Carcinoembryonic antigen-expressing oncolytic measles virus derivative in recurrent glioblastoma: a phase 1 trial.

Measles virus (MV) vaccine strains have shown significant preclinical antitumor activity against glioblastoma (GBM), the most lethal glioma histology. In this first in human trial (NCT00390299), a carcinoembryonic antigen-expressing oncolytic measles virus derivative (MV-CEA), was administered in recurrent GBM patients either at the resection cavity (Group A), or, intratumorally on day 1, followed by a second dose administered in the resection cavity after tumor resection on day 5 (Group B). A total of 22 patients received study treatment, 9 in Group A and 13 in Group B. Primary endpoint was safety and toxicity: treatment was well tolerated with no dose-limiting toxicity being observed up to the maximum feasible dose (2×107 TCID50). Median OS, a secondary endpoint, was 11.6 mo and one year survival was 45.5% comparing favorably with contemporary controls. Other secondary endpoints included assessment of viremia, MV replication and shedding, humoral and cellular immune response to the injected virus. A 22 interferon stimulated gene (ISG) diagonal linear discriminate analysis (DLDA) classification algorithm in a post-hoc analysis was found to be inversely (R = -0.6, p = 0.04) correlated with viral replication and tumor microenvironment remodeling including proinflammatory changes and CD8 + T cell infiltration in post treatment samples. This data supports that oncolytic MV derivatives warrant further clinical investigation and that an ISG-based DLDA algorithm can provide the basis for treatment personalization.

Preclinical safety assessment of MV-s-NAP, a novel oncolytic measles virus strain armed with an H . pylori immunostimulatory bacterial transgene.

Despite recent therapeutic advances, metastatic breast cancer (MBC) remains incurable. Engineered measles virus (MV) constructs based on the attenuated MV Edmonston vaccine platform have demonstrated significant oncolytic activity against solid tumors. The Helicobacter pylori neutrophil-activating protein (NAP) is responsible for the robust inflammatory reaction in gastroduodenal mucosa during bacterial infection. NAP attracts and activates immune cells at the site of infection, inducing expression of pro-inflammatory mediators. We engineered an MV strain to express the secretory form of NAP (MV-s-NAP) and showed that it exhibits anti-tumor and immunostimulatory activity in human breast cancer xenograft models. In this study, we utilized a measles-infection-permissive mouse model (transgenic IFNAR KO-CD46Ge) to evaluate the biodistribution and safety of MV-s-NAP. The primary objective was to identify potential toxic side effects and confirm the safety of the proposed clinical doses of MV-s-NAP prior to a phase I clinical trial of intratumoral administration of MV-s-NAP in patients with MBC. Both subcutaneous delivery (corresponding to the clinical trial intratumoral administration route) and intravenous (worst case scenario) delivery of MV-s-NAP were well tolerated: no significant clinical, laboratory or histologic toxicity was observed. This outcome supports the safety of MV-s-NAP for oncolytic virotherapy of MBC. The first-in-human clinical trial of MV-s-NAP in patients with MBC (ClinicalTrials.gov: NCT04521764) was subsequently activated.

Immunostimulatory bacterial antigen-armed oncolytic measles virotherapy significantly increases the potency of anti-PD1 checkpoint therapy.

Clinical immunotherapy approaches are lacking efficacy in the treatment of glioblastoma (GBM). In this study, we sought to reverse local and systemic GBM-induced immunosuppression using the Helicobacter pylori neutrophil-activating protein (NAP), a potent TLR2 agonist, as an immunostimulatory transgene expressed in an oncolytic measles virus (MV) platform, retargeted to allow viral entry through the urokinase-type plasminogen activator receptor (uPAR). While single-agent murine anti-PD1 treatment or repeat in situ immunization with MV-s-NAP-uPA provided modest survival benefit in MV-resistant syngeneic GBM models, the combination treatment led to synergy with a cure rate of 80% in mice bearing intracranial GL261 tumors and 72% in mice with CT-2A tumors. Combination NAP-immunovirotherapy induced massive influx of lymphoid cells in mouse brain, with CD8+ T cell predominance; therapeutic efficacy was CD8+ T cell dependent. Inhibition of the IFN response pathway using the JAK1/JAK2 inhibitor ruxolitinib decreased PD-L1 expression on myeloid-derived suppressor cells in the brain and further potentiated the therapeutic effect of MV-s-NAP-uPA and anti-PD1. Our findings support the notion that MV strains armed with bacterial immunostimulatory antigens represent an effective strategy to overcome the limited efficacy of immune checkpoint inhibitor-based therapies in GBM, creating a promising translational strategy for this lethal brain tumor.

Parameters of immunoglobulin extraction from dried blood spot cards and immunoassays for detection of antibody response to pathogens including the novel SARS-CoV-2.

Dried blood spots (DBS) are routinely used in screening newborns for treatable disorders. Immunoglobulin extraction from DBS, serum or other biological fluids loaded on filter paper cards could represent a valuable method of specimen preservation in monitoring immune response against pathogens as well as vaccination efficiency. In this study using different sources including serum, and monoclonal antibodies we established parameters for antibody extraction from the filter cards to assess antibody reactivity against Helicobacter pylori, measles virus (MV) and the novel coronavirus SARS-CoV-2 antigens. We demonstrated that DBS and dried undiluted serum result in completely preserved antibody activity for immunoassays, including in virus neutralization assays against MV. Extraction efficiency was determined by IgG concentration measurements. The plaque-reduction neutralization titer 50% of dried human serum spots remained stable after more than 10-day storage - 1:359 vs. 1:345 for the corresponding frozen sample. DBSs could be used to monitor immune response to bacterial and viral antigens following natural exposure or immunization. Mice immunized with recombinant spike protein receptor-binding domain of SARS-CoV-2 developed a strong antibody response by day 14 and reached titers above 1:64,000 on day 21 following the secondary boost immunization as measured on DBS samples in antigen-mediated ELISA. Variability in IgG concentration of eluted DBS could be influenced by factors involved in sample application, extraction process and sample characteristics. Adjustment of antibody specific activity to the eluted IgG concentration can increase accuracy of the result interpretation, including in SARS-CoV-2 serological diagnostics.

Live Attenuated Measles Virus Vaccine Expressing Helicobacter pylori Heat Shock Protein A.

Measles virus (MV) Edmonston derivative strains are attractive vector platforms in vaccine development and oncolytic virotherapy. Helicobacter pylori heat shock protein A (HspA) is a bacterial heat shock chaperone with essential function as a Ni-ion scavenging protein. We generated and characterized the immunogenicity of an attenuated MV strain encoding the HspA transgene (MV-HspA). MV-HspA showed faster replication within 48 h of infection with >10-fold higher titers and faster accumulation of the MV proteins. It also demonstrated a superior tumor-killing effect in vitro against a variety of human solid tumor cell lines, including sarcoma, ovarian and breast cancer. Two intraperitoneal (i.p.) doses of 106 50% tissue culture infectious dose (TCID50) MV-HspA significantly improved survival in an ovarian cancer xenograft model: 63.5 days versus 27 days for the control group. The HspA transgene induced a humoral immune response in measles-permissive Ifnarko-CD46Ge transgenic mice. Eight of nine animals developed a long-term anti-HspA antibody response with titers of 1:400 to 1:12,800 without any negative impact on development of protective anti-MV immune memory. MV-HspA triggered an immunogenic cytopathic effect as measured by an HMGB1 assay. The absence of significant elevation of PD-L1 expression indicated that vector-encoded HspA could act as an immunomodulator on the immune check point axis. These data demonstrate that MV-HspA is a potent oncolytic agent and vaccine candidate for clinical translation in cancer treatment and immunoprophylaxis against H. pylori.

Diet Mimicking "Fast Food" Causes Structural Changes to the Retina Relevant to Age-Related Macular Degeneration.

Introduction: Metabolic syndrome is a disorder characterized by a constellation of findings including truncal obesity, elevated blood pressure, abnormal cholesterol levels, and high blood glucose. Recent evidence suggests that metabolic syndrome may be associated with increased risk of age-related macular degeneration (AMD) and other eye diseases. Recently, C57BL/6J wild-type mice fed with a "fast food" diet consisting of high fat, cholesterol, and fructose-supplemented water showed unique systemic pathology consistent with metabolic syndrome and nonalcoholic steatohepatitis. Additionally, these mice showed higher levels of fibrosis, inflammation, endoplasmic reticulum stress, and mitochondrial dysfunction compared to mice fed with only a high-fat diet alone. Since similar pathways are activated in AMD, we sought to determine whether mice fed a "fast food" diet exhibited retinal changes.Methods: 3-month-old wild-type mice were randomized to a standard chow (n = 11) or a "fast food" (n = 18) diet and fed for 9 months. At 1 year of age, tissues were collected and retinas were analyzed using transmission electron microscopy. Quantitative measures of Bruch's membrane thickness and retinal pigment epithelium (RPE) cell counts were performed.Results: "Fast food" fed mice showed ocular pathology relevant to various stages of AMD including basal laminar deposits, focal thickening of Bruch's membrane, and a significant loss of RPE cells.Discussion/conclusion: A wild-type mouse model of metabolic syndrome fed a "fast food" diet developed changes to the retina similar to some of the pathologic features seen in AMD. Further investigations into this and similar animal models as well as further epidemiological studies are needed to more clearly define the association between metabolic syndrome and AMD.

Stanniocalcin-1 Is an Ocular Hypotensive Agent and a Downstream Effector Molecule That Is Necessary for the Intraocular Pressure-Lowering Effects of Latanoprost.

Purpose: To identify downstream signaling molecules through which intraocular pressure (IOP) is lowered following treatment with the prostaglandin analog latanoprost. Methods: Total RNA and protein isolated from primary human Schlemm's canal cells (n = 3) treated with latanoprost (free acid; 100 nM) were processed for quantitative PCR and Western blot analysis. IOP was evaluated in stanniocalcin-1 (STC-1-/-) and wild-type mice following treatment with latanoprost or Rho kinase inhibitor Y27632. Human anterior segment pairs (n = 8) were treated with recombinant STC-1 (5, 50, or 500 ng/mL) and pressure was recorded using custom-designed software. The effect of recombinant STC-1 (0.5 mg/mL) on IOP was evaluated in wild-type mice. Tissue morphology was evaluated by light and transmission electron microscopy. Results: Increased STC-1 mRNA (4.0- to 25.2-fold) and protein expression (1.9- to 5.1-fold) was observed within 12 hours following latanoprost treatment. Latanoprost reduced IOP in wild-type mice (22.0% ± 1.9%), but had no effect on STC-1-/- mice (0.5% ± 0.7%). In contrast, Y27632 reduced IOP in both wild-type (12.5% ± 1.2%) and in STC-1-/- mice (13.1% ± 2.8%). Human anterior segments treated with STC-1 (500 ng/mL) showed an increase in outflow facility (0.15 ± 0.03 to 0.27 ± 0.09 µL/min/mm Hg) while no change was observed in paired vehicle-treated controls. Recombinant STC-1 reduced IOP in wild-type mice by 15.2% ± 3.0%. No observable morphologic changes were identified between treatment groups when evaluated by microscopy. Conclusions: Latanoprost-induced reduction of IOP is mediated through the downstream signaling molecule STC-1. When used by itself, STC-1 exhibits ocular hypotensive properties.

Analogs of the ATP-Sensitive Potassium (KATP) Channel Opener Cromakalim with in Vivo Ocular Hypotensive Activity.

ATP-sensitive potassium (KATP) channel openers have emerged as potential therapeutics for the treatment of glaucoma, lowering intraocular pressure (IOP) in animal models and cultured human anterior segments. We have prepared water-soluble phosphate and dipeptide derivatives of the KATP channel opener cromakalim and evaluated their IOP lowering capabilities in vivo. In general, the phosphate derivatives proved to be more chemically robust and efficacious at lowering IOP with once daily dosing in a normotensive mouse model. Two of these phosphate derivatives were further evaluated in a normotensive rabbit model, with a significant difference in activity observed. No toxic effects on cell structure or alterations in morphology of the aqueous humor outflow pathway were observed after treatment with the most efficacious compound, (3S,4R)-2, suggesting that it is a strong candidate for development as an ocular hypotensive agent.

Correlation of chromosomal instability, telomere length and telomere maintenance in microsatellite stable rectal cancer: a molecular subclass of rectal cancer.

INTRODUCTION: Colorectal cancer (CRC) tumor DNA is characterized by chromosomal damage termed chromosomal instability (CIN) and excessively shortened telomeres. Up to 80% of CRC is microsatellite stable (MSS) and is historically considered to be chromosomally unstable (CIN+). However, tumor phenotyping depicts some MSS CRC with little or no genetic changes, thus being chromosomally stable (CIN-). MSS CIN- tumors have not been assessed for telomere attrition. EXPERIMENTAL DESIGN: MSS rectal cancers from patients ≤50 years old with Stage II (B2 or higher) or Stage III disease were assessed for CIN, telomere length and telomere maintenance mechanism (telomerase activation [TA]; alternative lengthening of telomeres [ALT]). Relative telomere length was measured by qPCR in somatic epithelial and cancer DNA. TA was measured with the TRAPeze assay, and tumors were evaluated for the presence of C-circles indicative of ALT. p53 mutation status was assessed in all available samples. DNA copy number changes were evaluated with Spectral Genomics aCGH. RESULTS: Tumors were classified as chromosomally stable (CIN-) and chromosomally instable (CIN+) by degree of DNA copy number changes. CIN- tumors (35%; n=6) had fewer copy number changes (<17% of their clones with DNA copy number changes) than CIN+ tumors (65%; n=13) which had high levels of copy number changes in 20% to 49% of clones. Telomere lengths were longer in CIN- compared to CIN+ tumors (p=0.0066) and in those in which telomerase was not activated (p=0.004). Tumors exhibiting activation of telomerase had shorter tumor telomeres (p=0.0040); and tended to be CIN+ (p=0.0949). CONCLUSIONS: MSS rectal cancer appears to represent a heterogeneous group of tumors that may be categorized both on the basis of CIN status and telomere maintenance mechanism. MSS CIN- rectal cancers appear to have longer telomeres than those of MSS CIN+ rectal cancers and to utilize ALT rather than activation of telomerase.

Lumican, an extracellular matrix proteoglycan, is a novel requisite for hepatic fibrosis.

Lumican, an extracellular matrix proteoglycan was previously shown to be upregulated with increasing severity of nonalcoholic steatohepatitis (NASH). Although lumican is involved in collagen fibrillogenesis in extra-hepatic tissues, little is known about the role of lumican in hepatic disease. We therefore determined lumican expression in etiologies other than clinical NASH. Our results indicated that lumican is upregulated in clinical samples of hepatitis C virus infection, in experimental rodent models of chronic and acute liver injury and could additionally be induced in vitro in response to the pro-fibrotic cytokine transforming growth factor ß1 (TGFß1) and to lipotoxic palmitic acid. Together, these results suggested a role for lumican in hepatic fibrosis. To investigate the functional role of lumican in hepatic fibrosis, lumican null (Null) and wild-type (WT) littermates were administered carbon tetrachloride intra-peritoneally. Serum and liver tissue were analyzed for indices of liver injury, fibrosis, matrix turnover, and proliferation. Hepatic fibrosis was greatly reduced in null animals (P<0.05). Paradoxically, gene expression of fibrosis-related genes such as TGFß1 and collagen 1 was numerically higher in null animals though statistically insignificant from WT animals. On the other hand, α smooth muscle actin expression (α-SMA), a marker for activated fibroblasts, the main contributors of collagen production was significantly higher (P<0.05) in null animals as compared with WT littermates. Among the matrix metalloproteases (MMP), MMP13 was significantly increased (P<0.05) in null animals. Ultra-structural imaging indicated differences in the organization and spatial distribution of hepatic collagen fibrils of null and WT mice. Cell proliferation was significantly increased (P<0.05) in null animals. We conclude that lumican is a prerequisite for hepatic fibrosis. The protective effect of lumican deficiency in hepatic fibrosis appears to be downstream of collagen production and mediated through the combined effects of impaired collagen fibrillogenesis, increased matrix turnover, and an enhanced proliferative response.

Fast food diet mouse: novel small animal model of NASH with ballooning, progressive fibrosis, and high physiological fidelity to the human condition.

Although there are small animal platforms that recapitulate some of the histological features of nonalcoholic fatty liver disease, there are no small animal models of nonalcoholic steatohepatitis (NASH) with consistent hepatocellular ballooning and progressive fibrosis that also exhibit fidelity to the human condition physiologically. We examined the metabolic and histological effects of a diet on the basis of the composition of "fast food" (high saturated fats, cholesterol, and fructose). Mice (n = 8 in each group) were assigned to diets as follows: 1) standard chow (SC), i.e., 13% energy as fat [1% saturated fatty acids (SFA)], 2) high fat (HF), i.e., 60% energy as fat (1% SFA), and 3) fast food (FF), i.e., 40% energy as fat (12% SFA, 2% cholesterol). All three diets were supplemented with high fructose. All diets produced obesity. The HF and FF diets produced insulin resistance. Liver histology was normal in animals fed the SC diet. Steatohepatitis with pronounced ballooning and progressive fibrosis (stage 2) was observed in mice fed the FF diet. Although the HF diet produced obesity, insulin resistance, and some steatosis; inflammation was minimal, and there was no increase in fibrosis. The FF diet produced a gene expression signature of increased fibrosis, inflammation, and endoplasmic reticulum stress and lipoapoptosis. A diet based on high cholesterol, high saturated fat, and high fructose recapitulates features of the metabolic syndrome and NASH with progressive fibrosis. This represents a novel small animal model of fibrosing NASH with high fidelity to the human condition. These results highlight the contribution of dietary composition to the development of nonalcoholic fatty liver disease and NASH.

Noninvasive diagnosis of acute cellular rejection in liver transplant recipients: a proteomic signature validated by enzyme-linked immunosorbent assay.

The diagnosis of acute cellular rejection (ACR) requires liver biopsy with its attendant expense and risk. Our first aim was to prospectively determine in an exploratory analysis whether there is a serum proteome signature associated with histologically confirmed ACR. Our second aim was to use simpler and faster enzyme-linked immunosorbent assay (ELISA)-based assays for proteins identified as differentially abundant in the proteomic analysis to identify patients with ACR in a separate validation cohort. We used sequential high-abundance protein depletion and isobaric tag for relative and absolute quantitation liquid chromatography-tandem mass spectrometry to characterize the serum proteome in serum samples of patients with or without ACR. Seven of the 41 proteins identified as differentially abundant [serum amyloid A, complement component 4 (C4), fibrinogen, complement component 1q (C1q), complement component 3, heat shock protein 60 (HSP60), and HSP70] could be measured with ELISA-based assays in a validation cohort consisting of patients with ACR (n = 25) and patients without ACR (n = 21). The mean alanine aminotransferase (ALT) levels in patients with ACR and in patients without ACR were 198 ± 27 and 153 ± 34 U/L, respectively. Among the 7 proteins for which ELISA assays were available, C4 and C1q were both independent predictors of ACR. C4 had the greatest predictivity for differentiating patients with or without ACR. A C4 level ≤ 0.31 g/L had a sensitivity of 97%, a specificity of 62%, a positive predictive value of 74%, and a negative predictive value of 94%. A C4 level ≤ 0.31 g/L and an ALT level ≥ 70 IU/mL together had a sensitivity of 96%, a specificity of 81%, a positive predictive value of 86%, and a negative predictive value of 94%. In summary, in this exploratory analysis, serum C4 and ALT levels were highly predictive of ACR in liver transplant recipients. Confirmation in a prospective, larger, and diverse population is needed.

NAFLD and insulin resistance do not increase the risk of postoperative complications among patients undergoing bariatric surgery--a prospective analysis.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) and insulin resistance are common consequences of obesity and are highly prevalent among patients undergoing bariatric surgery. Insulin resistance and NAFLD have been reported to be associated with postoperative complications following major surgery. METHODS: We prospectively evaluated complications in a cohort of 437 consecutive patients undergoing bariatric surgery. Detailed metabolic profile was obtained prior to surgery, and liver biopsies were taken routinely during surgery. RESULTS: Mean age was 47.8 years (20-77), and mean body mass index (BMI) was 48.5 kg/m(2) (32-94). Common co-morbid conditions were metabolic syndrome (79%), obstructive sleep apnea (73%), and hypertension (60%). Seventy-seven percent underwent Roux-en-Y gastric bypass, 15% biliopancreatic diversion with duodenal switch, and 8% adjustable gastric banding. The operative approach was laparoscopic in 81% of patients. Liver histology was normal in 22% of patients. Seventy-eight percent of patients had NAFLD. Nonalcoholic steatohepatitis (NASH) was present in 18%. Advanced fibrosis (stage 3-4) was present in 5%. Complications were observed in 25% of the cohort, the most frequent being infection, occurring in 14%. The wound was the most frequent site of infection (9%). There were no cases of postoperative hepatic decompensation. Reoperation was necessary in 7%. In univariate analysis, the factors associated with complications were male gender (p = 0.009), type and approach of surgery (p = 0.023 and p = 0.0001, respectively), BMI (p = 0.000), serum creatinine (p = 0.023), and serum albumin (p = 0.0001). In multivariate analysis, the independent factors associated with complications in bariatric surgery were BMI (OR 1.039, 95% CI 1.010-1.068; p = 0.008), surgical approach (OR 2.696, 95% CI 1.547-4.698; p = 0.000), and serum albumin (OR 0.416, 95% CI 0.176-0.978; p = 0.044). NASH was not predictive of complications. CONCLUSIONS: Bariatric surgery is a safe and efficient treatment for obesity. The occurrence of NAFLD or NASH without portal hypertension should not preclude the procedure.

Differential expression of lumican and fatty acid binding protein-1: new insights into the histologic spectrum of nonalcoholic fatty liver disease.

UNLABELLED: The basis of hepatocellular injury and progressive fibrosis in a subset of patients with nonalcoholic fatty liver disease (NAFLD) is poorly understood. We sought to identify hepatic proteins that are differentially abundant across the histologic spectrum of NAFLD. Hepatic protein abundance was measured in liver samples from four groups (n = 10 each) of obese (body mass index >30 kg/m(2)) patients: (1) obese normal group (normal liver histology), (2) simple steatosis (SS), (3) nonalcoholic steatohepatitis (NASH)-mild (steatohepatitis with fibrosis stage 0-1), and (4) NASH-progressive (steatohepatitis with fibrosis stage 2-4). Hepatic peptides were analyzed on an API Qstar XL quadrupole time-of-flight mass spectrometer using Analyst QS software. Linear trends tests were performed and used to screen for differential abundance. Nine known proteins were expressed with differential abundance between study groups. For seven proteins differential abundance is likely to have been on the basis increased hepatic lipid content and/or inflammation. Lumican, a 40-kDa keratin sulfate proteoglycan that regulates collagen fibril assembly and activates transforming growth factor-beta and smooth muscle actin, was expressed similarly in obese normal and SS but was overexpressed in a progressive manner in NASH-mild versus SS (124%, P < 0.001), NASH-progressive versus NASH-mild (156%, P < 0.001) and NASH-progressive versus obese normal (178%, P < 0.001). Fatty acid binding protein-1 (FABP-1), which is protective against the detergent effects of excess free fatty acids, facilitates intracellular free fatty acid transport and is an important ligand for peroxisome proliferator-activated receptor-mediated transcription, was overexpressed in SS when compared to the obese normal group (128%, P < 0.001), but was paradoxically underexpressed in NASH-mild versus SS (73%, P < 0.001), NASH-progressive versus NASH-mild (81%, P < 0.001), and NASH-progressive versus obese normal (59%, P < 0.001). CONCLUSION: Histologically progressive NAFLD is associated with overexpression of lumican, an important mediator of fibrosis in nonhepatic tissues, whereas FABP-1 is paradoxically underexpressed in NASH, suggesting a new potential mechanism of lipotoxicity in NAFLD. Further studies are needed to determine the biologic basis of lumican and/or FABP-1 dysregulation in NAFLD.

Low circulating levels of dehydroepiandrosterone in histologically advanced nonalcoholic fatty liver disease.

UNLABELLED: The biological basis of variability in histological progression of nonalcoholic fatty liver disease (NAFLD) is unknown. Dehydroepiandrosterone (DHEA) is the most abundant steroid hormone and has been shown to influence sensitivity to oxidative stress, insulin sensitivity, and expression of peroxisome proliferator-activated receptor alpha and procollagen messenger RNA. Our aim was to determine whether more histologically advanced NAFLD is associated with low circulating levels of DHEA. Serum samples were obtained prospectively at the time of liver biopsy in 439 patients with NAFLD (78 in an initial and 361 in validation cohorts) and in controls with cholestatic liver disease (n = 44). NAFLD was characterized as mild [simple steatosis or nonalcoholic steatohepatitis (NASH) with fibrosis stage 0-2] or advanced (NASH with fibrosis stage 3-4). Serum levels of sulfated DHEA (DHEA-S) were measured by enzyme-linked immunosorbent assay. Patients with advanced NAFLD had lower plasma levels of DHEA-S than patients with mild NAFLD in both the initial (0.25 +/- 0.07 versus 1.1 +/- 0.09 microg/mL, P < 0.001) and validation cohorts (0.47 +/- 0.06 versus 0.99 +/- 0.04 microg/mL, P < 0.001). A "dose effect" of decreasing DHEA-S and incremental fibrosis stage was observed with a mean DHEA-S of 1.03 +/- 0.05, 0.96 +/- 0.07, 0.83 +/- 0.11, 0.66 +/- 0.11, and 0.35 +/- 0.06 microg/mL for fibrosis stages 0, 1, 2, 3, and 4, respectively. All patients in both cohorts in the advanced NAFLD group had low DHEA-S levels, with the majority in the hypoadrenal range. The association between DHEA-S and severity of NAFLD persisted after adjusting for age. A relationship between disease/fibrosis severity and DHEA-S levels was not seen in patients with cholestatic liver diseases. CONCLUSION: More advanced NAFLD, as indicated by the presence of NASH with advanced fibrosis stage, is strongly associated with low circulating DHEA-S. These data provide novel evidence for relative DHEA-S deficiency in patients with histologically advanced NASH.

Prolonged engraftment of human hepatocytes in mice transgenic for the deleted form of human hepatocyte growth factor.

AIM: Small animal models chimeric for human hepatocytes have provided valuable insights into the biology of hepatotropic viral infection and provided a platform for the study of therapeutic agents. Existing models of human hepatocyte transplantation are limited by phenotypic fragility and impaired immunity. We hypothesized that mice transgenic for human hepatocyte growth factor (HGF), a potent human hepatocyte mitogen, would engraft human hepatocytes in the absence of immunodeficiency. METHODS: A plasmid construct containing the 2.3 kb coding region of the 723 amino acid isoform of HGF cDNA under the transcriptional control of the mouse albumin promoter/enhancer was used to generate transgenic mice. Cryopreserved human hepatocytes were transplanted into nine transgenic and six non-transgenic mice. Engraftment of human hepatocytes was followed for a period of 12 weeks by immunoblotting for human albumin in mouse serum samples. RESULTS: In six out of the nine transgenic mice, abundance of human albumin, following an initial decline, increased andpeaked at > 70 days post transplantation, demonstrating sustained engraftment of transplanted human hepatocytes. In all the non-transgenic mice, post-transplant human albumin levels declined sequentially without evidence of sustained engraftment. Immunostaining of mouse liver sections indicated the presence of human hepatocytes adjacent to clusters of non-staining murine hepatocytes. CONCLUSION: These results demonstrate that sustained engraftment of human hepatocytes in mice is facilitated by expression of the human dHGF transgene. Human hepatocyte engraftment in this model has been achieved on an immunocompetent strain background and merits further study as a candidate for the study of hepatotropic viral infections.