Ethanol Increases GABAA Responses in Cells Stably Transfected with Receptor Subunits

Ethanol enhancement of GABA_A receptor function has been found in some, but not all, studies. These results suggest the existence of ethanol-sensitive and -resistant receptors that may differ in subunit composition, although methodological differences (e.g., ³⁸Cl^- flux versus membrane currents) could also contribute to the different results. To examine these possibilities, we used mouse L(tk^-) cells stably transfected with α₁+β or α+β₁+γ_2L GABA_A receptor subunit DNAs and compared ³⁸Cl flux with whole-cell, patch-clamp measurements of GABA_A receptor function. Both techniques detected a similar modulation of the GABA receptor by ethanol, flunitrazepam, and pentobarbital. The potentiating action of ethanol required the -γ-subunit and was maximal at a concentration of 10 m m . Similar ethanol potentiation was obtained with brief (20 msec) or long (2 sec) applications of GABA. Analysis of data obtained from individual cells expressing α₁β_1-γ_2L subunits showed considerable variability in sensitivity to ethanol, particularly with concentrations of 30 and 100 m m . Ethanol potentiated GABA action if the cells were grown on coverslips coated with polylysine, but had no effect on GABA_A receptors of cells grown on uncoated coverslips. Thus, ethanol action was influenced by the growth matrix. Taken together, these data indicate that a y-subunit is necessary, but not sufficient, for ethanol sensitivity in this cell system. We suggest that posttranslational processing, particularly receptor phosphorylation, may also be important and that stably transfected cells will be useful in elucidating these events.     

Human GABA, Receptor αl and α3 Subunits Genes and Alcoholism

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain. GABA effects are largely mediated by binding to the postsynaptic GABA_A receptor, causing the opening of an integral chloride-ion channel. The GABA_A antagonists picrotoxin and bicuculline reduce some ethanol-induced behaviors, such as motor impairment, sedation, and hypnosis. The role of this receptor in alcoholism is further supported by effective alleviation of alcohol withdrawal symptoms by GABA_A agonists. To determine the role of the GABA, receptor (GABR) genes in the development of alcoholism, we have used α₁ and α ₃ simple sequence repeat polymorphisms in a sample of unrelated alcoholics, alcoholic probands with both parents, and psychiatrically normal controls. For the GABRα₁ gene, the differences between allele frequencies, when all alleles were compared together, were not significant between total alcoholics, subtypes of alcoholics, and normal controls. However, for GABRα₃, the differences between total alcoholics and normal controls were significant when all alleles were compared together. The differences between subtypes of alcoholics and normal controls were not significant. The results of haplotype relative risk analysis for both genes, GABRα₁ and GABRα₃, were also negative. It is possible that the sample size in the haplotype relative risk is too small to have power to detect the differences in transmitted versus nontransmitted alleles. There is a need for a replication study in a large family sample, that will allow haplotype relative risk or affected sib-pair analysis.     

Effects of Ethanol on Recombinant Glycine Receptors Expressed in Mammalian Cell Lines

We examined the effects of acute ethanol exposure on recombinant human glycine receptors transiently transfected into HEK 293 cells and stably transfected into Ltk⁻ fibroblast-like cells. In our study of the effects of ethanol, we used the whole-cell patch-clamp configuration. Relatively low concentrations of ethanol (25 mM and 50 mM) did not affect glycine-gated currents in any of the cell lines studied. Higher concentrations of ethanol (100 mM and 200 mM) significantly potentiated glycine responses only in stably transfected Ltk⁻ cells expressing α₁ and α₂ subunits and in HEK 293 cells transiently expressing α₂ subunits. Cells stably expressing α₁ versus α₂ glycine receptors were modulated equally by ethanol. Both glycine α₁ and glycine α₁β receptors transiently expressed in HEK 293 cells were insensitive to all concentrations of ethanol tested; however, there was a trend toward potentiation at 100 and 200 mM ethanol concentrations. A population of cells (41–87%) that was sensitive to the potentiating effects of 100 and 200 mM ethanol (defined as more than 10% potentiation) was identified in both cell lines tested. In these sensitive cells, ethanol (100 and 200 mM) produced significant potentiation, independent of the cell line and the glycine receptor sub-unit tested. Together with published results from studies with Xenopus oocytes, these data indicate that the sensitivity of recombinant glycine receptors to ethanol depends upon the expression system.     

Regional Differences in the Effects of Chronic Ethanol Administration on [3H]Zolpidem Binding in Rat Brain

A strong association has been observed between [³H]zolpidem binding and the presence of γ-aminobutyric acid (GABA_A) receptor mRNA for α₁-, β₂-, and γ₂-subunits in specific brain regions. This correlates with observed sensitivity of individual neurons to zolpidem and ethanol in these same regions. Previous studies using homogenate binding approaches showed small alterations in [³H] zolpidem binding levels after chronic ethanol exposure. This study was undertaken to ascertain if there is regional specificity of the effects of chronic ethanol administration on [³H] zolpidem binding levels. Chronic ethanol administration induced small, but significant alterations in [³H] zolpidem (5 nM) binding in the Inferior colliculus, substantia nigra, and the medial septum. [³H]Zolpidem binding was increased in the inferior colliculus and substantia nigra, and decreased in the medial septum. No significant differences in [³H] zolpidem binding were noted in any other brain area analyzed, including the cortex and cerebellum. These findings show that chronic ethanol administration has small effects on [³H] zolpidem binding, although they occur in a site-specific and bidirectional manner. Moreover, there is no correlation between changes in [³H] zolpidem binding and alterations In GABA_A receptor subunit expression.     

Altering the Relative Abundance of GABAA Receptor Subunits Changes GABA- and Ethanol-Responses in Xenopus Oocytes

Background:  Variations in GABRA2 and GABRG3 , genes encoding the α2 and γ3 subunits of the pentameric GABA_A receptor, are associated with the risk of developing alcoholism in adults, conduct disorder at younger ages, and with differences in electroencephalographic power in the β frequency range. The SNPs associated with alcoholism did not alter the coding of these genes, and extensive DNA sequencing of GABRA2 did not find coding changes in the high-risk haplotypes. Therefore, we hypothesize that the associations arise from differences in gene expression.
Methods:  Here we report studies in Xenopus oocytes to examine the functional effects of altering the relative abundance of these 2 receptor subunits on GABA current and response to ethanol, as a model of potential effects of regulatory differences.
Results:  When human α2β2γ3 subunits are co-expressed, increasing the amount of the α2 subunit mRNA increased GABA current; in contrast, increasing the amount of the γ3 subunit decreased GABA currents. Acute ethanol treatment of oocytes injected with a 1:1:1 or 2:2:1 ratio of α2:β2:γ3 subunit mRNAs resulted in significant potentiation of GABA currents, whereas ethanol inhibited GABA currents in cells injected with a 6:2:1 ratio. Overnight treatment with ethanol significantly reduced GABA currents in a manner dependent on the ratio of subunits.
Conclusions:  These studies demonstrate that changes in relative expression of GABA_A receptor subunits alter the response of the resulting channels to GABA and to ethanol.     

Direct Antagonism of Ethanol's Effects On GABAA Receptors by Increased Atmospheric Pressure

Previous studies have shown that exposure to 12 times normal atmospheric pressure of helium-oxygen gas (heliox) directly antagonizes a range of ethanol's acute and chronic behavioral effects. The present study extends the investigation to the biochemical level by investigating the effects of pressure on ethanol-induced potentiation of GABA_A receptor function in mouse membrane vesicles (micro-sacs). Exposure to 12 atmospheric pressure heliox significantly antagonized ethanol (25 to 100 mM) potentiation of GABA-activated ³⁶Cl^- uptake, but did not significantly alter baseline GABA_A receptor function measured by the response of the system to GABA (10 to 100 μM), bicuculline (3 and 100 μM), or picrotoxin (100 μM). These findings add essential support for the hypothesis that hyperbaric exposure is a direct ethanol antagonist that can be used as a tool to help identify ethanol's initial cellular and molecular sites of action that cause its behavioral effects. Taken in context with previous behavioral studies, the present results also provide important new evidence for a cause-effect relationship between ethanol potentiation of GABA_A receptor function and ethanol's anesthetic and behavioral effects.     

Efficient RT-PCR on platelet mRNA after long-term storage

We have developed a procedure permitting RT-PCR from mRNA even after a long-term storage (1 year) of platelet samples in ethanol (EtOH-platelets) at −80°C. To validate our method, we have analysed the human platelet alloantigen system (HPA-1) which is coded by β₃ mRNA. We have also demonstrated the efficiency of amplification of part of the coding region for (i) α_IIb subunit mRNA, (ii) α_v subunit mRNA, and (iii) the seven transmembrane domain thrombin receptor mRNA.     

THE ASSAY OF 1α, 25-DIHYDROXYVITAMIN D3: PHYSIOLOGIC AND PATHOLOGIC MODULATION OF CIRCULATING HORMONE LEVELS

A sensitive radioreceptor assay for 1α,25-dihydroxyvitamin D₃ (1α,25-(OH)₂D₃) is utilized to quantitate the circulating concentration of this sterol in experimental animals and humans. When weanling rats are grown for 2 weeks on low calcium or low phosphate diets, limited availability of either ion elicits a five-fold increase in the plasma level of 1α,25-(OH)₂D₃. The enhancement of 1α,25-(OH)₂D₃ in calcium deficiency is dependent upon the presence of the parathyroid and/or thyroid glands, which is consistent with parathyroid hormone (PTH) mediation of this effect. In contrast, the response to phosphate deficiency is independent of these glands and may result from a direct action of low phosphate on the renal synthesis of 1α,25-(OH)₂D₃. Studies in humans indicate that the normal level of 1α,25-(OH)₂D is 2.1-4.5 ng/100 ml plasma. Patients with chronic renal failure have markedly lower circulating 1α,25-(OH)₂D and this kidney hormone is undectectable in anephirc subjects, but returns to normal within 1 day after successful renal transplantation. Hypoparathyroidism and pseudohypoparatghyroidism are associated with reduced plasma 1α,25-(OH)₂D while patients with primary hyperparathyroidism have significantly elevated sterol hormone levels. Thus, from measurements in rats and humans, it appears that circulating 1α,25-(OH)₂D₃ is regulated by PTH and/or phosphate and that abnormal plasma 1α,25-(OH)₂D₃ is a part of the pathophysiology of renal osteodystrophy and parathyroid disorders.     

Attenuation of the Stimulant Response to Ethanol is Associated with Enhanced Ataxia for a GABAA, but not a GABAB, Receptor Agonist

Background:  The γ-aminobutyric acid (GABA) system is implicated in the neurobiological actions of ethanol, and pharmacological agents that increase the activity of this system have been proposed as potential treatments for alcohol use disorders. As ethanol has its own GABA mimetic properties, it is critical to determine the mechanism by which GABAergic drugs may reduce the response to ethanol (i.e., via an inhibition or an accentuation of the neurobiological effects of ethanol).
Methods:  In this study, we examined the ability of 3 different types of GABAergic compounds, the GABA reuptake inhibitor NO-711, the GABA_A receptor agonist muscimol, and the GABA_B receptor agonist baclofen, to attenuate the locomotor stimulant response to ethanol in FAST mice, which were selectively bred for extreme sensitivity to ethanol-induced locomotor stimulation. To determine whether these compounds produced a specific reduction in stimulation, their effects on ethanol-induced motor incoordination were also examined.
Results:  NO-711, muscimol, and baclofen were all found to potently attenuate the locomotor stimulant response to ethanol in FAST mice. However, both NO-711 and muscimol markedly increased ethanol-induced ataxia, whereas baclofen did not accentuate this response.
Conclusions:  These results suggest that pharmacological agents that increase extracellular concentrations of GABA and GABA_A receptor activity may attenuate the stimulant effects of ethanol by accentuating its intoxicating and sedative properties. However, selective activation of the GABA_B receptor appears to produce a specific attenuation of ethanol-induced stimulation, suggesting that GABA_B receptor agonists may hold greater promise as potential pharmacotherapies for alcohol use disorders.     

The Binding of Vitamin B12 by Serum Proteins in Normal and B12-Deficient Subjects

Endogenous B₁₂ in normal serum has been shown to be associated with α globulins (Pitney, Beard and Van Loon, 1954; Heinrich and Erdmann-Oehlecker, 1956; Mendelsohn, Watkin, Horbett and Fahey, 1958). When B₁₂ has been added to normal serum, however, most or all of the added B₁₂ has been associated with the β- and α₂-globulins (Miller, 1958). In the present investigation with radioactive ₅₇cobalt cyanocobalamin (B₁₂*) added to serum, it was possible to define at least two B₁₂-binding proteins (BP), a β-globulin, and an α₁ globulin. B₁₂* added to the serum of normal subjects migrated mainly with the β-globulins on electrophoresis, but when added to the serum of B₁₂-deficient subjects, a small fraction of the B₁₂* was also associated with the α₂-globulins.     

Combination low-dose lymphoblastoid interferon and thymosin α1 therapy in the treatment of chronic hepatitis B

SUMMARY. This open label study was initiated to assess the safety and efficacy of lymphoblastoid interferon-α (IFN-α) and thymosin α₁ (Tα₁) in the treatment of 11 patients with chronic hepatitis B, who had failed to respond to standard IFN-α2b therapy, and in four interferon naive patients. These fifteen hepatitis B surface antigen (HBsAg) positive and serum hepatitis B virus (HBV) DNA positive patients were given Tα₁ (1 mg) subcutaneously (sc) on 4 consecutive days. Low-dose lymphoblastoid IFN-α (3 MU) was administered intramuscularly (IM) on the fourth day. Beginning with the second and for the subsequent 25 weeks, patients self-administered Tα₁ twice weekly in the morning followed, 12 h later, by 3 million units (MU) lymphoblastoid IFN-α. Patients were followed-up for 12 months. Nine (60%) of the 15 patients, including six (55%) of the 11 patients previously treated with IFN-α2b, responded by losing serum HBV DNA and normalizing alanine aminotransferase (ALT) values. Six of the nine responders seroconverted to HBsAg negativity. Significant improvements in the Knodell histological activity index were observed in the responders and no significant adverse effects were observed. Combination low-dose lymphoblastoid IFN-α and Tα₁ treatment may provide a safe and potentially effective therapeutic approach in chronic hepatitis B. These results require confirmation in future randomized controlled studies.     

The Neurosteroid, 3α-Hydroxy-5α-pregnan-20-one, Protects against Bicuculline-Induced Seizures during Ethanol Withdrawal in Rats

Prolonged alcohol consumption leads to the development of tolerance to and dependence on ethanol, resulting in a decreased response to the sedative/hypnotic effects of ethanol, and by negative symptomatology following abrupt termination of use. One symptom associated with ethanol withdrawal in humans, as well as laboratory animals, is enhanced susceptibility to seizures. This study investigated the effects of the neurosteroid, 3α-hydroxy-5α-pregnan-20-one (3α-5α-THP), on alterations in seizure sensitivity associated with ethanol withdrawal. 3α-5α-THP is a potent anxiolytic and anticonvulsant agent that acts via selective interactions with GABA_A receptors. Extensive evidence suggests that some aspects of ethanol dependence and withdrawal are mediated by alterations in GABA_A receptor function. Withdrawal from chronic ethanol exposure elicited dramatic increases in seizure susceptibility in male and female rats. Administration of 3α-5α-THP just before seizure threshold determinations blocked the increased seizure susceptibility induced by ethanol withdrawal. Ethanol-withdrawn animals were protected by 3α-5α-THP at a dose that had no effect on control animal seizure thresholds. Moreover, male and female rats displayed differential responses to the seizure-threshold lowering effects of ethanol withdrawal, as well as the protection by 3α-5α-THP pretreatment. These findings suggest that there are gender differences associated both with ethanol withdrawal as well as the protection by 3α-5α-THP in ethanol-dependent rats.     

Electrophysiological Interactions of Ethanol with GABAergic Mechanisms in the Rat Cerebellum in Vivo

Biochemical studies indicate that ethanol (EtOH) will facilitate the activation of the GABA_A/CI^– channel, and behavioral studies demonstrate that EtOH-induced sedative and incoordinating effects can be potentiated by GABA mimetics and blocked by GABA antagonists. It has been difficult, however, to demonstrate an EtOH-induced potentiation of the depressant electrophysiological effects of locally applied GABA in mammalian brain in vivo. Similarly, in this study, local EtOH applications only infrequently caused potentiations of the depressant effects of microiontophoretically applied GABA on cerebellar Purkinje neurons, and this interaction was modest when present. The predominant interaction of locally applied EtOH was an antagonism of GABA-induced depressions of neuronal activity. However, the GABA_A receptor antagonist bicuculline reversibly and apparently competitively blocked the depressant effects of locally applied EtOH on single cerebellar Purkinje neurons. Our data suggest that EtOH potentiation of GABA responses alone is insufficient to account for EtOH-induced depressions of cerebellar Purkinje neurons. However, these data clearly imply that activation of a GABA_A receptor is required for the expression of EtOH-induced depressions of neuronal activity in this brain area. It is less clear how lower, nondepressant doses of EtOH interact with GABA mechanisms. We hypothesize that either the GABA_A receptor mechanism must be sensitized to the potentiative effects of EtOH through the influences of neuromodulatory and/or hormonal regulation, or that EtOH interacts directly with these regulatory processes.     

Effects of Chronic Ethanol Exposure on GABA Receptors and GABAB Receptor Modulation of 3H-GABA Release in the Hippocampus

Chronic ethanol treatment (CET), sufficient for decreasing long-term potentiation (LTP) in rats, also enhances ³H-GABA release from hippocampal slices in these same animals. The mechanism for an increase in GABA release may involve changes in presynaptic receptors. Therefore, we characterized presynaptic autoreceptor modulation of ³H-GABA release in hippocampal slices from control and CET rats. The effects of a GABA_B receptor agonist (baclofen) and antagonist [2-hydroxy (OH)-saclofen] were tested for their ability to modulate electrically stimulated ³H-GABA release from superfused hippocampal slices. Baclofen decreased stimulated release in a dose-dependent manner and 2-OH-saclofen increased release consistent with the existence of presynaptic GABA_B autoreceptors in hippocampus. The GABA_A antagonist bicuculline did not significantly modulate basal or stimulated release. When the effects of baclofen and 2-OH-saclofen were measured in animals 48 hr after withdrawal from CET, presynaptic modulation of release by baclofen and 2-OH-saclofen was decreased. In addition, we examined the density of ³H-baclofen and ³H-bicuculline binding in the hippocampal formation using quantitative autoradiographic techniques. We found that the density of ³H-baclofen binding sites was not affected by CET, whereas the density of ³H-bicuculline binding sites was increased by 28% in ethanol-treated rats. These data may explain how CET increases presynaptic regulation of GABA release from hippocampus that may contribute to the decrease in LTP seen in rats after CET.     

Alcohol Actions on GABAA Receptors: From Protein Structure to Mouse Behavior

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were R. Adron Harris and Susumu Ueno. The presentations were (1) Protein kinase Cɛ-regulated sensitivity of γ-aminobutyric acid type A (GABA_A) receptors to allosteric agonists, by Robert O. Messing, A. M. Sanchez-Perez, C. W. Hodge, T. McMahon, D. Wang, K. K. Mehmert, S. P. Kelley, A. Haywood, and M. F. Olive; (2) Genetic and functional analysis of a GABA_A receptor γ2 subunit variant: A candidate for quantitative trait loci involved in alcohol sensitivity and withdrawal, by Kari J. Buck and Heather M. Hood; (3) Tryptophan-scanning mutagenesis in GABA_A receptor subunits: Channel gating and alcohol actions, by Susumu Ueno; and (4) Can a single binding site account for actions of alcohols on GABA_A and glycine receptors? by R. Adron Harris, Yuri Blednov, Geoffrey Findlay, and Maria Paola Mascia.     

BK channel subunit composition modulates molecular tolerance to ethanol

Background:  The large conductance calcium-activated potassium channel (also called BK channel or Slo channels) is a well-studied target of alcohol action, and plays an important role in behavioral tolerance.
Methods:  Using patch clamp electrophysiology, we examined human BK channels expressed in HEK293 cells to test whether tolerance to ethanol occurs in excised patches and whether it is influenced by subunit composition. Three combinations were examined: hSlo, hSlo + β₁, and hSlo + β₄.
Results:  The 2 components of BK alcohol adaptation (Component 1: rapid tolerance to acute potentiation, and Component 2: a more slowly developing decrease in current density) were observed, and varied according to subunit combination. Using a 2-exposure protocol, Component 1 tolerance was evident in 2 of the 3 combinations, because it was more pronounced for hSlo and hSlo + β₄.
Conclusions:  Thus, rapid tolerance in human BK occurs in cell-free membrane patches, independent of cytosolic second messengers, nucleotides or changes in free calcium. Alcohol pretreatment for 24 hours altered subsequent short-term plasticity of hSlo + β₄ channels, suggesting a relationship between classes of tolerance. Finally, Component 2 reduction in current density showed a striking dependency on channel composition. Twenty-four hour exposure to 25 mM ethanol resulted in a down-regulation of BK current in hSlo and hSlo + β₄ channels, but not in hSlo + β₁ channels. The fact that hSlo + β₁ channels show less sensitivity to acute challenge, in conjunction with less Component 1 and Component 2 tolerance, suggests subunit composition is an important factor for these elements of alcohol response.     

The Role of α1 Protease Inhibitor (α1 Antitrypsin) in the Regulation of Immunologic and Inflammatory Reactions

Summary: Twenty-six different alleles have been identified for α₁ protease inhibitor (α₁ antitrypsin), each designated by a letter of the alphabet. In any individual two alleles co-dominantly determine the characteristics of α₁ protease inhibitor (Pi), including mobility on electrophoresis, serum concentration and acute phase response. Recent evidence has linked some mildly deficient phenotypes of Pi with a variety of chronic immunologic and inflammatory disorders, such as rheumatoid arthritis, juvenile chronic arthritis, systemic lupus erythematosus, ankylosing spondylitis, uveitis, asthma and fibrosing alveolitis, in addition to the well recognised association of severe deficiency with emphysema and chronic liver disease. This disease susceptibility in phenotypes associated with reduced serum levels may be due to alteration in lymphocyte responses, complement activation and leukocyte migration. Pi can also influence the autolytic effects of leukocytic enzymes on tissues and may inhibit some aspects of coagulation and fibrinolysis. Therefore patients with deficient Pi phenotypes are likely to have exaggerated immunologic and inflammatory responses.     

Compartmentalization regulates the interaction between the platelet integrin αIIbβ3 and ICln

The volume-regulating protein, ICln, interacts with the conserved KxGFFKR α-integrin signature motif. ICln is an abundant protein (4455 ± 650 molecules/platelet) found exclusively in the soluble cytosolic fraction of unactivated platelets. In contrast, its binding partner, the platelet integrin α_IIbβ₃, is present in detergent-insoluble fractions associated with membrane and cytoskeleton subcellular localizations. This study investigated factors that regulate the interaction of ICln with α_IIbβ₃ during platelet activation. His-tagged recombinant ICln bound equally to purified α_IIbβ₃ and to integrin from resting or activated platelets. Binding was not affected by direct integrin activation with Mn⁺⁺ or by inhibitors of integrin occupancy (abciximab, RGD). However, the capacity for interaction between integrin and recombinant ICln was slowly downregulated following prolonged platelet activation for >300 s. In parallel, ICln redistributed to membrane and cytoskeletal platelet subcellular fractions. The time-course of this redistribution preceded the downregulation of integrin binding capacity and suggests that only a short window of opportunity exists for ICln interaction with α_IIbβ₃ to occur. Thus, although ICln has the inherent capacity to bind to α_IIbβ₃ regardless of its activation state, it can only do so following platelet activation. Activation-dependent subcellular redistribution of ICln represents a novel, temporally-regulated mechanism for control of integrin function in platelets.     

Thymosin alpha1 treatment of chronic hepatitis B: results of a phase III multicentre, randomized, double-blind and placebo-controlled study

Previous clinical trials have suggested that thymosin α₁ (Tα₁), an immunomodulatory peptide, may be effective in the treatment of chronic hepatitis B (CHB). The aim of this study was to determine the efficacy of Tα₁ in a multicentre, placebo-controlled and double-blind study of 97 patients with serum hepatitis B virus (HBV) DNA- and hepatitis B e antigen (HBeAg)-positive CHB. Patients who had been hepatitis B surface antigen (HBsAg) positive for at least 12 months entered a 3-month screening period prior to randomization. Forty-nine patients received Tα₁ (1.6 mg) and 48 patients received placebo, twice weekly for 6 months, and were followed-up for an additional 6 months. At inclusion, both groups were comparable for age, gender, histological grading, and aminotransferase and HBV DNA levels. A complete response to treatment, defined as a sustained serum HBV DNA-negative status (two negative results at least 3 months apart) during the 12-month study, with negative HBV DNA and HBeAg values at month 12, was seen in seven (14%) patients given Tα₁ and in two (4%) patients treated with placebo ( P = 0.084). Five (10%) patients given Tα₁ and four (8%) patients given placebo exhibited a delayed response (defined as sustained serum HBV DNA negativity achieved after the 12-month study period with negative HBV DNA and HBeAg values at the last assessment). A total of 12 (25%) patients given Tα₁ and six (13%) patients given placebo showed a sustained loss of HBV DNA with a negative HBeAg value during or following the 12-month study period ( P < 0.11). These results do not confirm observations of treatment efficacy reported in other clinical studies.