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Activated T lymphocytes are abundant in the airway during lung allograft rejection. Based on respiratory viral studies, it is the current paradigm that T cells cannot divide in the airway, and that their accumulation in the lumen of the respiratory tract is the exclusive result of recruitment from other sites, such as mediastinal lymph nodes. Here, we show that CD8(+) T cell activation and proliferation can occur in the airway after orthotopic lung transplantation. We also demonstrate that airway epithelium expresses major histocompatibility class I predominantly on the apical surface, both in vitro and in vivo, and initiates CD8(+) T cell responses in a polarized fashion, favoring luminal activation. Our data identify a unique site for CD8(+) T cell activation after lung transplantation, and suggest that attenuating these responses may provide a clinically relevant target.

Activated T lymphocytes are abundant in the airway during lung allograft rejection. Based on respiratory viral studies, it is the current paradigm that T cells cannot divide in the airway, and that their accumulation in the lumen of the respiratory tract is the exclusive result of recruitment from other sites, such as mediastinal lymph nodes. Here, we show that CD8+ T cell activation and proliferation can occur in the airway after orthotopic lung transplantation. We also demonstrate that airway epithelium expresses major histocompatibility class I predominantly on the apical surface, both in vitro and in vivo, and initiates CD8+ T cell responses in a polarized fashion, favoring luminal activation. Our data identify a unique site for CD8+ T cell activation after lung transplantation, and suggest that attenuating these responses may provide a clinically relevant target.

The interaction of CD4+ T cells with MHC class II (MHCII)-expressing hematopoietic APCs plays a critical role in both the generation of protective immune responses and maintenance of tolerance in the lung. The functional significance of MHCII expression by nonhematopoietic stromal cells, however, has not been defined in vivo. Using a novel mouse model of orthotopic left lung transplantation, we demonstrate that selective elimination of MHCII expression on nonhematopoietic cells leads to an inflammatory response as a result of reduced peripheral generation of regulatory CD4+ T cells. Absence of MHCII expression on nonhematopoietic cells also inhibits local growth of metastatic pulmonary tumor. These findings indicate that nonhematopoietic cells play a previously unrecognized role in downregulating inflammatory responses in nonlymphoid tissues.

Unlike transplantation of other solid organs, vascularized mouse lung transplantation has only recently been developed. In this protocol, we describe a detailed method for performing a vascularized and aerated mouse orthotopic lung transplant, which to date represents the most physiological mouse model of lung transplantation. The procedure is divided into two separate portions consisting of donor harvest followed by implantation using the cuff technique for bronchovascular anastomoses. After a training period spanning several months, the procedure can be successfully mastered and, in experienced hands, requires approximately 90 min to perform. After an initial learning curve, perioperative survival is close to 100%. As the donor hematopoietic cells in the transplanted lung are replaced by those of the host over time, thereby creating a 'chimeric lung,' this model represents a novel research tool for the study of transplantation biology as well as other disease processes, such as malignancies.

Acute rejection continues to present a major obstacle to successful lung transplantation. Although CD4+ T lymphocytes are critical for the rejection of some solid organ grafts, the role of CD4+ T cells in the rejection of lung allografts is largely unknown. In this study, we demonstrate in a novel model of orthotopic vascularized mouse lung transplantation that acute rejection of lung allografts is independent of CD4+ T cell-mediated allorecognition pathways. CD4+ T cell-independent rejection occurs in the absence of donor-derived graft-resident hematopoietic APCs. Furthermore, blockade of the CD28/B7 costimulatory pathways attenuates acute lung allograft rejection in the absence of CD4+ T cells, but does not delay acute rejection when CD4+ T cells are present. Our results provide new mechanistic insight into the acute rejection of lung allografts and highlight the importance of identifying differences in pathways that regulate the rejection of various organs.

Graft rejection remains a formidable problem contributing to poor outcomes after lung transplantation. Blocking chemokine pathways have yielded promising results in some organ transplant systems. Previous clinical studies have demonstrated upregulation of CCR2 ligands following lung transplantation. Moreover, lung injury is attenuated in CCR2-deficient mice in several inflammatory models. In this study, we examined the role of CCR2 in monocyte recruitment and alloimmune responses in a mouse model of vascularized orthotopic lung transplantation. The CCR2 ligand MCP-1 is upregulated in serum and allografts following lung transplantation. CCR2 is critical for the mobilization of monocytes from the bone marrow into the bloodstream and for the accumulation of CD11c(+) cells within lung allografts. A portion of graft-infiltrating recipient CD11c(+) cells expresses both recipient and donor MHC molecules. Two-photon imaging demonstrates that recipient CD11c(+) cells are associated with recipient T cells within the graft. While recipient CCR2 deficiency does not prevent acute lung rejection and is associated with increased graft infiltration by T cells, it significantly reduces CD4(+) T(h)1 indirect and direct allorecognition. Thus, CCR2 may be a potential target to attenuate alloimmune responses after lung transplantation.

It is the prevailing view that adaptive immune responses are initiated in secondary lymphoid organs. Studies using alymphoplastic mice have shown that secondary lymphoid organs are essential to initiate allograft rejection of skin, heart, and small bowel. The high immunogenicity of lungs is well recognized and allograft rejection remains a major contributing factor to poor outcomes after lung transplantation. We show in this study that alloreactive T cells are initially primed within lung allografts and not in secondary lymphoid organs following transplantation. In contrast to other organs, lungs are acutely rejected in the absence of secondary lymphoid organs. Two-photon microscopy revealed that recipient T cells cluster predominantly around lung-resident, donor-derived CD11c+ cells early after engraftment. These findings demonstrate for the first time that alloimmune responses following lung transplantation are initiated in the graft itself and therefore identify a novel, potentially clinically relevant mechanism of lung allograft rejection.

The use of stable isotopes of water as tracers in characterising the source of water in vegetation is tried in three field situations. Initially, methodological aspects of the uptake of water into plant tissue, water movement from roots to tops and recovery from stem-wood by distillation are considered. Using tracers of deuterium and oxygen-18, diffusion of water into lettuce leaf tissue was demonstrated to occur as the whole molecule, equilibrating in 24 h. It is considered from the similar tissue density that diffusion of water into roots would be similar. Distillation in toluene or kerosene to recover water from various materials found small decreases in deuterium composition of the recovered water: − 2.3‰ when recovering water from pine wood, −1.4‰ from potting mix and −3.2‰ from wood of native plants, while water recovered from lettuce leaf was not subject to such effects. These decreases are correlated to the proportion of organic matter in the materials, and may relate to decomposition of a component of this. Uptake of water by potted Hordeum vulgare, Atriplex nummularia, A. rhagodiodes, A. vesicaria. Callitris preissii, Casuarina glauca, Eucalyptus oleosa, E. citriodora and Maireana sedifolia was found to be relatively free of further fractionation. In field studies, comparison of the isotopic composition of water from stems of Hordeum vulgare, Maireana sedifolia and Pinus radiata and from adjacent soil profiles suggested that the depth of extraction of water by roots varied with water availability and rooting depth. Examination of the stable isotopes of soil and sap water in addition to soil water contents provides a more realistic interpretation of plant water source than obtained by studying soil water content alone.

Lung allografts are considered to be more immunogenic than other solid organs. Little is known about the effectiveness of immunosuppressive regimens after lung transplantation. Herein, we describe a novel model of murine vascularized orthotopic lung transplantation we used to study the effects of costimulatory blockade on lung rejection. Transplants were performed in the Balb --> B6 strain combination. Recipients were either not immunosuppressed or received perioperative CD40/CD40L and CD28/B7 costimulatory blockade. Nonimmunosupressed Balb/c --> B6 lung transplants had severe acute rejection 7 days after transplantation and CD8(+) T cells outnumbered CD4(+) T cells within the allografts. Alternatively, B6 recipients that received perioperative costimulatory blockade had minimal inflammation and there were nearly equal numbers of CD8(+) and CD4(+) T cells in these grafts. Approximately one third of graft-infiltrating CD4(+) T cells expressed Foxp3. CD4(+) T cells isolated from these grafts induced apoptosis of alloreactive CD8(+) T cells that were stimulated with donor splenocytes in vitro. In contrast with wild-type B6 recipient mice, we observed severe rejection of Balb/c lungs 7 days after transplantation into Bcl-2 transgenic B6 recipients that had received costimulatory blockade. CD8(+) T cells outnumbered CD4(+) T cells in these immunosuppressed Bcl-2 transgenic recipients and, compared with immunosuppressed wild-type B6 recipients, a lower percentage of graft-infiltrating CD4(+) T cells expressed Foxp3, and a higher percentage of graft-infiltrating CD8(+) T cells expressed intereferon-gamma. Thus, our results show that perioperative blockade of the CD40/CD40L and CD28/B7 costimulatory pathways markedly ameliorates acute rejection of lung allografts in wild type but not Bcl-2 transgenic recipients.

Lung transplantation remains the only therapeutic option for many patients suffering from end-stage pulmonary disease. Long-term success after lung transplantation is severely limited by the development of bronchiolitis obliterans. The murine heterotopic tracheal transplantation model has been widely used for studies investigating pathogenesis of obliterative airway disease and immunosuppressive strategies to prevent its development. Despite its utility, this model employs proximal airway that lacks airflow and is not vascularized. We have developed a novel model of orthotopic vascularized lung transplantation in the mouse, which leads to severe vascular rejection in allogeneic strain combinations. Here we characterize differences in the fate of airway epithelial cells in nonimmunosuppressed heterotopic tracheal and vascularized lung allograft models over 28 days. Up-regulation of growth factors that are thought to be critical for the development of airway fibrosis and interstitial collagen deposition were similar in both models. However, while loss of airway epithelial cells occurred in the tracheal model, airway epithelium remained intact and fully differentiated in lung allografts, despite profound vascular rejection. Moreover, we demonstrate expression of the anti-apoptotic protein Bcl-2 in airway epithelial cells of acutely rejected lung allografts. These findings suggest that in addition to alloimmune responses, other stimuli may be required for the destruction of airway epithelial cells. Thus, the model of vascularized mouse lung transplantation may provide a new and more physiologic experimental tool to study the interaction between immune and nonimmune mechanisms affecting airway pathology in lung allografts.

Outcomes after lung transplantation are markedly inferior to those after other solid organ transplants. A better understanding of cellular and molecular mechanisms contributing to lung graft injury will be critical to improve outcomes. Advances in this field have been hampered by the lack of a mouse model of lung transplantation. Here, we report a mouse model of vascularized aerated single lung transplantation utilizing cuff techniques. We show that syngeneic grafts have normal histological appearance with minimal infiltration of T lymphocytes. Allogeneic grafts show acute cellular rejection with infiltration of T lymphocytes and recipient-type antigen presenting cells. Our data show that we have developed a physiological model of lung transplantation in the mouse, which provides ample opportunity for the study of nonimmune and immune mechanisms that contribute to lung allograft injury.

Ischemia reperfusion (I/R) injury following lung transplantation is exacerbated by the destruction of the endothelial cell barrier leading to pulmonary edema and dysregulated activated lymphocyte migration. Sphingosine 1-phosphate (S1P), a G-coupled protein receptor (GPCR) agonist, has been previously shown to promote endothelial cell tight junction formation and prevent monocyte chemotaxis. We asked if S1P treatment could improve pulmonary function and attenuate I/R injury following syngeneic rat lung transplantation. In comparison to vehicle-treated recipients, S1P administered before reperfusion significantly improved recipient oxygenation following transplantation. Improved graft function was associated with reduced inflammatory signaling pathway activation along with attenuated intragraft levels of MIP-2, TNF-alpha and IL-1beta. Moreover, S1P-treated recipients had significantly less apoptotic endothelial cells, pulmonary edema and graft accumulation of neutrophils than did vehicle-treated recipients. Thus our data show that S1P improves lung tissue homeostasis following reperfusion by enhancing endothelial barrier function and blunting monocytic graft infiltration and inflammation.

etiologies of respiratory failure. Charts of patients with emphysema were reviewed for NETT criteria that suggest favorable prognosis with LVRS including PFTs, CT scan, and comorbid conditions. Active tobacco use resulted in exclusion. Results: 162 patients were referred for lung transplant evaluation, 69 patients were listed. 93 patients deferred or denied, 56 carried the diagnosis of emphysema. 16 (29%) met NETT criteria for LVRS. 4 patients had ongoing tobacco use. 9 patients had cardiac contraindications, 6 other comorbid conditions. 17 patients had PFTs outside the strict NETT criteria. One patient had a previous talc pleurodesis. One patient had prior LVRS. Conclusions: LVRS has been shown to be beneficial in select patients with emphysema yet appears underutilized in the VA population. Almost 30% of deferred patients were potential candidates for volume reduction. LVRS as a bridge to transplant has been used minimally in the VA. This unique population with referral from throughout the United States gives insight into national trends in medical care . Our data suggests that changes in reimbursment coverage after NETT recommendations have not increased numbers of LVRS or its acceptance as alternative to transplantation in select patients.

A study was undertaken to determine how the moisture content of an aqueous foam affects its ability to attenuate an acoustic signal. The energy losses from an acoustic signal, for the series of foams considered in this study, are believed to arise principally from two causes: an increase in the density of the medium through which the signal passes (resulting in a decrease in the velocity of sound) and a viscous loss mechanism. The experimental results presented indicate that both these processes are dependent on the density or liquid content of the foam. In addition to this, the viscous loss mechanism displays a bubble size and liquid viscosity dependence. The incorporation of talc particles into the aqueous foams was found to greatly enhance the stability of the foam and its attenuation characteristics. This is believed to arise from an increase in the viscosity of the liquid phase of the foam and the enhanced signal/air and particle/liquid interactions, both of which result from the presence of the fine particles in the foam.