Clinical validation of a new molecular test (Seegene Allplex™ Vaginitis) for the diagnosis of vaginitis: a cross-sectional study.

OBJECTIVE: To validate the use of Seegene Allplex™ Vaginitis assay in the diagnosis of candidiasis, bacterial vaginosis (BV) and trichomoniasis. DESIGN: Cross-sectional, prospective study conducted in a single centre. SETTING: Outpatient clinic of a gynaecology department. POPULATION: Consecutive symptomatic and asymptomatic women (18-60 years of age). METHODS: Comparison of the assay test with the reference standards for the diagnosis of vaginitis (cultures for yeasts, Nugent for BV and nucleic acid amplification test for trichomoniasis). MAIN OUTCOME MEASURES: Performance of the investigational assay, in comparison with the reference standards for the diagnosis of the presence of Candida spp., Trichomonas vaginalis and BV. Secondary objectives are the evaluation of the performance of the test in postmenopausal women and in symptomatic women. RESULTS: A diagnosis of vaginitis was established in 14.0%. The global prevalences of BV, Candida spp. and T. vaginalis were 22.3%, 13.2% and 2.4%, respectively. The sensitivity and specificity of the assay test for those three causes of vaginitis were as follows: BV 91.7% and 86.6%; any Candida spp. 91.1% and 95.6%; Candida albicans 88.1% and 98.2%, non-albicans Candida 100% and 97.5%, and T. vaginalis 94.4 and 99.9%. The performance of the test was identical in the subgroup of women that reported vulvovaginal symptoms. The presence of multiple infections did not interfere with the performance of the test. CONCLUSIONS: The Seegene Allplex™ Vaginitis assay has an excellent performance in the diagnosis of the BV and presence of Candida; the results were good for trichomoniasis, but the study was underpowered for this outcome. TWEETABLE ABSTRACT: Seegene Allplex™ Vaginitis is an excellent option for screening and diagnosis of vaginitis.

Wernicke's encephalopathy mimicking neuropsychiatric symptoms in patients with systemic lupus erythematosus: a report of three cases and literature review.

Systemic lupus erythematosus (SLE) is a chronic inflammatory disease that involves many organs and systems. Nervous system involvement in SLE encompasses neurological and psychiatric disorders, and remains a diagnostic and therapeutic challenge. Wernicke's encephalopathy (WE) is a neurological disorder that occurs as a consequence of thiamine deficiency, and its clinical presentation resembles the neuropsychiatric events attributed to SLE (NPSLE). Differentiation between these two entities is crucial because their treatment differs greatly and can change prognosis. We describe three cases of patients with SLE who presented with initial clinical findings suggestive of NPSLE that, at the end of a thorough clinical investigation, were actually found to represent WE. In all of these cases, treatment with thiamine resulted in significant improvement. WE should be considered as a differential diagnosis in SLE patients with neuropsychiatric signs and symptoms, especially when risk factors for thiamine deficiency are present.

Bacterial vaginosis, aerobic vaginitis, vaginal inflammation and major Pap smear abnormalities.

The purpose of this investigation was to evaluate the impact of the vaginal milieu on the presence of abnormal Pap smears and a positive human papilloma virus (HPV) test. A cross-sectional study was conducted between June 2014 and May 2015, evaluating the vaginal discharge by fresh wet mount microscopy and comparing these data with Pap smear findings. Wet mount slides were scored for bacterial vaginosis (BV), aerobic vaginitis (AV), presence of Candida and Trichomonas vaginalis. Cytologic evaluation was done on all Pap smears according to the Bethesda criteria. The cobas© HPV Test (Roche) was performed for HPV detection. A total of 622 cases were evaluated. The mean age of the patients was 41.6 ± 10.65 years (range 21-75). Eighty-three women (13.3 %) had a cytology result worse than low-grade squamous intraepithelial lesion (LSIL). When comparing this group with the one with normal or minor [atypical squamous cells of undetermined significance (ASC-US) or LSIL] Pap smear abnormalities, there were no differences in the presence of Candida (32.5 % vs. 33.2 %, p = 1.0), absence of lactobacilli (38.6 % vs. 32.5 %, p = 0.32) or BV (20.5 % vs. 13.2 %, p = 0.09). On the other hand, moderate or severe inflammation (msI) (41.0 % vs. 28.8 %, p = 0,04), moderate or severe AV (msAV) (16.9 % vs. 7.2 %, p = 0.009) and msAV/BV (37.3 % vs. 20.0 %, p = 0.001) were more common in women with such major cervical abnormalities. No significant association was found between deviations of the vaginal milieu and high-risk HPV infection. The presence of msI or msAV, but not BV, is independently associated with an increased risk of major cervical cytological abnormalities, but not with HPV infection.

A Quantification of the Injury-Induced Changes in Central Aromatase, Oestrogenic Milieu and Steroid Receptor Expression in the Zebra Finch.

In songbirds and mammals, brain injury results in the up-regulation of aromatase (oestrogen synthase) expression in astroglia. The resulting presumed synthesis of neural oestradiol (E2 ) has neuroprotective effects including a decrease in neurodegeneration, neuroinflammation and apoptosis. The development of therapeutic tools that exploit oestrogenic neuroprotection in the treatment of neurotrauma requires a precise quantification of the endogenous changes in neural aromatase and E2 following brain injury. Surprisingly, the expected increase in neural oestrogens following brain injury has not been demonstrated. Furthermore, we are just beginning to unravel the mechanisms behind the protective effects of centrally synthesised E2 . In the present study, levels of aromatase immunoprotein, neural E2 and steroid receptor mRNA were quantified in adult male and female zebra finches 48 h following a unilateral penetrating brain injury. Both aromatase and E2 were up-regulated in the injured hemisphere of the brain compared to the uninjured hemisphere, demonstrating for the first time a robust increase in neural E2 levels following injury. We did not detect an effect of injury on mRNA expression of the oestrogen receptors (ER)-α, ER-ß or GPER-1, but observed a significant decrease in androgen receptor transcription in the injured lobe relative to the contralateral uninjured hemisphere. We conclude that mechanical damage causes a dramatic increase in local aromatisation, and the resultant high levels of central E2 are available to modulate steroid sensitive targets. Studies using alternate methods of receptor detection and/or time points may be necessary to understand the complete suite of mechanisms underlying the neuroprotective effects of induced oestrogen synthesis in this animal model.

Erythrocyte deformability and nitric oxide mobilization under pannexin-1 and PKC dependence.

The erythrocyte adenosine triphosphate (ATP) is utilised for protein phosphorylation and exported through the pannexin 1 hemichannel (Px1) in the microcirculation. The physiological stimuli for ATP release are dependent of blood shear rate level and of the tissue oxygen content. The deoxygenated and oxygenated states of haemoglobin are respectively bound and unbound to N terminal domain of the protein band 3 of the erythrocyte membrane in dependence of its degree of phosphorylation. The protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) contribute to the phosphorylation degree of band 3 and are modulated by protein kinase C (PKC). Chelerythrine (Che) is a competitive inhibitor of ATP for PKC and a negative modulator of erythrocyte deformability. The aim of this study was to assess the mobilization of nitric oxide (NO) in erythrocyte in absence and presence of Che and Px1 inhibitor (carbenoxolone). Erythrocyte deformability was evaluated in presence of carbenoxolone (Carb). Regarding the effects observed in the erythrocyte by presence of Che or Carb, the values of efflux of NO and the concentration of nitrosogluthatione are similar and with no changes in relation to their absence. Px1inhibition by Carb 10 µM ameliorates the erythrocyte deformability at a shear force of 0.6 and 1.2 Pa. The PKC inhibitor shows similar effects to the Carb on the mobilization of nitric oxide in erythrocyte. The blockage of ATP release by Carb from erythrocytes suggests a possible benefit to develop in ischemia reperfusion or in inflammatory response where will be needed to rescue the excess of NO present and ameliorate the red blood cell deformability at low shear rates.

Fibrinogen modulates leukocyte recruitment in vivo during the acute inflammatory response.

Besides playing an important role in blood hemostases, fibrinogen also regulates leukocyte function in inflammation. Our previous in vitro studies showed that the adhesive behaviour of the neutrophil is modulated by soluble fibrinogen when present at a physiological concentration. This led us to propose that this plasma glycoprotein might further influence leukocyte recruitment in vivo and thus contribute to the inflammatory response. To address this in vivo, leukocyte recruitment was here investigated under acute inflammatory conditions in the absence of soluble fibrinogen in the blood circulation. For such, intravital microscopy on mesentery post-capillary venules was performed on homozygous fibrinogen α chain-deficient mice ((α-/-) mice). Acute inflammatory states were induced by perfusing platelet activating factor (PAF) over the exposed tissue. As control animals, two groups of mice expressing soluble fibrinogen in circulation were used, namely, C57BL/6 wild type animals and heterozygous fibrinogen α chain-deficient mice ((α+/-) mice). Under acute inflammatory conditions, an abnormal pattern of recruitment was observed for leukocytes in homozygous (α-/-) mice in comparison to both control groups. In fact, the former exhibited a significantly decreased number of rolling leukocytes that nevertheless, migrated with increased rolling velocities when compared to leukocytes from control animals. Consistently, homozygous mice further displayed a diminished number of adherent leukocytes than the other groups. Altogether our observations led us to conclude that leukocyte recruitment in homozygous (α-/-) mice is compromised what strongly suggests a role for soluble fibrinogen in leukocyte recruitment in inflammation.

An in vitro study on the modulation of the neutrophil adhesive behavior by soluble fibrinogen.

Fibrinogen constitutes an important plasma glycoprotein involved in hemostasis and in inflammation. Previously, we have shown that at physiological concentrations, soluble fibrinogen is able to modulate the pattern of neutrophil activation. This led us to propose that under these conditions, fibrinogen could as well interfere with the adhesive behaviour of circulating neutrophils which is of utmost importance in their recruitment to the vascular wall during inflammatory processes. To address our working hypothesis, in vitro adhesion assays were here performed in a flow chamber by using primary cultures of human umbilical vein endothelial cells (HUVEC) and neutrophils isolated from peripheral venous blood of healthy human donors. In the presence of a physiological concentration of soluble fibrinogen (300 mg/dL), we observed that despite the number of neutrophils rolling on an activated endothelium was not affected, their rolling velocity was increased in comparison to that of non-activated neutrophils. Consequently as expected, the number of fibrinogen-treated neutrophils adhering to activated HUVEC monolayers was significantly diminished. Overall, we have here demonstrated that at least in vitro, soluble fibrinogen under physiological concentrations is able to modulate the interaction of neutrophils with the vascular endothelium. In vivo studies will enable us in the future to study the physiological relevance of these findings and further to understand the mechanisms underlying this function.

Induced synthesis of oestrogens by glia in the songbird brain.

Studies on birds have long provided landmarks and touchstones in the fields of neuroendocrinology, immunology and neuroplasticity. The passerine brain is an excellent model for studying the actions of hormones, including steroids, on a diversity of behavioural endpoints. Oestrogens, for example, have profound effects on avian neuroanatomy and neurophysiology throughout life and, importantly, are synthesised at high levels within neurones of the songbird brain. More recently, aromatisation in another set of neural cells has been identified. Specifically, aromatase expression is induced in astrocytes and radial glia following disruption of the neuropil by multiple forms of perturbation. The avian brain, therefore, can be provided with high levels of oestrogens constitutively or via induction, by aromatisation in neurones and glia, respectively. In this review, we begin with the initial discovery of aromatisation by non-neuronal cells and discuss the mechanisms underlying the induction of aromatase expression in glial cells. We then focus on the emerging interactions between the neuroendocrine and neuroimmune systems with respect to brain injury. Next, we briefly review the extensive literature on the influence of glial aromatisation on neuroplasticity, and end with some recent data on sex differences in the induction of glial aromatase in the zebra finch. Throughout this review, we consider the unanswered questions and future studies that may emerge from these findings.

Effects of acetylcholine on an animal mode of inflammation.

Acetylcholinesterase (AChE) is found both on the membranes of neuronal and non-neuronal cells. In this study we performed intravenous administrations of velnacrine (VLN) and acetylcholine (ACh), respectively, AChE inhibitor and substrate, in an animal model of lipopolysaccharide (LPS)-induced inflammation in Wistar rats. Using intravital microscopy the number of rolling and adherent leukocytes in post-capillary venules was monitorized and blood samples were collected for TNF-α plasma concentrations determination. Our results showed that in presence of LPS, ACh has an anti-inflammatory effect, seen by a decrease in TNF-α plasma levels and maintains the number of rolling and adherent leukocytes. The presence of VLN before LPS almost blocked the LPS-induced rolling and TNF-α releasing. Thereby VLN seems to have, like ACh, an anti-inflammatory effect by diminishing TNF-α concentrations.

Fibrinogen effects on erythrocyte nitric oxide mobilization in presence of acetylcholine.

AIMS: The objectives of this study were to evaluate the effects of high fibrinogen concentration on erythrocyte deformability on mobilization of nitric oxide (NO) and of its metabolites in the presence of acetylcholine (ACh) in healthy human blood samples. MAIN METHODS: Levels of NO were evaluated by amperometric method. Nitrite, nitrate and S-nitrosoglutathione (GSNO) were measured using the spectrophotometric Griess reaction. Erythrocyte deformability was determined using the Rheodyn SSD laser diffractometer. KEY FINDINGS: In the presence of high concentrations of fibrinogen and ACh (10 µM) in the blood samples from healthy humans the erythrocyte nitrites, nitrates and GSNO concentrations increased without significant changes in NO efflux. Mobilization of NO in erythrocytes' presence was enhanced in the presence of ACh and high fibrinogen levels. SIGNIFICANCE: These results suggest that during inflammation when both ACh and high levels of fibrinogen are present, NO delivery by erythrocytes might be compromised by their NO scavenging ability that acts as a compensatory mechanism against the overproduced NO by endothelial inducible nitric oxide synthase.

Differential effect of soluble fibrinogen as a neutrophil activator.

A fundamental paradigm involved in acute inflammatory responses to invading pathogens and tissue damage is the migration of specific leukocyte populations to the affected tissues to mount an initial innate response to the aggression. The recruitment of polymorphonuclear neutrophils (PMNs) from the blood is a central event in this respect. The aim of this study was to understand whether fibrinogen is able to modulate the pattern of neutrophil activation and thus contribute to neutrophil recruitment. We demonstrated that fibrinogen induces free radical production by neutrophils without modifying the activation status of Mac-1 (αMß2, CD11b/CD18), the previously identified neutrophil receptor for fibrinogen. This data indicates that fibrinogen must have an additional different binding site in the neutrophil membrane. Importantly, we propose that as Mac-1 activation was not affected by the binding of fibrinogen, activated neutrophils can further maintain their ability to marginate, roll and adhere to the endothelial walls.

Erythrocyte deformability dependence on band 3 protein in an in-vitro model of hyperfibrinogenemia.

Recent evidence has shown that plasma fibrinogen, a major cardiovascular risk factor, interacts with the erythrocyte membrane and acts to influence blood flow via erythrocyte nitric oxide (NO) modulation. In the present in-vitro study, whole blood samples were harvested from healthy subjects and aliquots were incubated in the absence (control aliquots) and presence of fibrinogen at different degrees of band 3 phosphorylation, and the erythrocyte deformability was determined. The present study shows that in the presence of higher fibrinogen concentrations, similar to those found in inflammatory conditions, erythrocyte deformability is increased only when band 3 is dephosphorylated by the presence of syk inhibitor and at low shear stress. On the contrary, no changes were verified in the presence of fibrinogen when band 3 is allowed to be phosphorylated by inhibiting the phosphotyrosine phosphatase enzyme activity with calpeptin. We also observed that the presence of fibrinogen at higher concentration does not induce changes in erythrocyte deformability in the absence of modulators of the band 3 phosphorylation degree. However, the mechanisms by which fibrinogen signalling modulates erythrocyte function remain to be clarified and are currently under study.

Cell-specific regulation of acetylcholinesterase expression under inflammatory conditions.

Acetylcholine (ACh) has been shown to exert an anti-inflammatory function by down-modulating the expression of pro-inflammatory cytokines. Its availability can be regulated at different levels, namely at its synthesis and degradation steps. Accordingly, the expression of acetylcholinesterase (AChE), the enzyme responsible for ACh hydrolysis, has been observed to be modulated in inflammation. To further address the mechanisms underlying this effect, we aimed here at characterizing AChE expression in distinct cellular types pivotal to the inflammatory response. This study was performed in the human acute leukaemia monocytyc cell line, THP-1, in human monocyte-derived primary macrophages and in human umbilical cord vein endothelial cells (HUVEC). In order to subject these cells to inflammatory conditions, THP-1 and macrophage were treated with lipopolysaccharide (LPS) from E.coli and HUVEC were stimulated with the tumour necrosis factor α (TNF-α). Our results showed that although AChE expression was generally up-regulated at the mRNA level under inflammatory conditions, distinct AChE protein expression profiles were surprisingly observed among the distinct cellular types studied. Altogether, these results argue for the existence of cell specific mechanisms that regulate the expression of acetylcholinesterase in inflammation.

Erythrocyte as a biological sensor.

The erythrocytes ability of sensing the local oxygen gradient through the hemoglobin conformation, along with changes in nitric oxide mobilization and vasomotor repercussions at the microcirculation, were reviewed in detail in this article. Different approachs trying to explain the erythrocyte death were additionally documented. Also, the influence of several types of molecules (vasoactive, oxidant/reductor) on the erythrocyte roles as sensor of (i) oxygen tissue needs, (ii) blood viscosity and myogenic environment, (iii) and inflammatory conditions were mentioned in order to highlight its physiologycal function and substitute the erroneous idea of the erythrocyte being simply a hemoglobin sac content.

Behaviour of human erythrocyte aggregation in presence of autologous lipoproteins.

The aim of this work was to evaluate in vitro the effect of autologous plasma lipoprotein subfractions on erythrocyte tendency to aggregate. Aliquots of human blood samples were enriched or not (control) with their own HDL-C, LDL-C, or VLDL-C fractions obtained from the same batch by density gradient ultracentrifugation. Plasma osmolality and erythrocyte aggregation index (EAI) were determined. Blood aliquots enriched with LDL-C and HDL-C showed significant higher EAI than untreated aliquots, whereas enrichment with VLDL-C does not induce significant EAI changes. For the same range of lipoprotein concentrations expressed as percentage of osmolality variation, the EAI variation was positive and higher in presence of HDL-C than upon enrichment with LDL-C (P < 0.01). Particle size, up to LDL diameter values, seems to reinforce erythrocyte tendency to aggregate at the same plasma osmolality (particle number) range of values.

Integrin-associated protein (CD47) is a putative mediator for soluble fibrinogen interaction with human red blood cells membrane.

Fibrinogen is a multifunctional plasma protein that plays a crucial role in several biological processes. Elevated fibrinogen induces erythrocyte hyperaggregation, suggesting an interaction between this protein and red blood cells (RBCs). Several studies support the concept that fibrinogen interacts with RBC membrane and this binding, due to specific and non-specific mechanisms, may be a trigger to RBC hyperaggregation in inflammation. The main goals of our work were to prove that human RBCs are able to specifically bind soluble fibrinogen, and identify membrane molecular targets that could be involved in this process. RBCs were first isolated from blood of healthy individuals and then separated in different age fractions by discontinuous Percoll gradients. After isolation RBC samples were incubated with human soluble fibrinogen and/or with a blocking antibody against CD47 followed by fluorescence confocal microscopy, flow cytometry acquisitions and zeta potential measurements. Our data show that soluble fibrinogen interacts with the human RBC membrane in an age-dependent manner, with younger RBCs interacting more with soluble fibrinogen than the older cells. Importantly, this interaction is abrogated in the presence of a specific antibody against CD47. Our results support a specific and age-dependent interaction of soluble fibrinogen with human RBC membrane; additionally we present CD47 as a putative mediator in this process. This interaction may contribute to RBC hyperaggregation in inflammation.

Rapid effects of aggressive interactions on aromatase activity and oestradiol in discrete brain regions of wild male white-crowned sparrows.

Testosterone is critical for the activation of aggressive behaviours. In many vertebrate species, circulating testosterone levels rapidly increase after aggressive encounters during the early or mid-breeding season. During the late breeding season, circulating testosterone concentrations did not change in wild male white-crowned sparrows after an aggressive encounter and, in these animals, changes in local neural metabolism of testosterone might be more important than changes in systemic testosterone levels. Local neural aromatisation of testosterone into 17ß-oestradiol (E(2)) often mediates the actions of testosterone, and we hypothesised that, in the late breeding season, brain aromatase is rapidly modulated after aggressive interactions, leading to changes in local concentrations of E(2). In the present study, wild male white-crowned sparrows in the late breeding season were exposed to simulated territorial intrusion (STI) (song playback and live decoy) or control (CON) for 30 min. STI significantly increased aggressive behaviours. Using the Palkovits punch technique, 13 brain regions were collected. There was high aromatase activity in several nuclei, although enzymatic activity in the CON and STI groups did not differ in any region. E(2) concentrations were much higher in the brain than the plasma. STI did not affect circulating levels of E(2) but rapidly reduced E(2) concentrations in the hippocampus, ventromedial nucleus of the hypothalamus and bed nucleus of the stria terminalis. Unexpectedly, there were no correlations between aromatase activity and E(2) concentrations in the brain, nor were aromatase activity or brain E(2) correlated with aggressive behaviour or plasma hormone levels. This is one of the first studies to measure E(2) in microdissected brain regions, and the first study to do so in free-ranging animals. These data demonstrate that social interactions have rapid effects on local E(2) concentrations in specific brain regions.

Evidence that the degree of band 3 phosphorylation modulates human erythrocytes nitric oxide efflux--in vitro model of hyperfibrinogenemia.

Recent evidence has shown that plasma fibrinogen, a major cardiovascular risk factor, interacts with the erythrocyte membrane and acts to influence blood flow via erythrocyte nitric oxide (NO) modulation. In the present pioneer in-vitro study, whole blood samples were harvested from healthy subjects and aliquots were incubated in the absence (control aliquots) and presence of fibrinogen at different degrees of band 3 phosphorylation, and the levels of NO, nitrite, nitrate and S-nitroglutathione (GSNO) were determined. Hyperfibrinogenemia interferes with erythrocyte NO mobilization without changing its efflux in a way that seems to be dependent of the degree of band 3 phosphorylation. In presence of higher fibrinogen concentrations the NO efflux is reinforced when band 3 is phosphorylated (p < 0.001). Higher levels of nitrite, nitrate and GSNO were documented (p < 0.05). However, the mechanisms by which fibrinogen signalling modulates erythrocyte function remain to be clarified and are currently under study. These conditions may be considered an approach to be followed in blood storage for transfusions.

Erythrocyte as a link between basic and clinical research.

We review the major hemorheological experimental studies that show the erythrocyte aggregation as a link between basic and clinical research. The results of the clinical cross-sectional and longitudinal studies presented here will highlight the possible association between erythrocyte aggregation and plasma fibrinogen. Basic studies conducted in vitro are also mentioned as for its relevance in answering questions raised in clinical settings, as well as and in understanding the underlying influent factors in the erythrocyte tendency to aggregate and disaggregate.