Platelet-derived endothelial cell growth factor expression and angiogenesis in cervical intraepithelial neoplasia and squamous cell carcinoma of the cervix

Growth and metastatic spread of invasive carcinoma depends on angiogenesis, the formation of new blood vessels. Platelet-derived endothelial cell growth factor (PD-ECGF) is an angiogenic growth factor for a number of solid tumors, including lung, bladder, colorectal, and renal cell cancer. Cervical intraepithelial neoplasia (CIN) is the precursor to squamous cell cervical carcinoma (SCC). Mean vessel density (MVD) increases from normal cervical tissue, through low- and high-grade CIN to SCC. We evaluated PD-ECGF immunoreactivity and correlated its expression with MVD in normal, premalignant, and malignant cervical tissue. PD-ECGF expression was assessed visually within the epithelial tissues and scored on the extent and intensity of staining. MVD was calculated by counting the number of vessels positive for von Willebrand factor per unit area subtending normal or CIN epithelium or within tumor hotspots for SCC. Cytoplasmic and/or nuclear PD-ECGF immunoreactivity was seen in normal epithelium. PD-ECGF expression significantly increased with histologic grade from normal, through low- and high-grade CIN, to SCC (P < .02). A progressive significant increase in the microvessel density was also seen, ranging from a mean of 28 vessels for normal tissue to 57 for SCC (P < .0005). No correlation was found between PD-ECGF expression and MVD (P = .45). We conclude that PD-ECGF expression and MVD increase as the cervix transforms from a normal to a malignant phenotype. PD-ECGF is thymidine phosphorylase, a key enzyme in the activation of fluoropyrimidines, including 5-fluorouracil. Evaluation of PD-ECGF thymidine phosphorylase expression may be important in designing future chemotherapeutic trials in cervical cancer. Copyright (C) 2000 by W.B. Saunders Company.

A comparative study of antibody expressions in primary biliary cirrhosis and autoimmune cholangitis using phage display

Primary biliary cirrhosis (PBC) and autoimmune cholangitis (AIC) are serologic expressions of an autoimmune liver disease affecting biliary ductular cells. Previously we screened a phage-displayed random peptide library with polyclonal IgG from 2 Australian patients with PBC and derived peptides that identified a single conformational (discontinuous) epitope in the inner lipoyl domain of the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), the characteristic autoantigen in PBC. Here we have used phage display to investigate the reactivity of PBC sera from 2 ethnically and geographically distinct populations, Japanese and Australian, and the 2 serologic expressions, PBC and AIC. Random 7-mer and 12-mer peptide libraries were biopanned with IgG from 3 Japanese patients with PBC and 3 with AIC who did not have anti-PDC-E2. The phage clones (phagotopes) obtained were tested by capture enzyme-linked immunosorbent assay (ELISA) for reactivity with affinity-purified anti-PDC-E2, and compared with those obtained from Australian patients with PBC. Peptide sequences of the derived phagotopes and sequences derived by biopanning with irrelevant antisera were aligned to develop a guide tree based on physicochemical similarity. Both Australian and Japanese PBC-derived phagotopes were distributed in branches of the guide tree that contained the peptide sequences MH and FV previously identified as part of an immunodominant conformational epitope of PDC-E2, indicating that epitope selection was not influenced by the racial origin of the PBC sera. Biopanning with either PBC or AIC-derived IgG yielded phagotopes that reacted with anti-PDC-E2 by capture ELISA, further establishing that there is a similar autoimmune targeting in PBC and AIC.

Temporal and functional changes in glycosaminoglycan expression during osteogenesis

Heparan sulfate proteoglycans (HSPGs) are complex and labile macromolecular moieties on the surfaces of cells that control the activities of a range of extracellular proteins, particularly those driving growth and regeneration. Here, we examine the biosynthesis of heparan sulfate (HS) sugars produced by cultured MC3T3-E1 mouse calvarial pre-osteoblast cells in order to explore the idea that changes in HS activity in turn drive phenotypic development during osteogenesis. Cells grown for 5 days under proliferating conditions were compared to cells grown for 20 days under mineralizing conditions with respect to their phenotype, the forms of HS core protein produced, and their HS sulfotransferase biosynthetic enzyme levels. RQ-PCR data was supported by the results from the purification of day 5 and day 20 HS forms by anionic exchange chromatography. The data show that cells in active growth phases produce more complex forms of sugar than cells that have become relatively quiescent during active mineralization, and that these in turn can differentially influence rates of cell growth when added exogenously back to preosteoblasts.

Disturbed microtubule function and induction of micronuclei by chelate complexes of mercury(II)

Interactions of mercury(II) with the microtubule network of cells may lead to genotoxicity. Complexation of mercury(II) with EDTA is currently being discussed for its employment in detoxification processes of polluted sites. This prompted us to re-evaluate the effects of such complexing agents on certain aspects of mercury toxicity, by examining the influences of mercury(II) complexes on tubulin assembly and kinesin-driven motility of microtubules. The genotoxic effects were studied using the micronucleus assay in V79 Chinese hamster fibroblasts. Mercury(II) complexes with EDTA and related chelators interfered dose-dependently with tubulin assembly and microtubule motility in vitro. The no-effect-concentration for assembly inhibition was 1 μM of complexed Hg(II), and for inhibition of motility it was 0.05 μM, respectively. These findings are supported on the genotoxicity level by the results of the micronucleus assay, with micronuclei being induced dose-dependently starting at concentrations of about 0.05 μM of complexed Hg(II). Generally, the no-effect-concentrations for complexed mercury(II) found in the cell-free systems and in cellular assays (including the micronucleus test) were identical with or similar to results for mercury tested in the absence of chelators. This indicates that mercury(II) has a much higher affinity to sulfhydryls of cytoskeletal proteins than to this type of complexing agents. Therefore, the suitability of EDTA and related compounds for remediation of environmental mercury contamination or for other detoxification purposes involving mercury has to be questioned.

A novel human leucocyte antigen-DRB1 genotyping method based on multiplex primer extension reactions

We have developed and validated a semi-automated fluorescent method of genotyping human leucocyte antigen (HLA)-DRB1 alleles, HLA-DRB1*01-16, by multiplex primer extension reactions. This method is based on the extension of a primer that anneals immediately adjacent to the single-nucleotide polymorphism with fluorescent dideoxynucleotide triphosphates (minisequencing), followed by analysis on an ABI Prism 3700 capillary electrophoresis instrument. The validity of the method was confirmed by genotyping 261 individuals using both this method and polymerase chain reaction with sequence-specific primer (PCR-SSP) or sequencing and by demonstrating Mendelian inheritance of HLA-DRB1 alleles in families. Our method provides a rapid means of performing high-throughput HLA-DRB1 genotyping using only two PCR reactions followed by four multiplex primer extension reactions and PCR-SSP for some allele groups. In this article, we describe the method and discuss its advantages and limitations.

Mid-gestation intra-amniotic infection with Ureaplasma parvum is resolved within spiny mice (Acomys cahirinus) by term delivery: but caused chronic infection of fetal lungs and placentae

Background: Ureaplasma species in amniotic fluid at the time of second-trimester amniocentesis increases the risk of preterm birth, but most affected pregnancies continue to term (Gerber et al. J Infect Dis 2003). We aimed to model intra-amniotic (IA) ureaplasma infection in spiny mice, a species with a relatively long gestation (39 days) that allows investigation of the disposition and possible clearance of ureaplasmas in the feto-placental compartment. Method: Pregnant spiny mice received IA injections of U. parvum serovar 6 (10µL, 1x104 colony-forming-units in PBS) or 10B media (10µL; control) at 20 days (d) of gestation (term=39d). At 37d fetuses (n=3 ureaplasma, n=4 control) were surgically delivered and tissues were collected for; bacterial culture, ureaplasma mba and urease gene expression by PCR, tissue WBC counts and indirect fluorescent antibody (IFA) staining using anti-ureaplasma serovar 6 (rabbit) antiserum. Maternal and fetal plasma IgG was measured by Western blot. Results: Ureaplasmas were not detected by culture or PCR in fetal or maternal tissues but were visualized by IFA within placental and fetal lung tissues, in association with inflammatory changes and elevated WBC counts (p<0.0001). Anti-ureaplasma IgG was detected in maternal (2/2 tested) and fetal (1/2 tested) plasma but not in controls (0/3). Conclusions: IA injection of ureaplasmas in mid-gestation spiny mice caused persistent fetal lung and placental infection even though ureaplasmas were undetectable using standard culture or PCR techniques. This is consistent with resolution of IA infection, which may occur in human pregnancies that continue to term despite detection of ureaplasmas in mid-gestation.

Approximate Bayesian computation using indirect inference

We present a novel approach for developing summary statistics for use in approximate Bayesian computation (ABC) algorithms by using indirect inference. ABC methods are useful for posterior inference in the presence of an intractable likelihood function. In the indirect inference approach to ABC the parameters of an auxiliary model fitted to the data become the summary statistics. Although applicable to any ABC technique, we embed this approach within a sequential Monte Carlo algorithm that is completely adaptive and requires very little tuning. This methodological development was motivated by an application involving data on macroparasite population evolution modelled by a trivariate stochastic process for which there is no tractable likelihood function. The auxiliary model here is based on a beta–binomial distribution. The main objective of the analysis is to determine which parameters of the stochastic model are estimable from the observed data on mature parasite worms.

Automatic classification of human epithelial type 2 cell indirect immunofluorescence images using cell pyramid matching

This paper describes a novel system for automatic classification of images obtained from Anti-Nuclear Antibody (ANA) pathology tests on Human Epithelial type 2 (HEp-2) cells using the Indirect Immunofluorescence (IIF) protocol. The IIF protocol on HEp-2 cells has been the hallmark method to identify the presence of ANAs, due to its high sensitivity and the large range of antigens that can be detected. However, it suffers from numerous shortcomings, such as being subjective as well as time and labour intensive. Computer Aided Diagnostic (CAD) systems have been developed to address these problems, which automatically classify a HEp-2 cell image into one of its known patterns (eg. speckled, homogeneous). Most of the existing CAD systems use handpicked features to represent a HEp-2 cell image, which may only work in limited scenarios. We propose a novel automatic cell image classification method termed Cell Pyramid Matching (CPM), which is comprised of regional histograms of visual words coupled with the Multiple Kernel Learning framework. We present a study of several variations of generating histograms and show the efficacy of the system on two publicly available datasets: the ICPR HEp-2 cell classification contest dataset and the SNPHEp-2 dataset.

Mimotopes identified by phage display for the monoclonal antibody CII- C1 to type II collagen

The characterization of B cell epitopes has been advanced by the use of random peptide libraries displayed within the coat protein of bacteriophage. This technique was applied to the monoclonal antibody (mAb) C1 to type II collagen (CII-C1). CII-C1 is known to react with a conformational epitope on type II collagen that includes residues 359-363. Three rounds of selection were used to screen two random nonameric phage libraries and 18 phagotopes were isolated. CII-C1 reacted by ELISA with 17 of the 18 phagotopes: one phagotope contained a stop codon. Of the eight most reactive phage, seven inhibited the reactivity by ELISA of CII-C1 with type II collagen. Of the 18 phage isolated, 11 encoded the motif F-G-x-Q with the sequence F-G-S-Q in 6, 2 encoded F-G-Q, and one the reverse motif Q-x-y-F. Most phagotopes that inhibited the reactivity of CII-C1 encoded two particular motifs consisting of two basic amino acid residues and a hydrophobic residue in the first part of the insert and the F-G-x-Q or F-G-Q motif ill the second part; phagotopes which contained only one basic residue in the first part of the sequence were less reactive. These motifs are not represented in the linear sequence of type II collagen and thus represent mimotopes of the epitope for CII-C1 on type II collagen. There were five phagotopes with peptide inserts containing the sequence RLPFG occurring in the Epstein-Barr virus nuclear antigen, EBNA- 1. This is of interest because EBV has been implicated in the initiation of rheumatoid arthritis (RA) by reason of increased reactivity to EBNA-1 in RA sera. In conclusion, the phage display technique disclosed mimotopes for a conformational epitope of type II collagen, and revealed an interesting homology with a sequence of the EBNA-1 antigen from Epstein Barr virus.

Fine mapping of the MHC Class III region demonstrates association of AIF1 and rheumatoid arthritis

Objective. The heritability of RA has been estimated to be ∼55%, of which the MHC contributes about one-third. HLA-DRB1 alleles are strongly associated with RA, but it is likely that significant non-DRB1 MHC genetic susceptibility factors are involved. Previously, we identified two three-marker haplotypes in a 106-kb region in the MHC class III region immediately centromeric to TNF, which are strongly associated with RA on HLA-DRB1*0404 haplotypes. In the present study, we aimed to refine these associations further using a combination of genotyping and gene expression studies. Methods. Thirty-nine nucleotide polymorphisms (SNPs) were genotyped in 95 DRB1*0404 carrying unrelated RA cases, 125 DRB1*0404 - carrying healthy controls and 87 parent-case trio RA families in which the affected child carried HLA-DRB1*04. Quantitative RT-PCR was used to assess the expression of the positional candidate MHC class III genes APOM, BAT2, BAT3, BAT4, BAT5, AIF1, C6orf47, CSNK2β and LY6G5C, and the housekeeper genes, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and β2-microglobulin (B2M) in 31 RA cases and 21 ethnically, age- and sex-matched healthy controls. Synovial membrane specimens from RA, PsA and OA cases were stained by an indirect immunoperoxidase technique using a mouse-anti-human AIF1 monoclonal antibody. Results. Association was observed between RA and single markers or two marker haplotypes involving AIF1, BAT3 and CSNK. AIF1 was also significantly overexpressed in RA mononuclear cells (1.5- to 1.9-fold difference, P = 0.02 vs HPRT, P = 0.002 vs B2M). AIF1 protein was clearly expressed by synovial macrophages in all the inflammatory synovial samples in contrast to the non-inflammatory OA samples. Conclusions. The results of the genotyping and expression studies presented here suggest a role for AIF1 in both the aetiology and pathogenesis of RA.

Clinical Accuracy of the MedGem Indirect Calorimeter for Measuring Resting Energy Expenditure in Cancer Patients

OBJECTIVE: To compare, in patients with cancer and in healthy subjects, measured resting energy expenditure (REE) from traditional indirect calorimetry to a new portable device (MedGem) and predicted REE. DESIGN: Cross-sectional clinical validation study. SETTING: Private radiation oncology centre, Brisbane, Australia. SUBJECTS: Cancer patients (n = 18) and healthy subjects (n = 17) aged 37-86 y, with body mass indices ranging from 18 to 42 kg/m(2). INTERVENTIONS: Oxygen consumption (VO(2)) and REE were measured by VMax229 (VM) and MedGem (MG) indirect calorimeters in random order after a 12-h fast and 30-min rest. REE was also calculated from the MG without adjustment for nitrogen excretion (MGN) and estimated from Harris-Benedict prediction equations. Data were analysed using the Bland and Altman approach, based on a clinically acceptable difference between methods of 5%. RESULTS: The mean bias (MGN-VM) was 10% and limits of agreement were -42 to 21% for cancer patients; mean bias -5% with limits of -45 to 35% for healthy subjects. Less than half of the cancer patients (n = 7, 46.7%) and only a third (n = 5, 33.3%) of healthy subjects had measured REE by MGN within clinically acceptable limits of VM. Predicted REE showed a mean bias (HB-VM) of -5% for cancer patients and 4% for healthy subjects, with limits of agreement of -30 to 20% and -27 to 34%, respectively. CONCLUSIONS: Limits of agreement for the MG and Harris Benedict equations compared to traditional indirect calorimetry were similar but wide, indicating poor clinical accuracy for determining the REE of individual cancer patients and healthy subjects.