Analysis of Post-Liver Transplant Hepatitis C Virus Recurrence Using Serial Cluster of Differentiation Antibody Microarrays.

BACKGROUND: Hepatitis C virus (HCV) reinfection of the liver allograft after transplantation is universal, with some individuals suffering severe disease recurrence. Predictive markers of recurrent disease severity are urgently needed. In this study, we used a cluster of differentiation (CD) microarray to predict the severity of HCV recurrence after transplantation. METHODS: The CD antibody microarray assays of live leukocytes were performed on peripheral blood taken in the first year after transplantation. The results were grouped into phases defined as; Pre-transplant (day 0), Early (day 3 to week 2), Mid (week 4 to week 10), and Late (week 12 to week 26). Hepatitis C virus severity was based on fibrosis stages in the first 2 years (F0-1 mild and F2-4 severe). RESULTS: Serial blood samples from 16 patients were taken before and after liver transplantation. A total of 98 assays were performed. Follow-up was 3 years or longer. Comparing recurrence severity, significantly greater numbers of CD antigens were differentially expressed on the pretransplant samples compared to any posttransplant timepoints. Five differentially expressed CD antigens before transplantation (CD27 PH, CD182, CD260, CD41, and CD34) were significantly expressed comparing severe to mild recurrence, whereas expression of only CD152 was significant in the late phase after transplantation. No relationship was observed between the donor or recipient interleukin-28B genotypes and HCV recurrence severity. CONCLUSIONS: This study shows that circulating leukocyte CD antigen expression has utility in assessing recurrent HCV disease severity after liver transplantation and serves as a proof of principle. Importantly, pretransplant CD antigen expression is most predictive of disease outcome.

Analysis of human liver disease using a cluster of differentiation (CD) antibody microarray.

BACKGROUND: A CD antibody microarray has been previously developed allowing semi-quantitative identification of greater than 80 CD antigens on circulating leucocytes from peripheral blood samples. This assay, which uses a live cell-capture technique, enables an extensive leucocyte immunophenotype determination in a single analysis and to date this has been used successfully to characterise diseases including human leukaemias and HIV infection. AIMS: To determine CD antigen expression profiles for patients with various liver diseases and to look for preserved disease-specific signatures. METHODS: Three liver disease groups including hepatitis C (HCV) (n = 35), non-alcoholic steatohepatitis (NASH) (n = 21) and alcohol-related liver disease (n = 14) were compared with a normal group (n = 23). Hierarchal Clustering (HCL) and Principal Component Analysis (PCA) of the data revealed distinct binding patterns for patients with and without cirrhosis. RESULTS: Patients with cirrhosis and portal hypertension compared with those without cirrhosis had significantly reduced expression of several markers of T-cell function including CD45, CD8, CD28 and TCR α/ß. Disease prediction algorithms based on the expression data were able to discriminate cirrhotics from non-cirrhotics with 71% overall success, which improved to 77% when only patients with HCV were considered. CONCLUSIONS: These results demonstrate disease-specific consensus patterns of expression of CD antigens for patients with chronic liver disease, suggesting that the CD antibody array is a promising tool in the analysis of human liver disease, and with further refinement may have future research and clinical utility.

Preclinical evaluation of novel, all-in-one formulations of 5-fluorouracil and folinic acid with reduced toxicity profiles.

5-Fluorouracil (5-FU) in combination with its synergistic biomodulator folinic acid maintains a pivotal position in cancer chemotherapy. However, clinical limitations such as phlebitis and catheter blockages persist with the administration of these drugs in combination, and are associated with reduced efficacy and/or quality of life for patients. We have reported earlier on the novel, all-in-one, pH neutral, parenteral 5-FU and folinic acid formulations (termed Fluorodex) incorporating ß-cyclodextrins. Fluorodex maintains potency while overcoming the accepted incompatibility of 5-FU and folinic acid. We carried out toxicological, pharmacokinetic and biodistribution, and efficacy evaluations of Fluorodex compared with 5-FU:folinic acid using several administration routes and schedules in two rodent models. These were compared with the dose-matched sequential administration of 5-FU:folinic acid. Fluorodex showed bioequivalence to 5-FU:folinic acid as assessed by the tissue distribution and pharmacokinetic studies of 5-FU, but was generally better tolerated as determined by weight loss, hematological, and other clinical parameters. Compared with 5-FU:folinic acid, Fluorodex was also associated with reduced phlebitis using a rabbit ear vein model. Furthermore, using human carcinoma tumor models in mice, Fluorodex resulted in equivalent or improved efficacy profiles compared with 5-FU:folinic acid. In conclusion, these novel, all-in-one formulations represent a superior injectable form of 5-FU that allows codelivery of folinic acid. This should translate into improved patient tolerability with potential for enhanced efficacy.

The application of CD antigen proteomics to pharmacogenomics.

The advent of multiplexing technologies has raised the possibility that disease states can be defined using discrete genomic and proteomic patterns or signatures. However, this emerging area has been limited by the 'content problem', arising from the uncertainty of which molecules to focus on. The human cluster of differentiation (CD) antigens are expressed on cells of the human immune system (leukocytes) and on other cell types. These heterogeneous molecules perform a host of roles essential to immune function and to the physiology of other lineages. The 339 defined CD antigens and their, as yet, undefined counterparts constitute key components of the expressed human cell surface proteome. We propose that CD antigen expression patterns will form the basis of a rational, discrete and generalized diagnostic and prognostic system. Furthermore, disease-specific CD antigen proteomic signatures are likely to be more robust than corresponding genomic signatures and will also help to identify molecular targets for therapeutic intervention.

Analysis of human leukaemias and lymphomas using extensive immunophenotypes from an antibody microarray.

A novel antibody microarray has been developed that provides an extensive immunophenotype of leukaemia cells. The assay is a solid phase cell-capture technique in which 82 antigens are studied simultaneously. This paper presents the analysis of 733 patients with a variety of leukaemias and lymphomas from peripheral blood and bone marrow. Discriminant Function Analysis of the expression profiles from these 733 patients and 63 normal subjects were clustered and showed high levels of consistency with diagnoses obtained using conventional clinical and laboratory criteria. The overall levels of consensus for classification using the microarray compared with established criteria were 93.9% (495/527 patients) for peripheral blood and 97.6% (201/206 patients) for bone marrow aspirates, showing that the extensive phenotype alone was frequently able to classify the disease when the leukaemic clone was the dominant cell population present. Immunophenotypes for neoplastic cells were distinguishable from normal cells when the leukaemic cell count was at least 5 x 10(9) cells/l in peripheral blood, or 20% of cells obtained from bone marrow aspirates. This technique may be a useful adjunct to flow cytometry and other methods when an extensive phenotype of the leukaemia cell is desired for clinical trials, research and prognostic factor analysis.

Screening microarrays of novel monoclonal antibodies for binding to T-, B- and myeloid leukaemia cells.

We have developed a microarray (DotScan) that enables rapid immunophenotyping and classification of leukaemias and lymphomas by measuring the capture of cells by immobilized dots of 82 CD antibodies [Belov, L., de la Vega, O., dos Remedios, C.G., Mulligan, S.P., 2001. Immunophenotyping of leukemia using a cluster of differentiation antibody microarray. Cancer Res. 61, 4483; Belov, L., Huang, P., Barber, N., Mulligan, S.P., Christopherson, R.I., 2003. Identification of repertoires of surface antigens on leukemias using an antibody microarray. Proteomics 3, 2147]. The DotScan technology has been used to investigate the properties of 498 new antibodies submitted to the HLDA8 Workshop. These antibodies have been applied as 10 nl dots to a film of nitrocellulose on a microscope slide to make an HLDA8 microarray. After blocking the remaining nitrocellulose surface, individual arrays were incubated with each of 7 cell types from a human leukaemia cell panel consisting of three cell lines, CCRF-CEM (a T-cell acute lymphocytic leukaemia), MEC-1 (derived from B-cell chronic lymphocytic leukaemia) and HL-60 (a promyelocytic leukaemia), and four leukaemias from patients: a T-cell prolymphocytic leukaemia, a B-cell chronic lymphocytic leukaemia, and two acute myeloid leukaemias. Leukaemia cells were captured by those immobilized antibodies for which they expressed the corresponding surface molecule. Unbound cells were gently washed off, bound cells were fixed to the arrays and dot patterns were recorded using a DotScan array reader and quantified using DotScan data analysis software. The data obtained show the unique expression profiles of the 7 cell types in the leukaemia cell panel obtained with the DotScan microarray, and the differential capture patterns for these 7 cell types screened against the 498 antibodies in the HLDA8 microarray constructed for this study.

Conservation of unique cell-surface CD antigen mosaics in HIV-1-infected individuals.

Cluster of differentiation (CD) antigens are expressed on cells of myeloid and lymphoid lineages. As most disease processes involve immune system activation or suppression, these antigens offer unique opportunities for monitoring host responses. Immunophenotyping using limited numbers of CD antigens enables differentiation states of immune system cells to be determined. Extended phenotyping involving parallel measurement of multiple CD antigens may help identify expression pattern signatures associated with specific disease states. To explore this possibility we have made a CD monoclonal antibody array and scanner, enabling the parallel immunophenotyping of leukocyte cell suspensions in a single and rapid analysis. To demonstrate this approach, we used the specific example of patients infected with human immunodeficiency virus type-1 (HIV-1). An invariant HIV-induced CD antigen signature has been defined that is both robust and independent of clinical outcome, composed of a unique profile of CD antigen expression levels that are both increased and decreased relative to internal controls. The results indicate that HIV-induced changes in CD antigen expression are disease specific and independent of outcome. Their invariant nature indicates an irreversible component to retroviral infection and suggests the utility of CD antigen expression patterns in other disease settings.