Interindividual variation in the responses of cultured human lymphocytes to exposure from DNA damaging chemical agents: interindividual variation to carcinogen exposure.

Human population variability to standardized doses of N-acetoxy-2-acetylaminofluorene (NA-AAF) and 7, 12-dimethylbenz(a) anthracene (DMBA) was determined in cultured lymphocytes by measuring (a) differential stimulation of unscheduled DNA synthesis after 1 h induction of DNA damage by 10 micrometer NA-AAF, (b) the level of NA-AAF induced chromosome aberrations remaining after 8 h of DNA-repair synthesis, and (c) the level of [3H]DMBA bound to DNA after 18 h incubation of resting lymphocytes in 5 micrometer DMBA. All 3 parameters indicated individual variation to carcinogen exposure and were correlated to the population differences in age, sex, blood pressure and mortality rates. Males always had a greater potential to accumulate DNA-damage than did females regardless of the sampled population. DNA-damage potentials increased with increasing age, blood pressure or mortality rates. There was always proportionally greater DNA-damage potentials in the males than in females. The in vitro response of mature granulocytes to a 10 micrometer NA-AAF dose, as estimated by [3H] thymidine incorporation from unscheduled DNA synthesis, was much lower than lymphocyte response. Nevertheless, individual variations in granulocyte NA-AAF induced unscheduled DNA synthesis paralleled the inter-individual fluctuations observed in the lymphocyte responses to NA-AAF.     

Interindividual variation in the responses of cultured human lymphocytes to exposure from DNA damaging chemical agents: Interindividual variation to carcinogen exposure

Human population variability to standardized doses of N-acetoxy-2-acetylaminofluorene (NA-AAF) and 7,12-dimenthylbenz(a)anthracene (DMBA) was determined in cultured lymphocytes by measuring (a) differential stimulation of unscheduled DNA synthesis after 1 h induction of DNA damage by 10 μM NA-AAF, (b) the level of NA-AAF induced chromosome aberrations remaining after 8 h of DNA-repair synthesis, and (c) the level of [³H]DMBA bound to DNA after 18 h incubation of resting lymphocytes in 5 μM DMBA. All 3 parameters indicated individual variation to carcinogen exposure and were correlated to the population differences in age, sex, blood pressure and mortality rates. Males always had a greater potential to accumulate DNA-damage than did females regardless of the sampled population. DNA-damage potentials increased with increasing age, blood pressure of mortality rates. There was always proportionally greater DNA-damage potentials in the males than in females. The in vitro response of mature granulocytes to a 10 μM NA-AAF dose, as estimated by [³H]thymidine incorporation from unscheduled DNA synthesis, was much lower than lymphocyte response. Nevertheless, individual variations in granulocyte NA-AAF induced unscheduled DNA synthesis paralleled the inter-individual fluctuations observed in the lymphocyte responses to NA-AAF.     

Prior exposure to restraint stress enhances 7,12-dimethylbenz(a)anthracene (DMBA) induced DNA damage in rats

Over the years, several lines of evidence have emerged supporting the role of stress in the development and progression of cancer. Stress can cause an increase in the production of reactive oxygen species (ROS) and decrease in the in vivo antioxidant defense systems. A ROS-induced DNA damage in peripheral lymphocytes, liver and skin cells may be revealed by Comet assay. To test whether DNA is damaged by stress/DMBA/stress and DMBA, rats were exposed to multiple doses of DMBA in the presence and absence of restraint stress, and DNA damage was evaluated. Insignificant differences were detected in all the three cells tested (peripheral lymphocytes, liver and skin cells) between control and stress treatment in terms of frequencies of damaged DNA. The extent of DNA migration was enhanced in DMBA treated rats in a dose dependent manner. Pre-stress DMBA treatment showed still higher frequencies of damage in comparison with control, stress alone or DMBA alone groups. Thus, prior exposure to stress clearly enhanced the DMBA induced DNA damage, especially so in the skin cells (target organ of the carcinogen application) than liver and peripheral lymphocytes as observed on the basis of the extent of DNA migration (tail DNA) during single cell gel electrophoresis.     

Kinetics of excision repair of UV-induced DNA damage, measured using the comet assay

The kinetics of UV- (254 nm) irradiation-induced DNA single-strand breaks (SSBs), generated during the excision repair of UV-induced DNA damage, in leukemic lymphocytes and in normal blood mononuclear cells (MNCs) were studied using the alkaline comet assay. The cells were isolated by density gradient centrifugation from peripheral blood of patients with chronic lymphocytic leukemia (CLL) and from healthy study subjects. The cytotoxicity of UV irradiation was determined in vitro in peripheral blood mononuclear lymphocytes from 36 CLL patients and from eight healthy donors using the incorporation of radioactive leucine in 4-day cultures. A remarkable difference in excision repair capability was observed between normal and leukemic lymphocytes. In contrast to normal lymphocytes, there was always a subpopulation of CLL cells that did not complete the repair of UV-induced DNA damage during the 24-h repair period. Furthermore, differences were also recorded between UV-sensitive and UV-resistant CLL cases. The differences in DNA migration between the maximum increase (59-77 microm) and that at 24 h after irradiation (21-66 microm) was statistically significant in two of three patients exhibiting UV-resistance. Correspondingly, only in one of three patients exhibiting UV-sensitivity was the difference in DNA migration statistically significant (maximum increase: 44-107 microm, vs. 24 h after: 42-100 microm). Our results confirm an abnormal pattern of the CLL cell response to UV irradiation. Furthermore, we identified defective processing of UV-induced DNA damage in CLL versus normal lymphocytes, particularly in UV-sensitive cases.     

Quantification of DNA repair capacity in whole blood of patients with head and neck cancer and healthy donors by comet assay

Comet assay has been used to estimate cancer risk by quantification of DNA damage and repair in response to mutagen challenge. Our goal was to adopt best practices for the alkaline comet assay to measure DNA repair capacity of white blood cells in whole blood of patients with squamous cell carcinoma of the head and neck (HNSCC). The results show that initial damage by 10 Gy of gamma radiation expressed as percent DNA in comet tail was higher in stimulated lymphocytes (61.1+/-11.8) compared to whole blood (43.0+/-12.1) but subsequent repair was similar with comet tail of approximately 20% at 15 min and 13% at 45 min after exposure. Exposure of whole blood embedded in agarose from 5 to 10 Gy gamma radiation was followed by an approximately 70% repair of the DNA damage within 45 min with a faster repair phase in the first 15 min. Variability of the measurement was lower within repeated measurements of the same person compared to measurement of different healthy individuals. The repair during first 15 min was slower (p=0.01) in ex-/non-smokers (41.0+/-2.1%) compared to smokers (50.3+/-2.7%). This phase of repair was also slower (p=0.02) in HNSCC patients (36.8+/-2.1%) compared to controls matched on age and smoking (46.4+/-3.0%). The results of this pilot study suggest that quantification of repair in whole blood following a gamma radiation challenge is feasible. Additional method optimization would be helpful to improve the assay for a large population screening.     

Endothelial and smooth muscle cells from abdominal aortic aneurysm have increased oxidative stress and telomere attrition

Background

Abdominal aortic aneurysm (AAA) is a complex multi-factorial disease with life-threatening complications. AAA is typically asymptomatic and its rupture is associated with high mortality rate. Both environmental and genetic risk factors are involved in AAA pathogenesis. Aim of this study was to investigate telomere length (TL) and oxidative DNA damage in paired blood lymphocytes, aortic endothelial cells (EC), vascular smooth muscle cells (VSMC), and epidermal cells from patients with AAA in comparison with matched controls.

Methods

TL was assessed using a modification of quantitative (Q)-FISH in combination with immunofluorescence for CD31 or α-smooth muscle actin to detect EC and VSMC, respectively. Oxidative DNA damage was investigated by immunofluorescence staining for 7, 8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG).

Results and Conclusions

Telomeres were found to be significantly shortened in EC, VSMC, keratinocytes and blood lymphocytes from AAA patients compared to matched controls. 8-oxo-dG immunoreactivity, indicative of oxidative DNA damage, was detected at higher levels in all of the above cell types from AAA patients compared to matched controls. Increased DNA double strand breaks were detected in AAA patients vs controls by nuclear staining for γ-H2AX histone. There was statistically significant inverse correlation between TL and accumulation of oxidative DNA damage in blood lymphocytes from AAA patients. This study shows for the first time that EC and VSMC from AAA have shortened telomeres and oxidative DNA damage. Similar findings were obtained with circulating lymphocytes and keratinocytes, indicating the systemic nature of the disease. Potential translational implications of these findings are discussed.     

Isolation, identification and electrophoretic properties of DNA excreted by human lymphocytes

It was shown that the buoyant density of DNA isolated from lymphocyte incubation media as well as from blood plasma of healthy patients and patients with chronic lympholeukemia (CLL) using the SDS-phenol method with subsequent ultracentrifugation in a cesium trifluoroacetate density gradient is 1.59-1.60 and 1.60-1.63 g/ml, respectively. Using electrophoresis in 0.6% agarose gel, it was found that the DNA excreted by lymphocytes from healthy patients and CLL patients is a high molecular weight fraction comprising 21,000 nucleotide pairs. This DNA fragment contains 90-95% of [3H]thymidine used for the labeling of DNA excreted by lymphocytes from healthy patients and patients with CLL. No traces of [3H]thymidine were found in the middle part of the agarose gel containing the diffuse material bound to ethidium bromide.     

Hyperactive ENaC identifies hypertensive individuals amenable to amiloride therapy

Pathophysiological features of bothprimary aldosteronism and pseudohyperaldosteronism are hyperactiveamiloride-sensitive epithelial Na⁺ channels (ENaC) andrefractory hypertension. Peripheral blood lymphocytes express ENaC,which functions and is regulated similarly to ENaC expressed by renalprincipal cells. Thus it was hypothesized that individuals with eitherof these hypertensive etiologies could be identified by assessment ofthe function and regulation of peripheral blood lymphocyte ENaC, bywhole cell patch clamp. We also tested the hypothesis that specificinhibition of hyperactive ENaC with amiloride could ameliorate thehypertension. To test these hypotheses, we solicited blood samples fromnormotensive, controlled hypertensive, and refractory hypertensiveindividuals. Lymphocytes were examined electrophysiologically todetermine whether ENaC was hyperactive. All positive findings were from refractory hypertensive individuals. Nine refractory hypertensive patients had amiloride added to their hypertensive therapy. Amiloride normalized the blood pressure of four subjects. These individuals allhad hyperactive ENaC. Amiloride had no effect on individuals withnormal ENaC. These findings suggest that whole-cell patch clamp ofperipheral blood lymphocytes can be used to identify accurately andrapidly hypertensive individuals who will respond toamiloride therapy.

     

The inhibitory effect of oral administration of garlic on experimental carcinogenesis in hamster buccal pouches by DMBA painting

The inhibitory effect of oral administration of garlic on experimental carcinogenesis in buccal pouches induced by painting 9,10-dimethyl-1,2-benz(a)anthracene (DMBA) was studied on 40 golden Syrian hamsters. The animals were grouped at random into four experimental groups (oral administration of garlic, NTP, BP or mineral oil followed by DMBA painting on buccal pouches), three chemical control groups (oral administration of garlic, NTP or BP without DMBA painting) and a DMBA control group (only painted DMBA on buccal pouches). Starting from the fourth week after DMBA painting, the pouch mucosae were examined biweekly for its tumor formation and blood vessel architecture. Animals were sacrificed 25 weeks after DMBA application. Tumors and pouch mucosae were dissected to examine tumor nature and biochemical reactions of DNA synthesis and GGTase activity. The inhibitory efficacy of garlic, BP and NTP were evaluated according to the results of these examinations. Garlic was found to have a higher inhibitory efficacy than BP and NTP through the probable mechanism of competitive binding with nuclear DNA and diminishing the opportunity of DMBA to initiate carcinogenesis. Other factors related to cancer inhibition included insufficient local blood flow, low GGTase activity and lesser DNA synthesis. The inhibitory effect of fractions of garlic on experimental carcinogenesis should be a reasonable and necessary continuation in future studies of the series of cancer prevention by garlic.     

Frequency in hypertensives of alleles for a RFLP associated with the renin gene

The genetic basis of primary hypertension is not known. Renin is important in blood pressure and volume control and a HindIII restriction fragment length polymorphism (RFLP) is present within the human renin gene locus. To examine whether there is a relationship between this RFLP and primary hypertension, DNA and renin analyses were performed on leukocytes and plasma from hypertensive and normotensive individuals. In hypertensives the frequencies of alleles for the HindIII RFLP were found to be 0.55 and 0.45, compared with 0.60 and 0.40 in the total population of 231 subjects examined, a difference that was not statistically significant. There also appeared to be no significant difference in renin activity in plasma for hypertensive patients of each genotype, nor in their pre- or post-treatment blood pressures. We thus conclude that, within the limits of the present study, the suspected genetic abnormalities associated with primary hypertension in man do not appear to be related to a HindIII RFLP in the renin gene.     

Purifying human Y chromosomes by flow cytometry and sorting

A method of producing an enriched sample of human Y chromosomes from peripheral blood lymphocytes is described. Metaphase chromosomes were prepared from peripheral blood lymphocytes donated by 17 normal male individuals. A suspension of chromosomes in a polyamine buffer was produced from each sample, stained with the fluorescent dye Hoechst 33258, and passed through a flow cytometer and sorter. Following analysis of the 17 fluorescence distributions, a single donor was found giving a separate peak corresponding to the Y chromosome. Seventy percent of the chromosomes sorted from this peak were identified as Y chromosomes. Batches of a million Y chromosomes were produced from each of several 40 ml donations of peripheral blood. These were assessed for the amount of Y DNA present and used to construct a DNA library.     

Binding of polycyclic aromatic hydrocarbons to DNA of cells in culture: a rapid method for its analysis using hydroxylapatite column chromatography.

A rapid procedure to study the interaction of carcinogens with DNA in cultured cells has been developed. The cells, which are labeled with 7,12-[3H]dimethylbenz[a] anthracene ([3H]DMBA), are lysed with 0.24 M phosphate buffer (pH 6.8), 1% sodium dodecyl sulfate (SDS), 8 M urea and 0.01 M ethylenediamine-tetraacetate (EDTA) and sonicated. The cell lysates are fractionated on columns of hydroxylapatite. Proteins and RNA are removed with 8 M urea in 0.24 M phosphate buffer (pH 6.8). DMBA-bound DNA is eluted with 0.4 M phosphate buffer (pH 6.8). DMBA-DNA isolated by this procedure is virtually free from proteins and RNA. Thermal stability, ultraviolet spectra and the density of DNA is not altered by DMBA binding. The uptake of DMBA by mouse epidermal cells is rapid and the binding of DMBA to DNA is linear for the first 8 h of exposure. DMBA binds to DNA in all phases of the cell cycle. However, the highest binding occurs immediately following maximum DNA synthesis.     

Intercalative DNA binding of model compounds derived from metabolites of 7,12-dimethylbenz[a]anthracene

The DNA binding of nonreactive model compounds of metabolites of 7,12-dimethylbenz[a]-anthracene (DMBA)1 was studied in fluorescence quenching and fluorescence lifetime experiments. The model compounds examined were DMA and 8,9,10,11-tetrahydro-BA. DMA is a pi electron model of a highly carcinogenic bay region epoxide of DMBA, 8,9,10,11-tetrahydro-BA is a model compound of a less carcinogenic DMBA epoxide. The results indicate that the binding of DMA occurs primarily via intercalation. In 15% methanol the binding constant is 3.1 x 10(3) M-1. In 15% methanol and at DNA phosphate levels of 5.0 x 10(-4) M the intercalative binding of DMA is reduced by a factor of 6.2 when 5.0 x 10(-4) M Mg+2 is added. The DMA binding constant for intercalation is reduced by more than a factor of 4 when the methanol content of the solvent is increased from 0% to 20%. Finally DMA binding arising from pi interactions with the DNA bases is reduced more than 15 times when the DNA is denatured. For 8,9,10,11-tetrahydro-BA in 15% methanol the binding constant for intercalation is 6 times lower than that for DMA. These results along with previously reported binding data on other model compounds suggest that bay region metabolites of DMBA readily participate in physical pi stacking interactions with DNA.     

Functional and biochemical disposition of 7,12-dimethylbenz[a]anthracene in murine lymphoid cells

The metabolism of the polycyclic aromatic hydrocarbon (PAH) 7,12-dimethylbenz[a]anthracene (DMBA) was studied in murine lymphocytes. This carcinogen has previously been shown to be immunosuppressive to lymphocytes regardless of their ability to be induced via the Ah locus and receptor. Experiments were designed to quantify the generation of metabolites of DMBA by lymphocytes incubated with [14C]DMBA and to ascertain whether radioactivity was covalently bound to cellular macromolecules in DMBA-exposed lymphocytes. No significant metabolism of DMBA was detected in culture supernatants, except when cultures were incubated in the presence of Arochlor-induced rat liver 9000 x g supernatants (S9). Covalent binding of 14C to cellular macromolecules was enhanced approximately eightfold in the presence of S9. Inhibition of monooxygenase activity by alpha-naphthoflavone did not modulate the immunosuppressive character of DMBA. Furthermore, addition of S9 did not amplify or ablate DMBA-mediated suppression of lymphocyte proliferation to the mitogen concanavalin A (Con A). Selected metabolites of DMBA were evaluated for immunosuppressive effects in cultures stimulated with mitogens and cellular alloantigens. 7-Hydroxymethyl-12-methylbenz[a]anthracene (OHMe) and 5,6-dihydro-5,6-dihydroxybenz[a]anthracene (Diol) were found to cause only slightly greater suppression of lymphocyte responses than DMBA. Thus, it appears that metabolites of DMBA were not responsible for the immunosuppression observed in lymphocyte cultures and that lymphocytes were not equipped to metabolize any significant amount of DMBA. These data lend support to the hypothesis that parent compound alone is responsible for the immunosuppressive effects observed in murine lymphocyte culture.     

The binding of 7,12-dimethylbenz[a]anthracene to mammary gland macromolecules in the hamster:effects of Enovid feeding or transplacental exposure to diethylstilboestrol

The covalent binding of 7,12-[3H]dimethylbenz[a]anthracene ([3H--DMBA) to mammary gland macromolecules was studied in hamsters fed a contraceptive mixture, Enovid, those exposed transplacentally to diethylstilboestrol (DES), and controls. Compared with rats, hamsters are relatively resistant to DMBA mammary carcinogenesis, but susceptibility is increased by either of the above treatments with Enovid or DES. The amount of DMBA bound to DNA and protein ws 4-5 times greater than to RNA, but only DNA binding was persistent. Fifty-three percent of the DNA-bound DMBA was still present after 8 days. The amount of DMBA bound to hamster mammary DNA and its persistence was similar to that found in rats. Neither Enovid nor DES treatment altered the levels of binding to mammary macromolecules, nor their persistence. These results indicate that the species differences in the susceptibility to DMBA-induced mammary carcinogenesis in hamsters and rats, and modification of the former by hormones, is not due to differences in the activation of carcinogens. The role of hormones such as prolactin in the promotion phase of mammary gland carcinogenesis may explain these differences.     

Effectiveness of treatments for hypertension in a sample of Colombian patients

Hypertension represents a high health cost because of its prevalence, its low level of diagnosis and control, and its role as a primary risk factor for other cardiovascular diseases. According to the JNC 7 report, hypertensive individuals have blood pressures of 140/90 mm Hg or higher; recommended treatment reduces these values to below 120/80 mm Hg. Co-morbidity and the presence of other risk factors must also be considered. In a random sample of 458 hypertensive patients from 6 Colombian cities, the effectiveness, tolerance and adherence to treatment was compared in cases with treatment of at least one year's duration. During routine blood pressure examinations, trained nurses obtained patient consent and additional anthropometric data, such as including co-morbidity, risk factors, antihypertensive medication prescribed, dosages and usage of unrelated medications. Some of the data were retrieved from the patients' medical histories. The average age of the patients was 57.6 +/- 13 years, with 67.5% women; 92% with complete adherence to the treatment and 59% not reporting adverse events associated with the medication. Forty-four percent were treated with antihypertensive monotherapy with the most commonly prescribed medications as follows (in order): hydrochlorothiazide, verapamil, enalapril, metoprolol and propanolol. Forty-five percent (n=207) were control patients, 35% were in a hypertensive stage 1 and 19.7% were in stage 2. Multivariate analysis showed that uncontrolled hypertension was significantly associated with geriatrics receiving a combination of antihypertensive medication and residence in three cities--Ibagué, Barranquilla and Manizales--where smaller daily doses of hypertensive medications are prescribed. Health care teams are advised to adjust doses carefully to obtain clearly defined therapeutic objectives.     

Inhibition of stage-specific DNA synthesis in rat spermatogenic cells by polycyclic aromatic hydrocarbons

Changes in the rate of DNA synthesis in spermatogenic cells after treatment of segments of rat seminiferous tubule at defined stages of epithelial cycle with benzo[a]pyrene (BP) or 7,12-methylbenz[a]anthracene (DMBA) were studied. The incorporation of labeled thymidine into DNA was used as a measure of the rate of DNA synthesis. Very little or no inhibition of DNA synthesis at stages V and VIII of the cycle was observed at BP and DMBA concentrations lower than 100 microM. In contrast, in the presence of added mitochondria and/or microsomes from whole rat testis, 20 microM BP or DMBA inhibited DNA synthesis 5% and 80%, respectively. This inhibition of DNA synthesis was prevented by inhibitors of the cytochrome P-450 system and by free radical scavengers. These results suggest that polycyclic aromatic hydrocarbons (PAH) require metabolic activation in order to inhibit DNA replication in seminiferous tubules. The first step of this biotransformation is cytochrome P-450-dependent and occurs in Leydig cells. However, the metabolites produced in this step may be further metabolized to reactive metabolites by peroxidative pathways in the seminiferous tubules; these latter products may affect DNA replication.     

Particularities of blood lymphocyte response to irradiation in vitro in breast cancer patients

DNA breaks and their repair efficiency were analyzed in irradiated in vitro lymphocytes (at doses 1 Gy, gamma-radiation of 60Co, dose rate 1 Gy/min) isolated from peripheral blood of 41 untreated patients with breast cancer and 25 healthy donors using the DNA comet assay under non-denaturing conditions (mainly double-strand DNA breaks (DSB), as well as apoptotic cell death using the DNA halo assay. To estimate the expression of bystander effect, the cells were incubated in a culture medium obtained from lymphocytes irradiated in vitro at doses 1 Gy. The average DSB level in blood lymphocytes of breast cancer patients was shown to be significantly higher (p < 0.05) compared with that in control donors. In general, the following effects were observed in irradiated in vitro lymphocytes of cancer patients: (1) increased sensitivity to y-radiation-induced DNA DSBs compared with lymphocytes from healthy donors, (2) reduced repair efficiency of these damages. Incubation of irradiated blood lymphocytes in a medium from irradiated cells led to an increased relative number of DNA DSBs and an elevated fraction of cells dying through apoptotic pathway both in blood lymphocytes from cancer patients and control donors. However, these non-targeted effects were more expressed for the blood lymphocytes of breast cancer patients.     

The effect of topical instillation of 2.5% phenylephrine and 1.0% tropicamide on the blood pressure of hypertensive patients

Background: Pupillary dilation is necessary to complete a thorough examination of the internal ocular structures and perform threshold visual fields on the automated perimeter. In our clinic, the topical instillation of 2.5% phenylephrine and 1.0% tropicamide following one drop of topical anesthetic is used routinely for pupil dilation. The vasoconstrictive effects of phenylephrine can cause an increase in peripheral resistance resulting in elevation of systolic and diastolic blood pressures. A rise in systemic blood pressure has been shown to occur following topical instillation of phenylephrine (Heath, Arch Ophthalmol, 1936;16:839–846). This study investigates the effect of topical instillation of 2.5% phenylephrine and 1.0% tropicamide on the blood pressure of known hypertensive patients 30 and 70 min after instillation. Methods: 118 hypertensive patients, all of whom were being treated with anti-hypertensive medications, were involved in the study. Fifty-six patients were dilated with two drops 2.5% phenylephrine and two drops 1.0% tropicamide instilled 5 min apart after one drop of local anesthetic (proparacaine 0.5%). The remaining 62 patients were examined but not dilated. Blood pressure was measured using a sphygmomanometer and stethoscope (right arm sitting) prior to dilation and 30 and 70 min following drop instillation. Results: No clinically significant increase in blood pressure at 30 and 70 min after instillation was observed in the hypertensive group that was dilated. In addition, the change in blood pressure of the dilated group and undilated group was not statistically significant. Conclusion: This study shows that pupillary dilation with 2.5% phenylephrine and 1.0% tropicamide did not significantly increase systemic blood pressure in this population of hypertensive patients.     

In vivo cellular tropism of human T-cell leukemia virus type 1.

To establish the phenotype of human T-cell leukemia virus type 1 (HTLV-1)-infected cells in peripheral blood, the polymerase chain reaction was used to detect and quantitate viral DNA in subpopulations of leukocytes obtained from patients with tropical spastic paraparesis and asymptomatic carriers. HTLV-1 could not be detected in peripheral blood mononuclear cells thoroughly depleted of T lymphocytes (E- CD3-), nor could it be detected in highly enriched populations of B lymphocytes (E- CD19+), monocytes (E- CD14+), or natural killer cells (E- CD16+). T lymphocytes were strongly positive for HTLV-1, and fractionation of this population revealed that 90 to 99% of the HTLV-1 DNA segregated with the CD4+ CD8- and CD45RO+ subsets. No difference between the cell type distribution of HTLV-1 in the asymptomatic carrier and the subjects with tropical spastic paraparesis was evident. Southern hybridization of genomic DNA prepared from the peripheral blood of HTLV-1 carriers indicated that up to 10% of circulating leukocytes may carry the HTLV-1 provirus.