Donor-transmitted malignancies in organ transplantation: assessment of clinical risk.

The continuing organ shortage requires evaluation of all potential donors, including those with malignant disease. In the United States, no organized approach to assessment of risk of donor tumor transmission exists, and organs from such donors are often discarded. The ad hoc Disease Transmission Advisory Committee (DTAC) of the Organ Procurement and Transplantation Network/United Network for Organ Sharing (OPTN/UNOS) formed an ad hoc Malignancy Subcommittee to advise on this subject. The Subcommittee reviewed the largely anecdotal literature and held discussions to generate a framework to approach risk evaluation in this circumstance. Six levels of risk developed by consensus. Suggested approach to donor utilization is given for each category, recognizing the primacy of individual clinical judgment and often emergent clinical circumstances. Categories are populated with specific tumors based on available data, including active or historical cancer. Benign tumors are considered in relation to risk of malignant transformation. Specific attention is paid to potential use of kidneys harboring small solitary renal cell carcinomas, and to patients with central nervous system tumors. This resource document is tailored to clinical practice in the United States and should aid clinical decision making in the difficult circumstance of an organ donor with potential or proven neoplasia.

Donor-derived disease transmission events in the United States: data reviewed by the OPTN/UNOS Disease Transmission Advisory Committee.

Donor-derived disease transmission is increasingly recognized as a source of morbidity and mortality among transplant recipients. Policy 4.7 of the Organ Procurement and Transplantation Network (OPTN) currently requires reporting of donor-derived events. All potential donor-derived transmission events (PDDTE) reported to OPTN/UNOS were reviewed by the Disease Transmission Advisory Committee (DTAC). Summary data from January 1, 2005-December 31, 2007, were prepared for presentation. Reports of PDDTE have increased from 7 in 2005, the first full year data were collected, to 60 in 2006 and to 97 in 2007. More detailed information is available for 2007; a classification system for determining likelihood of donor-derived transmission was utilized. In 2007, there were four proven and one possible donor-derived malignancy transmissions and four proven, two probable and six possible donor-derived infectious diseases transmissions. There were nine reported recipient deaths attributable to proven donor transmissions events arising from eight donors during 2007. Although recognized transmission events resulted in significant morbidity and mortality, transmission was reported in only 0.96% of deceased donor donations overall. Improved reporting, through enhanced recognition and communication, will be critical to better estimate the transmission risk of infection and malignancy through organ transplantation.

Adaptive mechanisms that preserve cardiac function in mice without myoglobin.

Mice lacking myoglobin survive to adulthood and meet the circulatory demands of exercise and pregnancy without cardiac decompensation. In the present study, we show that many myoglobin-deficient embryos die in utero at midgestation with signs of cardiac failure. Fetal mice that survive to gestational day 12.5, however, suffer no subsequent excess mortality. Survival in the absence of myoglobin is associated with increased vascularity and the induction of genes encoding the hypoxia-inducible transcription factors 1alpha and 2, stress proteins such as heat shock protein 27, and vascular endothelial growth factor. These adaptations are evident in late fetal life, persist into adulthood, and are sufficient to maintain normal myocardial oxygen consumption during stressed conditions. These data reveal that myoglobin is necessary to support cardiac function during development, but adaptive responses evoked in some animals can fully compensate for the defect in cellular oxygen transport resulting from the loss of myoglobin.

HER2/neu-derived peptides are shared antigens among human non-small cell lung cancer and ovarian cancer.

Previously, we have reported a correlation between the expression of HER2/neu and sensitivity to HLA-A2-restricted cytotoxic T-cells (CTL) in ovarian cancer. To investigate the role of HER2/neu in human non-small cell lung cancer (NSCLC), we established autologous tumor-specific CTL from tumor-infiltrating lymphocytes of HLA-A2+ HER2/neu+ NSCLC patients. These CTL lines specifically recognized HLA-A2+ HER2/neu+ autologous and allogeneic NSCLC cell lines as well as HLA-A2+ HER2/neu+ heterologous ovarian cancer cell lines. Furthermore, these CTL recognized an overexpressed, HER2/neu-derived peptide. From these results, we conclude that HLA-A2 serves as a restriction element in NSCLC. More importantly, at least one HER2/neu-derived peptide is a tumor-associated antigen in NSCLC and ovarian cancer.

Adoptive transfer of cytotoxic T lymphocytes for the treatment of transplant-associated lymphoma.

BACKGROUND: Immunocompromised organ transplant recipients have a high incidence of B cell lymphomas (BCL). Severe combined immunodeficient (SCID) mice develop human BCL when engrafted with Epstein-Barr virus (EBV) transformed and immortalized B lymphoblastoid cell lines (BLCL). Because a lack of effective EBV-specific cytotoxic T lymphocytes (EBV-CTL) is thought to lead to lymphoma development, the SCID mouse model was used to determine the relationship between EBV-infected B cells and EBV-specific CTL in BCL development in vivo. METHODS: EBV-CTL were generated by in vitro stimulation of peripheral blood leukocytes with autologous BLCL. CD8+ CTL were isolated from CTL populations by depletion of CD4+ cells. SCID mice were engrafted with BLCL, EBV-CTL were adoptively transferred into engrafted SCID mice either immediately or 7 days after engraftment, and the animals were monitored for the development of BCL. Statistical significance was determined by the log rank test. RESULTS: SCID mice engrafted with BLCL rapidly developed BCL (mean, 20 days). SCID mice engrafted with BLCL and human leukocyte antigen-identical EBV-CTL or CD8+ EBV-CTL had a significant delay in BCL development (p < 0.05), whereas some mice did not develop BCL. In contrast, human leukocyte antigen-nonidentical EBV-CTL did not significantly delay BCL development. CONCLUSIONS: This study showed the role of EBV-CTL in inhibiting the development of BCL. A greater understanding of the cellular and viral interactions leading to B-cell transformation and malignancy may allow the development of specific interventional therapies in patients who have received immunosuppressants.

Directed enzyme pro-drug gene therapy for pancreatic cancer in vivo.

BACKGROUND: Directed enzyme pro-drug therapy incorporates the delivery of a gene to a cancer cell that will be specifically expressed and will confer sensitivity to a therapeutic agent. Tumor-specific gene expression can be achieved by coupling the promoter for the carcinoembryonic antigen (CEA) to a gene such as herpes simplex virus thymidine kinase (HSV-tk), which phosphorylates ganciclovir to a potent DNA synthesis inhibitor. METHODS: Retroviral vectors were constructed to contain the CEA promoter coupled to HSV-tk (LN-CEA-TK) and were used to transduce the CEA-expressing pancreatic carcinoma cell line BXPC3. Recombinant pancreatic carcinoma cell lines expressing HSV-tk (BXPC3CEA-TK) were then tested for sensitivity to the toxic effects on ganciclovir after engraftment into severe combined immunodeficient mice. Tumors were generated by subcutaneous inoculation of 20 x 10(6) tumor cells consisting of BXPC3 and/or BXPC3CEA-TK cells in ratios of 100:0, 90:10, 50:50, 10:90, and 0:100. After 3 days mice received daily ganciclovir (0.1 mg/kg) or phosphate-buffered saline solution by intraperitoneal injection and were monitored for tumor growth. RESULTS: All severe combined immunodeficient mice inoculated with BXPC3 or BXPC3CEA-TK cells in any proportion developed large pancreatic tumors. As expected, a significant reduction in tumor size was seen in the BXPC3CEA-TK engrafted mice receiving ganciclovir compared with mice receiving phosphate-buffered saline solution or mice engrafted with only BXPC3. In addition, all animals with any fraction of cells expressing HSV-tk exhibited a significant reduction in tumor growth, including animals with only 10% of cells expressing HSV-tk. CONCLUSIONS: These results suggest the potential utility of directed enzyme pro-drug therapy in patients with CEA-expressing pancreatic carcinoma.

Gene delivery systems in surgery.

Increased understanding of the genetic basis of human disease has led to a number of potential gene-based therapies for various medical and surgical disorders. The development of efficient methods for delivering genes to mammalian cells in vitro has increased the potential clinical utility of gene-based therapies; however, a major focus of research has been more efficient delivery to appropriate target cells, in vivo as well as in vitro, to establish gene therapy as an effective clinical modality for common disorders. Despite substantial progress, a number of critical technical issues to enhance and optimize not only gene transfer but also gene expression must be resolved. These future technological developments will be essential for the widespread clinical implementation of gene-based therapy.

Indications for using video-assisted thoracoscopic surgery to diagnose diaphragmatic injuries after penetrating chest trauma.

BACKGROUND: Video-assisted thoracoscopic surgery (VATS) has been shown to be an accurate method for identifying diaphragmatic injuries (DIs). The purpose of this investigation was to establish specific indications for the use of VATS after penetrating chest trauma. METHODS: A retrospective review of all patients undergoing VATS after penetrating chest trauma at a level 1 trauma center over an 8-year period was performed. Logistic regression was used in an attempt to identify independent predictors of DI. RESULTS: One hundred seventy-one patients underwent VATS assessment of a hemidiaphragm, and 60 patients (35%) were found to have a DI. Five independent risk factors for DI were identified from analyzing the patient records: abnormal chest radiograph, associated intraabdominal injuries, high-velocity mechanism of injury, entrance wound inferior to the nipple line or scapula, and right-sided entrance wound. CONCLUSIONS: In the largest published series of patients undergoing VATS to exclude a DI, this review identifies five independent predictors of DI after penetrating chest trauma. A diagnostic algorithm incorporating these five factors was designed with the goal of reducing the number of unrecognized DIs after penetrating chest trauma by using VATS for patients at greatest risk for such injuries.

The thoracic surgeon's role in the management of patients with HIV infection and AIDS.

The advent of improved medical therapy with multidrug HIV medicines including protease inhibitors has prolonged life expectation of patients with HIV infection. The risk of perinatal transmission has also decreased with education and antiviral medications. Education of all groups about precautions to decrease HIV transmission appears to have resulted in a decline in the disease incidence; however, this trend has not been mirrored in other countries. All of these factors combine to increase the probability that the surgeon's role to take care of patients with HIV-related illnesses could be significant in the future. The thoracic surgeon may be called upon to assist in the diagnosis and treatment of HIV-infected patients with thoracic complications including pulmonary, cardiac, and esophageal problems. The morbidity and mortality of procedures performed on these patients appear to be no different than patients without HIV. We use very aggressive treatment strategies in patients with AIDS and HIV infections. The use of VATS, especially in the treatment of pneumothoraces and empyemas, should be used and has been shown to be safe and efficacious.

T cells or active Epstein-Barr virus infection in the development of lymphoproliferative disease in human B cell-injected severe combined immunodeficient mice.

BACKGROUND: Severe combined immunodeficient (SCID) mice develop Epstein-Barr virus (EBV) containing human lymphoproliferative disease (LPD) tumors when reconstituted with human peripheral blood leukocytes (PBLs) from EBV-seropositive donors, but LPD tumors do not develop in the presence of immunosuppressive agents, such as cyclosporine A or corticosteroids. METHODS: Therefore, LPD development in SCID mice was used as a model to explore the relationship among B cells, T cells, and EBV in vivo. SCID mice were engrafted with PBLs isolated by leukapheresis from a single EBV-seropositive donor. Purified populations of CD3+ lymphocytes (T cells) or CD19+ lymphocytes (B cells) were isolated and engrafted into SCID mice. RESULTS: SCID mice engrafted with purified CD3+ lymphocytes (T cells) or CD19+ lymphocytes (B cells) did not develop LPD. In contrast, mice engrafted with purified B cells developed LPD if they were co-engrafted with purified T cells or if they were inoculated with infectious EBV. CONCLUSIONS: This study confirms the requirement of T cells or active EBV infection in the development of LPD in animals engrafted with B cells latently infected with EBV. A greater understanding of the cellular and viral interactions leading to transformation and malignancy may allow the development of specific interventional therapies for malignancies in the immunosuppressed host.

p21 is essential for normal myogenic progenitor cell function in regenerating skeletal muscle.

Despite the ability of myogenic progenitor cells (MPCs) to completely regenerate skeletal muscle following injury, little is known regarding the molecular program that regulates their proliferation and differentiation. Although mice lacking the cyclin-dependent kinase inhibitor p21 (p21-/-), develop normally, we report here that p21-/- MPCs display increased cell number and enhanced cell cycle progression compared with wild-type MPCs. Therefore, we hypothesized that p21-/- mice would demonstrate temporally enhanced regeneration following myotrauma. In response to cardiotoxin-induced injury, p21-/- skeletal muscle regeneration was significantly attenuated vs. regenerating wild-type muscle, contrary to the hypothesis. Regenerating p21-/- skeletal muscle displayed increased proliferative (PCNA positive) nuclei coincident with increased apoptotic nuclei (TUNEL positive) compared with wild-type muscle up to 3 wk after injury. Differentiation of p21-/- MPCs was markedly impaired and associated with increased apoptosis compared with wild-type MPCs, confirming that the impaired differentiation of the p21-/- MPCs was a cell autonomous event. No dysregulation of p27, p53, or p57 protein expression in differentiating p21-/- MPCs compared with wild-type MPCs was observed, suggesting that other compensatory mechanisms are responsible for the regeneration that ultimately occurs. On the basis of these findings, we propose that p21 is essential for the coordination of cell cycle exit and differentiation in the adult MPC population and that in the absence of p21, skeletal muscle regeneration is markedly impaired.

Phenotypic and functional consequences of herpesvirus saimiri infection of human CD8+ cytotoxic T lymphocytes.

Herpesvirus saimiri (HVS) was used to infect and transform human CD8+ cytotoxic T lymphocytes (CTL), and the phenotypic and functional consequences of HVS infection of CD8+ T lymphocytes were investigated. HVS-transformed CTL no longer require antigen restimulation yet maintain their phenotype and HLA-restricted cytolytic function and specificity. The ability of HVS to transform CTL may have an important role in the functional analysis of human antigen-specific CTL.

Generation of tumor-specific T lymphocytes for the treatment of posttransplant lymphoma.

BACKGROUND: The incidence of lymphoproliferative disease, including B-cell lymphomas (BCL) in patients who have undergone heart or combined heart-lung transplants, has been reported to be as high as 15%. The majority of these tumors contain Epstein-Barr virus (EBV) DNA and regress when immunosuppressive agents are discontinued. This tumor regression is thought to be secondary to cytotoxic T lymphocytes (CTL) reactive to EBV-infected cells whose function is impaired in patients receiving immunosuppressive agents. We hypothesize that EBV-CTL expanded in the absence of these agents may demonstrate an antitumor effect against an EBV-expressing human BCL in vitro and in vivo. METHODS AND RESULTS: An EBV-expressing BCL from a heart transplant recipient was isolated and expanded in culture. EBV-CTL were generated by stimulation of peripheral blood leukocytes with irradiated autologous tumor cells in low-dose interleukin-2. Autologous BCL, HLA-mismatched BCL, lymphokine-activated killer target cell line (Daudi), and the natural killer target cell line (K562) were used in a standard 4-hour cytotoxicity assay using 51CrO4 after 7, 14, and 28 days of stimulation. There was significant percent specific lysis of autologous BCL targets (78%) at an effector-to-target ratio as low as 20:1 as compared with control cells. EBV-CTL were then adoptively transferred into SCID mice (provided by Duke University Vivarium) that had been engrafted with autologous BCL 7 days before. There was a significant survival advantage to those mice engrafted with EBV-CTL as compared with control cells. CONCLUSIONS: The results indicate that ex vivo expansion of EBV-CTL in the absence of immunosuppressive agents results in a population that has significant antitumor activity. This strategy may be useful in the generation of EBV-CTL that might be effective antitumor agents in transplant recipients with EBV-associated lymphomas.

Activation of MEF2 by muscle activity is mediated through a calcineurin-dependent pathway.

Gene expression in skeletal muscles of adult vertebrates is altered profoundly by changing patterns of contractile work. Here we observed that the functional activity of MEF2 transcription factors is stimulated by sustained periods of endurance exercise or motor nerve pacing, as assessed by expression in trans genic mice of a MEF2-dependent reporter gene (desMEF2-lacZ). This response is accompanied by transformation of specialized myofiber subtypes, and is blocked either by cyclosporin A, a specific chemical inhibitor of calcineurin, or by forced expression of the endogenous calcineurin inhibitory protein, myocyte-enriched calcineurin interacting protein 1. Calcineurin removes phosphate groups from MEF2, and augments the potency of the transcriptional activation domain of MEF2 fused to a heterologous DNA binding domain. Across a broad range, the enzymatic activity of calcineurin correlates directly with expression of endogenous genes that are transcriptionally activated by muscle contractions. These results delineate a molecular pathway in which calcineurin and MEF2 participate in the adaptive mechanisms by which skeletal myofibers acquire specialized contractile and metabolic properties as a function of changing patterns of muscle contraction.