Discordant fibrin formation in hemophilia.

BACKGROUND: The conversion of fibrinogen to fibrin and its crosslinking to form a stable clot are key events in providing effective hemostasis. OBJECTIVES: To evaluate the relationship of fibrinopeptide (FP) release and factor (F) XIII activation in whole blood from hemophiliacs. PATIENTS/METHODS: We investigated FPA and FPB release, FXIII activation and fibrin mass in tissue factor-initiated coagulation in whole blood from individuals with hemophilia and healthy subjects. RESULTS: In hemophiliacs, the rates of fibrin formation were delayed as compared to healthy individuals. FPA/FPB release and FXIII activation were decreased in hemophiliacs vs. healthy individuals: 5.4 +/- 0.7 microM min(-1) to 1.7 +/- 0.4 microM min(-1) (P = 0.003), 2.3 +/- 0.6 microM min(-1) to 0.5 +/- 0.1 microM min(-1) (P = 0.025), and 12.1 +/- 0.7 nM min(-1) to 3.1 +/- 0.7 nM min(-1) (P < 0.0005), respectively. More FPA was released in hemophiliacs (6.6 +/- 1.2 microM) prior to clot time (CT) than in healthy individuals (2.6 +/- 0.4 microM, P = 0.013), whereas FPB and activated FXIII levels remained comparable. FXIII activation, which normally coincides with FPA release, was delayed in hemophiliacs. At CT in normal blood, the FPA concentration was 2.6-fold higher than that of FPB (P = 0.003), whereas in hemophiliacs this ratio was increased to 6.6-fold (P = 0.001). CONCLUSIONS: These data suggest that essential dynamic correlations exist between the presentations of fibrin I, fibrin II, and FXIIIa. The 'discordance' of fibrin formation in hemophiliacs results in clots that are more soluble than normal (43% lower mass; P = 0.02). The resulting poor physical clot strength probably plays a crucial role in the pathology of hemophilia.

Oral anticoagulation thresholds.

BACKGROUND: Monitoring patients on oral anticoagulation is essential to prevent hemorrhage and recurrent thrombosis. We studied tissue factor-induced whole-blood coagulation in patients on warfarin therapy with similar international normalized ratios (INRs). METHODS AND RESULTS: Contact pathway-suppressed whole-blood coagulation initiated with tissue factor was studied in 8 male subjects (group W) and in 1 individual multiple times (subject A). Coagulation profiles for group W showed that subjects with similar INRs had widely varying clot times (6.2 to 23 minutes) and thrombin-antithrombin III (TAT) profiles with rates of 25 to 40 nmol. L(-1). min(-1) and maximum levels varying from 192 to 349 nmol/L. The normal control group exhibited clot times of 5.7+/-0.3 minutes and TAT rates of 57+/-13 nmol. L(-1). min(-1), reaching maximum levels of 742+/-91 nmol/L. Subject A, who was stably anticoagulated at an INR of 2.1+/-0.4 for 6 months, had widely ranging profiles with clot times of 9.0 to 22.7 minutes, TAT maximums varying from 141 to 345 nmol/L, and TAT formation rates of 10 to 57 nmol. L(-1). min(-1). INR did not correlate with TAT formation. Platelet activation was decreased by anticoagulants but also displayed variability. Fibrinopeptide A generation showed threshold variability independent of the INR. Factor VIII levels were increased (P=0.03) in group W (204+/-34.4%) compared with normal control subjects (149.4+/-37.4%). A significant correlation was identified between increasing factor VIII levels and years on warfarin therapy (r=0.78, P=0.01), suggesting a possible factor VIII compensatory mechanism. CONCLUSIONS: These results suggest that control of anticoagulation in patients to a set INR therapeutic range may be less secure than anticipated. Patients with similar INRs show significant individual variability in their tissue factor coagulation response, suggesting different risks to anticoagulation when confronted with underlying vascular anomalies.

Platelets and phospholipids in tissue factor-initiated thrombin generation.

The influence of platelets on tissue factor (TF)-initiated thrombin generation in a reconstituted model of blood coagulation and in whole blood was evaluated. No thrombin generation was observed over 15 min in the reconstituted model when either TF or platelets and phospholipids were omitted. At 25 pM TF, the rates of thrombin generation were platelet and PCPS concentration-dependent and achieved maximum (1.0 nM/s) in the physiological range of platelet concentration. Similar rates were achieved in the absence of platelets when 1-2 microM phospholipid was used. However, the maximum rates of thrombin generation (5.2-6.0 nM/s) and the shortest initiation phase (1 min) were attained between 25 and 100 microM phospholipid. In the reconstituted model, an increase in platelet concentration from 0.125 x 10(8)/ml to 0.5 x 10(8)/ml decreased the duration of the initiation phase (in the absence of phospholipids) from 4.3 min to 2 min. Further increases in platelet concentration did not affect this phase. Sequential whole blood studies were conducted in blood of a chemotherapy patient who developed reduced platelet counts. The TF (12.5 pM) initiated clotting of patient's blood was accelerated from approximately 10 min to 5 min when the platelet concentration increased from 0.05 x 10(8)/ml to 0.11 x 10(8)/ml. Clotting times were essentially unchanged for platelet concentrations exceeding 0.5 x 10(8)/ml (range 0.5-3.1 x 10(8)/ml). Similarly, clotting of whole blood obtained from healthy volunteers was not affected by the platelet count, which varied from 1.5 x 10(8)/ml to 3.1 x 10(8)/ml (4.0+/-0.5 min). The data obtained in both models are consistent with in vivo observations that clinical bleeding is most likely to occur at platelet counts <0.1 x 10(8)/ml.

Molecular description of three macro-deletions and an Alu-Alu recombination-mediated duplication in the HPRT gene in four patients with Lesch-Nyhan disease.

Mutations in the HPRT gene cause a spectrum of diseases that ranges from hyperuricemia alone to hyperuricemia with profound neurological and behavioral dysfunction. The extreme phenotype is termed Lesch-Nyhan syndrome. In 271 cases in which the germinal HPRT mutation has been characterized, 218 different mutations have been found. Of these, 34 (13%) are large- (macro-) deletions of one exon or greater and four (2%) are partial gene duplications. The deletion breakpoint junctions have been defined for only three of the 34 macro-deletions. The molecular basis of two of the four duplications has been defined. We report here the breakpoint junctions for three new deletion mutations, encompassing exons 4-8 (20033bp), exons 4 and 5 (13307bp) and exons 5 and 6 (9454bp), respectively. The deletion breakpoints were defined by a combination of long polymerase chain reaction (PCR) amplifications, and conventional PCR and DNA sequencing. All three deletions are the result of non-homologous recombinations. A fourth mutation, a duplication of exons 2 and 3, is the result of an Alu-mediated homologous recombination between identical 19bp sequences in introns 3 and 1. In toto, two of three germinal HPRT duplication mutations appear to have been caused by Alu-mediated homologous recombination, while only one of six deletion mutations appears to have resulted from this type of recombination mechanism. The other five deletion mutations resulted from non-homologous recombination. With this admittedly limited number of characterized macro-mutations, Alu-mediated unequal homologous recombinations account for at least 8% (3 of 38) of the macro-alterations and 1% (3 of 271) of the total HPRT germinal mutations.

The effect of folate deficiency on the cytotoxic and mutagenic responses to ethyl methanesulfonate in human lymphoblastoid cell lines that differ in p53 status.

Folic acid deficiency acts synergistically with alkylating agents to increase genetic damage at the HPRT locus in Chinese hamster ovary cells in vitro and in rat splenocytes in vivo. The present studies extend these observations to human cells and, in addition, investigate the role of p53 activity on mutation induction. The human lymphoblastoid cell lines TK6 and WTK1 are derived from the same parental cell line (WI-L2), but WTK1 expresses mutant p53. Treatment of folate-replete or deficient WTK1 and TK6 cells with increasing concentrations (0-50microg/ml) of ethyl methanesulfonate (EMS) resulted in significantly different HPRT mutation dose-response relationships (P<0.01), indicating that folate deficiency increased the EMS-induced mutant frequency in both cell lines, but with a greater effect in TK6 cells. Molecular analyses of 152 mutations showed that the predominant mutation (65%) in both cell types grown in the presence or absence of folic acid was a G>A transition on the non-transcribed strand. These transitions were mainly at non-CpG sites, particularly when these bases were flanked 3' by a purine or on both sides by G:C base pairs. A smaller number of G>A transitions occurred on the transcribed strand (C>T=14%), resulting in 79% total G:C>A:T transitions. There were more genomic deletions in folate-deficient (15%) as compared to replete cells (4%) of both cell types. Mutations that altered RNA splicing were common in both cell types and under both folate conditions, representing 33% of the total mutations. These studies indicate that cells expressing p53 activity exhibit a higher rate of mutation induction but are more sensitive to the toxic effects of alkylating agents than those lacking p53 activity. Folate deficiency tends to reduce toxicity but increase mutation induction after EMS treatment. The p53 gene product did not have a major influence on the molecular spectrum after treatment with EMS, while folate deficiency increased the frequency of deletions in both cell types.

The effect of folate deficiency on the hprt mutational spectrum in Chinese hamster ovary cells treated with monofunctional alkylating agents.

Folic acid deficiency acts synergistically with alkylating agents to increase DNA strand breaks and mutant frequency at the hprt locus in Chinese hamster ovary (CHO) cells. To elucidate the mechanism of this synergy, molecular analyses of hprt mutants were performed. Recently, our laboratory showed that folate deficiency increased the percentage of clones with intragenic deletions after exposure to ethyl methanesulfonate (EMS) but not N-nitroso-N-ethylurea (ENU) compared to clones recovered from folate replete medium. This report describes molecular analyses of the 37 hprt mutant clones obtained that did not contain deletions. Folate deficient cells treated with EMS had a high frequency of G>A transitions at non-CpG sites on the non-transcribed strand, particularly when these bases were flanked on both sides by G:C base pairs. Thirty-three percent of these mutations were in the run of six G's in exon 3. EMS-treated folate replete cells had a slightly (but not significantly) lower percentage of G>A transitions, and the same sequence specificity. Treatment of folate deficient CHO cells with ENU resulted in predominantly T>A transversions and C>T transitions relative to the non-transcribed strand. These findings suggest a model to explain the synergy between folate deficiency and alkylating agents: (1) folate deficiency causes extensive uracil incorporation into DNA; (2) greatly increased utilization of base excision repair to remove uracil and to correct alkylator damage leads to error-prone DNA repair. In the case of EMS, this results in more intragenic deletions and G:C to A:T mutations due to impaired ligation of single-strand breaks generated during base excision repair and a decreased capacity to remove O6-ethylguanine. In the case of ENU additional T>A transversions and C>T transitions are seen, perhaps due to mis-pairing of O2-ethylpyrimidines. Correction of folate deficiency may reduce the frequency of these types of genetic damage during alkylator therapy.

Nutritional folate status influences the efficacy and toxicity of chemotherapy in rats.

The effect of folate status on the efficacy and toxicity of chemotherapy was investigated in weanling Fischer 344 rats maintained on diets of varying folate content or supplemented with daily injections of folic acid, 50 mg/kg, for 6 to 7 weeks. MADB106 rat mammary tumor growth rate was the same in folate replete and supplemented rats, but retarded in the low folate groups. The tumor growth inhibitions in low folate, replete and high folate rats treated with cyclophosphamide were: 53%, 98%, and 97% (P = .048); with 5-fluorouracil (5-FU): 46%, 49%, and 66%; and with doxorubicin: 25%, 55%, and 61%. Significant differences in survival were observed for cyclophosphamide (P = .0084) and 5-FU (P = .025) related to dietary folate content. Thus, folate deficiency impedes tumor growth rate, but supplementation does not accelerate it in folate replete animals. Correction of folate deficiency approximately doubles the efficacy of cyclophosphamide in rats with much less host toxicity. Folate repletion improves survival in 5-FU-treated animals. These studies indicate that nutritional folate status has an important influence on the efficacy and toxicity of some commonly used cancer chemotherapeutic drugs.

Nutritional folate deficiency augments the in vivo mutagenic and lymphocytotoxic activities of alkylating agents.

To investigate the interaction of folate deficiency and alkylating agents in vivo, weanling Fischer 344 rats were maintained for 5 weeks on a folate replete, moderately folate deficient, or a severely folate deficient diet. Mutant frequencies at the HPRT locus in splenic lymphocytes were 1.2+/-0.6, 1.9+/-1.1, and 6.4+/-4.0 x 10(-6), respectively (P < 0.01). N-nitroso-N-ethylurea (ENU), 100 mg/kg body weight, was much more mutagenic with progressive folate deficiency (5.0+/-2.4 vs. 16.2+/-7.3 vs. 39.2+/-21.0 x 10(-6)), suggesting a synergistic interaction (P << 0.01). Neither moderate nor severe folate deficiency significantly enhanced the mutagenic effects of cyclophosphamide, 50 mg/kg body weight (18.0+/-7.9 vs. 6.0+/-2.8 vs. 28.5+/-28.2 x 10(-6)). The number of cloning cells/ spleen were reduced 68% in moderately folate deficient rats and by 87% in severely deficient animals (P < 0.05). The combination of folate deficiency and cyclophosphamide reduced the total number of cloning cells further, but ENU alone, or in combination with folate deficiency, did not. These findings indicate that folate deficiency increases the risk of somatic mutations and is lymphocytotoxic in rats. Folate deficiency enhances the mutagenic but not the lymphotoxic effects of ENU, while it increases the lymphotoxic but not the mutagenic activity of cyclophosphamide. Correction of folate deficiency may decrease the immunologic and genetic damage caused by some alkylating agents.

Blood coagulation in hemophilia A and hemophilia C.

Tissue factor (TF)-induced coagulation was compared in contact pathway suppressed human blood from normal, factor VIII-deficient, and factor XI-deficient donors. The progress of the reaction was analyzed in quenched samples by immunoassay and immunoblotting for fibrinopeptide A (FPA), thrombin-antithrombin (TAT), factor V activation, and osteonectin. In hemophilia A blood (factor VIII:C <1%) treated with 25 pmol/L TF, clotting was significantly delayed versus normal, whereas replacement with recombinant factor VIII (1 U/mL) restored the clot time near normal values. Fibrinopeptide A release was slower over the course of the experiment than in normal blood or hemophilic blood with factor VIII replaced, but significant release was observed by the end of the experiment. Factor V activation was significantly impaired, with both the heavy and light chains presenting more slowly than in the normal or replacement cases. Differences in platelet activation (osteonectin release) between normal and factor VIII-deficient blood were small, with the midpoint of the profiles observed within 1 minute of each other. Thrombin generation during the propagation phase (subsequent to clotting) was greatly impaired in factor VIII deficiency, being depressed to less than 1/29 (<1.9 nmol TAT/L/min) the rate in normal blood (55 nmol TAT/L/min). Replacement with recombinant factor VIII normalized the rate of TAT generation. Thus, coagulation in hemophilia A blood at 25 pmol/L TF is impaired, with significantly slower thrombin generation than normal during the propagation phase; this reduced thrombin appears to affect FPA production and factor V activation more profoundly than platelet activation. At the same level of TF in factor XI-deficient blood (XI:C <2%), only minor differences in clotting or product formation (FPA, osteonectin, and factor Va) were observed. Using reduced levels of initiator (5 pmol/L TF), the reaction was more strongly influenced by factor XI deficiency. Clot formation was delayed from 11.1 to 15.7 minutes, which shortened to 9.7 minutes with factor XI replacement. The maximum thrombin generation rate observed ( approximately 37 nmol TAT/L/min) was approximately one third that for normal (110 nmol/L TAT/min) or with factor XI replacement (119 nmol TAT/L/min). FPA release, factor V activation, and release of platelet osteonectin were slower in factor XI-deficient blood than in normal blood. The data demonstrate that factor XI deficiency results in significantly delayed clot formation only at sufficiently low TF concentrations. However, even at these low TF concentrations, significant thrombin is generated in the propagation phase after formation of the initial clot in hemophilia C blood.

Effect of folate deficiency on mutations at the hprt locus in Chinese hamster ovary cells exposed to monofunctional alkylating agents.

Multiplex PCR amplification of hprt exons from 113 Chinese hamster ovary cell clones selected for resistance to 6-thioguanine was performed to investigate the molecular basis for the synergistic mutagenic effects of nutritional folic acid deficiency and alkylating agents. In cells treated with ethyl methanesulfonate, intragenic deletions were detected in 9 of 46 (19.6%) clones derived from folate-deficient cells, but in none of 16 mutants grown in folate-replete medium. The number of deletions found in mutants generated by N-nitroso-N-ethylurea was low in both folate-deficient (1 of 25; 4%) and folate-replete (1 of 26; 3.8%) cells. Correction of folate deficiency may decrease the frequency of intragenic deletions caused by some alkylating agents.

Amplification of antibody production by phosphorothioate oligodeoxynucleotides.

A phosphorothioate oligodeoxynucleotide that is complementary (antisense) to the initiation region of the rev gene of HIV-1 causes hypergammaglobulinemia and splenomegaly in mice, and it induces B cell proliferation and differentiation in mouse spleen mononuclear cells (SMNCs) and human peripheral blood mononuclear cells in vitro. The current studies were performed to investigate the specificity of these immunomodulatory effects. Both the sense and antisense rev oligomers stimulated tritiated thymidine incorporation and secretion of immunoglobulin M (IgM) and immunoglobulin G (IgG) by mouse SMNCs in a concentration-dependent fashion, but the antisense oligomer produced greater immune effects. Studies comparing phosphorothioate oligomers (anti-rev, c-myc, and c-myb) either methylated or unmethylated at CpG dinucleotides showed that methylation effectively abrogated the proliferative effect and tended to reduce the immunoglobulin secretory activity, but the latter was not statistically significant except in the case of IgG in anti-rev oligomer-treated cultures. Mice were injected with the sense or antisense rev oligomers singly or in combination. The animals then were immunized with tetanus toxoid and received a booster 21 days later. Oligodeoxynucleotide-treated mice had significantly higher levels of IgM antibodies on days 28 and 35 and of IgG antibodies on days 14 and 35 as compared with mice that were immunized but received vehicle alone. There was no evidence for additive, synergistic, or antagonistic interactions of the sense and antisense rev oligomers. These results indicate that the unmethylated anti-rev oligomer is the most potent of the phosphorothioate oligomers tested at activating lymphocyte proliferation and differentiation and that a single intravenous injection of this oligodeoxynucleotide augments antibody production to a specific antigen as long as 35 days later.

B-cell proliferation and differentiation in common variable immunodeficiency patients produced by an antisense oligomer to the rev gene of HIV-1.

The immunostimulatory activity of a phosphorothioate oligodeoxynucleotide (27 mer) that is antisense to the rev gene of HIV-1 was studied on normal human lymphocytes and on cells from patients with common variable immunodeficiency (CVI). For peripheral blood mononuclear cells from nine normal individuals, the proliferation index (16.8 +/- 12.5) after anti-rev oligomer exposure was proportional to the percentage of peripheral B-cells (r = 0.76, P = 0.02). In five experiments, enriched B- or T-cell populations had proliferation indices of 47.2 +/- 32.9 and 2.4 +/- 1.9, respectively. The addition of T-cells to anti-rev oligomer treated B-cells had no effect (proliferation index = 47.5 +/- 38.1). After anti-rev oligomer stimulation, autoradiography, and counterstaining for B- and T-cell markers, all detectable [3H]thymidine uptake was by CD19-positive cells. Eight of the 14 CVI patients had a proliferation index and secreted levels of IgM and IgG comparable to cells from normal individuals. In contrast to normal cells, the direct correlation between proliferation of peripheral blood mononuclear cells and the percentage of peripheral B-cells was weak in samples from 13 CVI patients (r = 0.4, P = 0.2). These findings indicate that peripheral blood B-cells from about half of CVI patients proliferate and produce immunoglobulin after exposure to anti-rev oligomer. These data demonstrate that under the appropriate circumstances, B-cells of some CVI patients can proliferate and differentiate normally.

Effect of dietary components on hprt mutant frequencies in human T-lymphocytes.

The 6-thioguanine resistance (TGr) assay in human T-lymphocytes, which detects mutations at the hprt locus, identifies exposures to environmental mutagens. However, the ability of this assay to detect small increases in mutation rates is limited by the broad range of mutant frequencies (Mf) in healthy individuals. While subject age, lymphocyte cloning efficiency, and cigarette smoking history have been shown to influence the Mf, these factors account for only a portion of the variability in the Mf in human populations. To investigate the influence of dietary differences on hprt Mf, 70 women with breast masses were asked to complete a nutritional questionnaire and submit a peripheral blood sample for a TGr assay. Multivariate analyses, adjusted for age, cloning efficiency and total caloric intake, showed significant positive correlations between vitamin A and iron and InMf (p = 0.03), and a negative correlation between total fat and InMf (p = 0.004). Positive correlations between dietary fiber and copper and InMf, and a negative correlation between alcohol and InMf were of borderline significance (0.05 < or = p < or = 0.07). These results suggest that nutritional components may modulate the hprt Mf. Dietary differences may account for a part of the variability observed in hprt Mf in human populations.

The effect of T-lymphocyte 'clonality' on the calculated hprt mutation frequency occurring in vivo in humans.

The frequency of 6-thioguanine resistant (TGr) mutant T-lymphocytes arising in vivo in humans can be quantified with a cell cloning assay. However, the in vivo proliferation of T-lymphocytes that may include TGr mutant cells can distort the relationship between mutation events and the resulting frequency of mutant cells. The T-cell receptor (TCR) gene rearrangement pattern of T-cell colonies can be used as an independent measure of clonality. Analysis of T-cell 'clonality' in 413 wild type and 1736 TGr mutant isolates from 58 individuals shows that mutant clonality is a frequent occurrence (35/58 individuals = 60.3%). However, a major effect on the mutant frequency corrected for clonality (the calculated 'mutation frequency') was found only in nine samples all of which had mutant frequencies greater than 40 x 10(-6).

Folate deficiency increases genetic damage caused by alkylating agents and gamma-irradiation in Chinese hamster ovary cells.

The effect of folate deficiency on genetic damage caused by alkylating agents and gamma-irradiation was studied in Chinese hamster ovary (CHO) cells. Mutant frequencies of 6-thioguanine-resistant and diphtheria toxin-resistant cells were not significantly increased by incubation in low-folate medium. In contrast, folate deficiency increased the mutant frequencies of 6-thioguanine-resistant cells caused by N-ethyl-N-nitrosourea or ethyl methanesulfonate by about 3-fold. Folate deficiency was associated with a 70% increase of diphtheria toxin-resistant cells after exposure to ethyl methanesulfonate. Folate deficiency alone caused DNA strand breaks equivalent to 26 cGy, as monitored by alkaline filter elution. Following 400 cGy of gamma-irradiation, folate-deficient cells manifested strand breaks equivalent to a dose of 710 cGy. CHO cells in folate-containing medium repaired breaks within 3 h, while cells in low-folate medium had an increased break frequency (P = 0.02) at 3 h and were unable to fully repair radiation-induced damage even after 9 h. These studies indicate that folate deficiency acts synergistically with alkylating agents to increase somatic mutation and with gamma-irradiation to promote DNA strand breaks in CHO cells. Folate deficiency appears to potentiate the genetic damage caused by mutagens/carcinogens by limiting DNA repair.

Measurement of chromosomal aberrations, sister chromatid exchange, hprt mutations, and DNA adducts in peripheral lymphocytes of human populations at increased risk for cancer.

Using a multidisciplinary approach, we have measured various indicators of DNA damage in peripheral lymphocytes of human populations potentially at increased risk for cancer. Sister chromatid exchanges (SCE) and polycyclic aromatic hydrocarbon (PAH)-DNA adducts were evaluated in a group of firefighters; chromosomal aberrations and hprt mutations were evaluated in a group of cancer patients undergoing radioimmunoglobulin therapy (RIT); SCE and acrolein-modified DNA were measured in cancer chemotherapy patients and in pharmacists preparing chemotherapy prescriptions; and SCE and PAH-DNA adducts are being measured in U.S. army troops stationed in Kuwait. Our results indicate that both SCE and PAH-DNA adduct levels were not elevated in firefighters, but that other factors such as smoking status and race were risk factors for increased SCE and PAH-DNA adducts. RIT was found to increase background rates of chromosome-type aberrations and frequencies of hprt mutations and there was a strong correlation between levels of therapy-induced chromosome damage sustained in vivo and in vitro sensitivity to radiation-induced chromosome damage. Peripheral blood lymphocytes of cancer patients treated with cyclophosphamide showed higher levels of SCE and had a higher incidence of acrolein adducts in DNA. Lymphocytes from pharmacists preparing antineoplastic drugs were found to acquire increased in vitro sensitivity to SCE induction by phosphoramide mustard with increased lifetime duration of drug handling. A prospective, longitudinal study was performed to identify environmental factors that modulate genetic damage in breast cancer patients. Women with benign breast masses and no apparent disease served as controls. Mutant frequency, cloning efficiency, and chromosomal aberration frequency did not differ significantly among the three groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Immune stimulation by an antisense oligomer complementary to the rev gene of HIV-1.

Mice developed massive splenomegaly and polyclonal hypergammaglobulinemia within 2 days after intravenous injection of a phosphorothioate oligomer that is antisense to a portion of the rev region of the HIV-1 genome. Histologic examination of spleens from injected animals showed marked expansion of a uniform-appearing population of small lymphocytes and many mitoses. Spleen mononuclear cells (SMNCs) from injected animals showed approximately a 10-fold-increased uptake of [3H]thymidine and production of IgM and IgG. Flow cytometry analysis indicated that the responding cells were predominantly B-lymphocytes. The anti-rev oligomer also was mitogenic in vitro and stimulated immunoglobulin production by normal mouse SMNCs and human peripheral blood mononuclear cells. Similar immunologic effects were observed with an anti-rev 21-mer phosphorothioate, truncated at the 3' end, but not with a 20-mer human p53 antisense phosphorothioate or a 28-mer anti-rev phosphodiester. These observations are consistent with the possibility that DNA sequences homologous to the rev gene participate in the regulation of mammalian lymphocyte activation, proliferation and maturation.

In vivo mutations in human blood cells: biomarkers for molecular epidemiology.

Mutations arising in vivo in recorder genes of human blood cells provide biomarkers for molecular epidemiology by serving as surrogates for cancer-causing genetic changes. Current markers include mutations of the glycophorin-A (GPA) or hemoglobin (Hb) genes, measured in red blood cells, or mutations of the hypoxanthine-guanine phosphoribosyltransferase (hprt) or HLA genes, measured in T-lymphocytes. Mean mutant frequencies (variant frequencies) for normal young adults are approximately: Hb (4 x 10(-8)) < hprt (5 x 10(-6)) = GPA (10 x 10(-6)) < HLA (30 x 10(-6)). Mutagen-exposed individuals show decided elevations. Molecular mutational spectra are also being defined. For the hprt marker system, about 15% of background mutations are gross structural alterations of the hprt gene (e.g., deletions); the remainder are point mutations (e.g., base substitutions or frameshifts). Ionizing radiations result in dose-related increases in total gene deletions. Large deletions may encompass several megabases as shown by co-deletions of linked markers. Possible hprt spectra for defining radiation and chemical exposures are being sought. In addition to their responsiveness to environmental mutagens/carcinogens, three additional findings suggest that the in vivo recorder mutations are relevant in vivo surrogates for cancer mutations. First, a large fraction of GPA and HLA mutations show exchanges due to homologous recombination, an important mutational event in cancer. Second, hprt mutations arise preferentially in dividing T-cells, which can accumulate additional mutations in the same clone, reminiscent of the multiple hits required in the evolution of malignancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of HPRT mutant frequencies in T-lymphocytes from healthy human populations.

Somatic cell mutant frequencies at the hprt locus of the X-chromosome were measured with the T-lymphocyte cloning technique in healthy human populations. A statistical analysis was performed of assays from 232 individuals (77 males and 155 females) ranging in age from 19 to 80 years. Data from 4 donor groups were compiled: (a) 132 participants in a study of identical and fraternal twins; (b) 17 health care workers studied as part of an assessment of the risks of handling chemotherapeutic drugs; (c) 62 women with benign breast masses; and (d) 21 normal laboratory and office personnel. The relationship between age and mutant frequency (MF) was expressed by the equation: ln MF = 1.46 + 0.018 age (P < 0.001). Thus, MF increased by about 2% per year. Increases in cloning efficiency (CE) reduced the MF, as shown in the equation: ln MF = 2.91 - 1.32 CE (P < 0.001). CE was significantly related to age (CE = 0.47 - 0.002 age, P = 0.038), and the interdependent relationship between MF, age and CE expressed by the equation: ln MF = 1.99 - 1.13 CE + 0.016 age was significant at the P < 0.001 level. There was no statistically significant effect of donor gender or smoking history on MF in our population, but CE was significantly lower in males (P < 0.001). These findings confirm the importance of age and CE as factors which influence the thioguanine-resistant MF in circulating T-lymphocytes from normal adults.

Factors influencing mutation at the hprt locus in T-lymphocytes: studies in normal women and women with benign and malignant breast masses.

A prospective, longitudinal study was performed to test the hypothesis that environmental factors (e.g., diet or cigarette smoking) modulate genetic damage caused by treatment for breast cancer and render these women more susceptible to developing second malignancies. A total of 107 women (49 with breast cancer, 52 with benign breast masses, and 6 normal women) were enrolled. This report describes initial studies at the time of enrollment and disease presentation. Mutant frequency at the hprt locus and cloning efficiency of peripheral blood lymphocytes did not differ significantly among the 3 groups. Mutant frequency increased with age, with a history of cigarette smoking, and with the number of years that current smokers used cigarettes. There was no correlation in women with benign masses between mutant frequency and the incidence of chromosome aberrations (28 women) or sister chromatid exchanges (23 women). A maternal history of breast cancer did not influence mutant frequency. There was no significant relationship between dietary intake of vitamins A, B12, C and E, folacin, selenium, calcium, caffeine, or multivitamin pills, and mutant frequency. Serum folate levels in the deficient range were associated (P = 0.02) with elevated mutant frequencies, whereas SCE rates inversely correlated with serum vitamin B12 levels. These results confirm the importance of age and, less so, cigarette smoking as factors that influence mutant frequency and suggest that a micronutrient, folic acid, may modify genetic damage at the hprt locus. To the extent that somatic mutation contributes to carcinogenesis, these environmental factors may enhance the risk of developing malignant transformation.