Delta-Like Protein 3 Expression and Targeting in Merkel Cell Carcinoma.

PURPOSE: Delta-like protein 3 (DLL3) is being developed as a predictive biomarker for DLL3-targeting antibody-drug conjugate and other therapies. Given the neuroendocrine features of Merkel cell carcinoma (MCC), we sought to evaluate DLL3 expression and its role in MCC. EXPERIMENTAL DESIGN: Formalin-fixed and paraffin-embedded MCC cases were consecutively selected. Immunohistochemistry was performed for DLL3 (SC16.65 antibody) and polyomavirus large T-antigen (sc-136172 antibody). Slides were read out for percentage of positive tumor cells. Cox proportional hazards model was applied to assess the association between DLL3 expression and overall survival (OS). A patient with a DLL3-expressing MCC was treated with rovalpituzumab tesirine (Rova-T) in the "other tumor" cohort of NCT02709889 and assessed for response. RESULTS: The median H-score of DLL3 expression of 65 patients included was 60 (interquartile range, 30-100). Fifty-eight cases (89%) had ≥1% tumor cells positive for DLL3 expression with any intensity, of which the median DLL3 expression was 50% (interquartile range, 25%-70%). Thirty-four cases (52%) had ≥50% tumor cells positive for DLL3 expression with any intensity. Higher H-score of DLL3 expression was associated with higher polyomavirus nuclear expression (p = .003) when it was dichotomized to negative versus positive. H-score of DLL3 expression did not predict OS of patients with MCC (p = .4) after being adjusted for common clinicopathological factors. A patient treated with Rova-T for refractory metastatic MCC achieved partial response. CONCLUSIONS: DLL3 overexpression is very common in MCC by immunohistochemistry. The response to treatment suggests that DLL3 expression may have predictive relevance for DLL3-targeting therapies in MCC. IMPLICATIONS FOR PRACTICE: Delta-like protein 3 (DLL3) is being developed as a predictive biomarker to identify patients for treatment with DLL3-targeting agents. Merkel cell carcinoma (MCC) is an aggressive neuroendocrine carcinoma of the skin. It was found that DLL3 overexpression is very common in MCC by immunohistochemistry and significantly associated with Merkel cell polyomavirus expression. Despite the lack of prognostic significance in this cohort, DLL3 expression may have predictive relevance for DLL3-targeting therapies in MCC. The high levels of DLL3 expression in a subset of MCC may potentially be used to select patients to receive DLL3-targeting therapies.

IRF-1 promotes liver transplant ischemia/reperfusion injury via hepatocyte IL-15/IL-15Rα production.

Ischemia and reperfusion (I/R) injury following liver transplantation (LTx) is an important problem that significantly impacts clinical outcomes. IFN regulatory factor-1 (IRF-1) is a nuclear transcription factor that plays a critical role in liver injury. Our objective was to determine the immunomodulatory role of IRF-1 during I/R injury following allogeneic LTx. IRF-1 was induced in liver grafts immediately after reperfusion in both human and mouse LTx. IRF-1 contributed significantly to I/R injury because IRF-1-knockout (KO) grafts displayed much less damage as assessed by serum alanine aminotransferase and histology. In vitro, IRF-1 regulated both constitutive and induced expression of IL-15, as well as IL-15Rα mRNA expression in murine hepatocytes and liver dendritic cells. Specific knockdown of IRF-1 in human primary hepatocytes gave similar results. In addition, we identified hepatocytes as the major producer of soluble IL-15/IL-15Rα complexes in the liver. IRF-1-KO livers had significantly reduced NK, NKT, and CD8(+) T cell numbers, whereas rIL-15/IL-15Rα restored these immune cells, augmented cytotoxic effector molecules, promoted systemic inflammatory responses, and exacerbated liver injury in IRF-1-KO graft recipients. These results indicate that IRF-1 promotes LTx I/R injury via hepatocyte IL-15/IL-15Rα production and suggest that targeting IRF-1 and IL-15/IL-15Rα may be effective in reducing I/R injury associated with LTx.

Tissue-type plasminogen activator suppresses activated stellate cells through low-density lipoprotein receptor-related protein 1.

Hepatic stellate cell (HSC) activation and trans-differentiation into myofibroblast (MFB)-like cells is key for fibrogenesis after liver injury and a potential therapeutic target. Recent studies demonstrated that low-density lipoprotein receptor-related protein 1 (LRP1)-dependent signaling by tissue-type plasminogen activator (t-PA) is a pro-fibrotic regulator of the MFB phenotype in kidney. This study investigated whether LRP1 signaling by t-PA is also relevant to HSC activation following injury. Primary and immortalized rat HSCs were treated with t-PA and assayed by western blot, MTT, and TUNEL. In vitro results were then verified using an in vivo, acute carbon tetrachloride (CCl4) injury model that examined the phenotype and recovery kinetics of MFBs from wild-type animals vs mice with a global (t-PA) or HSC-targeted (LRP1) deletion. In vitro, in contrast to kidney MFBs, exogenous, proteolytically inactive t-PA suppressed, rather than induced, activation markers in HSCs following phosphorylation of LRP1. This process was mediated by LRP1 as inhibition of t-PA binding to LRP1 blocked the effects of t-PA. In vivo, following acute injury, phosphorylation of LRP1 on activated HSCs occurred immediately prior to their disappearance. Mice lacking t-PA or LRP1 retained higher densities of activated HSCs for a longer time period compared with control mice after injury cessation. Hence, t-PA, an FDA-approved drug, contributes to the suppression of activated HSCs following injury repair via signaling through LRP1. This renders t-PA a potential target for exploitation in treating patients with fibrosis.

Breast tumor kinase/protein tyrosine kinase 6 (Brk/PTK6) activity in normal and neoplastic biliary epithelia.

BACKGROUND & AIMS: Breast tumor kinase (BRK) augments proliferation and promotes cell survival in breast cancers via interactions with SH2 and SH3 ligand-containing proteins, such as receptor tyrosine kinases (RTK; e.g. EGFR, ErbB2/neu). Since RTK contribute to cholangiocarcinoma (CC) evolution we probed BRK protein expression and function in normal and CC livers. METHODS: Immunohistochemical staining of normal livers and CC (n=93) in a tissue microarray and three CC and an immortalized human cholangiocyte cell lines (real-time PCR, Western blotting, siRNA) were used to study the functional relationships between BRK, EGFR, ErbB2, SAM68, and SPRR2a. RESULTS: BRK protein was expressed in normal human intrahepatic bile ducts; all CC cell lines and a majority of CC showed strong BRK protein expression. Multiplex immunostaining/tissue cytometry and immunoprecipitation studies showed: 1) BRK co-localized with EGFR and ErbB2/neu; 2) BRK(high)/EGFR(high)-co-expressing CC cells had significantly higher Ki67 labeling and; 3) stronger BRK protein expression was seen in perihilar and distal CC than intrahepatic CC and directly correlated with CC differentiation. In cell lines, BRK expression augmented proliferation in response to exogenous EGF, whereas BRK siRNA significantly reduced growth. The SH3 ligand-containing, SPRR2A activated pTyr342 BRK, which in turn, phosphorylated SAM68, causing nuclear localization and increased cell proliferation similar to observations in breast cancers. CONCLUSION: BRK expression in a majority of CC can interact with RTK, augmenting growth and interfering with proliferation inhibitors (SAM68). Therapeutically targeting BRK function (in addition to RTK) should be of benefit for CC treatment.

Small proline rich protein 2a in benign and malignant liver disease.

UNLABELLED: STAT3-driven expression of small proline rich protein 2a (SPRR2a), which acts as an src homology 3 (SH3) domain ligand, induces biliary epithelial cell (BEC) epithelial-mesenchymal transition (EMT), which, in turn, promotes wound healing. SPRR2a also quenches free radicals and protects against oxidative stress and DNA damage in nonneoplastic BEC. Sprr2a-induced EMT also increases local invasiveness of cholangiocarcinomas (CC), but prevents metastases. Understanding SPRR2a regulation of EMT has potential for therapeutic targeting in both benign and malignant liver disease. Molecular mechanisms responsible for SPRR2a-induced EMT were characterized, in vitro, and then evidence for utilization of these pathways was sought in human intrahepatic CC, in vivo, using multiplex labeling and software-assisted morphometric analysis. SPRR2a complexes with ZEB1 and CtBP on the microRNA (miR)-200c/141 promoter resulting in synergic suppression of miR-200c/141 transcription, which is required for maintenance of the BEC epithelial phenotype. SPRR2a induction promotes dephosphorylation and nuclear translocation of the SH3-domain containing protein GRB2 and an SH3-domain ligand in ZEB1 is required for SPRR2a-induced synergic suppression of miR-200c/141. Multiplex protein labeling of CC and morphometric analyses showed: 1) up-regulation of ZEB-1, and 2) down-regulation of CK19 in intrahepatic CC compared to nonneoplastic BEC, consistent with previous CC proteomic studies showing EMT during cholangiocarcinogenesis. CONCLUSION: SPRR2a modulates ZEB-1 signaling by way of miR-200c/141-associated EMT through SH3-domain networks and contributes to benign and malignant BEC wound-healing responses.

Low TCR signal strength induces combined expansion of Th2 and regulatory T cell populations that protect mice from the development of type 1 diabetes.

AIMS/HYPOTHESIS: Weak stimulation of CD4(+) T cells induces expansion of CD4(+) forkhead box P3(+) regulatory T cells (Tregs) and can also promote T helper (Th) 2 responses, which have demonstrable beneficial effects on autoimmune diabetes. This study explored the feasibility of combined Treg/Th2 expansion for immunotherapy of type 1 diabetes in NOD mice. METHODS: We compared Treg and Th responses to dendritic cells (DC) presenting scaled antigen doses to islet-specific NOD CD4(+) T cells. Flow cytometric and Luminex analyses were performed to determine the phenotype and cytokine profile of expanded T cells. The ability of expanded T cells to prevent type 1 diabetes was tested in an adoptive transfer model. RESULTS: In vitro studies revealed a hierarchical, selective expansion of Treg and T effector (Teff) populations at different antigen doses. Thus, a single low dose produced a mixture of Tregs Th2 and type 1 regulatory (Tr1) cells, which prevented diabetes in NOD-SCID mice and increased the ratio of Treg/Teff cells infiltrating the pancreatic islets. Subcutaneous injection of DC, previously shown to prevent diabetes in NOD mice, induced expansion of the same mixture of Tregs Tr1 and Th2 cells. Low-dose expansion of Treg required MHC-T cell receptor interaction and was partly dependent on T cell derived TGF-ß and IL-2. Autocrine IFN-γ was required for the promotion of diabetogenic Th1 cells at high antigen doses. CONCLUSIONS/INTERPRETATION: Weak stimulation of CD4(+) T cells with DC and low-dose antigen expands a combination of antigen-specific Tregs Th2 and Tr1 cells that prevent autoimmunity, without the need to target or purify specific Treg populations.

Acute liver allograft antibody-mediated rejection: an inter-institutional study of significant histopathological features.

Acute antibody-mediated rejection (AMR) occurs in a small minority of sensitized liver transplant recipients. Although histopathological characteristics have been described, specific features that could be used (1) to make a generalizable scoring system and (2) to trigger a more in-depth analysis are needed to screen for this rare but important finding. Toward this goal, we created training and validation cohorts of putative acute AMR and control cases from 3 high-volume liver transplant programs; these cases were evaluated blindly by 4 independent transplant pathologists. Evaluations of hematoxylin and eosin (H&E) sections were performed alone without knowledge of either serum donor-specific human leukocyte antigen alloantibody (DSA) results or complement component 4d (C4d) stains. Routine histopathological features that strongly correlated with severe acute AMR included portal eosinophilia, portal vein endothelial cell hypertrophy, eosinophilic central venulitis, central venulitis severity, and cholestasis. Acute AMR inversely correlated with lymphocytic venulitis and lymphocytic portal inflammation. These and other characteristics were incorporated into models created from the training cohort alone. The final acute antibody-mediated rejection score (aAMR score)--the sum of portal vein endothelial cell hypertrophy, portal eosinophilia, and eosinophilic venulitis divided by the sum of lymphocytic portal inflammation and lymphocytic venulitis--exhibited a strong correlation with severe acute AMR in the training cohort [odds ratio (OR) = 2.86, P < 0.001] and the validation cohort (OR = 2.49, P < 0.001). SPSS tree classification was used to select 2 cutoffs: one that optimized specificity at a score > 1.75 (sensitivity = 34%, specificity = 86%) and another that optimized sensitivity at a score > 1.0 (sensitivity = 81%, specificity = 71%). In conclusion, the routine histopathological features of the aAMR score can be used to screen patients for acute AMR via routine H&E staining of indication liver transplant biopsy samples; however, a definitive diagnosis requires substantiation by DSA testing, diffuse C4d staining, and the exclusion of other insults.

Gut bacteria drive Kupffer cell expansion via MAMP-mediated ICAM-1 induction on sinusoidal endothelium and influence preservation-reperfusion injury after orthotopic liver transplantation.

Bacteria in the gut microbiome shed microbial-associated molecule patterns (MAMPs) into the portal venous circulation, where they augment various aspects of systemic immunity via low-level stimulation. Because the liver is immediately downstream of the intestines, we proposed that gut-derived MAMPs shape liver immunity and affect Kupffer cell (KC) phenotype. Germ-free (GF), antibiotic-treated (AVMN), and conventional (CL) mice were used to study KC development, function, and response to the significant stress of cold storage, reperfusion, and orthotopic transplantation. We found that a cocktail of physiologically active MAMPs translocate into the portal circulation, with flagellin (Toll-like receptor 5 ligand) being the most plentiful and capable of promoting hepatic monocyte influx in GF mice. In MAMP-deficient GF or AVMN livers, KCs are lower in numbers, have higher phagocytic activity, and have lower major histocompatibility complex II expression. MAMP-containing CL livers harbor significantly increased KC numbers via induction of intercellular adhesion molecule 1 on liver sinusoidal endothelium. These CL KCs have a primed yet expected phenotype, with increased major histocompatibility complex class II and lower phagocytic activity that increases susceptibility to liver preservation/reperfusion injury after orthotopic transplantation. The KC number, functional activity, and maturational status are directly related to the concentration of gut-derived MAMPs and can be significantly reduced by broad-spectrum antibiotics, thereby affecting susceptibility to injury.

Preexisting epithelial diversity in normal human livers: a tissue-tethered cytometric analysis in portal/periportal epithelial cells.

UNLABELLED: Routine light microscopy identifies two distinct epithelial cell populations in normal human livers: hepatocytes and biliary epithelial cells (BECs). 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 preexisting 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 preexist 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., scatterplots) while still tethered to the WSI and tissue structure. We employed biomarkers specifically associated with mature epithelial forms (HNF4α for hepatocytes, CK19 and HNF1ß for BEC) and explored for the presence of cells with hybrid biomarker phenotypes. The results showed abundant hybrid cells in portal bile duct BEC, canals of Hering, and immediate periportal hepatocytes. These bipotential 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. CONCLUSION: Novel imaging and computational tools enable increased information extraction from tissue samples and quantify the considerable preexistent hybrid epithelial diversity in normal human liver. This computationally enabled tissue analysis approach offers much broader potential beyond the results presented here.

Optimal lung inflation techniques in a rat lung transplantation model: a revisit.

BACKGROUND: Most of the experimental work assessing optimal lung inflation during lung graft preservation was performed in the late 1990s. Since that time, lung preservation before transplantation has been more standardized, and the optimal lung inflation techniques used during lung preservation in the current clinical setting remain undefined. Nonetheless, lung inflation during storage may play a pivotal role in optimal lung preservation. MATERIALS AND METHODS: Lewis rat lungs were perfused with and stored in cold, low-potassium dextran solution (Perfadex, Vitrolife, Göteborg, Sweden) for 6 hours at different levels of lung inflation (25, 50, 75, or 100% of vital capacity [VC]). Orthotopic left lung transplantation using cuff techniques was performed in syngeneic Lewis rats. Posttransplant allograft function, expression of proinflammatory mediators, and expression of lung surfactants were evaluated. RESULTS: Lungs inflated to 75 or 100% VC showed a significantly better oxygenation in blood gas analysis than lungs inflated to 25 or 50% VC. The levels of mRNAs for tumor necrosis factor-α, pro-interleukin-1ß, intracellular adhesion molecule 1 were attenuated in lungs inflated to 75 or 100% VC as compared with deflated lungs, suggesting reduced ischemia/reperfusion injury. In addition, transmission electron microscopy demonstrated better preserved lung surfactants in the alveolar space in the lungs inflated to 75 or 100% VC. CONCLUSIONS: Inflating lungs to 75 or 100% VC during preservation may be beneficial and result in better posttransplant allograft function through attenuated reperfusion injury and better preserved lung surfactants.

Human dominant-negative class II transactivator transgenic pigs - effect on the human anti-pig T-cell immune response and immune status.

Swine leucocyte antigen (SLA) class II molecules on porcine (p) cells play a crucial role in xenotransplantation as activators of recipient human CD4(+) T cells. A human dominant-negative mutant class II transactivator (CIITA-DN) transgene under a CAG promoter with an endothelium-specific Tie2 enhancer was constructed. CIITA-DN transgenic pigs were produced by nuclear transfer/embryo transfer. CIITA-DN pig cells were evaluated for expression of SLA class II with/without activation, and the human CD4(+) T-cell response to cells from CIITA-DN and wild-type (WT) pigs was compared. Lymphocyte subset numbers and T-cell function in CIITA-DN pigs were compared with those in WT pigs. The expression of SLA class II on antigen-presenting cells from CIITA-DN pigs was significantly reduced (40-50% reduction compared with WT; P < 0·01), and was completely suppressed on aortic endothelial cells (AECs) even after activation (100% suppression; P < 0·01). The human CD4(+) T-cell response to CIITA-DN pAECs was significantly weaker than to WT pAECs (60-80% suppression; P < 0·01). Although there was a significantly lower frequency of CD4(+) cells in the PBMCs from CIITA-DN (20%) than from WT (30%) pigs (P < 0·01), T-cell proliferation was similar, suggesting no significant immunological compromise. Organs and cells from CIITA-DN pigs should be partially protected from the human cellular immune response.

IL-33 expands suppressive CD11b+ Gr-1(int) and regulatory T cells, including ST2L+ Foxp3+ cells, and mediates regulatory T cell-dependent promotion of cardiac allograft survival.

IL-33 administration is associated with facilitation of 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 (Tregs) in mice. IL-33 expands functional myeloid-derived suppressor cells, CD11b(+) cells that exhibit intermediate (int) levels of Gr-1 and potent T cell suppressive function. Furthermore, IL-33 administration causes an St2-dependent expansion of suppressive CD4(+) Foxp3(+) Tregs, including an ST2L(+) population. IL-33 monotherapy after 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-1(hi) cells, IL-33-treated recipients exhibited increased CD11b(+) Gr-1(int) 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 Tregs. 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(+) Tregs that underlie IL-33-mediated cardiac allograft survival.

Tissue biopsy monitoring of operational tolerance in liver allograft recipients.

PURPOSE OF REVIEW: Highly selected, long-surviving, liver allograft recipients with normal/near normal liver injury tests can be weaned from immunosuppression. Baseline biopsies document changes before weaning and can help stratify risk of rejection or dysfunction after weaning; biopsies after weaning are used to study mechanisms of operational tolerance and to monitor for subclinical events. RECENT FINDINGS: Clinicopathological features associated with successful weaning include a lack of sensitization [negative donor-specific antibodies (DSA) and lack of tissue C4d deposits]; 'inexperienced' recipient immune system with limited potential for cross-reactivity (less immunological memory; infant recipients); noninflamed allograft in those with nonviral, nonimmunological original diseases; upregulation of liver genes associated with iron metabolism; allograft colonization with 'immunosuppressive' cells (Treg and γδ-1>γδ-2); and longer time on immunosuppression, which might signal slow clonal deletion or silencing. The differential diagnosis of histopathological findings detected before and after weaning includes emerging infections, typical and atypical cellular rejection, indolent antibody-mediated rejection, 'autoimmunity', and other causes of progressive fibrosis. SUMMARY: Operationally tolerant liver allograft recipients can be successfully managed with very low, and sometimes no immunosuppression, but challenges exist. Newer approaches to tissue pathology and tissue, serum, and cross-platform analytics are needed to predict successful weaning and to monitor for subclinical events.

SPRR2A enhances p53 deacetylation through HDAC1 and down regulates p21 promoter activity.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Profiling molecular changes induced by hydrogen treatment of lung allografts prior to procurement.

BACKGROUND: We previously demonstrated that donor treatment with inhaled hydrogen protects lung grafts from cold ischemia/reperfusion (I/R) injury during lung transplantation. To elucidate the mechanisms underlying hydrogen's protective effects, we conducted a gene array analysis to identify changes in gene expression associated with hydrogen treatment. METHODS: Donor rats were exposed to mechanical ventilation with 98% oxygen and 2% nitrogen or 2% hydrogen for 3 h before harvest; lung grafts were stored for 4h in cold Perfadex. Affymetrix gene array analysis of mRNA transcripts was performed on the lung tissue prior to implantation. RESULTS: Pretreatment of donor lungs with hydrogen altered the expression of 229 genes represented on the array (182 upregulated; 47 downregulated). Hydrogen treatment induced several lung surfactant-related genes, ATP synthase genes and stress-response genes. The intracellular surfactant pool, tissue adenosine triphosphate (ATP) levels and heat shock protein 70 (HSP70) expression increased in the hydrogen-treated grafts. Hydrogen treatment also induced the transcription factors C/EBPα and C/EBPß, which are known regulators of surfactant-related genes. CONCLUSION: Donor ventilation with hydrogen significantly increases expression of surfactant-related molecules, ATP synthases and stress-response molecules in lung grafts. The induction of these molecules may underlie hydrogen's protective effects against I/R injury during transplantation.

Hepatic B7 homolog 1 expression is essential for controlling cold ischemia/reperfusion injury after mouse liver transplantation.

UNLABELLED: Ischemia/reperfusion (I/R) injury remains a key risk factor significantly affecting morbidity and mortality after liver transplantation (LT). B7 homolog 1 (B7-H1), a 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 using B7-H1 knockout (KO) liver grafts, we tested this hypothesis in the mouse LT model with 24 hours of cold storage. Cold I/R injury in wild type (WT)-to-WT LT 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 alanine aminotransferase (ALT) and graft necrosis levels were significantly higher than those after WT-to-WT LT. Augmented tissue injury in B7-H1 KO grafts was associated with increased frequencies and absolute numbers of graft CD3(+) T cells (particularly CD8(+) T cells). B7-H1 KO grafts had significantly fewer annexin V(+) CD8(+) T cells, and this indicated a failure to delete infiltrating CD8(+) T cells. To evaluate the relative contributions of parenchymal cell and bone marrow-derived cell (BMDC) B7-H1 expression, we generated and transplanted into WT recipients chimeric liver grafts lacking B7-H1 on parenchymal cells or BMDCs. A selective B7-H1 deficiency on parenchymal cells or BMDCs resulted in similar levels of ALT and liver injury, and this suggested that parenchymal cell and BMDC B7-H1 expression was involved in liver damage control. Human livers up-regulated B7-H1 expression after LT. CONCLUSION: The study demonstrates that graft tissue expression of B7-H1 plays a critical role in regulating inflammatory responses during LT-induced hepatic I/R injury, and negative coregulatory signals may have an important function in hepatic innate immune responses.

ABBV-011, A Novel, Calicheamicin-Based Antibody-Drug Conjugate, Targets SEZ6 to Eradicate Small Cell Lung Cancer Tumors.

In the past year, four antibody-drug conjugates (ADC) were approved, nearly doubling the marketed ADCs in oncology. Among other attributes, successful ADCs optimize targeting antibody, conjugation chemistry, and payload mechanism of action. Here, we describe the development of ABBV-011, a novel SEZ6-targeted, calicheamicin-based ADC for the treatment of small cell lung cancer (SCLC). We engineered a calicheamicin conjugate that lacks the acid-labile hydrazine linker that leads to systemic release of a toxic catabolite. We then screened a patient-derived xenograft library to identify SCLC as a tumor type with enhanced sensitivity to calicheamicin ADCs. Using RNA sequencing (RNA-seq) data from primary and xenograft SCLC samples, we identified seizure-related homolog 6 (SEZ6) as a surface-expressed SCLC target with broad expression in SCLC and minimal normal tissue expression by both RNA-seq and IHC. We developed an antibody targeting SEZ6 that is rapidly internalized upon receptor binding and, when conjugated to the calicheamicin linker drug, drives potent tumor regression in vitro and in vivo. These preclinical data suggest that ABBV-011 may provide a novel treatment for patients with SCLC and a rationale for ongoing phase I studies (NCT03639194).

Evaluation of the DLL3-targeting antibody-drug conjugate rovalpituzumab tesirine in preclinical models of neuroblastoma.

Neuroblastomas have neuroendocrine features and often show similar gene expression patterns to small cell lung cancer including high expression of delta-like ligand 3 (DLL3). Here we determine the efficacy of rovalpituzumab tesirine (Rova-T), an antibody drug conjugated (ADC) with a pyrrolobenzodiazepine (PBD) dimer toxin targeting DLL3, in preclinical models of human neuroblastoma. We evaluated DLL3 expression in RNA sequencing data sets and performed immunohistochemistry (IHC) on neuroblastoma patient derived xenograft (PDX), human neuroblastoma primary tumor and normal childhood tissue microarrays (TMAs). We then evaluated the activity of Rova-T against 11 neuroblastoma PDX models using varying doses and schedules and compared anti-tumor activity to expression levels. DLL3 mRNA was differentially overexpressed in neuroblastoma at comparable levels to small cell lung cancer, as well as Wilms and rhabdoid tumors. DLL3 protein was robustly expressed across the neuroblastoma PDX array, but membranous staining was variable. The human neuroblastoma array, however, showed staining in only 44% of cases, whereas no significant staining was observed in the normal childhood tissue array. Rova-T showed a clear dose response effect across the 11 models tested, with a single dose inducing a complete or partial response in 3/11 and stable disease in another 3/11 models. No overt signs of toxicity were observed, and there was no treatment-related mortality. Strong membranous staining was necessary, but not sufficient, for anti-tumor activity. Rova-T has activity in a subset of neuroblastoma preclinical models, but heterogeneous expression in these models and the near absence of expression seen in human tumors suggests that any DLL3-targeting clinical trial should be only performed with a robust companion diagnostic to evaluate DLL3 expression for patient selection.

Estrogen stimulates female biliary epithelial cell interleukin-6 expression in mice and humans.

UNLABELLED: Females are more susceptible than males to several biliary tract diseases. Interleukin-6 (IL-6) is critical to triggering autoimmune reactions and contributes substantially to biliary epithelial cell (BEC) barrier function and wound repair, and estrogen differentially regulates IL-6 expression in various cell types. We hypothesized that estrogen might stimulate BEC IL-6 production. Exposure to physiologic levels of estradiol, in vitro, increased female mouse BEC (mBEC) IL-6 messenger RNA (mRNA) and protein expression, but either inhibited or had no effect on male mBECs. Female mBECs expressed higher concentrations of estrogen receptor-alpha (ERalpha) mRNA and protein and were also more dependent on estradiol for survival, in vitro. In vivo, elevated estrogen during estrous cycling in mice, and estrogen treatment of mice harboring an ERalpha(+) human cholangiocarcinoma resulted in increased BEC IL-6 mRNA and tumor viability, respectively. Both responses could be blocked by an ERalpha antagonist. Human cholangiocarcinoma cell lines differentially expressing ERalpha were treated with specific ERalpha and ERbeta agonists/antagonists to further test the relationship between estrogen stimulation, ERalpha expression, and IL-6 production. Results show that ERalpha, and not the underlying BEC sex, was responsible for estrogen-induced IL-6 production. Estrogen-induced proliferation of ERalpha-expressing cholangiocarcinoma was blocked by anti-IL-6 antibodies, indicating that at least some of the estrogen-trophic effects are mediated via IL-6. Finally, an association between ERalpha, IL-6, and phosphorylated signal transducer and activator of transcription 3 (pSTAT3) signaling was shown in female-predominant polycystic livers using immunohistochemical analyses, including multiplex quantum dot labeling. CONCLUSION: Estrogens stimulate IL-6 production in non-neoplastic female BECs and in neoplastic BECs expressing ERalpha. An association between these signaling pathways was demonstrated for female-predominant polycystic livers and might also influence autoimmune hepatitis, primary biliary cirrhosis, and cholangiocarcinogenesis.

Cyclooxygenase-2 prevents fas-induced liver injury through up-regulation of epidermal growth factor receptor.

UNLABELLED: Cyclooxygenase-2 (COX-2)-derived prostaglandins participate in a number of pathophysiological responses such as inflammation, carcinogenesis, and modulation of cell growth and survival. This study used complementary approaches of COX-2 transgenic (Tg) and knockout (KO) mouse models to evaluate the mechanism of COX-2 in Fas-induced hepatocyte apoptosis and liver failure in vivo. We generated Tg mice with targeted expression of COX-2 in the liver by using the albumin promoter-enhancer-driven vector. The COX-2 Tg, COX-2 KO, and wild-type mice were treated with the anti-Fas antibody Jo2 (0.5 microg/g of body weight) for 4 to 6 hours, and the extent of liver injury was assessed by histopathology, serum aminotransferases, TUNEL staining, and caspase activation. The COX-2 Tg mice showed resistance to Fas-induced liver injury in comparison with the wild-type mice; this was reflected by the lower alanine aminotransferase (ALT) and aspartate aminotransferase (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, caspase-9, and caspase-3 activity than the wild-type mice (P < 0.01). The COX-2 Tg livers expressed higher levels of epidermal growth factor receptor (EGFR) than the wild-type controls; the COX-2 KO livers expressed the lowest levels of EGFR. Pretreatment with a COX-2 inhibitor (NS-398) or an EGFR inhibitor (AG1478) exacerbated Jo2-mediated liver injury and hepatocyte apoptosis. CONCLUSION: These findings demonstrate that COX-2 prevents Fas-induced hepatocyte apoptosis and liver failure at least in part through up-regulation of EGFR.