Nox2 Is a Mediator of Ischemia Reperfusion Injury

Delayed graft function (DGF) results from ischemia‐reperfusion injury (IRI) and the generation of reactive oxygen species. We hypothesized that NADPH oxidase 2 (Nox2) plays an important role in pathways leading to DGF. We tested this hypothesis in vitro, in an animal model of IRI using wild type and Nox2^?/? mice, and in patients with DGF. Under hypoxic conditions, primary tubular epithelial cells from Nox2^?/? mice had reduced expression of MMP2, vimentin, and HSP27. BUN and creatinine levels were significantly increased in both Nox2^?/? and WT mice at 4 weeks and 6 months after IRI, suggesting the development of acute and chronic kidney injury. At 4 weeks, kidney fibrosis (α‐SMA, picrosirius) and oxidative stress (dihydroethidine, HNE) were significantly reduced in Nox2^?/? mice, confirming the oxidative and pro‐fibrotic effects of Nox2. The molecular signature of IRI using genomic analyses demonstrated a significant decline in hypoxia reponse, oxidative stress, fibrosis, and inflammation in Nox2^?/? mice. Immunohistochemical analyses of pre‐implanatation kidney allograft biopsies from patients with subsequent DGF showed significantly greater Nox2 levels and vascular injury compared with patients without DGF. These studies demonstrate that Nox2 is a modulator of IRI and its absence is associated with reduced inflammation, OS, and fibrosis.

     

An Extraperitoneal Technique for Murine Heterotopic Cardiac Transplantation

The mouse heterotopic cardiac transplantation model has been used extensively by investigators in the field of organ transplantation to study the rejection process, test new antirejection treatments, tolerance induction protocols or to understand basic immunological principles. Due to its extensive use, any small refinement of the technique would have a major impact on replacement, reduction and refinement (commonly known as the 3Rs). Here, we describe a novel approach to refine this model. The donor aorta and pulmonary artery are anastomosed peripherally to the femoral artery and vein of the recipient, respectively. The technical success rate is comparable to the conventional abdominal site, but it avoids a laparotomy and handling of the bowels making it less invasive method. As a result, recipients recover faster and require less postoperative analgesia. It is a major refinement under one of the 3Rs and would represent an advance in animal welfare in scientific research.     

CD4 T Cells but Not Th17 Cells Are Required for Mouse Lung Transplant Obliterative Bronchiolitis

Lung transplant survival is limited by obliterative bronchiolitis (OB), but the mechanisms of OB development are unknown. Previous studies in a mouse model of orthotopic lung transplantation suggested a requirement for IL‐17. We have used this orthotopic mouse model to investigate the source of IL‐17A and the requirement for T cells producing IL‐17A. The major sources of IL‐17A were CD4⁺ T cells and γδ T cells. Depletion of CD4⁺ T cells led to a significantly decreased frequency and number of IL‐17A⁺ lymphocytes and was sufficient to prevent acute rejection and OB. However, mice with STAT3‐deficient T cells, which are unable to differentiate into Th17 cells, rejected lung allografts and developed OB similar to control mice. The frequency of IL‐17A⁺ cells was not decreased in mice with STAT3‐deficient T cells due mainly to the presence of IL‐17A⁺ γδ T cells. Deficiency of γδ T cells also did not affect the development of airway fibrosis. Our data suggest that CD4⁺ T cells are required for OB development and expansion of IL‐17A responses in the lung, while Th17 and γδ T cells are not absolutely required and may compensate for each other.     

Imaging Tolerance Induction in the Classic Medawar Neonatal Mouse Model: Active Roles of Multiple F1‐Donor Cell Types

The immature immune system is uniquely susceptible to tolerance induction and thus an attractive target for immunomodulation strategies for organ transplantation. Newborn mice injected with adult semi‐allogeneic lymphohematopoietic cells accept transplants without immunosuppressive drugs. Early in vivo/in situ events leading to neonatal tolerance remain poorly understood. Here, we show by whole body/organ imaging that injected cells home to lymphoid organs and liver where various F1‐donor cell types selectively alter neonatal immunity. In host thymus, F1‐donor dendritic cells (DC) interact with developing thymocytes and regulatory T cells suggesting a role in negative selection. In spleen and lymph nodes, F1‐donor regulatory T/B cells associate with host alloreactive cells and by themselves prolong cardiac allograft survival. In liver, F1‐donor cells give rise to albumin‐containing hepatocyte‐like cells. The neonatal immune system is lymphopenic, Th‐2 immunodeviated and contains immature DC, suggesting susceptibility to regulation by adult F1‐donor cells. CD8a T cell inactivation greatly enhances chimerism, suggesting that variable emerging neonatal alloreactivity becomes a barrier to tolerance induction. This comprehensive qualitative imaging study systematically shows contribution of multiple in vivo processes leading simultaneously to robust tolerance. These insights into robust tolerance induction have important implications for development of strategies for clinical application.     

Exploiting the power of OMICS approaches to produce E. coli O157 vaccines

Enterohemorrhagic Escherichia coli (EHEC) strains are well-documented human pathogens and causative agents of diarrheal episodes and hemorrhagic colitis. The serotype O157:H7 is highly virulent and responsible for both outbreaks and sporadic cases of diarrhea. Because antibiotic treatment is contraindicated against this pathogen, development of a human vaccine could be an effective intervention in public health. In our recent Infection and Immunity paper, we applied integrated approaches of in silico genome wide search combined with bioinformatics tools to identify and test O157 vaccine candidates for their protective effect on a murine model of gastrointestinal infection. Using genomic/immunoinformatic approaches that are further described here, we categorized vaccine candidates as high, medium, and low priorities, and demonstrate that some high priority candidates were able to significantly induce Th2 cytokines and reduce EHEC colonization. Using the STRING database, we have recently evaluated the vaccine candidates and predict functional protein interactions, determining whether correlations exist for the development of a multi-subunit vaccine, targeting different pathways against EHEC O157:H7. The overall approach is designed to screen potential vaccine candidates against EHEC; however, the methodology can be quickly applied to many other intestinal pathogens.     

Markers of potential malignancy in chronic hyperplastic candidiasis

Aim: To examine the presence of markers associated with malignancy, including p53, p21 cyclin‐dependent kinase inhibitor 1A, murine double minutes‐2, and others, in chronic hyperplastic candidiasis. Methods: Immunohistochemical methods were used to examine the expression of p53, murine double minutes‐2, p21 cyclin‐dependent kinase inhibitor 1A, metallothionein, and proliferating cell nuclear antigen in 42 chronic hyperplastic candidiasis lesions and 11 non‐infected control tissues. Terminal deoxynucleotidyl transferase‐mediated digoxigenin‐dUTP nick‐end labeling was used to examine apoptosis, which was correlated with p53 expression. These markers were measured in lesions of chronic hyperplastic candidiasis that did not show any epithelial dysplasia or histological signs of malignancy. Results: p53 scores were higher in chronic hyperplastic candidiasis than in controls (P = 0.0046). Murine double‐minutes 2 levels were not elevated. p21 cyclin‐dependent kinase inhibitor 1A was increased in parabasal (P < 0.0001) and basal epithelial cells. Chronic hyperplastic candidiasis lesions showed a similar basal/parabasal metallothionein staining pattern to that seen in normal squamous epithelium. Proliferating cell nuclear antigen was increased (P = 0.0007), as was apoptosis (P = 0.0033). Conclusion: Increased p53 in oral chronic hyperplastic candidiasis suggests an increased potential for malignant change in the epithelium, above that of normal tissues. Further functional investigation is required, as well as clinical follow‐up studies.     

Deoxycytidine kinase promotes the migration and invasion of fibroblast-like synoviocytes from rheumatoid arthritis patients

Rheumatoid arthritis (RA) is a complex, multi-system disease whose primary site of inflammatory tissue damage is the joint. The increasing evidences indicate that activated RA fibroblast-like synoviocytes (FLS) play a critical role in the development of pannus by migrating into cartilage and bone. Furthermore FLS and T cells can activate each other in vitro and in vivo, which is crucial for the progress of RA. Deoxycytidine kinase (DCK) has been linked to peripheral T cell homeostatic proliferation and survival, which is very important for RA. Yet, the function of DCK in FLS is still unknown. Here, we present a story that DCK could regulate the migration and invasion of FLS through AKT pathway in RA patients. Moreover, DCK seems to be the upstream of AKT and FAK, and AKT inhibitor exerted the similar effect on FLS motility. In summary, our study characterized the new role of DCK in human primary FLS cells, and figured out the possible pathway DCK involved in, and these findings might propose DCK as a novel target for controlling joint destruction of RA.     

Use of Imatinib in the Prevention of Heterotopic Ossification

Background

Heterotopic ossification (HO) is a common complication following orthopedic and trauma surgery, which may have substantial negative effects on the postoperative outcome. Angiogenesis appears to play a critical role in heterotopic ossification. One of the involved signaling molecules is platelet-derived growth factor (PDGF) which may be inhibited by imatinib.

Questions/Purposes

Our goal was to prevent HO by pharmacologically interfering with the molecular signaling pathways involved in the developmental process. We hypothesized that by administering a proven inhibitor of PDGF expression, heterotopic bone formation may be prevented.

Methods

The effect of imatinib on HO formation was studied in a murine model which reliably produces islets of HO within the soft tissue following Achilles tenotomy. The control group underwent Achilles tenotomy only. The imatinib group received imatinib mesylate. After trial completion, the limbs were harvested and scanned by micro-CT. Heterotopic bone volume was then identified and quantified.

Results

The mean volume of heterotopic bone formed in the control group was 0.976mm³ compared to 0.221 mm³ in the imatinib group. The volume of HO in the treatment group was reduced by 85% compared to the control group.

Conclusions

The administration of imatinib was associated with a significantly reduced volume of HO. This may be due to the inhibitory effect of imatinib on the PDGF signaling pathway during development of HO.

Clinical Relevance

The successful reduction of HO formation following imatinib administration has led to further insight concerning the pathogenesis of HO which in the future may lead to new clinical approaches towards the prevention of HO.

Electronic supplementary material

The online version of this article (doi:10.1007/s11420-013-9335-y) contains supplementary material, which is available to authorized users.