[In-utero antiretroviral exposure and mitochondrial toxicity in a cohort of uninfected infants born to HIV-1-infected women]. / Exposición a antirretrovirales y toxicidad mitocondrial en una cohorte de niños no infectados hijos de madres VIH positivas.

INTRODUCTION: In this study, we attempt to find out the percentage of uninfected infants born to HIV-infected women and exposed in-utero and perinatally to Antiretroviral Treatment (ART) that show high lactate levels, or any other mitochondrial damage markers (such as hypertransaminasaemia or hyperamylasaemia), during the first three months of age. We shall also establish whether certain drugs used in-utero are associated with higher lactate, transaminase or amylase levels. METHODS: We analysed the available data from 623 uninfected infants born in the Spanish FIPSE cohort that were born in the period 2000-2005. The normal values for lactate, transaminases and amylase were set according to AIDS Clinical Groups Trials toxicity tables for infants. RESULTS: The percentages of children with high lactate levels at 0.5; 1.5 and 3 months of age were 48%, 51.4% and 43% among those infants with available data. Respectively, the percentages of children with high AST values were 13.2; 10.4 and 17.2%. The values for high ALT were 3.3%; 3.4% and 5%. The percentages for hyperamylasaemia were 0%; 0.6% and 2.6%. We found no significant difference among the drugs used in utero for the four analysed biochemical markers along the first three months of age. CONCLUSIONS: We have found a high proportion of hyperlactataemia among infants exposed in-utero to ART, as shown in other cohorts of similar characteristics. No morbidity or mortality was communicated to the cohort analysis group. No ART drug among those used in-utero was statistically associated with a higher proportion of high lactate levels in these infants.

[Growth of uninfected infants exposed to antiretrovirals born to HIV-infected woman]. / Exposición a antirretrovirales y crecimiento en una cohorte de niños no infectados, hijos de madre con VIH positivo.

INTRODUCTION: Recent reports show that Antiretroviral Treatment (ART) during pregnancy does not affect somatic growth of children born to HIV-infected mothers, are reassuring. The aim of this study is to perform an anthropometric analysis of the uninfected children followed in the Spanish FIPSE cohort during their first 18 months of life, and to describe the possible risk factors during pregnancy that may influence low birth weight. METHODS: The FIPSE cohort includes 8 public hospitals in Madrid, and prospectively follows children born to HIV-infected women at these hospitals. We collected data on 601 uninfected children, following standardised protocols, during their first 2 years of life. A P value<0.05 was considered statistically significant. Data from the Pablo Orbegozo Foundation were used to compare the means of our population with the standard weight, longitude an occipitofrontal circumference (OFC) of the Spanish population during the first 18 months of life. RESULTS: The mean weight was 2766g (+/-590), and 2967g (+/-427) when premature neonates were excluded. The proportion of Intrauterine Growth Restriction among non- premature neonates was 19.8% (95% CI: 16.3-23.8). Children born to mothers that used illicit drugs weighed less: 2752g (+/-325) vs. 3002g (+/ 435), P<0.001, as did children born to mothers who smoked during pregnancy: 2842g (+/-363) vs. 3018g (+/-444), P>0.001. Maternal anaemia did not influence the low birth weight of the children when premature neonates were excluded. We found no statistically significant differences depending on the ART received during pregnancy. Children born to mothers who had CD4 > 500 cell /mm were heavier (2834g +/-503) than those whose mothers had CD4 of less than 200 cell/mm (2565g +/-702), P=0.008. These differences disappeared when premature neonates were excluded. Children born to mothers with undetectable viral load were heavier (2866g +/-532 vs. 2704g +/-588, P=0.005), but these differences also disappeared when the prematures were excluded from the analysis. Mean weight, length, and OFC of our population at birth (excluding premature neonates) were lower than the Spanish standards. (z for weight=-0.83; z for length =-1.02; z for OFC=-1.00), but these differences are not statistically significant and disappear at 18 months of age (z for weight=-0.08; z for height=-0.32; z for OFC=-0.31). The type of ART did not have any significant influence. DISCUSSION: There is a very significant difference between the weight of the children born to mothers addicted to illicit drugs and the rest of the children. Similarly, the weight of the children born to smoking mothers is significantly lower. There was no association between maternal anaemia and the type of ART. The children of our population have lower weights, length and OFC at birth, but this may due to the high number of scheduled caesarean births, practised at 38 weeks of pregnancy (54.5%). Our children catch-up with anthropometric measurements during the first and second year of life, and these are similar to Spanish standards at 18 months old.

[Birth defects in a cohort of uninfected children born to HIV-infected women]. / Malformaciones congénitas en una cohorte de niños no infectados, hijos de madres infectadas por el virus de la inmunodeficiencia humana.

INTRODUCTION: Mother-to-Child HIV transmission is now just 1% in western countries, due to prevention measures. Antiretroviral Treatment (ART) drugs do have adverse effects, anaemia and myelosupression caused by cidovudina being the most commonly observed effects. In the present study, we have analysed the proportion and characteristics of congenital malformations (CM) or birth defects (BD) in a cohort of uninfected children born to HIV-infected women. METHODS: A total of 623 uninfected children belonging to the FIPSE cohort were followed up according to standardised protocols. This cohort includes 8 public hospitals from Madrid and follows up HIV-infected pregnant women and their children. Children were classified according to prematurity, ethnic origin, birth weight, withdrawal syndrome, in-utero treatment. Birth defects were described and defined according to the EUROCAT, the European registry for BD. Mild errors of morphogenesis were excluded from the analysis. Categorical variables were compared with the X(2) or the Fisher test. RESULTS: A total of 78% (486) of the mothers were of Caucasian origin; 18.8% (117) used some illicit drug (heroine, cocaine or methadone) during gestation; 51 mothers (8.1%) received no ART, 10 (1.6%) received monotherapy and 469 (75.3%) received HAART. BD were seen in 52 children, with the most frequent being genitourinary and cardiological. Anaemia in the first trimester was an associated risk for BD (17.9% vs. 8.1%, P = 0,04). Similarly, mothers who used any illicit drug (plus methadone), had a slightly higher risk for BD in their offspring (13.8% vs. 7.6%, P = 0,04) There was no increased risk for BD significantly associated with any of the in-utero used antiretrovirals, although Nevirapine use in-utero showed a protective effect. Children born to mothers who received ART in the first trimester had the same rate of BD (7.4%) as those whose mothers started ART in the second trimester (8.8%), P = 0,67. CONCLUSIONS: The proportion of BD that we have observed seems higher than those shown in other European teratogenicity studies and also higher than those shown in cohorts with HIV and antiretroviral exposed infants. This may be due to the fact that our series show the results of an active surveillance system (that includes ultrasound), where BD classically appear in a higher proportion. Immunovirological characteristics of the mother did not influence the proportion of BD, but anaemia in the fist trimester and the use of illicit drugs (or methadone) did. No specific antiretroviral drug was associated with an increase in BD, although Nevirapine showed a possible protective effect in the statistical analysis. Mothers who started antiretrovirals in the first trimester do not have more BD in their offspring than mothers who started on antiretrovirals later on.

[Anaemia and neutropenia in a cohort of non-infected children of HIV-positive mothers]. / Anemia y neutropenia en una cohorte de niños no infectados hijos de madres positivas para el virus de la inmunodeficiencia humana.

INTRODUCTION: Mother-to-child HIV transmission is currently around 1% in western countries, due to prevention measures. Antiretroviral drugs do have adverse effects, anaemia and myelosupression caused by AZT being the most observed effects. In the present study, we analyse the prevalence of anaemia and neutropenia in an uninfected children cohort born to HIV-infected women. MATERIAL AND METHODS: We followed up 623 uninfected children belonging to the FIPSE cohort according to standardised protocols. This cohort groups 8 hospitals from Madrid and follows up HIV infected pregnant women and their children. Anaemia and neutropenia were defined according to the ACTG (AIDS Clinical Trails Group) toxicity tables. Children were classified according to prematurity, ethnic origin, birth weight, withdrawal syndrome, in-utero treatment and neonatal prophylaxis. Categorical variables were compared with the chi2 or the Fisher tests. RESULTS: Anaemia was observed in 188 (30.1%) children during follow-up and 161 (25.8%) had anaemia grade 2 or higher. Prematurity (p < 0.001), low birth weight (p = 0.005) and Highly Active Antiretroviral Treatment (HAART) with Protease Inhibitors (p = 0.016) were associated with higher percentages of anaemia in children. Nadir haemoglobin values were reached by 6 weeks of life and anaemia was transient and disappeared by six months of age. Neutropenia was present in 41.9% (261 children) and 22.7% of the children had moderate-severe neutropenia. Prematurity was again associated with neutropenia (p = 0.01) and low birth weigh was associated only with moderate-severe neutropenia (p = 0.023). African infants had a higher percentage of neutropenia than the rest of the children (50% vs. 44%), although the differences were not significant. The type of in-utero treatment did not appear to influence the neutropenia. Neutropenia was still present in 12.5% of infants at 18 months of age. The type of neonatal prophylaxis to prevent mother-to-child transmission (monotherapy, dual therapy or triple therapy) did not influence either cytopenia. CONCLUSION: In our series, the proportion of children with anaemia is high: 30.1% Prematurity, low birth weight and HAART with IP were associated with a higher proportion of anaemia, which was transient and had little clinical relevance. The proportion of children with neutropenia was higher (41.9%) and was associated with prematurity, low birth weight and African origin. The type of neonatal prophylaxis does not seem to influence the development of cytopenias. Persistence of neutropenia (without clinical significance) was observed in a small percentage of the children 12.5%, at 18 months of age.

[Why are HIV-infected infants still being born in Spain?]. / Por qué se infectan aún niños con el virus de la inmunodeficiencia humana en España?

INTRODUCTION: Despite the success of preventive measures against mother-to-child transmission (MTCT) of human immunodeficiency virus-1 and -2 (HIV-1 and -2) in developed countries, HIV-infected infants continue to be born. The aim of this study was to evaluate failures in the prevention of MTCT and the clinical characteristics of infected infants. METHODS: The Foundation for the Investigation and Prevention of AIDS in Spain (FIPSE) Cohort in Madrid prospectively follows up children at risk of MTCT HIV born in eight public hospitals in Madrid. From May 2000 to December 2005, 632 children born to HIV-infected mothers were evaluated. Data from pregnancy follow-up, antiretroviral therapy (ART), and symptoms at diagnosis in infected infants were analyzed. RESULTS: Nine infants were infected. The rate of vertical transmission was 1.42 (95% CI 0.7-2.68). Of the nine mothers, seven had not received ART during pregnancy (and five had not received ART at delivery). Of the mothers who received ART, one had only done so for the last month of pregnancy. Two infants were given three drugs as prevention of MTCT, one received bitherapy and six received monotherapy. The median age at diagnosis was 2.4 months (range 7 days-2 years). The mean plasma viral load at diagnosis was 276,000 copies/ml (range: 11,900-1,000,000). Five of the infants were symptomatic at diagnosis (P. jirovaci pneumonia in two, sepsis in one, recurrent bacterial infections in one, hepatosplenomegaly in one). Four of the nine infants had been admitted to hospital prior to HIV diagnosis. DISCUSSION: Missed opportunities for the prevention of MTCT were identified in eight of the nine HIV-infected infants (89%). Administration of AZT during labor in HIV-infected mothers and triple therapy for the prevention of MTCT in high risk infants is not universal. Hospital admission in young infants at risk might lead to suspicion of infection in infants born to HIV-infected mothers. Improved implementation of all the preventive measures for MTCT should be encouraged.

[Incidence of bronchopulmonary carcinoma in Extremadura in 1998]. / Incidencia de carcinoma broncopulmonar en Extremadura durante el año 1998.

OBJECTIVES: Our main objective was to determine the incidence of bronchopulmonary cancer in Extremadura (Spain). We also studied the presence of risk factors such as smoking and exposure to other carcinogens. MATERIAL AND METHODS: This prospective study used a protocol followed by all hospitals in Extremadura; enrolled were patients with a diagnosis of bronchopulmonary carcinoma in 1998 who lived habitually in the region. Cyto-histological confirmation of the diagnosis was sought or, when such confirmation was unavailable, diagnostic agreement among researchers was based on clinical, radiological and/or endoscopic data. RESULTS: The incidences adjusted to world population were 53.4, 2.16 and 25.3 per 100,000 inhabitants for men, women and the entire population sample, respectively. Patterns in the provinces of Cáceres and Badajoz were very similar. Of 433 cases recorded, 95% were men and 78% were aged 60 years or older. Cyto-histological confirmation of diagnosis was available for 92.2%. Most tumors were epidermoid (41.1%). The time between the start of smoking and diagnosis was significantly shorter in heavier smokers.

Afferents to different layers of the dorsolateral isocortex in rats.

Fluorescent somatopetal tracers were used to infiltrate, by diffusion rather than injections, the dorsolateral cortex of one hemisphere in rats. In different animals the tracers penetrated into the cortex to different depths. We found several interesting features of the commissural system: first, there were no areas without commissural neurons. At least a few labelled cell bodies were present in a single-cell layer also in "acallosal" cortical areas. Secondly, there is a considerable variety of laminar distribution patterns of labelled perikarya in different areas. Thirdly, some cortical fields, which cytoarchitecturally appear uniform, can be subdivided according to different distributions of cell bodies with commissural projections. Fourthly, when only supragranular layers were infiltrated, labelled cell bodies were present mainly in the supragranular layers of the contralateral cortex. Infiltration of the first layer alone did not label any neurons in the contralateral cortex but did label neurons in layer VIb ipsilaterally. In the subcortex, the labelled perikarya were found in the structures already known to project directly to the cortex. In rats with the tracer restricted mainly to the supragranular layers, a conspicuously reduced labelling was found in the basal forebrain and the thalamus. In the thalami of those animals, labelled neurons were found only in paralamellar nuclei. The high sensitivity of the tracer used, together with infiltration of the entire dorsolateral cortex, allows us to conclude that probably all sources of innervation of the isocortex in rats have been seen.

Anti-inflammatory drugs suppress injury-induced NADPH-d activity in CA1 pyramidal neurones.

Inducibility of NADPH-diaphorase (NADPH-d) or nitric oxide synthase (NOS) has been demonstrated in pyramidal neurones of the hippocampus, but the mechanisms of this induction are not known. The present study aimed to assess the role of anti-inflammatory drugs in injury-induced production of NADPH-d/NOS in CA1 pyramidal neurones. We found that either a steroid, dexamethasone or a non-steroid, indomethacin, prevents induction of these enzymes. We also found that NO is not necessary for the induction. None of the three drugs used had detectable effect on the neurones which contain constitutive NADPH-d/NOS.

NADPH-diaphorase (NOS) is induced in pyramidal neurones of hippocampal slices.

We found NADPH-diaphorase (presumably identical with nitric oxide synthase) in pyramidal neurones of the hippocampus in slices that stayed in a chamber for 30 min or longer. In some instances parallel slices showed normal membrane properties when studied electrophysiologically. In freshly made slices the pyramidal neurones were not stained. Thus, after induction of the enzyme, the hippocampal pyramidal neurones can synthesize nitric oxide which may serve as a retrograde messenger in long-term potentiation. The enzyme may also play a role in cell loss seen in slices which stayed in a chamber for 9-22 h before fixation.

Bilateral induction of NADPH-diaphorase activity in neocortical and hippocampal neurons by unilateral injury.

Unilateral injury of the cerebral cortex or hippocampus induced a bilateral appearance of NADPH-diaphorase in some pyramidal neuronal and glial cells of both structures. Only in the cerebral cortex, near the needle track, did some V layer pyramidal neurons contain so much of this enzyme that they looked to be stained by the Golgi method. The induction of NADPH-diaphorase in a subset of cortical and hippocampal neurons in both hemispheres after unilateral injections of either cysteamine or artificial cerebro-spinal fluid suggests the existence in the isocortex and the hippocampus of a selective signalling system which may play a role in recovery of function following local brain damage.

Cortical projections of the thalamic mediodorsal nucleus in the rat. Definition of the prefrontal cortex.

Deposits of somatopetal tracers, that filled the entire dorsolateral cortex of one hemisphere, labelled only a few cell bodies in the thalamic mediodorsal nucleus (MD). Further experiments served to confirm the existence of strong projections from MD to the mesial and suprarhinal areas of the frontal cortex. Thus, only a very small proportion of the MD neurones in the rat projects outside the cortical areas described first by Leonard (1969). We conclude that at least in the rat, cortical projections of the MD are selective enough to be used as the basis for definition of the prefrontal cortex.

NOS neurones lie near branchings of cortical arteriolae.

We show that numerous neurones labelled for NADPH-diaphorase, which synthetize nitric oxide, lie near branching points of the arteriolae which descend through the cerebral cortex from its pial surface. This spatial relationship suggests the possibility of neural control of cortical blood flow by the NADPH-diaphorase neurones, possibly mediated by the rapid action of nitric oxide.

Bilateral thalamocortical projection in hedgehogs: evolutionary implications.

In adult hedgehogs with large unilateral cortical deposits of fluorescent somatopetal tracers, labelled perikarya were found not only in the ipsilateral but also contralateral thalamus. An exceptionally large number of contralaterally labelled neurons was seen in the ventrolateral nucleus, also at a considerable distance from the midline. Deposits of one of two different tracers in the frontoparietal cortex of each hemisphere appear to label different perikarya in each ventrolateral nucleus. This projection to the contralateral cortex in hedgehogs does not resemble thalamo-cortical connections in either adult or developing brains of other mammalian species. Among amniotes, only in pigeons have contralateral projections from the thalamus to the telencephalon been described. The somatosensorimotor system of hedgehogs may be the only known mammalian remnant of primitive vertebrate thalamocortical organization. Whether primitive or derived, the bilateral thalamocortical projection in hedgehogs shows that hedgehog brains cannot be uncritically taken to represent brains of primate ancestors.

Vertical ascending connections in the isocortex.

Different fluorescent tracers were applied to the surface of the cortex of rats, marmosets and one hedgehog. Irrespective of the kind of tracer and the depth of penetration, some perikarya of layer VI were labelled in each specimen and in all cortical regions. In the rat almost all labelled neurons were packed in sublayer VIb, in the marmoset such cells were dispersed throughout layer VI, whereas in the hedgehog the degree of their segregation to sublayer VIb was intermediate. Additional experiments in the rat indicated that most of the medium-sized neurons in the VIb layer project to layer I, that most of the perikarya projecting to the thalamus are localized in sublayer VIa, that different neurons project to the thalamus and to the surface of the cortex, and that only very few perikarya in deep parts of layers III and V and of sublayer VIa send axons or axon collaterals to layers I and II.

Architectonics of the thalamus in the echidna (Tachyglossus aculeatus): search for the mediodorsal nucleus.

Architectural characteristics of the thalamus in echidnas and rats were compared in sections stained to reveal cell bodies, myelin, acetylcholinesterase, succinate dehydrogenase and cytochrome oxidase. Numerous species differences were noticed: in general, the thalamus is architecturally more homogeneous in echidnas than in rats, especially anteriorly. In this report we emphasize the presence of a relatively large structure localized in the anteromediodorsal part of the thalamus in echidnas. This structure, previously shown to project to the frontal cortex, contains very small amounts of acetylcholinesterase and the oxidative enzymes; in this respect it resembles the mediodorsal nucleus of rats. The same properties make this formation different from the anterodorsal and anteroventral nuclei in rats, the equivalents of which could not be identified in echidnas. The anteromediodorsal region of the thalamus in echidnas consists chiefly of two cytoarchitecturally different regions: the medial, 'polymorphic' part contains relatively small, densely packed, multiform perikarya, whereas the lateral, 'monomorphic' part is characterised by larger, sparse neurons with little cytoplasm and round, large, empty-looking nuclei in which the nucleolus is clearly seen. We conclude tentatively that this brain structure of echidnas corresponds to the mediodorsal nucleus in placental species. Further studies of connections and chemical properties will be essential to determine the degree of correspondence of the presumed 'frontal lobe system' in echidnas to that in other mammals.

A method for the study of the spatial distribution of the neuronal dendritic tree using a universal stage.

The use of a microscope equipped with a universal stage with 4 rotation axes, drawing tube and photographic system for the 3-dimensional study of neuronal morphology is considered. Two-dimensional projections of the neuronal tree are obtained by rotating the stage, and the application of coordinate transformations results in 3-dimensional mapping of neuronal topography. Algorithms used for these transformations are presented and the method applied to Golgi-impregnated neurons of the cerebral cortex of adult lizards. The advantages, limitations and sources of error of this method are discussed.