Alejandro C. Bribriesco

Intravital 2-photon imaging of leukocyte trafficking in beating heart

Two-photon intravital microscopy has substantially broadened our understanding of tissue- and organ-specific differences in the regulation of inflammatory responses. However, little is known about the dynamic regulation of leukocyte recruitment into inflamed heart tissue, largely due to technical difficulties inherent in imaging moving tissue. Here, we report a method for imaging beating murine hearts using intravital 2-photon microscopy. Using this method, we visualized neutrophil trafficking at baseline and during inflammation. Ischemia reperfusion injury induced by transplantation or transient coronary artery ligation led to recruitment of neutrophils to the heart, their extravasation from coronary veins, and infiltration of the myocardium where they formed large clusters. Grafting hearts containing mutant ICAM-1, a ligand important for neutrophil recruitment, reduced the crawling velocities of neutrophils within vessels, and markedly inhibited their extravasation. Similar impairment was seen with the inhibition of Mac-1, a receptor for ICAM-1. Blockade of LFA-1, another ICAM-1 receptor, prevented neutrophil adherence to endothelium and extravasation in heart grafts. As inflammatory responses in the heart are of great relevance to public health, this imaging approach holds promise for studying cardiac-specific mechanisms of leukocyte recruitment and identifying novel therapeutic targets for treating heart disease.

Lung transplant acceptance is facilitated by early events in the graft and is associated with lymphoid neogenesis

Early immune responses are important in shaping long-term outcomes of human lung transplants. To examine the role of early immune responses in lung rejection and acceptance, we developed a method to retransplant mouse lungs. Retransplantation into T-cell-deficient hosts showed that for lungs and hearts alloimmune responses occurring within 72 h of transplantation are reversible. In contrast to hearts, a 72-h period of immunosuppression with costimulation blockade in primary allogeneic recipients suffices to prevent rejection of lungs upon retransplantation into untreated allogeneic hosts. Long-term lung acceptance is associated with induction of bronchus-associated lymphoid tissue, where Foxp3+ cells accumulate and recipient T cells interact with CD11c+ dendritic cells. Acceptance of retransplanted lung allografts is abrogated by treatment of immunosuppressed primary recipients with anti-CD25 antibodies. Thus, events contributing to lung transplant acceptance are established early in the graft and induction of bronchus-associated lymphoid tissue can be associated with an immune quiescent state.

DAP12 Expression in Lung Macrophages Mediates Ischemia/Reperfusion Injury by Promoting Neutrophil Extravasation

Neutrophils are critical mediators of innate immune responses and contribute to tissue injury. However, immune pathways that regulate neutrophil recruitment to injured tissues during noninfectious inflammation remain poorly understood. DAP12 is a cell membrane–associated protein that is expressed in myeloid cells and can either augment or dampen innate inflammatory responses during infections. To elucidate the role of DAP12 in pulmonary ischemia/reperfusion injury (IRI), we took advantage of a clinically relevant mouse model of transplant-mediated lung IRI. This technique allowed us to dissect the importance of DAP12 in tissue-resident cells and those that infiltrate injured tissue from the periphery during noninfectious inflammation. Macrophages in both mouse and human lungs that have been subjected to cold ischemic storage express DAP12. We found that donor, but not recipient, deficiency in DAP12 protected against pulmonary IRI. Analysis of the immune response showed that DAP12 promotes the survival of tissue-resident alveolar macrophages and contributes to local production of neutrophil chemoattractants. Intravital imaging demonstrated a transendothelial migration defect into DAP12-deficient lungs, which can be rescued by local administration of the neutrophil chemokine CXCL2. We have uncovered a previously unrecognized role for DAP12 expression in tissue-resident alveolar macrophages in mediating acute noninfectious tissue injury through regulation of neutrophil trafficking.

The American Association for Thoracic Surgery consensus guidelines for the management of empyema.

Objective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .e2 Methods of Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .e2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .e2 Clinical Presentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . .e3 Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . .e3 Reasoning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .e3 Imaging Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .e4 Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . .e4 Reasoning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .e4 Chest X-ray . . . . . . . . . . . . . . . . . . . . . . . . . . . .e4 Pleural US . . . . . . . . . . . . . . . . . . . . . . . . . . . . .e4

The price of innovation: a primer on high risk, high reward surgery

Difficult problems stimulate the development of novel therapies which more often than not are high-risk due to the nature of the disease process. Rare diseases offer few opportunities to refine a technique or protocol. Diseases that leave patients in critical and unstable conditions leave little margin for error.

Surgical technique for lung retransplantation in the mouse

Microsurgical cuff techniques for orthotopic vascularized murine lung transplantation have allowed for the design of studies that examine mechanisms contributing to the high failure rate of pulmonary grafts. Here, we provide a detailed technical description of orthotopic lung retransplantation in mice, which we have thus far performed in 144 animals. The total time of the retransplantation procedure is approximately 55 minutes, 20 minutes for donor harvest and 35 minutes for the implantation, with a success rate exceeding 95%. The mouse lung retransplantation model represents a novel and powerful tool to examine how cells that reside in or infiltrate pulmonary grafts shape immune responses.

Atlas is not alone: sharing the burden of clinical challenge

In the field of oncology where the disease does not respect any boundaries, it is essential for the treating physicians to reciprocate through the multidisciplinary charge. The expertise of a particular group is derived from their familiarity with cutting edge and salvage approaches to challenging situations within their field.