Identification of the GlialCAM interactome: the G protein-coupled receptors GPRC5B and GPR37L1 modulate megalencephalic leukoencephalopathy proteins.

Megalencephalic Leukoencephalopathy with subcortical Cysts (MLC) is a type of vacuolating leukodystrophy, which is mainly caused by mutations in MLC1 or GLIALCAM. The two MLC-causing genes encode for membrane proteins of yet unknown function that have been linked to the regulation of different chloride channels such as the ClC-2 and VRAC. To gain insight into the role of MLC proteins, we have determined the brain GlialCAM interacting proteome. The proteome includes different transporters and ion channels known to be involved in the regulation of brain homeostasis, proteins related to adhesion or signaling as several G protein-coupled receptors (GPCRs), including the orphan GPRC5B and the proposed prosaposin receptor GPR37L1. Focusing on these two GPCRs, we could validate that they interact directly with MLC proteins. The inactivation of Gpr37l1 in mice upregulated MLC proteins without altering their localization. Conversely, a reduction of GPRC5B levels in primary astrocytes downregulated MLC proteins, leading to an impaired activation of ClC-2 and VRAC. The interaction between the GPCRs and MLC1 was dynamically regulated upon changes in the osmolarity or potassium concentration. We propose that GlialCAM and MLC1 associate with different integral membrane proteins modulating their functions and acting as a recruitment site for various signaling components as the GPCRs identified here. We hypothesized that the GlialCAM/MLC1 complex is working as an adhesion molecule coupled to a tetraspanin-like molecule performing regulatory effects through direct binding or influencing signal transduction events.

Role of astroglial Connexin 43 in pneumolysin cytotoxicity and during pneumococcal meningitis.

Streptococcus pneumoniae or pneumococcus (PN) is a major causative agent of bacterial meningitis with high mortality in young infants and elderly people worldwide. The mechanism underlying PN crossing of the blood brain barrier (BBB) and specifically, the role of non-endothelial cells of the neurovascular unit that control the BBB function, remains poorly understood. Here, we show that the astroglial connexin 43 (aCx43), a major gap junctional component expressed in astrocytes, plays a predominant role during PN meningitis. Following intravenous PN challenge, mice deficient for aCx43 developed milder symptoms and showed severely reduced bacterial counts in the brain. Immunofluorescence analysis of brain slices indicated that PN induces the aCx43-dependent destruction of the network of glial fibrillary acid protein (GFAP), an intermediate filament protein specifically expressed in astrocytes and up-regulated in response to brain injury. PN also induced nuclear shrinkage in astrocytes associated with the loss of BBB integrity, bacterial translocation across endothelial vessels and replication in the brain cortex. We found that aCx4-dependent astrocyte damages could be recapitulated using in vitro cultured cells upon challenge with wild-type PN but not with a ply mutant deficient for the pore-forming toxin pneumolysin (Ply). Consistently, we showed that purified Ply requires Cx43 to promote host cell plasma membrane permeabilization in a process involving the Cx43-dependent release of extracellular ATP and prolonged increase of cytosolic Ca2+ in host cells. These results point to a critical role for astrocytes during PN meningitis and suggest that the cytolytic activity of the major virulence factor Ply at concentrations relevant to bacterial infection requires co-opting of connexin plasma membrane channels.

Astrocytes in the regulation of cerebrovascular functions.

Astrocytes are the most numerous type of neuroglia in the brain and have a predominant influence on the cerebrovascular system; they control perivascular homeostasis, the integrity of the blood-brain barrier, the dialogue with the peripheral immune system, the transfer of metabolites from the blood, and blood vessel contractility in response to neuronal activity. These regulatory processes occur in a specialized interface composed of perivascular astrocyte extensions that almost completely cover the cerebral blood vessels. Scientists have only recently started to study how this interface is formed and how it influences cerebrovascular functions. Here, we review the literature on the astrocytes' role in the regulation of the cerebrovascular system. We cover the anatomy and development of the gliovascular interface, the known gliovascular functions, and molecular factors, the latter's implication in certain pathophysiological situations, and recent cutting-edge experimental tools developed to examine the astrocytes' role at the vascular interface. Finally, we highlight some open questions in this field of research.

Developing and user testing new pharmacy label formats-A study to inform labelling standards.

BACKGROUND: Dispensed prescription medicine labels (prescription labels) are important information sources supporting safe and appropriate medicines use. OBJECTIVE: To develop and user test patient-centred prescription label formats. METHODS: Five stages: developing 12 labels for four fictitious medicines of varying dosage forms; diagnostic user testing of labels (Round 1) with 40 consumers (each testing three labels); iterative label revision, and development of Round 2 labels (n = 7); user testing of labels (Round 2) with 20 consumers (each testing four labels); labelling recommendations. Evaluated labels stated the active ingredient and brand name, using various design features (eg upper case and bold). Dosing was expressed differently across labels: frequency of doses/day, approximate times of day (eg morning), explicit times (eg 7 to 9 AM), and/or explicit dosing interval. Participants' ability to find and understand medicines information and plan a dosing schedule were assessed. RESULTS: Participants demonstrated satisfactory ability to find and understand the dosage for all label formats. Excluding active ingredient and dosing schedule, 14/19 labels (8/12 in Round 1; 6/7 in Round 2) met industry standard on performance. Participants' ability to correctly identify the active ingredient varied, with clear medicine name sign-posting enabling all participants evaluating these labels to find and understand the active ingredient. When planning a dosing schedule, doses were correctly spaced if the label stated a dosing interval, or frequency of doses/day. Two-thirds planned appropriate dosing schedules using a dosing table. CONCLUSIONS: Effective prescription label formatting and sign-posting of active ingredient improved communication of information on labels, potentially supporting safe medicines use. PATIENT AND PUBLIC INVOLVEMENT: Consumers actively contributed to the development of dispensed prescription medicine labels. Feedback from consumers following the first round was incorporated in revisions of the labels for the next round. Patient and public involvement in this study was critical to the development of readable and understandable dispensed prescription medicine labels.

Circumventing 'free care' and 'shouting louder': using a health systems approach to study eye health system sustainability in government and mission facilities of north-west Tanzania.

BACKGROUND: Little is known about the contributions of faith-based organisations (FBOs) to health systems in Africa. In the specialist area of eye health, international and domestic Christian FBOs have been important contributors as service providers and donors, but they are also commonly critiqued as having developed eye health systems parallel to government structures which are unsustainable. METHODS: In this study, we use a health systems approach (quarterly interviews, a participatory sustainability analysis exercise and a social network analysis) to describe the strategies used by eye care practitioners in four hospitals of north-west Tanzania to navigate the government, church mission and donor rules that govern eye services delivery there. RESULTS: Practitioners in this region felt eye care was systemically neglected by government and therefore was 'all under the NGOs', but support from international donors was also precarious. Practitioners therefore adopted four main strategies to improve the sustainability of their services: (1) maintain 'sustainability funds' to retain financial autonomy over income; (2) avoid granting government user fee exemptions to elderly patients who are the majority of service users; (3) expand or contract outreach services as financial circumstances change; and (4) access peer support for problem-solving and advocacy. Mission-based eye teams had greater freedom to increase their income from user fees by not implementing government policies for 'free care'. Teams in all hospitals, however, found similar strategies to manage their programmes even when their management structures were unique, suggesting the importance of informal rules shared through a peer network in governing eye care in this pluralistic health system. CONCLUSIONS: Health systems research can generate new evidence on the social dynamics that cross public and private sectors within a local health system. In this area of Tanzania, Christian FBOs' investments are important, not only in terms of the population health outcomes achieved by teams they support, but also in the diversity of organisational models they contribute to in the wider eye health system, which facilitates innovation.

Mapping human resources for eye health in 21 countries of sub-Saharan Africa: current progress towards VISION 2020.

BACKGROUND: Development of human resources for eye health (HReH) is a major focus of the Global Action Plan 2014 to 2019 to reduce the prevalence of avoidable visual impairment by 25% by the year 2019. The eye health workforce is thought to be much smaller in sub-Saharan Africa than in other regions of the world but data to support this for policy-making is scarce. We collected HReH and cataract surgeries data from 21 countries in sub-Sahara to estimate progress towards key suggested population-based VISION 2020 HReH indicators and cataract surgery rates (CSR) in 2011. METHODS: Routinely collected data on practitioner and surgery numbers in 2011 was requested from national eye care coordinators via electronic questionnaires. Telephone and e-mail discussions were used to determine data collection strategies that fit the national context and to verify reported data quality. Information was collected on six practitioner cadres: ophthalmologists, cataract surgeons, ophthalmic clinical officers, ophthalmic nurses, optometrists and 'mid-level refractionists' and combined with publicly available population data to calculate practitioner to population ratios and CSRs. Associations with development characteristics were conducted using Wilcoxon rank sum tests and Spearman rank correlations. RESULTS: HReH data was not easily available. A minority of countries had achieved the suggested VISION 2020 targets in 2011; five countries for ophthalmologists/cataract surgeons, four for ophthalmic nurses/clinical officers and two for CSR. All countries were below target for optometrists, even when other cadres who perform refractions as a primary duty were considered. The regional (sample) ratio for surgeons (ophthalmologists and cataract surgeons) was 2.9 per million population, 5.5 for ophthalmic clinical officers and nurses, 3.7 for optometrists and other refractionists, and 515 for CSR. A positive correlation between GDP and CSR as well as many practitioner ratios was observed (CSR P = 0.0042, ophthalmologists P = 0.0034, cataract surgeons, ophthalmic nurses and optometrists 0.1 > P > 0.05). CONCLUSIONS: With only a minority of countries in our sample having reached suggested ophthalmic cadre targets and none having reached targets for refractionists in 2011, substantially more targeted investment in HReH may be needed for VISION 2020 aims to be achieved in sub-Saharan Africa.

Trends and implications for achieving VISION 2020 human resources for eye health targets in 16 countries of sub-Saharan Africa by the year 2020.

BACKGROUND: Development of human resources for eye health (HReH) is a major global eye health strategy to reduce the prevalence of avoidable visual impairment by the year 2020. Building on our previous analysis of current progress towards key HReH indicators and cataract surgery rates (CSRs), we predicted future indicator achievement among 16 countries of sub-Saharan Africa by 2020. METHODS: Surgical and HReH data were collected from national eye care programme coordinators on six practitioner cadres: ophthalmologists, cataract surgeons, ophthalmic clinical officers, ophthalmic nurses, optometrists and 'mid-level refractionists' and combined them with publicly available population data to calculate practitioner-to-population ratios and CSRs. Data on workforce entry and exit (2008 to 2010) was used to project practitioner population and CSR growth between 2011 and 2020 in relation to projected growth in the general population. Associations between indicator progress and the presence of a non-physician cataract surgeon cadre were also explored using Wilcoxon rank sum tests and Spearman rank correlations. RESULTS: In our 16-country sample, practitioner per million population ratios are predicted to increase slightly for surgeons (ophthalmologists/cataract surgeons, from 3.1 in 2011 to 3.4 in 2020) and ophthalmic nurses/clinical officers (5.8 to 6.8) but remain low for refractionists (including optometrists, at 3.6 in 2011 and 2020). Among countries that have not already achieved target indicators, however, practitioner growth will be insufficient for any additional countries to reach the surgeon and refractionist targets by year 2020. Without further strategy change and investment, even after 2020, surgeon growth is only expected to sufficiently outpace general population growth to reach the target in one country. For nurses, two additional countries will achieve the target while one will fall below it. In 2011, high surgeon practitioner ratios were associated with high CSR, regardless of the type of surgeon employed. The cataract surgeon workforce is growing proportionately faster than the ophthalmologist. CONCLUSIONS: The HReH workforce is not growing fast enough to achieve global eye health targets in most of the sub-Saharan countries we surveyed by 2020. Countries seeking to make rapid progress to improve CSR could prioritise investment in training new cataract surgeons over ophthalmologists and improving surgical output efficiency.

In mice and humans, brain microvascular contractility matures postnatally.

Although great efforts to characterize the embryonic phase of brain microvascular system development have been made, its postnatal maturation has barely been described. Here, we compared the molecular and functional properties of brain vascular cells on postnatal day (P)5 vs. P15, via a transcriptomic analysis of purified mouse cortical microvessels (MVs) and the identification of vascular-cell-type-specific or -preferentially expressed transcripts. We found that endothelial cells (EC), vascular smooth muscle cells (VSMC) and fibroblasts (FB) follow specific molecular maturation programs over this time period. Focusing on VSMCs, we showed that the arteriolar VSMC network expands and becomes contractile resulting in a greater cerebral blood flow (CBF), with heterogenous developmental trajectories within cortical regions. Samples of the human brain cortex showed the same postnatal maturation process. Thus, the postnatal phase is a critical period during which arteriolar VSMC contractility required for vessel tone and brain perfusion is acquired and mature.

Providing community-based health practitioners with timely and accurate discharge medicines information.

BACKGROUND: Accurate and timely medication information at the point of discharge is essential for continuity of care. There are scarce data on the clinical significance if poor quality medicines information is passed to the next episode of care. This study aimed to compare the number and clinical significance of medication errors and omission in discharge medicines information, and the timeliness of delivery of this information to community-based health practitioners, between the existing Hospital Discharge Summary (HDS) and a pharmacist prepared Medicines Information Transfer Fax (MITF). METHOD: The study used a sample of 80 hospital patients who were at high risk of medication misadventure, and who had a MITF completed in the study period June - October 2009 at a tertiary referral hospital. The medicines information in participating patients' MITFs was validated against their Discharge Prescriptions (DP). Medicines information in each patient's HDS was then compared with their validated MITF. An expert clinical panel reviewed identified medication errors and omissions to determine their clinical significance. The time between patient discharge and the dispatching of the MITF and the HDS to each patient's community-based practitioners was calculated from hospital records. RESULTS: DPs for 77 of the 80 patients were available for comparison with their MITFs. Medicines information in 71 (92%) of the MITFs matched that of the DP. Comparison of the HDS against the MITF revealed that no HDS was prepared for 16 (21%) patients. Of the remaining 61 patients; 33 (54%), had required medications omitted and 38 (62%) had medication errors in their HDS. The Clinical Panel rated the significance of errors or omissions for 70 patients (16 with no HDS prepared and 54 who's HDS was inconsistent with the validated MITF). In 17 patients the error or omission was rated as insignificant to minor; 23 minor to moderate; 24 moderate to major and 6 major to catastrophic. 28 (35%) patients had their HDS dispatched to their community-based practitioners within 48 hours post discharge compared to 80 (100%) of MITFs. CONCLUSION: The MITF is an effective approach for the timely delivery of accurate discharge medicines information to community-based practitioners responsible for the patient's ongoing care.

Megalencephalic leukoencephalopathy with subcortical cysts is a developmental disorder of the gliovascular unit.

Absence of the astrocyte-specific membrane protein MLC1 is responsible for megalencephalic leukoencephalopathy with subcortical cysts (MLC), a rare type of leukodystrophy characterized by early-onset macrocephaly and progressive white matter vacuolation that lead to ataxia, spasticity, and cognitive decline. During postnatal development (from P5 to P15 in the mouse), MLC1 forms a membrane complex with GlialCAM (another astrocytic transmembrane protein) at the junctions between perivascular astrocytic processes. Perivascular astrocytic processes along with blood vessels form the gliovascular unit. It was not previously known how MLC1 influences the physiology of the gliovascular unit. Here, using the Mlc1 knock-out mouse model of MLC, we demonstrated that MLC1 controls the postnatal development and organization of perivascular astrocytic processes, vascular smooth muscle cell contractility, neurovascular coupling, and intraparenchymal interstitial fluid clearance. Our data suggest that MLC is a developmental disorder of the gliovascular unit, and perivascular astrocytic processes and vascular smooth muscle cell maturation defects are primary events in the pathogenesis of MLC and therapeutic targets for this disease.

Integrating pharmacists into care teams: a qualitative systematic review protocol.

OBJECTIVE: This review will systematically examine the qualitative literature reporting on strategies that have been used (or could be developed) by health care services to integrate pharmacists into a multidisciplinary health care team. INTRODUCTION: Delivery models of pharmaceutical care have been developed, trialed and refined since this concept was first defined more than 30 years ago. Delivery models that integrate pharmacists within a multidisciplinary team allow pharmacists to play a pivotal role in improving health outcomes for patients and contributing to patient self-management. Systematic reviews clearly demonstrate the effectiveness of these models; however, the attitudes, beliefs, expectations, understandings, perceptions and experiences of these multidisciplinary teams is less clear. INCLUSION CRITERIA: The populations of interest in this review are health care providers, including hospital specialists, general practitioners, nurses, health workers, pharmacists, allied health workers, aged care workers, Indigenous health workers and health promotion workers. The phenomena of interest are attitudes, beliefs, expectations, understandings, perceptions and experiences of the populations of interest arising from experiencing, developing or implementing strategies that have or could support the integration of pharmacists into multidisciplinary health care teams. METHODS: The databases to be searched include PubMed, Cochrane, EBSCO (CINAHL), Embase, MedNar, Trove and Australian Indigenous Health Infonet. Studies published from 2011 onwards and in English will be considered for inclusion. Selected studies will be assessed for methodological quality by two independent reviewers, using standardized critical appraisal instruments. Where possible, qualitative research findings will be pooled. Where textual pooling is not possible, the findings will be presented in narrative form.

Postnatal development of the astrocyte perivascular MLC1/GlialCAM complex defines a temporal window for the gliovascular unit maturation.

Astrocytes, the most abundant glial cells of the central nervous system are morphologically complex. They display numerous processes interacting with synapses and blood vessels. At the vascular interface, astrocyte endfeet-terminated processes almost entirely cover the blood vessel surface and participate to the gliovascular unit where important vascular properties of the brain are set such as the blood-brain barrier (BBB) integrity. How specific morphological and functional interactions between astrocytes and the vascular compartment develop has not been fully investigated. Here, we elaborated an original experimental strategy to study the postnatal development of astrocyte perivascular endfeet. Using purified gliovascular units, we focused on the postnatal expression of MLC1 and GlialCAM, two transmembrane proteins forming a complex enriched at the junction between mature astrocyte perivascular endfeet. We showed that MLC1 and GlialCAM were enriched and assembled into mature complexes in astrocyte perivascular endfeet between postnatal days 10 and 15, after the formation of astrocyte perivascular Aquaporin 4 water channels. These events correlated with the increased expression of Claudin-5 and P-gP, two endothelial-specific BBB components. These results illustrate for the first time that astrocyte perivascular endfeet differentiation is a complex and progressive process which correlates with BBB maturation. Moreover, our results suggest that maturation of the astrocyte endfeet MLC1/GlialCAM complex between postnatal days 10 and 15 might be a key event in the gliovascular unit maturation.

Connexin 30 is expressed in a subtype of mouse brain pericytes.

Pericytes are mural cells of blood microvessels which play a crucial role at the neurovascular interface of the central nervous system. They are involved in the regulation of blood-brain barrier integrity, angiogenesis, clearance of toxic metabolites, capillary hemodynamic responses, and neuroinflammation, and they demonstrate stem cell activity. Morphological and molecular studies to characterize brain pericytes recently pointed out some heterogeneity in pericyte population. Nevertheless, a clear definition of pericyte subtypes is still lacking. Here, we demonstrate that a fraction of brain pericytes express Connexin 30 (Cx30), a gap junction protein, which, in the brain parenchyma, was thought to be exclusively found in astrocytes. Cx30 could thus be a candidate protein in the composition of the gap junction channels already described between endothelial cells and pericytes. It could also form hemichannels or acts in a channel-independent manner to regulate pericyte morphology, as already observed in astrocytes. Altogether, our results suggest that Cx30 defines a novel brain pericyte subtype.

Connexin 43 Controls the Astrocyte Immunoregulatory Phenotype.

Astrocytes are the most abundant glial cells of the central nervous system and have recently been recognized as crucial in the regulation of brain immunity. In most neuropathological conditions, astrocytes are prone to a radical phenotypical change called reactivity, which plays a key role in astrocyte contribution to neuroinflammation. However, how astrocytes regulate brain immunity in healthy conditions is an understudied question. One of the astroglial molecule involved in these regulations might be Connexin 43 (Cx43), a gap junction protein highly enriched in astrocyte perivascular endfeet-terminated processes forming the glia limitans. Indeed, Cx43 deletion in astrocytes (Cx43KO) promotes a continuous immune recruitment and an autoimmune response against an astrocyte protein, without inducing any brain lesion. To investigate the molecular basis of this unique immune response, we characterized the polysomal transcriptome of hippocampal astrocytes deleted for Cx43. Our results demonstrate that, in the absence of Cx43, astrocytes adopt an atypical reactive status with no change in most canonical astrogliosis markers, but with an upregulation of molecules promoting immune recruitment, complement activation as well as anti-inflammatory processes. Intriguingly, while several of these upregulated transcriptional events suggested an activation of the γ-interferon pathway, no increase in this cytokine or activation of related signaling pathways were found in Cx43KO. Finally, deletion of astroglial Cx43 was associated with the upregulation of several angiogenic factors, consistent with an increase in microvascular density in Cx43KO brains. Collectively, these results strongly suggest that Cx43 controls immunoregulatory and angiogenic properties of astrocytes.

Isolation and differential transcriptome of vascular smooth muscle cells and mid-capillary pericytes from the rat brain.

Brain mural cells form a heterogeneous family which significantly contributes to the maintenance of the blood-brain barrier and regulation of the cerebral blood flow. Current procedures to isolate them cannot specifically separate their distinct subtypes, in particular vascular smooth muscle cells (VSMCs) and mid-capillary pericytes (mcPCs), which differ among others by their expression of smooth muscle actin (SMA). We herein describe an innovative method allowing SMA+ VSMCs and SMA- mcPCs to be freshly isolated from the rat cerebral cortex. Using differential RNA-Seq analysis, we then reveal the specific gene expression profile of each subtype. Our results refine the current description of the role of VSMCs in parenchymal cortical arterioles at the molecular level and provide a unique platform to identify the molecular mechanisms underlying the specific functions of mcPCs in the brain vasculature.

Impact of pain on cognitive functions in primary Sjögren syndrome with small fiber neuropathy: 10 cases and a literature review.

Primary Sjögren syndrome (pSS) is a chronic systemic autoimmune disease characterized by xerophthalmia, xerostomia, and potential peripheral or central neurological involvement. In pSS, the prevalence of cognitive disorders is generally sparse across literature and the impact of pain on cognitive profile is unclear. The aim of this study was to determine the relation between pain, cognitive complaint, and impairment in a very homogenous population of 10 pSS patients with painful small fiber neuropathy (PSFN) and spontaneous cognitive complaint. Neurological exam, neuropsychological assessment, clinical evaluation measuring pain level, fatigue, anxiety, depression, and cognitive complaint were performed. Our results showed that 100% of patients had cognitive dysfunction especially in executive domain (80%). The most sensitive test was the Wisconsin Card Sorting Test (WCST), abnormal in 70% of our population. Moreover, we found clear cut significant correlations between pain levels and 3 measures of WCST: the number of errors (R = -0.768, P = .0062), perseverations (R = 0.831, P = .0042), and categories (R = 0.705, P = .02). In the literature review, the impact of pain is underexplored and results could be discordant. In a homogeneous cohort of pSS patients with PSFN, a cognitive complaint seems to be a valid reflection of cognitive dysfunction marked by a specific executive profile found with the WCST. In this preliminary study, this profile is linked to the level of pain and highlights that an appropriate management of pain control and a cognitive readaptation in patients could improve the quality of life.

Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface.

Astrocytes send out long processes that are terminated by endfeet at the vascular surface and regulate vascular functions as well as homeostasis at the vascular interface. To date, the astroglial mechanisms underlying these functions have been poorly addressed. Here we demonstrate that a subset of messenger RNAs is distributed in astrocyte endfeet. We identified, among this transcriptome, a pool of messenger RNAs bound to ribosomes, the endfeetome, that primarily encodes for secreted and membrane proteins. We detected nascent protein synthesis in astrocyte endfeet. Finally, we determined the presence of smooth and rough endoplasmic reticulum and the Golgi apparatus in astrocyte perivascular processes and endfeet, suggesting for local maturation of membrane and secreted proteins. These results demonstrate for the first time that protein synthesis occurs in astrocyte perivascular distal processes that may sustain their structural and functional polarization at the vascular interface.