Autoantibodies in sera of pancreatic cancer patients identify recombination factor Rad51 as a tumour-associated antigen.

PURPOSE: The recombination factor Rad51 is highly expressed in human pancreatic adenocarcinoma. In this study we asked whether high-level expression of Rad51 antigen stimulates a B-cell response leading to Rad51-specific autoantibodies in human pancreatic cancer patients. METHODS: Sera of patients suffering from pancreatic cancer (57) as well as sera of healthy donors (86) were screened for Rad51 autoantibodies by Western-blot analysis. Rad51 over-expressing cell lines were used as antigen source. RESULTS: Four out of 57 (7%) sera tested were found positive for Rad51 autoantibodies of IgG subclass, while all 86 control sera were negative. CONCLUSION: This observation identifies Rad51 as a tumour-associated antigen in pancreatic adenocarcinoma. Since high-level expression of Rad51 is restricted to tumour cells, Rad51 is also a tumour-specific antigen. Further analyses should reveal whether Rad51 might also function as a tumour-specific transplantation antigen (TSTA) and whether it might serve as a target for new immunotherapeutical approaches.

DNA repair and recombination factor Rad51 is over-expressed in human pancreatic adenocarcinoma.

Molecular processes that could contribute to differences in chemo- and radioresistance include variations in DNA repair mechanisms. In mammalian cells, the product of the rad51 gene mediates DNA repair via homologous recombination. We describe that in contrast to conventional monolayer cell systems Rad51 protein accumulates to high-levels in three-dimensional cell culture models as well as in orthotopic xeno-transplants of human pancreatic cancer cells. Strikingly, over-expression of wild-type Rad51 was also found in 66% of human pancreatic adenocarcinoma tissue specimens. Functional analysis revealed that Rad51 over-expression enhances survival of cells after induction of DNA double strand breaks. These data suggest that perturbations of Rad51 expression contribute to the malignant phenotype of pancreatic cancer. Oncogene (2000).

Enhancement of hepatitis B virus infection by noninfectious subviral particles.

The biological function of the huge excess of subviral particles over virions in hepatitis B virus infections is unknown. Using the duck hepatitis B virus as a model, we unexpectedly found that subviral particles strongly enhance intracellular viral replication and gene expression. This effect is dependent on the multiplicity of infection, the ratio of virions over subviral particles, and the time point of addition of subviral particles. Most importantly, we show that the pre-S protein of the subviral particles triggers enhancement and requires the presence of the binding regions for putative cell-encoded virus receptor proteins. These data suggest that enhancement is due either to the recently described transactivation function of the pre-S protein or to signalling pathways which become activated upon binding of subviral particles to cellular receptors. The findings are of clinical importance, since they imply that infectivity of sera containing hepadnaviruses depends not only on the amount of infectious virions but also decisively on the number of particles devoid of nucleic acids. A similarly dramatic enhancing effect of noninfectious particles in other virus infections is well conceivable.

Differentiation of core gene products of the hepatitis B virus in infected liver tissue using monoclonal antibodies.

Hepatitis B virus (HBV) core gene translational products were localised previously in the cytoplasm and/or in the nuclei of infected cells. We investigated in naturally infected human hepatocytes whether this variation in the subcellular expression is due to differences in the presence of assembled core particles and other core gene derived proteins, the expression of HBeAg and the processing of liver tissue. By immunostaining of liver specimens infected with HBeAg-positive and HBeAg-minus variants of HBV, using monoclonal antibodies specific for assembled core particles and for various epitopes on denatured core protein, it was shown that virtually all immunoreactive core gene products are assembled into core particles. The latter are present both in the nuclei and in the cytoplasm of hepatocytes, independent of the infecting virus strain. A marked reduction or absence of immunoreactivity, observed with some monoclonal antibodies, was shown to result from nucleotide sequence variations within or close to the corresponding epitope. These results demonstrate that immunoreactive products, derived from the HBV core gene, in the nuclei and cytoplasm of human hepatocytes represent assembled core particles and that monoclonal antibodies with known recognition sites can reveal region-specific core gene variation of the infecting HBV population.

Efficacy of venlafaxine and placebo during long-term treatment of depression: a pooled analysis of relapse rates.

The objective of this analysis was to determine the efficacy of venlafaxine in comparison with that of placebo during long-term treatment. A pooled analysis of relapse rates in outpatients with major depression continuing long-term treatment (up to 12 months) after responding to short-term treatment (6 weeks) was performed combining the data from four randomized, double-blind, placebo-controlled clinical trials. Relapses were defined as two consecutive Clinical Global Impression (CGI) severity scores greater than 3 (mildly ill), as a CGI severity score greater than 3 at withdrawal regardless of the reason for withdrawal, or as withdrawal due to lack of efficacy. Data from 304 patients (185 venlafaxine, 119 placebo) well balanced for baseline characteristics were included in the pooled analysis. Percentages of patients completing the long-term phase were 38% venlafaxine and 26% placebo (p = 0.034). Cumulative relapse rates by 6 months of long-term treatment were 11% venlafaxine and 23% placebo (p = 0.019). Cumulative relapse curves for the venlafaxine and placebo groups over the 1-year long-term treatment differed significantly (p = 0.022). The results from this analysis indicate that long-term treatment with venlafaxine in patients with major depressive disorder is effective in maintaining the initial response compared with placebo and suggest that venlafaxine will be effective in the prevention of relapse.

A novel method for efficient amplification of whole hepatitis B virus genomes permits rapid functional analysis and reveals deletion mutants in immunosuppressed patients.

Current knowledge of hepatitis B virus (HBV) sequence heterogeneity is based mainly on sequencing of amplified subgenomic HBV fragments. Here, we describe a method which allows sensitive amplification and simplified functional analysis of full-length HBV genomes with or without prior cloning. By this method, a large number of HBV genomes were cloned from sera of six immunosuppressed kidney transplant patients. Two size classes of HBV genomes, one 3.2 kb and another about 2.0 kb in size, were found in all patients. The genome population from one serum sample was studied in detail by size analysis of subgenomic PCR fragments and sequencing. Regions with deletions and insertions were mapped in the C gene and pre-S region. Up to 100% of HBV genomes in all other immunosuppressed patients also had deletions in the C gene. Our results demonstrate the potential of the established method for the structural and functional characterization of heterogeneous populations of complete virion-encapsidated HBV DNAs and suggest that HBV genomes with C gene deletions can have a selective advantage in immunosuppressed patients.

Structure and selection of hepatitis B virus variants during the natural course of infection and interferon therapy.

The consequences of hepatitis B virus (HBV) infection range from asymptomatic transient and chronic infections to mild and severe forms of hepatitis up to the development of liver cirrhosis and hepatocellular carcinoma. Immune-mediated destruction of infected liver cells plays a major role in HBV pathogenesis. Escape from immune-recognition by changing the antigenic make-up of HBV or by preventing the expression of some viral proteins leads to the emergence of viral variants. Variants can harbor mutations in the pre-core/core gene (pre C/C) or the preS/S gene, either of which can play a role in immune escape, viral persistence, and pathogenesis. A novel method has been developed to facilitate identification and functional analysis of full-length variant genomes of HBV, with implications for HBV diagnosis and therapy.

Heterogeneity of hepatitis B virus C-gene sequences: implications for amplification and sequencing.

Occasionally direct sequencing of amplified hepatitis B virus DNA leads to weak signals on autoradiograms. Using amplified C-gene sequences we investigated whether this is due to sequence heterogeneity of virus populations and use of inappropriate primers for direct sequencing. High C-gene sequence heterogeneity (point mutations, stop codons and a one codon deletion) was observed in HBV genomes from serum of a chronic carrier who underwent interferon treatment. The type of C-gene mutations detected by direct sequencing depended on the type of primers used. Cloning and sequencing of amplified C-gene sequences demonstrated that this was due to mutations in the region complementary to the sequencing primer. These data demonstrate the existence of novel HBV C-gene mutants and imply that multiple or degenerate sequencing and amplification primers are essential for accurate evaluation of the extent of HBV C-gene heterogeneity. Based on comparative sequence analysis of all available completely or incompletely sequenced C-genes, guidelines for optimal primer design are proposed for similar studies.

Epitopes recognized by antibodies to denatured core protein of hepatitis B virus.

Particulate and denatured core protein as well as e-antigen (HBe) of hepatitis B virus (HBV) differ in part immunologically but this has not been studied in sufficient detail. Therefore, in this study the B-cell immune response to native and denatured HBV core protein which both can exhibit HBe-specific epitopes was examined using a panel of mouse MABs and rabbit polyclonal antibodies to native and denatured core protein and polyclonal anti-HBe/anti-HBc antibodies from sera of infected patients. Epitope mapping was performed using a set of partially overlapping synthetic HBc peptides, carboxy-terminally truncated HBc proteins and various HBc fusion proteins. A major immunogenic region between amino acids 134-140 and two less immunogenic regions, one spanning amino acids 2-10 and one with three partially overlapping epitopes between amino acid positions 138 and 154, were defined by mouse MABs. Polyclonal rabbit antibodies to denatured HBc, woodchuck and ground squirrel hepatitis core proteins (WHc and GSHc) recognized similar epitopes but in addition occasionally region 61-85, and the latter was also recognized on particulate HBc. Two antigenic regions (amino acid positions 2-10 and 138-145) were found to be exposed on HBe from human serum, and were recognized by mouse anti-HBe but not by anti-HBc antibodies from sera of infected patients. This study demonstrates a more complex pattern of HBc and HBe epitopes than detected previously and provides tools to study conformational changes which may take place during HBc/HBe processing, transport and core particle assembly.

Hepatitis B virus C-gene variants.

The heterogeneity of hepatitis B virus (HBV) is increasingly believed to play a role in viral persistence, pathogenesis, and the type of response to antiviral therapy. One of the best studied parts of the HBV genome is the C-gene which codes for the nucleocapsid protein (HBc) and the e-antigen (HBeAg). Here we attempt to review the recent data on the sequence heterogeneity of this region and its possible implications.

Frequent and rapid emergence of mutated pre-C sequences in HBV from e-antigen positive carriers who seroconvert to anti-HBe during interferon treatment.

Hepatitis B virus (HBV) variants which cannot express e-antigen (HBeAg) are characteristic for many viremic anti-HBe positive chronic carriers who often have particularly severe and fluctuating hepatitis. Whether such variants are selected for and are less amenable to interferon treatment is under dispute. Therefore, by DNA amplification and direct sequencing we have investigated the emergence of HBV pre-C sequence variants in nine e-antigen positive chronic carriers, all of whom seroconverted to anti-HBe or lost HBeAg during interferon treatment, and in three of whom no viral DNA was detectable after interferon treatment. In most, but not all of the patients we found newly emerging pre-C sequences in a subpopulation of the viral genomes that included silent point mutations, amino acid changes, start and stop codon and frameshift mutations. The emergence of these mutations was paralleled by a drastic decrease of viremia during treatment. The observed mutations appeared most frequently during interferon treatment. Some of the mutations appeared or disappeared late after interferon treatment concomitant with anti-HBe antibody development. The appearance or lack of mutations in the pre-C region of a subpopulation of HBV of these patients was independent of successful virus elimination. These data indicate that interferon treatment is frequently associated with the simultaneous fall in titer of viral DNA by several orders of magnitude and the emergence of novel pre-C sequences, some of them preventing HBeAg expression. However, the presumably immune-mediated selection for pre-C mutant viruses and decrease in viremia under interferon treatment appears not to be prognostic for successful or unsuccessful virus elimination.

Precore mutant hepatitis B virus infection and liver disease.

The type of hepatitis B virus ("wild-type" or precore mutant) in anti-e antigen antibody-positive carriers, viral DNA levels in the serum, and core and e antigen expression in the liver were investigated to search for a possible correlation of these factors with the severity of liver damage. Two major groups of patients were found: the patients in group A were predominantly infected with precore mutant virus and had chronic active hepatitis, expressed nuclear/cytoplasmic core and e antigens in liver biopsy specimens, and usually had high levels of viral DNA in their serum; patients in group B were infected with a mixture of wild-type and mutant viruses, had predominantly chronic persistent hepatitis, showed weaker expression of nuclear core antigen with no cytoplasmic core or e antigen, and had low viremia. A few patients were infected with viruses without precore stop-codon mutation. These data indicate a high prevalence of precore mutant viruses in anti-e carriers with chronic liver disease and suggest that monitoring of virus sequence type and DNA level may be of prognostic value for liver disease sequelae.

Prevalence and type of pre-C HBV mutants in anti-HBe positive carriers with chronic liver disease in a highly endemic area.

The sequence variability in the pre-C region of the hepatitis B virus (HBV) genome in the serum of 42 anti-HBe antibody positive carriers with chronic hepatitis B was studied by PCR and direct sequencing to determine prevalence and type of HBV pre-C mutants in a highly endemic area. Except for one, all patients were infected with viruses containing mutations in the pre-C region which prevent precore and e-antigen (HBeAg) expression: 33 were infected predominantly or exclusively with variants containing a stop codon; two had a mixture of wild-type and a pre-C stop codon mutant virus; three had precore variants with mutations of the pre-C initiation codon and two of them an additional stop codon; four had a frameshift mutation; and one had two stop codons. One patient was infected with viruses which contained a mutation creating an amino acid exchange which should not prevent precore and HBeAg expression. These data demonstrate that in an endemic area a higher prevalence and even broader spectrum of pre-C HBV mutants are found than has been recognized previously in anti-HBe positive patients with chronic hepatitis B.

High prevalence and heterogeneity of HBV preC mutants in anti-HBe-positive carriers with chronic liver disease in southern Italy.

In this study we have investigated the prevalence and type of preC mutants in anti-HBe/HBV-DNA-positive patients with chronic hepatitis B in an endemic area. HBV-DNA from sera of 42 anti-HBe chronic HBV carriers was amplified by PCR and the preC region was directly sequenced. With one exception, all patients tested were found to be infected with viruses containing mutations in the preC region that predictably prevent precore and e antigen expression. Thirty-one patients were infected with HBV containing a stop codon; two had a mixture of wild-type and preC stop codon mutant; three had preC mutants with mutations in the translation initiation codon and two of them an additional stop codon; four had a frameshift mutation, and one had two stop codons. One patient was infected with a virus showing a mutation creating only an amino acid exchange which could not prevent HBeAg expression. The data obtained indicate a higher prevalence and heterogeneity of preC mutants in our geographical area than recognized so far in anti-HBe-positive carriers with chronic hepatitis B.

Effect of phenytoin on rat bone resorption in vitro.

Effects of phenytoin (DPH) on parathyroid hormone (PTH)- and 1,25-dihydroxyvitamin D3(1,25DHCC)-mediated bone resorption in bone organ culture were evaluated: it markedly inhibited the actions of both. However, PTH tended to overcome the initial effects of DPH, whereas bone resorption due to 1,25DHCC remained depressed as if DPH treatment had continued. Thus DPH-induced bone abnormalities may involve a greater direct effect on 1,25DHCC regulation of bone homeostasis than on PTH.

Ontogeny of cardiac ornithine decarboxylase and its beta adrenergic responsiveness in the rat.

Ornithine decarboxylase (ODC) is a marker of tissue growth and development and, because sympathetic stimulation of beta adrenergic receptors acutely increases ODC in the adult rat heart, measurement of this enzyme can be used to indicate the functional intactness of the beta adrenergic receptor system in the heart. Changes in the postnatal ontogenetic pattern of this enzymatic activity may also indicate abnormal development and ODC appears to be particularly useful in evaluating the effects of prenatal insult on cardiac development. The present study examines the pattern of basal ODC activity and its developing sensitivity to beta adrenergic stimulation during the perinatal period in order to establish a data base for studies on the effect of various environmental agents on the developing cardiovascular system. ODC activity was measured in rat hearts on gestation day (GD) 20 through postnatal day (PND) 28 under saturating conditions of L-ornithine and pyridoxal 5-phosphate. Basal ODC activity fell from 3 nmol of CO2/hr/mg of protein at GD 20 to less than 0.5 nmol of CO2/hr/mg of protein at PND 18, rising again to nearly 1 nmol of CO2/hr/mg of protein at PND 22. The beta adrenergic agonist isoproterenol (10 mg/kg s.c.) resulted in peak ODC stimulation at 4 hr postinjection on PNDs 6, 14 and 21; however, no response was seen at PND 1 at this dose or at GD 20 (300 micrograms/kg s.c.). In dose-response studies, isoproterenol produced a maximal response at 10 mg/kg s.c., resulting in increases from control of 67, 230 and 1700% at PNDs 6, 14 and 21, respectively, indicating that the sensitivity of the heart to beta adrenergic stimulation increases with age, during the perinatal period.