increased systemic flow during ECLS in piglets with systemic-to-pulmonary shunt. Four piglets were placed on ECLS with right and left atrial drainage. A shunt was created between the bicarotid trunk and pulmonary artery, using 5-mm ePTFE tubing. Inspired oxygen was reduced to

Background: Aortic surgery in neonates often uses a patch of cryopreserved allogeneic artery. Drawbacks include alloimmunization in infants who may later need heart transplant. We hypothesized that the umbilical cord (UC) may be a non-immunogenic, viable vascular patch suited to neonatal aortic surgery. This was evaluated in ex vivo studies of human UC, and in vivo study in piglets. Method: Human UC (20) were collected after vaginal birth, were dilated and measured. Swab cultures were taken. For final culture UC was put in Thiol, and held for 4 wk. Disinfection was in dilute hypochlorite for 30 sec to 5 min. UC segments were stored in HTK solution with antibiotic/antifungal agent at 4 deg for 3 to 9 d. Samples were fixed for histology. At 9 d, samples were put in tissue culture to check viability. Then, 18 farm piglets in three litters were delivered by hysterotomy. Each UC was sanitized, cultured, and stored as described for human UC. At 7 d, each piglet underwent laparotomy. A defect made in the abdominal aorta was patched with autologous UC or with PTFE. Piglets were killed after 8 and 12 wk to examine the patched aorta. Result: Human UC diameter was 8-11 mm. Cultures of 5 UC in Dakins for 30 sec, 1, 2, 3, and 4 min were all negative. All later UC were treated for 30 sec only. One culture from one UC segment after 9 d was positive; final culture from this UC wwas negative. Histology did not differ at 9 d from day of delivery (n=3). Cells grew from 9 d UC in tissue culture (n=2). Piglet UC at 7 d also held viable cells. Piglet UC patch worked well in surgery. Hemostasis was rapid with UC, and was longer with PTFE. Three piglets were killed before 8 wk due to hernia. There was no dehiscence, infection, or aneurysm in either group. UC-patched aorta lengthened 114%, PTFE grew 21%. Patched aorta was non-stenotic in both groups, 92% of normal in UC vs. 95% in PTFE. By histology the UC did not remain viable, but calcified and broke into fragments. Beneath both UC and PTFE patches, fibroblast proliferation spanned the aortic defect. In UC piglets, this layer enlarged after UC fragmentation. Conclusion: UC can be sterilized and kept viable after vaginal birth . UC patch worked well for repair of an aortic defect in piglets, but UC viability and growth were not seen. UC needs further study before trial in human aortic surgery.

In this study, a nonlinear finite element analysis was implemented on the balloon-expandable stent deployment in stenotic artery with asymmetric plaque to investigate the stent stenotic artery interaction. Uniform pressure loading was applied onto the inner surface of stent to expand it. The result showed that the stent restores the patency of the stenotic artery lumen; however, non-uniform expansion (i.e. dogbone shape) was observed on stent, which indicates a possibility of the injury to arterial wall at the ends of stent. The stress on arterial wall induced by stent expansion is higher than blood pressure induced stress, which may initiate the proliferation of smooth muscle cells and lead to the restenosis. The stent design was alternated by increasing the thickness of distal strut. With this improved design, the dogboning effect was alleviated dramatically and stress level on arterial wall was also decreased. This FEM work provided a better understanding of the coronary stenting and its effect on the arterial response from biomechanical view, which can facilitate new stent design.

The ABO histo-blood group system is the most important antigen system in transplantation medicine, yet no small animal model of the ABO system exists. To determine the feasibility of developing a murine model, we previously subcloned the human alpha-1,2-fucosyltransferase (H-transferase, EC 2.4.1.69) cDNA and the human alpha-1,3-N-acetylgalactosaminyltransferase (A-transferase, EC 2.4.1.40) cDNA into lentiviral vectors to study their ability to induce human histo-blood group A antigen expression on mouse cells. Herein we investigated the optimal conditions for human A and H antigen expression in murine cells. We determined that transduction of a bicistronic lentiviral vector (LvEF1-AH-trs) resulted in the expression of A antigen in a mouse endothelial cell line. We also studied the in vivo utility of this vector to induce human A antigen expression in mouse liver. After intrahepatic injection of LvEF1-AH-trs, A antigen expression was observed on hepatocytes as detected by immunohistochemistry and real-time RT-PCR. In human group A erythrocyte-sensitized mice, A antigen expression in the liver was associated with tissue damage, and deposition of antibody and complement. These results suggest that this gene transfer strategy can be used to simulate the human ABO blood group system in a murine model. This model will facilitate progress in the development of interventions for ABO-incompatible transplantation and transfusion scenarios, which are difficult to develop in clinical or large animal settings.

Proper characterization of the material properties of pulmonary arterial tissue is needed for many medical applications. The objective of this study was to investigate the stress-strain relationship and characterize the nonlinear elastic behavior of porcine pulmonary arteries; thus, uniaxial tension tests and cyclic loading-unloading tests were conducted on healthy porcine pulmonary arterial tissue. In these experiments, pulmonary arteries from different piglets and a commercial pulmonary valved conduit, called “Contegra 200”, were subjected to uniaxial tension. Results demonstrated a higher stiffness along the circumferential direction than the axial direction. The “Contegra 200” was much suffer than real pulmonary arterial tissue along the axial direction and had a similar stiffness to natural tissue along the circumferential direction within physiological stretch ranges, which is less than 40% strain. Elastic hysteresis was observed from cyclic loading-unloading tests, which indicates that more energy was required during the loading than the unloading. A nonlinear hyperelastic model based on second order polynomial constitutive equation was derived from average values of the test data along both axial and circumferential directions. The material model could be used in numerical analysis of pulmonary arterial response and facilitate the design of intravascular devices.Copyright © 2010 by ASME