Amalia Rubio

Prevalence of genotypic resistance to nucleoside analogues in antiretroviral-naive and antiretroviral-experienced HIV-infected patients in Spain.

Objective: To determine the prevalence of genotypic resistance to nucleoside analogues (NA) in a large group of HIV‐infected individuals in Spain, some of whom had no previous treatment with antiretroviral drugs (antiretroviral‐naive) and some of whom had such experience (antiretroviral‐experienced). Setting: Cross‐sectional study in outpatient clinics in three reference hospitals for HIV/AIDS located in Barcelona, Madrid and Seville, Spain. Patients and methods: Primary mutant genotypes were examined in plasma HIV RNA collected from 150 antiretroviral‐naive subjects, half in 1993 and the other half in 1997. Furthermore, drug resistance mutations were analysed in plasma collected from another 150 antiretroviral‐experienced patients who had received 2 NA for longer than 1 year, either in sequence as monotherapy or as combination therapy. A line probe assay was used for recognizing mutations conferring resistance to zidovudine (ZDV), didanosine (ddI), zalcitabine (ddC), and lamivudine (3TC). A point‐mutation nested‐PCR assay was used for examining a codon 151 mutation associated with multiple drug resistance. Results: One or more mutations associated with primary resistance to NA were seen in 10 antiretroviral‐naive (13.3%) patients in 1993 and in nine (12%) in 1997. In all but two cases, they were associated with ZDV resistance. In contrast, all but six (96%) of the antiretroviral‐experienced subjects harboured drug‐resistant mutant viruses. The codon 184 mutation (associated with resistance to 3TC) was detected in 92% of patients treated with 3TC, but also in 18% of those treated with only ddI or ddC. The codon 215 mutation was found in 67.3% of patients who had been exposed to ZDV; the codon 69 mutation was found in 15% of patients treated with ddC; and the codon 74 mutation was found in only 7.2% of patients treated with ddI. Finally, the codon 151 multidrug resistant mutation was found in four (2.7%) of 150 patients with a longterm exposure to NA. Conclusions: Overall, the prevalence of drug‐resistant HIV‐1 genotypes was 12.7% in antiretroviral‐naive patients, most of whom had ZDV‐resistant mutants. There is no evidence of an increase during the last 5 years. However, multidrug‐resistant HIV genotypes are currently circulating in Spain.

Prevalence of genotypic resistance to nucleoside analogues and protease inhibitors in Spain.

ObjectiveTo examine the prevalence of resistance mutations to nucleoside reverse transcriptase inhibitors (NRTI) and protease inhibitors (PI) in a representative HIV-1 population in Spain. MethodsA 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. ResultsPrimary resistance to NRTI was recognized in nine out of 52 (17%) naïve 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. ConclusionThe 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.

Drug resistance in patients experiencing early virological failure under a triple combination including indinavir.

ObjectiveTo assess the pattern of drug resistance mutations selected in HIV-1-infected patients failing a first line triple combination therapy including indinavir. Patients and methodsPlasma samples from 87 patients collected at the time of the first virological rebound (> 50 HIV-RNA copies/ml) were examined for the presence of drug-resistant genotypes. ResultsThe mean level of plasma viraemia at rebound was 7824 HIV-1 RNA copies/ml in 73 subjects with good compliance, whereas it was 359 460 HIV-1 RNA copies/ml in 14 patients who admitted to poor adherence. Genetic sequence analysis yielded results for 51 (70%) of the patients having good adherence. More than half of them (26/51, 51%) carried primary mutations associated with resistance to nucleoside analogues. In contrast, primary protease inhibitor resistance mutations were recognized less frequently (14/51, 27%; P < 0.05). Moreover, in 23 (45%) patients there was no evidence of drug-resistant viruses at all. The most frequent drug-resistant genotypes in the reverse transcriptase gene were at codons 184 (n = 19), 215 (n = 14) and 41 (n = 8), whereas for the protease they were at codons 46 (n = 10), 82 (n = 9) and 90 (n = 7). No resistance genotypes were found among non-compliant patients. ConclusionThe overall rate of drug-resistant HIV genotypes was 38% (28/73) in patients with good adherence and who were experiencing a first virological failure under a triple combination regimen including indinavir; resistance to nucleoside analogues was more frequent than resistance to indinavir. Therefore, treatment intensification in those patients without resistance, or a selective substitution of nucleosides in those with resistance limited to these compounds, might be justified.

Melatonin biosynthesis in the thymus of humans and rats

Abstract.Melatonin is an indoleamine widely distributed in the evolution that shows a great functional versatility, playing an important role as a transmitter of photoperiodic information and exhibiting antioxidant, oncostatic, anti-aging and immunomodulatory properties. In vertebrates, this molecule is produced by the pineal gland and other extrapineal sites. The present study was carried out to investigate the presence of melatonin in thymus and the possibility of an endogenous melatonin synthesis in this organ, in which T cells are matured. In this work, we demonstrate in humans and rats that thymus contains melatonin, expresses the mRNAs encoding N-acetyltransferase and hydroxyindol-O-methyltransferase, the two key enzymes of the melatonin synthesis, and has this biosynthetic machinery activated. In addition, rat thymocytes cultured for 24 h exhibited high levels of melatonin. The results presented here suggest that human and rat thymuses are able to synthesize melatonin, which could have intracrine, autocrine and paracrine functions.

CD4+ and CD8+ T lymphocyte regeneration after anti-retroviral therapy in HIV-1-infected children and adult patients

Previous studies have shown a slow recovery of naive CD4+ T cell counts after anti‐retroviral therapy in HIV‐1‐infected adults, which is in accordance with thymus atrophy after puberty. Here we investigate whether or not different patterns of naive CD4+ and CD8+ T cell repopulation are present in adult and child patients undergoing anti‐retroviral treatment. Thus, 25 adults under highly active anti‐retroviral therapy and 10 children under combined anti‐retroviral therapy were retrospectively analysed for T cell subpopulations at baseline (T0) and around week 12 (T1) and week 24 (T2) of anti‐retroviral treatment. Mean serum HIV‐1 RNA levels dropped in both groups. Recovery of T cells in adults was characterized by a heterogeneous response between patients, with only 44% of them increasing their naive CD4+ and CD8+ T cell counts at T1, and changes in mean total CD4+ T cells were mainly shaped by memory cells. Otherwise, children were characterized by an early increase in naive T cells. Thus, at T1, all children analysed had a strong rise in CD4+ (from 389 ± 116 to 569 ± 121 cells/μl; P < 0·01), and nine out of 10 also in naive CD8+ T cells (from 244 ± 58 to 473 ± 85 cells/μl; P < 0·05). However, no significant correlation between age and naive repopulation was observed (P= 0·22) in children. Thus, children had the earlier and greater increases in naive T cell subsets than adults, probably due to a more active thymus, with the potential for immune reconstitution when HIV‐1 replication is controlled.

Melatonin synthesized by T lymphocytes as a ligand of the retinoic acid-related orphan receptor.

Abstract:  Melatonin modulates a wide array of physiological events with pleiotropic effects on the immune system. While the relevance of specific melatonin membrane receptors has been well established for several biological functions, retinoic acid‐related orphan receptor alpha (RORα) has been suggested as a mediator of nuclear melatonin signalling by results obtained from pharmacological approaches. However, a melatonin‐mediated downstream effect cannot be ruled out, and further evidence is needed to support a direct interaction between melatonin and RORα. Here, we show that RORα is mainly located in human Jurkat T‐cell nucleus, and it is co‐immunoprecipitated with melatonin. Moreover, immunocytochemistry studies confirmed the co‐localization of melatonin and RORα. Melatonin promoted a time‐dependent decrease in nuclear RORα levels, suggesting a role in the RORα transcriptional activity. Interestingly, RORα acts as a molecular switch implicated in the mutually exclusive generation of Th17 and Treg cells, both involved in the harm/protection balance of immune conditions such as autoimmunity or acute transplant rejection. Therefore, the identification of melatonin as a natural modulator of RORα gives it a tremendous therapeutic potential for a variety of clinical disorders.

Role of early cell-free DNA levels decrease as a predictive marker of fatal outcome after severe traumatic brain injury.

INTRODUCTION Circulating cell-free DNA levels are increased after trauma injury. This increase is higher since the first hours after trauma and may be related with primary outcome. A sensitive and reliable biomarker for patients at higher risk is needed to identify these patients to initiate early intervention. In this way, circulating DNA may be a possible biological marker after severe TBI. MATERIALS AND METHODS We investigated DNA plasma concentrations after severe traumatic brain injury and during the next 96 h in the Intensive Care Unit (ICU) by real time PCR. 65 patients suffering severe TBI were included in the study. RESULTS Cell-free DNA levels were considerably higher in patients samples compared with voluntary control ones. After the following four days we observed a 51% decrease during the first 24h and a 71% fall from 48 h. TBI population was stratified for the primary outcome (survivors/non-survivor) and DNA levels decrease ratio was calculated for the first 48 h. A higher decrease in the survivors from 0 h to 24h compared with the non-survivors was found. A cut-off point of 1.95 ratio was established for the detection of the highest proportions of patients after the TBI that will not survive after the injury with a sensitivity of 70% and specificity of 66%. CONCLUSIONS In summary we showed that severe TBI is associated with elevated cf-DNA levels and we propose that cf-DNA decrease during the first 24h may predict patient outcome.

Characterization of melatonin binding sites in the harderian gland and median eminence of the rat.

The characterization of specific melatonin binding sites in the Harderian gland (HG) and median eminence (ME) of the rat was studied using [125I]melatonin. Binding of melatonin to membrane crude preparations of both tissues was dependent on time and temperature. Thus, maximal binding was obtained at 37 degrees C after 30-60 min incubation. Binding was also dependent on protein concentration (up to 1.5 mg/ml). The specific binding of [125I]melatonin was saturable, exhibiting only one class of binding sites in both tissues. The dissociation constants (Kd) were 170 and 190 pM for ME and HG, respectively. The concentration of the binding sites in ME was 8 fmol/mg protein, and in the HG 4 fmol/mg protein. In competition studies, binding of [125I]melatonin to ME or HG was inhibited by increasing concentration of native melatonin; 50% inhibition was observed at about 702 and 422 nM for ME and HG, respectively. Additionally, the [125I]melatonin binding to the crude membranes was not affected by the addition of different drugs such as norepinephrine, isoproterenol, phenylephrine, propranolol, or prazosin. The results confirm the presence of melatonin binding sites in median eminence and show, for the first time, the existence of melatonin binding sites in the Harderian gland.

Changes in the rate of genotypic resistance to antiretroviral drugs in Spain.

Genotypic resistance to antiretroviral drugs was examined in 684 individuals attending 18 outpatient clinics distributed across Spain in June 2000. Compared with similar surveys conducted before 1998, the prevalence of resistance to nucleosides has declined significantly among naive chronic HIV carriers. In contrast, resistance among pre-treated patients has increased; resistance to all three drug families having been recognized in nearly a quarter of patients.

A novel interplay between membrane and nuclear melatonin receptors in human lymphocytes : significance in IL-2 production

Abstract.Human lymphocyte melatonin, through membrane and nuclear receptors binding, acts as an activator in IL-2 production. Antagonism of membrane melatonin receptors using luzindole exacerbates the drop of the IL-2 production induced by PGE2 in peripheral blood mononuclear and Jurkat cells. This paper studies the melatonin membrane and nuclear receptors interplay in PGE2-diminished IL-2 production. The decrease in IL-2 production after PGE2 and/or luzindole administration correlated with downregulation in the nuclear receptor RORα. We also highlighted a role of cAMP in the pathway, because forskolin mimicked the effects of luzindole and/or PGE2 in the RORα expression. Finally, a significant RORα downregulation was observed in T cells permanently transfected with inducible MT1 antisense. In conclusion, we show a novel connection between melatonin membrane receptor signalling and RORα expression, opening a new way to understand melatonin regulation in lymphocyte physiology.