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Purpose: More effective therapy is needed for intrahepatic cholangiocarcinoma (ICC). The encouraging clinical results obtained with checkpoint molecule-specific monoclonal antibodies (mAb) have prompted us to investigate whether this type of immunotherapy may be applicable to ICC. The aims of this study were to determine whether (i) patients mount a T-cell immune response to their ICC, (ii) checkpoint molecules are expressed on both T cells and tumor cells, and (iii) tumor cells are susceptible to recognition by cognate T cells.Experimental Design: Twenty-seven ICC tumors were analyzed for (i) lymphocyte infiltrate, (ii) HLA class I and HLA class II expression, and (iii) PD-1 and PD-L1 expression by T cells and ICC cells, respectively. The results of this analysis were correlated with the clinicopathologic characteristics of the patients investigated.Results: Lymphocyte infiltrates were identified in all tumors. PD-L1 expression and HLA class I antigen expression by ICC cells was observed in 8 and 11, respectively, of the 27 tumors analyzed. HLA class I antigen expression correlated with CD8+ T-cell infiltrate. Furthermore, positive HLA class I antigen expression in combination with negative/rare PD-L1 expression was associated with favorable clinical course of the disease.Conclusions: ICC patients are likely to mount a T-cell immune response against their own tumors. Defects in HLA class I antigen expression in combination with PD-L1 expression by ICC cells provide them with an immune escape mechanism. This mechanism justifies the implementation of immunotherapy with checkpoint molecule-specific mAbs in patients bearing ICC tumors without defects in HLA class I antigen expression. Clin Cancer Res; 22(2); 470–8. ©2015 AACR.

Figure S2. Representative staining patterns of formalin-fixed, paraffinembedded primary ICC lesions with HLA-DR, DQ and DP β chain-specific mAb LGII- 612.14.

Objective A national robotic surgery curriculum is still developing for general surgery residents as robotic surgery becomes increasingly accessible. One general surgery residency program utilized a Delphi process to optimize a robotic surgery curriculum and to determine key factors that might affect robotic proficiency and intraoperative independence. Design Delphi methodology was used to gain consensus amongst robotic surgery faculty and trainees. Consensus was defined as agreement of 66.7% or above in factors that would allow a resident to independently operate in a robotic case. A panel of diverse representatives proposed factors that might affect resident robotic learning and operative experience. In a subsequent round, questions were sent through an anonymous online survey for respondents to identify factors that affect resident robotic independence. Respondents were also given the ability to write in pertinent factors. Setting This study was conducted from July 2020 to September 2020 via anonymous web-based questionnaires for education researchers, faculty members, and residents of a university-affiliated independent general surgery residency program. Participants The initial panel consisted of a robotic surgeon, a 2020 graduate, a 2019 graduate and/or robotic fellow, a research resident, and a current resident. The subsequent survey was completed by 8 faculty members, 6 recent graduates, and 15 current residents within the general surgery residency program. Results Proposed items fell into 3 categories: institutional resources, individual qualities, and curricular elements. Consensus within groups was achieved in the following items: dual robotic console models, robotic-focused faculty, resident interest, PGY level, and resident time spent on a simulator. Conclusions This Delphi exercise has informed this general surgery residency program in the development of a robotic surgery curriculum, through contribution from multiple stakeholders. While curricular elements for baseline robotic knowledge are necessary, institutional resources, deliberate practice, resident entrustment and faculty teaching proficiency warrant further study.

BackgroundNeoadjuvant folinic acid, fluorouracil, irinotecan, and oxaliplatin (FOLFIRINOX) and chemoradiation have been used to downstage borderline and locally advanced pancreatic ductal adenocarcinoma (PDAC). Whether neoadjuvant therapy-induced tumor immune response contributes to the improved survival is unknown. Therefore, we evaluated whether neoadjuvant therapy induces an immune response towards PDAC.MethodsClinicopathological variables were collected for surgically resected PDACs at the Massachusetts General Hospital (1998-2016). Neoadjuvant regimens included FOLFIRINOX with or without chemoradiation, proton chemoradiation (25 Gy), photon chemoradiation (50.4 Gy), or no neoadjuvant therapy. Human leukocyte antigen (HLA) class I and II expression and immune cell infiltration (CD4+, FoxP3+, CD8+, granzyme B+ cells, and M2 macrophages) were analyzed immunohistochemically and correlated with clinicopathologic variables. The antitumor immune response was compared among neoadjuvant therapy regimens. All statistical tests were 2-sided.ResultsTwo hundred forty-eight PDAC patients were included. The median age was 64 years and 50.0% were female. HLA-A defects were less frequent in the FOLFIRINOX cohort (P = .006). HLA class II expression was lowest in photon and highest in proton patients (P = .02). The FOLFIRINOX cohort exhibited the densest CD8+ cell infiltration (P < .001). FOLFIRINOX and proton patients had the highest CD4+ and lowest T regulatory (FoxP3+) cell density, respectively. M2 macrophage density was statistically significantly higher in the treatment-naïve group (P < .001) in which dense M2 macrophage infiltration was an independent predictor of poor overall survival.ConclusionsNeoadjuvant FOLFIRINOX with or without chemoradiation may induce immunologically relevant changes in the tumor microenvironment. It may reduce HLA-A defects, increase CD8+ cell density, and decrease T regulatory cell and M2 macrophage density. Therefore, neoadjuvant FOLFIRINOX therapy may benefit from combinations with checkpoint inhibitors, which can enhance patients’ antitumor immune response.

The impact of HLA matching on graft survival has been well characterized in renal transplantation, with a higher degree of matching associated with superior graft survival. Additionally, living donor grafts are known to confer superior survival compared to those from deceased donors. The purpose of this study is to report our multi-decade institutional experience and outcomes for patients who received HLA-identical living donor grafts, which represent the most favorable scenario in kidney transplantation. We conducted a retrospective analysis of these graft recipients performed at a Duke University Medical Center between the years of 1965 and 2002. The recipients demonstrated excellent graft and patient survival outcomes, superior to a contemporary cohort, with median patient and graft survival of 24.2 and 30.9 years, respectively, among Duke recipients vs. 16.1 and 16.0 years in a cohort derived from national data. This study offers a broad perspective on the importance of HLA matching and graft type, and demonstrates a historical best-case-scenario in renal transplantation.

The rheology of liquid blends based on PDMS has been studied by varying the components molecular weight, the temperature and the concentration of the curing agent, at values well below the gelation threshold. The formulated lightly-cured blends showed interesting unusual behaviors. An interpretative model based on the nanocomposite polymer, the relaxation of dangling chains and the free-volume variation has been proposed. At low temperatures both the M/H (Medium-in-High molecular weights) and L/H (Low-in-High) blends confirm the previously observed anomalous rheology weakening at very low crosslinker concentrations. This is explained by means of the nanosized random coils of the grafted prepolymer nucleated in situ. As the concentration of curing agent increases, crosslinked isolated domains will be sufficiently wide to give the expected increase in rheological properties. At the temperature of 70 °C, the M/H blend shows a reverse creep behavior: the most crosslinked material exhibits an increase in compliance and the less one a decrease. This behavior has been explained taking into account the entanglement onset with a free-volume reduction (Free-Volume Change model); in contrast, the shorter chains of the micro-domains due to thermal agitation would expand the free-volume interface, with the drop of rheological performance. Moving from the M/H blend to the L/H one, at low temperatures we observe a strong decrease in compliance due to the trapping of the short chains on the entanglement sites, with the increase of the entanglement stability (Entanglement Swelling Tube model). The creep experiments show an unexpected shrinkage phenomenon for liquid blends at higher crosslinker concentration: the crosslinked domains enough extended should aggregate by non-bonding interactions giving a prestressed Bingham fluid. The rheology in rotational mode are confirmed in oscillating regime by the dissipation tanδ maximum at high frequency and the peak broadening with the curing agent concentration.

Viscoelasticity of chemically modified Cedrus deodara by means of pure solvent or solution treatments has been evaluated using dynamic-mechanical analysis in bending mode at 30 °C. The samples are treated up to equilibrium conditions in solvents with different polarity (water, ethanol and acetone), and corresponding solutions with bifunctional chemicals (acyl dichloride and aliphatic-diamine). Then, in all cases the samples are air-dried and temperature equilibrated before to be characterized. Mechanical properties have been explored by means of stress–strain plots at different strain rate, creep and step-strain experiments. Dynamic-mechanical properties have been studied by frequency-strain spectra and E1, E2 and tanδ have been investigated. Outcomes have been compared with those of a previous paper determined for seasoned or thermocured wood. The samples treated with the pure solvents exhibit an improvement of mechanical properties, due to the extraction of low molecular weight compounds acting as plasticizers of the lignin-based matrix: an increase of the elastic modulus, a reduction of the compliance and longer stress-relaxation time are observed. For the samples treated with the solutions of dichloride or diamine, the viscoelasticity behaviour is the resultant of plasticizers extraction and cross-linking of the polymeric matrix by means of the bifunctional reagents.

We previously reported that transplantation (Tx) of prevascularized donor islets as composite islet-kidneys (IK) reversed diabetic hyperglycemia in both miniature swine and baboons. In order to enhance this strategy's potential clinical applicability, we have now combined this approach with hematopoietic stem cell (HSC) Tx in an attempt to induce tolerance in nonhuman primates. IKs were prepared by isolating islets from 70% partial pancreatectomies and injecting them beneath the autologous renal capsule of five rhesus monkey donors at least 3 months before allogeneic IK Tx. HSC Tx was performed after mobilization and leukapheresis of the donors and conditioning of the recipients with total body irradiation, T cell depletion, and cyclosporine. One IK was harvested for histologic analysis and four were transplanted into diabetic recipients. IK Tx was performed either 20-22 (n = 3) or 208 (n = 1) days after HSC Tx. All animals accepted IKs without rejection. All recipients required >20 U/day insulin before IK Tx to maintain

Viscoelasticity of air-dried or thermo-treated Cedrus deodara and Alnus cordata has been evaluated by means of Dynamic Mechanical Analysis (DMA) in the bending mode at 30 °C. E1, E2 and tanδ have been investigated by frequency-strain spectra. Mechanical properties have been explored by means of creep, stress-relaxation experiments and stress–strain plots at different strain-rates. Relaxation times and Young’s modulus E have been evaluated. The morphology of the samples has been examined by means of optical microscopy. Rheological differences either among air-dried woods, or between air-dried and thermo-treated woods, have been observed. In particular, air-dried wood (and especially Cedrus) shows a significant plastic behaviour. In contrast, the thermo-treatment makes the materials mainly elastic and structurally stable. The plasticity of the air-dried woods seems to be due to the shear of the hemicellulose interface and to plasticizers of lignin matrix; the increased elasticity of the thermo-treateds would be due to the additional lignin cross-linking, and loss of hemicellulose and plasticizers. Air-dried woods, especially the softwood, have a thermoplastic-like behaviour, where the non-bonded intermolecular interactions prevail, in contrast, the thermo-treated woods are thermosetting-like due to the higher degree of cross-link of the polymeric matrix. The study demonstrates the possibility of characterizing wood through viscoelasticity and dynamic mechanical analysis.

BACKGROUND. The staging of pancreatic neuroendocrine tumors (PNETs) is continuously evolving. Mitotic count, as measured by hematoxylin and eosin (H&E) or Ki67 labeling index (Ki67LI), is the best predictor of disease biology. However, both of these methods have several limitations. Phosphorylated histone H3 (PHH3), a novel mitotic marker, is potentially more accurate and easier to evaluate. This study aimed to evaluate the prognostic impact of PHH3 on patients with PNETs. METHODS. Clinicopathologic data and paraffin-embedded tissue were evaluated for 100 of the 247 PNET patients whose tumors were resected between 1998 and 2010. Mitotic counts were analyzed on H&E-, Ki67-, and PHH3- stained slides by two independent pathologists. Kaplan–Meier curves, log-rank tests, Cox regression models, and time-dependent receiver operative characteristics (ROC) curves were used to evaluate the prognostic power of these markers. An internal data cross-validation was performed to select the best cutoff. RESULTS. Of the 100 PNET patients resected, 53 were men. The median age of the patients was 59 years (range 19–96 years). The median follow-up period was 68 months (range 3–186 months). The median time for evaluation of an H&E- or PHH3-stained slide was 3 min, relative to 15 min for Ki67. The findings showed H&E, Ki67, and PHH3 all to be excellent predictors of disease-specific survival (DSS). However, PHH3 was superior to H&E and Ki67 in predicting both disease-free survival (DFS) (p = 0.006) and DSS (p = 0.001). Evaluation of the PHH3 mitotic count showed 7 mitoses per 10 high-power fields (HPFs) to be the optimal cutoff for differentiating between low- and high-risk PNET patients. CONCLUSIONS. PHH3 is a better predictor of both DFS and DSS than H&E or Ki67 in PNET. In addition, PHH3 appears to be both easier to interpret and more accurate when compared to current prognostic markers.

Purpose: More effective therapy is needed for intrahepatic cholangiocarcinoma (ICC). The encouraging clinical results obtained with checkpoint molecule-specific monoclonal antibodies (mAb) have prompted us to investigate whether this type of immunotherapy may be applicable to ICC. The aims of this study were to determine whether (i) patients mount a T-cell immune response to their ICC, (ii) checkpoint molecules are expressed on both T cells and tumor cells, and (iii) tumor cells are susceptible to recognition by cognate T cells. Experimental Design: Twenty-seven ICC tumors were analyzed for (i) lymphocyte infiltrate, (ii) HLA class I and HLA class II expression, and (iii) PD-1 and PD-L1 expression by T cells and ICC cells, respectively. The results of this analysis were correlated with the clinicopathologic characteristics of the patients investigated. Results: Lymphocyte infiltrates were identified in all tumors. PD-L1 expression and HLA class I antigen expression by ICC cells was observed in 8 and 11, respectively, of the 27 tumors analyzed. HLA class I antigen expression correlated with CD8+ T-cell infiltrate. Furthermore, positive HLA class I antigen expression in combination with negative/rare PD-L1 expression was associated with favorable clinical course of the disease. Conclusions: ICC patients are likely to mount a T-cell immune response against their own tumors. Defects in HLA class I antigen expression in combination with PD-L1 expression by ICC cells provide them with an immune escape mechanism. This mechanism justifies the implementation of immunotherapy with checkpoint molecule-specific mAbs in patients bearing ICC tumors without defects in HLA class I antigen expression. Clin Cancer Res; 22(2); 470–8. ©2015 AACR.

Background/Aims: There is increasing evidence showing that innate immune responses and inflammatory processes play an important role in the development and progression of diabetic nephropathy (DN). The potential effect of innate immunity in the early stage of DN is still unclear. Toll-Like-Receptor 4 (TLR4) is vigorously involved in the progress of kidney diseases in a sterile environment. The activation of the interleukin 17 (IL-17) pathway produces inflammatory cytokines, appearing in various kidney diseases. Unfortunately the relationship between TLR4 and IL-17 has not been investigated in diabetic nephropathy to date. The aim of this study is to investigate whether mammalian target of rapamycin (mTOR) inhibition may be dependent on TLR4 signaling and the pro-inflammatory factor IL-17 to delay the progression of DN. Methods: Streptozotocin (STZ)-induced diabetic rats were randomly assigned to 3 experimental groups: a diabetic nephropathy group (DN, n = 6); and a diabetic nephropathy treated with rapamycin group (Rapa, n = 6) and a control group (Control, n =6). Body weight, fasting blood sugar, and 24h urine albumin were assessed at week 2, week 4 and week 8. Renal tissues were harvested for H&E, PAS staining, as well as an immunohistochemistry assay for TLR4 and IL-17. TLR4 quantitative expression was measured by Western-Blot analysis and RT-PCR. Results: Our results demonstrated that the expression of both TLR4 and IL-17 were upregulated in early stage DN and reduced by rapamycin. TLR4 and IL-17 both increased and positively related to 24h urinary albumin and kidney/weight ratio. However, neither TLR4 nor IL-17 made a significant difference on fasting blood sugar. Conclusions: Taken together, our results confirm and extend previous studies identifying the significance of the TLR4 and Th17 pathways in development of early stage DN. Furthermore, we suggest this overexpression of TLR4 might be involved in the immunopathogenesis of DN through activation of Th17 cells. Rapamycin may attenuate DN via reduction of the TLR4 signaling pathway and Th17 cells signaling. Although the underlying mechanisms need to be explored, the observed increase of TLR4 and IL-17 during the early stages of DN and their suppression with rapamycin treatment suggest the importance of TLR4 and IL-17 in DN pathophysiology.

Since α-1,3-galactosyltransferase knockout (GalT-KO) pigs became available, there has been an increasing interest in non-Gal natural antibody (nAb)-mediated xenograft rejection. To better understand mechanisms of non-Gal nAb-mediated rejection, a simple small animal model without gene manipulation would be extremely valuable. Here, we tested whether the Chinese tree shrew (CTS), which is a small-sized mammal that is phylogenetically close to primates, could serve as a model for discordant xenograft rejection. Methods Study 1: Expression of α-Gal antigens in hearts and kidneys of CTSs and rats was assessed by IB4 lectin binding. Presence of anti-Gal and anti-non-Gal IgM and IgG nAb in CTS sera was tested by FACS using Gal+ and GalTKO PBMC as well as BSA-ELISA. Study 2: Rat hearts were transplanted into CTS recipients (group 1, n = 7), and CTS hearts were transplanted in rats [n = 10; seven received no immunosuppression (group 2) and three received FK506 + leflunomide (group 3)]. Results Study 1: Both CTSs and rats had α-Gal expression in hearts and kidneys. ELISA showed CTSs do not have anti-Gal nAb, and flow cytometry indicated CTSs have anti-non-Gal IgM and IgG nAb in serum. Study 2: Rat hearts in CTSs were uniformly rejected within 35 mins, while CTS hearts in rats continued beating until day 5 without immunosuppression, and up to day 8 with immunosuppression. Conclusion Rat-to-CTS heart transplantation is a discordant xenotransplant model, CTS-to-Rat heart transplantation is a concordant xenotransplant model. CTSs are valuable small animals to study mechanisms and strategies to avoid non-Gal nAb-mediated xenograft rejection.

In 1954, plastic surgeon Dr Joseph Murray performed the first successful organ transplant between identical twins. Today solid organ transplants are performed routinely and offer a curative option for patients with end stage organ failure. For his pioneering work in transplantation biology, Dr Murray remains the only plastic surgeon to have been awarded the Nobel Prize in Medicine.The success of modern day transplants has allowed transplantation to be applied more broadly. In particular, vascularized composite allografts (VCAs), including face and hand transplants, have emerged over the past 15 y. These have been used for reconstruction of patients with extensive disfigurements. Despite these great successes, the use of chronic immunosuppression by transplant recipients is accompanied by the risk of malignancy, organ toxicity and infection. Current research efforts by transplant physicians and plastic surgeons are aimed at reducing and subsequently eliminating the burden of life-long immunosuppression.Thi...

In addi-tion, they are chemo- and radio-resistant, and play a major role in disease recurrence and metastatic spread, the two major causes of patients’ mortality and morbid-ity. Therefore to effectively ‘cure’ a patient from a malignancy, CSCs need to be eradi -cated along with differentiated tumor cells, as the latter have been shown to de-differ -entiate into CSCs

Previous attempts of α-1,3-galactocyltransferase knockout (GalTKO) pig bone marrow (BM) transplantation (Tx) into baboons have demonstrated a loss of macro-chimerism within 24 h in most cases. In order to achieve improved engraftment with persistence of peripheral chimerism, we have developed a new strategy of intra-bone BM (IBBM) Tx. Six baboons received GalTKO BM cells, with one-half of the cells transplanted into the bilateral tibiae directly and the remaining cells injected intravenously (IBBM/BM-Tx) with a conditioning immunosuppressive regimen. In order to assess immune responses induced by the combined IBBM/BM-Tx, three recipients received donor SLA-matched GalTKO kidneys in the peri-operative period of IBBM/BM-Tx (Group 1), and the others received kidneys 2 months after IBBM/BM-Tx (Group 2). Peripheral macro-chimerism was continuously detectable for up to 13 days (mean 7.7 days; range 3–13) post-IBBM/BM-Tx and in three animals, macro-chimerism reappeared at days 10, 14 and 21. Pig CFUs, indicating porcine progenitor cell engraftment, were detected in the host BM in four of six recipients on days 14, 15, 19 and 28. In addition, anti-pig unresponsiveness was observed by in vitro assays. GalTKO/pCMV-kidneys survived for extended periods (47 and 60 days). This strategy may provide a potent adjunct for inducing xenogeneic tolerance through BM-Tx.

Organ transplantation represents a unique method of treatment to cure people with end-stage organ failure. Since the first successful organ transplant in 1954, the field of transplantation has made great strides forward. However, despite the ability to transform and save lives, transplant surgery is still faced with a fundamental problem the number of people requiring organ transplants is simply higher than the number of organs available. To put this in stark perspective, because of this critical organ shortage 18 people every day in the United States alone die on a transplant waiting list (U.S. Department of Health & Human Services, http://organdonor.gov/about/data.html). To address this problem, attempts have been made to increase the organ supply through xenotransplantation and more recently, bioengineering. Here we trace the development of both fields, discuss their current status and highlight limitations going forward. Ultimately, lessons learned in each field may prove widely applicable and lead to the successful development of xenografts, bioengineered constructs, and bioengineered xeno-organs, thereby increasing the supply of organs for transplantation.

We have previously shown that 12days of high-dose calcineurin inhibition induced tolerance in MHC inbred miniature swine receiving MHC-mismatched lung, kidney, or co-transplanted heart/kidney allografts. However, if lung grafts were procured from donation after brain death (DBD), and transplanted alone, they were rejected within 19-45days. Here, we investigated whether donor brain death with or without allograft ischemia would also prevent tolerance induction in kidney or heart/kidney recipients. Four kidney recipients treated with 12days of calcineurin inhibition received organs from donors rendered brain dead for 4hours. Six heart/kidney recipients also treated with calcineurin inhibition received organs from donors rendered brain dead for 4hours, 8hours, or 4hours with 4 additional hours of cold storage. In contrast to lung allograft recipients, all isolated kidney or heart/kidney recipients that received organs from DBD donors achieved long-term survival (>100days) without histologic evidence of rejection. Proinflammatory cytokine gene expression was upregulated in lungs and hearts, but not kidney allografts, after brain death. These data suggest that the deleterious effects of brain death and ischemia on tolerance induction are organ-specific, which has implications for the application of tolerance to clinical transplantation. Donor brain death prevents tolerance induction in lung, but not isolated kidney or heart plus kidney, allograft recipients, suggesting that the deleterious effects of brain death on tolerance induction are organ specific.

Background Hydrogen sulfide (H2S) has recently been reported to demonstrate both antiinflammatory and cytoprotective effects; however, its efficacy has not been well documented in large animal models. In this study, we examined whether the administration of H2S offers cytoprotective effects on renal ischemia-reperfusion injury (IRI) in a preclinical miniature swine model. Methods Major histocompatibility complex-inbred, CLAWN miniature swine (n = 9) underwent a right nephrectomy, followed by induction of a 120-min period of warm ischemia via placement of clamps on the left renal artery and vein. Group 1 (n = 3) underwent renal ischemia without H2S administration. Groups 2 (n = 3) and 3 (n = 3) received Na2S (prodrug of H2S) 10 min before reperfusion of the ischemic kidneys followed by a 30-min of Na2S postreperfusion intravenously (group 2) or selective administration of Na2S via the left renal artery (group 3). IRI was assessed by kidney biopsies, levels of inflammatory cytokines in sera and kidney tissue. Results Animals in group 1 had significantly higher serum creatinine levels compared with animals in groups 2 and 3 (P Conclusions Na2S administration, especially via an organ selective approach, appears to potentially offer cytoprotective and antiinflammatory effects following renal IRI.

BACKGROUND: The clinical significance of antibodies directed against antigens other than major histocompatibility complex (MHC) antigens is poorly understood, and there are few large animal models in which such antibodies can be examined. We studied, both retrospectively and prospectively, the development of antibodies to non-MHC antigens in tolerant miniature swine. METHODS: Our database was assessed for cases of antidonor antibody formation in tolerant animals over the last 20 years. Flow cytometry, absorption assays, and familial analyses for inheritance pattern of the gene(s) potentially responsible for the antibody reactivities were carried out, and an animal determined to be negative for this reactivity was immunized by a skin graft and subcutaneous injections of peripheral blood monocyte cells from an antigen-positive donor. RESULTS: Sixteen of 469 tolerant animals tested were found to have developed antidonor antibodies. These antibodies were found to be specific for the same, presumably single, non-MHC antigen. Familial analyses indicated that the gene encoding this antigen was expressed in an autosomal-dominant manner in approximately 95% of the herd. In a prospective study, antidonor antibodies with the same specificity as those observed retrospectively were successfully induced in an antigen-negative animal after immunization with peripheral blood monocyte cells. CONCLUSION: To our knowledge, this is the first report of the development of antibodies to a highly prevalent, non-MHC antigen present on peripheral blood mononuclear cells and developing in tolerant animals without signs of graft dysfunction. Considering the concern often raised by the appearance of antidonor antibodies in transplant recipients, these data could have important implications for clinical transplantation.

BACKGROUND Various durations of survival have been observed in the xenotransplantation of life-supporting α-1,3-galactosyltransferase knockout (GalT-KO) porcine kidneys into nonhuman primates. Although others have demonstrated loss of GalT-KO-transplanted kidneys within 2 weeks, we have reported an average survival of 51 days with the cotransplantation of the kidney and vascularized thymus and an average of 29 days with the kidney alone. To determine the factors responsible for this difference in survival time, we performed xenogeneic kidney transplantations into cynomolgus monkeys with an anti-CD40L-based regimen using two different strains of GalT-KO swine, one derived from MGH miniature swine and the other obtained from Meji University. MATERIALS AND METHODS Eight cynomolgus moneys received GalT-KO kidneys. Three kidney grafts were from Massachusetts General Hospital (MGH)-Nippon Institute for Biological Science (NIBS) GalT-KO pigs and five GalT-KO grafts were from MEIJI GalT-KO swine. All cynomolgus recipients were treated identically. RESULTS Recipients of kidneys from the MGH GalT-KO kidneys swine, produced by nuclear transfer in Japan, survived an average of 28.7 days, whereas recipients of MEIJI GalT-KO kidneys swine survived an average of 9.2 days. Among the differences between these two groups, one potentially revealing disparity was that the MEIJI swine were positive for porcine cytomegalovirus, whereas the MGH-derived swine were negative. CONCLUSION This is the first study comparing renal xenotransplantation from two different sources of GalT-KO swine into nonhuman primates at a single center. The results demonstrate that porcine cytomegalovirus may be responsible for early loss of GalT-KO swine kidney xenografts.

The mechanisms of H-transfer in termination reactions, and the role of fluorine interactions to achieve the living polymerization of olefins, are investigated. Mecking's bis(enolatoimine)Ti catalysts are considered, with or without ortho-F substituents. DFT calculations are performed on a parallel platform. The stationary structures are localized and energy barriers of activation are determined. Normal mode analysis is carried out and free energies are calculated. Different mechanisms can be involved in the termination: in all cases, the presence of F atoms rise the energy barrier of activation, according to experimental observations of living polymerization. The stabilizing role of the fluorine o-F · · · H bonds in the equilibrium species, and destabilizing o-F · · · H and o-F · · · o-F interactions in the transition states, are discussed.

Class I mismatched kidney transplantation in Massachusetts General Hospital MHC-defined miniature swine has been studied extensively as a model for induction of systemic allograft tolerance. In a large series of juvenile swine, long-term graft acceptance has been observed consistently following a 12-day course of cyclosporine. It was therefore surprising when three of five recipients in one of our studies rejected their grafts. Examination of the origins of the rejecting animals revealed that they were derived from a subline of the SLA(dd) miniature swine herd that was intentionally being inbred toward full homozygosity and had been inbred for eight generations prior to these experiments. A blinded study of additional class I mismatched renal transplants into animals from this subline confirmed the genetic basis of this rejection. We present here preliminary evidence suggesting that a likely explanation for this phenomenon is that the rejectors in this subline are homozygous for a recessive mutant allele of a gene normally involved in the induction of tolerance. Subsequent studies will be directed toward identification and characterization of the gene(s) involved, since existence of a similar genetic locus in humans might have implications for assessing an individual's likelihood of graft rejection versus tolerance induction prior to organ transplantation.

Background We have previously reported that Massachusetts General Hospital miniature swine, which had accepted class I-mismatched kidneys long-term after 12 days of high-dose cyclosporine A, uniformly accepted donor-major histocompatibility complex (MHC)-matched kidneys without immunosuppression but rejected donor MHC-matched split-thickness skin grafts by day 25, without changes in renal graft function or antidonor in vitro responses. We have now tested whether this "split tolerance" would also be observed for the primarily vascularized skin of vascularized composite allografts (VCAs). Methods Group 1 animals (n=3) received donor MHC-matched VCAs less than 70 days after primary kidney transplant (KTx). Group 2 animals (n=3) received a second donor-matched kidney transplant followed by a donor-matched VCA more than 200 days after primary KTx. Results Animals in Group 1 lost the epidermis on days 28, 30, and 40, with all other components of the VCAs remaining viable. Histology showed cellular infiltration localized to dermal-epidermal junction. One of three recipients of VCAs in Group 2, accepted all components of the VCA, including epidermis (>200 days). The other two recipients lost only the epidermis on days 45 and 85, with survival of the remainder of the VCA long-term. Conclusions All tissues of a VCA are accepted long-term on animals tolerant of class I-mismatched kidneys, with the exception of epidermis, the survival of which is markedly prolonged compared with split-thickness skin grafts but not indefinite. Exposure of tolerant animals to second donor-matched kidneys before VCA increases the longevity of the VCA epidermis, suggesting an increase in the immunomodulatory mechanisms associated with tolerance of the kidney.

Thymic involution is associated with age-related changes of the immune system. Utilizing our innovative technique of transplantation of a thymus as an isolated vascularized graft in MHC-inbred miniature swine, we have previously demonstrated that aged thymi are rejuvenated after transplantation into juvenile swine. Here we have studied the role of insulin-like growth factor (IGF) and forkhead-box protein-N1 (FOXN1) as well as bone marrow (BM) in thymic rejuvenation and involution. We examined thymic rejuvenation and involution by means of histology and flow cytometry. Thymic function was assessed by the ability to induce tolerance of allogeneic kidneys. Aged thymi were rejuvenated in a juvenile environment, and successfully induced organ tolerance, while juvenile thymi in aged recipients involuted and had a limited ability to induce tolerance. However, juvenile BM inhibited the involution process of juvenile thymi in aged recipients. An elevated expression of both FOXN1 and IGF1 receptors (IGF-1R) was observed in juvenile thymi and rejuvenated thymi. Juvenile BM plays a role in promoting the local thymic milieu as indicated by its ability to inhibit thymic involution in aged animals. The expression of FOXN1 and IGF-1R was noted to increase under conditions that stimulated rejuvenation, suggesting that these factors are involved in thymic recovery.

Reaction mechanisms of both propagation and termination steps in homogeneous polyolefin catalysis are investigated at DFT level. Starting from existent catalysts, we propose the design of a new Ti-bis(enolato-azo) catalytic family. We considered a number of hydrogenated or fluorinated models in aromatic group and chelating ring. Fluorination in the aromatic rings is able to control the propagation steps by activating the metal site, while hydrogenation of the ligand can inhibit the termination transfers. So, we have optimized the structure of the catalyst to give it the highest reactivity towards polymerization and the lowest one towards termination, by means of the meta-F substitutions in the aromatic group, and –CH2CF3 on the chelating ring. Thus, the developed bis(enolato-azo) models seem to be interesting for experimental investigations as new living catalysts.

Learning curves are believed to resemble an "idealized" model, in which continuous improvement occurs until a plateau is reached. We hypothesized that this "idealized" model would not adequately describe the learning process for a complex surgical technique, specifically laparoscopic liver resection (LLR).We analyzed the first 150 LLRs performed by a surgeon with expertise in hepatobiliary/laparoscopic surgery but with no previous LLR experience. We divided the procedures performed in 5 consecutive groups of 30 procedures, then compared groups in terms of complications, operative time, length of stay, and estimated blood loss.We observed an increase in operative complexity (3.3% major operations in Group 1 vs. 23.3% in Group 5, p = 0.05). Complications decreased from Group 1 to Group 2 (20%-3%), but increased again as more complex procedures were performed (3% in Group 2-13% in Group 5). Similar improvement/regression patterns were observed for operative time and EBL.The "true" learning curve for LLR is more appropriately described as alternating periods of improvement and regression until mastery is achieved. Surgeons should understand the true learning curves of procedures they perform, recognizing and mitigating the increased risk they assume by taking on more complex procedures.

BACKGROUND BRAF(V600E)-mediated MAPK pathway activation is associated in melanoma cells with IFNAR1 downregulation. IFNAR1 regulates melanoma cell sensitivity to IFNα, a cytokine used for the adjuvant treatment of melanoma. These findings and the limited therapeutic efficacy of BRAF-I prompted us to examine whether the efficacy of IFNα therapy of BRAF(V600E) melanoma can be increased by its combination with BRAF-I. METHODS BRAF/NRAS genotype, ERK activation, IFNAR1, and HLA class I expression were tested in 60 primary melanoma tumors from treatment-naive patients. The effect of BRAF-I on IFNAR1 expression was assessed in three melanoma cell lines and in four biopsies of BRAF(V600E) metastases. The antiproliferative, pro-apoptotic and immunomodulatory activity of BRAF-I and IFNα combination was tested in vitro and in vivo utilizing three melanoma cell lines, HLA class I-MA peptide complex-specific T-cells and immunodeficient mice (5 per group for survival and 10 per group for tumor growth inhibition). All statistical tests were two-sided. Differences were considered statistically significant when the P value was less than .05. RESULTS The IFNAR1 level was statistically significantly (P < .001) lower in BRAF(V600E) primary melanoma tumors than in BRAF wild-type tumors. IFNAR1 downregulation was reversed by BRAF-I treatment in the three melanoma cell lines (P ≤ .02) and in three out of four metastases. The IFNAR1 level in the melanoma tumors analyzed was increased as early as 10 to 14 days following the beginning of the treatment. These changes were associated with: 1) an increased susceptibility in vitro of melanoma cells to the antiproliferative (P ≤ .04), pro-apoptotic (P ≤ .009) and immunomodulatory activity, including upregulation of HLA class I antigen APM component (P ≤ .04) and MA expression as well as recognition by cognate T-cells (P < .001), of BRAF-I and IFNα combination and 2) an increased survival (P < .001) and inhibition of tumor growth of melanoma cells (P < .001) in vivo by BRAF-I and IFNα combination. CONCLUSIONS The described results provide a strong rationale for the clinical trials implemented in BRAF(V600E) melanoma patients with BRAF-I and IFNα combination.

The stability of weak fluorine bonds in homogeneous catalyst is studied at the ab initio level for an arylpyridine post-metallocene complex. The Ti···F···H β interaction scheme, involving the agostic bond with titanium and the H-bond with an H β in the growing chain, is found to be the the most stable one. A possible optimal overlap involving the fluorine lone pairs is proposed. This three-center interaction could play a role in the inhibition of H β transfer to the metal or to the monomer in the termination reactions.

The thermal behaviour of nanocomposites of linear-low-density-poly(ethylene) (LLDPE) and carbon nanotubes (CNT), prepared by the method of High Energy Ball Milling (HEBM) has been in-depth investigated. The CNT affect the nucleation of LLDPE, in that they act as nucleating agents for LLDPE. Moreover, as it is shown by self-nucleation experiments, in the presence of CNT a direct transition from domain I to domain III, with lack of domain II takes place. Isothermal experiments show that the crystallization rate of the nanocomposites is much higher than that of neat LLDPE. It is worthy noting that such effect is very high even for low contents of CNT in the sample (i.e. 1% by weight), then additions of CNT do not have any further effect. Lastly, the dynamic-mechanical properties of LLDPE are slightly modified in the nanocomposites.

Postoperatively, transplant recipients receive immunosuppressants, as well as sedatives and analgesics. The immunomodulatory effects of these other agents during the induction period following transplantation remain unclear. We aimed to determine whether the agents dexmedetomidine hydrochloride (Dex) and fentanyl (Fen) have immunomodulatory effects during the induction period following heart transplantation (HTx).Fifty mice were used for antinociception tests after administration of Dex and Fen, and T cells from 3 naive animals were used for in vitro lymphocyte transformation test (study 1). Fifty-four B6 mice received HTx from BALB/c mice and were treated with Dex, Fen, or neither (study 2). Thirty-six recipients were used for graft survival data and were humanely killed at the time of cessation of heart graft contraction. The remaining 18 were humanely killed at either postoperative day (POD) 4 or 6 for histologic examination of graft survival, as well as in vitro analysis.Based on the results of study 1, daily intraperitoneal administration of Dex at 30 μg/kg or Fen at 0.25 mg/kg was determined to be the optimal dose to induce analgesia without oversedation following HTx. Graft survivals in both Dex- or Fen-treated animals were statistically prolonged compared with control (P0.01). Graft survival of Fen-treated recipients was increased up to 15 days, and graft survival of Dex-treated animals was also increased up to 10 days, whereas control mice rejected heart grafts by POD 7. Mixed lymphocyte reaction responses on POD 4 showed statistically lower responses in Dex-treated recipients and Fen-treated recipients when compared with controls (P0.01). Cytokine profiles of splenocytes showed markedly fewer interferon γ-positive splenocytes in Fen-treated recipients on POD 4.These data suggest that both Dex and Fen have immunomodulatory properties in the induction period following transplantation.

Lignin powder, obtained from an abundant and low-cost source, straw, through a low-environmental-impact process, steam explosion, was used for the preparation of blends with a biodegradable polyester, poly(e-caprolactone) (PCL), with an innovative technique, high-energy ball milling. Lignin strongly stabilized PCL against UV radiation. The modulus of the blends increased with the addition of lignin; nevertheless, the elongation at breaking decreased. Through thermal characterization (differential scanning calorimetry and dynamic mechanical analysis), lignin and PCL were found to be immiscible. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008

Novel composites based on poly(e-caprolactone) (PCL) and an organically modified layer double hydroxide (LDH) obtained using the melt-extrusion technique have been characterized through structural, thermal, and mechanical analyses. Although exfoliation has not been achieved and despite the very low content of filler (from 1 to 3% by weight), significant enhancements are obtained in the physical and mechanical properties of the composites with respect to neat PCL. As a consequence, LDHs can substitute other nanofillers, in particular, cationic clays for polymeric matrices. They can be modified by a large number of organic anions, generally more numerous than the cationic ones, and can be mixed in very simple ways with polymers. This makes such nanofillers suitable to obtain new hybrid materials for a series of applications, from active food packaging to intelligent materials for biomedical device, for example, controlled drug release. © 2007 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 45: 945–954, 2007

For the first time the kinetics of formation of syndiotactic polypropylene mesophase close to 0 °C through differential scanning calorimetry has been investigated. The calorimetric data have been analyzed through the well-known Avrami analysis. We have obtained Avrami coefficients less or at most equal to 2, which are likely to indicate a nearly bidimensionality of the crystallites. The morphology of the mesophase, not known up to now, has been examined through atomic force microscopy (AFM): lamellar structures have been observed. The fractal dimension of the AFM images has been evaluated through the box counting method. It resulted about 2, in a very good agreement with the calculated Avrami coefficients. An infrared and diffractometer analysis on samples kept for increasing times at 0 °C has also been performed to confirm the calorimetric data. The experimental results allow one to better clarify the kinetics of mesophase-formation. It has been found that a time of 3 h at 0 °C is actually necessary for the complete crystallization of the trans-planar mesophase, a longer time is needed to stabilize the mesophase domains when the sample is extracted from 0 °C at room temperature.

Our recent studies in an inbred swine model demonstrated that both peripheral and intra-graft regulatory cells were required for the adoptive transfer of tolerance to a second, naive donor-matched kidney. Here, we have asked whether both peripheral and intra-graft regulatory elements are required for adoptive transfer of tolerance when only a long-term tolerant (LTT) kidney is transplanted. Nine highly-inbred swine underwent a tolerance-inducing regimen to prepare LTT kidney grafts which were then transplanted to histocompatible recipients, with or without the peripheral cell populations required for adoptive transfer of tolerance to a naive kidney. In contrast to our previous studies, tolerance of the LTT kidney transplants alone was achieved without transfer of additional peripheral cells and without strategies to increase the number/potency of regulatory T cells in the donor. This tolerance was systemic, since most subsequent, donor-matched challenge kidney grafts were accepted. These results confirm the presence of a potent tolerance-inducing and/or tolerance-maintaining cell population within LTT renal allografts. They suggest further that additional peripheral tolerance mechanisms, required for adoptive transfer of tolerance to a naive donor-matched kidney, depend on peripheral cells that, if not transferred with the LTT kidney, require time to develop in the adoptive host.

Background Achieving tolerance of vascularized composite allografts (VCAs) would improve the risk-to-benefit ratio in patients who undergo this life-enhancing, though not lifesaving, transplant. Kidney cotransplantation along with a short course of high-dose immunosuppression enables tolerance of heart allografts across a full major histocompatibility complex (MHC) mismatch. In this study, we investigated whether tolerance of VCAs across full MHC disparities could be achieved in animals already tolerant of heart and kidney allografts. Methods Miniature swine that were tolerant of heart and/or kidney allografts long term underwent transplantation of myocutaneous VCA across the same MHC barrier. Before VCA transplant, group 1 (n = 3) underwent class I-mismatched kidney transplantation; group 2 (n = 3) underwent 2 sequential class I-mismatched kidney transplantations; group 3 (n = 2) underwent haploidentical MHC-mismatched heart/kidney transplantation; and group 4 (n = 2) underwent full MHC-mismatched heart/kidney transplantation. Results All 3 animals in group 1 and 2 of 3 animals in group 2 showed skin rejection within 85 days; 1 animal in group 2 showed prolonged skin survival longer than 200 days. Animals in groups 3 and 4 showed skin rejection within 30 days and regained in vitro evidence of donor responsiveness. Conclusions This is the first preclinical study in which hearts, kidneys, and VCAs have been transplanted into the same recipient. Despite VCA rejection, tolerance of heart and kidney allografts was maintained. These results suggest that regulatory tolerance of skin is possible but not generally achieved by the same level of immunomodulation that is capable of inducing tolerance of heart and kidney allografts. Achieving tolerance of skin may require additional immunomodulatory therapies.

The beneficial clinical effects of immunotherapy with GD2-specific monoclonal antibodies (mAbs) in melanoma and neuroblastoma patients have stimulated interest in characterizing the mechanisms underlying their antitumor effects. Previous studies have shown that GD2-specific mAbs mediate complement- and cell-dependent cytotoxicity and induce caspase-dependent apoptosis of tumor cells. In this study, we showed that GD2-specific mAb 3F8, which is undergoing clinical evaluation, inhibited the in vitro growth and induced apoptosis of melanoma cells. This effect was dose- and time-dependent, mediated by the interaction of mAb 3F8 combining site with GD2 ganglioside, associated with GD2 expression level on the cell surface, mAb internalization and increase of GD2 containing endosomes triggered by mAb 3F8. The induction of apoptosis by mAb 3F8 was mediated by caspase 3-, 7-, and 8-dependent pathways, downregulation of the anti-apoptotic molecules survivin and cytochrome c, and caspase 9 independent-AIF release from mitochondria. In addition, analyses of signaling pathway components demonstrated that mAb 3F8 strongly inhibited AKT and FAK activation and increased cleaved PARP expression. These results indicated that multiple mechanisms played a role in the antitumor activity of mAb 3F8 in melanoma cells. This information should provide a mechanistic basis for the optimization of the rational design of immunotherapeutic strategies in the mAb-based treatment of GD2 positive tumors.

With differential scanning calorimetry, we have demonstrated a peculiar behavior under equilibrium conditions of neat poly(e-caprolactone) and its organophilic montmorillonite nanocomposites. In particular, in the determination of the equilibrium melting temperature by the extrapolation of the data of the melting temperature (Tm) versus the crystallization temperature (Tc), a bimodal trend has been observed. At the lower Tc's, the data of Tm follow a constant trend, whereas at the higher ones, the usual increasing trend has been obtained. Morphological observations by atomic force microscopy (AFM) have provided evidence of two different crystalline morphologies for the lower and higher Tc ranges. Moreover, AFM has shown that the thermal treatments strongly influence the clay dispersion in the polymer matrix. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 44: 22–32, 2006

Lignin powder, obtained from an abundant and low cost source, straw, through a low environmental impact process, the steam explosion, is used for the preparation of blends with low-density polyethylene (LDPE), linear low-density polyethylene (LLDPE), high-density polyethylene (HDPE) and atactic polystyrene (PS). The obtained blends are processable through the conventional techniques used for thermoplastics; the modulus slightly increases for most lignin-polymer blends, while the tensile stress and elongation reduce. Moreover, lignin acts as a stabilzer against the UV radiation for PS, LDPE and LLDPE.

Temperature-modulated differential scanning calorimetry (TMDSC) in the quasi-isothermal mode is applied to investigate melting and crystallization of poly(tetrafluoroethylene) (PTFE) obtained from aqueous dispersion, both melt-crystallized and native (as-polymerized). The differences, shown in the past between the melting behavior of melt-crystallized and native PTFE, have been confirmed and further evidenced through this technique. A large reversing heat capacity is present in the melting and crystallization regions. As proposed by many authors, the presence of the reversing heat capacity can be related to surface melting and crystallization. It can occur on the growth and/or fold surfaces and it has been shown that it is larger for those macromolecules having higher chain mobility that allows rearrangements on the crystal surface. In the present case, the large observed reversing heat capacity can be related to the very high sliding ability of PTFE chains in the pseudohexagonal phase, which is much larger than that of most semicrystalline polymers. Due to the crystal–crystal transition at 30 °C, which can be described as a fusion in the longitudinal direction, melting of PTFE can be considered intermediate between the irreversible melting of macromolecules and the completely reversible isotropization of liquid crystalline polymers.

A new algorithm is proposed for the determination of the maximum entropy state of complex molecular systems, as flexible macromolecules in solution. This method is called relaxed annealing (RAN) and is based on molecular dynamics simulations. In this paper it is applied to an elastin (the Rubber protein of vertebrates) polypeptide in aqueous solution, where the mobility of the peptide is increased gradually by decreasing an external force applied to the ends of the chain. The goal of the method is to optimize a thermodynamic potential by the relaxation of an external constraint that enhances the convergence velocity of the algorithm. The tetrapeptide Ac–Gly–Leu–Gly–Gly–NMe has been simulated for 2 ns, starting from the fully extended conformation with the ends constrained by harmonic potentials. The external force constants were reduced exponentially, and finally 2 ns of the free-running molecule were considered. Thus, the elasticity recoil process of the stretched chain has been simulated and the resilience of the elastin peptide to the experimented reversible deformations verified. The global dynamics of the peptide has been examined by the tools of nonlinear complex systems analysis. The evolution has been observed toward increasing entropy states, beginning from the essentially linear dynamics of the extended conformation, to the high entropy fractional Brownian chaotic behavior of the final folded state, typical of bounded diffusive fluctuations below the percolation threshold. In this way, the transition-to-chaos mechanism of the entropic driving force of elasticity and the soft-solution model of aggregation state of rubber proteins are confirmed.

Temperature modulated differential calorimetry (TMDSC) is used to examine the crystal–crystal transitions of poly(tetrafluoroethylene). This study gives new information about the dynamic thermal behavior of such transitions. The involvement of reversible and irreversible processes during the phenomenon is observed, which are related to the order–disorder changes occurring during the transition. This study adds a new example to the response of TMDSC during first order transitions.