In vitro immune modulation by antibodies coupled to tumour cells.

Modification of autologous tumour cells to express the immune costimulator B7.1 is a potential strategy for immunotherapy of cancer. Previously, this has involved introduction of genetic material into cells, in vitro culture, and confirmation of the protein product on the cell surface.

Plasma levels and hepatic mRNA expression of transforming growth factor-beta1 in patients with fulminant hepatic failure.

Background/aims: Transforming growth factor-beta1 is an important cytokine involved in cell growth and inflammation which has been shown to be inhibitory to hepatic DNA synthesis. The aim of this study was to investigate the plasma levels and hepatic mRNA expression of transforming growth factor-beta1 in patients with fulminant hepatic failure in whom liver regeneration may be impaired.

Methods: Plasma levels of transforming growth factor-beta1 and human hepatocyte growth factor were measured in 57 fulminant hepatic failure patients and 20 healthy volunteers by ELISA.

p53 alterations and HPV infections are common in oral SCC: p53 gene mutations correlate with the absence of HPV 16-E6 DNA.

To examine the association between HPV infections and p53 gene aberrations, a panel of 28 oral squamous cell carcinomas (SCC) and 12 potentially malignant oral mucosal lesions were analysed for p53 mutations in exons 2-9. p53 protein was analysed by immunocytochemistry using DO7 antibody. The same panel was also examined for the possible presence of HPV infection.

Transcriptional repression by the promyelocytic leukemia protein, PML.

Acute promyelocytic leukemia is characterized by the presence of a t(15; 17) chromosomal translocation which results in the expression of a chimeric gene product, PMLRAR alpha, consisting of an N-terminal-truncated retinoic acid receptor-alpha fused to a C-terminal-truncated PML. Several structural features, and regions of homology to known transcription factors, suggest that PML may be involved in the regulation of gene expression. In this study we have analyzed the transcriptional regulatory activity of PML using chimeric GAL4/PML constructs and GAL4-responsive reporter plasmids.

Cardiac expression of sarcoplasmic reticulum calcium ATPase in fetuses with trisomy 21 and trisomy 18 presenting with nuchal translucency.

At 10-14 weeks of gestation about 80% of fetuses with chromosomal defects have abnormal accumulation of subcutaneous fluid in the nuchal region that is visualized by ultrasonography as nuchal translucency. A possible cause for this translucency is cardiac dysfunction due to the associated defects in the heart and great arteries. The aim of this study was to investigate whether in cardiac tissue from trisomic fetuses, compared to normals, there is an alteration in the steady state levels of expression of the genes encoding sarcoplasmic reticulum calcium ATPase (calcium ATPase), which is known to be downregulated in postnatal heart failure.

Molecular cloning of human GATA-6 DNA binding protein: high levels of expression in heart and gut.

Human GATA-6 has been cloned from foetal heart by a combination of PCR-based methods and cDNA library screening. The 3.8 kbp cDNA has a coding sequence of 1347 bp the 449 aa protein is virtually identical in the two zinc-finger binding domains to other human GATA sequences, but varies considerably in the amino and carboxy terminal regions.

Enhanced immune costimulatory activity of primary acute myeloid leukaemia blasts after retrovirus-mediated gene transfer of B7.1.

Gene modification of malignant cells to express immune stimulators (cytokines and immune costimulators) has provided the basis for a novel form of immunotherapy. Using a MPSV-based retroviral vector with hygromycin resistance gene as a selectable marker, we have studied retrovirus-mediated gene transfer of an immune costimulator, B7.1, into primary human acute myeloid leukaemia (AML) cells and the subsequent induction of immune costimulatory function.

Irradiated NC adenocarcinoma cells transduced with both B7.1 and interleukin-2 induce CD4+-mediated rejection of established tumors.

Previous studies have shown that expression of the immune co-stimulator B7.1 reduces the tumorigenicity of some, but not all, malignant cell lines. However, B7.

Alcohol dehydrogenase: a target of humoral autoimmune response in liver disease.

Background & Aims: Liver-specific membrane lipoprotein (LSP) is a heterogeneous liver preparation that has been widely used to study autoreactivity in liver disease. The aim of this study was to identify autoantigens in LSP.

Methods: Guinea pig anti-LSP serum was used to screen a human liver complementary DNA (cDNA) library.

Identification of 3 beta-hydroxysteroid dehydrogenase as a novel target of steroid cell autoantibodies: association of autoantibodies with endocrine autoimmune disease.

Autoantibodies directed against steroid hormone-producing cells (SCA) detectable by immunofluorescence are typically found in a small proportion of patients with premature ovarian failure (POF) as well as in other endocrine autoimmune diseases. The SCA pattern stains cells in the outer zones of the adrenal cortex, ovary, and testis. To identify the molecular target of SCA, an adrenal complementary DNA expression library was screened using SCA-positive serum, and the steroid enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) was identified.

Expression of genes involved in iron metabolism in mouse intestine.

Intestinal nonheme iron levels and mRNA levels of genes implicated in iron metabolism were measured in mice with altered iron metabolism [chronic (4 wk) and acute (4 days) dietary iron deficiency; iron overload and hypoxia] to investigate their role in the process and regulation of intestinal iron absorption. Mucosal nonheme iron levels were decreased by both chronic and acute iron deficiency and increased by iron overload but were not affected by hypoxia. There was evidence of a gradient of mucosal nonheme iron along the small intestine (duodenum, jejunum > ileum).

Comparison of IL-2- and IL-4-transfected B16-F10 cells with a novel oil-microemulsion adjuvant for B16-F10 whole cell tumour vaccine.

The use of whole cell tumour vaccines in the treatment of malignant melanoma has given mixed results. Cytokine-transfected tumour cells as vaccine have shown efficacy in animal models but need to be compared with other means of enhancing a systemic anti-tumour immune response. A new generation of immunological adjuvants claimed to be more effective than the conventional adjuvants is now available for assessment.

Regulation of pancreatitis-associated protein (HIP/PAP) mRNA levels in mouse pancreas and small intestine.

1. In the present study we have examined the expression of pancreatitis-associated protein mRNA in mouse pancreas and small intestine and determined the effect of a number of factors on the steady-state level of the RNA. 2.

The effect of combined expression of interleukin 2 and interleukin 4 on the tumorigenicity and treatment of B16F10 melanoma.

The recent use of interleukin 2 (IL-2) and interleukin 4 (IL-4) single cytokine modified tumour cells in rodent models has demonstrated a potential use of these cytokines to produce autologous cancer cell vaccines. Here we compare the potential therapeutic benefit of transduction with IL-2 or IL-4 alone, and combined IL-2 + IL-4 in B16F10 cells, a murine malignant melanoma of poor immunogenicity. Transduction of B16F10 cells (MHC class I and II negative) to express either IL-2 or IL-4 alone delays the formation of tumours, IL-4 being more effective than IL-2.

Loss of heterozygosity on chromosome 5q in ovarian cancer is frequently accompanied by TP53 mutation and identifies a tumour suppressor gene locus at 5q13.1-21.

Forty-nine ovarian tumours were examined for loss of heterozygosity (LOH) on chromosome 5 using eight microsatellite markers spanning both arms, including one at the APC locus. LOH on 5q was a frequent event, detectable in 23 of 49 (47%) tumours, whereas 5p LOH was detected in only 1 of 22 tumours (5%). Six tumours showed partial LOH on 5q, enabling the candidate region to be localised to a 22 cM region proximal to APC, flanked by D5S424 and D5S644.

Placental mRNA expression of alpha and beta human chorionic gonadotrophin in early trisomy 18 pregnancies.

The placental expression of human chorionic gonadotrophin (HCG) I- and beta-subunits was investigated in eight pregnancies presenting with trisomy 18 and in 30 normal pregnancies at 11-15 weeks gestation. In the control group, the median densitometric scores of placental beta-HCG and I-HCG mRNA were 1.23 and 1.

Efficient retroviral infection of mammalian cells is blocked by inhibition of poly(ADP-ribose) polymerase activity.

Integration of proviral DNA into the host cell genome is a characteristic feature of the retroviral life cycle. This process involves coordinate DNA strand break formation and rejoining reactions. The full details of the integration process are not yet fully understood.

Novel costimulators in the immune gene therapy of cancer.

One of the major goals of cancer immunotherapy is the induction of tumour-specific T-lymphocyte responses that will be effective in the rejection of established tumours. The prospects for such therapy rely on the identification of tumour antigens, and although there is persuasive evidence for the presence of such antigens,1,2 the occurrence of the disease does illustrate that the immune system is at least, on some occasions, unable to recognise and destroy these targets. Tumour antigens may be novel proteins (from genetic lesions or viral infections), modified existing antigens (eg, abnormally glycosylated cell surface proteins), or inappropriately expressed normal gene products (eg, CA125, carcinoembryonic antigen, and alpha-fetoprotein).

Cardiac gene expression of atrial natriuretic peptide and brain natriuretic peptide in trisomic fetuses.

Objective: To investigate whether the increased nuchal translucency of trisomic fetuses is the consequence of heart failure by examining cardiac expression of atrial natriuretic peptide and brain natriuretic peptide genes.

Methods: Cardiac atrial natriuretic peptide and brain natriuretic peptide messenger RNA (mRNA) levels were measured in fetal hearts from 15 pregnancies affected by trisomy 21 or 18 at 12-16 weeks' gestation and from 30 normal controls at 10-20 weeks.

Results: In normal fetuses, mRNA levels of atrial natriuretic peptide decreased, but levels of brain natriuretic peptide did not change significantly with gestation.

Fetal hepatic alpha-fetoprotein mRNA expression in fetuses with trisomy 21 and 18 at 12-15 weeks gestation.

In this study we examined alpha-fetoprotein (AFP) mRNA expression in fetal liver at 12-15 weeks of gestation in trisomy 21 (n = 13), trisomy 18 (n = 5) and control fetuses (n = 24). No significant difference was found in the steady-state level of fetal liver AFP mRNA levels in either of the two trisomy groups studied. These findings suggest that the decrease in maternal serum AFP concentration found in trisomic pregnancies is unlikely to be the consequence of impaired transcription of the AFP gene by the fetal liver.

Gene expression of human pregnancy-associated plasma protein-A in placenta from trisomic pregnancies.

Placental pregnancy-associated plasma protein-A (PAPP-A) mRNA expression, placental PAPP-A protein concentration and maternal serum levels of PAPP-A were examined in pregnancies affected by trisomy 21 (n=8), trisomy 18 (n=7) and 15 normal controls at 12-15 weeks of gestation. The maternal serum concentration of PAPP-A in the trisomic group of pregnancies was significantly lower than in the normal controls. However there were no significant differences between the three groups in PAPP-A mRNA expression or PAPP-A protein concentration in the placental tissues.

Quantitative multiwell myeloid differentiation assay using dichlorodihydrofluorescein diacetate (H2DCF-DA) or dihydrorhodamine 123 (H2R123).

It is well established that the fluorescent probes dichlorodihydrofluorescein diacetate (H2DCF-DA) and dihydrorhodamine 123 (H2R123) can be used to detect the respiratory burst response of mature myeloid cells. We describe a simple, fast and quantitative assay for myeloid differentiation based on the oxidation of these probes, which can be performed from start to finish in 96-well dishes. A bis(acetoxymethyl) ester of H2DCF-DA, 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (CODCF-DA) is also capable of detecting the respiratory burst, but is less suitable than H2DCF-DA or H2R123 in our system.

Placental expression of alpha and beta subunits of human chorionic gonadotrophin in early pregnancies with Down's syndrome.

This study examines the expression of alpha and beta subunits of human chorionic gonadotrophin (HCG) in samples of placental and decidual tissue obtained at 11-15 weeks of gestation from 30 control pregnancies and 11 pregnancies with trisomy 21. In the placental tissue, the concentrations of beta-HCG mRNA and alpha-HCG mRNA were augmented in six and seven of the trisomy 21 cases respectively and in 16 and 14 of the control pregnancies. The median values of beta-HCG mRNA and alpha-HCG mRNA in the two groups were not significantly different.