Effect of surface treatment and cement maturation on the bond strength of resin-modified glass ionomers to dentin.

This study investigated the effect of surface treatment and cement maturation on the bond strength of resin-modified glass ionomer cements (RMGICs) to dentin. Forty-two freshly extracted premolars were embedded and horizontally sectioned at a level 2 mm from the central fossa to obtain a flat dentin surface. The premolars were randomly divided into three groups of 14 teeth and treated as follows: Group 1 (control)--no surface treatment, Group 2--conditioned with 20% polyacrylic acid for 10 seconds and Group 3--etched with 37% phosphoric acid for 15 seconds. RMGIC (Fuji II LC, GC) columns (3 mm diameter; 2 mm high) were applied to the dentin surface and shear bond testing was carried out after one week (n = 7) and one month (n = 7) storage in distilled water at 37 degrees C using an Instron Universal testing machine with a cross-head speed of 0.6 mm/minute. The failure mode was examined at 40x magnification and scored with imaging software. The results were analyzed using ANOVA/Scheffe's post-hoc test and Kruskal-Wallis/Mann-Whitney test at a significance level of 0.05. The effect of surface treatment on shear bond strength to dentin was time dependent. Mean strengths ranged from 3.16 to 5.81 MPa at one week and 5.00 to 14.95 MPa at one month. Although no significant difference in strengths was observed among the groups at one week, significant differences (Group 2 > Group 1 > Group 3) were detected at one month. At one month, conditioned and untreated specimens exhibited significantly less adhesive failure than etched specimens.

Flow cytometric enumeration of micronucleated reticulocytes: high transferability among 14 laboratories.

This laboratory previously described a single-laser flow cytometric method, which effectively resolves micronucleated erythrocyte populations in rodent peripheral blood samples. Even so, the rarity and variable size of micronuclei make it difficult to configure instrument settings consistently and define analysis regions rationally to enumerate the cell populations of interest. Murine erythrocytes from animals infected with the malaria parasite Plasmodium berghei contain a high prevalence of erythrocytes with a uniform DNA content. This biological model for micronucleated erythrocytes offers a means by which the micronucleus analysis regions can be rationally defined, and a means for controlling interexperimental variation. The experiments described herein were performed to extend these studies by testing whether malaria-infected erythrocytes could also be used to enhance the transferability of the method, as well as control intra- and interlaboratory variation. For these studies, blood samples from mice infected with malaria, or treated with vehicle or the clastogen methyl methanesulfonate, were fixed and shipped to collaborating laboratories for analysis. After configuring instrumentation parameters and guiding the position of analysis regions with the malaria-infected blood samples, micronucleated reticulocyte frequencies were measured (20,000 reticulocytes per sample). To evaluate both intra- and interlaboratory variation, five replicates were analyzed per day, and these analyses were repeated on up to five separate days. The data of 14 laboratories presented herein indicate that transferability of this flow cytometric technique is high when instrumentation is guided by the biological standard Plasmodium berghei.

Comparison of mutant frequencies at the transgenic lambda LacI and cII/cI loci in control and ENU-treated Big Blue mice.

We compared the lambda cII/cI transgenic mutation assay described by Jakubczak et al. [(1996): Proc Natl Acad Sci USA 93:9073-9078] to the previously established Big Blue assay. Genomic DNA isolated from liver, spleen, and lung tissue of control or ethylnitrosourea (ENU)-treated Big Blue mice (100 mg/kg i.p., single dose) was packaged into phage (five animals, two packagings per DNA sample) which were simultaneously plated for lacI and cII/cI mutant frequency (MF) and titer. Mean MF of control animals was higher for cII/cI than lacI for all three tissues examined (spontaneous cII/cI MF divided by spontaneous lacI MF = 2.9, 3.1, and 1.7 for liver, spleen, and lung, respectively). The differences were statistically significant for liver and spleen, but not lung. The ENU-induced MF measured by subtracting control MFs from ENU-treated MFs was higher in the cII/cI assay than lacI (liver = 23.0 x 10(-5) for cII/cI vs. 15.1 x 10(-5) for lacI; spleen = 64.8 x 10(-5) for cII/cI vs. 36.1 x 10(-5) for lacI; lung = 17.1 x 10(-5) for cII/cI vs. 15.8 x 10(-5) for lacI). Fold increase over control values measured by dividing MF of ENU-treated animals by appropriate control values was higher for lacI than cII/cI (liver = 4.4-fold for lacI vs. 2.7 for cII/cI; spleen = 13.1-fold for lacI vs. 8.4 for cII/cI; and lung = 5.6-fold for lacI vs. 4.0 for cII/cI). Despite these differences, overall results were similar for the two mutational endpoints. These results suggest that the cII/cI assay may be an acceptable alternative to lacI where transgenic mutation studies are indicated.

Lack of response to multiple genotoxic agents at the hprt locus in peripheral blood T-lymphocytes of cynomolgus monkeys.

We tested the ability of a series of known genotoxic agents to cause mutations at the hprt locus in peripheral blood T-lymphocytes of cynomolgus monkeys as measured by the ability to form clones in the presence of 6-thioguanine. Ethylmethane sulfonate (EMS, 300 mg/kg i.p.), chloroethylmethane sulfonate (CI-EMS, 35 or 50 mg/kg i.p.), and the Pharmacia & Upjohn antitumor agents adozelesin (1.6, 4, 6, or 8 microg/kg i.v.) and CC-1065 (6 microg/kg i.v.) were all negative in the hprt mutation test. Results with cyclophosphamide (CP, 75 mg/kg i.v.) were equivocal. Adozelesin, CC-1065, and CI-EMS treatments increased the percentage of T-lymphocytes with chromosome aberrations, as well as inducing types of aberrations not seen in control cells. EMS and CP were not tested for chromosome aberrations. We have previously shown that treatment of monkeys with 77 mg/kg ENU substantially increased the hprt mutant frequency, with a lag time of approximately 77 days between treatment and peak MF values. The results of the present study suggest a low sensitivity of the hprt mutation assay to certain classes of genotoxic agents in cynomolgus monkeys.

Spontaneous mutation spectrum at the lambda cII locus in liver, lung, and spleen tissue of Big Blue transgenic mice.

Big Blue mice harbor a recoverable transgene in a lambda/LIZ shuttle vector. In the standard assay, in vivo mutations are measured in the bacterial lacI gene using a labor-intensive color plaque assay. Applying a simpler assay [Jakubczak et al. (1996): Proc Natl Acad Sci USA 93:9073-9078], we measured mutations in the lambda cII gene portion of the transgene. Spontaneous clear plaque mutants were analyzed from liver, lung, and spleen of five untreated mice. Of 314 mutants, 182 (58%) had independent mutations, 74 (23.5%) appeared clonal, and 58 (18.5%) showed no cII mutations. Of 182 independent cII mutations, 156 (85.7%) were base substitutions, 20 (10.9%) were frameshifts, and 6 (3.2%) were multiple substitutions and one deletion. G:C --> A:T transitions were the predominant base substitution (78% of these at CpG sites). The major mutation hotspot, a six G run and its 3' flanking T at bases 179 to 185, comprised 18.7% of the independent mutations. Other hotspots were positions 103, 196, and 212. The in vivo cII spectrum had a significantly higher proportion of G --> A and G --> T mutations and fewer frameshifts than reported in vitro. The cII and published lacI spectra are similar, though G --> A transitions and deletions were fewer in the cII gene. The cI gene was sequenced in 48 mutants with no cII mutations and most had cI mutations: 81.3% base substitutions and 18.7% frameshifts. We conclude that the cII/cI system is insensitive to deletion events, but is useful for detecting point mutations.

Validation of intratracheal instillation as an alternative for aerosol inhalation toxicity testing.

In collecting inhalation toxicity data for the evaluation of the health hazard from occupational exposure to the aerosols of a drug or a chemical, the determination of the inhaled dose in relation to the animal response is most desirable. Intratracheal administration is most likely to deliver an exact dose of a compound to the lungs of an experimental animal. In a series of tests, microliter (microl) quantities of a solution or a suspension of a test material were nebulized into the trachea of an anesthetized rat using an intratracheal fast instillation (ITFI) method. The dose-response in terms of the minimal effective dose (MED) and the median lethal dose (LD50) were determined. The ITFI dose-response for four drugs, five chemicals or chemical intermediates and four pesticides were compared with those obtained via inhalation (IH) and ingestion (p.o.). In addition, the dose-responses of the four pesticides were compared with two additional parameters, intranasal instillation (IN) and intravenous injection (i.v.). The MED end-points for studies via the respiratory administration route were no pharmacotoxic signs other than transient respiratory rales and/or dyspnea and no gross lesions, whereas those for the intranasal, oral and the intravenous administration routes were transient and slight body weight loss and no pharmacotoxic signs and/or gross lesions. The MED ratios between ITFI, IH and p.o. were 1 : 9.3+/-6.5 : 201.4+/-133.3, respectively, for the drugs, chemicals and chemical intermediates. The MED ratios for ITFI, IH, IN, i.v. and p.o. for the four pesticides were 1 : 2.2+/-1.4 : 2.1+/-1.3 : 1.1+/-0.7 : 1.4+/-0.9. The MED ratios for the two categories of test materials were fairly consistent between different routes of administration. Thus, the ITFI dose can be used for extrapolating the IH dose. The simplicity of the ITFI procedure and its requirement of only microliters of a compound to generate a meaningful and reliable dose-response suggests that ITFI may be an alternative method for acute inhalation toxicity evaluation of materials that may present inhalation hazards from liquid or solid aerosols.

DNA sequence analysis of hprt mutants persisting in peripheral blood of cynomolgus monkeys more than two years after ENU treatment.

We have been studying in vivo mutagenesis at the hypoxanthine phosphoribosyl transferase (hprt) locus in cynomolgus monkey T-lymphocytes. This primate model allows us to study mutations and their kinetics under well-controlled conditions. Previously, we reported mutations detected at various times after intraperitoneal treatment with ethylnitrosourea (ENU, 77 mg/kg). At 832 days after that first treatment, the monkey received a second dose of 77 mg/kg ENU. Up to 1,331 days after the second treatment, the T-cell mutant frequency (44.2 x 10(-6)) was still 26-fold higher than background (1.7 x 10(-6)), suggesting that mutants persisted in the peripheral blood. Mutant clones from Days 974, 1,164, and 1,311 after the second treatment were selected in thioguanine. Hprt cDNA was prepared from a cell lysate, PCR-amplified, and sequenced. Of 45 mutants, 30 yielded PCR product and 26 were sequenced. Base substitutions were found in 21 (81%) of the 26 mutants and consisted of one G:C --> A:T and five A:T --> G:C transitions, one G:C --> C:G, eight A:T --> T:A, and six A:T --> C:G transversions. Therefore, most base substitutions occurred at A:T basepairs, characteristic of ENU-induced mutations in vivo, and were detected up to 3.6 years after the second treatment. Deletions of exons 2 and 3 occurred in two mutants and exon 7 was deleted in one mutant. There were two insertion mutants: one was a single base insertion and the other contained an insertion of 277 basepairs which was nearly identical to a simian retroviral sequence.

Quantitative morphometric analysis of pulmonary deposition of aerosol particles inhaled via intratracheal nebulization, intratracheal instillation or nose-only inhalation in rats.

The effectiveness of three techniques to deliver a diazo dye suspension into the lungs of rats was compared. The intratracheal nebulization (ITN) technique delivered 10 microl of the suspension per 5-ml puff of air in 10 puffs as an aerosol. The intratracheal fast instillation (ITFI) technique delivered 100 microl of the suspension in a single 2-ml puff of air as droplets. The nose-only inhalation (NI) technique aerosolized the suspension at an analytical concentration that provided a calculated dose equivalent to 100 microl of the suspension in a 2-h inhalation period. Immediately after dosing, all the rats were killed by exsanguination. The trachea was tied and the lung was inflated in situ with air. After fixation, 5-microm thick slices were prepared from each lobe of the lung at a plane perpendicular to the axis of the lobar bronchus at levels proximal, medial and distal to the hilus. The numbers of bronchi, bronchioli and alveolar ducts within four ranges of diameters and the proportion of each selected area of lung tissue with and without dye particles were quantified using electronic imaging analyzers. The results indicated that ITN and ITFI dispersed the particles evenly throughout most of the airways and in patches in the alveoli. The NI technique dispersed the particles homogeneously throughout the airways and the alveoli in the lungs. The mean number-percentage and the mean area-percentage data revealed that the doses delivered by ITN and NI were approximately 60% and 10%, respectively, of the ITFI dose. Thus, the ITFI technique appeared to be most suitable for pulmonary absorption and disposition studies where dosage precision is of primary concern. The ITN technique would need further improvement to meet the requirements for dose precision and particle distribution. For both ITFI and ITN, particle size was apparently not a critical determinant for deposition. The NI technique is suitable for inhalation toxicity studies where the pattern and uniformity of particle deposition is the primary concern.

Effect of plating medium and phage storage on mutant frequency and titer in the lambda cII transgenic mutation assay.

We examined several experimental parameters of the lambda cI/cII transgenic mutation assay. In the assay, clear plaque lambda phage mutants are identified in a positive selection scheme following rescue of the lambda/LIZ shuttle vector from frozen tissues of Big Blue" transgenic mice. Mutant frequency and titer of phage from various tissues of control and ENU-treated animals was essentially the same on LB or TB1 plating medium, and storage of isolated DNA at 4 degrees C for up to 4 months did not affect either mutant frequency or titer. Storage of packaged phage for 28 days at 4 degrees C did not affect titer. The mean mutant frequency of packaged phage stored 28 days at 4 degrees C was consistently higher than phage plated the same day as packaging (day 0), though the difference was statistically significant in only two of the four samples tested. Reconstruction experiments in which numerically defined titers of known cII mutants were plated on both G1217 and G1225 E. coli strains and incubated at 37 degrees C or 24 degrees C showed highest titers on G1217 at 37 degrees C. The fraction of the G1217, 37 degrees C titer seen in the other strains and conditions varied widely with the cII mutation.

In vivo mutagenesis in the cynomolgus monkey: time course of HPRT mutant frequency at long time points following ethylnitrosourea exposure.

We have monitored mutant frequency at the HPRT locus in peripheral blood lymphocytes of cynomolgus monkeys using a clonal assay in which mutants are selected by resistance to 6-thioguanine. Among untreated animals, the mean spontaneous mutant frequency was 2.9 +/- 2.9 x 10(-6) (standard deviation, based on 131 determinations in 33 animals), in good agreement with HPRT mutant frequencies in other species. In four animals treated with a single intraperitoneal dose of 77 mg/kg ethylnitrosourea, mutant frequency increased with time, peaking 70 to 100 days after treatment. Mutant frequency in two of the four animals was monitored at intervals for 6 years, and a second identical treatment was given about 830 days after the first. Mutant frequency again peaked in these two animals 70 days after the second dose and decreased following peak values, declining to a plateau that was higher than the predose mutant frequency in both animals. This pattern was repeated following the second ethylnitrosourea treatment. Fractionating the dose of ethylnitrosourea into five equal daily injections had no effect on mutant frequency in two animals when compared to a single dose.

Contribution of serum protein association to discrepancy between the in vivo and in vitro UDS results for 6,7-dimethyl-2,4-di-1-pyrrolidinyl-7H-pyrrolo[2,3-d]pyrimidine (U-89843).

U-89843 has been shown to undergo biotransformation, both in vitro and in vivo, to form U-97924 as a major primary metabolite. U-89843 was found to be positive in an in vitro UDS mutagenesis screen conducted with primary rat hepatocytes in serum-free media. In contrast to in vitro results, no evidence of genetic toxicity of U-89843 was observed in rats in the in vivo/in vitro version of the UDS test with single oral doses up to 1400 mg/kg. The negative results may be related to more robust in vivo detoxification mechanisms or relatively lower exposure to reactive metabolites formed by bioactivation of U-89843 as compared to that observed in the serum-free in vitro hepatocyte test system. Further studies showed rat serum suppressed the in vitro metabolism of U-89843 as well as the formation of the corresponding hydroxylated metabolite, U-97924, the putative precursor of proposed reactive electrophilic metabolite. The measured in vivo systemic clearance of U-89843 (0.53 l/h/kg) in rats was about 1000-fold slower than the in vitro intrinsic clearance (606 l/h/kg) estimated by measuring the formation of U-97924 in rat liver microsomal incubations. Since U-89843 is extensively associated with serum proteins a poor extraction ratio into the liver may account for the slower biotransformation of U-89843 in vivo as compared to that exhibited in in vitro serum-free hepatocyte incubations. Addition of bovine serum albumin (1-40 mg/ml) to the in vitro UDS assay medium decreased the UDS mean net grains per nucleus response of U-89843. These results suggest that the effect of serum protein should be considered when comparing serum-free in vitro UDS and in vivo UDS results for highly serum protein bound compounds.

Bioactivation of 6,7-dimethyl-2,4-di-1-pyrrolidinyl-7H-pyrrolo[2,3-d]pyrimidine (U-89843) to reactive intermediates that bind covalently to macromolecules and produce genotoxicity.

U-89843 is a novel pyrrolo[2,3-d]pyrimidine antioxidant with prophylactic activity in animal models of lung inflammation. During preclinical safety evaluation, U-89843 was found to give a positive response in the in vitro unscheduled DNA synthesis (UDS) assay, an assay which measures DNA repair following chemically-induced DNA damage in metabolically competent rat hepatocytes. Incubation of [14C]U-89843 with liver microsomes resulted in covalent binding of radioactive material to macromolecules by a process that was NADPH-dependent. U-89843 has been shown to undergo C-6 methylhydroxylation to give U-97924, in rat both in vivo and in vitro, in a reaction catalyzed by cytochrome P450 2C11. Synthetical U-97924 is chemically reactive and undergoes dimerization in aqueous solution. The dimerization of U-97924 was significantly inhibited by addition of nucleophiles such as methanol, glutathione, and N-acetylcysteine. Characterization of the corresponding methanol, glutathione, and N-acetylcysteine adducts of U-97924 supported the hypothesis of a reaction pathway involving reactive iminium species formed via dehydration of U-97924. The metabolism-dependent irreversible covalent binding of radioactive material to liver microsomal protein and DNA also is dramatically reduced in the presence of reduced glutathione (GSH). A trifluoromethyl analog of U-89843 was prepared in an effort to block the corresponding metabolic hydroxylation pathway. This new compound (U-107634) was found to be negative in the in vitro UDS assay, and its metabolic susceptibility toward hydroxylation at the C-6 methyl group was eliminated. These observations suggest that the positive in vitro UDS results of U-89843 are mediated by the bioactivation of U-89843, leading to reactive electrophilic intermediates derived from the (hydroxymethyl)pyrrole metabolite U-97924.

End points for biomonitoring: assay sensitivity/selectivity.

Estimation of population exposure and biological impact of potential hazards are central reasons for performing biomonitoring. The sensitivity of the biomonitoring methods and the linkage of the measured phenomenon to human disease are also important, but often overlooked, considerations. We are conducting experiments to evaluate the sensitivity of hprt mutation measurement in the nonhuman primate, the cynomolgus monkey. Our findings demonstrate in the monkey that hypoxanthine guanine phosphoribosyltransferase (hprt) mutations produced in vivo can be detected using technique originally worked out using human cells; cynomolgus monkeys were chosen to avoid many of the complications encountered in studying humans. Sequencing of mutants from the monkey using reverse transcriptase polymerase chain reaction methods has led us to conclude that there is similarity of the spectra observed between the spontaneous mutations detected in the two species. However, more recent data suggest that due to low sensitivity, the method is probably not appropriate for routine biomonitoring of randomly selected populations. For example, the inability of the hprt mutation assay to detect some very potent mutagens in the monkey and the effects of the time-dependent pattern of mutant occurrence serve to urge caution in interpretation of elevation or lack of elevation in mutant frequency. Mechanisms for splitting and archiving samples of human tissues/blood from populations at risk may prove valuable as methods improve.

Spontaneous and ethylnitrosourea-induced mutation fixation and molecular spectra at the lacI transgene in the Big Blue rat-2 embryo cell line.

Big Blue Rat-2 cells were evaluated for mutagenesis and mutational spectra (spontaneous and ethylnitrosourea [ENU]-induced). Survival, mutant frequency, population doubling time, and kinetics of mutant increase (to 120 hr) were determined. Exposures were 100, 200, 400, 600, and 1,000 micrograms ENU/ml. The spontaneous mutant frequency was similar to that previously reported in vivo, i.e., 5 X 10(5). Dose-related increases in mutant frequency were observed following ENU treatment. Kinetics (time course) of mutant frequency increase, population doubling, and mutational spectra were investigated following treatment at 1,000 micrograms ENU/ml. Among 39 spontaneous mutants, 26 independent mutations were found as follows: nine (34.6%) G:C-->A:T transitions (five at CpG sites), six (23%) G:C-->T:A transversions, three (11.5%) G:C-->C:G transversions (two at CpG sites), two (7.7%) frameshifts, five (19%) deletions or insertions, and one (3.8%) complex (deletion+insertion) mutation. Among 46 ENU-induced mutants, 37 independent mutations (all base substitutions) were found as follows: 15 (40.5%) G:C-->A:T transitions (four at CpG sites), five (13.5%) A:T-->G:C transitions, four (10.8%) G:C-->T:A transversions, 11 (30%) A:T-->T:A transversions, and two (5.4%) A:T-->C:G transversions. Nearly 50% of the base substitutions in the ENU-treated cells were at A:T base pairs, in contrast to the spontaneous mutants where none was found. Both the spontaneous and the ENU-induced mutational spectra were similar to that reported in vivo and for other cells. An important aspect of the experiment is that all mutations sequenced following ENU treatment (1,000 micrograms/ml) occurred under conditions which our experiments show corresponded to very little mitotic activity.

Lung tumor induction in A/J mice and clastogenic effects in CD-1 mice of the sequence-selective DNA alkylating agents (+)-CC-1065 and (-)-CC-1065.

The in vivo genotoxic effects of the antitumor antibiotic, (+)-CC-1065, and its unnatural enantiomer, (-)-CC-1065, were investigated in two mouse models. These two compounds alkylate AT-rich regions of double stranded DNA with distinct sequence selectivities. (+)-CC-1065 dose-dependently increased the chromosomal aberration frequency in bone marrow cells of CD-1 mice from 1.2 +/- 0.8% in vehicle control animals to 5.0 +/- 1.2%, 11.4 +/- 3.9%, and 20.6 +/- 2.3% 24 hours following single intravenous doses of 2, 4, and 8 micrograms/kg, respectively. (-)-CC-1065 was significantly less potent with a maximal response at 8 micrograms/kg approximately one-third of that observed for (+)-CC-1065. (+)-CC-1065 induced a significant (P < or = 0.05), three-fold increase in the number of lung tumors/mouse in strain A/J mice from 0.27 +/- 0.15 for vehicle control animals to 0.83 +/- 0.15 24 weeks following a single intravenous dose of 8 micrograms/kg. This effect was paralleled by corresponding threefold increases in the percentage of mice with tumors and the percentage of mice with multiple tumors, compared to vehicle controls. (-)-CC-1065 at 8 micrograms/kg induced 0.67 +/- 0.15 tumors/mouse and resulted in slightly smaller increases in the tumor incidence and multiple tumor incidence, compared to (+)-CC-1065. The above results demonstrate that single intravenous doses of (+)- CC-1065 and (-)-CC-1065 which cause chromosomal damage in CD-1 mice also induce an increased incidence of lung tumors in A/J mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Southern blot analysis of T-cell receptor gene rearrangements in cynomolgus monkeys, and identification of a progenitor cell HPRT mutation.

Increases in peripheral blood T-lymphocyte HPRT mutant frequency may reflect either a number of independent HPRT gene mutational events or clonal proliferation of a single HPRT mutant. Sequence analysis of HPRT mutations in conjunction with T-cell receptor (TCR) gene rearrangement pattern analysis can distinguish these possibilities. Our laboratory previously characterized a nonhuman primate model for in vivo mutation studies using the clonal HPRT mutation assay. In the present study we report the use of probes for human TCR beta and gamma genes to characterize TCR rearrangements in cynomolgus monkeys. Together, these methods were used to examine a monkey which exhibited a mean spontaneous HPRT mutant frequency (MF) of 16.4 x 10(-6), compared to the normal mean MF of 3.03 x 10(-6). The elevated MF resulted from the occurrence of a single HPRT mutation in a lymphocyte progenitor cell or stem cell, since T-cell clones isolated from the monkey exhibited a G to T transversion at base pair 539 in the HPRT coding region, and had unique rearrangements of TCR gamma along with an apparent germline TCR beta configuration. In a preliminary in vivo mutation study, the animal was treated with the investigational potent mutagen and antitumor agent adozelesin (U-73975). No increase in HPRT mutant frequency was observed. The HPRT mutant clones isolated after treatment showed rearrangement of both TCR gamma and beta genes. Possible explanations for these findings are discussed.