Long-term outcome in 306 males with alcoholism.

The subjects of this study were 306 male alcoholics who lived in Osaka, Japan, and who were initially diagnosed with alcoholism at a psychiatric institution between 1972 and 1983. Follow-up studies were done on three occasions: 1 March 1985 (Time 1), 1 November 1988 (Time 2) and 1 March 1992 (Time 3). We followed up 232 (75.8%) of the 306 male alcoholics. By the end of the study period 110 (35.9%) of the subjects were deceased. Regarding cross-sectional sobriety status, from Time 1 to Time 3 the complete abstinence rate changed from 16.0 to 18.6%, excessive drinking rate was from 13.1 to 9.8%, and controlled drinking rate was from 6.9 to 9.8%. The longitudinal sobriety status of 122 living patients during the 5 years before the close of this study were: rate of stable abstinence, 28.7%; unstable abstinence, 21.3%; controlled drinking, 12.3%; and relapse 37.7%. Such variables as being without public assistance at the time of the initial diagnosis of alcoholism and attending a self-help group soon after the initial treatment were associated with stable abstinence. Age (20-39 years) and receiving outpatient treatment at the time of the initial treatment also emerged as predictors of survival. However, those variables, except attending a self-help group soon after the initial treatment, might merely indicate severity of alcoholism. For improving treatment results, it may be most important to provide a treatment environment within the residential area so that alcoholics may receive treatment at an early stage of alcoholism and attend a self-help group.

Genetic divergence of the dihydrofolate reductase and dihydropteroate synthase genes in Pneumocystis carinii from 7 different host species.

To investigate the phylogenetic and therapeutic implications of the genetic divergence in the dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genes among different Pneumocystis carinii strains, these 2 genes in P. carinii obtained from 7 different host species were sequenced. Pairwise comparison of the DHPS sequences demonstrated 6%-24% and 6%-30% divergence in the nucleotide and deduced amino acid sequences, respectively. The DHFR gene was even more divergent, with differences of 15%-34% and 18%-42% in the nucleotide and deduced amino acid sequences, respectively. Phylogenetic analysis of DHFR and DHPS sequences revealed that all P. carinii strains were confined within a distinct group that was closely related to ascomycete fungi and that human-derived P. carinii was most closely related to monkey-derived P. carinii. Recognizing the substantial differences in the DHFR and DHPS genes among P. carinii from different host species has important implications for drug discovery and the development of new diagnostic methods.

Cause-specific mortality risk among male alcoholics residing in the Osaka metropolitan area.

We evaluated the mortality risk among 306 male alcoholics living in Osaka, Japan, at the time of initial diagnosis between 1972 and 1983, with regard to the cause of death, length of time from diagnosis, and participation in an alcohol abstinence self-help group. By the closing date on 1 March 1992, 110 of the 306 alcoholics had died, yielding an observed-to-expected (O/E) ratio of 4.5 [95% confidence interval (CI) = 3.7-5.4]. The alcoholics had significantly elevated mortality risks from all malignant neoplasms (O/E = 2.1, 95%CI = 1.2-3.3), esophageal cancer (O/E = 8.4, 95%CI = 1.7-24.5), diseases of the circulatory system (O/E = 4.4, 95%CI = 3.0-6.2), liver cirrhosis (O/E = 15.9, 95%CI = 10.2-23.6), diseases of the genitourinary system (O/E = 6.3, 95%CI = 1.3-18.5), and external death (O/E = 10.3, 95%CI = 6.3-15.8). The mortality risk from all causes still remained significantly high beyond the tenth year following initial diagnosis (O/E = 2.6, 95%CI = 1.0-6.2). The mortality risks from liver cirrhosis and external death (such as suicide) were highest within the first year following diagnosis, and were still high beyond the tenth year. A significantly high mortality risk from diseases of the circulatory system was observed between the first and ninth years, and the mortality risk from all malignant neoplasms was significantly elevated beyond 10 years following diagnosis. Alcoholics who did not join a self-help group soon after the initial institutional treatment had different cause-specific and time-specific mortality risks from those who did join a self-help group. These findings show the importance of long-term clinical follow-up of male alcoholics, taking into consideration the cause-specific mortality.

Amino acid deletion at codon 67 and Thr-to-Gly change at codon 69 of human immunodeficiency virus type 1 reverse transcriptase confer novel drug resistance profiles.

The potential roles of an amino acid deletion at codon 67 (Delta67) and a Thr-to-Gly change at codon 69 (T69G) in the reverse transcriptase of human immunodeficiency virus (HIV) type 1 in drug sensitivity and relative replication fitness were studied. Our results suggest that the Delta67 and T69G changes can be categorized as mutations associated with multidrug resistance. The combination of both mutations with an L74I change (Delta67+T69G/L74I) leads to a novel 3'-azido-3'-deoxythymidine resistance motif and compensates for impaired HIV replication.

Human immunodeficiency virus type 1 quasi species that rebound after discontinuation of highly active antiretroviral therapy are similar to the viral quasi species present before initiation of therapy.

In an effort to identify the sources of the viruses that emerge after discontinuation of therapy, analyses of human immunodeficiency virus (HIV) quasi species were done for 3 patients with sustained levels of HIV RNA of <50 copies/mL for 1-3 years. The sequences found in the rebounding plasma virus were closely related to those of the actively replicating form of viruses present before the initiation of combination therapy. All quasi species found in the rebounding plasma virus were also present in proviral DNA, cell-associated RNA in peripheral blood mononuclear cells (PBMC), and virion RNA derived from PBMC coculture during periods when plasma HIV RNA levels were <50 copies/mL. These findings suggest that the rapid resurgence of plasma viremia observed after discontinuation of therapy and the viruses cocultured from PBMC are derived from a relatively stable pool of the replicating form of virus rather than from activation of a previously latent pool.

Relative replication fitness of a high-level 3'-azido-3'-deoxythymidine-resistant variant of human immunodeficiency virus type 1 possessing an amino acid deletion at codon 67 and a novel substitution (Thr-->Gly) at codon 69.

The combination of an amino acid deletion at codon 67 (delta 67) and Thr-to-Gly change at codon 69 (T69G) in the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is associated with high-level resistance to multiple RT inhibitors. To determine the relative contributions of the delta 67 and T69G mutations on viral fitness, we performed a series of studies of HIV replication using recombinant variants. A high-level 3'-azido-3'-deoxythymidine (AZT)-resistant variant containing delta 67 plus T69G/K70R/L74I/K103N/T215F/K219Q in RT replicated as efficiently as wild-type virus (Wt). In contrast, the construct without delta 67 exhibited impaired replication (23% of growth of Wt). A competitive fitness study failed to reveal any differences in replication rates between the delta 67+T69G/K70R/L74I/K103N/T215F/+ ++K219Q mutant and Wt. Evaluation of proviral DNA sequences over a 3-year period in a patient harboring the multiresistant HIV revealed that the T69G mutation emerged in the context of a D67N/K70R/T215F/K219Q mutant backbone prior to appearance of the delta 67 deletion. To assess the impact of this stepwise accumulation of mutations on viral replication, a series of recombinant variants was constructed and analyzed for replication competence. The T69G mutation was found to confer 2',3'-dideoxyinosine resistance at the expense of fitness. Subsequently, the development of the delta 67 deletion led to a virus with improved replication and high-level AZT resistance.

Effects of intermittent interleukin-2 therapy on plasma and tissue human immunodeficiency virus levels and quasi-species expression.

To characterize the effects of intermittent interleukin (IL)-2 therapy on human immunodeficiency virus (HIV), 11 patients underwent detailed virological evaluation during a year of IL-2 therapy. Six patients showed a >0.5 log increase in plasma HIV during at least 1 IL-2 cycle, with 2 experiencing an increase in >50% of cycles. Three of the remaining 5 patients had a >0.5 log decrease during at least 1 IL-2 cycle, and the remaining patients exhibited <0.5 log changes. No changes in lymphoid (tonsil) levels of HIV were seen during the year. Quasi-species analysis in a separate cohort demonstrated that the virus induced by IL-2 most commonly resembled pre-IL-2 plasma quasi species. Thus, intermittent IL-2 does not result in sustained increases in either plasma or tissue levels of HIV and does not result in sustained expression of a previously silent quasi species.

An association study between alcoholism and the serotonergic receptor genes.

BACKGROUND: Linkage and association studies of alcoholism using DNA makers have been conducted without conclusive results. The comorbidity of alcoholism with affective disorder indicates that dysfunction of the serotonergic system may play an important role in developing alcoholism. METHODS: We studied the genetic association between alcoholism and alleles of the HTR1A, HTR2A, and HTR2C genes. The subjects were 91 biologically unrelated alcoholics and 90 controls. Polymorphisms of these genes were determined by polymerase chain reaction restriction fragment length polymorphisms, and the data were analyzed by chi2 tests. RESULTS: We found no significant association between alcoholism and the HTR1A, HTR2A, and HTR2C genes. CONCLUSIONS: The study results suggest that these serotonergic receptor genes may not directly contribute to the etiology of alcoholism.

Resistance to replication of human immunodeficiency virus challenge in SCID-Hu mice engrafted with peripheral blood mononuclear cells of nonprogressors is mediated by CD8(+) T cells and associated with a proliferative response to p24 antigen.

High levels of resistance to challenge with human immunodeficiency virus type 1 SF162 were observed in animals engrafted with peripheral blood mononuclear cells of four long-term nonprogressors (LTNPs). Resistance was abrogated by depletion of CD8(+) T cells in vivo and was observed only in LTNPs with proliferative responses to p24. In a subgroup of nonprogressors, CD8(+) T cells mediated restriction of challenge viruses, and this response was associated with strong proliferative responses to p24 antigen.

High-level resistance to 3'-azido-3'-deoxythimidine due to a deletion in the reverse transcriptase gene of human immunodeficiency virus type 1.

A variant of human immunodeficiency virus type 1 (HIV-1) possessing a deletion in the reverse transcriptase (RT) gene at codon 67 was identified in a patient who had failed combination antiretroviral therapy. This deletion initially emerged under the selective pressure of combination therapy with 3'-azido-3'-deoxythymidine (AZT) plus 2',3'-dideoxyinosine. It has persisted for more than 3 years in association with the accumulation of a variety of other well-described drug resistance mutations and an uncharacterized mutation at RT codon 69 (T69G). Phenotypic studies demonstrated that the codon 67 deletion by itself had little effect on AZT sensitivity. However, in the context of the T69G mutation and three other mutations known to be associated with AZT resistance (K70R, T215F, and K219Q), this deletion led to a increase in AZT resistance from 8. 5-fold to 445-fold. A further increase in resistance (up to 1, 813-fold) was observed when two mutations associated with nonnucleoside RT inhibitor resistance (K103N and L74I) were added to the deletion T69G K70R T215F K219Q construct. Hence, these results establish that a deletion at RT codon 67 may be selected for in the presence of antiretroviral therapy and may lead to high-level resistance to AZT.

Parallel evolution of drug resistance in HIV: failure of nonsynonymous/synonymous substitution rate ratio to detect selection.

Parallel or convergent evolution at the molecular level has been difficult to demonstrate especially when rigorous statistical criteria are applied. We present sequence data from the protease gene from eight patients infected with the human immunodeficiency virus (HIV-1). These patients have been on multiple drug therapies for at least 2 years. We present sequence data from two timepoints: time zero--the initiation of drug therapy--and a subsequent timepoint between 59 and 104 weeks after the initiation of drug therapy. In addition to the sequence data, we present viral load data from both initial and final timepoints. Our phylogenetic analyses indicate significant evolution of virus from initial to final time points, even in three of eight patients who show low viral loads. Of the five patients who escaped drug therapy, identical amino acid replacements were seen in all five patients at two different codon positions, an indication of parallel evolution. We also measured genetic diversity for these patients and found no correlation between genetic diversity and viral load. Finally, we calculated the nonsynonymous and synonymous substitution rates and showed that the ratio of nonsynonymous to synonymous substitution compared to the value of one may be a poor indicator of natural selection.

Cholesterol hydroperoxides in erythrocyte membranes of alcoholic patients.

Evidence for the presence of 5alpha-hydroperoxycholest-6-en-3beta-ol (cholesterol 5alpha-hydroperoxide, Ch 5alpha-OOH) and 7alpha- and 7beta-hydroperoxycholest-5-en-3beta-ols (cholesterol 7-hydroperoxides: Ch 7alpha-OOH and Ch 7beta-OOH, respectively) in human erythrocyte membrane was found. Blood samples were collected from alcoholic patients and healthy volunteers (controls), and their cholesterol hydroperoxides were analyzed by high-performance liquid chromatography postcolumn chemiluminescence and roughly identified by liquid chromatography-mass spectrometry. Ch 7alpha-OOH and Ch 7beta-OOH were present in each sample, being significantly higher in alcoholic samples than in control samples. Ch 5alpha-OOH was present in some alcoholic samples, but not in the control ones. The accumulation of cholesterol hydroperoxides suggests enhanced lipid peroxidation by active oxygen species and/or a reduced elimination system for lipid peroxide in alcoholic patients.

Abnormality of very long-chain fatty acids of erythrocyte membrane in alcoholic patients.

Profiles of very long-chain fatty acids were studied in the erythrocyte membrane of five alcoholic patients. We identified three fatty acids as cis-16-pentacosenoic acid (C25:1), cis-17-hexacosenoic acid (C26:1), and hexacosenoic acid (C26:1), and hexacosanoic acid (C26:0) by gas chromatography-mass spectrometry. The ratios of C26:1/C22:0, C26:0/C22:0, C24:1/C22:0, and C24:0/C22:0 were increased. These findings suggest that active oxygen species or free radicals generated by chronic alcohol consumption in alcohol patients interrupt the peroxisomal beta-oxidation of fatty acids, because very long-chain fatty acids are mainly metabolized by the peroxisomal beta-oxidation system. This is the first study showing accumulation of very long-chain fatty acids in the erythrocyte membrane of alcoholic patients.

Drug resistance during indinavir therapy is caused by mutations in the protease gene and in its Gag substrate cleavage sites.

Two different responses to the therapy were observed in a group of patients receiving the protease inhibitor indinavir. In one, suppression of virus replication occurred and has persisted for 90 weeks (bDNA, < 500 human immunodeficiency virus type 1 [HIV-1] RNA copies/ml). In the second group, a rebound in virus levels in plasma followed the initial sharp decline observed at the start of therapy. This was associated with the emergence of drug-resistant variants. Sequence analysis of the protease gene during the course of therapy revealed that in this second group there was a sequential acquisition of protease mutations at amino acids 46, 82, 54, 71, 89, and 90. In the six patients in this group, there was also an identical mutation in the gag p7/p1 gag protease cleavage site. In three of the patients, this change was seen as early as 6 to 10 weeks after the start of therapy. In one patient, a second mutation occurred at the gag p1/p6 cleavage site, but it appeared 18 weeks after the time of appearance of the p7/p1 mutation. Recombinant HIV-1 variants containing two or three mutations in the protease gene were constructed either with mutations at the p7/p1 cleavage site or with wild-type (WT) gag sequences. When recombinant HIV-1-containing protease mutations at 46 and 82 was grown in MT2 cells, there was a 68% reduction in its rate of replication compared to the WT virus. Introduction of an additional mutation at the gag p7/p1 protease cleavage site compensated for the partially defective protease gene. Similarly, rates of replication of viruses with mutations M46L/I, I54V, and V82A in protease were enhanced both in the presence and in the absence of Indinavir when combined with mutations in the gag p7/p1 and the gag p1/p6 cleavage sites. Optimal rates of virus replication require protease cleavage of precursor polyproteins. A mutation in the cleavage site that enhanced the availability of a protein that was rate limiting for virus maturation would confer on that virus a significant growth advantage and may explain the uniform emergence of viruses with alterations at the p7/p1 cleavage site. This is the first report of the emergence of mutations in the gag p7/p1 protease cleavage sites in patients receiving protease therapy and identifies this change as an important determinant of HIV-1 resistance to protease inhibitors in patient populations.

Association between early-onset alcoholism and the dopamine D2 receptor gene.

We examined the allelic association between the dopamine D2 receptor (DRD2) gene and alcoholism in 100 biologically unrelated Japanese alcoholics and 93 unrelated controls. Genomic DNA was prepared from peripheral white blood cells using the phenol-chloroform method. A 310-bp region surrounding the TaqA site at the DRD2 locus was amplified by polymerase chain reaction (PCR), and the PCR product was incubated with TaqI. The A1 allele remained intact while the A2 allele was cut. The frequency of the A1/A1 genotype and the frequency of the A1 allele were higher in early-onset alcoholics than in controls, P < 0.05 and P < 0.01, respectively. Moreover, the frequency of the A1/A1 genotype and the frequency of the A1 allele were higher in early-onset alcoholics with family histories of alcohol dependence than in controls, P < 0.01 and P < 0.01, respectively. The results indicate that the DRD2 gene is associated with susceptibility to early-onset alcoholism, and that each additional A1 allele shifts onset of alcoholism to an earlier age.

Emergence of protease inhibitor resistance mutations in human immunodeficiency virus type 1 isolates from patients and rapid screening procedure for their detection.

Patient human immunodeficiency virus type 1 (HIV-1) isolates that are resistant to protease inhibitors may contain amino acid substitutions L10I/V, M46L/I, G-48V, L63P, V82A/F/T, I84V, and L90M in the protease gene. Substitutions at positions 82 and/or 90 occur in variants that display high levels of resistance to certain protease inhibitors. Nucleotide substitutions at these two sites also lead to the loss of two HindII restriction enzyme digestion sites, and these changes make possible a rapid procedure for the detection of drug-resistant variants in patients on protease inhibitor therapy. This procedure was used to detect the emergence of mutated viruses at various times after the initiation of therapy with the HIV-1 protease inhibitor indinavir. The method includes viral RNA isolation from plasma and reverse transcription PCR amplification of the protease gene with fluorescence-tagged primers. The PCR product is digested with HindII, the cleavage products are separated on a urea-acrylamide gel in a DNA sequencer, and the extent of cleavage is automatically analyzed with commercially available software. In viruses from 34 blood samples from four patients, mutations leading to an amino acid change at residue 82 appeared as early as 6 weeks after the start of therapy and persisted throughout the course of the study period (48 weeks). Mutations leading to double substitutions at residues 82 and 90 were seen at a lower frequency and appeared later than the change at position 82. The changes detected by restriction enzyme cleavage were confirmed by DNA sequencing of the cloned protease genes by reverse transcription PCR amplification of viral RNA from isolates in plasma. In addition to the changes at positions 82 and 90, we have identified M46L/I, G48V, and I54V substitutions in isolates derived from indinavir-treated patients. HindII analysis of uncloned, PCR-amplified DNA offers a rapid screening procedure for the detection of virus isolates containing mutations at amino acid residues 82 and 90 in the HIV-1 protease gene. By using other restriction enzymes, the same method can be used to detect additional protease drug-resistant variants and is generally applicable for the detection of mutations.

Effect of the cytochrome P-450IIE1 genotype on ethanol elimination rate in alcoholics and control subjects.

We studied an influence of genetic polymorphisms in the cytochrome P-450IIE1 (CYP2E1) gene on ethanol elimination rate in alcoholic patients and healthy subjects. The CYP2E1 genotype was determined by polymerase chain reaction-restriction fragment length polymorphism method for 124 alcoholics and 54 healthy subjects. There was no significant difference in the gene frequency of CYP2E1 between alcoholics and healthy control subjects. Blood ethanol concentrations in the 65 alcoholics on admission ranged from 0.32 to 4.22 mg/ml. In the patients with the c1/c2 genotype, the elimination rate was significantly correlated with blood ethanol concentration. In each of the three genotypes of CYP2E1, the patients were divided into three groups based on ethanol concentrations. The average of the ethanol elimination rate in the patients with c1/c2 having blood ethanol levels of > or = 2.5 mg/ml was significantly higher than the rates in the two other groups of c1/c2. When blood ethanol levels were > or = 2.5 mg/ml, the elimination rate in the patients with c1/c2 was significantly higher than that in those with c1/c1. Regardless of the CYP2E1 genotype, the elimination rate in the alcoholics was higher than that in the control subjects when blood ethanol levels were < 1.0 mg/ml. These results suggest the possibility that the c2 allele of CYP2E1 Influences the rate of ethanol elimination at high ethanol levels. The rate of ethanol elimination was independent of liver disorder judged by serum total bilirubin values.

Identification of cholesta-3,5-dien-7-one by gas chromatography-mass spectrometry in the erythrocyte membrane of alcoholic patients.

Lipids and oxidized lipids were analyzed by gas chromatography-mass spectrometry in the erythrocyte membranes of alcoholic and control subjects. Cholesta-3,5-dien-7-one and cholesta-trienes were detected in alcoholic samples examined, but not in significant amounts in controls. Levels of polyunsaturated fatty acids (arachidonic acid, 20:4; docosahexaenoic acid, 22:6; and docosatetraenoic acid, 22:4) in alcoholic samples declined significantly, whereas cholesta-3,5-dien-7-one levels increased. A high level of total bilirubin was observed in most patients. A possible mechanism of the accumulation of cholesta-3,5-dien-7-one in the erythrocyte membrane of alcoholics is discussed.