Efficacy of tenofovir and efavirenz in combination with lamivudine or emtricitabine in antiretroviral-naive patients in Europe.

BACKGROUND: The combination of tenofovir and efavirenz with either lamivudine or emtricitabine (TELE) has proved to be highly effective in clinical trials for first-line treatment of HIV-1 infection. However, limited data are available on its efficacy in routine clinical practice. METHODS: A multicentre cohort study was performed in therapy-naive patients initiating ART with TELE before July 2009. Efficacy was studied using ITT (missing or switch = failure) and on-treatment (OT) analyses. Genotypic susceptibility scores (GSSs) were determined using the Stanford HIVdb algorithm. RESULTS: Efficacy analysis of 1608 patients showed virological suppression to <50 copies/mL at 48 weeks in 91.5% (OT) and 70.6% (ITT). Almost a quarter of all patients (22.9%) had discontinued TELE at week 48, mainly due to CNS toxicity. Virological failure within 48 weeks was rarely observed (3.3%, n = 53). In multilevel, multivariate analysis, infection with subtype B (P = 0.011), baseline CD4 count <200 cells/mm³ (P < 0.001), GSS <3 (P = 0.002) and use of lamivudine (P < 0.001) were associated with a higher risk of virological failure. After exclusion of patients using co-formulated compounds, virological failure was still more often observed with lamivudine. Following virological failure, three-quarters of patients switched to a PI-based regimen with GSS <3. After 1 year of second-line therapy, viral load was suppressed to <50 copies/mL in 73.5% (OT). CONCLUSIONS: In clinical practice, treatment failure on TELE regimens is relatively frequent due to toxicity. Virological failure is rare and more often observed with lamivudine than with emtricitabine. Following virological failure on TELE, PI-based second-line therapy was often successful despite GSS <3.

Differences of nine drug resistance interpretation systems in predicting short-term therapy outcomes of treatment-experienced HIV-1 infected patients: a retrospective observational cohort study.

OBJECTIVE: Drug resistance interpretation systems are used to select the optimal antiretroviral therapy in HIV-infected patients. It is unclear how the systems perform in predicting therapy success and failure and in how far the interpretations are affected by insufficient drug levels. METHODS: The accuracy of nine different interpretation systems in predicting therapy outcomes was evaluated using virological, immunological, pharmacological, and clinical data of 130 patients treated at 13 outpatient centers. Individual susceptibility scores of the interpretation systems were converted into active drug scores (ADS) and correlated with therapy success and failure, defined as viral load reduction of equal to or more (n=66) and less than 1 log10 copies/ml (n=64) at three months after drug resistance testing. RESULTS: Three interpretation systems considered the respective therapies as more active compared to the other interpretation systems (p<0.01). These systems predicted therapy success better than the other systems, while the others performed better in predicting therapy failure. Thus, the overall rate of correctly predicted treatment outcomes was comparable between the different systems (73.1-80.0 %). Univariate and multivariate regression analysis revealed significant correlations between the ADS of all interpretation systems and virological therapy outcomes (p<0.0001). In contrast, only three interpretation systems were significantly correlated with immunological therapy outcomes in univariate and just one in multivariate models (p<0.05). Among 128 determinations of drug levels in 64 patient samples, 19.4 % revealed no detectable drug levels. The consideration of insufficient drug levels significantly improved the prediction accuracy of all interpretation systems (p<0.005). CONCLUSION: Differences between interpretation systems in predicting therapy failures and success need to be considered for future consensus algorithms. The prediction accuracy of interpretation systems can be improved by consideration of plasma drug levels.

Resistance-related mutations in the HIV-1 protease gene of patients treated for 1 year with the protease inhibitor ritonavir (ABT-538).

OBJECTIVE: To define genotypic and phenotypic resistance patterns following prolonged therapy with the protease inhibitor ritonavir (ABT-538). DESIGN: Seven HIV-1-infected patients, all but one previously treated with dideoxynucleoside analogues (zidovudine, didanosine, zalcitabine), were treated for 1 year with ritonavir. METHODS: Direct solid-phase sequencing of the protease gene starting from plasma derived viral RNA followed by comparison to phenotypic drug resistance data. RESULTS: The most frequent amino-acid substitutions occurring upon administration of the protease inhibitor were V82A/F (substrate binding site), I54V (flap region), A71V and L10I. Additional mutations found in more than one patient were I15V, M36I, I84V and I93L. Mutation L63P was found both in pre- and post-ritonavir samples. Phenotypic drug resistance assays confirmed resistance to ritonavir in post-treatment samples (approximately 170-fold) and showed cross-resistance to indinavir (approximately 30-fold) and partially to saquinavir (approximately fivefold). At 1 year of treatment, one patient without known resistance-associated mutations in the protease gene still showed a substantial rise in CD4 cell count accompanied by a more than 2.4 log decrease in RNA viral load. However, at week 78, mutations R8Q, E34K, R57K, L63P and I84V were detected and the treatment benefit was partially lost. CONCLUSIONS: Long-term treatment with ritonavir is associated with the emergence of multiple mutations in the HIV-1 protease gene. The mutations L10I, I54V, L63P, A71V, V82A/F and I84V correspond to known drug-resistance mutations for ritonavir and other protease inhibitors. Phenotypic resistance to ritonavir was detected in a majority of ritonavir-treated patients at 1 year of treatment. In addition, long-term ritonavir treatment selects for cross-resistance to the protease inhibitors indinavir and saquinavir. This argues against sequential therapy with several protease inhibitors. Delayed resistance in one patient was accompanied with a prolonged increase in CD4 cell count and decrease in viral load suggesting a temporary benefit of treatment.

Mutations in the non-nucleoside binding-pocket interfere with the multi-nucleoside resistance phenotype.

OBJECTIVES: To investigate the genotypic and phenotypic effects of in vitro resistance selection with lamivudine and/or the second generation non-nucleoside reverse transcriptase inhibitor (NNRTI) quinoxaline HBY097 using HIV-1 isolates carrying the multi-nucleoside resistance pattern linked to the Q151M mutation. METHODS: Virus strains were selected in C8166 cells in the presence of increasing concentrations of lamivudine or HBY097. In parallel control experiments, the virus was cultured in C8166 cells in the absence of drugs. The entire reverse transcriptase encoding region was amplified using polymerase chain reaction and was subsequently sequenced. Antiviral activities of drugs were evaluated in C8166 cells. RESULTS: High-level resistant viruses were selected rapidly in the presence of lamivudine and quinoxaline (less than 10 passages). The multi-nucleoside resistance mutations were stable during in vitro resistance selection. Lamivudine elicited the acquisition of the M184I mutation. Phenotypic resistance to all nucleoside-analog reverse transcriptase inhibitors (NRTIs) was increased when M184I was added to the multi-nucleoside resistance background in the absence of NNRTI-resistance mutations. In most cases of HBY097 resistance selection, at least two mutations associated with NNRTI resistance resulted in high-level NNRTI resistance. The NNRTI resistance-related mutations partially reversed the phenotypic resistance to most NRTIs, except to abacavir. The addition of the M184I mutation to the NNRTI-multi-nucleoside resistance set abolished this antagonizing effect for didanosine, zalcitabine and lamivudine, but further potentiated the phenotypic reversal for zidovudine and stavudine. CONCLUSION: Changes in the non-nucleoside binding pocket must affect the conformation of residues at the dNTP binding site, and can result in a partial phenotypic reversal of the multi-nucleoside resistance phenotype.

Phase I/II dose escalation and randomized withdrawal study with add-on azodicarbonamide in patients failing on current antiretroviral therapy.

BACKGROUND: Azodicarbonamide (ADA), a HIV-1 zinc finger inhibitor, targets a new step in viral replication and cell infectivity. OBJECTIVE: A first phase I/II clinical study of ADA. METHODS: ADA was administered at escalating doses concomitantly with current antiviral therapy during a 3-month open-label period in patients with advanced AIDS and documented virological failure. After 3 months, patients were randomized in a double-blind placebo-controlled withdrawal, ADA being given at the highest tolerated dosage. RESULTS: Fifteen patients with advanced disease failing on combined antiretroviral therapy, 75% of them with proven phenotypic resistance, had a median baseline CD4 cell count of 85 x 10(6) cells/l, CD4/CD8 cell ratio of 0.09 and median plasma RNA viral load of 4.2 log10 copies/ml. Tolerance to ADA was dose dependent and some patients developed nephrolithiasis, glucose intolerance or showed an ADA-related cytotoxicity towards CD4 cells at higher dosages. No patient died during the study period. ADA increased CD4 cell percentage, increased the CD4/CD8 cell ratio and decreased plasma RNA viral load from baseline. At the end of the double-blind period, the ADA group, but not the placebo group, showed a significant response (P < 0.05). No phenotypic resistance to ADA was observed. Overall, 3/11 patients (27%) had consistent viral load reductions > 0.5 log10 copies/ml compared with baseline and 5/ 11 (45%) showed a CD4 cell recovery from baseline > 33%. In responders, ADA induced a median peak increase in CD4 cell percentage change from baseline of 65% (range 47-243%), and viral load decrease of 1.04 log10 copies/ml (range 0.52-1.23). CONCLUSIONS: The maximal tolerated dosage of ADA appears to be 2 g (three times daily). This study provides safety results that will allow larger clinical trials to confirm the preliminary efficacy data.

Efficacy of triple combination therapy with zidovudine (ZDV) plus zalcitabine (ddC) plus lamivudine (3TC) versus double (ZDV+3TC) combination therapy in patients previously treated with ZDV+ddC.

OBJECTIVE: To evaluate the immunological and virological efficacy of triple combination therapy with zidovudine (ZDV) plus zalcitabine (ddC) plus lamivudine (3TC) and a double (ZDV+3TC) combination therapy in patients previously treated with ZDV plus ddC. DESIGN: A 6-month follow-up open-label randomized study was undertaken in 46 HIV-1-infected patients previously treated for at least 6 months with ZDV plus ddC, who were allocated to receive either ZDV/ddC/3TC (n = 15) or ZDV/3TC (n = 15) or to continue with the ZDV/ddC regimen (control group; n = 16). METHODS: Changes in CD4+ cell counts and plasma viral load (VL) were analysed with analysis of variance. Sequencing of the reverse transcriptase gene was performed in a subset of 3TC-treated patients. RESULTS: Mean CD4+ cell counts increased significantly above baseline in both 3TC regimens whereas counts decreased in the control group. Significant plasma VL reduction was achieved in both 3TC combination therapy groups at weeks 4 and 24 compared with the control group. Coexistence of mutations conferring resistance to ZDV and 3TC were found in patients from both 3TC treatment groups. CONCLUSIONS: Both therapy strategies, switching ddC to 3TC or adding 3TC, significantly improved the virological and immunological efficacy compared with continuing ZDV/ddC. Our results support the use of 3TC in patients previously treated with the ZDV/ddC combination.

Multiple dideoxynucleoside analogue-resistant (MddNR) HIV-1 strains isolated from patients from different European countries.

OBJECTIVE: To study the prevalence of multiple dideoxynucleoside (ddN)-resistant (MddNR) HIV-1 in European patients under treatment with multiple ddN analogues, and to characterize MddNR strains genotypically and phenotypically. DESIGN AND METHODS: Blood samples from patients after > or = 6 months of treatment with multiple ddN were screened for the MddNR mutation Q151M. After confirmation of MddNR in 15 patients from five European countries, genotypic resistance was evaluated by DNA sequencing of the reverse transcriptase (RT) gene. Phenotypic resistance was measured by the recombinant virus assay. Results were compared with the clinical evolution of the patients. RESULTS: The prevalence of MddNR strains in European patients treated with multiple ddN analogues was 3.5%. Viruses typically contained amino acid substitutions V75F, F77L, F116Y and Q151M in the RT gene. A new mutation, S68G, was frequently associated with MddNR. Phenotypically, viruses displayed high-level resistance to zidovudine (ZDV), didanosine (ddl), zalcitabine (ddC), stavudine (d4T) and partial resistance to lamivudine (3TC) once multiple mutations were present. Under in-vivo treatment pressure, some MddNR strains additionally developed resistance to protease inhibitors or non-nucleoside RT inhibitors (NNRTI). Clinically, most patients had advanced HIV disease with low CD4 cell counts, high viral loads and a rapid progression, but two patients harbouring MddNR virus responded well to dual protease inhibitor associations. CONCLUSIONS: MddNR resistant HIV-1 can be found in European patients. MddNR is characterized by a specific set of drug resistance mutations, cross-resistance to most ddN analogues and a fast clinical progression. MddNR can be associated with protease inhibitor or NNRTI resistance.

Genotypic correlates of resistance to HIV-1 protease inhibitors on longitudinal data: the role of secondary mutations.

Direct sequencing of the pol gene was assessed retrospectively with protease inhibitor susceptibility in a longitudinal study. A total of 134 samples from 26 patients were analysed at regular intervals up to 2 years. Patients were included in virological failure despite indinavir, ritonavir or saquinavir based triple-drug therapy. Both the type and number of certain secondary protease mutations modulated the effect of primary mutations on phenotypic resistance. This was notably applicable to L101/V, and to lesser extents to A711V/T. However, combinations of primary mutations, including 154V could predict resistance to the drug used and nelfinavir in more than 80%. In contrast, in vitro cross-resistance to amprenavir was rarely encountered. In addition, there was a relationship between a higher number of key mutations and poorer virological and clinical outcomes, respectively, from 6 and 3 months on. The key mutations were the protease mutations independently conferring phenotypic resistance and/or the reverse transcriptase mutations predicting treatment outcome. This relationship was independent from drug history, viral load and CD4 cell count measurements. In summary, even on a small sample size, sequence-based genotyping seems to be a good prognostic marker when performed longitudinally. In the context of primary resistance mutations, including additional secondary mutations, it may be useful in the prediction of phenotypic and clinical resistance. This should be assessed to optimize treatment monitoring before emergence of broadly cross-resistant virus.

Laboratory guidelines for the practical use of HIV drug resistance tests in patient follow-up.

HIV drug resistance is one of the major limitations in the successful treatment of HIV-infected patients using currently available antiretroviral combination therapies. When appropriate, drug susceptibility profiles should be taken into consideration in the choice of a specific combination therapy. Guidelines recommending resistance testing in certain circumstances have been issued. Many clinicians have access to resistance testing and will increasingly use these results in their treatment decisions. In this document, we comment on the different methods available, and the relevant issues relating to the clinical application of these tests. Specifically, the following recommendations can be made: (i) genotypic and phenotypic HIV-1 drug resistance analyses can yield complementary information for the clinician. However, insufficient information currently exists as to which approach is preferable in any particular clinical setting; (ii) when HIV-1 drug resistance testing is required, it is recommended that testing be performed on plasma samples obtained before starting, stopping or changing therapy, on samples that have a viral load above the detection limit of the resistance test; (iii) the panel recommends that genotypic and phenotypic HIV-1 drug resistance testing for clinical purposes be performed in a certified laboratory under strict quality control and quality assurance standards; and (iv) the panel recommends that resistance testing laboratories provide clinicians with resistance reports that include a list of drug-related resistance mutations (genotype) and/or a list of drug-related fold resistance values (phenotype), with interpretations of each by an experienced virologist. The interpretation of genotypic and phenotypic analysis is a complex and developing science, and in order to understand HIV-1 drug resistance reports, communication between the requesting clinician and the expert that interpreted the resistance report is recommended.

Updated European recommendations for the clinical use of HIV drug resistance testing.

In most European countries, HIV drug resistance testing has become a routine clinical tool. However, its practical implementation in a clinical context is demanding. The European HIV Drug Resistance Panel was established to make recommendations to clinicians and virologists on this topic and to propose quality control measures. The panel recommends resistance testing for the following indications: i) drug-naive patients with acute or recent infection; ii) therapy failure, including suboptimal treatment response, when treatment change is considered; iii) pregnant HIV-1-infected women and paediatric patients with detectable viral load when treatment initiation or change is considered; and iv) genotype source patient when post-exposure prophylaxis is considered. In addition, for drug-naive patients with chronic infection in whom treatment is to be started, the panel suggests that resistance testing should be strongly considered and recommends testing the earliest sample for drug resistance if suspicion of resistance is high or prevalence of resistance in this population exceeds 10%. The panel does not favour genotyping over phenotype, however it is anticipated that genotyping will be used more often because of its greater accessibility, lower cost and faster turnaround time. For the interpretation of resistance data, clinically validated systems should be used to the greatest extent possible. It is mandatory that laboratories performing HIV resistance tests take regular part in quality assurance programs. Similarly, it is necessary that HIV clinicians and virologists take part in continuous education and meet regularly to discuss problematic clinical cases. Indeed, resistance test results should be used in the context of all other clinically relevant information for predicting therapy response. The panel also encourages the timely collection of epidemiological information to estimate the impact of transmission of resistant HIV and the prevalence of HIV-1 non-B subtypes in the different European countries.

Cell type-dependent effect of sodium valproate on human immunodeficiency virus type 1 replication in vitro.

Sodium valproate (VPA), a simple branched-chain fatty acid that has anticonvulsant activity and is used in the treatment of many forms of epilepsy, has been reported to stimulate human immunodeficiency virus (HIV) type 1 replication in acutely infected CEM and chronically infected U1 cells (Chemico-Biological Interactions 1994;91:111-121). When attempting to reproduce and extend these findings, we confirmed that VPA is able to stimulate HIV-1(IIIB) replication in acutely infected CEM and C8166 T lymphocytic cell lines and chronically infected ACH-2 and U937/IIIB/LAI cells in a concentration-dependent manner. The stimulatory effect of VPA on HIV replication in CEM cells was not increased by pretreatment of the cells with VPA for 24 hr before infection. However, we could not detect any stimulatory effect of VPA on HIV-1(IIIB) replication in acutely infected peripheral blood mononuclear cells (PBMCs), MT-4, MT-2, HUT-78, and MOLT-4 (clone 8) cells and in chronically infected HUT-78/IIIB/LAI cells. The stimulatory effect by VPA under certain conditions (see above) may be ascribed to an enhanced HIV transcription, as VPA was found to enhance the HIV long terminal repeat (LTR)-directed expression of beta-galactosidase in transiently transfected HLtat, P4, and COS7 cells. VPA did not enhance beta-galactoside expression mediated by the cytomegalovirus (CMV) promoter. VPA did not affect HIV-induced syncytium formation. Nor had VPA any direct inactivating effect on HIV.

Baseline HIV type 1 genotypic resistance to a newly added nucleoside analog is predictive of virologic failure of the new therapy.

We evaluated the predictive value of baseline HIV-1 genotypic resistance mutations for failure of a nucleoside reverse transcriptase inhibitor (NRTI) containing therapy. The change in therapy of 88 HIV-1-infected patients was analyzed retrospectively, relating the genotypic resistance profile at baseline to the evolution of viral load and CD4+ T cell counts. Genotypic resistance at baseline and at 6 months was evaluated with the LiPA HIV-1 RT, which detects mutations at codons 41, 69, 70, 74, 184, and 215. At 1 to 3 months after change in therapy, patients without preexisting resistance mutations to the new drug (group S) had a significantly better evolution in viral load (reduction of 0.37 log(10)) compared with patients with known preexisting resistance mutation(s) (group R) (increase of 0.08 log(10)). This difference was particularly striking for patients with the baseline M184V mutation and whose treatment was modified by the addition of lamivudine. After 6 months the median difference in viral load evolution between the two groups increased to 0.61 log(10): the viral load of patients of group S was still 0.18 log(10) below baseline while patients of group R had an increase of 0.43 log(10) in viral load above baseline. Changes in CD4+ T cell counts were not significantly different. The evolution in viral load in HIV-1-infected patients with and without baseline resistance mutation(s) toward a newly added NRTI is significantly different at 1-3 months and at 6 months after changing or adding one NRTI.

Initiation of HAART in drug-naive HIV type 1 patients prevents viral breakthrough for a median period of 35.5 months in 60% of the patients.

The introduction of potent combinations of antiviral drugs is a major breakthrough in the treatment of HIV. We investigated the long-term virologic outcome and the development of resistance after initiating highly active antiretroviral therapy (HAART) in drug-naive patients in daily clinical practice. Twenty-five treatment-naive HIV-1 patients were started on HAART. Fifteen patients responded with a drop in viral load below the limit of detection during 35.5 (interquartile range: 7) months of therapy. In 6 of 10 patients with virologic failure, virus with resistance-related mutations against the received drugs emerged. Compared with responders (R), nonresponding (NR) patients were in a later disease stage at therapy start (p = 0.0089) with lower CD4 cell counts at baseline (p = 0.040), and a lower proportion of nonresponders showed protease inhibitor (PI) levels above C(min) (p = 0.049). More NR patients showed secondary PI mutations at baseline (p = 0.079), and the CCR2-64I coreceptor polymorphism was absent among NR patients, compared with 38.5% of R patients displaying CCR2-64I (p = 0.053), although the differences were not significant. In conclusion, starting HAART in antiretroviral drug-naive HIV-infected patients followed in daily clinical practice prevented viral breakthrough for up to 44 months in 60% of the patients. Virologic failure was associated with the development of resistance-related mutations, a later stage of disease at start of therapy and lower PI drug levels.

Presence of 2',5'-Bis-O-(tert-butyldimethylsilyl)-3'-spiro-5"-(4"-amino-1",2"-oxath iole-2",2"-dioxide) (TSAO)-resistant virus strains in TSAO-inexperienced HIV patients.

HIV-1 samples from six patients undergoing diverse anti-HIV therapies possessed the E138A mutation in their reverse transcriptase (RT) genome. Patients were receiving the following therapies: TIBO monotherapy (one patient); zidovudine plus didanosine combination therapy (one); zidovudine monotherapy (one); sequential therapy with zidovudine, then stavudine and finally zalcitabine plus didanosine (one); and two were drug naive. E138K, not E138A, is a known TSAO-specific resistance mutation, emerging under selective pressure in vitro. Our phenotypic data on the patient isolates, confirmed by data on an E138A mutant acquired through in vitro mutagenesis, indicated that an alanine substitution for glutamate at codon 138 of the HIV-1 RT renders the virus TSAO resistant, confirming the importance of this amino acid residue in the activity of TSAO derivatives. In addition, we have demonstrated through phenotypic analysis of the E138A and A98S mutants (after in vitro mutagenesis) that the mutation A98S, found in one of these patients, could be partially responsible for the phenotypic reversal of TSAO resistance. This reversal could be explained by the restoration of a hydrogen bond between 98S and the main-chain residue L349, which compensates for the loss of the E138-G99 main-chain hydrogen bond. As TSAO derivatives have not been used in the clinical setting, the presence of the E138A mutation at a frequency of 6.7% in our study of 90 TSAO-inexperienced HIV-seropositive individuals implies that 138A of the RT must be a natural variant and that the mutant virus is replication competent. Our observations suggest that the E138A mutation may likely arise in patients under the selective pressure of TSAO or related compounds that show a decreased antiviral potency toward the E138A variant.

Antiviral activity of the bicyclam derivative JM3100 against drug-resistant strains of human immunodeficiency virus type 1.

Bicyclams have recently been identified as potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) replication. The prototype of this series, JM3100 exhibits anti-HIV potency at concentrations ranging from 0.001 to 0.01 micrograms/ml. JM3100 proved to be active when tested against HIV strains resistant to the reverse transcriptase (RT) inhibitors 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (DDI), 3TC, alpha APA and TIBO, at roughly the same concentrations as for the wild-type strain. The virus was passaged in vitro in the presence of increasing concentrations of either TIBO or alpha APA alone or in combination with JM3100. The combination between TIBO, or alpha APA, and JM3100 delayed the development of TIBO- and alpha APA-resistant strains, without emergence of resistance to JM3100. In separate experiments, it took more than 60 passages (300 days) in MT-4 cells and 20 passages (140 days) in peripheral blood lymphocyte (PBL) cells for the virus to become resistant to JM3100. The JM3100-resistant virus showed cross-resistance to sulfated polysaccharides such as dextran sulfate (DS), pentosan sulfate (PS), heparin and cyclodextrin sulfate (CDS), suggesting that these compounds may share a common mechanism of action. Furthermore, the inhibitory effect of JM3100 on virus-induced syncytium formation was enhanced in the presence of heparin. The results presented here provide further support for the bicyclams as attractive candidate drugs for the chemotherapy of HIV infections.

Stable interaction between G-actin and neurofilament light subunit in dopaminergic neurons.

Excessive accumulation of neurofilaments in the cell bodies and proximal axons of motor neurons is a major pathological hallmark of motor neuron diseases. In this communication we provide evidence that the neurofilament light subunit (68 kDa) and G-actin are capable of forming a stable interaction. Cytochalasin B, a cytoskeleton disrupting agent that interrupts actin-based microfilaments, caused aggregation of neurofilaments in cultured mesencephalic dopaminergic neurons, suggesting a possible interaction between neurofilaments and actin; which was tested further by using crosslinking reaction and affinity chromatography techniques. In the cross-linking experiment, G-actin interacted with individual neurofilament subunits and covalently cross-linked with disuccinimidyl suberate, a homobifunctional cross-linking reagent. Furthermore, G-actin was extensively cross-linked to the light neurofilament subunit with this reagent. The other two neurofilament subunits showed no cross-linking to G-actin. Moreover, neurofilament subunits were retained on a G-actin coupled affinity column and were eluted from this column by increasing salt concentration. All three neurofilament subunits became bound to the G-actin affinity column. However, a portion of the 160 and 200 kDa neurofilament subunits did not bind to the column, and the remainder of these two subunits eluted prior to the 68 kDa subunit, suggesting that the light subunit exhibited the highest affinity for G-actin. Moreover, neurofilaments demonstrated little or no binding to F-actin coupled affinity columns. The phosphorylation of neurofilament proteins with protein kinase C reduced its cross-linking to G-actin. The results of these studies are interpreted to suggest that the interaction between neurofilaments and actin, regulated by neurofilament phosphorylation, may play a role in maintaining the structure and hence the function of dopaminergic neurons in culture.

Prevalence of nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance-associated mutations and polymorphisms in NNRTI-naïve HIV-infected patients.

BACKGROUND: The efficacy of treatment containing nonnucleoside reverse transcriptase inhibitors (NNRTIs) could be compromised in NNRTI-naïve patients already harboring a virus resistant to NNRTIs. On the contrary, hypersusceptibility to NNRTIs in patients having failed nucleoside reverse transcriptase inhibitor (NRTI)-containing regimens has been described and has been associated with improved outcome. METHOD: We assessed the prevalence of NNRTI resistance-associated mutations or polymorphisms in 146 antiretroviral-naïve patients and in 181 HIV-infected patients who were given an NNRTI-based regimen. We phenotypically evaluated the NNRTI susceptibility of 41 strains presenting with amino acid substitutions at positions involved in NNRTI resistance. RESULTS: In the 268 genotypically analyzable samples, the overall prevalence of NNRTI resistance-associated mutations was 2% (6/268 patients). The prevalence of strains with amino acid substitutions at reverse transcriptase (RT) gene positions (A98, K101, K103, V106, V108, V179) involved in NNRTI resistance was 15%. Hypersusceptibility to NNRTI was rare (2%, 1/41) in those samples. RT substitutions at positions involved in NNRTI resistance were not associated with a significantly worse virologic outcome in NNRTI-treated patients. Our understanding of small shifts in IC50 values (higher or lower) toward NNRTI is very limited. The significance of many RT mutations on NNRTI susceptibility is not clear. CONCLUSION: In contrast to resistance mutations, RT substitutions at positions involved in NNRTI resistance are frequent. They are not associated with a worse virologic outcome or with decreased phenotypic susceptibility to NNRTIs. It may be prudent not to rule out the use of NNRTIs in patients with small shifts in IC50 values or poorly understood mutations.

Three-year effectiveness of highly active antiretroviral treatment in the Luxembourg HIV cohort.

UNLABELLED: Clinical trials have shown that highly active antiretroviral treatment (HAART) is able to reduce HIV plasma viral loads to undetectable in 70% to 90% of patients and to increase CD4 cell counts. HAART in community settings (i.e., nonclinical trial situations) is reported to be much less effective. STUDY DESIGN: Observational study. PURPOSE: The aim of our study was to evaluate the effectiveness of protease inhibitor (PI)-based HAART in the Luxembourg HIV cohort after 36 months of treatment in previously treated and untreated patients. The secondary aim was to identify surrogate markers associated with long-term virologic and immunologic outcomes. PATIENTS AND METHOD: Seventy-three PI-naive patients, who started on HAART, combining one PI and two nucleoside reverse transcriptase inhibitors (NRTIs),with a follow-up of 3 years, were evaluated with plasma viral load and CD4 cell counts every 3 months and were analyzed retrospectively. Patients who had been treated previously with NRTI (n = 48) were at a more advanced stage of disease. RESULTS: Overall, there was a mean decrease in viral load compared to baseline of -1.89 log RNA copies/mL (SD = 1.40) that persisted at month 36. Sixty-two percent (62%) of patients reached an undetectable viral load (i.e., below 500 copies/mL): 82% and 53% of NRTI-naive and NRTI-experienced patients, respectively (p =.013). CD4 cell counts increased progressively in both groups with a sustained effect (mean increase of 146 cells/mL +/- 241) at month 36. NRTI-naive patients had a mean increase of 257 cells/mL (SD = 305), in contrast to experienced patients who had an increase of 108 cells/mL (SD = 206) at 3 years. Proportions of patients with a CD4 count under 200 cells/mL fell after 3 years for NRTI-naive (from 66% to 43%) and for experienced patients (from 32% to 13%). Predictors of short duration of viral load response were in decreasing order of importance: clinical AIDS, the use of saquinavir hard gel formulation as initial PI, and the number of NRTIs previously used. Viral load response was the only significant predictor of CD4 changes. CONCLUSION: In a community setting, effectiveness of PI-based HAART at 3 years is still achieved for most patients. NRTI-experienced patients have a good long-term response rate even if it is lower than NRTI-naive patients. A poor treatment response is associated with a more advanced stage of disease before HAART is introduced.

Detection of HIV-1 RNA in plasma and serum samples using the NASBA amplification system compared to RNA-PCR.

The presence of HIV-1 RNA in the plasma and serum of European and African patients was monitored using RNA-polymerase chain reaction (RNA-PCR) and the new isothermal NASBA nucleic acid amplification system encompassing a gel-based detection assay (ELGA). Identical RNA extraction procedures, provided by the NASBA amplification system, were used for both methods. The detection limit for HIV-1 RNA, measured on a 10-fold dilution series of spiked HIVIIIB in negative plasma, was about 0.05 CCID50 per test for both methods. Both NASBA and RNA-PCR were more sensitive than a p24 assay for the detection of circulating HIV-1 virus in blood: 17 of the 34 (50%) p24 antigen-tested seropositives were p24-positive while 32 (94%) were positive by NASBA and 30 (88%) by RNA-PCR. Among the 45 seropositives, 34 of which were tested for p24 antigen, 43 (96%) were positive by NASBA and 41 (91%) by RNA-PCR. Almost all seropositives had a detectable viral load in 100 microliters plasma. Lower viral loads were only encountered in some healthy seropositives with a higher CD4 count. There was no cross-reactivity with HIV-2 or HIV-I with both the RNA-PCR and NASBA. The extraction method used permitted the detection of HIV-1 RNA equally well in serum and in plasma with heparin or EDTA.

Comparison of the LiPA HIV-1 RT test, selective PCR and direct solid phase sequencing for the detection of HIV-1 drug resistance mutations.

The performance to detect drug resistance mutations in the reverse transcriptase gene of HIV-1 was compared for direct solid phase sequencing, selective polymerase chain reaction (PCR) using the amplification refractory mutation system (ARMS) and the new line probe assay (LIPA) HIV-1 RT. The three tests were undertaken on 50 plasma samples from 25 treatment-experienced patients under combination therapy with dideoxynucleoside analogues. LiPA HIV-1 RT gave interpretable results in 80 to 96% of the samples depending on the codon of interest. In 2% of the samples a failure to amplify resulted in uninterpretable results for sequencing. ARMS gave no result in seven samples (14%). Overall, there was a 73 to 100% concordance between the three methods. In this study, LiPA HIV-1 RT proved to be an accurate and reliable alternative to DNA sequencing for the detection of drug resistance mutations in patient samples.