Peripheral Blood Leukocyte DNA Research Articles

Detection and quantification of 8-hydroxydeoxyguanosine adducts in peripheral blood of people exposed to ionizing radiation.

Ionizing radiation produces a variety of damaging insults to nucleic acids, including the promutagenic lesion 8-hydroxydeoxyguanosine. In the present study, the 8-hydroxydeoxyguanosine content of peripheral blood leukocyte DNA isolated from individuals exposed to therapeutic doses of ionizing radiation was determined by a HPLC-coupled 32P-postlabeling assay. Peripheral blood leukocyte DNA from individuals irradiated with 180-200 cGy were observed to contain 2-4.5 times as much 8-hydroxydeoxyguanosine as that from unexposed individuals. These results were confirmed by the use of a HPLC-coupled electrochemical detection system. Thus, human exposure to ionizing radiation significantly increased the circulating leukocyte DNA content of 8-hydroxydeoxyguanosine.

Biomarkers of carcinogen exposure and cancer risk in a coke plant.

To evaluate the association between an indicator of carcinogen exposure (peripheral blood leukocyte DNA adducts of polycyclic aromatic hydrocarbons) and an early indicator of neoplastic transformation (sputum epithelial cell membrane antigens binding by monoclonal antibodies against small cell lung cancer and against nonsmall cell lung cancer), a survey of 350 coke-oven workers and 100 unexposed workers was planned. This paper reports a pilot investigation on a subgroup of 23 coke-oven workers and 8 unexposed controls. A "gas regulator" worker with positive tumor antigen binding was identified. Results show that smokers, subjects with decreased pulmonary function (forced expiratory volume in 1 sec/forced vital capacity% < 80), and those with morphological dysplasia of sputum cells have higher levels of DNA adducts. The gas regulators showed the highest values for adducts; however, no significant difference of adduct levels was found between the coke-oven group and unexposed controls.

Correlation between PAH airborne concentration and PAH-DNA adducts levels in coke-oven workers

In order to evaluate the correlation between peripheral blood leukocyte DNA adducts as an indicator of exposure to polycyclic aromatic hydrocarbons (PAH) and the airborne contamination of PAH at the work places, a survey of a cohort of coke-oven workers has been carried out. In each workplace, total and specific PAH airborne concentrations were measured. Among the job title groups, the highest proportion of subjects with levels of adducts above the detection limit and the highest mean value were observed in the door-operators. The correlation between median values of environmental benzo(a)pyrene concentration and mean values of adducts concentration support the fact that the maintenance workers group has a higher relative risk (RR) to have detectable level of PAH-adducts, which is estimated to be 1.84 for an increase of 1 micrograms/m3 of benzo(a)pyrene.

Failure to detect human cytomegalovirus DNA in peripheral blood leukocytes of healthy blood donors by the polymerase chain reaction.

The transmission of cytomegalovirus (CMV) by blood transfusion may have a major effect on certain immunocompromised patients. To protect susceptible blood recipients from infection, it is advisable to use blood components from CMV-seronegative donors. However, serologic tests are not capable of indicating which blood component actually harbors infectious virus and can transfer it to the recipient. Therefore, a sensitive method is needed for the detection of the virus itself. There have been three reports on the detection of CMV in healthy volunteer blood donors by the polymerase chain reaction (PCR). CMV DNA was found in all seropositive and most seronegative blood donors. However, many other authors have failed to confirm these data. A highly sensitive and specific PCR assay was developed for the detection of CMV DNA in peripheral blood leukocytes. With this protocol, blood samples from 116 volunteer blood donors were investigated. None of these samples proved to be positive for CMV DNA. In contrast, CMV DNA was detected in 10 of 10 renal transplant patients early in the course of active CMV infection. It can be concluded that the CMV genome copy number in the peripheral blood leukocytes of healthy individuals is beyond the detection limit of current PCR technology.

Genetic Factors in Autoimmune Thyroid Disease Analyzed by Restriction Fragment Length Polymorphisms of Candidate Genes*

Possible genetic effects on the development of autoimmune thyroid disease were studied by analysis of restriction fragment length polymorphisms (RFLPs) of candidate genes. Peripheral blood leukocyte DNA was obtained from 65 caucasian patients with Graves' disease, 63 caucasian patients with Hashimoto's thyroiditis, and 65 caucasian controls. RFLP analysis was carried out on genomic DNA, using probes for DR beta, DQ alpha, DQ beta, DP alpha, DP beta, T-cell receptor TCR alpha, and thyroid peroxidase. The methodology allowed HLA-DR and DQ typing and provided information on specific RFLP patterns related to T cell receptor (TCR)alpha, DP alpha and -beta, and -beta, and thyroid peroxidase. HLA-DR3 frequency was significantly increased in patients with Graves' disease, as reported previously by others, but neither DNA-derived subtype of DR3 was differentially increased. HLA-DQw2 was also present in increased frequency because of its linkage disequilibrium with HLA-DR3. It is uncertain whether the primary susceptibility is with DQ, DR, or another nearby locus. Susceptibility was not related in these studies to genetic loci recognized in these studies involving DQ alpha, DP, TCR, or thyroid peroxidase. A significant linkage disequilibrium between DR3 and a specific DX alpha RFLP was observed in Graves' disease, but is believed to be representative of a generalized linkage disequilibrium between DR3 and DX alpha, rather than a specific abnormality in Graves' disease. Previous studies indicating association with specific TCR RFLPs could not be reproduced. The relative risk for carriers of HLA-DR3 subtype A in this study was 7.37-fold. RFLP analysis offers the possibility of investigating linkage in a variety of candidate genes as well as established genetic relationships for potentially important subtypes. While the significant relationship with HLA-DR3/DQw2 was reconfirmed, the involvement of other genes or haplotypes could not be established.

Evidence for acrolein-modified DNA in peripheral blood leukocytes of cancer patients treated with cyclophosphamide

Monitoring human populations for specific DNA modifications has been made possible by developing highly sensitive immunoassays employing antibodies specific for carcinogen-DNA adducts. While these techniques have been used to follow occupationally and environmentally exposed populations, results have been limited by the lack of exposure data with which to correlate adduct formation. Cancer patients treated with precisely known doses of anticancer drugs can be studied to examine the association between drug dose and adduct formation. This study examined acrolein-modified DNA in patients treated with the anticancer drug cyclophosphamide (CP) and in newly diagnosed patients prior to treatment. Employing 2 different detection methods, enzyme-linked immunosorbent assay (ELISA) and immuno-dot blot (IDB), acrolein-modified DNA was identified in a total of 6 of 12 (50%) treated patients and in 0 of 15 untreated patients. Formation of acrolein-modified DNA was examined as a function of lifetime CP dose, recent CP dose, time since last treatment, regime of treatment, and smoking history; however no clear trends were observed.

Mitochondrial DNA Deletion in Oculoskeletal Myopathy

The case of a patient showing bilateral ophthalmoplegia with proximal limb weakness, severe dysphagia and short stature, without family history, is described. The diagnosis of Kearns-Sayre syndrome was excluded because of the absence of pigmentary retinopathy and of all other common manifestations except short stature. The analysis of mitochondrial DNA of the patient's muscle revealed a deleted form accounting for 65% of the total mitochondrial DNA. The deletion, undetectable in the mitochondrial DNA of peripheral blood leukocytes, was apparently indistinguishable from that already described by others in a far more severe form of classic Kearns-Sayre syndrome.

Mitogenic activity of selenoorganic compounds in human peripheral blood leukocytes

A variety of organoselenium compounds were originally described as antiinflammatory, antioxidant or glutathione-peroxidase-like agents, and as inhibitors of prostaglandin and leukotriene synthesis. Recently, the compounds have also been found to be inducers of interferon gamma and tumor necrosis factor in human peripheral blood leukocytes (PBL). We evaluated the effects of bis [2-(N-phenylcarboxamido)phenyl] diselenide and Ebselen; 2-phenyl-1,2-benzisoselenazol-3(2H)one, on the incorporation of tritiated thymidine into the DNA of PBL cultured in vitro. Both compounds were mitogenic and this effect was correlated with the expression of interleukin 2 receptor in T-lymphocytes. Therefore, we suggest that the selenoorganic compounds may induce mitogenic cytokines.

Hepatitis B virus DNA in peripheral blood leukocytes: A brief review

Despite being essentially hepatotropic, hepatitis B virus has frequently been detected in peripheral blood leukocytes. In most cases, viral DNA has been detected by hybridisation, though, occasionally, evidence of viral RNA and virus-specific proteins has been reported. Though definitive evidence is lacking, it appears likely that the virus is able to replicate in at least a subfraction of these cells. The possible consequences with respect to the transmission and reactivation of virus infections are discussed.

Changes in DNA and surface properties of peripheral blood leukocytes in monkeys after whole-body ?-irradiation

Changes of Macaca nemestrina and Rhesus blood DNA have been studied up to 5 days after the total uniform gamma-irradiation in doses 6.2 and 6.5 Gy. The content of nucleotide ATrich DNA has been evaluated in the fractions of leucocytes with the various surface adherence properties. The dynamics of the content nucleotide AT-DNA and blood leucocytes were similar at the both monkey species. The evaluation of structural state DNA in the blood nucleotide with the fluorescent dyes (ethidium bromide and 4; 6-diamidine-2-phenylindole) demonstrated that the important changes in the polynucleotide structure occurred from 6 to 24 h after irradiation and maintained up to 5 days. Adhesive capacity changes were reversible but they preceded the DNA structural changes. At 24 h postirradiation non-adhesive cells with relative higher AT-DNA content were found.

RAPID DETECTION OF HUMAN CYTOMEGALOVIRUS DNA IN PERIPHERAL BLOOD LEUKOCYTES OF VIREMIC TRANSPLANT RECIPIENTS BY THE POLYMERASE CHAIN REACTION

Peripheral blood leukocyte samples (n = 458) of 24 bone marrow transplant and 52 kidney transplant patients were examined weekly for the presence of human cytomegalovirus (HCMV) using an improved culture technique (DEAFF; detection of early antigen fluorescent foci). In total 5 (21%) bone marrow transplant and 11 (21%) kidney transplant patients developed a viremia. Patients' samples were investigated for the presence of HCMV DNA using an in vitro DNA amplification technique, the polymerase chain reaction (PCR). From the statistically evaluable viremic patients (n = 13), 110 blood samples were analyzed. In 5 of these patients, the DEAFF and PCR led to identical results. In 8 patients however the PCR was more sensitive, i.e. HCMV DNA was detected for a longer period of time. Applying statistical analysis using the McNemar test, this result was significant (P less than 0.05). The PCR applied on leukocyte samples did not detect HCMV DNA in viruric patients without viremia. Moreover, the current PCR never led to positive results with peripheral blood leukocyte samples of healthy seropositive or seronegative controls. Since the PCR can be performed in 6 hr, this technique will contribute to rapid detection of HCMV DNA in peripheral blood leukocytes and therefore to optimal clinical management of HCMV-infected transplant recipients.

DNA polymorphism of the HLA-B35 gene associated to different HLA-C locus alleles

Peripheral blood leukocyte DNA from 31 donors, previously typed as HLA-B35, was digested with EcoRV and analyzed by Southern transfer and hybridization to an HLA class I probe. Out of 31 HLA-B35 positive (+) individuals, 24 showed a 4.6-kb band, previously associated with the B35 allele by Cohen et al. (Proc Natl Acad Sci USA 80: 6289–6292). The HLA-B35 allele has a strong linkage disequilibrium (70%) with the Cw4 allele. All samples having the 4.6-kb band corresponded to HLA-B35+,Cw4 + cells, whereas those lacking the band came from HLA-B35+,Cw4-negative (−) cells. Five HLA-B35−,Cw4+ samples were also studied and none showed the band. An HLA-B locus-specific probe gave a strong hybridization signal to this fragment, whereas an HLA-C locus-specific probe revealed different bands. The results suggest that the 4.6-kb fragment contains the B35 gene. Digestion with other restriction enzymes, located the polymorphic site to the 3′ end of the gene. Analysis of 18 additional individuals with other specificities of the “4c” antigenic cluster (HLA-B18, B51, B52 and B53) showed that the EcoRV 14.6 kb-band was also present in 5/5 B52 cells and 4/4 B53 samples.

The site of the breakpoint within the bcr is a prognostic factor in Philadelphia-positive CML patients [see comments

The Philadelphia (Ph1) chromosome, characteristic of chronic myelogenous leukemia (CML), arises from a reciprocal translocation between chromosomes 9 and 22. The site of the breakpoint on chromosome 22 is within a small region called the breakpoint cluster region (bcr). We have mapped the breakpoint within the bcr in peripheral blood leukocyte DNA from 22 Ph1-positive CML patients. No correlation between the site of the breakpoint and the clinical phase of the disease was found. However, a striking correlation between the site of the breakpoint and the length of time between presentation and onset of acute phase was observed: on average, patients with a 5′ break-point had a fourfold longer chronic phase (median, 203 weeks) than those with a 3′ breakpoint (median, 52 weeks).

Harvey-ras oncogene restriction fragment alleles in familial melanoma kindreds.

Unique and uncommon BamHI allelic restriction fragments of the Ha-ras locus have been reported in the genomes of patients with cancer and of three affected members of a familial melanoma kindred (Krontiris et al., 1986). Analysis of the BamHI and Msp/HpaII restriction fragments of peripheral blood leucocyte DNA from the members of two families with hereditary melanoma (HM)/familial dysplastic naevus syndrome (DNS) revealed that the only Ha-ras allele common to four affected members of one kindred and two from a second kindred, was the 6.7kb allele which is found in 66% of the normal population. This allele was found equally in affected and non-affected family members, and in one affected case was inherited from an unaffected homozygous parent. It was absent in two affected sisters in a third kindred. In the first kindred the karyotype of all three melanoma sufferers was 46XX 9qh+, while six unaffected members had a normal karyotype. BamHI polymorphism of the Ha-ras gene does not identify the affected members in the HM/DNS families studied.