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author:("iss, kamiko")
1.  Pre-existing Epithelial Diversity in Normal Human Livers: A Tissue-tethered Cytometric Analysis in Portal/Periportal Epithelial Cells 
Hepatology (Baltimore, Md.)  2013;57(4):1632-1643.
Routine light microscopy identifies two distinct epithelial cell populations in normal human livers: hepatocytes and biliary epithelial cells (BEC). Considerable epithelial diversity, however, arises during disease states when a variety of hepatocyte-BEC hybrid cells appear. This has been attributed to activation and differentiation of putative hepatic progenitor cells (HPC) residing in the Canals of Hering and/or metaplasia of pre-existing mature epithelial cells. A novel analytic approach consisting of multiplex labeling, high resolution whole slide imaging (WSI), and automated image analysis was used to determine if more complex epithelial cell phenotypes pre-existed in normal adult human livers, which might provide an alternative explanation for disease-induced epithelial diversity. “Virtually digested” WSI enabled quantitative cytometric analyses of individual cells displayed in a variety of formats (e.g. scatter plots) while still tethered to the WSI and tissue structure. We employed biomarkers specifically-associated with mature epithelial forms (HNF4α for hepatocytes, CK19 and HNF1β for BEC) and explored for the presence of cells with hybrid biomarker phenotypes. Results showed abundant hybrid cells in portal bile duct BEC, canals of Hering, and immediate periportal hepatocytes. These bi-potential cells likely serve as a reservoir for the epithelial diversity of ductular reactions, appearance of hepatocytes in bile ducts, and the rapid and fluid transition of BEC to hepatocytes, and vice versa.
Novel imaging and computational tools enable increased information extraction from tissue samples and quantify the considerable pre-existent hybrid epithelial diversity in normal human liver. This computationally-enabled tissue analysis approach offers much broader potential beyond the results presented here.
PMCID: PMC3612393  PMID: 23150208
Digital imaging; progenitor cells; Canals of Hering; hepatocyte nuclear factors; fluorescence microscopy
2.  STAT1-Regulated Lung MDSC-like Cells Produce IL-10 and Efferocytose Apoptotic Neutrophils With Relevance In Resolution of Bacterial Pneumonia 
Mucosal immunology  2012;6(1):189-199.
Bacterial pneumonia remains a significant burden worldwide. Although an inflammatory response in the lung is required to fight the causative agent, persistent tissue-resident neutrophils in non-resolving pneumonia can induce collateral tissue damage and precipitate acute lung injury. However, little is known about mechanisms orchestrated in the lung tissue that remove apoptotic neutrophils to restore tissue homeostasis. In mice infected with Klebsiella pneumoniae, a bacterium commonly associated with hospital-acquired pneumonia, we show that interleukin-10 is essential for resolution of lung inflammation and recovery of mice after infection. Although IL-10−/− mice cleared bacteria, they displayed increased morbidity with progressive weight loss and persistent lung inflammation in the later phase after infection. A source of tissue IL-10 was found to be resident CD11b+Gr1intF4/80+ cells resembling myeloid-derived suppressor cells that appeared with a delayed kinetics after infection. These cells efficiently efferocytosed apoptotic neutrophils, which was aided by IL-10. The lung neutrophil burden was attenuated in infected STAT1−/− mice with concomitant increase in the frequency of the MDSC-like cells and lung IL-10 levels. Thus, inhibiting STAT1 in combination with antibiotics may be a novel therapeutic strategy to address inefficient resolution of bacterial pneumonia.
PMCID: PMC3505806  PMID: 22785228
3.  Long-term effects of Alemtuzumab on regulatory and memory T cell subsets in kidney transplantation 
Transplantation  2012;93(8):813-821.
Induction with lymphocyte-depleting antibodies is routinely employed to prevent rejection but often skews T cells towards memory. It is not fully understood which memory and regulatory T cell subsets are most affected and how they relate to clinical outcomes.
We analyzed T cells from 57 living-donor renal transplant recipients (12 reactive and 45 quiescent) 2.8±1.4 years after Alemtuzumab induction. 34 healthy subjects and 9 patients with acute cellular rejection (ACR) were also studied.
We found that Alemtuzumab caused protracted CD4>CD8 T lymphocyte deficiency, increased proportion of CD4+ memory T cells (TM), and decreased proportion of CD4+ regulatory T cells (TREG). Reactive patients exhibited higher proportions of CD4+ effector memory (TEM) and CD8+ terminally differentiated effector memory (TEMRA), with greater CD4+ TEM and CD8+ TEMRA to TREG ratios, than quiescent patients or healthy controls. Patients with ongoing ACR had profound reduction in circulating CD8+ TEMRA. Mixed lymphocyte assays showed significantly lower T cell proliferation to donor than third party antigens in the quiescent group, while reactive and ACR patients exhibited increased effector molecules in CD8+ T cells.
Our findings provide evidence that T cell skewing towards effector memory may be associated with anti-graft reactivity long after lymphodepletion. Further testing of TEM and TEMRA subsets as rejection predictors is warranted.
PMCID: PMC3323763  PMID: 22343334
kidney transplantation; memory T cells; regulatory T cells; alemtuzumab
4.  Digital Transplantation Pathology: Combining Whole Slide Imaging, Multiplex Staining, and Automated Image Analysis 
Conventional histopathology is the gold standard for allograft monitoring, but its value proposition is increasingly questioned. “-Omics” analysis of tissues, peripheral blood and fluids and targeted serologic studies provide mechanistic insights into allograft injury not currently provided by conventional histology. Microscopic biopsy analysis, however, provides valuable and unique information: a) spatial-temporal relationships; b) rare events/cells; c) complex structural context; and d) integration into a “systems” model. Nevertheless, except for immunostaining, no transformative advancements have “modernized” routine microscopy in over 100 years.
Pathologists now team with hardware and software engineers to exploit remarkable developments in digital imaging, nanoparticle multiplex staining, and computational image analysis software to bridge the traditional histology - global “–omic” analyses gap. Included are side-by-side comparisons, objective biopsy finding quantification, multiplexing, automated image analysis, and electronic data and resource sharing. Current utilization for teaching, quality assurance, conferencing, consultations, research and clinical trials is evolving toward implementation for low-volume, high-complexity clinical services like transplantation pathology. Cost, complexities of implementation, fluid/evolving standards, and unsettled medical/legal and regulatory issues remain as challenges. Regardless, challenges will be overcome and these technologies will enable transplant pathologists to increase information extraction from tissue specimens and contribute to cross-platform biomarker discovery for improved outcomes.
PMCID: PMC3627485  PMID: 22053785
Digital image; Fibrosis; Quantitative analysis; Allograft pathology; C4d; 3-dimensional imaging; Quantum dots
5.  IL-33 expands suppressive CD11b+ Gr-1int and regulatory T cells (Treg), including ST2L+ Foxp3+ cells, and mediates Treg-dependent promotion of cardiac allograft survival 
IL-33 administration is associated with facilitation of Th type-2 (Th2) responses and cardioprotective properties in rodent models. However, in heart transplantation, the mechanism by which IL-33, signaling through ST2L, the membrane-bound form of ST2, promotes transplant survival is unclear. We report that IL-33 administration, while facilitating Th2 responses, also increases immunoregulatory myeloid cells and CD4+ Foxp3+ regulatory T cells (Treg) in mice. IL-33 expands functional myeloid-derived suppressor cells (MDSC), -CD11b+ cells that exhibit intermediate (int) levels of Gr-1 and potent T cell suppressive function. Furthermore, IL-33 administration causes a St2-dependent expansion of suppressive CD4+ Foxp3+ Treg, including a ST2L+ population. IL-33 monotherapy following fully allogeneic mouse heart transplantation resulted in significant graft prolongation, associated with increased Th2-type responses and decreased systemic CD8+ IFN-γ+ cells. Also, despite reducing overall CD3+ cell infiltration of the graft, IL-33 administration markedly increased intragraft Foxp3+ cells. Whereas control graft recipients displayed increases in systemic CD11b+ Gr-1hi cells, IL-33-treated recipients exhibited increased CD11b+ Gr-1int cells. Enhanced ST2 expression was observed in the myocardium and endothelium of rejecting allografts, however the therapeutic effect of IL-33 required recipient St2 expression and was dependent on Treg. These findings reveal a new immunoregulatory property of IL-33. Specifically, in addition to supporting Th2 responses, IL-33 facilitates regulatory cells, particularly functional CD4+ Foxp3+ Treg that underlie IL-33-mediated cardiac allograft survival.
PMCID: PMC3197898  PMID: 21949025
Cytokines; Dendritic cells; Monocytes/Macrophages; Transplantation; Tolerance/Suppression/Anergy; T cells
6.  Hepatic B7-H1 expression is essential to control cold ischemia/reperfusion injury after mouse liver transplantation 
Hepatology (Baltimore, Md.)  2011;54(1):216-228.
Ischemia/reperfusion (I/R) injury remains a key risk factor significantly affecting morbidity and mortality after liver transplantation (LTx). B7-H1, recently identified member of the B7 family, is known to play important roles in regulating local immune responses. We hypothesized that B7-H1 plays crucial roles during innate immune responses induced by hepatic I/R injury and tested this hypothesis in the mouse LTx model using B7-H1 KO liver grafts with 24 hr cold storage. Cold I/R injury in WT to WT LTx enhanced constitutive B7-H1 expression on dendritic cells and sinusoidal endothelial cells, and promptly induced B7-H1 on hepatocytes. When B7-H1 KO liver grafts were transplanted into WT recipients, serum ALT levels and graft necrosis were significantly higher than WT to WT LTx. Augmented tissue injury in B7-H1 KO grafts was associated with increased frequencies and absolute numbers of graft CD3+ T cells, in particular CD8+ T cells. B7-H1 KO grafts had significantly lower incidences of Annexin V+ CD8+ T cells, indicating the failure to delete infiltrating CD8+ T cells. To evaluate the relative contribution of parenchymal and bone marrow-derived cell (BMDC) B7-H1 expression, chimeric liver grafts lacking B7-H1 on parenchymal cells or BMDC were generated and transplanted into WT recipients. Selective B7-H1 deficiency on parenchymal cells or BMDC resulted in similar levels of ALT and liver injury, suggesting that both parenchymal and BMDC B7-H1expression is involved in the control of liver damage. Human livers upregulated B7-H1 expression after LTx. Conclusion: The study demonstrates that graft tissue expression of B7-H1 plays critical roles in regulating inflammatory responses during LTx-induced hepatic I/R injury, and suggests that negative coregulatory signals may have an important function in hepatic innate immune responses.
PMCID: PMC3125416  PMID: 21503939
apoptosis; T cells; hepatocyte; nonparenchymal cells; dendritic cells
7.  SPRR2A enhances p53 deacetylation through HDAC1 and down regulates p21 promoter activity 
BMC Molecular Biology  2012;13:20.
Small proline rich protein (SPRR) 2A is one of 14 SPRR genes that encodes for a skin cross-linking protein, which confers structural integrity to the cornified keratinocyte cell envelope. New evidence, however, shows that SPRR2A is also a critical stress and wound repair modulator: it enables a variety of barrier epithelia to transiently acquire mesenchymal characteristics (EMT) and simultaneously quench reactive oxygen species during wound repair responses. p53 is also widely recognized as the node in cellular stress responses that inhibits EMT and triggers cell-cycle arrest, apoptosis, and cellular senescence. Since some p53-directed processes would seem to impede wound repair of barrier epithelia, we hypothesized that SPRR2A up regulation might counteract these effects and enable/promote wound repair under stressful environmental conditions.
Using a well characterized cholangiocarcinoma cell line we show that levels of SPRR2A expression, similar to that seen during stressful biliary wound repair responses, disrupts acetylation and subsequent p53 transcriptional activity. p53 deacetylation is accomplished via two distinct, but possibly related, mechanisms: 1) a reduction of p300 acetylation, thereby interfering with p300-p53 binding and subsequent p300 acetylation of K382 in p53; and 2) an increase in histone deacetylase 1 (HDAC1) mRNA and protein expression. The p300 CH3 domain is essential for both the autoacetylation of p300 and transference of the acetyl group to p53 and HDAC1 is a component of several non-p300 complexes that enhance p53 deacetylation, ubiquitination, and proteosomal degradation. HDAC1 can also bind the p300-CH3 domain, regulating p300 acetylation and interfering with p300 mediated p53 acetylation. The importance of this pathway is illustrated by showing complete restoration of p53 acetylation and partial restoration of p300 acetylation by treating SPRR2A expressing cells with HDAC1 siRNA.
Up-regulation of SPRR2A, similar to that seen during barrier epithelia wound repair responses reduces p53 acetylation by interfering with p300-p53 interactions and by increasing HDAC1 expression. SPRR2A, therefore, functions as a suppressor of p53-dependent transcriptional activity, which otherwise might impede cellular processes needed for epithelial wound repair responses such as EMT.
PMCID: PMC3495018  PMID: 22731250
8.  Cooperation of p300 and PCAF in the Control of MicroRNA 200c/141 Transcription and Epithelial Characteristics 
PLoS ONE  2012;7(2):e32449.
Epithelial to mesenchymal transition (EMT) not only occurs during embryonic development and in response to injury, but is an important element in cancer progression. EMT and its reverse process, mesenchymal to epithelial transition (MET) is controlled by a network of transcriptional regulators and can be influenced by posttranscriptional and posttranslational modifications. EMT/MET involves many effectors that can activate and repress these transitions, often yielding a spectrum of cell phenotypes. Recent studies have shown that the miR-200 family and the transcriptional suppressor ZEB1 are important contributors to EMT. Our previous data showed that forced expression of SPRR2a was a powerful inducer of EMT and supports the findings by others that SPRR gene members are highly upregulated during epithelial remodeling in a variety of organs. Here, using SPRR2a cells, we characterize the role of acetyltransferases on the microRNA-200c/141 promoter and their effect on the epithelial/mesenchymal status of the cells. We show that the deacetylase inhibitor TSA as well as P300 and PCAF can cause a shift towards epithelial characteristics in HUCCT-1-SPRR2a cells. We demonstrate that both P300 and PCAF act as cofactors for ZEB1, forming a P300/PCAF/ZEB1 complex on the miR200c/141 promoter. This binding results in lysine acetylation of ZEB1 and a release of ZEB1 suppression on miR-200c/141 transcription. Furthermore, disruption of P300 and PCAF interactions dramatically down regulates miR-200c/141 promoter activity, indicating a PCAF/P300 cooperative function in regulating the transcriptional suppressor/activator role of ZEB1. These data demonstrate a novel mechanism of miRNA regulation in mediating cell phenotype.
PMCID: PMC3284570  PMID: 22384255
9.  Initial In Vitro Investigation of the Human Immune Response to Corneal Cells from Genetically Engineered Pigs 
This study is the first to demonstrate the humoral and cellular immune response in humans to corneal endothelial cells from genetically engineered pigs. The results suggest that pig corneas may provide an acceptable alternative to human corneas for clinical transplantation.
To compare the in vitro human humoral and cellular immune responses to wild-type (WT) pig corneal endothelial cells (pCECs) with those to pig aortic endothelial cells (pAECs). These responses were further compared with CECs from genetically engineered pigs (α1,3-galactosyltransferase gene-knockout [GTKO] pigs and pigs expressing a human complement-regulatory protein [CD46]) and human donors.
The expression of Galα1,3Gal (Gal), swine leukocyte antigen (SLA) class I and class II on pCECs and pAECs, with or without activation by porcine IFN-γ, was tested by flow cytometry. Pooled human serum was used to measure IgM/IgG binding to and complement-dependent cytotoxicity (CDC) to cells from WT, GTKO, and GTKO/CD46 pigs. The human CD4+ T-cell response to cells from WT, GTKO, GTKO/CD46 pigs and human was tested by mixed lymphocyte reaction (MLR).
There was a lower level of expression of the Gal antigen and of SLA class I and II on the WT pCECs than on the WT pAECs, resulting in less antibody binding and reduced human CD4+ T-cell proliferation. However, lysis of the WT pCECs was equivalent to that of the pAECs, suggesting more susceptibility to injury. There were significantly weaker humoral and cellular responses to the pCECs from GTKO/CD46 pigs compared with the WT pCECs, although the cellular response to the GTKO/CD46 pCECs was greater than to the human CECs.
These data provide the first report of in vitro investigations of CECs from genetically engineered pigs and suggest that pig corneas may provide an acceptable alternative to human corneas for clinical transplantation.
PMCID: PMC3176056  PMID: 21596821
Hepatology (Baltimore, Md.)  2009;50(3):834-843.
COX-2-derived PGs participate in a number of pathophysiological responses such as inflammation, carcinogenesis, and modulation of cell growth and survival. This study utilized complementary approaches of COX-2 transgenic and knockout mice models to evaluate the mechanism of COX-2 in Fas-induced hepatocyte apoptosis and liver failure, in vivo. We generated transgenic mice with targeted expression of COX-2 in the liver by using the albumin promoter-enhancer driven vector. The COX-2 transgenic (Tg), COX-2 knockout (KO), and wild type mice were treated with the anti-Fas antibody Jo2 (0.5 µg/g body weight) for 4–6 hours and the extent of liver injury was assessed by histopathology, serum transaminases, TUNEL staining and caspase activation. The COX-2 Tg mice showed resistance to Fas-induced liver injury when compared to the wild type mice, as reflected by the lower ALT and AST levels, less liver damage and less hepatocyte apoptosis (p<0.01). In contrast, the COX-2 KO mice showed significantly higher serum ALT and AST levels, more prominent hepatocyte apoptosis, and higher levels of caspase-8, 9, 3 activities than the wild type mice (p<0.01). The COX-2 Tg livers express higher levels of epidermal growth factor receptor (EGFR) than the wild type controls; the COX-2 KO livers express lowest levels of EGFR. Pretreatment with the COX-2 inhibitor (NS-398) or the EGFR inhibitor (AG1478) exacerbated Jo2-mediated liver injury and hepatocyte apoptosis. These findings demonstrate that COX-2 prevents Fas-induced hepatocyte apoptosis and liver failure at least in part through upregulation of EGFR.
PMCID: PMC2758493  PMID: 19585617
Cyclooxygenase-2; liver; Fas; apoptosis; epidermal growth factor receptor

Results 1-10 (10)