An accessible pharmacodynamic transcriptional biomarker for notch target engagement.

γ-Secretase mediates amyloid production in Alzheimer's disease (AD) and oncogenic activity of Notch. γ-Secretase inhibitors (GSIs) are thus of interest for AD and oncology. A peripheral biomarker of Notch activity would aid determination of the therapeutic window and dosing regimen for GSIs, given toxicities associated with chronic Notch inhibition. This study examined the effects of GSI MK-0752 on blood and hair follicle transcriptomes in healthy volunteers. The effects of a structurally diverse GSI on rhesus blood and hair follicles were also compared. Significant dose-related effects of MK-0752 on transcription were observed in hair follicles, but not blood. The GSI biomarker identified in follicles exhibited 100% accuracy in a clinical test cohort, and was regulated in rhesus by a structurally diverse GSI. This study identified a translatable, accessible pharmacodynamic biomarker of GSI target engagement and provides proof of concept of hair follicle RNA as a translatable biomarker source.

The effects of a novel histamine-3 receptor inverse agonist on essential tremor in comparison to stable levels of alcohol.

Essential tremor (ET) is a common movement disorder. Animal studies show that histaminergic modulation may affect the pathological processes involved in the generation of ET. Histamine-3 receptor inverse agonists (H3RIA) have demonstrated attenuating effects on ET in the harmaline rat model. In this double-blind, three-way cross-over, single-dose, double-dummy study the effects of 25 mg of a novel H3RIA (MK-0249) and a stable alcohol level (0.6 g L(-1)) were compared with placebo, in 18 patients with ET. Tremor was evaluated using laboratory tremorography, portable tremorography and a clinical rating scale. The Leeds Sleep Evaluation Questionnaire (LSEQ) and a choice reaction time (CRT) test were performed to evaluate potential effects on sleep and attention, respectively. A steady state of alcohol significantly diminished tremor as assessed by laboratory tremorography, portable tremorography and clinical ratings compared with placebo. A high single MK-0249 dose was not effective in reducing tremor, but caused significant effects on the LSEQ and the CRT test. These results suggest that treatment with a single dose of MK-0249 does not improve tremor in alcohol-responsive patients with ET, whereas stable levels of alcohol as a positive control reproduced the commonly reported tremor-diminishing effects of alcohol.

Acute alertness-promoting effects of a novel histamine subtype-3 receptor inverse agonist in healthy sleep-deprived male volunteers.

The alertness-promoting effect of MK-0249 (10 or 50 mg), a histamine subtype-3 receptor (HRH3) inverse agonist (IA), was evaluated in the stimulant reference sleep deprivation model (SRSDM) using a double-blind, double-dummy, placebo- and modafinil- (200 mg) controlled, four-period crossover design in 24 healthy young men. The two primary hypotheses were related to sleep latency (first appearance of one epoch of stage 2, 3, or 4 or REM sleep, as detected using polysomnography (PSG)) at 8:00 AM on day 2. Statistically significant increases in sleep latency were observed in association with the use of modafinil 200 mg (9.07 min; P < 0.0001), MK-0249 50 mg (5.17 min; P = 0.008), and MK-0249 10 mg (5.45 min; P = 0.005) at the maintenance of wakefulness test (MWT) at 8:00 AM. Sleep latency was higher when averaged over all MWT time points (P < 0.0001 for modafinil and for both doses of MK-0249). The alertness-promoting effect with the use of MK-0249 in the SRSDM suggests that HRH3 IAs may be effective in disorders involving excessive somnolence.

Unrelated donor bone marrow transplantation for children with severe aplastic anemia: minimal GVHD and durable engraftment with partial T cell depletion.

Both increased graft rejection and increased graft vs host disease (GVHD) remain obstacles to success for unrelated donor (URD) BMT for patients with SAA. Partial T cell depletion (PTCD) may decrease the risk of severe GVHD, while still maintaining sufficient donor T lymphocytes to ensure engraftment. We report on 12 patients with SAA who underwent PTCD URD BMT. All patients had failed medical therapy or relapsed following initial responses, and were transfusion dependent. The median age was 6 years, and there were five males. Donors were matched for four patients, and mismatched for eight. All patients received total body irradiation with either Ara-C or thiotepa and cyclophosphamide. PTCD was accomplished using monoclonal antibody T10B9 or OKT3 and complement. All patients engrafted, with a median time of 18 days to ANC >500. Only one patient had greater than grade II acute GVHD; two patients had limited and one patient extensive chronic GVHD. Nine patients are alive and transfusion independent at a median months post BMT. Three patients died from infection or renal failure. This series suggests that an aggressive immunosuppressive conditioning regimen with PTCD results in successful engraftment and minimal GVHD in pediatric patients with SAA, even with HLA mismatched donors.

Durable engraftment of major histocompatibility complex-incompatible cells after nonmyeloablative conditioning with fludarabine, low-dose total body irradiation, and posttransplantation cyclophosphamide.

Treatment of leukemia by myeloablative conditioning and transplantation of major histocompatibility complex (MHC)-mismatched stem cells is generally avoided because of the high risk of graft rejection or lethal graft-versus-host disease (GVHD). This study shows that MHC-incompatible cells can engraft stably after nonmyeloablative conditioning with immunosuppressive chemotherapy and low-dose total body irradiation (TBI). Long-term mixed hematopoietic chimerism, clonal deletion of donor-reactive T cells, and bidirectional cytotoxic T-cell tolerance were achieved by transplanting MHC-mismatched marrow cells into recipients conditioned with pretransplantation fludarabine or cyclophosphamide (Cy), 50 to 200 cGy TBI on day -1, and Cy 200 mg/kg intraperitoneally on day 3. In this model, long-term donor chimerism was proportional to the dose of TBI or donor marrow cells. Pretransplantation fludarabine and posttransplantation Cy were both required for alloengraftment, but the drugs had additional effects. For example, fludarabine sensitized host stem cells to the toxicity of TBI, because animals conditioned with both agents had higher chimerism than animals conditioned with TBI alone (P <.05). Also, posttransplantation Cy attenuated lethal and nonlethal GVH reactions, because F(1) recipients of host-reactive, parental spleen cells survived longer (P <.05) and had lower donor cell chimerism (P <.01) if they received posttransplantation Cy than if they did not. Finally, delayed infusions of donor lymphocytes into mixed chimeras prolonged survival after leukemia challenge (P <.0001) without causing lethal GVHD. These results indicate that stable engraftment of MHC-incompatible cells can be induced after fludarabine-based, nonmyeloablative conditioning and that it serves as a platform for adoptive immunotherapy with donor lymphocyte infusions.

Effects of mixed hematopoietic chimerism in a mouse model of bone marrow transplantation for sickle cell anemia.

Sickle cell anemia (SCA) is an inherited disorder of beta-globin, resulting in red blood cell rigidity, anemia, painful crises, organ infarctions, and reduced life expectancy. Allogeneic blood or marrow transplantation (BMT) can cure SCA but is associated with an 8% to 10% mortality rate, primarily from complications of marrow-ablative conditioning. Transplantation of allogeneic marrow after less intensive conditioning reduces toxicity but may result in stable mixed hematopoietic chimerism. The few SCA patients who inadvertently developed mixed chimerism after BMT remain symptom free, suggesting that mixed chimerism can reduce disease-related morbidity. However, because the effects of various levels of mixed chimerism on organ pathology have not been characterized, this study examined the histologic effects of an increasing percentage of normal donor hematopoiesis in a mouse model of BMT for SCA. In lethally irradiated normal mice that were reconstituted with varying ratios of T-cell-depleted marrow from normal and transgenic "sickle cell" mice, normal myeloid chimerism in excess of 25% was associated with more than 90% normal hemoglobin (Hb). However, 70% normal myeloid chimerism was required to reverse the anemia. Organ pathology, including liver infarction, was present in mice with sickle Hb (HbS) levels as low as 16.8% (19.6% normal myeloid chimerism). Histologic abnormalities increased in severity up to 80% HbS, but were less severe in mice with more than 80% HbS than in those with 40% to 80% HbS. Therefore, stable mixed chimerism resulting from nonmyeloablative BMT may reduce the morbidity from SCA, but prevention of all disease complications may require minimizing the fraction of circulating sickle red cells. (Blood. 2001;97:3960-3965)

EMX2 protein in the developing mouse brain and olfactory area.

The distribution of EMX2, the protein product of the homeobox gene Emx2, was analyzed in the developing mouse CNS by means of a polyclonal antibody we raised against it. The protein is present in the rostral brain, the olfactory area and a set of scattered cells lying between the nasal pits and the telencephalon. In the cortical neuroepithelium EMX2 is expressed all along the rostro-caudal axis in a graded distribution with a caudal-medial maximum and a rostral-lateral minimum. Anti-EMX2 immunoreactivity is also detectable in Cajal-Retzius cells as well as in apical dendrites of marginal neurons of the cortical plate. We also observe that the EMX2 and EMX1 homeoproteins display complementary expression patterns in olfactory bulbs and amygdaloid complex. Here, they demarcate different neuronal populations, involved in processing olfactory information coming from the vomero-nasal organ and from the main olfactory epithelium, respectively. EMX2 is also detectable in mesencephalic structures, such as the optic tectum and tegmentum. The graded distribution of EMX2 along antero-posterior and medial-lateral axes of the primitive cortex prefigures a role of this protein in the subdivision of the cortex in cytoarchitectonic regions and possibly functional areas, whereas its presence in Cajal-Retzius cells suggests a role in the process of cortical lamination.

Mutation spectra analysis suggests that N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-induced lesions are subject to transcription-coupled repair in Escherichia coli.

To determine the influence of some bacterial DNA repair pathways on the mutagenic and the lethal effects of N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (CCNU), pZ189 plasmids treated in vitro with 2 mM CCNU were transfected into Escherichia coli strains with different repair capacities (uvr+ada+ogt+, uvr-ada+ogt+, and uvr-ada-ogt-). Despite the differences in repair capacities, no statistically significant difference in survival and mutability was observed among the tested strains. One hundred and sixty-six CCNU-induced supF mutants were isolated and sequenced. All mutants were characterized by single base-pair substitutions, most of which (more than 96%) were GC-->AT transitions (the mutated G being almost exclusively preceded 5' by a purine). Mutation distribution was not random. Position 160 (5'-GGT-3', nontranscribed (NT) strand) was a uvr+ada+ogt(+)-specific hot-spot. Position 123 (5'-GGG-3', NT strand) was a common hot-spot but significantly more mutable in repair-proficient strains than in repair-deficient strains. Conversely, position 168 (5'-GGA-3', transcribed (T) strand) was significantly more mutable in repair-deficient strains than in repair-proficient strains. By applying a computer program for comparison of mutational spectra, we found that the uvr+ mutational spectrum was significantly different from those obtained in uvr- strains, whereas in the uvr- background, no difference was observed between mutation spectra in ada+ogt+ versus ada-ogt- strains. Our results are consistent with the hypothesis that O6-alkylguanine is responsible for most mutations observed in all strains. The results also indicate that excision repair modulates the distribution of GC-->AT transitions. The fact that mutations at G lesions on the T strand were significantly less frequent in uvr+ than in uvr- strains suggests that CCNU-induced premutational lesions are susceptible to strand-preferential repair in E. coli.

Determining mutational fingerprints at the human p53 locus with a yeast functional assay: a new tool for molecular epidemiology.

In order to isolate experimentally induced p53 mutations, a yeast expression vector harbouring a human wild-type p53 cDNA was treated in vitro with the antineoplastic drug chloroethyl-cyclohexyl-nitroso-urea (CCNU) and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. p53 mutations were identified in 32 out of 39 plasmids rescued from independent ade- transformants. Ninety-two percent of CCNU induced mutations were GC-targeted single base pair substitutions, and GC > AT transitions represented 73% of all single base pair substitutions. In 70% of the cases the mutated G was preceded 5' by a purine. The distribution of the mutations along the p53 cDNA was not random: positions 734 and 785 appeared as CCNU mutational hotspots (n=3, P<0.0003) and CCNU induced only GC > AT transitions at those positions. The features of these CCNU-induced mutations are consistent with the hypothesis that O6-alkylguanine is the major causative lesion. One third of the CCNU-induced mutants were absent from a huge collection of 4496 p53 mutations in human tumours and cell lines, thus demonstrating that CCNU has a mutational spectrum which is uniquely different from that of naturally selected mutations. This strategy allows direct comparison of observed natural mutation spectra with experimentally induced mutation spectra and opens the way to a more rigorous approach in the field of molecular epidemiology.

Mutational specificity of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea in Escherichia coli: comparison of in vivo with in vitro exposure of the supF gene.

Forward mutations induced by 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in the supF gene of Escherichia coli were recovered from bacteria deficient in nucleotide excision repair and in DNA-alkyltransferase activity. Bacteria were exposed to 0.4 mM CCNU (in vivo supF mutagenesis), increasing the overall mutation frequency 15.7-fold above the spontaneous value. A total of 73 independent supF- mutants were sequenced. The resulting mutation spectrum was compared with those obtained in bacteria and mammalian cells following the classical shuttle-vector approach (in vitro supF mutagenesis). In vivo CCNU mutagenesis in E. coli yielded a large number of deletions (20/73), in agreement with mammalian data but distinct from in vitro bacterial spectra, which are almost exclusively composed of G:C-->A:T transitions. A substantial proportion (6/18) of CCNU-induced deletions (> 3 bp) involved repeated DNA sequences, suggesting a contribution of a slippage-misalignment process in the generation of this mutation class. Substitutions occurred primarily at G:C base pairs (44/53) and were predominantly G:C-->A:T transitions (39/53). This mutational change was attributed to the mispair potential of the O6-chloroethylguanine lesion with thymine. Most G:C-->A:T transitions (34/39) were located at three 5'-GG-3' hotspot sites (positions 123, 160, and 168). The distribution of hotspot sites for G:C-->A:T substitutions differed as a function of the in vivo or in vitro chemical modification of the supF-bearing plasmids and revealed significant differences in the DNA strand distribution of this mutational event. Our data suggest that the transcriptional status of the target gene has strong influence on the probability of O6-chloroethylguanine formation, reducing its incidence in the transcribed DNA strand.

EMX1 homeoprotein is expressed in cell nuclei of the developing cerebral cortex and in the axons of the olfactory sensory neurons.

We analyzed the distribution of EMX1 during mouse development. EMX1 is a homeoprotein encoded by Emx1, a regulatory homeobox gene expressed in the developing forebrain. Its distribution essentially overlaps the expression domains of Emx1 transcripts. The EMX1 protein is present in the developing dorsa telencephalon, that is in the cerebral cortex, olfactory bulb and hippocampus. In the cerebral cortex EMX1 is present in nuclei of proliferating, differentiating and most mature neurons belonging to all cortical layers. In the olfactory bulb it is present in all proliferating cells during development, whereas postnatally it is faintly expressed in some mitral cells. Non-cerebral localizations include a transient expression in branchial pouches, in the apical ectodermal ridge of the developing limbs and in the developing kidney. Of particular interest is the presence of EMX1 in the olfactory nerve from its first appearance during embryogenesis to birth. The protein is present in axons of olfactory sensory neurons along their entire length, including their terminals in spherical regions of neuropil in the olfactory bulb called glomeruli.

Study on aneuploidy and p53 mutations in astrocytomas.

To determine whether a correlation exists between aneuploidy and p53 status in astrocytic tumors we analyzed 48 astrocytomas with different grades of malignancy for the presence of p53 mutations and aneuploidy of chromosomes 10 and 17 (Ch10, Ch17), known to be particularly involved with this type of tumor. We used polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis on exons 5-8 of the p53 gene, and fluorescence in situ hybridization (FISH) analysis on interphase nuclei using chromosome specific pericentromeric probes, respectively. Our results showed that Ch10/Ch17 aneuploidy is a common early event in astrocytomas (90% of low grade tumors are aneuploid). p53 mutations and Ch17 aneuploidy are early events, but their incidence is not dependent on tumor grade. Loss of Ch10 is the only alteration that significantly correlates with tumor progression. No significant correlation between the presence of Ch10/Ch17 aneuploidy and p53 mutations was found. However, the coexistence of p53 mutations and aneuploidy, was observed in a subset of cases. The presence of p53 mutations appeared to be a significant predictor of a poor prognosis. In conclusion, genomic instability may or may not be associated with p53 mutations in astrocytomas, thus suggesting that other cellular determinants can also be responsible for the aneuploidy observed.

Mutational fingerprint induced by the antineoplastic drug chloroethyl-cyclohexyl-nitrosourea in mammalian cells.

Using the pZ189 shuttle vector approach, we determined two chloroethyl-cyclohexyl-nitrosourea (CCNU)-induced mutation spectra (3 and 6 mM) in African green monkey kidney cells (CV1). One hundred and twenty-one independent clones (101 CCNU induced, 45 at 3 mM and 56 at 6 mM; 20 spontaneous) showing functional inactivation of the supF gene were analyzed. One hundred and five plasmids (91 CCNU induced, 41 at 3 mM and 50 at 6 mM; 14 spontaneous), showing no large deletion/rearrangements, were sequenced. Ninety mutants (81 CCNU induced and 9 spontaneous) showed at least one mutation in the supF region. The analysis of the 122 CCNU-induced mutations (56 and 66 at 3 and 6 mM, respectively) revealed that: (a) the majority of the mutations were GC-targeted base pair substitutions; (b) AT-targeted mutations were significantly more frequent in the CCNU-induced (6 mM) than in the spontaneous mutational spectrum (P < 0.0006, Fisher's exact test); (c) mutational spectra obtained at 3 and 6 mM CCNU were significantly different (P < 0.008); (d) induced mutations were nonrandomly located in both spectra and generated either a common hot spot (position 123, 5'-GGG-3') or hot spots exclusive for each CCNU concentration (3 mM: position 159, 5'-AGG-3'; 6 mM: position 109, 5'-GGG-3'); (e) the occurrence of GC-->AT transitions was significantly different as a function of CCNU concentration (P < 0.02, Fisher's exact test), the mutated G being almost exclusively preceded by a purine (5'Pu G) at 6 mM and by either Pu or Py at 3 mM; and (f) by applying Calladine's rules, we found that sequences encompassing the three CCNU hot spots shared identical helix parameters for no more than 2 bp steps 5' (or 3 bp steps 3') to the mutated G. Our results are consistent with the hypothesis that O6-alkylguanine is responsible, either directly or indirectly, for the majority of GC-targeted mutations, while O4-alkylthymine and/or N3-alkyladenine are probably responsible for AT-targeted mutations. The results suggest also that, in CV1 cells, the efficiency of the repair mechanism(s) involved in the removal of O6-alkylguanine is influenced by the DNA sequence context. All of these factors determine the CCNU mutational fingerprint. CCNU has been implicated in the induction of therapy-related leukemias.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of human T cell leukemia/lymphoma virus type I antibodies, DNA, and protein in patients with polymyositis.

OBJECTIVE: To investigate a possible association between human T cell leukemia/lymphoma virus type I (HTLV-I) and polymyositis (PM). METHODS: Sera and muscle biopsy samples from 9 Jamaican PM patients were compared with specimens from American HTLV-I-positive PM patients and normal controls. Sera were evaluated for HTLV antibodies by enzyme-linked immunosorbent assay and Western blot. The biopsy samples were analyzed for HTLV-I/II DNA by polymerase chain reaction and were also immunohistochemically stained for HTLV gp46 envelope protein. RESULTS: Seven of the 8 Jamaican PM patients from whom sera were available were HTLV-I seropositive. The muscle biopsies of all 9 Jamaican patients demonstrated severe lymphocytic infiltration, cellular degeneration, myofiber atrophy, and fibrosis. Each muscle biopsy specimen contained HTLV-I DNA. Two of 6 samples demonstrated intense staining for HTLV-I gp46 in many of the invading mononuclear cells and weak staining for HTLV-I gp46 in many of the invading mononuclear cells and weak staining in the adjacent myocytes. Two other specimens were weakly positive for gp46 in rare mononuclear cells. All control specimens were negative for the presence of HTLV-I DNA and protein. CONCLUSION: HTLV-I is associated with an inflammatory muscle disease characterized by direct invasion of the affected muscle by HTLV-I-infected mononuclear cells.

Mutation spectrum of 4-nitroquinoline 1-oxide-damaged single-stranded shuttle vector DNA transfected into monkey cells.

4-Nitroquinoline 1-oxide (4NQO) is a potent mutagen and carcinogen which induces two main guanine adducts at positions C8 and N2. We recently determined the mutation spectrum induced by the ultimate metabolite of 4NQO, acetoxy-4-aminoquinolone 1-oxide in the M13lacZ'/E. coli lacZ delta M15 alpha-complementation assay. Our data suggested that dGuo-C8-AQO induces (per se or via AP sites) G to Pyr transversions. Here we report our study on 4NQO mutagenesis in monkey cells. 4NQO lesions were induced in vitro on a single-stranded (ss) DNA shuttle vector carrying the supF tRNA gene. This vector was able to replicate both in mammalian cells and in bacteria. The mutations induced in monkey cells were screened by the white/blue beta-galactosidase activity assay in E. coli. We took advantage of the peculiar feature of ss supF DNA in which the extent of secondary structure may be a function of the temperature, with the dependence of the 4NQO-specific adduct spectrum on DNA secondary structure. We reasoned that mutational spectra derived from damage induced in the presence (20 degrees C) or absence (70 degrees C) of DNA secondary structure should be different. The result of sequencing a total of 89 induced and spontaneous mutants confirmed that the spectra are statistically different. These data suggest that the two 4NQO guanine adducts may induce different mutations.

Defective splicing induced by 4NQO in the hamster hprt gene.

The molecular analysis of mutations affecting mRNA processing may contribute to a better understanding of the splicing mechanism through the identification of genomic sequences necessary for the recognition of splice sites. In this paper we report the sequence analysis of 14 splice mutants induced by 4-nitroquinoline 1-oxide (4NQO) at the hamster hypoxanthine-guanine-phosphoribosyltransferase (hprt) locus. We show that mutations at the 3' acceptor splice site or at the first or fifth base of the 5' donor splice site are responsible for exon skipping. In addition, mutations in exon sequences also determine the skipping of one or more exons. Our data indicate that point mutations in intron regions at either side of an internal exon may induce the skipping of the same exon, supporting a model where the exon is the unit of early spliceosome assembly. Furthermore, they suggest that the splicing of hprt mRNA precursors may proceed through a clustering of exons 2, 3 and 4 which are then spliced in a concerted way.

Analysis of 4-nitroquinoline-1-oxide induced mutations at the hprt locus in mammalian cells: possible involvement of preferential DNA repair.

Mutation spectra induced by 4-nitroquinoline 1-oxide (4NQO) at the hprt locus for both normal (AA8) and 4NQO-sensitive (UV5) Chinese hamster ovary cells were determined to investigate the effect of DNA repair on the nature of induced mutations. The UV5 cell line is three times more sensitive to 4NQO than the AA8 parental cell line. In UV5 cells, the dGuo-N2-AQO adduct, which is considered to be the most toxic and mutagenic adduct in Escherichia coli, is poorly repaired. The molecular nature of 30 hprt mutants isolated from AA8 and 20 isolated from UV5 cells was determined by sequence analysis of in vitro amplified hprt cDNA. Both similarities and differences emerged. In both cell lines we found that (i) 4NQO is basically a base substitution mutagen acting almost exclusively at G residues and (ii) G transversions are prevalent over G transitions in both cell lines, independently from the ability to repair dGuo-N2-AQO. A high proportion (13/25) of splice mutations was observed in AA8 cells, statistically different (P < 0.04, Fisher's exact test) from the incidence of splice mutants in UV5 cells (4/20). In AA8 mutants, all but two of the point mutations were due to lesions localized on the non-transcribed strand, suggesting preferential repair of the transcribed strand. Compared with AA8, the proportion of mutants due to lesions present on the transcribed strand was higher in UV5 cells, as expected if a preferential repair mechanism was impaired in the sensitive cell line. Our data are consistent with the molecular defect in DNA repair recently characterized in UV5.

Sequence analysis of an immunogenic and neutralizing domain of the human T-cell lymphoma/leukemia virus type I gp46 surface membrane protein among various primate T-cell lymphoma/leukemia virus isolates including those from a patient with both HTLV-I-associated myelopathy and adult T-cell leukemia.

Human T-cell lymphoma/leukemia virus type I (HTLV-I) causes adult T-cell leukemia/lymphoma and HTLV-I-associated myelopathy. Specific regions within the outer envelope proteins of other retroviruses, e.g., human immunodeficiency virus type 1, are highly immunogenic and, because of the selective pressure of the host immune system, quite variable. Mutations in the external envelope protein gene of murine retroviruses and human immunodeficiency virus type 1 influence cellular tropism and disease pathogenesis. By contrast, no disease-specific viral mutations have been identified in HTLV-I-infected patients. However, all isolates studied thus far have originated from leukemic cell lines, peripheral blood mononuclear cells, or cerebrospinal fluid lymphocytes from patients with HTLV-I-associated myelopathy and adult T-cell leukemia/lymphoma and, therefore, may not truly reflect tissue-associated variation. The midregion of the HTLV-I gp46 external envelope glycoprotein (amino acids 190-209) induces an antibody response in 90% of infected individuals, and a hexapeptide in this region (amino acids 191-196) elicits antibodies in rabbits which inhibit syncytia formation and infection of target lymphocytes. Because of the above, we expected the neutralizing domain of the gp46 env gene of HTLV-I to possess disease or organ-associated mutations selected by the infected host's immune system. Hence, we amplified, cloned, and sequenced HTLV-I DNA directly from in vivo central nervous system, spleen, and kidney specimens, and a leukemic cell line from a patient (M. J.) with both HTLV-I-associated myelopathy and adult T-cell leukemia/lymphoma to discern the possibility of tissue- and/or disease-specific variants. In addition, we sequenced several HTLV-I isolates from different regions of the world, including Papua New Guinea, Bellona, and Liberia, and compared them to other previously published HTLV-I and related retroviral sequences. The 239-base pair sequence corresponding to amino acids 178 to 256 in gp46 displayed minor tissue-specific variation in clones derived from central nervous system tissues from patient M. J., but overall was highly conserved at both the DNA and amino acid levels. Variation was observed in this region among the other HTLV-I, simian T-cell lymphoma virus type I, and HTLV-II isolates in a pattern that was consistent with their known phylogenetic relationship. No consistent disease-related changes were observed.(ABSTRACT TRUNCATED AT 400 WORDS)