[Pilot tests using molecular diagnostic assay cervicovaginal infection during pregnancy]. / Ensayo piloto del uso de pruebas moleculares para el diagnóstico de infecciones cervicovaginales en pacientes embarazadas.

Background: The prevalence of cervicovaginal infections during pregnancy has been associated with adverse perinatal outcomes however, the actual approach used for diagnosis is not effective. The aim of this study was to compare the diagnosis of vaginal infections in pregnant women using clinical, molecular diagnostic and traditional microbiological culture in a pilot study, to determine the prevalence and association with the development of preterm labor. Materials and methods: We performed a nested cross-sectional study composed by 54 women in a cohort of pregnant women in Mexico City. Cervicovaginal infections were evaluated by clinical methods, microbiology culture and a commercially available molecular biology test. Results: Prevalence of cervicovaginal infections during pregnancy was estimated between 28% and 50% according to methodologies. Considering the clinical diagnosis of preterm labor as the gold standard, all diagnostic tests were poor as predictors of preterm labor. Conclusion: Traditional approaches to establish the significance of cervicovaginal infection in pregnancy are exhausted, so be sought new ways to understand this complex relationship. Meanwhile it is recommended to continue to use traditional methods to identify infections during pregnancy in both knowledge of new methods aimed at understanding these relationships are sophisticated.

Klf9 is necessary and sufficient for Purkinje cell survival in organotypic culture.

During their phase of developmental programmed cell death (PCD), neurons depend on target-released trophic factors for survival. After this period, however, they critically change as their survival becomes target-independent. The molecular mechanisms underlying this major transition remain poorly understood. Here, we investigated, which transcription factors (TFs) might be responsible for the closure of PCD. We used Purkinje cells as a model since their PCD is restricted to the first postnatal week in the mouse cerebellum. Transcriptome analysis of Purkinje cells during or after PCD allowed the identification of Krüppel like factor 9 (Klf9) as a candidate for PCD closure, given its high increase of expression at the end of the 1st postnatal week. Klf9 function was tested in organotypic cultures, through lentiviral vector-mediated manipulation of Klf9 expression. In absence of trophic factors, the Purkinje cell survival rate is of 40%. Overexpression of Klf9 during PCD dramatically increases the Purkinje cell survival rate from 40% to 88%, whereas its down-regulation decreases it to 14%. Accordingly, in organotypic cultures of Klf9 knockout animals, Purkinje cell survival rate is reduced by half as compared to wild-type mice. Furthermore, the absence of Klf9 could be rescued by Purkinje cell trophic factors, Insulin growth factor-1 and Neurotrophin3. Altogether, our results ascribe a clear role of Klf9 in Purkinje cell survival. Thus, we propose that Klf9 might be a key molecule involved in turning off the phase of Purkinje PCD.

Adverse cardiovascular, obstetric and perinatal events during pregnancy and puerperium in patients with heart disease.

BACKGROUND AND OBJECTIVES: cardiovascular changes during pregnancy carry greater risk in heart disease. We analyze cardiovascular, obstetric and perinatal adverse effects associated with congenital and acquired heart disease during pregnancy and postpartum. MATERIALS AND METHODS: Cross-sectional and retrospective study, which included the 2017-2023 registry of pregnant or postpartum patients hospitalised with diagnosis of congenital or acquired heart disease. Adverse events (heart failure, stroke, acute pulmonary edema, maternal death, obstetric haemorrhage, prematurity and perinatal death) were compared with the clinical variables and the implemented treatment. RESULTS: 112 patients with a median age of 28 years (range 15-44) were included. Short circuits predominated 28 (25%). Thirty-six patients (32%) were classified in class IV of the modified WHO scale for maternal cardiovascular risk. Heart failure occurred in 39 (34.8%), acute lung edema 12 (10.7%), stroke 2 (1.8%), maternal death 5 (4.5%), obstetric haemorrhage 4 (3.6%), prematurity 50 (44.5%) and perinatal death 6 (5.4%). Shunts were associated with prematurity (adjusted odds ratio 4; 95% CI: 1.5-10, p = 0.006). Peripartum cardiomyopathy represented higher risk of pulmonary edema (adjusted OR 34; 95% CI: 6-194, p = 0.001) and heart failure (adjusted OR 16; 95% CI: 3-84, p = 0.001). An increased risk of obstetric haemorrhage was observed in patients with prosthetic valves (adjusted OR 30; 95% CI: 1.5-616, p = 0.025) and with the use of acetylsalicylic acid (adjusted OR 14; 95% CI: 1.2-16, p = 0.030). Furthermore, the latter was associated with perinatal death (adjusted OR 9; 95% CI: 1.4-68, p = 0.021). CONCLUSIONS: severe complications were found during pregnancy and postpartum in patients with heart disease, which is why preconception evaluation and close surveillance are vital.

Study of the immunologic response of marine-derived collagen and gelatin extracts for tissue engineering applications.

The host immunologic response to a specific material is a critical aspect when considering it for clinical implementation. Collagen and gelatin extracted from marine sources have been proposed as biomaterials for tissue engineering applications, but there is a lack of information in the literature about their immunogenicity. In this work, we evaluated the immune response to collagen and/or gelatin from blue shark and codfish, previously extracted and characterized. After endotoxin evaluation, bone marrow-derived macrophages were exposed to the materials and a panel of pro- and anti-inflammatory cytokines were evaluated both for protein quantification and gene expression. Then, the impact of those materials in the host was evaluated through peritoneal injection in C57BL/6 mice. The results suggested shark collagen as the less immunogenic material, inducing low expression of pro-inflammatory cytokines as well as inducible nitric oxide synthase (encoded by Nos2) and high expression of Arginase 1 (encoded by Arg1). Although shark gelatin appeared to be the material with higher pro-inflammatory expression, it also presents a high expression of IL-10 (anti-inflammatory cytokine) and Arginase (both markers for M2-like macrophages). When injected in the peritoneal cavity of mice, our materials demonstrated a transient recruitment of neutrophil, being almost non-existent after 24 hours of injection. Based on these findings, the studied collagenous materials can be considered interesting biomaterial candidates for regenerative medicine as they may induce an activation of the M2-like macrophage population, which is involved in suppressing the inflammatory processes promoting tissue remodeling. STATEMENT OF SIGNIFICANCE: Marine-origin biomaterials are emerging in the biomedical arena, namely the ones based in marine-derived collagen/gelatin proposed as cell templates for tissue regeneration. Nevertheless, although the major cause of implant rejection in clinical practice is the host's negative immune response, there is a lack of information in the literature about the immunological impact of these marine collagenous materials. This work aims to contribute with knowledge about the immunologic response to collagen/gelatin extracted from blue shark and codfish skins. The results demonstrated that despite some differences observed, all the materials can induce a macrophage phenotype related with anti-inflammation resolution and then act as immuno-modulators and anti-inflammatory inducible materials.

Human papillomavirus infection and lung adenocarcinoma: special benefit is observed in patients treated with immune checkpoint inhibitors.

BACKGROUND: Human papilloma virus (HPV) has been associated with the development and modulation of response in a series of neoplasms. In the case of lung adenocarcinoma, its role in etiology and pathogenesis is still controversial. Considering that this infection brings foreign epitopes, it could be of prognostic significance in patients with lung adenocarcinoma treated with immunotherapy. METHODS: In a retrospective cohort study we evaluated the presence of HPV genomic material in lung adenocarcinoma primary lesions with the INNO-LiPA platform. Viral replication was also evaluated by detecting the presence of oncoprotein E6/E7 messenger RNA (mRNA) by quantitative RT-PCR. To confirm possible hypotheses regarding viral oncogenesis, vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1 (HIF1) were evaluated with stromal fibrosis and immunoscore. RESULTS: A total of 133 patients were included in the analysis, of whom 34 tested positive for HPV, reaching an estimated prevalence of 25.6% [95% confidence interval (CI) 18.2% to 32.9%]. E6/7 mRNA was identified in 28 out of the 34 previously positive cases (82.3%). In immune checkpoint inhibitor (ICI)-treated patients, the median overall survival reached 22.3 months [95% CI 19.4 months- not reached (NR)] for HPV-negative and was not reached in HPV-positive (HPV+) ones (95% CI 27.7-NR; P = 0.008). With regard to progression-free survival, HPV- patients reached a median of 9.2 months (95% CI 7.9-11.2 months) compared to 14.3 months (95% CI 13.8-16.4 months) when HPV was positive (P = 0.001). The overall response rate for HPV+ patients yielded 82.4% compared to 47.1% in negative ones. No differences regarding programmed death-ligand 1, VEGF, HIF1, stromal fibrosis, or immunoscore were identified. CONCLUSIONS: In patients with HPV+ lung adenocarcinoma, a significant benefit in overall response and survival outcomes is observed.

THE FINE STRUCTURE OF THE LATERAL VESTIBULAR NUCLEUS IN THE RAT : I. Neurons and Neuroglial Cells.

The lateral vestibular nucleus consists of multipolar isodendritic neurons of various sizes The distal segments of some dendrites display broad expansions packed with slender mitochondria and glycogen particles. These distinctive formations are interpreted as being growing tips of dendrites, and the suggestion is advanced that they are manifestations of architectonic plasticity in the mature central nervous system. Unlike large neurons elsewhere, the giant cells (Deiters) contain small Nissl bodies interconnected in a dense mesh-work. The Nissl substance is characterized by randomly arranged cisterns of the endoplasmic reticulum and by a high proportion of free ribosomes. Whether attached or free, ribosomes usually cluster in groups of four to six, and larger polysomal arrays are rare. Free ribosomal clusters also occur in the axon hillock and the initial segment. The neuronal perikarya contain distinctive inclusions consisting of a ball of neurofilaments enveloped by a complex honeycombed membrane. The failure of these fibrillary inclusions to stain with silver suggests that the putative argyrophilia of neurofilaments may reside in an inconstant matrix surrounding them. Giant cells of Deiters are in intimate contact with two kinds of cellular elements-astroglial processes and synaptic terminals. Oligodendroglial cells are only rarely satellites of giant cells; in contrast, they are frequently satellites of small and medium-sized cells.

Specialized membrane junctions between neurons in the vertebrate cerebellar cortex.

"Gap" junctions, the morphological correlate for low-resistance junctions, are demonstrated between some mossy fiber terminals and granule cell dendrites in some lower vertebrate cerebella (gymnotid and frog). Most of the gap junctions (GJs) seen in the gymnotid-fish cerebellum exhibit an asymmetrical configuration, the electron-opaque cytoplasmic material underlying the junction being more extensive in the dendritic than in the axonal side. In the frog cerebellum, the GJs have a symmetrical distribution of such electron-opaque material. In both species the GJs are encountered at the same synaptic interface as the conventional synaptic zone (CSZ), constituting "mixed synapses" in a morphological sense. The axonal surface covered by CSZs is larger than that covered by GJs. In mammalian cerebellum, GJs are observed only in the molecular layer, between perikarya, dendrites, or perikarya and dendrites of the inhibitory interneurons. These GJs are intermixed with attachment plates and intermediary junctions interpreted as simply adhesive. In the mammalian cerebellum, a new type of junction which resembles the septate junctions (SJs) of invertebrate epithelia is observed between axonal branches forming the tip of the brush of basket fibers around the initial segment of the Purkinje cell axon. It is suggested that such junctions may be modified forms of septate junctions. The physiological implications of the possible existence of high-resistance cross-bridges between basket cell terminals, which may compartmentalize the extracellular space and thus regulate extracellular current flow, must be considered.

The axon hillock and the initial segment.

Axon hillocks and initial segments have been recognized and studied in electron micrographs of a wide variety of neurons. In all multipolar neurons the fine structure of the initial segment has the same pattern, whether or not the axon is ensheathed in myelin. The internal structure of the initial segment is characterized by three special features: (a) a dense layer of finely granular material undercoating the plasma membrane, (b) scattered clusters of ribosomes, and (c) fascicles of microtubules. A similar undercoating occurs beneath the plasma membrane of myelinated axons at nodes of Ranvier. The ribosomes are not organized into Nissl bodies and are too sparsely distributed to produce basophilia. They vanish at the end of the initial segment. Fascicles of microtubules occur only in the axon hillock and initial segment and nowhere else in the neuron. Therefore, they are the principal identifying mark. Some speculations are presented on the relation between these special structural features and the special function of the initial segment.

Inferior olive: its role in motor learing.

Specific chemical lesion of the rat inferior olive by intraperitoneal administration of 3-acetylpyridine prevents recuperation from motor abnormalities generated by unilateral labyrinthine lesion. Moreover, in animals that have recuperated from the balyrinthine lesion, 3-acetylpyridine produces a reversal of the symptoms within 2 hours of administration. These results indicate that the integrity of the olivo-cerebellar system is necessary for the acquisition and retention of this form of motor learning, but that the cerebellum itself is not the seat of such learning.

Comparison of real-time PCR methods for quantification of European hake (Merluccius merluccius) in processed food samples.

The quantification of species in commercial products is limited by analytical shortcomings, as most of them provide semiquantitative results. An exception is real-time PCR, which can provide quantitative results using hybridization probes. In the present work, this technique has been applied to the absolute, absolute-relative and relative quantification of the most valued hake species in European markets, Merluccius merluccius (European Hake). The best quantification results for this species in binary mixtures with non-target species (Merluccius capensis) and using a species-specific real-time PCR MMER_VIC system was achieved using a relative quantification approach (MLL as reference system). Absolute quantification using the MLL nuclear system has been demonstrated as appropriate for the quantification of the Merluccius genus in food model samples. This study reveals the impact of different reference systems (MLL and HAKE) in the absolute-relative and relative quantification approaches, showing that the nuclear MLL system performed better than the mitochondrial HAKE system.

Lack of barrels in the somatosensory cortex of monoamine oxidase A-deficient mice: role of a serotonin excess during the critical period.

In a transgenic mouse line (Tg8) deficient for the gene encoding monoamine oxidase A (MAOA), we show that the primary somatosensory cortex (S1) lacks the characteristic barrel-like clustering of layer IV neurons, whereas normal pattern formation exists in the thalamus and the trigeminal nuclei. No barrel-like patterns were visible with tenascin or serotonin immunostaining or with labeling of thalamocortical axons. An excess of brain serotonin during the critical period of barrel formation appears to have a causal role in these cortical abnormalities, since early administration of parachlorophenylalanine, an inhibitor of serotonin synthesis, in Tg8 pups restored the formation of barrels in S1, whereas inhibition of catecholamine synthesis did not. Transient inactivation of MAOA in normal newborns reproduced a barrelless phenotype in parts of S1.

Cajal-Retzius cells regulate the radial glia phenotype in the adult and developing cerebellum and alter granule cell migration.

Studies on the reeler mutation have shown that pioneer Cajal-Retzius (CR) cells are involved in neuronal migration in the developing cortex. Here, we use grafting and coculture experiments to investigate the mechanisms by which CR cells govern migration. We show that transplantation of embryonic CR cells, but not other cortical neurons, into adult cerebella induces a transient rejuvenation of host Bergmann glia into a radial glia phenotype. Similarly, CR cells sustain the phenotype of developing radial glia in postnatal cerebellar slices and induce the organization of a glial scaffold inside the CR cell explants. Studies with semipermeable inserts show that these effects are mediated by diffusible signals. We also show that CR cells adjacent to the surface of cerebellar slices reverse the direction of the migration of granule cells. Finally, CR cells from reeler mutant embryos elicited similar effects. These observations imply a role for CR cells in the regulation of the radial glia phenotype, a key step for neuronal migration, and suggest that these pioneer neurons may also exert a chemoattractive influence on migrating neurons.

Slit2-Mediated chemorepulsion and collapse of developing forebrain axons.

Diffusible chemorepellents play a major role in guiding developing axons toward their correct targets by preventing them from entering or steering them away from certain regions. Genetic studies in Drosophila revealed a novel repulsive guidance system that prevents inappropriate axons from crossing the CNS midline; this repulsive system is mediated by the Roundabout (Robo) receptor and its secreted ligand Slit. In rodents, Robo and Slit are expressed in the spinal cord and Slit can repel spinal motor axons in vitro. Here, we extend these findings into higher brain centers by showing that Robo1 and Robo2, as well as Slit1 and Slit2, are often expressed in complementary patterns in the developing forebrain. Furthermore, we show that human Slit2 can repel olfactory and hippocampal axons and collapse their growth cones.

Expression of X-chromosome linked inhibitor of apoptosis protein in mature Purkinje cells and in retinal bipolar cells in transgenic mice induces neurodegeneration.

Transgenic mice with overexpression of the caspase-inhibitor, X-chromosome-linked inhibitor of apoptosis protein (XIAP) in Purkinje cell (PC) and in retinal bipolar cells (RBCs) were produced to study the regulation of cell death. Unexpectedly, an increased neurodegeneration was observed in the PCs in these L7-XIAP mice after the third postnatal week with the mice exhibiting severe ataxia. The loss of PCs was independent of Bax as shown by crossing the L7-XIAP mice with Bax gene-deleted mice. Electron microscopy revealed intact organelles in PCs but with the stacking of ER cisterns indicative of cell stress. Immunostaining for cell death proteins showed an increased phosphorylation of c-Jun in the PCs, suggesting an involvement in cell degeneration. Apart from PCs, the number of RBCs was decreased in adult retina in line with the expression pattern for the L7 promoter. The data show that overexpression of the anti-apoptotic protein XIAP in vulnerable neurons leads to enhanced cell death. The mechanisms underlying this neurodegeneration can be related to the effects of XIAP on cell stress and altered cell signaling.

The reconstruction of cerebellar circuits.

Repair of adult 'point-to-point' systems by neural grafting is possible only when grafted neurons succeed in synaptically replacing the host's missing neurons, thus re-establishing the anatomical and functional integrity of the impaired circuits. Grafting experiments carried out on the cerebellum of the adult pcd (Purkinje-cell-degeneration) mutant mouse (an animal model of hereditary degenerative ataxia) reveal that embryonic Purkinje cells, by some unknown sorting mechanism, selectively invade the deprived cerebellar cortex. These neurons migrate to their proper domains and, inducing axonal sprouting of specific populations of host neurons, they become integrated synaptically within the pcd cerebellar cortex. However, the re-establishment of the corticonuclear projection is achieved only rarely, and this is the current experimental limit for the complete reconstruction of the cerebellar circuit.

Neuronal activity and brain-derived neurotrophic factor regulate the density of inhibitory synapses in organotypic slice cultures of postnatal hippocampus.

Hippocampal interneurons inhibit pyramidal neurons through the release of the neurotransmitter GABA. Given the importance of this inhibition for the proper functioning of the hippocampus, the development of inhibitory synapses must be tightly regulated. In this study, the possibility that neuronal activity and neurotrophins regulate the density of GABAergic inhibitory synapses was investigated in organotypic slice cultures taken from postnatal day 7 rats. In hippocampal slices cultured for 13 d in the presence of the GABA(A) receptor antagonist bicuculline, the density of glutamic acid decarboxylase (GAD) 65-immunoreactive terminals was increased in the CA1 area when compared with control slices. Treatment with the glutamate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione decreased the density of GAD65-immunoreactive terminals in the stratum oriens of CA1. These treatments had parallel effects on the density of GABA-immunoreactive processes. Electron microscopic analysis after postembedding immunogold labeling with antibodies against GABA indicated that bicuculline treatment increased the density of inhibitory but not excitatory synapses. Application of exogenous BDNF partly mimicked the stimulatory effect of bicuculline on GAD65-immunoreactive terminals. Finally, antibodies against BDNF, but not antibodies against nerve growth factor, decrease the density of GAD65-immunoreactive terminals in bicuculline-treated slices. Thus, neuronal activity regulates the density of inhibitory synapses made by postnatal hippocampal interneurons, and BDNF could mediate part of this regulation. This regulation of the density of inhibitory synapses could represent a feedback mechanism aimed at maintaining an appropriate level of activity in the developing hippocampal networks.

Floor plate and netrin-1 are involved in the migration and survival of inferior olivary neurons.

During their circumferential migration, the nuclei of inferior olivary neurons translocate within their axons until they reach the floor plate where they stop, although their axons have already crossed the midline to project to the contralateral cerebellum. Signals released by the floor plate, including netrin-1, have been implicated in promoting axonal growth and chemoattraction during axonal pathfinding in different midline crossing systems. In the present study, we report experiments that strongly suggest that the floor plate could also be involved in the migration of inferior olivary neurons. First, we show that the pattern of expression of netrin receptors DCC (for deleted in colorectal cancer), neogenin (a DCC-related protein), and members of the Unc5 family in wild-type mice is consistent with a possible role of netrins in directing the migration of precerebellar neurons from the rhombic lips. Second, we have studied mice deficient in netrin-1 production. In these mice, the number of inferior olivary neurons is remarkably decreased. Some of them are located ectopically along the migration stream, whereas the others are located medioventrally and form an atrophic inferior olivary complex: most subnuclei are missing. However, axons of the remaining olivary cell bodies located in the vicinity of the floor plate still succeed in entering their target, the cerebellum, but they establish an ipsilateral projection instead of the normal contralateral projection. In vitro experiments involving ablations of the midline show a fusion of the two olivary masses normally located on either side of the ventral midline, suggesting that the floor plate may function as a specific stop signal for inferior olivary neurons. These results establish a requirement for netrin-1 in the migration of inferior olivary neurons and suggest that it may function as a specific guidance cue for the initial steps of the migration from the rhombic lips and then later in the development of the normal crossed projection of the inferior olivary neurons. They also establish a requirement for netrin-1, either directly or indirectly, for the survival of inferior olivary neurons.

Chemoattraction and chemorepulsion of olfactory bulb axons by different secreted semaphorins.

During development, growth cones can be guided at a distance by diffusible factors, which are attractants and/or repellents. The semaphorins are the largest family of repulsive axon guidance molecules. Secreted semaphorins bind neuropilin receptors and repel sensory, sympathetic, motor, and forebrain axons. We found that in rat embryos, the olfactory epithelium releases a diffusible factor that repels olfactory bulb axons. In addition, Sema A and Sema IV, but not Sema III, Sema E, or Sema H, are able to orient in vitro the growth of olfactory bulb axons; Sema IV has a strong repulsive action, whereas Sema A appears to attract those axons. The expression patterns of sema A and sema IV in the developing olfactory system confirm that they may play a cooperative role in the formation of the lateral olfactory tract. This also represents a further evidence for a chemoattractive function of secreted semaphorins.

Diversity and specificity of actions of Slit2 proteolytic fragments in axon guidance.

The Slits are secreted proteins that bind to Robo receptors and play a role in axon guidance and neuronal migration. In vertebrates, Slit2 is a major chemorepellent for developing axons and is involved in the control of midline crossing. In vivo, Slit2 is cleaved into 140 kDa N-terminal (Slit2-N) and 55-60 kDa C-terminal (Slit2-C) fragments, although the uncleaved/full-length form can also be isolated from brain extract. We explored the functional activities of Slit2 fragments by engineering mutant and truncated versions of Slit2 representing the N-, C-, and full/uncleavable (Slit2-U) fragments. Only Slit2-N and Slit2-U bind the Robo proteins. We found that in collagen gel, olfactory bulb (OB) but not dorsal root ganglia (DRG) axons are repelled by Slit2-N and Slit2-U. Moreover, only Slit2-N membranes or purified protein-induced OB growth cones collapse. Finally, we found that only recombinant Slit2-N could induce branching of DRG axons and that this effect was antagonized by Slit2-U. Therefore, different axons have distinct responses to Slit2 fragments, and these proteins have different growth-promoting capacities.

Relationships between neuronal birthdates and cytoarchitecture in the rat inferior olivary complex.

The correlation between birthdates of neurons and their ultimate location within the inferior olivary nucleus (ION) was investigated in the rat by the 5-bromodeoxyuridine (BrdU) method. We performed injections every 4 hours throughout the ION generation period, and were thus able to demonstrate that 1) neurons are distributed in the adult ION following characteristic gradients that define subdivisions identical to those established by hodological studies; and 2) ION neurons born at the same time tend to be arrayed in small clusters in the adult structure. Implications of these findings for the mechanisms of olivary neuron migration, selective aggregation, and elaboration of projectional topography are discussed. This study provides direct evidence that one of the factors governing the elaboration of the cytoarchitecture of a neuronal nucleus is the temporal sequence of generation of its neurons.