Comparative characterisation of extracellular vesicles from canine and human plasma: a necessary step in biomarker discovery.

Extracellular Vesicles (EV) have become an interesting focus as novel biomarkers of disease and are increasingly reported upon in humans and other species. The Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018) guidelines were published to improve rigor and standardisation within the EV field and provide a framework for the reliable isolation and characterisation of EV populations. However, this rigor and standardisation has been challenging in the area of comparative medicine. Herein we present the successful isolation of EVs from human and canine plasma using Size Exclusion Chromatography and characterise these EVs according to best international practice. This study provides evidence for the reliable comparison of human and canine EVs isolated by this approach, and a baseline description of the EVs from healthy dogs to inform future biomarker studies. This work also demonstrates that the MISEV2018 guidelines can be successfully applied to EVs isolated from canine plasma.

Gene expression profiling in melanoma: A view from the clinic.

The treatment of Melanoma, one of the most aggressive human malignancies, has been revolutionised by the advent of novel targeted and immuno-therapies. However, methods utilised to detect early presentations, and to stratify risk for patients diagnosed with invasive melanoma in the clinical setting are lagging. The primary prognostic indicator is restricted to Breslow Thickness, or depth the tumour invades into the dermis. Gene Expression Profiling (GEP), the analysis of molecular gene signatures of an individual tumour, has been implemented with great success in other malignancies, such as breast and prostate cancer. In the setting of melanoma, commercial GEP panels are becoming available, offering patients a personalised approach, though yet to enter widespread clinical use. This short perspective seeks to describe how GEP is currently employed in practice, and its current clinical impact. We also suggest the potential roles for GEP in meeting the key clinical challenges faced by clinicians in melanoma treatment, such as decisions around adjuvant therapy, sentinel lymph node biopsy (SLNB) and surgical resection , thus highlighting areas for future potential research.

Referral of patients to plastic surgeons following self-harm: Opportunities for suicide prevention.

Self-harm is a common source of referral to plastic and hand surgery services. Appropriate management of these patients is complex and includes the need for close liaison with mental health services. Self-harm is the single biggest risk factor for completed suicide, thereby increasing the risk by a factor of 66.1 This study aimed to analyse the clinical pathway and demographics of patients referred to plastic surgeons following self-harm. This 6-year retrospective series included patients referred to plastic surgeons following self-harm within the Galway University Hospital group. Patients were identified through the Hospital inpatient enquiry system, cross-referenced with data from the National Suicide Research Foundation. Data collected included demographics, psychiatric history, details of self-harm injury, admission pathway and operative intervention. Forty-nine patients were referred to plastic surgery services during the study period, accounting for 61 individual presentations. The male-to-female ratio was 26 (53%) to 23 (47%). Mean age was 40 years (range 21-95 years). Alcohol or illicit substance use was recorded in 17 of 61 (28%) presentations. Mortality from suicide occurred in 4 patients (8%). Mental health assessment was not carried out in 9 presentations (15%). Documentation of need for close or one-to-one observation was made in 11 cases (20%) and was not referred to in 43 cases (83%) following mental health assessment. This study demonstrates significant diversity in the management of this vulnerable patient group and may inform development of referral pathways to improve the safety of transfer, surgical admission and discharge of patients following self-harm, in consultation with mental health services.

Reliability of functioning free muscle transfer and vascularized ulnar nerve grafting for elbow flexion in complete brachial plexus palsy.

We compared outcomes of primary vascularized ulnar nerve grafts from the C5 root neurotizing biceps and brachialis muscles, and gracilis functioning free muscle transfer neurotized by the distal spinal accessory nerve, as a primary or salvage procedure after complete brachial plexus injury. At 45 months, three of eight primary vascularized ulnar nerve graft patients regained grade 4 elbow flexion, while one regained grade 3. All 13 primary gracilis transfer patients regained grade 4 elbow flexion. Four patients with vascularized ulnar nerve grafts failed and subsequently had salvage functioning free muscle transfer procedures resulting in delayed recovery. Although vascularized ulnar nerve graft-based primary reconstructions can provide useful elbow flexion, this was achieved in less than half the cases. We consider primary gracilis functioning free muscle transfer neurotized by the distal spinal accessory nerve as the most reliable reconstruction for the restoration of elbow flexion in complete brachial plexus injury. LEVEL OF EVIDENCE: IV.

Relationship between CCL5 and transforming growth factor-ß1 (TGFß1) in breast cancer.

PURPOSE: Investigate circulating CCL5 in breast cancer patients and healthy controls, along with gene expression levels in corresponding tumour tissue and isolated primary stromal cells. Hormonal control of CCL5, and a potential relationship with TGFß1, was also investigated. METHODS: Circulating levels of CCL5 and TGFß1 were measured in 102 breast cancer patients and 66 controls using ELISA. Gene expression levels (CCL5, CCR5, TGFß1, TGFßRII) were quantified in corresponding tumour tissue (n = 43), normal tissue (n = 16), and isolated tumour (n = 22) and normal (n = 3) stromal cells using RQ-PCR. CCL5 and circulating menstrual hormones (LH, FSH, Oestradiol, Progesterone) were analysed in serum samples from healthy, premenopausal volunteers (n = 60). RESULTS: TGFß1 was significantly higher in breast cancer patients (Mean(SEM) 27.4(0.9)ng/ml) compared to controls (14.9(0.9)ng/ml). CCL5 levels decreased in the transition from node negative (59.6(3.7)ng/ml) to node positive disease (40.5(6.3)ng/ml) and increased again as the number of positive lymph nodes increased (⩾3 positive 50.95(9.8)ng/ml). A significant positive correlation between circulating CCL5 and TGFß1 (r = 0.423, p<0.0001) was observed, and mirrored at the gene expression level in tumour tissue from the same patients (r = 0.44, p<0.001). CCL5, CCR5 and TGFß1 expression was significantly higher in tumour compared to normal breast tissue (p < 0.001). A significant negative correlation was observed between circulating CCL5, Oestradiol and Progesterone (r = -0.50, r = -0.39, respectively, p < 0.05). CONCLUSION: CCL5 expression is elevated in the tumour microenvironment. The data support a role for hormonal control of circulating CCL5 and also highlight a potentially important relationship between CCL5 and TGFß1 in breast cancer.

An assessment of skin preparation in upper limb surgery.

Postoperative wound infections remain a major source of upper limb morbidity. The effectiveness of peri-operative human upper limb preparation was determined using a clear fluid antiseptic and an iodine-based solution over 60 and 90 seconds. Less area was missed using iodine over both times and increasing clear solution preparation time from 60 to 90 seconds improved coverage. Surgical experience had little outcome relevance and a 90-second preparation time with either solution was insufficient, with fingers being the sites most commonly missed.

Control of pathogenic CD8+ T cell migration to the brain by IFN-gamma during experimental cerebral malaria.

Previous studies have shown that IFN-gamma is essential for the pathogenesis of cerebral malaria (CM) induced by Plasmodium berghei ANKA (PbA) in mice. However, the exact role of IFN-gamma in the pathway (s) leading to CM has not yet been described. Here, we used 129P2Sv/ev mice which develop CM between 7 and 14 days post-infection with PbA. In this strain, both CD4(+) and CD8(+) T cells were involved in the effector phase of CM. When 129P2Sv/ev mice deficient in the IFN-gamma receptor alpha chain (IFN-gammaR1) were infected with PbA, CM did not occur. Migration of leucocytes to the brain at the time of CM was observed in wild type (WT) but not in deficient mice. However, in the latter, there was an accumulation of T cells in the lungs. Analysis of chemokines and their receptors in WT and in deficient mice revealed a complex, organ-specific pattern of expression. Up-regulation of RANTES/CCL5, IP-10/CCL3 and CCR2 was associated with leucocyte migration to the brain and increased expression of MCP-1/CCL2, IP-10/CCL3 and CCR5 with leucocyte migration to the lung. This shows that IFN-gamma controls trafficking of pathogenic T cells in the brain, thus providing an explanation for the organ-specific pathology induced by PbA infection.

Precisely timed spatiotemporal patterns of neural activity in dissociated cortical cultures.

Recurring patterns of neural activity, a potential substrate of both information transfer and transformation in cortical networks, have been observed in the intact brain and in brain slices. Do these patterns require the inherent cortical microcircuitry of such preparations or are they a general property of self-organizing neuronal networks? In networks of dissociated cortical neurons from rats--which lack evidence of the intact brain's intrinsic cortical architecture--we have observed a robust set of spontaneously repeating spatiotemporal patterns of neural activity, using a template-matching algorithm that has been successful both in vivo and in brain slices. The observed patterns in cultured monolayer networks are stable over minutes of extracellular recording, occur throughout the culture's development, and are temporally precise within milliseconds. The identification of these patterns in dissociated cultures opens a powerful methodological avenue for the study of such patterns, and their persistence despite the topological and morphological rearrangements of cellular dissociation is further evidence that precisely timed patterns are a universal emergent feature of self-organizing neuronal networks.

Co-infection of malaria with HIV: an immunological perspective.

Human immunodeficiency virus (HIV) and Plasmodium parasites are pathogens that induce significant perturbation and activation of the immune system. Due to their geographical overlap, there have been concerns that co-infection with the two pathogens may be a factor in the modification of their development, and in the severity and rate of disease progression that they induce. In this article, we have reviewed some of the studies that have addressed this topic and we have tried to provide immunological mechanisms to explain these potential interactions.

Mycoplasma iguanae sp. nov., from a green iguana (Iguana iguana) with vertebral disease.

Strain 2327T, first cultured from vertebral abscesses of green iguanas (Iguana iguana) collected in Florida, USA, was readily distinguished from all previously described mollicutes by 16S rRNA gene sequence comparisons. Strain 2327T lacks a cell wall, ferments glucose, does not hydrolyse arginine, aesculin or urea and is sensitive to digitonin. Western blots distinguished the novel isolate serologically from the most closely related members of the Mycoplasma neurolyticum cluster. On the basis of these data, the isolate represents a novel species for which the name Mycoplasma iguanae sp. nov. is proposed. The type strain is strain 2327T (=ATCC BAA-1050T = NCTC 11745T).

Phagocyte-derived reactive oxygen species do not influence the progression of murine blood-stage malaria infections.

Phagocyte-derived reactive oxygen species have been implicated in the clearance of malaria infections. We investigated the progression of five different strains of murine malaria in gp91(phox-/-) mice, which lack a functional NADPH oxidase and thus the ability to produce phagocyte-derived reactive oxygen species. We found that the absence of functional NADPH oxidase in the gene knockout mice had no effect on the parasitemia or total parasite burden in mice infected with either resolving (Plasmodium yoelii and Plasmodium chabaudi K562) or fatal (Plasmodium berghei ANKA, Plasmodium berghei K173 and Plasmodium vinckei vinckei) strains of malaria. This lack of effect was apparent in both primary and secondary infections with P. yoelii and P. chabaudi. There was also no difference in the presentation of clinical or pathological signs between the gp91(phox-/-) or wild-type strains of mice infected with malaria. Progression of P. berghei ANKA and P. berghei K173 infections was unchanged in glutathione peroxidase-1 gene knockout mice compared to their wild-type counterparts. The rates of parasitemia progression in gp91(phox-/-) mice and wild-type mice were not significantly different when they were treated with l-N(G)-methylarginine, an inhibitor of nitric oxide synthase. These results suggest that phagocyte-derived reactive oxygen species are not crucial for the clearance of malaria parasites, at least in murine models.

Effects of thyrotropin-releasing hormone on human myometrium and umbilical vasculature in vitro.

OBJECTIVE: The purpose of this study was to investigate the direct effects of thyrotropin-releasing hormone on isolated human myometrium that was obtained during pregnancy and on human umbilical vasculature in vitro. STUDY DESIGN: Isolated human myometrial strips were dissected from biopsy specimens that were obtained at elective cesarean delivery and suspended for isometric recording under physiologic conditions. The effects of cumulative additions of thyrotropin-releasing hormone (10(-9)-10(-4) mol/L) on oxytocin-induced myometrial contractility were evaluated. The effects of thyrotropin-releasing hormone (10(-9)-10(-4) mol/L) on umbilical vessel (artery and vein) resistance in vitro were investigated with the use of isolated ring preparations. RESULTS: Thyrotropin-releasing hormone exerted a significant concentration-dependent relaxant effect on pregnant human myometrial tissue, which ranged from 3.54% (10(-9) mol/L, P=.935) to a net cumulative total of 21.06% (10(-4) mol/L, P<.001). Thyrotropin-releasing hormone also exerted a concentration-dependent relaxant effect on human umbilical vasculature that ranged from 12.51% (10(-9) mol/L, P=.994) to a net cumulative total of 23.27%+/-4.87% (SEM, 10(-4) mol/L, P<.01) in umbilical artery. For umbilical vein, the relaxant effect ranged from 1.80% (10(-9) mol/L, P=.998) to a net cumulative total of 14.64% (10(-4) mol/L, P<.009). CONCLUSION: Thyrotropin-releasing hormone exerts a significant relaxant effect in human myometrium and in human umbilical vasculature and highlights a potential physiologic role for this neuropeptide in these tissues. These findings have clinical implications for the therapeutic use of thyrotropin-releasing hormone antenatally.

Corticosteroids and fetal vasculature: effects of hydrocortisone, dexamethasone and betamethasone on human umbilical artery.

OBJECTIVE: To investigate the direct effects of corticosteroids on human umbilical artery resistance, in vitro. DESIGN: Prospective laboratory study. SETTING: University teaching hospital. SAMPLES AND METHODS: Umbilical artery samples were obtained following normal, term deliveries (n = 50) and dissected rings were suspended for isometric recording under physiological conditions. The effects of hydrocortisone (10(-9) - 10(-4) M), dexamethasone (10(-9) - 10(-4) M) and betamethasone (10(-9) - 10(-4) M) on umbilical artery resistance were measured in vitro. MAIN OUTCOME MEASURES: Changes in umbilical artery resistance, in vitro. RESULTS: Hydrocortisone (n = 12) exerted a vasodilatory effect on human umbilical artery at all concentrations studied compared with vehicle control experiments (n = 12) (P < 0.0001). The mean net relaxant effect of hydrocortisone ranged from 11.77% (10(-9) M) to 57.01% (10(-4)). Both exogenous compounds, dexamethasone (n = 12) and betamethasone (n = 12), similarly exerted a significant relaxant effect on human umbilical artery tone (P < 0.05-0.01), compared with vehicle control experiments (n = 12). The mean net relaxant effect of dexamethasone ranged from 14.43% (10(-9) M) to 38.12% (10(-4)) and that of betamethasone ranged from 6.02% (10(-9) M) to 42.30% (10(-4)), in a cumulatively increasing fashion. There was a non-significant trend towards a greater vasodilatory effect of dexamethasone than betamethasone at lower bath concentrations studied. CONCLUSION: Corticosteroids exert a direct and potent vasodilatory effect on human umbilical artery resistance in vitro, thus providing an explanation for the previously unexplained vascular effects associated with antenatal administration of corticosteroids.

Structure and emergence of specific olfactory glomeruli in the mouse.

Olfactory sensory neurons (OSNs) expressing a given odorant receptor (OR) gene project their axons to a few specific glomeruli that reside at recognizable locations in the olfactory bulb. Connecting approximately 1000 populations of OSNs to the approximately 1800 glomeruli of the mouse bulb poses a formidable wiring problem. Additional progress in understanding the mechanisms of neuronal connectivity is dependent on knowing how these axonal pathways are organized and how they form during development. Here we have applied a genetic approach to this problem. We have constructed by gene targeting novel strains of mice in which either all OSNs or those that express a specific OR gene, M72 or M71, also produce green fluorescent protein (GFP) or a fusion of tau with GFP. We visualized OSNs and their axons in whole mounts with two-photon laser scanning microscopy. The main conclusion we draw from the three-dimensional reconstructions is the high degree of morphological variability of mature glomeruli receiving axonal input from OR-expressing OSNs and of the pathways taken by the axons to those glomeruli. We also observe that axons of OR-expressing OSNs do not innervate nearby glomeruli in mature mice. Postnatally, a tangle of axons from M72-expressing OSNs occupies a large surface area of the bulb and coalesces abruptly into a protoglomerulus at a reproducible stage of development. These results differ in several aspects from those reported for the development of glomeruli receiving input from OSNs expressing the P2 OR, suggesting the need for a more systematic examination of OR-specific glomeruli.

A new approach to neural cell culture for long-term studies.

We have developed a new method for culturing cells that maintains their health and sterility for many months. Using conventional techniques, primary neuron cultures seldom survive more than 2 months. Increases in the osmotic strength of media due to evaporation are a large and underappreciated contributor to the gradual decline in the health of these cultures. Because of this and the ever-present likelihood of contamination by airborne pathogens, repeated or extended experiments on any given culture have until now been difficult, if not impossible. We surmounted survival problems by using culture dish lids that form a gas-tight seal, and incorporate a transparent hydrophobic membrane (fluorinated ethylene-propylene) that is selectively permeable to oxygen (O(2)) and carbon dioxide (CO(2)), and relatively impermeable to water vapor. This prevents contamination and greatly reduces evaporation, allowing the use of a non-humidified incubator. We have employed this technique to grow dissociated cortical cultures from rat embryos on multi-electrode arrays. After more than a year in culture, the neurons still exhibit robust spontaneous electrical activity. The combination of sealed culture dishes with extracellular multi-electrode recording and stimulation enables study of development, adaptation, and very long-term plasticity, across months, in cultured neuronal networks. Membrane-sealed dishes will also be useful for the culture of many other cell types susceptible to evaporation and contamination.

Effects of feeding 4 levels of soy protein for 3 and 6 wk on blood lipids and apolipoproteins in moderately hypercholesterolemic men.

BACKGROUND: Replacing animal protein with soy protein has been shown to reduce total and LDL-cholesterol concentrations in humans. However, the minimum amount of soy protein required for significant reduction of blood lipids is not known. OBJECTIVE: We evaluated the amount of soy protein needed to reduce blood lipids in moderately hypercholesterolemic men. DESIGN: Eighty-one men with moderate hypercholesterolemia (total cholesterol concentration between 5.70 and 7.70 mmol/L) were studied. After a 3-wk lead-in on a Step I diet, total cholesterol was measured and subjects were randomly divided into 5 groups. For 6 wk, each group received 50 g protein/d, which included isolated soy protein (ISP) and casein, respectively, in the following amounts: 50:0, 40:10, 30:20, 20:30, and 0:50 (control group) g. Blood was collected at baseline and weeks 3 and 6 of the intervention. RESULTS: At week 6, significant reductions (P < 0.05) from baseline compared with the control group were found for non-HDL and total cholesterol and apolipoprotein (apo) B for all ISP groups (except total cholesterol with 40 g ISP). At week 3, significant reductions (P < 0.05) were found in apo B for the groups that consumed >/=30 g ISP and in non-HDL cholesterol for the groups that consumed >/=40 g ISP. HDL-cholesterol, apo A-I, lipoprotein(a), and triacylglycerol concentrations were not significantly affected by dietary treatment. CONCLUSION: Our findings show that consuming as little as 20 g soy protein/d instead of animal protein for 6 wk reduces concentrations of non-HDL cholesterol and apo B by approximately 2.6% and 2.2%, respectively. 2000;71:-84.