Richard F. Branda

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.

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.

Discordant fibrin formation in hemophilia

Summary.  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 μm min−1 to 1.7 ± 0.4 μm min−1 (P = 0.003), 2.3 ± 0.6 μm min−1 to 0.5 ± 0.1 μm 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 μm) prior to clot time (CT) than in healthy individuals (2.6 ± 0.4 μm, 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.

Nutritional folate-deficiency in Chinese hamster ovary cells : chromosomal abnormalities associated with perturbations in nucleic acid precursors

Folate deficiency is known to induce chromosomal abnormalities. We used a nutritionally folate-deficient Chinese hamster ovary (CHO) cell culture system to examine modulation of chromosome damage by purine or pyrimidine supplementation. The cells were cultured in folate-deficient (Fol-) medium or Fol- medium supplemented with thymidine (dT) or hypoxanthine (Hx) until population growth arrest. The cultures were then switched to complete medium, permitting the cells to begin cell division. Cell-cycle progression was followed by flow cytometry to identify the first mitosis, when samples for analysis were collected. The mitotic index, frequency of chromosomal aberrations in mitotic cells, and relative distribution of different types of aberrations were determined. Cells grown in Fol- medium supplemented with Hx entered the G2/M phase of the cell cycle at 14 hours after media change as compared with 16 hours for Fol- cultures or 24 hours for Fol- cultures supplemented with dT. Cells cultured in Fol- medium alone or supplemented with dT showed similar frequencies of damage, averaging 20-22%, as compared with 2% for control cultures. In contrast, cells grown in Hx-supplemented medium exhibited a lower frequency of damaged mitoses (15%), as well as a reduction in certain types of abnormalities. This latter finding is surprising in light of our previous work showing that the presence of Hx during folate deficiency produced a more severe perturbation of phenotype (cellular enlargement) and growth control (S-phase delay and cell killing) than did dT supplementation or the absence of both nucleotide precursors.

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.

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 × 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 × 10−6), suggesting a synergistic interaction (P ≪ 0.01). Neither moderate nor severe folate deficiency significantly enhanced the mutagenic effects of cyclophosphamide, 50 mg/kgbody weight (18.0 ± 7.9 vs. 6.0 ± 2.8 vs. 28.5 ± 28.2 × 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. Environ. Mol. Mutagen. 32:33–38, 1998

Cellular pharmacology of chloroquinoxaline sulfonamide and a related compound in murine B16 melanoma cells

Chloroquinoxaline sulfonamide (CQS), a chlorinated derivative of sulfaquinoxaline (SQ), inhibited proliferation of murine B16 melanoma cells, but only when relatively high drug concentrations (1 mM) were used. The inhibition of cell growth by CQS was at least partially reversible by incubation in drug-free medium. Incubation of melanoma cells with CQS was associated with an arrest of the cell cycle in G0/G1 as measured by flow cytometry. The drug slightly decreased uptake of radiolabeled deoxyuridine and thymidine after 24- and 48-hr incubation periods but increased nucleoside incorporation at 72 hr. No evidence of intercalation with DNA was found. Because SQ previously was reported to inhibit an aspect of folate metabolism, we investigated the possibility that CQS limits tumor cell growth by altering folate homeostasis. This appears unlikely, however, in view of the following observations: (1) the cytotoxic effects of CQS could not be reversed by folinic acid; (2) deoxyuridine suppression of thymidine incorporation was not affected by CQS treatment; (3) CQS did not inhibit dihydrofolate reductase from mammalian or bacterial sources; and (4) CQS toxicity in mice was not reduced by folinic acid. Experiments performed with analogues modified in the quinoxaline and para-amino phenyl functions indicated that tumor cell inhibition did not require preservation of the conventional sulfonamide structure.

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 Gs 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.

Immunosuppressive properties of chloroquinoxaline sulfonamide

Chloroquinoxaline sulfonamide (CQS), a sulfanilamide derivative with antitumor activity, was found to be toxic to lymphoid tissue during preclinical studies. The mechanism of this toxicity appears to involve profound inhibition of lymphocyte activation. Incubation of human peripheral blood mononuclear cells (PBMNCs) with CQS decreased cellular incorporation of thymidine and deoxyuridine in a dose-dependent manner. Analysis of cell cycle distribution by flow cytometry indicated that CQS blocked movement out of the G0/G1 phase. Drug-treated cells were smaller and expressed fewer receptors for interleukin-2 (IL-2) and transferrin than untreated mitogen-stimulated lymphocytes. These observations support the notion that CQS has cell cycle specificity in regulating lymphocyte proliferation. As little as 10 microM CQS markedly inhibited both human lymphocyte and murine CTLL cell replication in response to IL-2 containing growth factors. However, CQS did not block secretion of IL-2 into culture supernatant fractions by human PBMNCs. Finally, CQS inhibited in vitro production of immunoglobulins G and M by mitogen-stimulated lymphocytes, primarily by causing cytotoxicity. In all of these drug effects, CQS was approximately one to two logs more potent than the parent compound, sulfaquinoxaline (SQ). These studies indicate that CQS inhibits essential basic processes in human lymphocytes. This agent may find use as an immunosuppressive drug.

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.