CT angiographic features of symptom-producing plaque in moderate-grade carotid artery stenosis.

BACKGROUND AND PURPOSE: Emerging evidence indicates that plaque imaging can improve stroke risk stratification in patients with carotid artery atherosclerosis. We studied the association between soft and hard (calcified) plaque thickness measurements on CTA and symptomatic disease status (ipsilateral stroke or TIA) in patients with moderate-grade carotid artery stenosis. MATERIALS AND METHODS: We measured soft-plaque and hard-plaque thickness on CTA axial source images in each carotid artery plaque in subjects with NASCET 50%-69% ICA stenosis. We used logistic regression and receiver operating characteristic analyses to assess the strength of the association between thickness measurements and prior stroke or TIA. RESULTS: Twenty of 72 vessels studied (27.7%) had ischemic symptoms ipsilateral to the side of moderate-grade carotid stenosis. Each 1-mm increase in soft plaque resulted in a 3.7 times greater odds of a prior ipsilateral ischemic event (95% CI, 1.9-7.2). Conversely, for each 1-mm increase in hard plaque, the odds of being symptomatic decreased by approximately 80% (OR, 0.22; 95% CI, 0.10%-0.48%). Receiver operating characteristic analysis showed an area under the curve of 0.88 by using soft-plaque thickness measurements to discriminate between asymptomatic and symptomatic plaques. Sensitivity and specificity were optimized by using a maximum soft-plaque thickness of 2.2 mm, which provided a sensitivity of 85% and a specificity of 83%. CONCLUSIONS: Simple CTA plaque-thickness measurements might differentiate symptomatic and asymptomatic moderate-grade carotid artery plaque. With further prospective validation, CTA plaque measures could function as an easily implementable tool for risk stratification in carotid artery disease.

[Results and participation factors to the European Randomized study of Screening for Prostate Cancer (ERSPC) with Prostate Specific Antigen: French departments of Tarn and Hérault]. / Etat des lieux et facteurs de participation à l'étude européenne ERSPC de dépistage randomisé du cancer de la prostate par dosage sérique de l'antigène prostatique spécifique: départements français du Tarn et de l'Hérault.

INTRODUCTION: Mass screening modalities remained controversial and made necessary large studies. The European Randomized study of Screening for Prostate cancer (ERSPC) was initiated in 1994. Eight countries including France are participating. METHODS: ERSPC is a multicentric randomised study and started with the aim to determine whether a 20% reduction in prostate cancer mortality can be achieved with PSA-based screening. Men aged 50-74 and living in the Tarn or Hérault were included. After randomization and exclusion of men who died or had a prostate cancer were invited to participate by giving their consent and had a PSA test. In case of PSA greater than or equal to 3 ng/ml, biopsy was recommended. Included men in both screening and control group were followed through cancer registries. Objective was to present first round results of French participation to ERSPC, to determine factors of participation and to compare detected cancers cases between both groups. RESULTS: Population of men included was 84,781 and were randomized in screening (n=42,590) or control (n=42,191) group. Participation rate was 36.9% in Tarn and 24.3% in Hérault. PSA was greater than or equal to 3 ng/ml in 15,4% of cases (n=1812) and 45.9% of men (n=832) who were biopsied. Age, previous PSA performed within two years prior to invitation, health insurance and department of residence were significantly associated to participation rate. Cumulated incidence with a four years follow-up was 2.48% (n=1053) in screening and 1.99% (n=840) in control group, with a relative risk (RR) of 1.242. Corresponding RR for Tarn and Hérault were 1.37 and 1.20 respectively. Clinical parameters and treatments modalities were similar between both screening and control groups (radical prostatectomy 68% and radiation therapy 20%). CONCLUSION: Participation rate at first round was modest. Profile of men who participated compared to men who did not were different. The control group was probably contaminated by PSA testing outside study protocol. Consequences at ERSPC level of this low participation rate on final analysis remain to be determined.

WITHDRAWN: Sex differences in the expression of sodium/calcium exchanger influence the arrhythmia phenotype in the long QT syndrome type 2.

The paper entitled "Sex differences in the expression of sodium/calcium exchanger influence the arrhythmia phenotype in the long QT syndrome type 2" by Salama et al, which was published online on 19 May 2008, has been withdrawn at the authority of the editor and the publisher.

Properties of new, long-wavelength, voltage-sensitive dyes in the heart.

Membrane potential measurements using voltage-sensitive dyes (VSDs) have made important contributions to our understanding of electrophysiological properties of multi-cellular systems. Here, we report the development of long wavelength VSDs designed to record cardiac action potentials (APs) from deeper layers in the heart. The emission spectrum of styryl VSDs was red-shifted by incorporating a thienyl group in the polymethine bridge to lengthen and retain the rigidity of the chromophore. Seven dyes, Pittsburgh I to IV and VI to VIII (PGH I-VIII) were synthesized and characterized with respect to their spectral properties in organic solvents and heart muscles. PGH VSDs exhibited 2 absorption, 2 excitation and 2 voltage-sensitive emission peaks, with large Stokes shifts (> 100 nm). Hearts (rabbit, guinea pig and Rana pipiens) and neurohypophyses (CD-1 mice) were effectively stained by injecting a bolus (10-50 microl) of stock solution of VSD (2-5 mM) dissolved in in dimethylsulfoxide plus low molecular weight Pluronic (16% of L64). Other preparations were better stained with a bolus of VSD (2-5 mM) Tyrode's solution at pH 6.0. Action spectra measured with a fast CCD camera showed that PGH I exhibited an increase in fractional fluorescence, DeltaF/F = 17.5 % per AP at 720 nm with 550 nm excitation and DeltaF/F = - 6% per AP at 830 nm with 670 nm excitation. In frog hearts, PGH1 was stable with approximately 30% decrease in fluorescence and AP amplitude during 3 h of intermittent excitation or 1 h of continuous high intensity excitation (300 W Xe-Hg Arc lamp), which was attributed to a combination of dye wash out > photobleaching > dynamic damage > run down of the preparation. The long wavelengths, large Stokes shifts, high DeltaF/F and low baseline fluorescence make PGH dyes a valuable tool in optical mapping and for simultaneous mapping of APs and intracellular Ca(2+).

The distribution of refractory periods influences the dynamics of ventricular fibrillation.

The spatial and dynamic properties of ventricular fibrillation (VF) may be random or related to cellular electrical properties of the normal heart. Local activation intervals (AIs) in VF may depend on the local refractory period (RP), and sustained VF may require a steep action potential (AP) restitution curve. In guinea pig hearts, AP durations (APDs) and RPs on the epicardium are shorter at the apex and progressively longer toward the base, producing gradients of RPs that may influence the spatial organization of VF. In the present study, the influence of APDs on VF dynamics is investigated in perfused guinea pig hearts stained with a voltage-sensitive dye by comparing APD gradients to the dynamics of VF elicited by burst pacing. In VF, AIs had no clear periodicity, but average AIs were shorter at the apex (57.5+/-8.1 ms) than the base (76.1+/-1.5 ms, n=6, P<0.05) and had gradients similar to APD gradients (correlation coefficient 0.71+/-0.04). Analysis of local velocity vectors showed no preferential directions, and fast Fourier transform (FFT) power spectra were broad (10 to 24 Hz) with multiple peaks (n=6). However, the selective inhibition of delayed K(+) rectifying currents, I(Kr) (E4031; 0.5 micromol/L, n=3), shifted FFT spectra from complex to a lower dominant frequency (10 Hz) and altered repolarization but retained the correlation between mean AIs and RPs. Thus, VF dynamics are consistent with a multiple wave-make and wave-break mechanism, and the local RP influences VF dynamics by limiting the range of VF frequencies and AIs at each site. The full text of this article is available at http://www.circresaha.org.

High levels of cytokeratin 19 fragments but no evidence of cytokeratins 1, 2, 10/11, 14 or filaggrin in the serum of squamous cell lung carcinoma patients.

The CYFRA 21-1 assay detects circulating fragments of cytokeratin 19, which is a sensitive marker for the diagnosis of lung cancers, particularly squamous cell carcinomas and adenocarcinomas. Epidermis-type proteins, such as cytokeratins 1, 2, 10/11 and 14 or filaggrin, are also expressed in squamous cell carcinomas. These could also be pertinent tumor markers, ideally as sensitive as CYFRA 21-1 and more specific for squamous cell lung cancer. To verify this hypothesis, using monoclonal antibodies produced in our laboratory, we developed immunoassays specific for these proteins. After optimization, the immunoassays were evaluated in sera from 91 controls and 138 patients with squamous cell lung cancer and compared to conventional tumor markers (CEA, SCC Ag and CYFRA 21-1). Less than 14% of the sera were above the lower limit of detection of the cytokeratin- and filaggrin-specific immunoassays. Moreover, part of these positive sera were induced by the presence of interfering heterophilic antibodies in sera. Thus, in patients with squamous cell lung cancer, we confirmed the high diagnostic sensitivity of CYFRA 21-1 (55.6%) but were unable to detect significant levels of epidermis-type cytokeratins or filaggrin.

Cardiac ischemia oxidizes regulatory thiols on ryanodine receptors: captopril acts as a reducing agent to improve Ca2+ uptake by ischemic sarcoplasmic reticulum.

We tested the hypothesis that ischemia alters sarcoplasmic reticulum (SR) Ca2+ transport by oxidizing regulatory thiols on ryanodine receptors (RyRs), and that membrane-permeable sulfhydryl-containing angiotensin-converting enzyme (ACE) inhibitors protect against ischemia-induced oxidation and explain in part, the therapeutic actions of captopril. Ca2+ uptake and adenosine triphosphatase (ATPase) activity was measured from SR vesicles isolated from control or ischemic dog and human ventricles and compared with or without sulfhydryl reductants. The rate and amount of Ca2+ uptake was lower for canine ischemic SR compared with control (6.5 +/- 0.2 --> 18.5 +/- 1.1 nmol Ca2+/mg/min and 123.1 +/- 4.7 --> 235.0 +/- 17.3 nmol Ca2+/mg; n = 8 each). Captopril, dithiothreitol (DTT), glutathione (GSH), and L-cysteine increased the rate and amount of Ca2+ uptake by canine and human ischemic SR vesicles by approximately 50%. Reducing agents had no effect on Ca2+- ATPase activity in either canine control or ischemic (approximately 40% less than control) SR. Captopril was as potent as DTT at reversing the oxidation of skeletal and cardiac RyRs induced by reactive disulfides (RDSs) or nitric oxide (NO). In neonatal rat myocytes, RDSs or NO triggered SR Ca2+ release and increased cytosolic Ca2+, an effect reversed by captopril and DTT but not GSH or cysteine. Pretreatment of myocytes with captopril (exposure and then wash) inhibited Ca2+ elevation elicited by RDSs or NO, indicating that captopril is an effective, membrane-permeable intracellular reducing agent. Thus, net SR Ca2+ accumulation is reduced by ischemia in part due to the oxidation of thiols that gate RyRs, an effect reversed by captopril.

Simultaneous maps of optical action potentials and calcium transients in guinea-pig hearts: mechanisms underlying concordant alternans.

1. The mechanisms underlying electro-mechanical alternans caused by faster heart rates were investigated in perfused guinea-pig hearts stained with RH237 and Rhod-2 AM to simultaneously map optical action potentials (APs) and intracellular free Ca2+ (Ca2+i). 2. Fluorescence images of the heart were focused on two 16 x 16 photodiode arrays to map Ca2+i (emission wavelength (lamdda;em) = 585 +/- 20 nm) and APs (lamdda;em > 715 nm) from 252 sites. Spatial resolution was 0.8 mm x 0.8 mm per diode and temporal resolution 4000 frames s-1. 3. The mean time-to-peak for APs and [Ca2+]i was spatially homogeneous (8.8 +/- 0.5 and 25.6 +/- 5.0 ms, respectively; n = 6). The durations of APs (APDs) and Ca2+i transients were shorter at the apex and progressively longer towards the base, indicating a gradient of ventricular relaxation. 4. Restitution kinetics revealed increasingly longer delays between AP and Ca2+i upstrokes (9.5 +/- 0.4 to 11.3 +/- 0.4 ms) with increasingly shorter S1-S2 intervals, particularly at the base, despite nearly normal peak [Ca2+]i. 5. Alternans of APs and Ca2+i transients were induced by a decrease++ in cycle length (CL), if the shorter CL captured at the pacing site and was shorter than refractory periods (RPs) near the base, creating heterogeneities of conduction velocity. 6. Rate-induced alternans in normoxic hearts were concordant (long APD with large [Ca2+]i) across the epicardium, with a magnitude (difference between odd-even signals) that varied with the local RP. Alternans were initiated by gradients of RP, producing alternans of conduction that terminated spontaneously without progressing to fibrillation.

Low N-ethylmaleimide concentrations activate ryanodine receptors by a reversible interaction, not an alkylation of critical thiols.

Previous studies proposed that N-ethylmaleimide (NEM) alkylates 3 classes of thiols on skeletal muscle ryanodine receptors (RyRs) producing 3 phases of channel modification, as function of time and concentration. NEM (5 mm) decreased, increased, and then decreased the open probability (P(o)) of the channel by thiol alkylation, a reaction not reversed by reducing agents. We now show that low NEM concentrations (20-200 microm) elicit Ca(2+) release from sarcoplasmic reticulum (SR) vesicles, but contrary to expectations, the effect was fully reversed by reducing agents or by washing SR vesicles. In bilayers, NEM (0.2 mm) increased P(o) of RyRs within seconds when added to the cis (not trans) side, and dithiothreitol (DTT; 1 mm) decreased P(o) in seconds. High (5 mm) NEM concentrations elicited SR Ca(2+) release that was not reversed by DTT, as expected for an alkylation reaction. A non-sulfhydryl reagent structurally related to NEM, N-ethylsuccinimide (0.1-0.5 mm), also elicited SR Ca(2+) release that was not reversed by DTT (1 mm). Other alkylating agents elicited SR Ca(2+) release, which was fully (N-methylmaleimide) or partially (iodoacetic acid) reversed by DTT and inhibited by ruthenium red. Nitric oxide (NO) donors at concentrations that did not activate RyRs inhibited NEM-induced Ca(2+) release, most likely by an interaction of NO with NEM rather than an inactivation of RyRs by NO. Thus, at low concentrations, NEM does not act as a selective thiol reagent and activates RyRs without alkylating critical thiols indicating that the multiple phases of ryanodine binding are unrelated to RyR activity or to NEM alkylation of RyRs.

Hepatitis C virus infection in French hemodialysis units: a multicenter study.

The aims of the study were: (i) to evaluate the prevalence of hepatitis C virus (HCV) antibodies (third generation tests) and RNA (standardized ultrasensitive RT-PCR assay) in a large cohort of hemodialysis patients, and (ii) to correlate HCV markers with bioclinical features and alanine-aminotransferase (ALT) activity. Antibodies were assayed by two methods in 1,323 patients (60% men, median age 65 years, median hemodialysis duration 3 years) attending 25 French hemodialysis centers including 9 self-care units. RNA was assayed using the Cobas Amplicor 2.0 method in pooled samples from 10 anti-HCV(-/-) patients and on individual samples from the other patients. Of the 16.3% patients (range 0-44%) tested (+/+) for HCV antibodies (anti-HCV), 2.3% tested (+/-) and 81.4% tested (-/-). 70% of the anti-HCV(+/+) patients and 3% of the HCV(+/-) patients were RNA(+). Pooled analysis revealed that 5/1077 anti-HCV(-/-) patients (0.5%) were RNA(+); all 5 displayed subsequently an increase in ALT and became anti-HCV(+/+). Mean ALT was higher (multiple of normal) in anti-HCV(+/+) RNA(+) patients than in anti-HCV(+/+) RNA(-) patients (0.46 +/- 0.08 vs. 0.22 +/- 0.07, P < 0.0001) and similar in all the RNA(-) patients, whatever their HCV antibody status. Multivariate analysis demonstrated that HCV status was linked to hemodialysis duration, previous kidney transplantation and positive anti-HBc. To summarize, the determination of the RNA status of anti-HCV(+/-) patients may have clinical relevance if a policy of isolation is contemplated. Standardized ultrasensitive RT-PCR assay combined with a pooling strategy is a promising method for use in epidemiological studies.

Enhanced dispersion of repolarization and refractoriness in transgenic mouse hearts promotes reentrant ventricular tachycardia.

The heterogeneous distribution of ion channels in ventricular muscle gives rise to spatial variations in action potential (AP) duration (APD) and contributes to the repolarization sequence in healthy hearts. It has been proposed that enhanced dispersion of repolarization may underlie arrhythmias in diseases with markedly different causes. We engineered dominant negative transgenic mice that have prolonged QT intervals and arrhythmias due to the loss of a slowly inactivating K(+) current. Optical techniques are now applied to map APs and investigate the mechanisms underlying these arrhythmias. Hearts from transgenic and control mice were isolated, perfused, stained with di-4-ANEPPS, and paced at multiple sites to optically map APs, activation, and repolarization sequences at baseline and during arrhythmias. Transgenic hearts exhibited a 2-fold prolongation of APD, less shortening (8% versus 40%) of APDs with decreasing cycle length, altered restitution kinetics, and greater gradients of refractoriness from apex to base compared with control hearts. A premature impulse applied at the apex of transgenic hearts produced sustained reentrant ventricular tachycardia (n=14 of 15 hearts) that did not occur with stimulation at the base (n=8) or at any location in control hearts (n=12). In transgenic hearts, premature impulses initiated reentry by encountering functional lines of conduction block caused by enhanced dispersion of refractoriness. Reentrant VT had stable (>30 minutes) alternating long/short APDs associated with long/short cycle lengths and T wave alternans. Thus, optical mapping of genetically engineered mice may help elucidate some electrophysiological mechanisms that underlie arrhythmias and sudden death in human cardiac disorders.

Molecular interaction between nitric oxide and ryanodine receptors of skeletal and cardiac sarcoplasmic reticulum.

In striated muscle, the sarcoplasmic reticulum (SR) is the major storage compartment of intracellular Ca2+ that controls cytosolic free Ca2+ (Cai) and developed force by sequestering and releasing Ca2+ during each contraction. Ca2+ release from the SR occurs through high-conductance Ca2+ release channels or ryanodine receptors (RyR), which are regulated by various signaling processes. Over the last 15 years, there has been a growing consensus that critical sulfhydryl sites on RyRs can be oxidized and reduced, respectively, to open and close the release channels. The pharmacological actions of various classes of sulfhydryl reagents have demonstrated the existence of hyperreactive thiols on RyRs, which could play a role in the regulation of normal contractile function and explain contractile dysfunctions in pathological conditions. More recent studies show that redox regulation of release channels may occur by nitric oxide (NO), a physiological signaling mechanism. This article is intended to review current concepts in thiol regulation of RyRs and present new data on the possible identification of the primary cysteine residues, which may be the site of oxidation and S-nitrosylation involved in channel opening.

Evaluation of seven tumour markers in pleural fluid for the diagnosis of malignant effusions.

Carcinoembryonic antigen (CEA), carbohydrate antigens 15-3, 19-9 and 72-4 (CA 15-3, CA 19-9 and CA 72-4), cytokeratin 19 fragments (CYFRA 21-1), neuron-specific enolase (NSE) and squamous cell carcinoma antigen (SCC) were evaluated in pleural fluid for the diagnosis of malignant effusions. With a specificity of 99%, determined in a series of 121 benign effusions, the best individual diagnostic sensitivities in the whole series of 215 malignant effusions or in the subgroup of adenocarcinomas were observed with CEA, CA 15-3 and CA 72-4. As expected, a high sensitivity was obtained with SCC in squamous cell carcinomas and with NSE in small-cell lung carcinomas. CYFRA and/or CA 15-3 were frequently increased in mesotheliomas. Discriminant analysis showed that the optimal combination for diagnosis of non-lymphomatous malignant effusions was CEA + CA 15-3 + CYFRA + NSE: sensitivity of 94.4% with an overall specificity of 95%. In malignant effusions with a negative cytology, 83.9% were diagnosed using this association. The association CYFRA + NSE + SCC was able to discriminate adenocarcinomas from small-cell lung cancers. Regarding their sensitivity and their complementarity, CEA, CA 15-3, CYFRA 21-1, NSE and SCC appear to be very useful to improve the diagnosis of malignant pleural effusions.

Using RT-PCR and bDNA assays to measure non-clade B HIV-1 subtype RNA.

The performance of the new version of RT-PCR assay (Amplicor HIV-1 Monitor v1.5) was assessed. The quantification of non-B subtype HIV-1 plasma RNA (30A, 1C, 1D, 3E, 2F, 3G) obtained using Monitor v1.5 was compared to the former version of this assay (Monitor v1.0) and to the Quantiplex v2.0 bDNA assay. The new primers used in Monitor v1.5 were similar to the former version in both specificity and sensitivity. The new primers corrected the detection and quantification defect observed previously for HIV-1 non-B subtypes and gave slightly higher RNA concentrations than those measured using the bDNA assay (+0.39 log copies/ml).

Nitric oxide prevents myoglobin/tert-butyl hydroperoxide-induced inhibition of Ca2+ transport in skeletal and cardiac sarcoplasmic reticulum.

Interaction of hydrogen peroxide or organic hydroperoxides with hemoproteins is known to produce oxoferryl hemoprotein species that act as very potent oxidants. Since skeletal and cardiac muscle cells contain high concentrations of myoglobin this reaction may be an important mechanism of initiation or enhancement of oxidative stress, which may impair their Ca2+ transport systems. Using skeletal and cardiac sarcoplasmic reticulum (SR) vesicles, we demonstrated by EPR the formation of alkoxyl radicals and protein-centered peroxyl radicals in the presence of myoglobin (Mb) and tert-butyl hydroperoxide (t-BuOOH). The low temperature EPR signal of the radicals was characterized by major feature at g = 2.016 and a shoulder at g = 2.036. In the presence of SR vesicles, the magnitude of the protein-centered peroxyl radical signal decreased, suggesting that the radicals were involved in oxidative modification of SR membranes. This was accompanied by SR membrane oxidative damage, as evidenced by accumulation of 2-thiobarbituric acid-reactive substances (TBARS) and the inhibition of Ca2+ transport. We have shown that nitric oxide (NO), reacting with redox-active heme iron, can prevent peroxyl radical formation activated by Mb/t-BuOOH. Incubation of SR membranes with an NO donor, PAPA/NO (a non-thiol compound that releases NO) at 200-500 microM completely prevented the t-BuOOH-dependent production of peroxyl radicals and formation of TBARS, and thus protected against oxidative inhibition of Ca2+ transport.

Risperidone-induced increase of plasma norepinephrine is not correlated with symptom improvement in chronic schizophrenia.

BACKGROUND: Previous studies have shown an increase in plasma levels of norepinephrine (NE) after clozapine treatment of schizophrenia. This effect has been suggested to relate to improvement in symptoms. METHODS: To test whether other novel antipsychotic drugs have such an effect, the present experiment examined schizophrenic symptoms and plasma levels of NE before and after 5 weeks of treatment with risperidone or haloperidol. RESULTS: Risperidone, but not haloperidol, significantly increased plasma NE; however, there was no correlation of this effect with clinical improvement on any symptom scale. CONCLUSIONS: This finding suggests that risperidone shares similar properties with clozapine in enhancing peripheral NE, but that these changes in plasma NE may not be a consistent indicator of atypical antipsychotic drug efficacy.

Evaluation of alpha-fetoprotein assay in ascitic fluid for the diagnosis of hepatocellular carcinoma.

Ascites and hepatocellular carcinoma are frequently associated. We evaluated the usefulness of alpha-fetoprotein assay in ascitic fluid versus the serum assay, for the diagnosis of hepatocellular carcinoma, in 125 patients with peritoneal effusions (31 patients with hepatocellular carcinoma, 14 with extra-hepatic malignancies and 80 with a benign effusion). Albumin and total protein were also assayed and cytological analysis of the ascitic fluid performed. Alpha-fetoprotein appeared to be lower in ascitic fluid than in serum. For a diagnostic specificity of 95%, the thresholds were 18.9 microg/l in serum and 4 microg/l in ascitic fluid and the diagnostic sensitivity of alpha-fetoprotein was identical in serum and ascitic fluid (67.7%). Various ratios between alpha-fetoprotein and albumin or total protein did not enhance the diagnostic performance. Thus alpha-fetoprotein concentration in ascitic fluid reflected the serum concentration and proved to be of similar value for the diagnosis of hepatocellular carcinoma, providing that the appropriate thresholds are considered.

Decay of HIV-1 DNA in patients receiving suppressive antiretroviral therapy.

We have examined the effect of potent antiretroviral regimens on the latent reservoirs of HIV-1. The HIV-1 DNA in the peripheral blood mononuclear cells (PBMC) of 10 patients with undetectable plasma HIV-1 RNA (<20 copies/ml) who had received combination antiretroviral therapy was assayed every 12 weeks. No evidence of residual viral replication was found in the PBMC after 24 weeks of treatment. Although HIV-1 DNA remained detectable in all patients, it decreased significantly from 3.5 log copies/10(6) cells (range, 1.8-4.7 log copies/10(6) cells) to 2.3 log copies/10(6) cells (range, 0.6-3.1 log copies/10(6) cells) after 60 weeks of suppressive therapy. Analysis based on 6 patients who reached 60 weeks showed a slow decline with an estimated half-life of 40 weeks (range, 26-163 weeks). Genotypic analysis by sequencing the HIV-1 pol gene revealed no changes in the reverse transcriptase or protease coding regions after 48 to 60 weeks of therapy. The findings suggest that, in addition to potent antiretroviral regimens, new strategies must be developed to ensure eradication of the latent reservoir of provirus, and hence of the virus itself.