Primary resistance to integrase strand transfer inhibitors in Spain using ultrasensitive HIV-1 genotyping.

BACKGROUND: Transmission of resistance mutations to integrase strand transfer inhibitors (INSTIs) in HIV-infected patients may compromise the efficacy of first-line antiretroviral regimens currently recommended worldwide. Continued surveillance of transmitted drug resistance (TDR) is thus warranted. OBJECTIVES: We evaluated the rates and effects on virological outcomes of TDR in a 96 week prospective multicentre cohort study of ART-naive HIV-1-infected subjects initiating INSTI-based ART in Spain between April 2015 and December 2016. METHODS: Pre-ART plasma samples were genotyped for integrase, protease and reverse transcriptase resistance using Sanger population sequencing or MiSeq™ using a ≥ 20% mutant sensitivity cut-off. Those present at 1%-19% of the virus population were considered to be low-frequency variants. RESULTS: From a total of 214 available samples, 173 (80.8%), 210 (98.1%) and 214 (100.0%) were successfully amplified for integrase, reverse transcriptase and protease genes, respectively. Using a Sanger-like cut-off, the overall prevalence of any TDR, INSTI-, NRTI-, NNRTI- and protease inhibitor (PI)-associated mutations was 13.1%, 1.7%, 3.8%, 7.1% and 0.9%, respectively. Only three (1.7%) subjects had INSTI TDR (R263K, E138K and G163R), while minority variants with integrase TDR were detected in 9.6% of subjects. There were no virological failures during 96 weeks of follow-up in subjects harbouring TDR as majority variants. CONCLUSIONS: Transmitted INSTI resistance remains rare in Spain and, to date, is not associated with virological failure to first-line INSTI-based regimens.

Retinol-binding protein 4 (RBP4), a potential biomarker of frailty in HIV-infected people on stable antiretroviral therapy.

OBJECTIVES: A quantitative biomarker for identification of pre-frail and frail persons is still lacking. This study aimed to identify biomarker predictors of frailty in HIV-infected patients. METHODS: A cross-sectional study of HIV-infected patients who had been on antiretroviral therapy (ART) for at least 1 year and who presented an undetectable viral load (< 50 HIV-1 RNA copies/mL) at baseline was carried out. For each frail patient, up to four pre-frail and robust patients were randomly selected. The frailty status assessment was based on the five-item criteria described by Fried et al. Sociodemographic, anthropometric, biochemical and HIV-related characteristics were evaluated. Multiple potential biomarkers of frailty and a biological age biomarker were analysed. RESULTS: A total of 73 HIV-infected patients on ART for at least 1 year were evaluated. The patients were categorized as robust (n = 33), pre-frail (n = 32) and frail (n = 8) using the Fried criteria. All patients were on ART, with 100% undetectable viral load (< 50 copies/mL) at baseline. No significant differences in demographic, clinical or analytical characteristics were observed among patients in the different categories based on Fried criteria, with the exception of the veterans aging cohort study index (VACS). Similarly, no differences were observed in HIV-related characteristics, although nucleoside reverse transcriptase inhibitor (NRTI) use was less common in frail persons. The distribution of biomarker values varied according to frailty status, with frail persons having higher levels of interleukin (IL)-8, IL-18, CXC chemokine ligand 10 (CXCL10) and retinol-binding protein 4 (RBP4). In multivariable analysis, the assocation of frailty with RBP4 showed a tendency to statistical significance (odds ratio 1.0; 95% confidence interval 0.99-1.00; P < 0.05). CONCLUSIONS: Differential biomarker expression was present according to Fried status. Longitudinal studies will clarify the utility of these biomarkers as targets for diagnostic or therapeutic intervention.

[The microbiota-gut-brain axis and its great projections]. / El eje microbiota-intestino-cerebro y sus grandes proyecciones.

INTRODUCTION: The microbiota is the set of millions of microorganisms that coexist in a symbiotic way in our body. It is mainly located in the digestive tract, being distributed in function of the chemical properties and the functions of the different organs. The factors that influence its composition are multiple (diet, individual habits, diseases or drugs). It also participates in several functions of the organism such as metabolism, immunity or even the function of the central nervous system. DEVELOPMENT: This last interrelationship is called: gut-brain axis. For years the relationship between the microbiota and the central nervous system has been known and how they influence one over the other. It is postulated that communication occurs through three systems: the vagus nerve, the systemic pathway (with the release of hormones, metabolites and neurotransmitters) and the immune system (by the action of cytokines). CONCLUSIONS: There are still many unknowns to be clarified in this field, but this microbiota-intestine-brain relationship is postulated as a possible pathogenic basis for neurological diseases of great health impact such as Alzheimer, Parkinson or multiple sclerosis. There are currently studies with probiotics with hopeful results in patients with Alzheimer's disease.

TITLE: El eje microbiota-intestino-cerebro y sus grandes proyecciones.Introduccion. Se denomina microbiota al conjunto de millones de microorganismos que conviven de manera simbiotica en nuestro organismo. Este conjunto bacteriano, que se localiza principalmente en el tracto digestivo, se distribuye a lo largo de los diferentes organos en funcion de las propiedades quimicas. Los factores que influyen en su composicion son multiples (dieta, habitos individuales, farmacos). La microbiota colabora en varias funciones, como pueden ser el metabolismo o la inmunidad. Desarrollo. En los ultimos años se ha puesto de relieve el papel bidireccional de la microbiota del tracto digestivo y del sistema nervioso central, es el denominado eje intestino-cerebro. En lo que a este eje se refiere, se cree que la 'comunicacion' se produce a traves de tres vias: el nervio vago, la via sistemica (mediante la liberacion de hormonas, metabolitos y neurotransmisores) y el sistema inmune (por la accion de las citocinas). Conclusiones. Aunque aun quedan muchas incognitas por esclarecer, este eje se postula como una posible base patogena para numerosos trastornos neurologicos de gran impacto sanitario, como la enfermedad de Alzheimer, la enfermedad de Parkinson o la esclerosis multiple. En el momento actual se estan llevando a cabo estudios que intentan evaluar el impacto de los probioticos sobre algunas de estas enfermedades neurologicas.

The beneficial effect of probiotics as a supplementary treatment in drug-resistant epilepsy: a pilot study.

Epilepsy is a neurological disease with high global prevalence. Despite the range of drug-based treatments currently available to control the condition, one in 3 patients experiences epileptic seizures. Therapeutic alternatives for these patients include the ketogenic diet, surgery or the cerebral implantation of neurostimulators; however these are benefits with limits. The target of this study is to find a new complementary treatment for these patients, studying the effectiveness of probiotics for controlling epileptic seizures in patients with drug-resistant epilepsy. A prospective study was designed in which a group of patients with drug-resistant epilepsy was administered a probiotic mixture for 4 months. Patients were assessed before and after taking the probiotics; among other variables, number of seizures and patients' quality of life (QOLIE-10) were monitored. Levels of cD-14, interleukin 6, and γ-aminobutyric acid were also analysed throughout the study. 45 patients were included in the study. In an intention-to-treat analysis, 28.9% of all patients displayed a greater than 50% reduction in the number of seizures (the parameter required in clinical trials). A significant improvement was also observed in patients' quality of life. We found that probiotics may be an option for supplementary therapy. Since the use of probiotics is safe, they may contribute to improving seizure control, and therefore quality of life, in patients with drug-resistant epilepsy. The study has been registered in https://clinicaltrials.gov with number NCT03403907.

Role of mitochondria in HIV infection and associated metabolic disorders: focus on nonalcoholic fatty liver disease and lipodystrophy syndrome.

Highly active antiretroviral therapy (HAART) has considerably improved the prognosis of HIV-infected patients. However, prolonged use of HAART has been related to long-term adverse events that can compromise patient health such as HIV-associated lipodystrophy syndrome (HALS) and nonalcoholic fatty liver disease (NAFLD). There is consistent evidence for a central role of mitochondrial dysfunction in these pathologies. Nucleotide reverse transcriptase inhibitors (NRTIs) have been described to be mainly responsible for mitochondrial dysfunction in adipose tissue and liver although nonnucleoside transcriptase inhibitors (NNRTIs) or protease inhibitors (PIs) have also showed mitochondrial toxicity, which is a major concern for the selection and the long-term adherence to a particular therapy. Several mechanisms explain these deleterious effects of HAART on mitochondria, and evidence points to other mechanisms beyond the "Pol- γ hypothesis." HIV infection has also direct effects on mitochondria. In addition to the negative effects described for HIV itself and/or HAART on mitochondria, HIV-infected patients are more prone to develop a premature aging and, therefore, to present an increased oxidative state that could lead to the development of these metabolic disturbances observed in HIV-infected patients.

[Treatment of human infections caused by Bartonella spp.]. / Tratamiento de las infecciones por Bartonella spp.

Infections by Bartonella spp. include a wide spectrum of emerging and re-emerging infectious diseases. There is not a universal therapy for this infection, therefore treatment should be chosen individually. The aim of this review is to update the therapeutics aspects of this kind of infections.

Estrogen receptor regulates MyoD gene expression by preventing AP-1-mediated repression.

Cell growth and differentiation are opposite events in the myogenic lineage. Growth factors block the muscle differentiation program by inducing the expression of transcription factors that negatively regulate the expression of muscle regulatory genes like MyoD. In contrast, extracellular clues that induce cell cycle arrest promote MyoD expression and muscle differentiation. Thus, the regulation of MyoD expression is critical for muscle differentiation. Here we show that estrogen induces MyoD expression in mouse skeletal muscle in vivo and in dividing myoblasts in vitro by relieving the MyoD promoter from AP-1 negative regulation through a mechanism involving estrogen receptor/AP-1 protein-protein interactions but independent of the estrogen receptor DNA binding activity.

Rickettsia slovaca infection: DEBONEL/TIBOLA.

This study describes the epidemiological, clinical, and microbiological characteristics of a new tick-borne disease in Spain-Dermacentor-borne necrosis erythema lymphadenopathy (DEBONEL). The clinical presentations include an eschar at the site of the tick bite, surrounded by an erythema and painful regional lymphadenopathy. The disease appears during the colder months and its vector is Dermacentor marginatus (D. marginatus). From January 1990 to December 2004, 54 patients presented at Hospital of La Rioja with these clinical and epidemiological data. The ratio of females to males was 32/22. The average age was 37 years. In all cases tick bites were located on the upper body (90% on the scalp). The median incubation period was 4.7 days. Signs and symptoms were mild in all cases. Only a small number of patients presented mild and nonspecific abnormalities in a complete blood cell count and mild elevation of erythrocyte sedimentation rates and C-protein reactive and liver enzyme levels. Serological evidence of acute rickettsiosis was observed in 19 patients (61%). In 29% sera tested by polymerase chain reactions (PCRs) were positive. The sequence obtained from a PCR product revealed 98% identity with Rickettsia sp. strains RpA4, DnS14, and DnS28. All ticks removed from patients were PCR-positive. Sequencing showed 8 of them identified as R. slovaca and 2 as Rickettsia sp. strains RpA4, DnS14, and DnS28.

Prevalence of spotted fever group Rickettsia species detected in ticks in La Rioja, Spain.

Our objective was to learn the prevalence of spotted fever group (SFG) Rickettsia detected in ticks in La Rioja, in the north of Spain. From 2001 to 2005, 496 ticks representing 7 tick species were analysed at the Hospital de La Rioja. Ticks were removed from humans with or without rickettsial syndrome (n = 59) or collected from mammals (n = 371) or from vegetation by dragging (n = 66). The presence of SFG Rickettsia in these ticks was investigated by semi-nested PCR (ompA gene) and sequencing. A phylogenetic tree using Clustal method (neighbor-joining) was constructed with these data. Only 3 of 170 Hyalomma marginatum ticks carried SFG Rickettsia. Sequencing analysis demonstrated the presence of Rickettsia aeschlimannii (1.8%). Furthermore, Rickettsia massiliae and BAR29 were found in 3 of 120 Rhipicephalus sanguineus specimens (2.5%). In contrast, 81 of 83 tested Dermacentor marginatus ticks were PCR-positive (97%). Rickettsia slovaca (40.6%) and Rickettsia sp. strains RpA4, DnS14, DnS28 and JL-02 (59.3%) were found within this tick species. No SFG Rickettsia was detected using ompA primers when Ixodes ricinus, Rhipicephalus bursa, Rhipicephalus turanicus, Rhipicephalus eversti eversti, Hyalomma detritum scupense and Rhipicephalus sp. were analyzed. We detected 17.5% of ticks associated with different SFG Rickettsia: R. aeschlimannii, R. massiliae, BAR29, R. slovaca and Rickettsia sp. strains RpA4, DnS14, DnS28 and JL-02. Their presence has to be taken into account since most of them have been recognized as human pathogens.

Dexamethasone represses cAMP rapid upregulation of TRH gene transcription: identification of a composite glucocorticoid response element and a cAMP response element in TRH promoter.

Hypothalamic proTRH mRNA levels are rapidly increased (at 1 h) in vivo by cold exposure or suckling, and in vitro by 8Br-cAMP or glucocorticoids. The aim of this work was to study whether these effects occurred at the transcriptional level. Hypothalamic cells transfected with rat TRH promoter (-776/+85) linked to the luciferase reporter showed increased transcription by protein kinase (PK) A and PKC activators, or by dexamethasone (dex), but co-incubation with dex and 8Br-cAMP decreased their stimulatory effect (as observed for proTRH mRNA levels). These effects were also observed in NIH-3T3-transfected cells supporting a characteristic of TRH promoter and not of hypothalamic cells. Transcriptional regulation by 8Br-cAMP was mimicked by noradrenaline which increased proTRH mRNA levels, but not in the presence of dex. PKA inhibition by H89 avoided 8Br-cAMP or noradrenaline stimulation. TRH promoter sequences, cAMP response element (CRE)-like (-101/-94 and -59/-52) and glucocorticoid response element (GRE) half-site (-210/-205), were analyzed by electrophoretic mobility shift assays with nuclear extracts from hypothalamic or neuroblastoma cultures. PKA stimulation increased binding to CRE (-101/-94) but not to CRE (-59/-52); dex or 12-O-tetradecanoylphorbol-13-acetate (TPA) increased binding to GRE, a composite site flanked by a perfect and an imperfect activator protein (AP-1) site in the complementary strand. Interference was observed in the binding of CRE or GRE with nuclear extracts from cells co-incubated for 3 h with 8Br-cAMP and dex; from cells incubated for 1 h, only the binding to GRE showed interference. Rapid cross-talk of glucocorticoids with PKA signaling pathways regulating TRH transcription constitutes another example of neuroendocrine integration.

Detection of a non-pathogenic variant of Anaplasma phagocytophilum in Ixodes ricinus from La Rioja, Spain.

Our aim was to identify variants of Anaplasma phagocytophilum 16S rRNA gene sequences among products amplified from Ixodes ricinus collected in La Rioja, Spain. A. phagocytophilum AP-variant 1, reported as non-pathogenic, was detected in 12 samples (two adults and ten nymphs). This finding could justify the low incidence of human anaplasmosis in our area, despite the high prevalence of A. phagocytophilum in ticks.

BDNF increases the early expression of TRH mRNA in fetal TrkB+ hypothalamic neurons in primary culture.

Known effects of neurotrophins in the developing central nervous system include induction or regulation of peptide expression. Hypothalamic postmitotic thyrotropin-releasing hormone (TRH)-producing neurons may require neurotrophins for survival and/or differentiation. This issue was investigated using primary cell cultures derived from 17-day-old fetal rat hypothalamus seeded in serum-free medium and analysed up to 4 days in vitro culture. Neurotrophin receptor (TrkB and TrkC) mRNA expression was detected by RT-PCR in fetal hypothalamus and throughout the culture period. Western blots confirmed the expression of the full-length proteins in vitro. Semi-quantitative RT-PCR showed that the addition of brain-derived neurotrophic factor (BDNF) increases TRH mRNA levels while the addition of neurotrophin-3 does not. TRH cell content was not modified. Studies on the effect of cell density or homologous conditioned medium demonstrated that endogenous factors probably contribute to determine TRH mRNA levels. One of these factors was BDNF because basal TRH mRNA levels were reduced by the addition of a Trk inhibitor or anti-BDNF. TrkB mRNA was expressed in 27% of cells and TRH mRNA in 2% of cells. The number of TRH+ cells was not affected by BDNF treatment. Forty-eight per cent of TRH neurons contained TrkB mRNA; these neurons had higher amounts of TRH mRNA than TrkB- neurons. Only TrkB+ cells responded to BDNF by increasing their TRH mRNA levels suggesting that BDNF may directly affect TRH biosynthesis. In conclusion, fetal hypothalamic TRH neurons are probably heterogeneous in regard to the neurotrophic factors enhancing peptide and mRNA levels. BDNF enhances TRH mRNA levels in a population of TrkB+ fetal hypothalamic TRHergic neurons in primary culture. However, additional influences may be necessary for the establishment of peptide phenotype in the TrkB+ neurons.

Development of pro-TRH gene expression in primary cultures of fetal hypothalamic cells.

Little is known about the temporal relationship and the sequential steps for peptide biosynthesis during the terminal differentiation of the peptide phenotype in central nervous system. Analysis of the TRH phenotype in primary cultures of rat fetal day 17 hypothalamic cells has shown that TRH levels start increasing only after a week in culture, in contrast with in vivo data showing a steady increase during late fetal life. The purpose of this study was to compare the developmental patterns of TRH and pro-TRH mRNA levels in vitro to determine whether the initial low and steady levels of TRH are due to deficient transcription. Pro-TRH mRNA levels were detected by semi-quantitative RT-PCR through the development of primary cultures of serum-supplemented hypothalamic fetal cells from 17 day old embryos. Pro-TRH mRNA levels per dish increased steadily since the beginning of the culture. In contrast, TRH levels per dish were low and stable during the first week increasing afterwards, but remaining low compared to equivalent in vivo values. Pro-TRH mRNA levels per hypothalamus increased between fetal day 17 and postnatal 14, suggesting that the in vitro pattern of pro-TRH mRNA development mimics that occurring in vivo. These data show that pro-TRH gene expression does not limit TRH accumulation in vitro suggesting that the transcriptional and post-transcriptional programs leading to peptide accumulation are established independently.

Acute ethanol administration induces changes in TRH and proenkephalin expression in hypothalamic and limbic regions of rat brain.

Thyrotropin releasing hormone (TRH) present in several brain areas has been proposed as a neuromodulator. Its administration produces opposite effects to those observed with acute ethanol consumption. Opioid peptides, in contrast, have been proposed to mediate some of the effects of alcohol intoxication. We measured TRH content and the levels of its mRNA in hypothalamic and limbic zones 1-24 h after acute ethanol injection. We report here fast and transient changes in the content of TRH and its mRNA in these areas. The levels of proenkephalin mRNA varied differently from those of proTRH mRNA, depending on the time and region studied. Wistar rats were administered one dose of ethanol (intraperitoneal, 3 g/kg body weight) and brains dissected in hypothalamus, hippocampus, amygdala, n. accumbens and frontal cortex, for TRH quantification by radioimmunoassay or for proTRH mRNA measurement by RT-PCR. After 1 h injection, TRH levels were increased in hippocampus and decreased in n. accumbens; after 4 h, it decreased in the hypothalamus, frontal cortex and amygdala, recovering to control values in all regions at 24 h. ProTRH mRNA levels increased at 1 h post-injection in total hypothalamus and hippocampus, while they decreased in the frontal cortex. The effect of ethanol was also studied in primary culture of hypothalamic cells; a fast and transient increase in proTRH mRNA was observed at 1 h of incubation (0.001% final ethanol concentration). Changes in the mRNA levels of proTRH and proenkephalin were quantified by in situ hybridization in rats administered ethanol intragastrically (2.5 g/kg). Opposite alterations were observed for these two mRNAs in hippocampus and frontal cortex, while in n. accumbens and the paraventricular nucleus of the hypothalamus, both mRNA levels were increased but with different kinetics. These results give support for TRH and enkephalin neurons as targets of ethanol and, as possible mediators of some of its observed behavioral effects.

Extracellular matrix proteins increase the expression of pro-TRH and pro-protein convertase PC1 in fetal hypothalamic neurons in vitro.

External clues for neuron development include extracellular matrix (ECM) molecules. To explore ECM influence on the early development of peptide phenotype in the CNS, we have compared pro-TRH levels in primary cultures of rat hypothalamic cells plated either on poly-lysine (PL) (control) or on PL plus one of various ECM molecules at 10 microgram/ml. Fetal day 17 cells plated at a density of 1250/mm(2) were grown in a serum free medium made of Neurobasal medium supplemented with B27 (GIBCO). Cultures, consisting mainly of neurons, were analyzed at DIV 2. ECM proteins induced morphological effects in agreement with previously published studies. The amount of pro-TRH per dish, quantified by Western blotting, was increased to 275% for laminin, 191% for fibronectin and 173% for tenascin-C (control=100%); there was no effect of vitronectin. Laminin or fibronectin did not change pro-TRH mRNA or TRH levels but enhanced levels of the pro-protein convertase PC1 suggesting that the ECM molecules did regulate the translational status of pro-TRH. In conclusion, we have shown that some ECM proteins increased pro-TRH level in vitro; this may contribute to the enhancement of pro-TRH levels observed early in vivo in the hypothalamus.

Dexamethasone rapidly regulates TRH mRNA levels in hypothalamic cell cultures: interaction with the cAMP pathway.

The biosynthesis of thyrotropin-releasing hormone (TRH) in the hypothalamic paraventricular nucleus (PVN) is subject to neural and hormonal regulations. To identify some of the potential effectors of this modulation, we incubated hypothalamic dispersed cells with dexamethasone for short periods of time (1-3 h) and studied the interaction of this hormone with protein kinase C (PKC) and PKA signaling pathways. TRH mRNA relative changes were determined by the RT-PCR technique. One hour incubation with 10(-10)-10(-4) M dexamethasone produced a concentration-dependent biphasic effect: an inhibition was observed on TRH mRNA levels at 10(-10) M, an increase above control at 10(-8)-10(-6) M and a reduction at higher concentrations (10(-5)- 10(-4) M). The stimulatory effect of 10(-8) M dexamethasone on TRH mRNA was essentially independent of new protein synthesis, as evidenced by cycloheximide pretreatment. Changes in TRH mRNA levels were reflected by enhanced TRH cell content. Incubation with a cAMP analogue (8-bromo-cAMP, 8Br-cAMP) or with a PKC activator (12-O-tetradecanoylphorbol-13-acetate, TPA) increased TRH mRNA levels after 1 and 2 h, respectively. An increase in TRH mRNA expression was observed by in situ hybridization of dexamethasone or 8Br-cAMP-treated cells. The interaction of dexamethasone, PKA and PKC signaling pathways was studied by combined treatment. The stimulatory effect of 10(-7) M TPA on TRH mRNA levels was additive to that of dexamethasone; in contrast, coincubation with 10(-3) M 8-Br-cAMP and dexamethasone diminished the stimulatory effect of both drugs. An inhibition was observed when the cAMP analogue was coincubated with TPA or TPA and dexamethasone. These results demonstrate that dexamethasone can rapidly regulate TRH biosynthesis and suggest a cross talk between cAMP, glucocorticoid receptors and PKC transducing pathways.

Multifactorial modulation of TRH metabolism.

1. Thyrotropin releasing hormone (TRH), synthesized in the paraventricular nucleus of the hypothalamus (PVN), is released in response to physiological stimuli through median eminence nerve terminals to control thyrotropin or prolactin secretion from the pituitary. 2. Several events participate in the metabolism of this neuropeptide: regulation of TRH biosynthesis and release as well as modulation of its inactivation by the target cell. 3. Upon a physiological stimulus such as cold stress or suckling, TRH is released and levels of TRH mRNA increase in a fast and transient manner in the PVN; a concomitant increase in cfos is observed only with cold exposure. 4. Hypothalamic cell cultures incubated with cAMP or phorbol esters show a rise in TRH mRNA levels; dexamethasone produces a further increase at short incubation times. TRH mRNA are thus controlled by transsynaptic and hormonal influences. 5. Once TRH is released, it is inactivated by a narrow specificity ectoenzyme, pyroglutamyl peptidase II (PPII). 6. In adenohypophysis, PPII is subject to stringent control: positive by thyroid hormones and negative by TRH; other hypothalamic factors such as dopamine and somatostatin also influence its activity. 7. These combined approaches suggest that TRH action is modulated in a coordinate fashion.