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Biomedical subjects

L Ruiz

Publications and source records attributed to L Ruiz.

At least 37 records · Page 2Linked to original sources
PubMed2001

Phenotypic cross-resistance to nelfinavir: the role of prior antiretroviral therapy and the number of mutations in the protease gene.

Cross-resistance to nelfinavir (NFV) is observed in patients failing protease inhibitor (PI)-containing therapies. We performed a study with 111 patients who started an NFV-based salvage regimen after failing PI-based therapy to evaluate genotypic changes and to identify factors associated with resistance to NFV. Genotypic and phenotypic resistance data at entry (111 and 51 samples) and after NFV failure (74 and 31 samples) were available. Median CD4(+) cell count was 208 x 10(6)/liter, HIV RNA level was 4.6 log(10) copies/ml, and median number of mutations in the protease was 9. At baseline, 51 and 14% of viral isolates showed high or intermediate phenotypic resistance to NFV. Phenotypic data correlated with virological outcome, reaching undetectability at the third month in 40, 14, and 0% of those patients with susceptible, intermediate, or resistant viral isolates, respectively. Phenotypic resistance to NFV was associated with the presence of the L90M mutation: 46% for resistant vs. 6% in susceptible strains. The number of mutations in the protease correlated with the fold-increase in the IC(50)-NFV. The D30N mutation was detected in only 1 of 74 patients who failed. In a logistic regression analysis, the number of mutations in the protease was associated with NFV cross-resistance (RR, 2.09 per each additional mutation; 95% CI 1.23-3.55; p < 0.01). In conclusion, phenotypic cross-resistance to NFV for PI-experienced patients can be predicted by the number of mutations in the protease. The L90M mutation is significantly associated with the subsequent failure of NFV-containing regimens. The presence of the D30N mutation was rare and not useful in identifying NFV-resistant isolates.

Adult↗
PubMed2001

Actin cytoskeleton role in the structural response of epithelial (MDCK) cells to low extracellular Ca2+.

Kinetic and stereometric assessment of the mechanical responses of epithelial cells to variations in the concentration of extracellular Ca2+ was carried out in vivo at the single cell level. Continuous monitoring of individual MDCK cells in subconfluent cultures attested to an intense, immediately relaxable, and cytochalasin D-sensitive contraction, equivalent to that seen in confluent monolayers following depletion of external Ca2+ (<0.1 mM). Increasingly greater and less readily reversible contractions were performed upon repeated stimulation with short-term cycles of alternating normal (30 min) and low Ca2+ (30 min) media. Constriction of a narrow horizontal girdle corresponding in position to the major ring-like bundle of actin filaments eventually develops into a deep lateral furrow in intensely contracted cells. Substantial membrane infolding in the contracted state is indicated also by stereometric estimates of apparent bounding surface area. Irrespective of the contracted or relaxed cell condition, rhodamine-phalloidin labeling showed a marginal position of the ring-like bundle of microfilaments and other components of the actin cytoskeleton. These results suggest, contrary to prevalent views, that the actin-myosin system stays associated to the cortex and retains contractile capability in epithelial cells deprived of external Ca2+. Hence, the mechanical responses to variations of Ca2+ may be an overstrained expression of a physiological mechanism.

Actin Cytoskeleton↗
PubMed2001

delta(9)-Tetrahydrocannabinol increases nerve growth factor production by prostate PC-3 cells. Involvement of CB1 cannabinoid receptor and Raf-1.

Cannabinoids, the active components of marihuana, exert a variety of effects in humans. Many of these effects are mediated by binding to two types of cannabinoid receptor, CB1 and CB2. Although CB1 is located mainly in the central nervous system, it may also be found in peripheral tissues. Here, we study the effect of cannabinoids in the production of nerve growth factor by the prostate tumor cell line PC-3. We show that addition of Delta(9)-tetrahydrocannabinol to PC-3 cells stimulated nerve growth factor production in a dose-dependent and time-dependent manner. Maximal effect was observed at 0.1 microM Delta(9)-tetrahydrocannabinol and 72 h of treatment. Stimulation was reversed by the CB1 antagonists AM 251 and SR 1411716A. Pre-treatment of cells with pertussis toxin also prevented the effect promoted by Delta(9)-tetrahydrocannabinol. These results indicate that Delta(9)-tetrahydrocannabinol stimulation of nerve growth factor production in these cells was mediated by the cannabinoid CB1 receptor. The implication of Raf-1 activation in the mode of action of Delta(9)-tetrahydrocannabinol is also suggested.

Dose-Response Relationship, Drug↗
PubMed2001

Primary HIV-1 drug resistance in Spain before and after the introduction of protease inhibitors.

The prevalence of genotypic resistance to nucleoside analogues was examined in 150 naive individuals with chronic HIV-1 infection acquired before June 1996, when protease inhibitors began to be used in Spain, and in a group of 52 recent seroconverters infected since 1997. Overall, the prevalence of subjects carrying nucleoside-resistant genotypes was similar in both groups (12.6% and 15.4%, respectively). A polymorphism at codon 70 accounted for more than half of cases (63.1%) in naive individuals infected before protease inhibitors became available, in contrast most recent seroconverters carried genotypes with one or more key nucleoside resistance mutations, mainly associated with lack of sensitivity to zidovudine or lamivudine (prevalence 9.6% and 5.9%, respectively). When only key mutations were considered in each of the two periods, the prevalence of nucleoside-resistant genotypes was increased significantly among recent seroconverters (13.5% vs. 4.7%; P<0.05). The relatively high rate of resistance to nucleosides in Spain justifies the introduction of drug resistance testing in naive recent seroconverters before beginning antiretroviral therapy.

Anti-HIV Agents↗
PubMed2000

Prospective randomized two-Arm controlled study to determine the efficacy of a specific intervention to improve long-term adherence to highly active antiretroviral therapy.

BACKGROUND: Nearly perfect compliance seems to be indispensable to obtain the maximum benefit from highly active antiretroviral therapy (HAART). Interventions to ensure a high level of adherence during a relatively long-term period of therapy are necessary. METHODS: This is a prospective, randomized, two-arm controlled study including patients starting their first-or second-line HAART who were randomized to receive psychoeducative intervention to implement adherence (experimental group [EG]) or a usual medical follow-up (control group [CG]). We aimed to study the efficacy of a psychoeducative intervention to ensure long-term adherence to HAART, its relation with the virologic efficacy of treatment, and to determine the variables related to long-term adherence. Visits were made at weeks 0, 4, 24, and 48 for data collection. Self-reported adherence was registered at each visit and its veracity was tested by randomized blood analyses performed without previous warning to 40% of patients. Appropriate adherence was defined as the consumption of >/=95% of medication prescribed. Statistical analyses were performed both by the as treated (AT) and the intention to treat missing = failure (ITT) methods. RESULTS: In all, 116 patients were included. At week 48, 94% of patients in the EG versus 69% controls achieved adherence >/=95% (p =.008); 89% of patients in the EG versus 66% controls had HIV-1 RNA levels <400 copies/ml (p =.026). Overall, 85% of patients with adherence >/=95% but only 45% of those with adherence <95% had viral load (VL) <400 copies/ml (p =. 008). In multivariate analysis, variables significantly related to adherence were having received a psychoeducative intervention (odds ratio [OR], 6.58; p =.04), poor effort to take medication (OR, 5.38; p =.03), and high self-perceived capacity to follow the regimen (OR, 13.76; p =.04). Self-reported adherence and drug plasma levels coincided in 93% of cases. However, differences in adherence did not reach statistical significance in the ITT analysis although a clear tendency toward benefit was observed in EG. CONCLUSIONS: Specific and maintained psychoeducative interventions based on excellence on clinical practice are useful to keep high levels of adherence as well as high levels of viral suppression. There is a clear relation between high adherence levels and virologic success. Assessment of certain specific variables related to adherence may be helpful to monitor patient's compliance in the clinical setting.

Adult↗
PubMed2000

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.

Anti-HIV Agents↗
PubMed2000

Prevalence of genotypic resistance to nucleoside analogues and protease inhibitors in Spain. The ERASE-2 Study Group.

OBJECTIVE: To examine the prevalence of resistance mutations to nucleoside reverse transcriptase inhibitors (NRTI) and protease inhibitors (PI) in a representative HIV-1 population in Spain. METHODS: A cross-sectional study was conducted including 601 HIV-infected patients who attended 20 Spanish hospitals in June 1998. Drug resistant mutations were examined using hybridization line probe assays (LiPA). The 6 bp insert at position 69 and the codon 75 mutant were examined by sequencing analysis in specimens lacking reactivity to 69/70 and 74 bands on LiPA, respectively. RESULTS: Primary resistance to NRTI was recognized in nine out of 52 (17%) naive individuals, whereas primary resistance to PI was found in seven out of 126 (6%) PI-naïve patients. The codons most frequently involved in NRTI resistance were at positions 70 (66%), 184 (44%), 215 (33%), and 41 (11%), whereas the most common PI resistance mutation was at codon 82 (6/7 subjects). In pre-treated patients, the overall prevalence of resistant genotypes was 72.9% for NRTI and 27.2% for PI. The most frequent NRTI mutations occurred at codons 184 (38.5%), 215 (30.1%), and 41 (22.5%), whereas the most frequent PI mutations in pre-treated subjects were found at positions 82 (15.8%) and 84 (11.4%). Overall, patients who began triple combinations as initial therapy showed a lower number of key resistance mutations than those who began highly active antiretroviral therapy (HAART) after being exposed to NRTI for a period of time (mean number of mutations, 0.1 versus 1.8, P< 0.05). Codon 75 mutant was found in three out of 387 patients (0.7%), whereas no insertions at codon 69 were recognized. CONCLUSION: The prevalence of primary genotypic resistance to NRTI and PI in Spain was 17% and 6%, respectively. Zidovudine, lamivudine, indinavir and ritonavir were the drugs most frequently affected. These data support the use of resistance testing prior to the introduction of first-line antiretroviral therapies in Spain. Among pre-treated subjects, drug resistance genotypes were less prevalent in those who began HAART as initial therapy.

Adult↗
PubMed2000

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.

CD4 Lymphocyte Count↗
PubMed2000

Structured treatment interruption in chronically HIV-1 infected patients after long-term viral suppression.

OBJECTIVE: To investigate the virological and immunological impact of a structured treatment interruption (STI) in a cohort of HIV-1 chronically infected patients with a further long-lasting effective virus suppression. METHODS: Twelve HIV-1 chronically infected adults who had maintained viral suppression (< 20 copies/ml) for more than 2 years, as well as a CD4:CD8 ratio > 1 for a median time of 22 months, were included in the study. Participants interrupted their antiretroviral treatment during a maximum period of 30 days or until a viral load rebound > 3000 copies/ml was detected. The same prior antiretroviral regimen was resumed after STI. Kinetics of plasma viral rebound was evaluated every 2 days during the treatment interruption period. Flow cytometry and cell proliferation assays were performed before and after STI. Genotypic resistance was assessed at the time of treatment resumption. RESULTS: No adverse events occurred during the interruption period. In two patients no viral rebound was detected after 30 days of treatment interruption. In the remaining 10 patients, viral load became detectable (> 20 copies/ml) at a median time of 14 days after treatment interruption. Afterwards, viral load increased exponentially with a mean t1/2 of 1.6 days. Treatment was successfully resumed in all patients. No resistance-conferring mutations associated with the pre-interruption antiretroviral regimen were detected. The percentage of CD4 and CD8 lymphocytes did not vary during the STI period; however, the level of expression of T-cell activation antigen CD38 on CD8 T cells increased significantly in response to viral rebound. Four patients gained T-helper cell responses to recall antigens (tuberculin and tetanus toxoid), two of who developed an HIV-specific response to p24. CONCLUSIONS: STI in chronically HIV-1-infected patients is not associated with reductions in CD4 T lymphocytes or to clinical complications in this group of patients after 2 years of effective plasma viral suppression. Viral load rebounds in most but not all patients, without evidence of selection of resistance-conferring mutations. Thereafter, viraemia can be effectively controlled by antiretroviral agent reintroduction. HIV-specific T-helper cell responses may be achieved after one cycle of treatment interruption suggesting some degree of immune-stimulation. These data do not discard consecutive cycles of STI as a therapeutic strategy to boost HIV-specific immunity in order to maintain viral replication under effective control.

Adult↗
PubMed2000

Sustained high-level expression of full-length human factor VIII and restoration of clotting activity in hemophilic mice using a minimal adenovirus vector.

The successful prophylactic treatment of hemophilia A by frequent infusions of plasma concentrates or recombinant factor VIII (hFVIII) indicates that gene therapy may be a potential alternative for the treatment of the disease. For efficient delivery and long-term expression of the hFVIII gene, a novel minimal adenovirus (mini-Ad) vector, MiniAdFVIII, has been developed. The vector is devoid of all viral genes and carries the full-length hFVIII cDNA under the control of the human 12.5-kb albumin promoter. The MiniAdFVIII vector was propagated with the assistance of an ancillary vector in 293 cells and was purified by CsCl banding. Sustained expression of hFVIII at physiologic levels (100-800 ng/mL) was achieved in mice after a single intravenous injection of MiniAdFVIII. The expressed hFVIII had a structure identical to that of recombinant hFVIII, as determined by Western blot analysis. The functionality of the protein was confirmed by the restoration of blood coagulation capacity in MiniAdFVIII-treated hemophilic mice, as determined by tail clipping observations. Although antivector or antihuman FVIII antibodies at various levels were detected, long-term expression of the transgene was observed in the mice that did not generate antibodies against the transgene product. The vector DNA persisted in the liver tissues of the mice with long-term expression. No significant histopathologic findings or toxicities were observed to be associated with the vector in the MiniAdFVIII-treated C57BL/6 mice. These results support the further development of MiniAdFVIII for clinical trials toward the treatment of hemophilia A.

Adenoviridae↗
PubMed2000

Non-nucleoside reverse transcriptase inhibitor resistance among patients failing a nevirapine plus protease inhibitor-containing regimen.

OBJECTIVE: To determine the rate of nevirapine resistance in patients failing a nevirapine plus protease inhibitor (PI)-based regimen, and whether these isolates remain susceptible to other non-nucleoside reverse transcriptase inhibitors (NNRTI). DESIGN AND SETTING: A retrospective cohort study in two tertiary university hospitals. PATIENTS: Eighty-eight HIV-infected, NNRTI-naive patients receiving nevirapine plus PI as a rescue regimen after PI treatment failure. MAIN OUTCOME MEASURES: Genotypic and phenotypic resistance data at inclusion (73 and 60 plasma samples, respectively) and after 24 weeks (53 and 42 samples). RESULTS: Baseline phenotypic susceptibility to nevirapine was found in 70% of patients, and similar data were observed for efavirenz (91%) and delavirdine (71%). NNRTI resistance-associated mutations were found in 11 patients (12.5%). At 24 weeks, resistant isolates to nevirapine were found in 92% of patients, and correlated with similar resistance to efavirenz (68%) and delavirdine (73%). In the genotypic analysis, the Y181 C mutation was observed in 76% of mutants, and the most common changes were a combination of mutations at positions Y181C/K103N (23%) and the single mutation Y181C (15%). The development of nevirapine resistance was associated with baseline resistance to PI included in the regimen (P= 0.01). For isolates containing the single amino acid substitution Y181C, 29% remained fully susceptible to efavirenz, whereas 14% showed intermediate resistance to efavirenz and delavirdine. CONCLUSION: The failure of a nevirapine plus PI-containing regimen is associated with nevirapine resistance in most patients, with the most common mutation occurring at amino acid residue 181. Although there is a high degree of cross-resistance among NNRTI, nearly one third of resistant isolates carrying the single Y181C mutation remain susceptible to efavirenz.

Adult↗
PubMed2000

Prevalence and characteristics of multinucleoside-resistant human immunodeficiency virus type 1 among European patients receiving combinations of nucleoside analogues.

The prevalence and the genotypic and phenotypic characteristics of multinucleoside-resistant (MNR) human immunodeficiency virus type 1 (HIV-1) variants in Europe were investigated in a multicenter study that involved centers in nine European countries. Study samples (n = 363) collected between 1991 and 1997 from patients exposed to two or more nucleoside analogue reverse transcriptase inhibitors (NRTIs) and 274 control samples from patients exposed to no or one NRTI were screened for two marker mutations of multinucleoside resistance (the Q151M mutation and a mutation with a 2-amino-acid insertion at codon 69, T69S-XX). Q151M was identified in six of the study samples (1. 6%), and T69S-XX was identified in two of the study samples (0.5%; both of them T69S-SS), but both patterns were absent among control samples. Non-NRTI (NNRTI)-related changes were observed in viral strains from two patients, which displayed the Q151M resistance pattern, although the patients were NNRTI naive. The patients whose isolates displayed multinucleoside resistance had received treatment with zidovudine and either didanosine, zalcitabine, or stavudine. Both resistance patterns conferred broad cross-resistance to NRTIs in vitro and a poor response to treatment in vivo. MNR HIV-1 is found only among multinucleoside-experienced patients. Its prevalence is low in Europe, but it should be closely monitored since it seriously limits treatment options.

Amino Acid Substitution↗
PubMed1999

High-dose saquinavir plus ritonavir: long-term efficacy in HIV-positive protease inhibitor-experienced patients and predictors of virologic response.

The year-long antiviral efficacy of a high-dose salvage regimen consisting of saquinavir (800 mg twice daily) plus ritonavir (400 mg twice daily) was evaluated in 58 HIV-positive patients who had seen no improvement under first-line protease inhibitor-containing regimens, nor in baseline predictors of virologic response. The efficacy of therapy was determined by CD4+/CD8+ and HIV-1 RNA values. The primary endpoint of our study was the percentage of patients with HIV-1 RNA levels <200 copies/ml (virologic success) at 6 and 12 months of of follow-up. Secondary endpoints were log10 reduction in HIV-1 RNA levels and CD4+ increases through follow-up. Surrogate markers related with a lower HIV-1 RNA area under the curve were identified at baseline. Kaplan-Meier analysis and Cox proportional hazards models were applied to identify baseline predictors of achieving viral suppression at <200 copies/ml. All analyses were intention to treat-last observation carried forward. Patients achieved a median HIV-1 RNA level reduction of >0.5 log through 1 year (-0.59 log10 at 12 months), as well as CD4+ counts increased significantly (89 cells/mm3 at 12 months). Overall, 53% of patients were likely to achieve HIV-1 RNA levels <200 copies/ml at 6 months. Seventy-six percent of patients who started therapy at HIV-1 RNA levels <5000 copies/ml but only 42% with baseline viral load of 5000 to 30,000 copies/ml and 18.7% with baseline viral load >30,000 copies/ml were likely to achieve viral suppression at 6 months (p < .001, log-rank test). Patients with baseline HIV-1 RNA levels between 5000 and 30,000 copies/ml (relative hazard [RH], 0.39; 95% confidence interval [CI], 0.01 to 0.98; p = .0396) and patients with baseline HIV-1 RNA levels >30,000 copies/ml (RH, 0.20; 95% CI, 0.07-0.61; p = .0040) were less likely to reach undetectable HIV-1 RNA levels than those with baseline HIV-1 RNA levels <5000 copies/ml. Salvage highly active antiretroviral therapy (HAART) strategies including saquinavir (SQV) at high doses plus ritonavir (RTV) exert a significant long-term efficacy in more than half of PI-experienced patients without significant additional toxicity. This therapeutic efficacy is strongly implemented by a switch at the lower HIV-1 RNA levels.

Adult↗