Single Cell Analysis of High-Parameter Histology Images Using Histoflow Cytometry.

Immunofluorescence histology is commonly used to study immune cells in tissues where the number of fluorescence parameters is normally limited to four or less. This makes it impossible to interrogate multiple subsets of immune cells in tissue with the same precision as flow cytometry. The latter, however, dissociates tissues and loses spatial information. To bridge the gap between these technologies, we developed a workflow to expand the number of fluorescence parameters that can be imaged on widely available microscopes. We instituted a method for identifying single cells in tissue and exporting the data for flow cytometry-based analysis. This histoflow cytometry technique successfully separates spectrally overlapping dyes and identifies similar numbers of cells in tissue sections as manual cell counts. Populations identified through flow cytometry-like gating strategies are mapped to the original tissue to spatially localize gated subsets. We applied histoflow cytometry to immune cells in the spinal cords of mice with experimental autoimmune encephalomyelitis. We ascertained that B cells, T cells, neutrophils, and phagocytes differed in their frequencies in CNS immune cell infiltrates and were increased relative to healthy controls. Spatial analysis determined that B cells and T cells/phagocytes preferentially localized to CNS barriers and parenchyma, respectively. By spatially mapping these immune cells, we inferred their preferred interacting partners within immune cell clusters. Overall, we demonstrate the ease and utility of histoflow cytometry, which expands the number of fluorescent channels used in conventional immunofluorescence and enables quantitative cytometry and spatial localization of histological analyses.

Uncovering Novel Extracellular Matrix Transcriptome Alterations in Lesions of Multiple Sclerosis.

The extracellular matrix (ECM) of the central nervous system (CNS) is an interconnected network of proteins and sugars with critical roles in both homeostasis and disease. In neurological diseases, excessive ECM deposition and remodeling impact both injury and repair. CNS lesions of multiple sclerosis (MS), a chronic inflammatory and degenerative disease, cause prominent alterations of the ECM. However, there are a lack of data investigating how the multitude of ECM members change in relation to each other and how this affects the MS disease course. Here, we evaluated ECM changes in MS lesions compared to a control brain using databases generated in-house through spatial mRNA-sequencing and through a public resource of single-nucleus RNA sequencing previously published by Absinta and colleagues. These results underline the importance of publicly available datasets to find new targets of interest, such as the ECM. Both spatial and public datasets demonstrated widespread changes in ECM molecules and their interacting proteins, including alterations to proteoglycans and glycoproteins within MS lesions. Some of the altered ECM members have been described in MS, but other highly upregulated members, including the SPARC family of proteins, have not previously been highlighted. SPARC family members are upregulated in other conditions by reactive astrocytes and may influence immune cell activation and MS disease course. The profound changes to the ECM in MS lesions deserve more scrutiny as they impact neuroinflammation, injury, and repair.

Derivation of the Buffalo Concussion Physical Examination risk of delayed recovery (RDR) score to identify children at risk for persistent postconcussive symptoms.

OBJECTIVE: The Buffalo Concussion Physical Examination (BCPE) is a brief, but pertinent physical examination designed for the subacute, outpatient assessment of concussion. The purpose of this study was to perform the BCPE on a larger sample and derive a scoring system to identify children at risk for Persistent Post-Concussive Symptoms (PPCS, recovery ≥30 days). METHODS: This prospective, observational cohort study from September 2016 to March 2019 was performed at three university-affiliated concussion clinics. Male and female children (n=270, 14.92±1.86 years, range 8-18, 38% female) were diagnosed with a concussion within 14 days of injury and followed-up until recovery. Logistic regression was used with history and physical examination variables to predict PPCS and a weighted scoring metric was derived. RESULTS: Out of 15 predictor variables, the main effects of 1 preinjury variable (≥3 previous concussions), 2 injury characteristic variables (days-since-injury and type-of-injury), 3 physical examination variables (orthostatic intolerance (OI), vestibulo-ocular reflex (VOR) and tandem gait) and 2 interaction terms (OI/VOR and tandem gait/type-of-injury) produced a score that was 85% accurate for identifying children with low-risk, medium-risk and high-risk for PPCS on cross-validation. CONCLUSION: The Risk for Delayed Recovery (RDR)-Score allows physicians in an outpatient setting to more accurately predict which children are at greater risk for PPCS early after their injury, and who would benefit most from targeted therapies. The RDR-Score is intended to be used as part of a comprehensive assessment that should include validated symptom checklists, mental health history and adjunct testing (eg, cognitive or physical exertion) where clinically indicated.

Activated B Cells Participating in the Anti-Myelin Response Are Excluded from the Inflamed Central Nervous System in a Model of Autoimmunity that Allows for B Cell Recognition of Autoantigen.

Once activated, T cells gain the ability to access both healthy and inflamed nonlymphoid tissues. They are then reactivated to remain in the tissue and exert their effector function only if they encounter their specific Ag. In this study, we set out to determine if the same is true for B cells using a mouse model of CNS autoimmunity that incorporates both T and B cell recognition of a myelin autoantigen. Both T and B cells were common infiltrates of spinal cords in diseased mice. However, unlike T cells, anti-myelin B cells were excluded from the inflamed tissue. Further, CNS B cells did not have a phenotype consistent with Ag-specific activation as it occurs in lymphatic tissue. Instead, they expressed elevated levels of CD80, indicating that B cells may contribute to local inflammation through nonantigen-specific mechanisms.

An Unusual Cause of Headache and Fatigue in a Division 1 Collegiate Athlete.

Variegate porphyria (VP) is an autosomal dominant disorder of porphyrin metabolism. We report a case of a 21-year-old male collegiate athlete who complained of recurrent headache and fatigue. Extensive testing after initial presentation failed to identify a cause. Months later, his grandmother was diagnosed with VP after being hospitalized; hence, he was tested. He was positive for a heterozygous missense mutation, R168H, in one protoporphyrinogen oxidase allele. This case highlights a rare disorder of heme synthesis that should be considered in the differential diagnosis of exertional fatigue and headaches in athletes. When other more common causes of fatigue and/or headache are unable to be identified, a more focused history and examination may lead to a more unusual but crucial diagnosis. To our knowledge, there are no reported cases of this condition in Division I collegiate athletes.

Lasing transition at 1.06 µm emission in Nd3+ -doped borate-based tellurium calcium zinc niobium oxide glasses for high-power solid-state lasers.

The spectroscopic properties of Tellurium Calcium Zinc Niobium oxide Borate (TCZNB) glasses of composition (in mol%) 10TeO2  + 15CaO + 5ZnO + 10 Nb2 O5  + (60 - x)B2 O3  + Nd2 O3 (x = 0.1, 0.5, 1.0 or 1.5 mol%) have been investigated experimentally. The three phenomenological intensity parameters Ω2 , Ω4, Ω6 have been calculated using the Judd-Ofelt theory and in turn radiative properties such as radiative transition probabilities, emission cross-sections, branching ratios and radiative lifetimes have been estimated. The trend found in the JO intensity parameter is Ω2  > Ω6  > Ω4 If Ω6  > Ω4 , the glass system is favourable for the laser emission 4 F3/2  â†’ 4 I11/2 in the infrared (IR) wavelength. The experimental values of branching ratio of 4 F3/2  â†’ 4 I11/2 transition indicate favourable lasing action with low threshold power. The evaluated total radiative transition probabilities (AT ), stimulated emission cross-section (σe ) and gain bandwidth parameters (σe  × Δλp ) were compared with earlier reports. An energy level analysis has been carried out considering the experimental energy positions of the absorption and emission bands.

Evaluation of the Veterans Health Administration's Specialty Care Transformational Initiatives to Promote Patient-Centered Delivery of Specialty Care: A Mixed-Methods Approach.

INTRODUCTION: Veteran's Affairs Office of Specialty Care (OSC) launched four national initiatives (Electronic-Consults [e-Consults], Specialty Care Access Networks-Extension for Community Healthcare Outcomes [SCAN-ECHO], Mini-Residencies, and Specialty Care Neighborhood) to improve specialty care delivery and funded a center to evaluate the initiatives. METHODS: The evaluation, guided by two implementation frameworks, provides formative (administrator/provider interviews and surveys) and summative data (quantitative data on patterns of use) about the initiatives to OSC. RESULTS: Evaluation of initiative implementation is assessed through CFIR (Consolidated Framework for Implementation Research)-grounded qualitative interviews to identify barriers/facilitators. Depending on high or low implementation, factors such as receiving workload credit, protected time, existing workflow/systems compatibility, leadership engagement, and access to information/resources were considered implementation barriers or facilitators. Findings were shared with OSC and used to further refine implementation at additional sites. Evaluation of other initiatives is ongoing. CONCLUSIONS: The mixed-methods approach has provided timely information to OSC about initiative effect and impacted OSC policies on implementation at additional sites.

Veterans Affairs initiative to prevent methicillin-resistant Staphylococcus aureus infections.

BACKGROUND: Health care-associated infections with methicillin-resistant Staphylococcus aureus (MRSA) have been an increasing concern in Veterans Affairs (VA) hospitals. METHODS: A "MRSA bundle" was implemented in 2007 in acute care VA hospitals nationwide in an effort to decrease health care-associated infections with MRSA. The bundle consisted of universal nasal surveillance for MRSA, contact precautions for patients colonized or infected with MRSA, hand hygiene, and a change in the institutional culture whereby infection control would become the responsibility of everyone who had contact with patients. Each month, personnel at each facility entered into a central database aggregate data on adherence to surveillance practice, the prevalence of MRSA colonization or infection, and health care-associated transmissions of and infections with MRSA. We assessed the effect of the MRSA bundle on health care-associated MRSA infections. RESULTS: From October 2007, when the bundle was fully implemented, through June 2010, there were 1,934,598 admissions to or transfers or discharges from intensive care units (ICUs) and non-ICUs (ICUs, 365,139; non-ICUs, 1,569,459) and 8,318,675 patient-days (ICUs, 1,312,840; and non-ICUs, 7,005,835). During this period, the percentage of patients who were screened at admission increased from 82% to 96%, and the percentage who were screened at transfer or discharge increased from 72% to 93%. The mean (±SD) prevalence of MRSA colonization or infection at the time of hospital admission was 13.6±3.7%. The rates of health care-associated MRSA infections in ICUs had not changed in the 2 years before October 2007 (P=0.50 for trend) but declined with implementation of the bundle, from 1.64 infections per 1000 patient-days in October 2007 to 0.62 per 1000 patient-days in June 2010, a decrease of 62% (P<0.001 for trend). During this same period, the rates of health care-associated MRSA infections in non-ICUs fell from 0.47 per 1000 patient-days to 0.26 per 1000 patient-days, a decrease of 45% (P<0.001 for trend). CONCLUSIONS: A program of universal surveillance, contact precautions, hand hygiene, and institutional culture change was associated with a decrease in health care-associated transmissions of and infections with MRSA in a large health care system.

Generating and Analyzing High-Parameter Histology Images with Histoflow Cytometry.

The usage of histology to investigate immune cell diversity in tissue sections such as those derived from the central nervous system (CNS) is critically limited by the number of fluorescent parameters that can be imaged at a single time. Most immune cell subsets have been defined using flow cytometry by using complex combinations of protein markers, often requiring four or more parameters to conclusively identify, which is beyond the capabilities of most conventional microscopes. As flow cytometry dissociates tissues and loses spatial information, there is a need for techniques that can retain spatial information while interrogating the roles of complex cell types. These issues are addressed here by creating a method for expanding the number of fluorescent parameters that can be imaged by collecting the signals of spectrally overlapping fluorophores and using spectral unmixing to separate the signals of each individual fluorophore. These images are then processed using an analysis pipeline to take high-parameter histology images and extract single cells from these images so that the unique fluorescent properties of each cell can be analyzed at a single-cell level. Using flow cytometry-like gating strategies, cells can then be profiled into subsets and mapped back onto the histology sections to not only quantify their abundance, but also establish how they interact with the tissue environment. Overall, the simplicity and potential of using histoflow cytometry to study complex immune populations in histology sections is demonstrated.

A cadaveric study of arteriovenous trigone of heart: the triangle of Brocq and Mouchet.

Left coronary artery divides into anterior interventricular branch and circumflex branch. As both the arteries run in their corresponding grooves, an arteriovenous trigone is formed between conus arteriosus and left auricle called triangle of Brocq and Mouchet. The triangle base is formed by great cardiac vein. This study aims to describe the frequency of triangle and its type and relationship between various boundaries and content of triangle and to supplement the existing knowledge of clinicians. This observational and descriptive study was conducted on 40 formalin fixed cadaveric hearts in department of anatomy, Kalpana chawla government medical college. The triangle was found in 92.5% of specimen with most common type being closed (51.3%) which is followed by inferiorly open in 35.1%, superiorly open in 8.1% and completely open in 5.4% hearts. Most frequent content of triangle was median artery followed by diagonal branches of anterior interventricular and circumflex branches. The mean area of the triangle was 246.3 mm2. Relationship of vein with two arterial branches was either superficial or deep. The knowledge of different patterns of existence will be required for angiographic procedures. Further the triangle is a potential epicardial access route to left fibrous ring. Thus detailed knowledge of variations will help cardiologist to achieve better outcome in interventional procedures with minimal complications.

B cells in central nervous system disease: diversity, locations and pathophysiology.

B cells represent a relatively minor cell population within both the healthy and diseased central nervous system (CNS), yet they can have profound effects. This is emphasized in multiple sclerosis, in which B cell-depleting therapies are arguably the most efficacious treatment for the condition. In this Review, we discuss how B cells enter and persist in the CNS and how, in many neurological conditions, B cells concentrate within CNS barriers but are rarely found in the parenchyma. We highlight how B cells can contribute to CNS pathology through antibody secretion, antigen presentation and secretion of neurotoxic molecules, using examples from CNS tumours, CNS infections and autoimmune conditions such as neuromyelitis optica and, in particular, multiple sclerosis. Overall, understanding common and divergent principles of B cell accumulation and their effects within the CNS could offer new insights into treating these devastating neurological conditions.

Prognostic Parameters and Their Relevance in Outcome of Middle Ear Surgeries.

To study various parameters, including Middle ear risk index (MERI) and their correlation with outcome of middle ear surgery. The study was conducted from September 2015 to May 2017 in Department of Otolaryngology at our institute. It included 185 cases of safe type of chronic suppurative otitis media. These patients were admitted and treated surgically and record was kept for at least 3 months follow-up in postoperated period. The study concluded that a good correlation exist between MERI and result of tympanoplasty.

Expression of antioxidant enzymes in lesions of multiple sclerosis and its models.

Oxidative stress promotes tissue injury in the central nervous system in neurological disorders such as multiple sclerosis (MS). To protect against this, antioxidant enzymes including superoxide dismutase-1 (SOD1), heme oxygenase-1 (HO-1), peroxiredoxin-5 (PRDX5) and glutathione peroxidase-4 (GPX4) may be upregulated. However, whether antioxidant enzyme elevation in mouse models of neurodegeneration corresponds to their expression in human diseases such as MS requires investigation. Here, we analyzed and compared the expression of SOD1, HO-1, PRDX5 and GPX4 in the murine spinal cord of three models of MS: focal lesions induced by (1) oxidized phosphatidylcholine or (2) lysophosphatidylcholine (lysolecithin), and (3) diffuse lesions of experimental autoimmune encephalomyelitis. Notably, CD68+ microglia/macrophages were the predominant cellular populations that expressed the highest levels of the detected antioxidant enzymes. Overall, the expression patterns of antioxidant enzymes across the models were similar. The increase of these antioxidant enzymes was corroborated in MS brain tissue using spatial RNA sequencing. Collectively, these results show that antioxidant capacity is relatively conserved between mouse models and MS lesions, and suggest a need to investigate whether the antioxidant elevation in microglia/macrophages is a protective response during oxidative injury, neurodegeneration, and MS.

Status of Contralateral Ear in Patients with Unilateral Chronic Otitis Media.

This study was conducted to evaluate the existence of otoscopic abnormality, hearing status and radiological changes in contralateral ear of patients with chronic otitis media. 300 patients having unilateral Chronic Otitis Media attending OPD in the Department of Otorhinolaryngology, Institute of Medical Sciences, Banaras Hindu University, Varanasi during the period of March 2019 to March 2020 were selected. Otoscopy, Pure Tone Audiometry and Bilateral X-ray mastoids (lateral oblique view) and/or HRCT Temporal bone were done. Contralateral ear was affected in more than 30% cases. Out of 188 patients having Mucosal COM, 58 cases (30.9%) had abnormal TM. Out of 112 patients having Squamosal COM, 48 cases (42.9%) had abnormal CLE. Out of 300 cases, 231 (77.0%) of them had normal hearing in contralateral ear. It was followed by 65 cases (21.6%) with conductive hearing loss. Mixed hearing loss and SNHL were seen in 2 patients each. In contralateral ear of Mucosal COM, pneumatic pattern of pneumatisation was seen in 69.1% followed by Diploic pattern (30.9%). In squamosal COM, X-ray mastoid showed pneumatic pattern (64.3%) followed by Diploic pattern (33.9%) in the contralateral ear. Sclerotic pattern was seen in only 1.8% of cases in contralateral ear. Chronic otitis media as a disease is not limited to one ear. The precise and critical evaluation of both ears does not play a role in prognostic evaluation of the patient only, but it can also serve as a guide for early detection of probable evolution of the disease process in a patient in contralateral ear with unilateral chronic otitis media.

Single-cell and spatial RNA sequencing identify perturbators of microglial functions with aging.

Microglia are the immune sentinels of the central nervous system with protective roles such as the removal of neurotoxic oxidized phosphatidylcholines (OxPCs). As aging alters microglial function and elevates neurological disability in diseases such as multiple sclerosis, defining aging-associated factors that cause microglia to lose their custodial properties or even become injurious can help to restore their homeostasis. We used single-cell and spatial RNA sequencing in the spinal cord of young (6-week-old) and middle-aged (52-week-old) mice to determine aging-driven microglial reprogramming at homeostasis or after OxPC injury. We identified numerous aging-associated microglial transcripts including osteopontin elevated in OxPC-treated 52-week-old mice, which correlated with greater neurodegeneration. Osteopontin delivery into the spinal cords of 6-week-old mice worsened OxPC lesions, while its knockdown in 52-week-old lesions attenuated microglial inflammation and axon loss. Thus, elevation of osteopontin and other transcripts in aging disorders including multiple sclerosis perturbs microglial functions contributing to aging-associated neurodegeneration.

Versican promotes T helper 17 cytotoxic inflammation and impedes oligodendrocyte precursor cell remyelination.

Remyelination failure in multiple sclerosis (MS) contributes to progression of disability. The deficient repair results from neuroinflammation and deposition of inhibitors including chondroitin sulfate proteoglycans (CSPGs). Which CSPG member is repair-inhibitory or alters local inflammation to exacerbate injury is unknown. Here, we correlate high versican-V1 expression in MS lesions with deficient premyelinating oligodendrocytes, and highlight its selective upregulation amongst CSPG members in experimental autoimmune encephalomyelitis (EAE) lesions modeling MS. In culture, purified versican-V1 inhibits oligodendrocyte precursor cells (OPCs) and promotes T helper 17 (Th17) polarization. Versican-V1-exposed Th17 cells are particularly toxic to OPCs. In NG2CreER:MAPTmGFP mice illuminating newly formed GFP+ oligodendrocytes/myelin, difluorosamine (peracetylated,4,4-difluoro-N-acetylglucosamine) treatment from peak EAE reduces lesional versican-V1 and Th17 frequency, while enhancing GFP+ profiles. We suggest that lesion-elevated versican-V1 directly impedes OPCs while it indirectly inhibits remyelination through elevating local Th17 cytotoxic neuroinflammation. We propose CSPG-lowering drugs as potential dual pronged repair and immunomodulatory therapeutics for MS.

Diagnostic and therapeutic challenges in a critically ill patient in ICU with superior vena cava syndrome--case report.

PURPOSE: To highlight the diagnostic and therapeutic challenges associated with the treatment of a patient with superior vena cava syndrome and a coexisting coagulopathy. CLINICAL FEATURES: This case report describes a bone marrow transplant patient with graft versus host diseases (GVHD) who was admitted to our intensive care unit with bronchiectasis complicated with nosocomial pneumonia. When he was recovering from pneumonia after prolonged ventilatory support, he developed superior vena cava (SVC) syndrome due to mediastinal lymphadenopathy. The diagnosis was delayed due to associated confounding clinical factors. Because of the rapid deterioration in patient's condition, immediate tissue diagnosis of mediastinal lymph nodes and re-canalization of vena cava by stenting were our priority. He had many other medical problems such as thrombocytopenia, deranged coagulation profile, old cerebral infarction with hemiplegia, seizure disorder and cardiac arrhythmias which complicated the treatment plan. USG guided biopsy followed by stenting of the SVC was done after discussing the risks and benefits with patient's relatives. But, he had bleeding from biopsy site due to deranged coagulation profile. Again for the same reason, he was not given any anticoagulants. Within 24 hours the stent was blocked by clot which was diagnosed by the deteriorating clinical features and repeat CT scan. Then he was given enoxaparin in therapeutic dose and the clot cleared within a day possibly partly due to enoxaparin and partly coagulopathy. CONCLUSION: In a bone marrow transplant patient with GVHD, the associated complications can confound the diagnosis of SVC syndrome. Physician has to show high degree of suspicion as it may develop even if patient has coagulopathy due to other factors such mediastinal lymphadenopathy. SVC stent may clot even if the patient has coagulopathy. So, it is advisable to defer the invasive diagnostic procedures such as mediastinal lymph node biopsy till the patient is well stabilized after the stent placement in SVC as it will prevent further use of anticoagulants. Enoxaparin may be helpful in the treatment of stent thrombosis in such patients with multiple complications.

A new marker of carotid atherosclerosis in middle aged adults: cystatin C or microalbuminuria.

OBJECTIVE: Cystatin C and microalbuminuria, the new markers of atherosclerosis, have been shown to predict cardiovascular outcome in older individuals. However there is limited data regarding their role in middle aged individuals with preserved renal functions. Hence this study. METHODS: Patients aged 45 to 65 years irrespective of presence or absence of diabetes, hypertension or coronary artery disease were subjected to high resolution B mode ultrasonography for carotid artery intimal-medial complex thickness (IMT). Patients with maximal intimal-medial complex thickness greater than 800 microm at the far wall of the common carotid artery, excluding raised lesions and plaques, were selected for the study. Study participants were subjected to biochemical tests for serum cystatin-C, serum creatinine, glomerular filtration rate (GFR) and for presence of microalbuminuria. The relationship of carotid artery intimal-medial complex thickness with serum cystatin C and microalbuminuria was compared. RESULTS: The mean carotid IMT of the study group was 928 +/- 117 microm. Carotid IMT was significantly associated with advancing age, raised systolic blood pressure, ESR, GFR, LDL cholesterol and microalbuminuria but not with cystatin C. Carotid IMT has significant association with systolic blood pressure but not with diastolic blood pressure. There was a statistically significant difference in ESR across tertiles (p < 0.05). There was a statistically significant difference in GFR across tertiles (p < 0.05) and an inverse relationship was found between GFR and IMT. Carotid IMT was positively and significantly correlated with microalbuminuria but there was no significant correlation of carotid IMT with serum cystatin C. CONCLUSION: Microalbuminuria was found to be associated with carotid atherosclerosis in middle aged individuals. However cystatin C was not associated with carotid atherosclerosis in patients with preserved renal functions.

Prognostic Value of Neutrophil-Lymphocyte Ratio and Platelet-Lymphocyte Ratio in Head and Neck Malignancies.

To study the prognostic significance of neutrophil-lymphocyte ratio (NLR) and platelet-lymphocyte ratio (PLR) in head and neck cancers. The study included 170 cases of histopathologically diagnosed head and neck cancer patients and 80 control subjects. NLR and PLR of patients with head and neck cancers were compared to the control group. The correlation between NLR and PLR values and factors such as age, gender, duration of symptoms, site of tumour, histological type, histological grading, T-category, N-category and TNM stages in cancer patients were analysed. NLR and PLR were statistically higher in cancer patients compared to control. There was a non-significant increase in both NLR and PLR with advancing degree of differentiation and TNM Stages of the cancer patients. A significant increase in NLR and PLR with increasing T Categories and increasing N Categories of head and neck cancer patients was obtained. NLR and PLR can be used to estimate tumour prognosis in head and neck cancers. Increased NLR and PLR values can be used as a marker for poor prognosis. However further studies with larger study groups including treatment response and surveillance should be carried out to corroborate these results.