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Purpose: To investigate minisatellite germline mutation rates in survivors of childhood and young adult cancer who received radiotherapy., Materials and Methods: DNA samples from 100 families, where one parent was a cancer survivor, were analysed for mutations at eight hypervariable minisatellite loci (B6.7, CEB1, CEB15, CEB25, CEB36, MS1, MS31, MS32) by Southern hybridisation., Results: No significant difference was observed between the paternal mutation rate of 5.6% in exposed fathers with a mean preconceptional testicular dose of 1.23 Gy (56 mutations in 998 informative alleles) and that of 5.8% in unexposed fathers (17 in 295 informative alleles). Subgrouping the exposed fathers into dose groups of < 0.10 Gy, 0.10-0.99 Gy, 1.00-1.99 Gy, ≥ 2.00 Gy revealed no significant differences in paternal mutation rate in comparison with the unexposed fathers. Maternal mutation rates of 1.6% in cancer survivor mothers with a mean preconceptional ovarian dose of 0.58 Gy (five mutations in 304 informative alleles) and 2.1% in unexposed mothers (21 in 987 informative alleles) were not significantly different. There were no differences in minisatellite mutation rates associated with treatment with chemotherapeutic agents., Conclusions: This study provides evidence that preconception radiotherapy for childhood or early adulthood cancer does not increase the germline minisatellite mutation rate.

Purpose: To investigate the relationship between chromosomal radiosensitivity and early-onset cancer under the age of 35 years and to examine the heritability of chromosomal radiosensitivity., Materials and Methods: Peripheral blood lymphocytes were cultured for 72 hours prior to being irradiated with 0.5 Gy, 300 kV X-rays. Colcemid was added to cultures 30 min post-irradiation. Cultures were harvested 90 min post-irradiation and analysed for chromatid gaps and breaks. Heritability was estimated using Sequential Oligogenic Linkage Analysis Routines (SOLAR) software and by segregation analysis., Results: Elevated radiosensitivity was seen for seven out of 29 (24.1%) cancer survivors, three out of 29 (10.3%) partners and 10 out of 53 (20.8%) offspring. Although the proportion of individuals displaying enhanced radiosensitivity was twice as high in both the cancer survivor and offspring groups than the partner controls, neither reached statistical significance. Heritability analysis of the radiosensitive phenotype suggested 57.9-78.0% of the variance could be attributed to genetic factors., Conclusion: An association between G(2) chromosomal radiosensitivity and childhood and young adult cancer is suggested but was not statistically significant. In contrast, there is strong evidence for heritability of the radiosensitive phenotype. The cancer survivors included a broad range of malignancies and future studies should focus on specific cancers with known or likely faults in deoxyribonucleic acid (DNA) damage recognition and repair mechanisms.

Identification of de novo minisatellite mutations in the offspring of parents exposed to mutagenic agents offers a potentially sensitive measure of germ line genetic events induced by ionizing radiation and genotoxic chemicals. Germ line minisatellite mutations (GMM) are usually detected by hybridizing Southern blots of unamplified size-fractionated genomic DNA with minisatellite probes. However, this consumes a relatively large amount of DNA, requires several steps and may lack sensitivity. We have developed a polymerase chain reaction (PCR)-based GMM assay, which we applied to the hypermutable minisatellite, CEB1. Here, we compare the sensitivity and specificity of this assay with the conventional Southern hybridization method using DNA from 10 spouse pairs, one parent of each pair being a survivor of cancer in childhood, and their 20 offspring. We report that both methods have similar specificity but that the PCR method uses 250 times less DNA, has fewer steps and is better at detecting GMM with single repeats provided that specific guidelines for allele sizing are followed. The PCR GMM method is easier to apply to families where the amount of offspring DNA sample is limited.

Purpose: To investigate germline mutation rate at eight minisatellite loci in 24 Danish families, where one parent is the survivor of childhood or adolescent cancer treated with radiotherapy., Materials and Methods: Parents and offspring were profiled for eight hypervariable minisatellite loci (B6.7, CEB1, CEB15, CEB25, CEB36, MS1, MS31, MS32) by Southern blotting., Results: Seven paternal mutations were observed for 130 informative alleles in 18 offspring from 11 radiation-exposed fathers (mean preconceptional dose for offspring 0.29 Gy, range<0.01-1.2 Gy), compared to six mutations for 146 informative alleles in 21 offspring from 13 unexposed fathers. No statistically significant difference between the total paternal mutation rates was observed (5.4% for exposed fathers and 4.1% for unexposed fathers). Three maternal mutations were observed for 148 informative alleles in 21 offspring from 13 radiation-exposed mothers (mean preconceptional dose for offspring 0.71 Gy, range <0.01-9.2 Gy), compared to one mutation for 130 informative alleles in 18 offspring from 11 unexposed mothers. Again, no statistically significant difference was observed between the total maternal mutation rates (2.0% for exposed mothers and 0.8% for unexposed mothers)., Conclusions: The data from this pilot study demonstrate no statistically significant increase in germline minisatellite mutation rate associated with radiotherapy for childhood and adolescent cancer.

The comparison of genotype frequencies between neonates and elderly populations can aid in the identification of loci, and polymorphisms within those loci, that affect longevity. Here we have compared genotype frequencies of seven polymorphisms at four loci involved in DNA repair between a cohort of newborns (n = 290) and a retired population (average age at sampling 70.02 years; n = 430) who have suffered a lifetime of DNA damage from normal, metabolic processes, and on whom selection on DNA repair gene variants may be expected to have acted. No differences in genotype frequencies at the four SNP loci were seen, indicating that there is no evidence of association with longevity in this population. Significant differences in frequency of certain repeat sizes at three microsatellite loci were detected. However, since there is no known functional consequence of these repeat lengths, the action of selection cannot yet be ascribed.

In order to investigate the relationship between chromosomal radiosensitivity and early-onset cancer, the G(2) chromosomal radiosensitivity assay was undertaken on a group of 23 Danish survivors of childhood and adolescent cancer, a control group comprising their partners and a group of 38 of their offspring. In addition, the previously reported in-house control group from Westlakes Research Institute (WRI) was extended to 27 individuals. When using the 90th percentile cutoff for the WRI control group, the proportion of individuals with elevated radiosensitivity was 11, 35, 52 and 53% for the WRI control, partner control, cancer survivor and the offspring groups, respectively, with significant differences between the WRI control group and the cancer survivor group (P=0.002) and the offspring group (P<0.001). However, while the comparisons with the WRI control group support an association of chromosomal radiosensitivity with cancer predisposition, when the partner control group was used to define the radiosensitivity cutoff point, no significant differences in radiosensitivity profiles were found between the partner control group and either the cancer survivor group or the offspring group. The failure to distinguish between the G(2) aberration profiles of the apparently normal group of partners and the cancer survivor group suggests that any association with cancer should be viewed with caution, but also raises questions as to the suitability of the partners of cancer survivors to act as an appropriate control group. Heritability of the radiosensitive phenotype was examined by segregation analysis of the Danish families and suggested that 67.3% of the phenotypic variance of G(2) chromosomal radiosensitivity is attributable to a putative major gene locus with dominant effect.

Suggestions that the induction of genomic instability could play a role in radiation-induced carcinogenesis and heritable disease prompted the investigation of chromosome instability in relation to radiotherapy for childhood cancer. Chromosome analysis of peripheral blood lymphocytes at their first in vitro division was undertaken on 25 adult survivors of childhood cancer treated with radiation, 26 partners who acted as the non-irradiated control group and 43 offspring. A statistically significant increase in the frequency of dicentrics in the cancer survivor group compared with the partner control group was attributed to the residual effect of past radiation therapy. However, chromatid aberrations plus chromosome gaps, the aberrations most associated with persistent instability, were not increased. Therefore, there was no evidence that irradiation of the bone marrow had resulted in instability being transmitted to descendant cells. Frequencies of all aberration categories were significantly lower in the offspring group, compared to the partner group, apart from dicentrics for which the decrease did not reach statistical significance. The lower frequencies in the offspring provide no indication of transmissible instability being passed through the germline to the somatic cells of the offspring. Thus, in this study, genomic instability was not associated with radiotherapy in those who had received such treatment, nor was it found to be a transgenerational radiation effect.

Polymorphic variation in DNA repair genes was examined in a group of retired workers from the British Nuclear Fuels plc facility at Sellafield in relation to previously determined translocation frequencies in peripheral blood lymphocytes. Variation at seven polymorphisms in four genes involved in the base excision repair (XRCC1 R194W, R399Q and a [AC]n microsatellite in the 3' UTR) and double strand break repair (XRCC3 T241M and a [AC]n microsatellite in intron 3 of XRCC3, XRCC4 I134T, and a GACTAn microsatellite located 120 kb 5' of XRCC5) pathways was determined for 291 retired radiation workers who had received cumulative occupational external radiation doses of between 0 and 1873 mSv. When the interaction between radiation dose and each DNA repair gene polymorphism was examined in relation to translocation frequency there was no evidence for any of the polymorphisms studied influencing the response to occupational exposure. A positive interaction observed between genotype (individuals with at least one allele > or =20 repeat units) at a microsatellite locus in the XRCC3 gene and smoking status should be interpreted cautiously because interactions were investigated for seven polymorphisms and two exposures. Nonetheless, further research is warranted to examine whether this DNA repair gene variant might be associated with a sub-optimal repair response to smoking-induced DNA damage and hence an increased frequency of translocations.

Purpose: To investigate changes in immune cell subsets in the peripheral circulation of a male population occupationally exposed to ionizing radiation., Materials and Methods: Peripheral blood samples were taken from 194 male workers with cumulative exposures of >200 mSv (mean exposure 331.5 mSv, mean age 51 years) and from a reference population of 131 male workers with cumulative exposures of <27.5 mSv (mean exposure 13.9 mSv, mean age 47 years). Samples were analysed by flow cytometry for T- and B-cell total counts and for the T-cell subset percentages of CD4+ (helper T-cells), CD8+ (cytotoxic T-cells) and CD3+/HLA-DR+ (activated T-cells)., Results: Comparison of the >200 and <27.5 mSv exposure groups using linear regression analysis showed no statistically significant differences between the two groups for T-cell total count, B-cell total count or for percentages of the T-cell subsets CD4+, CD8+ or CD3+/HLA-DR+ and CD4+:CD8+. However, statistically significant increases in both T- and B-cell total counts were observed within the two exposure groups and data pooled from both groups when non-smokers (never and ex-smokers) were compared with current smokers. For pooled data T-cell total count increased in smokers by 35% (p=0.0001) and B-cell total count increased by 37% (p=0.0004)., Conclusions: No significant immunological effects were observed in male radiation workers with cumulative exposures of >200 mSv when compared with a reference population with cumulative exposures of <27.5 mSv, although highly significant increases in both T- and B-cell total counts were observed in smokers compared with non-smokers.

Tumour necrosis factor-alpha is a pro-inflammatory cytokine involved in many aspects of acute phase and immune responses. Species specificity in the biological action and receptor binding of TNF-alpha make it desirable to use homologous reagents in experimental models, both in vivo and in vitro. As the rat is the model of choice in many investigations on fever, trauma and pathology, there is a need for specific rat reagents. In this paper, we describe the production of recombinant rat TNF-alpha in milligram quantities, using a methylotrophic yeast expression system, Pichia pastoris. Recombinant TNF-alpha was produced intracellularly in a soluble form, cells were lysed and the protein purified by ammonium sulphate precipitation, Sephadex G75 fractionation and finally, ion-exchange chromatography. The purified recombinant rat TNF-alpha had a molecular mass of 17401.38 +/- 0.38 Da, which is within 1 Da of the value predicted by the sequence data, taking into account N-acetylation of the initial methionine residue and a single disulphide bridge between amino acids 70 and 101. Recombinant rat TNF-alpha was shown to be 20 x fold more biologically active in the WEHI cytotoxicity assay, than the human standard preparation. Polyclonal antibodies were raised against purified recombinant rat TNF-alpha, these reagents were used to develop a novel enzyme-linked immunosorbant assay (ELISA). The ELISA was sensitive to 10 pg.ml- 1 rat TNF-alpha and was specific for TNF-alpha, showing no cross-reactivity with rat IL-1alpha, rat IL-1beta, rat IL-1Ra or rat IL-6. The ELISA was used to measure TNF-alpha in the plasma of rats injected with bacterial endotoxin and in cultures of rat white blood cells. The ELISA was shown to be a robust method suitable for use in assaying samples generated in both in vivo or in vitro experiments.

Interleukin (IL)-6 has been proposed to mediate several sickness responses, including brain-mediated neuroendocrine, temperature, and behavioral changes. However, the exact mechanisms and sites of action of IL-6 are still poorly understood. In the present study, we describe the effects of central administration of species-homologous recombinant rat IL-6 (rrIL-6) on the induction of hypothalamic-pituitary-adrenal (HPA) activity, fever, social investigatory behavior, and immobility. After intracerebroventricular administration of rrIL-6 (50 or 100 ng/rat), rats demonstrated HPA and febrile responses. In contrast, rrIL-6 alone did not induce changes in social investigatory and locomotor behavior at doses of up to 400 ng/rat. Coadministration of rrIL-6 (100 ng/rat) and rrIL-1beta (40 ng/rat), which alone did not affect the behavioral responses, reduced social investigatory behavior and increased the duration of immobility. Compared with rhIL-6, intracerebroventricular administration of rrIL-6 (100 ng/rat) induced higher HPA responses and early-phase febrile responses. This is consistent with a higher potency of rrIL-6, compared with rhIL-6, in the murine B9 bioassay. We conclude that species-homologous rrIL-6 alone can act in the brain to induce HPA and febrile responses, whereas it only reduces social investigatory behavior and locomotor activity in the presence of IL-1beta.

Adenovirus-mediated gene transfer into the brain is associated with significant inflammation and activation of anti-vector and anti-transgene immune responses that curtail the gene delivery of adenoviruses and therapeutic efficacy. Elucidating the molecular mediators of inflammatory and immune responses to adenoviruses injected into the brain should allow us to inhibit their inflammatory actions, thereby reducing vector clearance and enhance adenoviral-mediated gene transfer into the CNS. Cytokines are primary mediators of the immune response and are released during inflammation. Here we report for the first time that injection of replication-deficient adenovirus vectors into the cerebral ventricles of rats causes a rapid increase in body temperature. This fever response precedes any vector-encoded transgene expression and occurs with vectors encoding no transgene, as well as with vectors encoding a therapeutic transgene i.e., HSV1-thymidine kinase. No fever is detected after infection of the striatum, an important brain target in studies on neurodegeneration. After infection of the brain ventricles, CSF levels of immunoreactive tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta increase significantly (up to 300-fold). In the hypothalamus, the locus of thermoregulation in the brain, only IL-1beta and IL-6 are significantly elevated. A neutralizing TNF-alpha antibody has no effect on adenovirus-induced fever. However, pretreatment with either the IL-1 receptor antagonist or the cyclooxygenase inhibitor flurbiprofen completely abolishes adenovirus-induced fever, suggesting that IL-1 and prostaglandins are direct mediators of this response. These results are the first to demonstrate that IL-1, but not TNF-alpha, is the main mediator of a very early inflammatory response to adenovirus in the brain.

Interleukin 6 (IL-6) is a cytokine involved in many aspects of the acute phase and immune responses. Cloning of rat IL-6 cDNA into the pET-21d expression plasmid under control of a bacteriophage T7 RNA polymerase promoter system allowed isopropylthio-galactopyranoside (IPTG)-inducible production of recombinant rat IL-6 in Escherichia coli. The cloning, expression and purification of rat IL-6 is described. In this expression system, rat IL-6 was produced in insoluble inclusion bodies. The protein was solubilized in 6 M guanidine hydrochloride and refolded in a glutathione redox system. Refolded rat IL-6 was purified to homogeneity using anion-exchange chromatography on SP-Trisacryl. The purified recombinant rat IL-6 had a molecular mass of 21 756.38+/-0.25 Da, which is within 0.01% of the predicted value, taking into account cleavage of the N-terminal methionine residue and the formation of two disulfide bridges. Recombinant rat IL-6 was 2-3-fold more bioactive than the human standard preparation in the B9 hybridoma bioassay. Purified rat IL-6 was used to raise polyclonal antibodies in sheep and these reagents were used to develop a novel rat IL-6 enzyme-linked immunosorbent assay (ELISA). The ELISA is sensitive to 10 pg/ml and has been shown to detect IL-6 in plasma from rats injected with lipopolysaccharide (LPS)., (Copyright 1999 Academic Press.)

An association between allelic variants in the vitamin D receptor gene and bone mineral density has been previously described. A bimodal variation in the rate of bone resorption (as measured by urinary deoxypyridinoline excretion rate) has also been reported. We have recruited male volunteers, to minimise variation associated with ovarian function, to investigate a possible connection between these observations. Allelic variants in the vitamin D receptor gene were identified as Taq1 restriction fragment length polymorphisms. The ratio of variants TT:Tt:tt occurred with a frequency of 34%:47%:17%. Excretion rates of urinary free deoxypyridinoline, measured by immunoassay, were compared in age-matched males from each genetic group. There were no significant differences based on the paired Student's t-test. Excretion rates declined with age (P = 0.04) and the best fit model fits the same regression line to each group. Genetic variation in the vitamin D receptor is not linked with differences in bone resorption rates.