Single‐fiber PCR in MELAS3243 patients: Correlations between intratissue distribution and phenotypic expression of the mtDNAA3243G genotype

We performed morphological, biochemical, and genetic studies, including single‐fiber PCR (sf PCR), on muscle biopsies obtained from a mother and daughter with MELAS syndrome due to the A3243G transition of mitochondrial DNA (mtDNA). The severity of muscle involvement appeared quite distinct, in spite of the fact that both patients segregated similar mutant mtDNA levels on total muscle DNA. The daughter did not show any clinical muscle involvement: muscle biopsy revealed many ragged red fibers (RRFs) mostly positive for cytochrome‐c oxidase (COX) activity. In contrast, her mother had developed a generalized myopathy without progressive external ophthalmoplegia (PEO), morphologically characterized by many COX‐negative RRFs. Single‐muscle fiber PCR demonstrated in both patients significantly higher percentages of wild‐type mtDNA in normal fibers (daughter: 23.25 ± 15.22; mother: 43.13 ± 26.11) than in COX‐positive RRFs (daughter: 11.25 ± 5.22, P < 0.005; mother: 9.12 ± 5.9, P < 0.001) and in COX‐negative RRFs (daughter: 8.9 ± 4.2, P < 0.001 mother: 4.8 ± 2.8, P < 0.001). Wild‐type mtDNA levels resulted higher also in COX‐positive vs. COX‐negative RRFs (daughter: P < 0.05; mother: P < 0.001). Our data confirm a direct correlation between A3243G levels and impairment of COX function at the single‐muscle fiber level. Moreover, the evidence of a clinical myopathy in the patient with higher amounts of COX‐negative RRFs bolsters the concept that a differential distribution of mutant mtDNAs at the cellular level may have effects on the clinical involvement of individual tissues. However, the occurrence of a similar morphological and biochemical muscle phenotype also in PEO³²⁴³ patients suggests that other genetic factors involved in the interaction between mitochondrial and nuclear DNA, rather than the stochastic distribution of mtDNA genomes during embryogenesis, are primarily implicated in determining the various clinical expressions of the A3243G of mtDNA. Am. J. Med. Genet. 94:201–206, 2000. © 2000 Wiley‐Liss, Inc.     

Complete restoration of a wild-type mtDNA genotype in regenerating muscle fibres in a patient with a tRNA point mutation and mitochondrial encephalomyopathy

Replicative segregation of mitochondrial DNA (mtDNA) can produce large differences in the proportions of wild-type and mutant mtDNAs in different cell types of patients with mitochondrial encephalomyopathy. This is particularly striking in the skeletal muscle of patients with Kearns-Sayre syndrome (KSS), a sporadic disease associated with large- scale mtDNA deletions, and in sporadic patients with tRNA point mutations. Although the skeletal muscle fibres of these patients invariably contain a large proportion of mutant mtDNAs, mutant mtDNAs are rare or undetectable in satellite cells cultured from the same muscle biopsy specimens. Since satellite cells are responsible for muscle fibre regeneration, restoration of the wild-type mtDNA genotype might be achieved in these patients by encouraging muscle regeneration. To test this concept, we re-biopsied a patient with a KSS phenotype and a mtDNA point mutation in the tRNAleu(CUN)gene and analysed muscle fibres regenerating at the site of the original muscle biopsy. Regenerating fibres were identified by morphological criteria and by expression of neural cell adhesion molecule (NCAM). All such fibers were positive for cytochrome c oxidase (COX) activity by cytochemistry and essentially homoplasmic for wild-type mtDNA, while the majority of non-regenerating fibres were COX-negative and contained predominantly mutant mtDNAs. These results demonstrate that it may be possible to improve muscle function in similar patients by methods that promote satellite cell incorporation into existing myofibres.      

A novel heteroplasmic tRNAleu(CUN) mtDNA point mutation in a sporadic patient with mitochondrial encephalomyopathy segregates rapidly in skeletal muscle and suggests an approach to therapy

A novel mtDNA point mutation was detected in the tRNAleu(CUN) gene (G to A at position 12315) in a sporadic patient with chronic progressive external ophthalmoplegia, ptosis, limb weakness, sensorineural hearing loss and a pigmentary retinopathy. The mutation disrupts base pairing in the T psi C stem at a site which has been conserved throughout evolution. Although the other mtDNA tRNAleu gene (UUR) is a hotspot for mutation, this is the first pathogenic mutation to be reported in the gene coding for tRNAleu(CUN). MtDNAs carrying the mutation constituted 94% of total mtDNAs in two separate muscle biopsies. Single fibre analysis showed that skeletal muscle fibres without detectable cytochrome c oxidase activity (COX-ve fibres) contained predominantly mutant mtDNAs (93-98%) while fibres with apparently normal COX activity had up to 90% mutant mtDNAs, demonstrating that the G12315A mutation is functionally recessive. Immunofluorescence studies with specific antibodies to mtDNA- or nuclear-encoded subunits of COX were consistent with a defect in mitochondrial protein translation. The mutation was not present in blood cells or cultured fibroblasts and surprisingly, it could not be detected in satellite cells cultured from the patient's muscle. This pattern, which may by typical of patients who have inherited new germline pathogenic mtDNA mutations, possibly reflects loss of the mutation by random genetic drift in mitotic tissues and proliferation of mitochondria containing the mutant mtDNA in post- mitotic cells. The absence of mtDNA carrying the mutation in satellite cells suggests that regeneration of skeletal muscle fibres from satellite cells could restore a wild-type mtDNA genotype and normal muscle function.      

Nuclear DNA origin of cytochrome c oxidase deficiency in Leigh's syndrome: genetic evidence based on patient's-derived rho{degrees} transformants

Defects of the respiratory chain carrying out oxidative phosphorylation(OXPHOS) are the biochemical hallmark of human mitochondrialdisorders. Faulty OXPHOS can be due to mutations in either nuclearor mitochondrial genes, that are involved in the synthesis ofindividual respiratory subunits or in their post-translationalcontrol. The most common mitochondrial disorder of infancy andchildhood is Leigh's syndrome, a severe encephalopathy, oftenassociated with a defect of cytochrome c oxidase (COX). In orderto demonstrate which genome is primarily involved in COX-deficient(COX(^–))-Leigh's syndrome, we generated two lines of transmitochondrialcybrids. The first was obtained by fusing nuclear DNA-less cytoplastsderived from normal fibroblasts, with mitochondrial DNA-less(rho°) transformant fibroblasts derived from a patient withCOX(^–)-Leigh's syndrome. The second cybrid line was obtainedby fusing rho° cells derived from 143B.TK^– human osteosarcomacells, with cytoplasts derived from the same patient. The firstcybrid line showed a specific and severe COX(-) phenotype, whilein the second all the respiratory chain complexes, includingCOX, were normal. These results indicate that the COX defectin our patient is due to a mutation of a nuclear gene. The useof cybrids obtained from ‘customized’, patient-derivedrho° cells can have wide applications in the identificationof respiratory chain defects originated by nuclear DNA—encodedmutations, and in the study of nuclear DNA-mitochondrial DNAinteractions.     

Single-fiber PCR in MELAS(3243) patients: correlations between intratissue distribution and phenotypic expression of the mtDNA(A3243G) genotype

We performed morphological, biochemical, and genetic studies, including single-fiber PCR (sf PCR), on muscle biopsies obtained from a mother and daughter with MELAS syndrome due to the A3243G transition of mitochondrial DNA (mtDNA). The severity of muscle involvement appeared quite distinct, in spite of the fact that both patients segregated similar mutant mtDNA levels on total muscle DNA. The daughter did not show any clinical muscle involvement: muscle biopsy revealed many ragged red fibers (RRFs) mostly positive for cytochrome-c oxidase (COX) activity. In contrast, her mother had developed a generalized myopathy without progressive external ophthalmoplegia (PEO), morphologically characterized by many COX-negative RRFs. Single-muscle fiber PCR demonstrated in both patients significantly higher percentages of wild-type mtDNA in normal fibers (daughter: 23.25 +/- 15.22; mother: 43.13 +/- 26.11) than in COX-positive RRFs (daughter: 11.25 +/- 5.22, P < 0.005; mother: 9.12 +/- 5.9, P < 0.001) and in COX-negative RRFs (daughter: 8.9 +/- 4.2, P < 0.001 mother: 4.8 +/- 2.8, P < 0.001). Wild-type mtDNA levels resulted higher also in COX-positive vs. COX-negative RRFs (daughter: P < 0.05; mother: P < 0.001). Our data confirm a direct correlation between A3243G levels and impairment of COX function at the single-muscle fiber level. Moreover, the evidence of a clinical myopathy in the patient with higher amounts of COX-negative RRFs bolsters the concept that a differential distribution of mutant mtDNAs at the cellular level may have effects on the clinical involvement of individual tissues. However, the occurrence of a similar morphological and biochemical muscle phenotype also in PEO(3243) patients suggests that other genetic factors involved in the interaction between mitochondrial and nuclear DNA, rather than the stochastic distribution of mtDNA genomes during embryogenesis, are primarily implicated in determining the various clinical expressions of the A3243G of mtDNA.     

Procarbazine is a potent mutagen at the heterozygous thymidine kinase (tk+/-) locus of mouse lymphoma assay

Procarbazine (Natulan®) is a potent inducer of gene mutationsat the heterozygous tk^+/– locus in L5178Y mouse lymphomacells in the presence of Aroclor-induced rat liver s9 metabolicactivation ({small tilde} 10^–3 mutant frequency at 10µg/ml) while exerting a far weaker effect in the absenceof s9. This mutagenicity is fairly robust with respect to thequantitative composition of the s9 mix and to variations inmouse lymphoma assay protocols (soft agar cloning versus ‘microwell’assays). The high proportion of small colony tk^–/–mutants induced by procarbazine together with the far weakermuta-genic response at the hemizygous hgprt locus in these samecells is interpreted in terms of a chromosomal or mufti-genemutational mechanism. Although procarbazine is clastogenic invivo, it does not appear to be so under standard protocols usingcultured human lymphocytes (±s9). It is not yet clearwhy this should be so, especially in light of its apparent clasto-genicityin mouse lymphoma cells.     

Micronucleus assays using cytochalasin-blocked MCL-5 cells, a proprietary human cell line expressing five human cytochromesP-450 and microsomal epoxide hydrolase

The MCL-5 cell line is a human lymphoblastoid TK^+/- cell linethat constitutively expresses a relatively high level of nativeCYP1A1, four other human cytochromes (CYP1A2, CYP2A6, CYP3A4and CYP2E1) and microsomal epoxide hydrolase, carried as cDNAsin plasmids. The aim of this study was to evaluate this cellline for its suitability for detecting chromosomal anomalies,employing micronucleus formation in cells blocked at cytokinesisas the indicator of clastogenicity. Results from two laboratories(‘ICR’ and ‘Swansea’) using differentprotocols are reported. In the ICR protocol, aflatoxin B¹, sterigmatocystin,benzo[     

Human oocyte activation after intracytoplasmic sperm injection

The concentration of acetoxymethyl ester of fluo-3 given inthe subsection ‘Measurement of [Ca²⁺]_i’ of the ‘Materialsand methods’ on page 512 of the above paper was incorrect.The corresponding sentence should read as follows: Oocytes were loaded with the [Ca²⁺]_i indicator fluo-3 (Mintaet al., 1989) by incubation for 45 min at 37°C with 2 µMacetoxymethyl ester of fluo-3 (fluo-3/AM, Sigma) dissolved inB2 medium.     

Metabolic consequences of a novel missense mutation of the mtDNA CO I gene

We have identified a novel heteroplasmic C6489A missense mutation in the mitochondrial DNA (mtDNA) CO I gene encoding the cytochrome c oxidase (COX) subunit I in a 17-year-old girl with epilepsia partialis continua. This point mutation leads to an exchange of the highly conserved Leu196 to Ileu196. Muscle biopsy showed in single fibers decreased COX activity and lowered binding of COX antibodies, indicating decreased stability of the mutated enzyme. The analysis of blood mtDNA revealed about 30% mutant mtDNA in the patients blood but about 90% mutant mtDNA in the blood of two non-affected family members. Quantitative analysis of the mutation gene dose effect on COX activity on single muscle fiber level revealed a very high threshold-a COX deficiency was observed only in fibers containing >95% mutant mtDNA. In apparent contrast to this high mutation gene dose threshold, in vivo investigations of mitochondrial function in saponin-permeabilized muscle fibers of the index patient containing approximately 90% mutated mtDNA showed decreased maximal rates of respiration and an increased sensitivity of fiber respiration to cyanide. This is due to a 2-fold increase of COX flux control on muscle fiber respiration and a 30% decrease of COX metabolic threshold, supporting the concept of tight COX control of oxidative phosphorylation in skeletal muscle.     

Do sequence variants in the major non-coding region of the mitochondrial genome influence mitochondrial mutations associated with disease?

Several different mutations in human mitochondrial DNA (mtDNA) have been associated with disease, but their origins and the basis of the wide phenotypic variability remain to be elucidated. We initially investigated three patients with heteroplasmic disease associated mutations of mtDNA for the presence of cis mutations in the major non- coding region that might influence their origins or pathology. A T --> C transition at nt 16 189 previously identified in one patient with the 3243 G:C mutation was associated with heteroplasmic length variation. Identical length variation was found in patient-derived cybrid lines containing 0-97.5% 3243 G:C. Similarly, heteroplasmic length variation was demonstrated in 2/6 other probands with both the 3243 mutation and the 16,189 polymorphism. The distribution of length variants in probands and in asymptomatic family members was identical in all cases. Thus length variation appears to be independent of the level of 3243 mutant mtDNA and hence probably arose within both 3243 G:C and 3243 A:T mtDNAs. We suggest that the 16,189 polymorphism reflects a predisposition to the formation or fixation of several different mutations in mitochondrial tRNA-LeuUUR.      

Physiology: Human follicular fluid maturity and endothelial cell mitogenesis

Follicular fluid, of varying maturity, (day 5–16 of cycle)was collected from the largest Graafian follicle of each of22 ovulatory patients during laparoscopic procedures. Threesamples were blood-stained and discarded. The mitogenic potentialof each sample was determined using bovine aortic endothelialcells in the CellTiter 96^TM Non-Radioactive Cell Proliferation/CytotoxicityAssay system. Intra- and inter-plate coefficients of variationwere <9%. The follicular fluid samples induced cell doublingtimes which varied from 12–24 h and final cell numberswhich, in the individual wells, ranged from 782–30 900(starting number 2000/well). Follicular fluid total proteincontent was unrelated to the mitogenic potential, (R² = 0%).Serum oestradiol was negatively correlated with the mitogenicpotential (R² = 26%). No correlation was found with day of themenstrual cycle (R² = 4.3%), maximum follicular diameter (R²= 1.8%), or serum concentration of progesterone (R² = 0.7%),luteinizing hormone (LH) (R² = 1.5%) or follicle stimulatinghormone (R² = 0.1%). Five subjects were in ‘early’and six in ‘mid’-follicular phase, six were in ‘early’and two in ‘late’ LH surge. There was no differencein the mitogenic response between these four groups by one-wayanalysis of variance (F = 0.21; P = 0.89). It is concluded thatthe mitogenic potential of human follicular fluid is not relatedto Graafian follicle maturity or, more particularly, to theLH surge.     

Decreased aminoacylation of mutant tRNAs in MELAS but not in MERRF patients

Mutations in human mitochondrial tRNA genes are associated witha number of multisystemic disorders. Using an assay that combinestRNA oxidation and circularization we have determined the relativeamounts and states of aminoacylation of mutant and wild-typetRNAs in tissue samples from patients with MELAS syndrome (mito-chondrial myopathy, encephalopathy, lactic acidosis, stroke-likeepisodes) and MERRF syndrome (myoclonus epilepsy with raggedred fibers), respectively. In most, but not all, biopsies fromMELAS patients carrying the A3243G substitution in the mitochondrialtRNA^Leu(UUR) gene, the mutant tRNA is under-represented amongprocessed and/or aminoacylated tRNAs. In contrast, in biopsiesfrom MERRF patients harboring the A8344G substitution in thetRNA^Lys gene neither the relative abundance nor the aminoacylationof the mutated tRNA is affected. Thus, whereas the A3243G mutationmay contribute to the pathogenesis of MELAS by reducing theamount of aminoacylated tRNA^Leu, the A8344G mutation does notaffect tRNA^Lys function in the same way. ⁺ To whom correspondence should be addressed. Tel: +1 617 4954396; Fax: +1 617 495 0758; Email: boerner@fas.harvard.edu     

A genetically engineered V79 cell line SD1 expressing rat CYP2B1 exhibits chromosomal instability at the integration site of the transfected DNA

The genetically engineered cell line SD1 was constructed byco-transfection of V79 Chinese hamster cells with two plasmids:one containing a full-length cDNA encoding rat CYP2B1 and thesecond incorporating a selective marker gene. This cell linehas been used in gene mutation tests and in cytokinesis-blockmicronucleus assays to identify procarcinogens which are metabolizedby CYP2B1 to reactive metabolites. An elevated frequency ofspontaneous micronuclei was recorded in SD1 cells compared toparental V79 cultures. Karyotypic analysis revealed a chromosomalinstability which was manifested by amplification of the p-armsof a chromosome designated ‘n’ (derived from chromosome8). This chromosome was variable in length and sometimes exhibiteda telomeric fusion which led to the formation of a dicentricchromosome. Fluorescence in situ hybridization with digoxigenin-labelledplasmid DNA showed the presence of pSV450 plasmid DNA co-amplifiedwith genomic DNA sequences located in the terminal region ofchromosome ‘n’. ¹Present address: Molecular Genetics Laboratory, Departmentof Pathology, Royal Devon and Exeter Healthcare NHS Trust, BarrackRoad, Exeter EX2 5DW, UK. ²To whom correspondence should be addressed      

Ovarian stimulation for in-vitro fertilization combining administration of gonadotrophins and blockade of the pituitary with D-Trp6-LHRH microcapsules: pilot studies with two protocols

In women undergoing in-vitro fertilization and embryo transfer(TVF-ET), a total of 408IVF cycles were stimulated using humanmenopausal gonadotrophin (HMG) or pure follicle stimulatinghormone (FSH) plus HMG in combination with a single injectionof D-Trp⁶-LHRH microcapsules in order to enhance the ovarianresponse to gonadotrophins and to avoid spontaneous LH surges.Sixty-seven pregnancies were achieved. Two protocols were employed.In protocol 1 (‘blocking protocol’, n = 268), thepituitary was first inhibited with a full dose (3.75 mg) ofD-Trp⁶-LHRH in microcapsules and ovarian stimulation was startedafter the hypogonadotrophic hypogonadal state was ascertained(Ej >50 pg/ml). In protocol 2 (‘flareup protocol’,n = 140), the treatment with D–Trp⁶LHRH microcapsules(half-dose = 1.80 mg) and the ovarian stimulation with gonadotrophinswere started at the same time. Higher doses of gonadotrophinswere needed (39.5 11.2 ampoules FSH and/or HMG) in protocol1, in which the pituitary was blocked prior to and during thestimulation, than in protocol 2 (209 ampoules) where the exogenousgonadotrophin stimulation appeared to be augmented by the initialagonistic effect of the injection of D-Trp⁶LHRH microcapsules.In patients with purely tubal infertility, under 38 years oldand no male factor, the results obtained with protocols 1 and2 were similar in terms of pregnancy rate per cycle or per embryotransfer: 22.6 versus 20.5% and 28.3 versus 27.4%, respectively.However, considering the cost benefit, ‘flare-up’protocols appeared to be a better choice and could be recommended.     

Histochemical and molecular genetic study of MELAS and MERRF in Korean patients

Mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episode (MELAS) and myoclonic epilepsy and ragged-red fibers (MERRF) are rare disorders caused by point mutation of the tRNA gene of the mitochondrial genome. To understand the pathogenetic mechanism of MELAS and MERRF, we studied four patients. Serially sectioned frozen muscle specimens with a battery of histochemical stains were reviewed under light microscope and ultrastructural changes were observed under electron microscope. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was performed and the tRNA genes were sequenced to confirm mutations. In two patients with MELAS, strongly succinyl dehydrogenase positive blood vessels (SSVs) and many cytochrome oxidase (COX) positive ragged-red fibers (RRFs) were observed, and A3243G mutations were found from the muscle samples. In two patients with MERRF, neither SSV nor COX positive RRFs were seen and A8344G mutations were found from both muscle and blood samples. In the two MERRF families, the identical mutation was observed among family members. The failure to detect the mutation in blood samples of the MELAS suggests a low mutant load in blood cells. The histochemical methods including COX stain are useful for the confirmation and differentiation of mitochondrial diseases. Also, molecular biological study using muscle sample seems essential for the confirmation of the mtDNA mutation.     

Risks and benefits of steroid replacement therapy

The recommendations of the ESHRE workshop on ‘Risks andBenefits of Steroid Replacement Therapy’, held in Anacapri,September 18–19, 1987 ^*The workshop was sponsored by Schering (Italy).     

The effect of stress on semen reduction in the marmoset monkey (Callithrix jacchus)

This study compared a number of semen parameters of two separategroups of the common marmoset monkey (Callithrix jacchus) inorder to determine the effect of a continuous potentially stressfulsituation on these parameters, and thus on the monkey's reproductiveability. The semen from 16 adult male marmoset monkeys was collectedand analysed to compare semen parameters between a ’normal‘(control) group (n = 9) and a ’blood withdrawn‘(’stress‘) group (n = 7). The semen parameter valuesobserved in the control were: pH 7.51 ± 0.22, volume40.2 ± 27.2 µ1, concentration 27.3 ± l4.8xl04/ul,motility 47.4 ± 15.9%, grade of velocity 3.5 ±1.2, and normal morphological forms 51.8 ± 13.7%. The’blood withdrawn‘ group of marmoset monkeys showedsignificantly lower semen volume and sperm concentration thanthe ’normal‘ group. In addition, total count ofspermatozoa, normal spermatozoa, motile spermatozoa and normalmotile spermatozoa per ejaculate per monkey was significantlyreduced in the ‘blood withdrawn’ group. The semenof these monkeys also revealed a significantly higher percentageof abnormally-shaped sperm heads than the normal group, andcases of impotence and sham ejaculation were recorded. Our studyrevealed that the continuous withdrawal of a small volume ofblood from a group of marmoset monkeys appeared to be stressfulto these monkeys and as a result, influenced their semen parameters,possibly making them less fertile. In addition, electroejaculationwas found to be possibly harmful to the monkey's reproductiveability.     

Enhanced reactivation and mutagenesis of UV-irradiated adenovirus in normal human fibroblasts

UV-enhanced reactivation (UVER) and UV-enhanced mutagenesis(UVEM) for two adenovirus temperature-sensitive mutants (Ad5ts35and Ad5tsl25) were examined following the infection of normalhuman fibroblasts. Fibroblast monolayers were either UV-irradiatedor left non-irradiated and subsequently infected with eithernon-irradiated or UV-irradiated virus. After incubation of theinfected cultures at the permissive temperature, the inductionof wild-type revertants in the viral progeny was determinedby plaquing at the permissive (33°C) and the non-permissive(39°C) temperatures on human HeLa or KB cells. UV-irradiationof the virus alone resulted in a dose-dependent increase inthe UV-induced reversion frequency (RF) of viral progeny anda dose-dependent exponential decrease in progeny survival, wheninfecting non-irradiated cells. Analysis of the slopes of theUV-induced reversion curves suggested that 2.5 ± 0.3and 2.4 ± 0.5 ‘hits’ were required to producea targeted reversion event among the viral progeny of Ad5ts36and Ad5tsl25 respectively. UV-irradiation of cells 24 h priorto infection resulted in a significant increase in progeny survivalfor UV-irradiated virus (UVER factor = 3.4 ± 0.8) concomitantwith a significant increase in RF for UV-irradiated virus (targetedincrease = 1.9 ± 0.3). The UV-induced RF per lethal hitto the virus was also significantly greater in UV-irradiatedcompared with non-irradiated cells. These results are consistentwith the existence of a UV-inducible error-prone DNA repairmechanism in normal human cells. ¹Present address: National Institute of Environmental HealthSciences, Cellular Genetic Toxicology Branch, Research TrianglePark, NC 27709, USA ²To whom correspondence should be addressed      

Evaluation of presence and functional activity of potentially self-reactive T cells in aged mice

Autoimmunity is known to increase in aging. A possible factorcould be an alteration in the T cell repertoire wIth advancingage. Antibodies to the variable region of the ß chainof the TCR activate T cells and can serve as probes for analysisof the T cell repertoire. We have used V_ß3 and V_ß17aantibodies to determine the presence and functionality of normallydeleted T cells bearing potentially self-reactive TCR in peripherallymphoid tissue and blood from aged (SJL/J x BALB/c) F₁ LAF₁and BALB/c mice. Although an occasional 20- to 24-month-oldmouse exhibited V_ß3⁺ or V_ß17a⁺ T cells intheir lymph nodes or peripheral blood lymphocytes (PBL) slightlyabove the range for normal young mice of these I-E⁺ strains,there was no striking ‘escape’ from the normal thymicdeletion process. However, responsiveness to anti-V_ß3and anti-V_ß17a was slightly higher In aged, and particularlyIn aged thymectomlzed (TX), than in young mice. This was incontrast to proliferative responses to stimulation with antibodyto the normally expressed V_ß8 which were lower inthe lymph nodes from aged than from young mice. The PBL of some30- to 36-month-old mice were also examined. Enhanced numbersof ‘forbidden’ V_ß bearing T cells wereseen more frequently at this age. In spite of the age-relateddecrease in overall CD4/CD8 T cell ratios in all organs, themice with relatively high V_ß17a⁺ T cells exhibitedproportionally more CD4⁺ cells in that V_ß population.We conclude that the ‘forbidden’ T cells that respondto anti-V_ß stimulation in the 20- to 24-month-oldmice are most likely of extra-thymic origin, since they weremore readily detectable in aged TX mice. Potentially self-reactiveCD4 (and CD8) single-positive T cells were detectable in PBLonly in very aged (30–36 months old) euthymic mice.