Ischemia of the lung causes extensive long-term pulmonary injury: an experimental study

Background: Lung ischemia-reperfusion injury (LIRI) is suggested to be a major risk factor for development of primary acute graft failure (PAGF) following lung transplantation, although other factors have been found to interplay with LIRI. The question whether LIRI exclusively results in PAGF seems difficult to answer, which is partly due to the lack of a long-term experimental LIRI model, in which PAGF changes can be studied. In addition, the long-term effects of LIRI are unclear and a detailed description of the immunological changes over time after LIRI is missing. Therefore our purpose was to establish a long-term experimental model of LIRI, and to study the impact of LIRI on the development of PAGF, using a broad spectrum of LIRI parameters including leukocyte kinetics.Methods: Male Sprague-Dawley rats (n = 135) were subjected to 120 minutes of left lung warm ischemia or were sham-operated. A third group served as healthy controls. Animals were sacrificed 1, 3, 7, 30 or 90 days after surgery. Blood gas values, lung compliance, surfactant conversion, capillary permeability, and the presence of MMP-2 and MMP-9 in broncho-alveolar-lavage flui

Lymphocyte Modulation with FTY720 Improves Hemorrhagic Shock Survival in Swine

The inflammatory response to severe traumatic injury results in significant morbidity and mortality. Lymphocytes have recently been identified as critical mediators of the early innate immune response to ischemia-reperfusion injury. Experimental manipulation of lymphocytes following hemorrhagic shock may prevent secondary immunologic injury in surgical and trauma patients. The objective of this study is to evaluate the lymphocyte sequestration agent FTY720 as an immunomodulator following experimental hemorrhagic shock in a swine liver injury model. Yorkshire swine were anesthetized and underwent a grade III liver injury with uncontrolled hemorrhage to induce hemorrhagic shock. Experimental groups were treated with a lymphocyte sequestration agent, FTY720, (n = 9) and compared to a vehicle control group (n = 9). Animals were observed over a 3 day survival period after hemorrhage. Circulating total leukocyte and neutrophil counts were measured. Central lymphocytes were evaluated with mesenteric lymph node and spleen immunohistochemistry (IHC) staining for CD3. Lung tissue infiltrating neutrophils were analyzed with myeloperoxidase (MPO) IHC staining. Relevant immune-related gene expression from liver tissue was quantified using RT-PCR. The overall survival was 22.2% in the vehicle control and 66.7% in the FTY720 groups (p = 0.081), and reperfusion survival (period after hemorrhage) was 25% in the vehicle control and 75% in the FTY720 groups (p = 0.047). CD3+ lymphocytes were significantly increased in mesenteric lymph nodes and spleen in the FTY720 group compared to vehicle control, indicating central lymphocyte sequestration. Lymphocyte disruption significantly decreased circulating and lung tissue infiltrating neutrophils, and decreased expression of liver immune-related gene expression in the FTY720 treated group. There were no observed infectious or wound healing complications. Lymphocyte sequestration with FTY720 improves survival in experimental hemorrhagic shock using a porcine liver injury model. These results support a novel and clinically relevant lymphocyte immunomodulation strategy to ameliorate secondary immune injury in hemorrhagic shock

Acute Liver Injury Is Independent of B Cells or Immunoglobulin M

Acute liver injury is a clinically important pathology and results in the release of Danger Associated Molecular Patterns, which initiate an immune response. Withdrawal of the injurious agent and curtailing any pathogenic secondary immune response may allow spontaneous resolution of injury. The role B cells and Immunoglobulin M (IgM) play in acute liver injury is largely unknown and it was proposed that B cells and/or IgM would play a significant role in its pathogenesis.Tissue from 3 models of experimental liver injury (ischemia-reperfusion injury, concanavalin A hepatitis and paracetamol-induced liver injury) and patients transplanted following paracetamol overdose were stained for evidence of IgM deposition. Mice deficient in B cells (and IgM) were used to dissect out the role B cells and/or IgM played in the development or resolution of injury. Serum transfer into mice lacking IgM was used to establish the role IgM plays in injury.Significant deposition of IgM was seen in the explanted livers of patients transplanted following paracetamol overdose as well as in 3 experimental models of acute liver injury (ischemia-reperfusion injury, concanavalin A hepatitis and paracetamol-induced liver injury). Serum transfer into IgM-deficient mice failed to reconstitute injury (p = 0.66), despite successful engraftment of IgM. Mice deficient in both T and B cells (RAG1-/-) mice (p<0.001), but not B cell deficient (μMT) mice (p = 0.93), were significantly protected from injury. Further interrogation with T cell deficient (CD3εKO) mice confirmed that the T cell component is a key mediator of sterile liver injury. Mice deficient in B cells and IgM mice did not have a significant delay in resolution following acute liver injury.IgM deposition appears to be common feature of both human and murine sterile liver injury. However, neither IgM nor B cells, play a significant role in the development of or resolution from acute liver injury. T cells appear to be key mediators of injury. In conclusion, the therapeutic targeting of IgM or B cells (e.g. with Rituximab) would have limited benefit in protecting patients from acute liver injury

Reactive oxygen species in oncogenic transformation

Ever since ROS (reactive oxygen species) were shown to meet the criteria of true signalling molecules, such as regulated production and a specific biological function, many efforts have been made to understand the precise role of ROS. The function of ROS in pathological mechanisms is taking a more and more central role in various fields of biomedical research, including neurobiology, cardiology and cancer. An elevated oxidative status has been found in many types of cancer cells, and the introduction of chemical and enzymological antioxidants can inhibit tumour cell proliferation, pointing to a critical role of ROS in mediating loss of growth control. The present review describes ROS-regulated mechanisms that are associated with cancer and tumour invasiveness. The cellular processes that are linked to these ROS functions are mitogenic signalling and cell motility, while ROS have also been implicated in apoptosis and cellular senescence, two mechanisms regarded as being anti-tumorigenic. This ‘two-faced’ character of free radicals will be discussed and placed in the context of the physiological conditions of the tumour cell, the different molecular backgrounds, and the specific ROS. More detailed understanding of the signalling pathways regulated by ROS in tumour cells will open up new prospects for chemo- or gene-therapeutic interventions.</jats:p

Reactive oxygen species in oncogenic transformation

Ever since ROS (reactive oxygen species) were shown to meet the criteria of true signalling molecules, such as regulated production and a specific biological function, many efforts have been made to understand the precise role of ROS. The function of ROS in pathological mechanisms is taking a more and more central role in various fields of biomedical research, including neurobiology, cardiology and cancer. An elevated oxidative status has been found in many types of cancer cells, and the introduction of chemical and enzymological antioxidants can inhibit tumour cell proliferation, pointing to a critical role of ROS in mediating loss of growth control. The present review describes ROS-regulated mechanisms that are associated with cancer and tumour invasiveness. The cellular processes that are linked to these ROS functions are mitogenic signalling and cell motility, while ROS have also been implicated in apoptosis and cellular senescence, two mechanisms regarded as being anti-tumorigenic. This 'two-faced' character of free radicals will be discussed and placed in the context of the physiological conditions of the tumour cell, the different molecular backgrounds, and the specific ROS. More detailed understanding of the signalling pathways regulated by ROS in tumour cells will open up new prospects for chemo- or gene-therapeutic interventions

GENE THERAPY FOR COLON CANCER

The enormous number of newly diagnosed cases of colorectal cancer that occur each year and the lack of agents that are highly effective for all patients underscore the need for novel approaches to combating the disease. Gene therapy as a developing treatment modality is already well established, with a number of trials ongoing and a vast range of other approaches being assessed in animal and cell culture experiments. In this brief review, we have discussed five gene therapy trials in colon carcinoma that are ongoing or in the approval process in the United States. The gene therapy approaches being employed can be divided into three major categories: (1) enzyme/prodrug systems (HSVtk/ganciclovir; CD/5-fluorocytosine); (2) tumor suppressor gene replacement therapy with wild-type p53; and (3) immune-gene therapy which is based on cytokine or tumor antigen expression to induce tumor immunity (e.g., CEA). Replication-deficient recombinant adenoviral vectors are predominantly used for colon cancer gene therapy, because they can be produced at high titer and they readily infect a number of different cell types. One trial uses polynucleotide therapy for antitumor immunization with intramuscular injection. All of these studies are phase I trials, principally designed to evaluate safety, but they will also provide data on gene delivery. Some trials may provide some insight into potential therapeutic effects. We have alluded to some of the concerns on toxicity related to the use of adenovirus, risks and side effects from transgenes, lack of tumor-specificity of transgene expression, and potential problems with efficient gene delivery to solid tumors. The clinical trials, however, will provide insight that will inform design of future studies with respect to dose, form, and frequency of administration, as well as to the value of biologic and clinical endpoints. The molecular analysis of the fundamental basis of colon cancer has moved at a remarkable pace and that progress seems set to continue. Thus, the basic foundations for gene therapy are undoubtedly in place: a clinical need; growing understanding of basic tumor biology; and ever-improving delivery systems. The field is at a very early stage in its evolution, and one concern is that the considerable hurdles that must be overcome are seen as examples of the failure of cancer gene therapy; however, we believe these challenges will be overcome. The authors also believe that colon cancer gene therapy is likely to take new directions, such as use as adjuvant to radical surgery, rather than attempts to treat end-stage disease when the liver is replaced by metastases. Other new directions might include prophylactic gene-based immunization against a panel of well-characterized tumor antigens, at least for persons shown to be at high risk of colon cancer because of genetic or other predisposition. A marriage between gene therapy approaches and conventional anticancer treatments such as radiotherapy and chemotherapy also seems likely. There is already evidence of this move with demonstration of synergism between p53 replacement and radiotherapy and chemotherapy. It is also likely that therapies will be developed that combine elements from the cancer gene therapies discussed previously, namely, suicide gene transfer, immune modulation, and modulation of defective cancer genes. Perhaps one of the main concerns is not that researchers in cancer gene therapy want to walk before they can run, but that the public and government agencies believe they can. The next 10 years will be an interesting time in the development of novel treatments against colon cancer

NF-?B kinetics predetermine TNF-? sensitivity of colorectal cancer cells

BackgroundTumour necrosis factor (TNF)-alpha has considerable anti-tumour activity and may have potential as a treatment for metastatic colorectal cancer. However, TNF-alpha responses in patients and cell lines are variable and TNF-alpha treatment is associated with dose limiting clinical toxicity. Activation of NF-kappaB is protective against TNF-alpha induced cell death, and this may explain tumour resistance.MethodsIn order to provide further understanding of determinants of TNF-alpha responses, we studied TNF-alpha induced NF-kappaB activation and variable tumour responses. We analysed the kinetics of TNF-alpha induced NF-kappaB activation in colorectal cancer cells and determined whether it is possible to sensitize colorectal tumour cells to TNF-alpha by modulation of NF-kappaB signalling.ResultsWe demonstrated that sustained NF-kappaB activation exceeding 16 h was observed in HRT18 and SW480 cells and was associated with TNF-alpha resistance. In contrast, transient NF-kappaB activation in HCT116 cells was associated with sensitivity to cytotoxic TNF-alpha effects, suggesting that NF-kappaB kinetics may have utility as clinical marker of TNF-alpha tumour resistance. Despite variable TNF-alpha responses and NF-kappaB kinetics, all three colorectal cancer cell lines were highly sensitive to treatment with the TNF-related apoptosis-inducing ligand (TRAIL) which induced only transient NF-kappaB activation. This further supports the notion of a pre-determined NF-kappaB response influencing receptor-mediated cell death. We also show that stable transfection and adenoviral-mediated expression of IkappaB(A32/36) can be used to confer TNF-alpha sensitivity to colorectal tumour cells previously resistant.ConclusionsThese findings indicate that a combined approach using gene therapy and recombinant TNF-alpha merits further appraisal. Furthermore, the kinetics of the TNF-alpha response could be determined using a 'test-dose' to indicate whether individual patients might benefit from this gene therapy approach

AAV-encoded expression of TRAIL in experimental human colorectal cancer leads to tumor regression

Gene transfer vectors based on the adeno-associated virus (AAV) are used for various experimental and clinical therapeutic approaches. In the present study, we demonstrate the utility of rAAV as a tumoricidal agent in human colorectal cancer. We constructed an rAAV vector that expresses tumor necrosis factor (TNF)-related apoptosis-inducing figand (TRAIL/Apo2L) and used it to transduce human colorectal cancer cells. TRAIL belongs to the TNF superfamily of cytokines that are involved in various immune responses and apoptotic processes. It has been shown to induce cell death specifically in cancer cells. Transduction with AAV.TRAIL gave rise to rapid expression of TRAIL, followed by induction of apoptosis, which could be inhibited by the caspase inhibitor z-VAD.fmk, in several human colon cancer cell lines. The apoptotic mechanism included activation of caspase-3, as well as cytochrome c release from mitochondria. The outgrowth of human colorectal tumors grown in mice was completely blocked by transduction with AAV.TRAIL in vitro, while in vivo transduction significantly inhibited the growth of established tumors. AAV vectors could provide a safe method of gene delivery and offer a novel method of using TRAIL as a therapeutic protein. © 2004 Nature Publishing Group All rights reserved