Publications by authors named "Jonathan R Lamb"

51 Publications

Down-regulation of STAT3 enhanced chemokine expression and neutrophil recruitment in biliary atresia.

Clin Sci (Lond) 2021 Apr;135(7):865-884

Provincial Key Laboratory of Research in Structure Birth Defect Disease and Department of Pediatric Surgery, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou, Guangdong, China.

Biliary atresia (BA) is an immune-related disorder and signal transducer and activator of transcription 3 (STAT3) is a key signalling molecule in inflammation. The present study was designed to clarify the function of STAT3 in BA. STAT3 expression was examined in patients and a mouse BA model in which STAT3 levels were further altered with a specific inhibitor or activator. Neutrophil accumulation and the levels of the neutrophil chemoattractants (C-X-C motif) ligand 1 (CXCL1) and IL-8 were determined. The effects of STAT3 inhibition on IL-8 expression were examined in human biliary epithelial cell (BEC) cultures. Functional changes in liver STAT3+ neutrophils in the mouse model were analysed with 10× single cell RNA-seq methods. Results showed STAT3 and p-STAT3 expression was reduced in BA liver tissue compared with control samples. Administration of a STAT3 inhibitor increased jaundice and mortality and reduced body weight in BA mice. In contrast, the STAT3 activator ameliorated BA symptoms. Extensive neutrophil accumulation together with CXCL1 up-regulation, both of which were suppressed by an anti-CXCL1 antibody, were observed in the STAT3 inhibitor-treated group. Recombinant IL-8 administration increased disease severity in BA mice, and the STAT3 activator had the reverse effect. Inhibiting STAT3 increased apoptosis of human BECs together with up-regulated IL-8 expression. RNA-seq analysis revealed reduced the numbers of STAT3 expressing neutrophil in BA which was accompanied by marked enhanced interferon-related antiviral activities. In conclusion, STAT3 reduction, enhanced IL-8 and CXCL1 expression and promoted the accumulation of interferon-responsive neutrophils resulting in BEC damage in BA.
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http://dx.doi.org/10.1042/CS20201366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8035628PMC
April 2021

The Role of Neonatal Gr-1 Myeloid Cells in a Murine Model of Rhesus-Rotavirus-Induced Biliary Atresia.

Am J Pathol 2018 11 8;188(11):2617-2628. Epub 2018 Sep 8.

Department of Pediatric Surgery, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou, China; Department of Surgery, the University of Hong Kong, Hong Kong SAR, China. Electronic address:

Activation of innate immunity together with cholangiocyte damage occurs in biliary atresia (BA). However, detailed information on the inflammatory cells involved is lacking. This study investigates both the pathophysiology of CD11bGr-1 cells in a mouse model of BA and their presence in BA patients. CD11bGr-1 cells were targeted by an anti-Ly6G antibody in murine BA induced by inoculation with rhesus rotavirus. Expression of the Ly6G homolog CD177 was examined in biopsies from BA patients. The symptoms of BA were ameliorated, and survival was prolonged in those mice receiving 5 to 10 μg of antibody per mouse every 3 days for four times compared with the mice treated with virus alone. However, the mice later developed chronic BA with persistent low body weight and jaundice. Hepatic inflammatory cells were reduced compared with acute BA. Blockade of extrahepatic bile ducts occurred, whereas intrahepatic ductules were partially preserved, and a progressive increase in liver fibrosis was observed. High levels of CD11bGr-1 cells were present in these mice. The administration of an anti-Ly6G antibody again in those chronic BA mice reduced jaundice and restored body weight. In BA patients CD177 cells were highly expressed in the liver. Our data suggest that the chronic mouse BA model shares key characteristics with clinical BA and indicates the importance of CD11bGr-1 cells in the initiation and progression of BA.
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http://dx.doi.org/10.1016/j.ajpath.2018.07.024DOI Listing
November 2018

Downregulation of Hes1 expression in experimental biliary atresia and its effects on bile duct structure.

World J Gastroenterol 2018 Aug;24(29):3260-3272

Department of Pediatric Surgery, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou 510623, Guangdong Province, China.

Aim: To analyze the expression and function of the Notch signaling target gene Hes1 in a rhesus rotavirus-induced mouse biliary atresia model.

Methods: The morphologies of biliary epithelial cells in biliary atresia patients and in a mouse model were examined by immunohistochemical staining. Then, the differential expression of Notch signaling pathway-related molecules was investigated. Further, the effects of the siRNA-mediated inhibition of Hes1 expression were examined using a biliary epithelial cell 3D culture system.

Results: Both immature (EpCAM) and mature (CK19) biliary epithelial cells were detected in the livers of biliary atresia patients without a ductile structure and in the mouse model with a distorted bile duct structure. The hepatic expression of transcripts for most Notch signaling molecules were significantly reduced on day 7 but recovered to normal levels by day 14, except for the target molecule Hes1, which still exhibited lower mRNA and protein levels. Expression of the Hes1 transcriptional co-regulator, RBP-Jκ was also reduced. A 3D gel culture system promoted the maturation of immature biliary epithelial cells, with increased expression of CK19 cells and the formation of a duct-like structure. The administration of Hes1 siRNA blocked this process. As a result, the cells remained in an immature state, and no duct-like structure was observed.

Conclusion: Our data indicated that Hes1 might contribute to the maturation and the cellular structure organization of biliary epithelial cells, which provides new insight into understanding the pathology of biliary atresia.
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http://dx.doi.org/10.3748/wjg.v24.i29.3260DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6079292PMC
August 2018

Silver nanoparticle treatment ameliorates biliary atresia syndrome in rhesus rotavirus inoculated mice.

Nanomedicine 2017 04 25;13(3):1041-1050. Epub 2016 Nov 25.

Department of Pediatric Surgery, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou, China. Electronic address:

Biliary atresia (BA) is a neonatal biliary system disease closely associated with viral infection and bile duct inflammation. Silver nanoparticles (AgNps) have previously revealed antiviral and anti-inflammatory properties. In this study, we have investigated the effects of AgNps in the treatment of the Rhesus rotavirus inoculation induced BA in mice. The morphology, liver histopathology, clinical biochemistry examination, and inflammatory cells were analyzed in BA mice. Results indicated that AgNps could significantly increase the survival rate of BA mice, and reduce jaundice and weight lost and the liver enzymes and bilirubin metabolism clinical parameters were close to the normal levels. Diminished numbers of NK cells were observed by flow cytometry analysis and immunohistochemical staining. Furthermore, the viral load was reduced and transcripts for TGF-β mRNA were augmented after AgNps treatment. Collectively, our results suggest that AgNps treatment has beneficial effects on the BA mouse model partially through upregulation of TGF-β.
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http://dx.doi.org/10.1016/j.nano.2016.11.013DOI Listing
April 2017

Role of CD56-expressing immature biliary epithelial cells in biliary atresia.

World J Gastroenterol 2016 Feb;22(8):2545-57

Rui-Zhong Zhang, Jia-Kang Yu, Jiao Peng, Yan Chen, Hui-Min Xia, Department of Pediatric Surgery, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou 510623, Guangdong Province, China.

Aim: To analyze the clinical and pathological parameters and expression of the neural cell adhesion molecule (CD56) in patients with biliary atresia (BA).

Methods: Established clinical laboratory markers of hepatic function, including enzyme activity, protein synthesis, and bilirubin metabolism, were evaluated in patients with BA and compared with those in patients with choledochal cysts and neonatal hepatitis. Pathological changes in tissue morphology and fibrosis were examined by histological and tissue collagen staining. Immunohistochemical staining for the biliary epithelial cell markers CD56 and CK19 together with the Notch signaling related molecules Notch1 and Notch2 was performed in the context of alterations in the structure of intrahepatic biliary ducts.

Results: Differences in some clinical laboratory parameters among the three diseases examined were observed, but they did not correlate with the pathological classification of fibrosis in BA. Immunohistochemical staining showed the presence of CD56-positive immature bile ducts in most patients (74.5%) with BA but not in patients with choledochal cysts or neonatal hepatitis. The number of CD56-expressing cells correlated with disease severity, with more positive cells present in the later stages of liver damage (81.8% vs 18.2%). Furthermore, bile plugs were mainly found in CD56-positive immature biliary ducts. Notch signaling was a key regulatory pathway in biliary duct formation and played a role in tissue fibrosis. Notch1 was co-expressed in CD56-positive cells, whereas Notch2 was found exclusively in blood vessels in the portal area of patients with BA.

Conclusion: The maturation of biliary epithelial cells and the expression of Notch may play a role in the pathogenesis of BA.
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http://dx.doi.org/10.3748/wjg.v22.i8.2545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4768200PMC
February 2016

Mucosal inflammation at the respiratory interface: a zebrafish model.

Am J Physiol Lung Cell Mol Physiol 2016 Mar 30;310(6):L551-61. Epub 2015 Dec 30.

Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London, United Kingdom; and

Inflammatory diseases of the respiratory system such as asthma and chronic obstructive pulmonary disease are increasing globally and remain poorly understood conditions. Although attention has long focused on the activation of type 1 and type 2 helper T cells of the adaptive immune system in these diseases, it is becoming increasingly apparent that there is also a need to understand the contributions and interactions between innate immune cells and the epithelial lining of the respiratory system. Cigarette smoke predisposes the respiratory tissue to a higher incidence of inflammatory disease, and here we have used zebrafish gills as a model to study the effect of cigarette smoke on the respiratory epithelium. Zebrafish gills fulfill the same gas-exchange function as the mammalian airways and have a similar structure. Exposure to cigarette smoke extracts resulted in an increase in transcripts of the proinflammatory cytokines TNF-α, IL-1β, and MMP9 in the gill tissue, which was at least in part mediated via NF-κB activation. Longer term exposure of fish for 6 wk to cigarette smoke extract resulted in marked structural changes to the gills with lamellar fusion and mucus cell formation, while signs of inflammation or fibrosis were absent. This shows, for the first time, that zebrafish gills are a relevant model for studying the effect of inflammatory stimuli on a respiratory epithelium, since they mimic the immunopathology involved in respiratory inflammatory diseases of humans.
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http://dx.doi.org/10.1152/ajplung.00323.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4796261PMC
March 2016

Modulatory effects of adiponectin on the polarization of tumor-associated macrophages.

Int J Cancer 2015 Aug 4;137(4):848-58. Epub 2015 Mar 4.

Guangzhou Women and Children's Medical Center, Guangzhou, China.

The plasticity of macrophages with selective functional phenotypes partially arises in respective to their microenvironment. Tumor-associated macrophages (TAMs) may promote disease progression with tumor specific manner. Here we report that in pediatric malignant soft-tissue tumors, the presence of TAMs and expression of adiponectin (APN) are heterogeneous. Both APN and TAMs had high expression in rhabdomyosarcoma, especially in the malignant subtype, alveolar rhabdomyosarcoma. To investigate the mode of action of APN on TAM activation, a murine MN/MCA1 sarcoma model was used. The Results revealed that exogenous APN had no effect on MN/MCA1 proliferation but tumor size was markedly reduced in apn(-/-) mice versus WT controls. The accumulation of TAMs in apn(-/-) mice was also reduced which correlated to downregulated serum levels of MCP-1. Likewise, TAMs in apn(-/-) mice exhibited a M1-like phenotype, characterized by increase in MHC II(high) population and M1 phenotypic markers, such as iNOS gene and serum TNF-α accompanied by a decrease in M2 markers, namely YM1 gene and serum IL-10. In addition, APN deficiency increased the number of CD4(+) T cells, CD8(+) T cells and NK cells in tumors and reduced tumor metastasis. The altered phenotype of TAMs in apn(-/-) mice was associated with a marked decrease in phospho-p38 and treatment with a p38 MAPK inhibitor significantly reduced tumor size and increased MHC II expression on TAMs in WT mice, implying p38 MAPK signaling pathway may contribute to APN-mediated TAM polarization. Collectively, our findings suggest that APN may have a potential role in regulating soft tissue sarcoma growth.
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http://dx.doi.org/10.1002/ijc.29485DOI Listing
August 2015

Dietary cholesterol directly induces acute inflammasome-dependent intestinal inflammation.

Nat Commun 2014 Dec 23;5:5864. Epub 2014 Dec 23.

Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, Sir Alexander Fleming Building, London SW7 2AZ, UK.

Prolonged ingestion of a cholesterol- or saturated fatty acid-enriched diet induces chronic, often systemic, auto-inflammatory responses resulting in significant health problems worldwide. In vivo information regarding the local and direct inflammatory effect of these dietary components in the intestine and, in particular, on the intestinal epithelium is lacking. Here we report that both mice and zebrafish exposed to high-fat (HFDs) or high-cholesterol (HCDs) diets develop acute innate inflammatory responses within hours, reflected in the localized interleukin-1β-dependent accumulation of myeloid cells in the intestine. Acute HCD-induced intestinal inflammation is dependent on cholesterol uptake via Niemann-Pick C1-like 1 and inflammasome activation involving apoptosis-associated Speck-like protein containing a caspase recruitment domain, which leads to Caspase-1 activity in intestinal epithelial cells. Extended exposure to HCD results in localized, inflammation-dependent, functional dysregulation as well as systemic pathologies. Our model suggests that dietary cholesterol initiates intestinal inflammation in epithelial cells.
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http://dx.doi.org/10.1038/ncomms6864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4284652PMC
December 2014

Identification of altered dipeptidyl-peptidase activities as potential biomarkers for unipolar depression.

J Affect Disord 2013 Nov 31;151(2):667-672. Epub 2013 Jul 31.

Clinical Pharmacology Unit, GlaxoSmithKline, Addenbrooke's Hospital, Cambridge CB2 2GG, UK; Cambridge Centre for Neuropsychiatric Research, Department of Chemical Engineering and Biotechnology, Cambridge University, Tennis Court Road, Cambridge CB2 1QT, UK. Electronic address:

Background: Changes in circulatory aminopeptidases [dipeptidyl-peptidase-IV (DPP-IV), Prolyl-oligopeptidase (POP) and Leucine aminopeptidase (LAP)] activities have been found to be associated with psychiatric illnesses and inflammatory diseases.

Methods: The discriminatory indices of aminopeptidases activities were assessed by enzymatic assays in plasma samples from 240 unipolar depression (UD) patients and 264 matched controls. In addition the relationship between soluble and cellular DPP-IV activity was determined in plasma and blood cells from healthy subjects.

Results: Greater than 95% of the plasma DPP-IV activity could be blocked by inhibitors, demonstrating the specificity of the assay. Also, DPP-IV protein and activity levels were strongly correlated. In contrast, only 50% of the membrane-bound activity in blood cells was inhibited, which suggested that other similar peptidases may be present in these cells. UD patients had decreased plasma levels of DPP-IV and POP activities compared to healthy controls with a concomitant increase in LAP activity. Finally, testing of the LAP/DPP-IV ratio resulted in good discrimination of UD patients from controls with an area under the curve-receiver operating characteristic of 0.70.

Limitations: Further biological validation studies using different cohorts are warranted.

Conclusions: The finding that plasma DPP-IV activity was decreased and LAP activity was increased in UD patients suggests the potential value for testing the levels of these enzymes for improved classification of patients. In addition, the changes in these enzymes, suggests that the proteolytic maturation of their proneuropeptide and prohormone subtrates may also be affected in UD, resulting in altered production of the associated bioactive peptides.
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http://dx.doi.org/10.1016/j.jad.2013.07.015DOI Listing
November 2013

Macrophage migration is controlled by Tribbles 1 through the interaction between C/EBPβ and TNF-α.

Vet Immunol Immunopathol 2013 Sep 6;155(1-2):67-75. Epub 2013 Jun 6.

Royal (Dick) School of Veterinary Studies and Roslin Institute, The University of Edinburgh, Easter Bush, Midlothian EH25 9RG, UK.

In mammals, three Tribbles gene family members have been identified, Tribbles 1, 2 and 3 (Trib1, Trib2 and Trib3). All family members are considered to be pseudokinases in that they contain domains homologous to serine/threonine kinase catalytic cores, but they lack several conserved residues in the ATP-binding pocket. Trib1 is implicated in the inflammatory response pathway through its ability to regulate mitogen-activated protein kinase (MAPK), nuclear factor kappa B (NF-κB) and CCAAT Enhancer Binding Protein (C/EBP). However, its role in macrophages function is unknown. Here, we investigated the functional role of Trib1 in Toll-like receptor-mediated inflammatory responses to IFN-γ in RAW264.7 cells. In gene knock-down experiments in macrophages using small interfering RNAs targeted to Trib1, it was observed that TNF-α production was increased following treatment with IFN-γ and/or TLR2 ligands. Finally, Trib1-silenced macrophages failed to show MCP-1 induced chemokinesis and indicating involvement of Trib1 in controlling of macrophage migration. This work demonstrates that Trib1 contributes to the pro-inflammatory response caused by TLR2 ligands and controls macrophage migration as well as being a biomarker in macrophage-related diseases in both human and veterinary medicine.
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http://dx.doi.org/10.1016/j.vetimm.2013.06.001DOI Listing
September 2013

P38 and JNK have opposing effects on persistence of in vivo leukocyte migration in zebrafish.

Immunol Cell Biol 2013 Jan 20;91(1):60-9. Epub 2012 Nov 20.

Department of Life Sciences, Division of Cell and Molecular Biology, Imperial College London, London, UK.

The recruitment and migration of macrophages and neutrophils is an important process during the early stages of the innate immune system in response to acute injury. Transgenic pu.1:EGFP zebrafish permit the acquisition of leukocyte migration trajectories during inflammation. Currently, these high-quality live-imaging data are mainly analysed using general statistics, for example, cell velocity. Here, we present a spatio-temporal analysis of the cell dynamics using transition matrices, which provide information of the type of cell migration. We find evidence that leukocytes exhibit types of migratory behaviour, which differ from previously described random walk processes. Dimethyl sulfoxide treatment decreased the level of persistence at early time points after wounding and ablated temporal dependencies observed in untreated embryos. We then use pharmacological inhibition of p38 and c-Jun N-terminal kinase mitogen-activated protein kinases to determine their effects on in vivo leukocyte migration patterns and discuss how they modify the characteristics of the cell migration process. In particular, we find that their respective inhibition leads to decreased and increased levels of persistent motion in leukocytes following wounding. This example shows the high level of information content, which can be gained from live-imaging data if appropriate statistical tools are used.
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http://dx.doi.org/10.1038/icb.2012.57DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3540327PMC
January 2013

Adiponectin mediated MHC class II mismatched cardiac graft rejection in mice is IL-4 dependent.

PLoS One 2012 14;7(11):e48893. Epub 2012 Nov 14.

Paediatric Surgery Division, Department of Surgery, The University of Hong Kong, Hong Kong SAR, China.

Background: Adiponectin regulates glucose and fatty-acid metabolism but its role in chronic graft rejection mediated by Th2 cytokines remains ill-defined.

Methodology/principal Findings: Wild type and adiponectin-null mice were used as graft recipients in mouse MHC class II disparate cardiac transplantation (bm12 toB6) and the graft rejection was monitored. In adiponectin-null mice we observed that the cellular infiltrate of eosinophils, CD4(+) and CD8(+) T cells was reduced in grafts compared to the controls as was collagen deposition and vessel occlusion. A similar outcome was observed for skin transplants except that neutrophil infiltration was increased. Low levels of IL-4 were detected in the grafts and serum. The effect of adiponectin signaling on IL-4 expression was further investigated. Treatment with AMPK and p38 MAPK inhibitors blocked adiponectin enhanced T cell proliferation in mixed lymphocyte reactions. Inhibition of AMPK reduced eosinophil infiltration in skin grafts in wild type recipients and in contrast AMPK activation increased eosinophils in adiponectin-null recipients. The addition of adiponectin increased IL-4 production by the T cell line EL4 with augmented nuclear GATA-3 and phospho-STAT6 expression which were suppressed by knockdown of adiponectin receptor 1 and 2.

Conclusions: Our results demonstrate a direct effect of adiponectin on IL-4 expression which contributes to Th2 cytokine mediated rejection in mouse MHC class II histoincompatible transplants. These results add to our understanding of the interrelationship of metabolism and immune regulation and raise the possibility that AMPK inhibitors may be beneficial in selected types of rejection.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0048893PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3498365PMC
May 2013

The role of Nfil3 in zebrafish hematopoiesis.

Dev Comp Immunol 2012 Sep 2;38(1):187-92. Epub 2012 May 2.

Division of Cell and Molecular Biology, Department of Life Sciences, Imperial College London, London, UK.

Nfil3, a transcription factor that has an array of functions in immune cells, has been described as key regulator of CD8α(+) dendritic cell and natural killer cell development in mice. In this report we show that Nfil3 is enriched in the myeloid compartment of adult zebrafish including eosinophils. Knockdown of Nfil3 in pu.1:GFP embryos resulted in a reduced number of myeloid cells as early as 24h post-fertilization, while erythropoiesis was unaffected. Using mpx and fms-fluorescent transgenic fish we found that all myeloid cell lineages, and in particular macrophages, had reduced numbers at 4days post-fertilization. This was reflected by less myeloid cells accumulating at a wound site. Pu.1, l-plastin, csf1r and mpx had reduced expression in Nfil3 morphants while runx1, gata1 and rag1 were unaffected. Collectively, these results describe a conserved expression pattern of Nfil3 in evolutionarily divergent species and indicate that Nfil3 is central to myeloid lineage commitment.
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http://dx.doi.org/10.1016/j.dci.2012.04.008DOI Listing
September 2012

Incorporation of an experimentally determined MTF for spatial frequency filtering and deconvolution during optical projection tomography reconstruction.

Opt Express 2012 Mar;20(7):7323-37

Photonics Group, Department of Physics, Imperial College London, SW7 2AZ, UK.

We demonstrate two techniques to improve the quality of reconstructed optical projection tomography (OPT) images using the modulation transfer function (MTF) as a function of defocus experimentally determined from tilted knife-edge measurements. The first employs a 2-D binary filter based on the MTF frequency cut-off as an additional filter during back-projection reconstruction that restricts the high frequency information to the region around the focal plane and progressively decreases the spatial frequency bandwidth with defocus. This helps to suppress "streak" artifacts in OPT data acquired at reduced angular sampling, thereby facilitating faster OPT acquisitions. This method is shown to reduce the average background by approximately 72% for an NA of 0.09 and by approximately 38% for an NA of 0.07 compared to standard filtered back-projection. As a biological illustration, a Fli:GFP transgenic zebrafish embryo (3 days post-fertilisation) was imaged to demonstrate the improved imaging speed (a quarter of the acquisition time). The second method uses the MTF to produce an appropriate deconvolution filter that can be used to correct for the spatial frequency modulation applied by the imaging system.
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http://dx.doi.org/10.1364/OE.20.007323DOI Listing
March 2012

Calibrating spatio-temporal models of leukocyte dynamics against in vivo live-imaging data using approximate Bayesian computation.

Integr Biol (Camb) 2012 Mar 10;4(3):335-345. Epub 2012 Feb 10.

Centre for Integrative Systems Biology and Bioinformatics, Division of Molecular Biosciences, Department of Life Sciences, Imperial College London, London, UK.

In vivo studies allow us to investigate biological processes at the level of the organism. But not all aspects of in vivo systems are amenable to direct experimental measurements. In order to make the most of such data we therefore require statistical tools that allow us to obtain reliable estimates for e.g. kinetic in vivo parameters. Here we show how we can use approximate Bayesian computation approaches in order to analyse leukocyte migration in zebrafish embryos in response to injuries. We track individual leukocytes using live imaging following surgical injury to the embryos' tail-fins. The signalling gradient that leukocytes follow towards the site of the injury cannot be directly measured but we can estimate its shape and how it changes with time from the directly observed patterns of leukocyte migration. By coupling simple models of immune signalling and leukocyte migration with the unknown gradient shape into a single statistical framework we can gain detailed insights into the tissue-wide processes that are involved in the innate immune response to wound injury. In particular we find conclusive evidence for a temporally and spatially changing signalling gradient that modulates the changing activity of the leukocyte population in the embryos. We conclude with a robustness analysis which highlights the most important factors determining the leukocyte dynamics. Our approach relies only on the ability to simulate numerically the process under investigation and is therefore also applicable in other in vivo contexts and studies.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5058438PMC
http://dx.doi.org/10.1039/c2ib00175fDOI Listing
March 2012

Effects of water uptake on melamine renal stone formation in mice.

Nephrol Dial Transplant 2012 Jun 10;27(6):2225-31. Epub 2011 Oct 10.

Department of Surgery, LKS Faculty of Medicine, the University of Hong Kong, Hong Kong, China.

Background: Melamine-tainted food can induce kidney stones both in humans and animals and in domestic animals, severe cases caused acute kidney failure and death. Although increasing water intake can ameliorate kidney stone formation, its effect on melamine (Mel)-induced kidney stones has not been studied.

Methods: We have analysed the effect of restricted ingestion of drinking water on melamine stone formation in mice. They were given melamine and cyanuric acid orally and received drinking water either freely or for a restricted time. Kidney stone formation and renal function were monitored.

Results: Mice receiving drinking water for a restricted 10-h period initially lost body weight, which returned to normal within 2 days. No other abnormalities were observed. Ingestion of melamine alone failed to induce kidney stones even under conditions of restricted drinking water. In mice treated with melamine together with cyanuric acid for 3 days, no renal stones were formed when the supply of drinking was normal. However, when drinking water was limited, stone formation was observed and accompanied by high levels of serum urea and creatinine. An increase in urine haemoglobin and glucose levels was also found. The administration resulted in up-regulated tissue osteopontin, kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin messenger RNA expression and macrophage infiltration.

Conclusions: Our results indicate the importance of water intake in the formation of melamine-induced renal stone formation in the mouse and provide new information on the mechanisms of melamine stone formation.
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http://dx.doi.org/10.1093/ndt/gfr577DOI Listing
June 2012

In vivo fluorescence lifetime optical projection tomography.

Biomed Opt Express 2011 Apr 26;2(5):1340-50. Epub 2011 Apr 26.

We demonstrate the application of fluorescence lifetime optical projection tomography (FLIM-OPT) to in vivo imaging of lysC:GFP transgenic zebrafish embryos (Danio rerio). This method has been applied to unambiguously distinguish between the fluorescent protein (GFP) signal in myeloid cells from background autofluorescence based on the fluorescence lifetime. The combination of FLIM, an inherently ratiometric method, in conjunction with OPT results in a quantitative 3-D tomographic technique that could be used as a robust method for in vivo biological and pharmaceutical research, for example as a readout of Förster resonance energy transfer based interactions.
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http://dx.doi.org/10.1364/BOE.2.001340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3087590PMC
April 2011

Mast cells in health and disease.

Clin Sci (Lond) 2011 Jun;120(11):473-84

Leukocyte Biology Section, Medical Research Council, Asthma UK Centre in Allergic Mechanisms of Asthma, National Heart and Lung Institute, Faculty of Medicine, Imperial College London, South Kensington, UK.

Although MCs (mast cells) were discovered over 100 years ago, for the majority of this time their function was linked almost exclusively to allergy and allergic disease with few other roles in health and disease. The engineering of MC-deficient mice and engraftment of these mice with MCs deficient in receptors or mediators has advanced our knowledge of the role of MCs in vivo. It is now known that MCs have very broad and varied roles in both physiology and disease which will be reviewed here with a focus on some of the most recent discoveries over the last year. MCs can aid in maintaining a healthy physiology by secreting mediators that promote wound healing and homoeostasis as well as interacting with neurons. Major developments have been made in understanding MC function in defence against pathogens, in recognition of pathogens as well as direct effector functions. Probably the most quickly developing area of understanding is the involvement and contribution MCs make in the progression of a variety of diseases from some of the most common diseases to the more obscure.
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http://dx.doi.org/10.1042/CS20100459DOI Listing
June 2011

Regulation of IL-17 in chronic inflammation in the human lung.

Clin Sci (Lond) 2011 Jun;120(12):515-24

Division of Cell and Molecular Biology, Department of Life Sciences, Imperial College London, London SW7 2AZ, UK.

The regulation of human Th17 cell effector function by Treg cells (regulatory T-cells) is poorly understood. In the present study, we report that human Treg (CD4(+)CD25(+)) cells inhibit the proliferative response of Th17 cells but not their capacity to secrete IL (interleukin)-17. However, they could inhibit proliferation and cytokine production by Th1 and Th2 cells as determined by IFN-γ (interferon-γ) and IL-5 biosynthesis. Currently, as there is interest in the role of IL-17-producing cells and Treg cells in chronic inflammatory diseases in humans, we investigated the presence of CD4(+)CD25(+) T-cells and IL-17 in inflammation in the human lung. Transcripts for IL-17 were expressed in mononuclear cells and purified T-cells from lung tissue of patients with chronic pulmonary inflammation and, when activated, these cells secrete soluble protein. The T-cell-specific transcription factors RORCv2 (retinoic acid-related orphan receptor Cv2; for Th17) and FOXP3 (forkhead box P3; for Treg cells) were enriched in the T-cell fraction of lung mononuclear cells. Retrospective stratification of the patient cohort into those with COPD (chronic obstructive pulmonary disease) and non-COPD lung disease revealed no difference in the expression of IL-17 and IL-23 receptor between the groups. We observed that CD4(+)CD25(+) T-cells were present in comparable numbers in COPD and non-COPD lung tissue and with no correlation between the presence of CD4(+)CD25(+) T-cells and IL-17-producing cells. These results suggest that IL-17-expressing cells are present in chronically inflamed lung tissue, but there is no evidence to support this is due to the recruitment or expansion of Treg cells.
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http://dx.doi.org/10.1042/CS20100417DOI Listing
June 2011

Enhanced effector function of cytotoxic cells in the induced sputum of COPD patients.

Respir Res 2010 Jun 11;11:76. Epub 2010 Jun 11.

COPD Research Group, Nottingham Respiratory Biomedical Research Unit, The University of Nottingham, UK.

Background: We have previously shown that NK (CD56+CD3-) and NKT-like (CD56+CD3+) cells are reduced in both numbers and cytotoxicity in peripheral blood. The aim of the present study was to investigate their numbers and function within induced sputum.

Methods: Induced sputum cell numbers and intracellular granzyme B and perforin were analysed by flow cytometry. Immunomagnetically selected CD56+ cells (NK and NKT-like cells) were used in an LDH release assay to determine cytotoxicity.

Results: The proportion of NK cells and NKT-like cells in smokers with COPD (COPD subjects) was significantly higher (12.7% and 3%, respectively) than in healthy smokers (smokers) (5.7%, p < 0.01; 1%, p < 0.001) and non-smoking healthy subjects (HNS) (4.2%, p < 0.001; 0.8%, p < 0.01). The proportions of NK cells and NKT-like cells expressing both perforin and granzyme B were also significantly higher in COPD subjects compared to smokers and HNS. CD56+ cells from COPD subjects were significantly more cytotoxic (1414 biological lytic activity) than those from smokers (142.5; p < 0.01) and HNS (3.8; p < 0.001) and were inversely correlated to FEV1. (r = -0.75; p = 0.0098).

Conclusion: We have shown an increased proportion of NK and NKT-like cells in the induced sputum of COPD subjects and have demonstrated that these cells are significantly more cytotoxic in COPD subjects than smokers and HNS.
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http://dx.doi.org/10.1186/1465-9921-11-76DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2891678PMC
June 2010

Altered effector function of peripheral cytotoxic cells in COPD.

Respir Res 2009 Jun 22;10:53. Epub 2009 Jun 22.

COPD Research Group, Institute of Infection, Immunity and Inflammation, The University of Nottingham, UK.

Background: There is mounting evidence that perforin and granzymes are important mediators in the lung destruction seen in COPD. We investigated the characteristics of the three main perforin and granzyme containing peripheral cells, namely CD8+ T lymphocytes, natural killer (NK; CD56+CD3-) cells and NKT-like (CD56+CD3+) cells.

Methods: Peripheral blood mononuclear cells (PBMCs) were isolated and cell numbers and intracellular granzyme B and perforin were analysed by flow cytometry. Immunomagnetically selected CD8+ T lymphocytes, NK (CD56+CD3-) and NKT-like (CD56+CD3+) cells were used in an LDH release assay to determine cytotoxicity and cytotoxic mechanisms were investigated by blocking perforin and granzyme B with relevant antibodies.

Results: The proportion of peripheral blood NKT-like (CD56+CD3+) cells in smokers with COPD (COPD subjects) was significantly lower (0.6%) than in healthy smokers (smokers) (2.8%, p < 0.001) and non-smoking healthy participants (HNS) (3.3%, p < 0.001). NK (CD56+CD3-) cells from COPD subjects were significantly less cytotoxic than in smokers (16.8% vs 51.9% specific lysis, p < 0.001) as were NKT-like (CD56+CD3+) cells (16.7% vs 52.4% specific lysis, p < 0.001). Both cell types had lower proportions expressing both perforin and granzyme B. Blocking the action of perforin and granzyme B reduced the cytotoxic activity of NK (CD56+CD3-) and NKT-like (CD56+CD3+) cells from smokers and HNS.

Conclusion: In this study, we show that the relative numbers of peripheral blood NK (CD56+CD3-) and NKT-like (CD56+CD3+) cells in COPD subjects are reduced and that their cytotoxic effector function is defective.
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http://dx.doi.org/10.1186/1465-9921-10-53DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2705911PMC
June 2009

Human tribbles homologue 2 is expressed in unstable regions of carotid plaques and regulates macrophage IL-10 in vitro.

Clin Sci (Lond) 2009 Feb;116(3):241-8

GlaxoSmithKline Clinical Unit, Addenbrooke's Centre for Clinical Investigation, Addenbrooke's Hospital, Cambridge CB2 2GG, U.K.

Mammalian orthologues of the Drosophila tribbles protein (Trb1, Trb2 and Trb3) are a recently described family of signalling molecules that regulate gene expression by modulation of protein kinase signalling pathways. In the present study, a screen for mRNA species specifically regulated in vulnerable regions of human atherosclerotic plaque demonstrated the up-regulation of both Trb1 and Trb2, the latter by more than 8-fold. In vitro experiments in primary human monocyte-derived macrophages showed that Trb2 expression was up-regulated by treatment with oxidized LDL (low-density lipoprotein), and that expression of recombinant Trb2 specifically reduced macrophage levels of IL-10 (interleukin-10) mRNA. Our results thus identify Trb2 as a highly regulated gene in vulnerable atherosclerotic lesions, and demonstrate inhibition of macrophage IL-10 biosynthesis as a potential pro-inflammatory consequence of high Trb2 expression, which may contribute to plaque instability.
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http://dx.doi.org/10.1042/CS20080058DOI Listing
February 2009

Killer cells in chronic obstructive pulmonary disease.

Clin Sci (Lond) 2008 Apr;114(8):533-41

COPD Research Group, Institute of Infection, Immunity and Inflammation, The University of Nottingham, Nottingham NG7 2RD, UK.

COPD (chronic obstructive pulmonary disease) is a treatable and preventable disease state, characterized by progressive airflow limitation that is not fully reversible. It is a current and growing cause of mortality and morbidity worldwide, with the WHO (World Health Organization) projecting that total deaths attributed to COPD will increase by more than 30% in the next 10 years. The pathological hallmarks of COPD are destruction of the lung parenchyma (pulmonary emphysema), inflammation of the central airways (chronic bronchitis) and inflammation of the peripheral airways (respiratory bronchiolitis). The destructive changes and tissue remodelling observed in COPD are a result of complex interactions between cells of the innate and adaptive immune systems. The focus of the present review is directed towards the role of CD8(+) T-lymphocytes, NK (natural killer) cells and NKT cells (NK T-cells). These three classes of killer cell could all play an important part in the pathogenesis of COPD. The observed damage to the pulmonary tissue could be caused in three ways: (i) direct cytotoxic effect against the lung epithelium mediated by the activities of perforin and granzymes, (ii) FasL (Fas ligand)-induced apoptosis and/or (iii) cytokine and chemokine release. The present review considers the role of these killer cells in COPD.
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http://dx.doi.org/10.1042/CS20070356DOI Listing
April 2008

Combined treatment with triptolide and rapamycin prolongs graft survival in a mouse model of cardiac transplantation.

Transpl Int 2008 May 4;21(5):483-94. Epub 2008 Feb 4.

Department of Surgery, University of Hong Kong Medical Centre, Queen Mary Hospital, Hong Kong, China.

Current immunosuppressive strategies for transplantation have failed to achieve long-term graft survival. In this study, we investigate the effects of combined treatment with triptolide (TPT) and rapamycin (Rapa) on graft survival as well as changes in pathology and immunological responses. Heterotopic heart transplantation was performed. TPT and Rapa were administered either alone or in combination. The mean survival time (MST) for the cardiac allografts in animals receiving the combination of TPT and Rapa was 93.5 +/- 6.7 days compared to treatment with TPT (MST: 23.5 +/- 5.3 days), Rapa (22 +/- 1.3 days) alone or no treatment (7.66 +/- 0.8 days). Histopathological evaluation showed that inflammatory cell infiltration was markedly reduced in grafts with combined treatment groups. Down-regulation of CCL19, CCR5, CCR7, interferon gamma and interleukin (IL)-12 in the combination treatment was accompanied by increased expression of IL-4, IL-10 and CD4(+)CD25(+)Foxp3(+) regulatory T (Tr) cells in spleen. Finally, dendritic cell (DC) maturation was impaired by treatment with TPT/Rapa. Our results demonstrate that combination therapy with TPT and Rapa markedly prolongs cardiac allograft survival. This effect is accompanied by inhibition of DCs maturation, conditioning DCs to adopt tolerogenic phenotype, and the expansion of Tr cells. These results add weight to the application of combination therapy in transplantation.
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http://dx.doi.org/10.1111/j.1432-2277.2007.00630.xDOI Listing
May 2008

Hypoxia modulates lipopolysaccharide induced TNF-alpha expression in murine macrophages.

Exp Cell Res 2008 Apr 16;314(6):1327-36. Epub 2008 Jan 16.

Division of Paediatric Surgery, Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, PR China.

The pro-inflammatory activity of Tumor necrosis factor-alpha (TNF-alpha) together with tissue hypoxia determine the clinical outcome in sepsis and septic shock. p38 MAPKinase is the primary intracellular signaling pathway that regulates lipopolysaccharide (LPS)-induced TNF-alpha biosynthesis, however, the effect of hypoxia on LPS mediated activation of p38 is not known. Here we report that SB203580, a specific p38 MAPK inhibitor, which completely abolished LPS-induced TNF-alpha expression by the mouse macrophage cell RAW264.7 in normoxic conditions, lost the inhibitory effect in hypoxic conditions. Hypoxia did not modulate expression of p38 MAPK, but increased that of p-MK2, a downstream target of p38 MAPK. In LPS induced endotoxemia mice model SB203580 had no inhibitory effect on the serum levels of TNF-alpha. Furthermore, hypoxia inducible factor-1alpha (HIF-1alpha) was detected in vivo after LPS administration but its expression was not affected by SB203580. Our data indicate that LPS induced p38 MAPK activation was enhanced by hypoxia and consequently increased TNF-alpha secretion. Furthermore, the induction of HIF-1alpha in mice with endotoxemia suggested a synergistic effect on p38 mediated TNF-alpha expression. These findings provide new insights on the pathophysiological effects of hypoxia in sepsis and septic shock.
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http://dx.doi.org/10.1016/j.yexcr.2008.01.007DOI Listing
April 2008

LPS-induced up-regulation of TGF-beta receptor 1 is associated with TNF-alpha expression in human monocyte-derived macrophages.

J Leukoc Biol 2008 May 5;83(5):1165-73. Epub 2008 Feb 5.

Division of Paediatric Surgery, Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, China.

The immunosuppressive activity of TGF-beta-mediated signaling is well documented, but in contrast, its ability to promote proinflammatory responses is less clear. In this study, we report that blockade of TGF-beta signaling by a specific inhibitor of the TGF-beta receptor I [activin receptor-like kinase 5 (ALK5)] SB431542 significantly reduces the production of TNF-alpha, a key proinflammatory cytokine, by LPS-stimulated human monocyte-derived macrophages. ALK5 protein was only detectable after LPS stimulation, and the failure of treatment with SB431542 to alter TNF-alpha mRNA expression indicates that regulation is post-transcriptional. The additive effect of blocking TGF-beta and p38 MAPK signaling on reducing TNF-alpha but not IL-6 production suggests that there is selectivity in pathway signaling. SB431542 had similar inhibitory effects on TNF-alpha production by human monocytes and endothelial cells as well as macrophages. Furthermore, treatment with SB431542 reduced plasma TNF-alpha levels and tissue damage and thereby, prevented the lethal effects of LPS in a mouse model of septic shock. Our data demonstrate a direct effect of TGF-beta signaling via ALK5 on the regulation of TNF-alpha synthesis.
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http://dx.doi.org/10.1189/jlb.0807521DOI Listing
May 2008

Triptolide, a component of Chinese herbal medicine, modulates the functional phenotype of dendritic cells.

Transplantation 2007 Dec;84(11):1517-26

Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, PR China.

Background: Triptolide (TPT) is a component of the Chinese herb Triptergium wilfordii and has potent immunosuppressive and anti-inflammatory effects. Since dendritic cells (DCs) play a critical role in the initiation of alloimmune responses to foreign grafts, the aim of the present study was to evaluate the effects of TPT in the functional phenotype of bone marrow (BM)-derived DCs.

Methods: BM-derived DCs were cultured with or without TPT from days 2 to 7 and then stimulated with lipopolysaccharide (LPS) for a further 2 days. In some experiments, TPT was added to the cultures from day 7 to day 9. Heterotopic cardiac transplantation was performed and animals received either no treatment or were injected systemically with different doses of TPT posttransplantation.

Results: TPT treatment inhibited LPS-induced DC maturation. The ability of DCs to stimulate allogeneic T-cell responses was also impaired by TPT treatment and accompanied by upregulated synthesis of interleukin-10 facilitating the expansion of regulatory T cells. Furthermore, we observed that TPT treatment led to increased cell surface expression of DC-SIGN and reduced expression of TLR4 on DCs.

Conclusions: These findings demonstrate that TPT can inhibit the maturation and allogenicity of DCs and promotes the expansion of regulatory T cells. These effects were confirmed by in vivo experiments though treatment of TPT can prolong mice heart allograft survival. Furthermore, modulation of the DC surface phenotype towards that of a tolerogenic phenotype could contribute to the ability of TPT to suppress productive immunity and maintain homeostasis.
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http://dx.doi.org/10.1097/01.tp.0000289990.55668.0dDOI Listing
December 2007

Interleukin-10 is upregulated by nanomolar rosiglitazone treatment of mature dendritic cells and human CD4+ T cells.

Cytokine 2007 Sep 5;39(3):184-91. Epub 2007 Sep 5.

Translational Medicine and Genetics, GlaxoSmithKline, ACCI, Addenbrooke's Hospital, Cambridge CB2 2GG, UK.

Activators of peroxisome proliferator-activated receptor (PPAR)-gamma are anti-inflammatory and have been proposed as therapeutic agents for the treatment of Th1-type inflammatory diseases. We report that nanomolar concentrations of rosiglitazone enhance the production of IL-10 from activated human mature monocyte-derived dendritic cells. Also, rosiglitazone specifically induces the production of IL-10 from TCR-activated human CD4+ T cells and that this effect is PPAR-gamma-dependent. We also demonstrate for the first time the presence of a functional PPAR response element (PPRE) in the human IL-10 promoter region. Finally we show that rosiglitazone can induce IL-10 in combination with 1,25 alpha-dihydroxyvitamin D3 to a greater extent than each treatment alone. In summary our findings demonstrate that IL-10 is upregulated by nanomolar TZDs in immune cells, and this may, in part, be responsible for the potential anti-inflammatory effects of PPAR-gamma in humans.
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http://dx.doi.org/10.1016/j.cyto.2007.07.191DOI Listing
September 2007

Rosiglitazone suppresses cyclosporin-induced chronic transplant dysfunction and prolongs survival of rat cardiac allografts.

Transplantation 2007 Jun;83(12):1602-10

Department of Surgery, Faculty of Medicine Building, University of Hong Kong, Pokfulam, Hong Kong, SAR China.

Background: The lack of effective treatment for chronic transplant dysfunction restricts the long-term survival of solid organ allografts. Peroxisome proliferator-activated receptor ligands can suppress vascular inflammation. The aim of this study was to analyze the effects of rosiglitazone on chronic transplant dysfunction in a rat cardiac transplant model.

Methods: Inbred male Fisher 344 (F344, RT1lvl) and Lewis (LEW, RT1(1)) rats were subjected to heterotopic abdominal heart transplantation according to standard procedures. Cyclosporine A was administered intraperitoneally to cover acute rejection, and rosiglitazone was administered orally by gavage daily from 3 days before the operation to the end of experiments.

Results: Rosiglitazone significantly prolonged the survival of cardiac allografts in rats (F344 to LEW) that had received a 10-day course of cyclosporin A compared to treatment with immunosuppressant alone. Analysis of allografts at 120 days posttransplantation showed that rosiglitazone reduced the inflammatory cell infiltrate in both the vessels and graft parenchyma as were neointimal formation, vascular occlusion, and fibrosis. Expression of transforming growth factor-beta and related proteins was less abundant after cyclosporin A/rosiglitazone treatment.

Conclusions: The findings reported here demonstrate that rosiglitazone given under the cover of short-term treatment with cyclosporin A prolongs cardiac allograft survival and reduces the severity of chronic transplant dysfunction. This may be mediated in part through the downregulation of transforming growth factor-beta and related proteins. The combined effects of rosiglitazone and immunosuppressive drugs are potentially beneficial to patients receiving organ transplants.
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http://dx.doi.org/10.1097/01.tp.0000266994.39480.42DOI Listing
June 2007

Inhibition of sonic hedgehog signaling reduces chronic rejection and prolongs allograft survival in a rat orthotopic small bowel transplantation model.

Transplantation 2007 May;83(10):1351-7

Division of Paediatric Surgery, Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, PR China.

Background: Although acute graft rejection can be successfully controlled by immunosuppressive agents, chronic rejection (CR), which is characterized by arteriosclerosis in the donor organ vessels, is a major hurdle to long-term allograft survival. Sonic hedgehog (Shh), a morphogen critical in embryogenesis, also promotes peripheral immunity, which prompted us to investigate if inhibition of Shh signaling could reduce CR and thereby enhance allograft survival.

Methods: In a rat orthotopic small bowel transplantation model, FK506 prevented acute rejection; however, recipients eventually lost their grafts by CR. Anti-Shh antibody or isotype control were administered to animals at day 30 postoperatively. Graft survival, tissue fibrosis, vascular occlusion, and expression of vascular endothelial growth factor (VEGF) were investigated.

Results: Immunostaining revealed that Shh and the Hedgehog receptor Patched 1 (Ptc1) are strongly expressed in CR grafts and that Ptc1 expression partially overlapped with that of ED-1, a macrophage marker. In contrast, only minimal expression of Shh and Ptc1 was detected in syngeneic grafts. Grafts survival was significantly prolonged after anti-Shh antibody treatment compared with the immunoglobulin G control (116 vs. 77.5 days). Collagen deposition and vascular occlusion in the mesentery were markedly reduced in recipients of the anti-Shh antibody. Specific transcripts and protein expression for VEGF, which was present mainly in the blood vessels, were reduced.

Conclusion: In a rat small bowel transplantation model, anti-Shh antibody treatment reduced CR and prolonged graft survival. These beneficial effects of Shh treatment may occur partly by reducing VEGF expression in the blood vessels of the allografts.
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http://dx.doi.org/10.1097/01.tp.0000262568.73590.81DOI Listing
May 2007