This study examined the effects of perforin, a pore-forming protein, on oligodendrocytes cultured from SJL mice. Oligodendrocytes were exposed to perforin for up to 2.5 hours and examined using microscopy. The results showed that the majority of oligodendrocytes were killed within 60-90 minutes via pore expansion and membrane disruption. Structural features included cell body swelling, fenestration and fragmentation of membranes and processes, cytoplasmic vacuolation, and breakdown of the nuclear envelope. These patterns of damage resembled those seen in multiple sclerosis lesions. The findings suggest that perforin may play an important role in demyelination in multiple sclerosis.
Prevalence of Gene Polymorphisms in Intervertebral Disc Degenerative Diseasesijtsrd
Intervertebral disc diseases is otherwise known as breakdown of one or more discs that separate the bone of the spine, Which cause pain in the back, neck as well as frequent pain in legs and arms. The general role of intervertebral discs is to provide the cushioning between vertebrae and absorb the pressure that is exerted on the spine. Generally low back pain LBP is considered as common disorder from disc degenerative diseases. The main reason of the LBP is due to contribution of intervertebral disc diseases and combination of genetic and environmental factors. Most of these factor identified , But some of these remain unidentified. However recent researches identified that variations in several genes may influence the risc of intervertebral disc diseases . Most of the variations associated with genes related with immune function leads to increase risk of intervertebral disc diseases . Most of these genes plays a vital role in immune response like inflammation and water loss dehydration of the discs, Which causes their degeneration. This review focuses on the evidence of genetic disposition, the genes or biological processes that are implicated, and the need to consolidate resources and clarify phenotype definition to take advantage of the new technologies in genetic analysis to enhance our understanding of this condition. Mr. C. Marimuthu | Dr. V. Pushpa Rani | Dr. V. Judia Harriet Sumathy "Prevalence of Gene Polymorphisms in Intervertebral Disc Degenerative Diseases" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-3 | Issue-2 , February 2019, URL: https://www.ijtsrd.com/papers/ijtsrd19153.pdf
Paper URL: https://www.ijtsrd.com/biological-science/molecular-biology/19153/prevalence-of-gene-polymorphisms-in-intervertebral-disc-degenerative-diseases/mr-c-marimuthu
Endothelial Cell Mediated Delay of Blood Brain Barrier Recovery Following Tra...Arthur Stem
TBI is the leading cause of death among young adults and children in the developed world, accounting for over 50,000 deaths per year. [12] TBI results in a sleuth of poor health outcomes, including hemorrhaging, seizures, neural edema, neural inflammation, and cognitive and emotional disabilities. All of these outcomes are a direct result of fundamental degradation of the BBB over a time course post TBI. [1] [12] The BBB is an integral structure that forms around the microvascular of the cerebral cavity. Endothelial cells form the basal membrane through which strictly controlled movement of molecules is observed between the extravascular and intravascular space across this basal membrane. This basal membrane is maintained by endothelial cells, having tight junctions between them to make up the pores through which transport of molecules can occur between the brain and microvasculature. These tight junctions are maintained through cross-talk between the endothelial cells and supporting neurons such as astrocytes and pericytes. [2] A multitude of proteins make up the tight junctions between the endothelial cells, including six main scaffolding structures Claudins 1, 3, and 5, ZO-1, Occludins, and Cadherins. [3] VEGF release following trauma induces endothelial cells to release matrix metalloproteinases (MMPs), in particular MMP9, which can catalyze the N-terminal amino acids that compose the tight junction protein ZO-1. [10] [11] MMP9 when in circulation is also known to activate tumor necrosis factor alpha (TNFɑ) which in turn upregulates transcription of MMP9, creating a positive feedback loop. [11] The management of MMP production is three fold, transcription, proenzyme activation, and substrate inhibition. [11] In our study, it is proenzyme activation via TP that is the focus and how that affects the overall transcription levels of the tight junction proteins within the endothelial cells and astrocytes.
Prevalence of Gene Polymorphisms in Intervertebral Disc Degenerative Diseasesijtsrd
Intervertebral disc diseases is otherwise known as breakdown of one or more discs that separate the bone of the spine, Which cause pain in the back, neck as well as frequent pain in legs and arms. The general role of intervertebral discs is to provide the cushioning between vertebrae and absorb the pressure that is exerted on the spine. Generally low back pain LBP is considered as common disorder from disc degenerative diseases. The main reason of the LBP is due to contribution of intervertebral disc diseases and combination of genetic and environmental factors. Most of these factor identified , But some of these remain unidentified. However recent researches identified that variations in several genes may influence the risc of intervertebral disc diseases . Most of the variations associated with genes related with immune function leads to increase risk of intervertebral disc diseases . Most of these genes plays a vital role in immune response like inflammation and water loss dehydration of the discs, Which causes their degeneration. This review focuses on the evidence of genetic disposition, the genes or biological processes that are implicated, and the need to consolidate resources and clarify phenotype definition to take advantage of the new technologies in genetic analysis to enhance our understanding of this condition. Mr. C. Marimuthu | Dr. V. Pushpa Rani | Dr. V. Judia Harriet Sumathy "Prevalence of Gene Polymorphisms in Intervertebral Disc Degenerative Diseases" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-3 | Issue-2 , February 2019, URL: https://www.ijtsrd.com/papers/ijtsrd19153.pdf
Paper URL: https://www.ijtsrd.com/biological-science/molecular-biology/19153/prevalence-of-gene-polymorphisms-in-intervertebral-disc-degenerative-diseases/mr-c-marimuthu
Endothelial Cell Mediated Delay of Blood Brain Barrier Recovery Following Tra...Arthur Stem
TBI is the leading cause of death among young adults and children in the developed world, accounting for over 50,000 deaths per year. [12] TBI results in a sleuth of poor health outcomes, including hemorrhaging, seizures, neural edema, neural inflammation, and cognitive and emotional disabilities. All of these outcomes are a direct result of fundamental degradation of the BBB over a time course post TBI. [1] [12] The BBB is an integral structure that forms around the microvascular of the cerebral cavity. Endothelial cells form the basal membrane through which strictly controlled movement of molecules is observed between the extravascular and intravascular space across this basal membrane. This basal membrane is maintained by endothelial cells, having tight junctions between them to make up the pores through which transport of molecules can occur between the brain and microvasculature. These tight junctions are maintained through cross-talk between the endothelial cells and supporting neurons such as astrocytes and pericytes. [2] A multitude of proteins make up the tight junctions between the endothelial cells, including six main scaffolding structures Claudins 1, 3, and 5, ZO-1, Occludins, and Cadherins. [3] VEGF release following trauma induces endothelial cells to release matrix metalloproteinases (MMPs), in particular MMP9, which can catalyze the N-terminal amino acids that compose the tight junction protein ZO-1. [10] [11] MMP9 when in circulation is also known to activate tumor necrosis factor alpha (TNFɑ) which in turn upregulates transcription of MMP9, creating a positive feedback loop. [11] The management of MMP production is three fold, transcription, proenzyme activation, and substrate inhibition. [11] In our study, it is proenzyme activation via TP that is the focus and how that affects the overall transcription levels of the tight junction proteins within the endothelial cells and astrocytes.
CYTOO launches the CYTOO Stories!
For the first issue, we have chosen a Nature paper from Sirio Dupont and Stefano Piccolo, from the Department of Biomedical Sciences of the University of Padua School of Medicine. They identified the first direct connection between gene regulation and extracellular matrix stiffness and geometry. Their study, conducted on CYTOO micropatterns, opens new perspectives in the approach of tissue morphogenesis, tissue homeostasis, and in oncology.
"Nuclear transcription factors YAP/TAZ like it stiff", what do you think about this story? We are waiting for your comments!
And in a few days, you will discover a new CYTOO story...
Amyotrophic Lateral Sclerosis (ALS) is a progressive motoneuron degenerative disease; its cause and mechanisms of pathogenesis remain obscure. Apoptosis may be one of the pathways responsible for motoneuron death in ALS. The present study is to further explore the role of apoptosis in ALS. Using a mutant Cu, Zn-superoxide dismutase(mSOD1) transgenic mouse model, we investigated the correlation between SOD1 mutation and neuronal and glial apoptosis in different brain regions. TUNEL-fl uorescence staining and ELISA quantification found significantly more DNA fragmentation in mSOD1 mice than in controls. Double staining with TUNEL + an anti-choline acetyltransferase antibody showed many TUNEL-positive motoneurons and glial cells in the spinal cord and brain stem of mSOD1 mice, but not in the controls. Transmission electron microscopy confirmed neuronal and glial apoptosis in the spinal cord, brain stem, and motor cortex by specific morphological features of apoptosis. Double staining with TUNEL + an anti-neuron-specifi cenolase antibody showed many TUNEL-positive neurons in both the motor and sensory cortices of mSOD1 mice, but not in the controls. Counting the number of TUNEL-positive neurons in the sections from the three mouse groups showed significantly more TUNEL-positive neurons in both the motor and sensory cortices of mSOD1 mice than in the corresponding regions of control mice. There is no signifi cant difference between motor and sensory cortices of mSOD1 mice. These results provide firm in vivo evidence that SOD1 mutation induced apoptosis of motoneurons and non-motoneuron cells in motoneuron-enriched brain regions. Therefore, apoptosis is not specifi c for motoneuron death, but a common pathway causing neuronal and glial degeneration and death in motoneuron-enriched brain regions. SOD1 mutation also induced apoptosis in sensory neurons of sensory cortex. Together, these findings imply that apoptosis might be one of pathways in which the non-neuronal cells and non-motoneuron-enriched brain regions involve in motoneuron death in ALS.
My list of scientific publications (at present over 100 peer reviewed publications, 2 books, and several book chapters in other books), and more than 40 invited presentations world wide.
Pten Deletion in Adult Neural Stem/Progenitor Cells Enhances Constitutive Neu...johnohab
Here we show that conditional deletion of Pten in a subpopulation of adult neural stem cells in the subependymal zone (SEZ) leads to persistently enhanced neural stem cell self-renewal without sign of exhaustion. These Pten null SEZ-born neural stem cells and progenies can follow the endogenous migration, differentiation, and integration pathways and contribute to constitutive neurogenesis in the olfactory bulb. As a result, Pten deleted animals have increased olfactory bulb mass and enhanced olfactory function. Pten null cells in the olfactory bulb can establish normal connections with peripheral olfactory epithelium and help olfactory bulb recovery from acute damage. Following a focal stroke, Pten null progenitors give rise to greater numbers of neuroblasts that migrate to peri-infarct cortex. However, in contrast to the olfactory bulb, no significant long-term survival and integration can be observed, indicating that additional factors are necessary for long-term survival of newly born neurons after stroke. These data suggest that manipulating PTEN-controlled signaling pathways may be a useful step in facilitating endogenous neural stem/progenitor expansion for the treatment of disorders or lesions in regions associated with constitutive neurogenesis.
A Critical Role of Erythropoietin Receptor in Neurogenesis and Post-Stroke Re...johnohab
Erythropoietin (EPO) is the principal growth factor regulating the production of red blood cells. Recent studies demonstrated that
exogenous EPO acts as a neuroprotectant and regulates neurogenesis. Using a genetic approach, we evaluate the roles of endogenous EPO
and its classical receptor (EPOR) in mammalian neurogenesis. We demonstrate severe and identical embryonic neurogenesis defects in
animals null for either the Epo or EpoR gene, suggesting that the classical EPOR is essential for EPO action during embryonic neurogenesis.
Furthermore, by generating conditional EpoR knock-down animals, we demonstrate that brain-specific deletion of EpoR leads to
significantly reduced cell proliferation in the subventricular zone and impaired post-stroke neurogenesis. EpoR conditional knockdown
leads to a specific deficit in post-stroke neurogenesis through impaired migration of neuroblasts to the peri-infarct cortex. Our results
suggest that both EPO and EPOR are essential for early embryonic neural development and that the classical EPOR is important for adult
neurogenesis and for migration of regenerating neurons during post-injury recovery.
Define epigenetics.
Describe important epigenetic mechanism and explain the implication of epigenetics in normal functions, disease and disease presentation.
Outline the heritability or epigenetic effects.
Explain the role of epigenetic in the development of cancer.
Outline the potentials of epigenetic intervention in battling cancer.
Speaker,
Dr. Md. Mohiuddin Masum
MS Anatomy, Phase-A, Year-1, Block-1
Guided by,
Prof. K M Shamim
Professor, Dept. of Anatomy, BSMMU
CYTOO launches the CYTOO Stories!
For the first issue, we have chosen a Nature paper from Sirio Dupont and Stefano Piccolo, from the Department of Biomedical Sciences of the University of Padua School of Medicine. They identified the first direct connection between gene regulation and extracellular matrix stiffness and geometry. Their study, conducted on CYTOO micropatterns, opens new perspectives in the approach of tissue morphogenesis, tissue homeostasis, and in oncology.
"Nuclear transcription factors YAP/TAZ like it stiff", what do you think about this story? We are waiting for your comments!
And in a few days, you will discover a new CYTOO story...
Amyotrophic Lateral Sclerosis (ALS) is a progressive motoneuron degenerative disease; its cause and mechanisms of pathogenesis remain obscure. Apoptosis may be one of the pathways responsible for motoneuron death in ALS. The present study is to further explore the role of apoptosis in ALS. Using a mutant Cu, Zn-superoxide dismutase(mSOD1) transgenic mouse model, we investigated the correlation between SOD1 mutation and neuronal and glial apoptosis in different brain regions. TUNEL-fl uorescence staining and ELISA quantification found significantly more DNA fragmentation in mSOD1 mice than in controls. Double staining with TUNEL + an anti-choline acetyltransferase antibody showed many TUNEL-positive motoneurons and glial cells in the spinal cord and brain stem of mSOD1 mice, but not in the controls. Transmission electron microscopy confirmed neuronal and glial apoptosis in the spinal cord, brain stem, and motor cortex by specific morphological features of apoptosis. Double staining with TUNEL + an anti-neuron-specifi cenolase antibody showed many TUNEL-positive neurons in both the motor and sensory cortices of mSOD1 mice, but not in the controls. Counting the number of TUNEL-positive neurons in the sections from the three mouse groups showed significantly more TUNEL-positive neurons in both the motor and sensory cortices of mSOD1 mice than in the corresponding regions of control mice. There is no signifi cant difference between motor and sensory cortices of mSOD1 mice. These results provide firm in vivo evidence that SOD1 mutation induced apoptosis of motoneurons and non-motoneuron cells in motoneuron-enriched brain regions. Therefore, apoptosis is not specifi c for motoneuron death, but a common pathway causing neuronal and glial degeneration and death in motoneuron-enriched brain regions. SOD1 mutation also induced apoptosis in sensory neurons of sensory cortex. Together, these findings imply that apoptosis might be one of pathways in which the non-neuronal cells and non-motoneuron-enriched brain regions involve in motoneuron death in ALS.
My list of scientific publications (at present over 100 peer reviewed publications, 2 books, and several book chapters in other books), and more than 40 invited presentations world wide.
Pten Deletion in Adult Neural Stem/Progenitor Cells Enhances Constitutive Neu...johnohab
Here we show that conditional deletion of Pten in a subpopulation of adult neural stem cells in the subependymal zone (SEZ) leads to persistently enhanced neural stem cell self-renewal without sign of exhaustion. These Pten null SEZ-born neural stem cells and progenies can follow the endogenous migration, differentiation, and integration pathways and contribute to constitutive neurogenesis in the olfactory bulb. As a result, Pten deleted animals have increased olfactory bulb mass and enhanced olfactory function. Pten null cells in the olfactory bulb can establish normal connections with peripheral olfactory epithelium and help olfactory bulb recovery from acute damage. Following a focal stroke, Pten null progenitors give rise to greater numbers of neuroblasts that migrate to peri-infarct cortex. However, in contrast to the olfactory bulb, no significant long-term survival and integration can be observed, indicating that additional factors are necessary for long-term survival of newly born neurons after stroke. These data suggest that manipulating PTEN-controlled signaling pathways may be a useful step in facilitating endogenous neural stem/progenitor expansion for the treatment of disorders or lesions in regions associated with constitutive neurogenesis.
A Critical Role of Erythropoietin Receptor in Neurogenesis and Post-Stroke Re...johnohab
Erythropoietin (EPO) is the principal growth factor regulating the production of red blood cells. Recent studies demonstrated that
exogenous EPO acts as a neuroprotectant and regulates neurogenesis. Using a genetic approach, we evaluate the roles of endogenous EPO
and its classical receptor (EPOR) in mammalian neurogenesis. We demonstrate severe and identical embryonic neurogenesis defects in
animals null for either the Epo or EpoR gene, suggesting that the classical EPOR is essential for EPO action during embryonic neurogenesis.
Furthermore, by generating conditional EpoR knock-down animals, we demonstrate that brain-specific deletion of EpoR leads to
significantly reduced cell proliferation in the subventricular zone and impaired post-stroke neurogenesis. EpoR conditional knockdown
leads to a specific deficit in post-stroke neurogenesis through impaired migration of neuroblasts to the peri-infarct cortex. Our results
suggest that both EPO and EPOR are essential for early embryonic neural development and that the classical EPOR is important for adult
neurogenesis and for migration of regenerating neurons during post-injury recovery.
Define epigenetics.
Describe important epigenetic mechanism and explain the implication of epigenetics in normal functions, disease and disease presentation.
Outline the heritability or epigenetic effects.
Explain the role of epigenetic in the development of cancer.
Outline the potentials of epigenetic intervention in battling cancer.
Speaker,
Dr. Md. Mohiuddin Masum
MS Anatomy, Phase-A, Year-1, Block-1
Guided by,
Prof. K M Shamim
Professor, Dept. of Anatomy, BSMMU
That’s important because right now, doctors have to rely on a set of 11 criteria, which can overlap with many other diseases, to try to make a diagnosis.
“It is one of the most complex clinical diagnoses,” says Pascual, who is also a practicing pediatric rheumatologist.
“It might lead to better diagnostic tests, but we don’t know that yet,” Pascual says. Other experts say the discoveries will most certainly lead to new drug targets.
Systemic lupus erythematosus (also called SLE or lupus)
is an autoimmune condition. The normal role of your
body’s immune system is to fight off infections and
diseases to keep you healthy. In an autoimmune disease
like lupus, your immune system starts attacking your
own healthy tissues. For some people lupus may just
affect the skin and/or joints. In other people the lungs,
kidneys, blood vessels,
Analysis of external adaptability (agility) as a measure of organizational Effectiveness in Higher Education and recommendation of diagnostic testing based on the performance triangle model
Zeine et al. 2011 Organizational Culture in Higher Education, in Kazeroony, H...Rana ZEINE, MD, PhD, MBA
ZEINE, R., Boglarsky, C.A., Blessinger, P., and Hamlet, M.T. Organizational Culture in Higher Education. Chapter 3 in Kazeroony, H. (Ed.), The Strategic Management of Higher Education Institutions: Serving Students as Customers for Institutional Growth, Business Expert Press, LLC, New York, NY USA, 2011, 1st edition, pp. 19-38.
DOI 10.4128/9781606491034, ISBN-13: 978-160649-366-3 (paperback),
978-1-60649-103-4 (e-book) http://www.businessexpertpress.com/books/strategic-management-higher-education-serving-students-customers-institutional-growth
Zeine Seminar 2010, Cancer Associated Fibroblasts and Microvascular Prolifera...Rana ZEINE, MD, PhD, MBA
World Cancer Congress 2010
Presence of Cancer-Associated Fibroblasts correlates with Microvascular Proliferation which is a Poor Prognostic Factor in Neuroblastoma Tumors
Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
Recomendações da OMS sobre cuidados maternos e neonatais para uma experiência pós-natal positiva.
Em consonância com os ODS – Objetivos do Desenvolvimento Sustentável e a Estratégia Global para a Saúde das Mulheres, Crianças e Adolescentes, e aplicando uma abordagem baseada nos direitos humanos, os esforços de cuidados pós-natais devem expandir-se para além da cobertura e da simples sobrevivência, de modo a incluir cuidados de qualidade.
Estas diretrizes visam melhorar a qualidade dos cuidados pós-natais essenciais e de rotina prestados às mulheres e aos recém-nascidos, com o objetivo final de melhorar a saúde e o bem-estar materno e neonatal.
Uma “experiência pós-natal positiva” é um resultado importante para todas as mulheres que dão à luz e para os seus recém-nascidos, estabelecendo as bases para a melhoria da saúde e do bem-estar a curto e longo prazo. Uma experiência pós-natal positiva é definida como aquela em que as mulheres, pessoas que gestam, os recém-nascidos, os casais, os pais, os cuidadores e as famílias recebem informação consistente, garantia e apoio de profissionais de saúde motivados; e onde um sistema de saúde flexível e com recursos reconheça as necessidades das mulheres e dos bebês e respeite o seu contexto cultural.
Estas diretrizes consolidadas apresentam algumas recomendações novas e já bem fundamentadas sobre cuidados pós-natais de rotina para mulheres e neonatos que recebem cuidados no pós-parto em unidades de saúde ou na comunidade, independentemente dos recursos disponíveis.
É fornecido um conjunto abrangente de recomendações para cuidados durante o período puerperal, com ênfase nos cuidados essenciais que todas as mulheres e recém-nascidos devem receber, e com a devida atenção à qualidade dos cuidados; isto é, a entrega e a experiência do cuidado recebido. Estas diretrizes atualizam e ampliam as recomendações da OMS de 2014 sobre cuidados pós-natais da mãe e do recém-nascido e complementam as atuais diretrizes da OMS sobre a gestão de complicações pós-natais.
O estabelecimento da amamentação e o manejo das principais intercorrências é contemplada.
Recomendamos muito.
Vamos discutir essas recomendações no nosso curso de pós-graduação em Aleitamento no Instituto Ciclos.
Esta publicação só está disponível em inglês até o momento.
Prof. Marcus Renato de Carvalho
www.agostodourado.com
Lung Cancer: Artificial Intelligence, Synergetics, Complex System Analysis, S...Oleg Kshivets
RESULTS: Overall life span (LS) was 2252.1±1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years – 64.8%, 20 years – 42.5%. 513 LCP lived more than 5 years (LS=3124.6±1525.6 days), 148 LCP – more than 10 years (LS=5054.4±1504.1 days).199 LCP died because of LC (LS=562.7±374.5 days). 5YS of LCP after bi/lobectomies was significantly superior in comparison with LCP after pneumonectomies (78.1% vs.63.7%, P=0.00001 by log-rank test). AT significantly improved 5YS (66.3% vs. 34.8%) (P=0.00000 by log-rank test) only for LCP with N1-2. Cox modeling displayed that 5YS of LCP significantly depended on: phase transition (PT) early-invasive LC in terms of synergetics, PT N0—N12, cell ratio factors (ratio between cancer cells- CC and blood cells subpopulations), G1-3, histology, glucose, AT, blood cell circuit, prothrombin index, heparin tolerance, recalcification time (P=0.000-0.038). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and PT early-invasive LC (rank=1), PT N0—N12 (rank=2), thrombocytes/CC (3), erythrocytes/CC (4), eosinophils/CC (5), healthy cells/CC (6), lymphocytes/CC (7), segmented neutrophils/CC (8), stick neutrophils/CC (9), monocytes/CC (10); leucocytes/CC (11). Correct prediction of 5YS was 100% by neural networks computing (area under ROC curve=1.0; error=0.0).
CONCLUSIONS: 5YS of LCP after radical procedures significantly depended on: 1) PT early-invasive cancer; 2) PT N0--N12; 3) cell ratio factors; 4) blood cell circuit; 5) biochemical factors; 6) hemostasis system; 7) AT; 8) LC characteristics; 9) LC cell dynamics; 10) surgery type: lobectomy/pneumonectomy; 11) anthropometric data. Optimal diagnosis and treatment strategies for LC are: 1) screening and early detection of LC; 2) availability of experienced thoracic surgeons because of complexity of radical procedures; 3) aggressive en block surgery and adequate lymph node dissection for completeness; 4) precise prediction; 5) adjuvant chemoimmunoradiotherapy for LCP with unfavorable prognosis.
New Drug Discovery and Development .....NEHA GUPTA
The "New Drug Discovery and Development" process involves the identification, design, testing, and manufacturing of novel pharmaceutical compounds with the aim of introducing new and improved treatments for various medical conditions. This comprehensive endeavor encompasses various stages, including target identification, preclinical studies, clinical trials, regulatory approval, and post-market surveillance. It involves multidisciplinary collaboration among scientists, researchers, clinicians, regulatory experts, and pharmaceutical companies to bring innovative therapies to market and address unmet medical needs.
Ozempic: Preoperative Management of Patients on GLP-1 Receptor Agonists Saeid Safari
Preoperative Management of Patients on GLP-1 Receptor Agonists like Ozempic and Semiglutide
ASA GUIDELINE
NYSORA Guideline
2 Case Reports of Gastric Ultrasound
Explore natural remedies for syphilis treatment in Singapore. Discover alternative therapies, herbal remedies, and lifestyle changes that may complement conventional treatments. Learn about holistic approaches to managing syphilis symptoms and supporting overall health.
Basavarajeeyam is an important text for ayurvedic physician belonging to andhra pradehs. It is a popular compendium in various parts of our country as well as in andhra pradesh. The content of the text was presented in sanskrit and telugu language (Bilingual). One of the most famous book in ayurvedic pharmaceutics and therapeutics. This book contains 25 chapters called as prakaranas. Many rasaoushadis were explained, pioneer of dhatu druti, nadi pareeksha, mutra pareeksha etc. Belongs to the period of 15-16 century. New diseases like upadamsha, phiranga rogas are explained.
Adv. biopharm. APPLICATION OF PHARMACOKINETICS : TARGETED DRUG DELIVERY SYSTEMSAkankshaAshtankar
MIP 201T & MPH 202T
ADVANCED BIOPHARMACEUTICS & PHARMACOKINETICS : UNIT 5
APPLICATION OF PHARMACOKINETICS : TARGETED DRUG DELIVERY SYSTEMS By - AKANKSHA ASHTANKAR
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
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2. Oligodendrocyte Lysis by Perforin 381
al., 1990; Selmaj et al., 1991a; Freedman et al., 1991; Antel toplasmic damage that are reminiscent of the oligodendro-
et al., 1998; Dal Canto et al., 2000). Several lines of cyte pathology encountered in MS lesions.
evidence support the hypothesis that oligodendrocyte pa-
thology and depletion from the CNS in MS may be METHODS AND MATERIALS
related to perforin-mediated lysis. This is particularly a Animals
propos to lysis of human oligodendrocytes by human Lactating female SJL mice with newborn pups were ob-
T-cells that has been shown to occur in vitro via a pre- tained from either Jackson Labs (Bar Harbor, ME) or Charles
dominantly perforin-based cytotoxicity mechanism (Zeine River (Wilmington, MA), and housed in an AAALAC-
et al., 1998). accredited facility at the Albert Einstein College of Medicine.
Perforin has been demonstrated in vivo in inflam-
matory lesions of a variety of autoimmune diseases (Liu et Oligodendrocyte Cultures
al., 1996; Kappeler and Mueller, 2000). In the CNS, Oligodendrocyte-enriched cultures were prepared as de-
perforin expression has been detected in astrocytes in scribed previously (Barbarese, 1991). Primary CNS cultures
normal brain tissue, and in astrocytes and infiltrating lym- were obtained by dissociation from the cerebral hemispheres of
phocytes within demyelinative lesions of MS and its 1–3-day-old SJL mouse pups and plated onto poly-L-lysine-
model, experimental autoimmune encephalomyelitis coated tissue culture dishes (Nunc, Fisher Scientific, Pittsburgh,
(EAE) (Held et al., 1993; Gasque et al., 1998; Zeine et al., PA), for 12 days in Dulbecco’s modified Eagle’s medium
1999). Furthermore, perforin mRNA has been detected at (DMEM) (Gibco BRL, Rockville, MD), supplemented with
elevated levels in the CSF of patients with MS (Matusevi- 10% newborn bovine serum (Gibco BRL). Enriched cultures of
cius et al., 1998; Kivisakk et al., 1999). This potent mem- oligodendrocytes were obtained by mild trypsinization of the
branolytic molecule, perforin, is a 70 kD pore-forming mixed cultures followed by depletion of adherent cells though
protein (PFP) that is stored within cytolytic granules of differential adhesion to plastic substrata. Oligodendrocyte-
cytotoxic T-cells and natural killer (NK) cells (Liu et al., enriched populations were seeded onto 24-well plates at 1 105
1995a). Perforin-mediated cytotoxicity is a calcium- cells per well and grown on either Thermanox plastic coverslips
dependent mechanism as calcium ions are required both (Nunc) or glass D coverslips (Fisher), precoated with poly-L-
for effective granule exocytosis and for the appropriate ornithine (Sigma, St. Louis, MO). Alternatively, Lab-Tek glass
perforin conformation. Mechanistically, perforin mono- chamber slides (Nunc) were used. The feeding medium
mers released by killer cells bind to phosphorylcholine (DMEM-N1-B104) was composed of a freshly prepared mix-
lipid moieties and insert into target cell membranes where ture of DMEM/F12 (Gibco BRL), with pyruvic acid, HEPES,
they polymerize and assemble to form homopolymeric albumin and the following N1 supplements: apo-transferrin
transmembrane pore structures (Tschopp et al., 1989; (0.05 mg/ml), sodium selenite (30 nM) and biotin (10 ng/ml).
Ojcius et al., 1991a). These pores perturb membrane Insulin (0.016 mg/ml), hydrocortisone (3.6 ng/ml), T3
permeability and result in target cell death by osmotic lysis, (15 nM), -ME and gentamicin (0.5%) were also added, and the
a mechanism most closely related to necrosis. final mixture was supplemented with B104 neuroblastoma-
conditioned medium in an 87:13 proportion. Chemicals and
Among the mouse models of demyelination that
hormones were obtained from Sigma, and B104 was supplied by
most closely mimic the human disease, those most applied
Dr. Barbarese. The final composition of these cultures was ca.
to MS have been established in inbred mouse strains (SJL, 80% oligodendrocytes, 15% astrocytes and 5% microglia plus
PL), strains that are susceptible to remitting-relapsing precursor cells.
forms of EAE (Brown et al., 1982; Cross et al., 1987). The
susceptibility of oligodendrocytes from mouse strains to Perforin-Mediated Lysis
perforin-mediated lysis has never been examined because Adherent oligodendrocyte-enriched cultures were incu-
mouse oligodendrocytes have been difficult to culture. In bated at 37°C with a preparation of the pore forming protein
the early 1990s, the susceptibility of rat oligodendrocytes (PFP) perforin, provided by Dr. C.-C. Liu, in the range of
to perforin-mediated injury was described (Scolding et al., 36 –72 hemolytic units (HU) in the presence of 2 mM Ca2 and
1990), and more recently, using a chromium-51 release in a final volume of 400 l serum-free medium. Control cul-
cytotoxicity assay (Malipiero et al., 1997). To explore tures received either Ca2 Cl2 alone or PFP alone. Cell lysis was
perforin-mediated CNS injury in a mouse model, we have evaluated over a period of 2.5 hr by phase contrast microscopy,
developed a method to culture oligodendrocytes from SJL indirect immuno-fluorescence, LM and EM, or time-lapse dig-
mice and have documented for the first time the selective ital imaging.
effects of perforin on these cells using chronologic digital
techniques. For this, we have examined the kinetics of Immunocytochemistry
perforin-induced osmotic lysis of oligodendrocytes and At 30 min intervals after exposure to perforin, propidium
have employed immunofluorescence, digital imaging and iodide (PI) (50 g/ml), an indicator of membrane disruption,
light and electron microscopy (LM and EM), to define the was added to duplicate cultures for 3 min and the cells washed
structural features of this process. Our analysis has revealed in PBS (Sigma) and fixed in 4% paraformaldehyde at 4°C
that SJL mouse oligodendrocytes in vitro display several overnight. Fixed cells were then rinsed in PBS and incubated
perforin-induced structural patterns of membrane and cy- with the following primary antibodies for 1 hr at room temper-
3. 382 Zeine et al.
ature: mouse monoclonal anti-galactocerebroside-GalC anti- glutaraldehyde in Millonig’s buffer, pH 7.4. The cultures were
body (IgG3; Roche/Boehringer-Mannheim, Indianapolis, IN) then post-fixed for 45 min in 1% OsO4 in PBS, pH 7.4,
at 1:10 dilution; mouse monoclonal anti-O4 antibody (IgM; dehydrated though a graded series of ethyl alcohol, washed in a
Roche/Boehringer-Mannheim) at 1:5 dilution; mouse mono- 1:1 mixture of absolute alcohol and Epon 812, and embedded in
clonal anti-myelin basic protein-MBP antibody (IgG1; Roche/ Epon 812. After polymerization, the flat sheets of epoxy were
Boehringer-Mannheim) at 1:100 dilution. After gentle rinsing in removed from the plastic coverslips. For sectioning perpendic-
PBS, coverslips were treated with the appropriate secondary ular to the plane of growth, the blocks were re-embedded
antibodies for 1 hr at room temperature at 1:100 dilution: end-on in capsules of fresh epoxy (Norton et al., 1983). One
FITC-conjugated goat anti-mouse IgG (Rockland, Gilberts- micron sections stained with toluidine blue were examined by
ville, PA), and FITC-conjugated goat anti-mouse IgM light microscopy and for electron microscopy, thin sections
(Rockland). The coverslips were washed and mounted on slides were double-stained with uranium and lead salts, carbon-coated
using 50% glycerol containing the anti-fading agent, n-propyl and scanned in an Hitachi H 600.
gallate (Sigma). The cells were viewed using a Nikon Optiphot
and an Olympus IX 70 inverted microscope, both equipped for RESULTS
epifluorescence with filters for rhodamine and FITC. SJL Mouse Oligodendrocytes in Culture
DiI Membrane Staining and Imaging Oligodendrocytes were successfully isolated from
Living cells were labeled with a fluorescent lipidophilic primary CNS cultures from newborn SJL mice and con-
membrane tracer, the long-chain dialkylcarbocyanine, DiI (Mo- sisted of bipolar, tripolar and multipolar populations.
lecular Probes, Eugene, OR). Oligodendrocyte-enriched cul- These cells differentiated and expressed the oligodendro-
tures were incubated with 10 M DiI for 2 hr at 37°C. The cyte markers, GalC (Fig. 1A), O4 (Fig. 1J) and MBP (Fig.
DiI-containing medium was then removed and the cells washed 1G). They extended multiple processes and elaborated flat
before exposure to perforin. Control cells and cells exposed to membranes. These membrane sheets expanded with time
perforin were viewed under an Olympus IX 70 microscope. and covered larger areas, remaining intact and exhibiting
The temperature of the specimen was maintained at 37°C an homogeneous consistency and a well-demarcated edge
within an environmental chamber equipped with air and stage (Figs. 1A,J and 2A). DiI revealed cytoplasmic ribs within
heaters (Olympus, Melville, NY). Images were collected at 0.5 the flat membrane veils (Fig. 2A). O4 and GalC gave
and 1 min intervals with a Censys-cooled CCD camera (Pho- intense membrane staining (Fig. 1A,J), whereas MBP
tometrics, Tucson, AZ), and the light was shuttered (Ludl, staining was more cytoplasmic, denser in perinuclear re-
Hawthorne, NY) between exposures. Software control was gions and diffuse over the membrane sheets (Fig. 1G).
provided by I.P. Lab Spectrum (Scanalytics, Fairfax, VA), and
deconvolution was performed at 80% to 90% haze reduction Perforin-Mediated Injury of SJL Mouse
with PowerHazebuster (Vaytek, Fairfield, IA) on a Macintosh Oligodendrocytes
G3. Oligodendrocyte-enriched cultures were exposed to
36, 48, 60 and 72 HU of perforin in the presence of 2 mM
TUNEL Method Ca2 . Light and phase contrast microscopy showed swell-
In some experiments, cell death by apoptosis was assessed ing of the cells within 15 min and rearrangement of the
using the In Situ Cell Detection Kit with Fluorescein, (Enzo/ cytoplasm within 30 min of exposure. Examination of
Boehringer-Mannheim, Indianapolis, IN), according to the membrane integrity in living cells stained with DiI re-
manufacturer’s instructions. vealed the formation and expansion of distinct membrane
holes, probably the result of coalescence of multiple sub-
Dead Cell Ratio microscopic (15–20 nm) pores (Liu et al., 1995a), between
The ratio of PI positive cells to total cells was calculated by 30 and 90 min after exposure to perforin (Fig. 2B,C). By
counting approximately 300 cells in each of seven randomly- indirect immunofluorescence, the nuclei of dying and
selected low power fields. Cells were exposed to 72 HU of dead cells stained bright red with PI after exposure to PFP
perforin for 1.5 hr and then stained with PI and counted unfixed for 0.5 hr (Fig. 1D,K), 1 hr (Fig. 1H), and 2.5 hr (Fig.
at 10 magnification. 1F,I,L). The pattern of distribution of O4 and GalC re-
vealed distortion of the cellular architecture suggestive of
Membrane Hole Expansion (Fenestration) swelling of the cell body and fragmentation of membranes
The size of membrane holes (fenestrae) was measured at and processes seen by 30 min (Fig. 1C,K) Complete lysis
the LM level by manually tracing with I.P. Lab Spectrum most frequently involved reduction of the cytoplasm to
(Scanalytics), the areas of 16 individual fenestrae at each of 30 fragmented processes and droplets (Fig. 2D). At 2.5 hr,
time points. The rate of hole expansion was determined by some cells could be found that had not proceeded though
plotting the average areas at each time point against time. completion of osmotic lysis, but had arrested at a stage that
exhibited marked swelling with abundant membrane holes
Light and Electron Microscopy (Figs. 1E,L; 2C). These structural features of perforin-
At 30 min intervals after exposure to perforin, monolayers induced oligodendrocyte damage were also clearly dem-
of oligodendrocyte- enriched cells were fixed for 30 min di- onstrated in experiments where cultures were fixed and
rectly on Thermanox coverslips by immersion in cold 2.5% stained with toluidine blue (Fig. 3). As compared to con-
4. Oligodendrocyte Lysis by Perforin 383
Fig. 1. Indirect immunofluorescence of oligodendrocytes in culture, godendrocytes that are negative for PI. H: One hour PFP exposure. A
before and after exposure to PFP. A: Control culture. Two GalC- group of MBP-positive , PI-positive oligodendrocytes can be seen.
positive oligodendrocytes are shown. B: Same field as (A), stained with I: PFP exposure (2.5 hr). A group of MBP-positive/PI-positive oligo-
PI. Note lack of PI staining in control oligodendrocytes shown in (A). dendrocytes. J: Control culture. Superimposition of O4 on a phase
C: Thirty-minute PFP exposure. Four oligodendrocytes stain positively contrast image reveals a normal oligodendrocyte with a flattened mem-
for GalC. D: Same field as (C) showing PI positivity of the four brane. K: Thirty minute PFP exposure. The pattern of O4 distribution
oligodendrocytes. E: PFP exposure (2.5 hr). A group of GalC-positive suggests membrane fragmentation. The nucleus is slightly PI positive.
oligodendrocytes is shown. F: Same field as (E) showing PI positivity L: PFP exposure (2.5 hr). O4 staining reveals multiple membrane holes
and distorted nuclei of the oligodendrocytes. G: Control culture. and fragmentation. The nucleus is intensely positive for PI. Magnifi-
Superimposition of MBP and PI staining showing MBP-positive oli- cation 450 in A–I; 700 in J–L.
5. 384 Zeine et al.
Fig. 2. Living oligodendrocytes stained with DiI. A: Control oligodendrocyte. Note the well-
demarcated edge and homogeneous nature of the membranous veil of this oligodendrocyte.
B: Forty-five minute exposure to PFP. By DiI staining, distinct membrane holes are visible. C: Same
cell, 90 min exposure to PFP. The number and size of the holes have increased considerably.
D: End-Stage lysis. A collection of cytoplasmic fragments is arranged around a distorted nucleus.
Magnification 800.
trols (Fig. 3A–C), cells exposed to PFP for 60 min man- areas of 16 individual fenestrae and the rate of expansion
ifested swelling, cytoplasmic disintegration and vacuola- was determined from the slope of the plot of average areas
tion (Fig. 3D–F). The nuclei became asymmetrically against time (data not shown). For this particular cell,
distorted but did not exhibit signs of apoptosis (Figs. 1F,L, fenestrae expanded at a rate of 55 m2/min over a period
3D–F). TUNEL staining revealed no increase in TUNEL of 5 min after which most ceased to change. This pattern
positive cells over control cultures (data not shown). of cellular arrest was not uncommon and probably repre-
sented death with incomplete lysis. Some oligodendro-
Rate of Membrane Fenestration cytes that were fixed as late as 2.5 hr after exposure to
With DiI staining, distinct holes (fenestrae) became perforin exhibited a pattern of O4 distribution suggestive
visible by EM in the membranes of oligodendrocytes after of numerous expanded fenestrae delineating vacuoles
exposure to perforin for 30 min. The progression of throughout the damaged cell (Fig. 1L).
fenestration was followed in one cell by time-lapse imag-
ing starting at 45 min and ending at 90 min (Figs. 2B–C). Pattern of End-Stage Lysis
Images were collected at 30 sec intervals. Hole size was A more severe pattern of destruction was evident by
measured by tracing with I.P. Lab Spectrum software, the LM in oligodendrocytes that proceeded rapidly to com-
6. Oligodendrocyte Lysis by Perforin 385
Fig. 3. Light microscopy of 1 micron toluidine blue-stained epoxy swollen nucleus surrounded by cytoplasmic remnants (arrow) lies ad-
sections. A: Control culture. A flattened oligodendrocyte attached to jacent to a cell with vacuolated cytoplasm. E: Sixty minutes PFP. Two
the substrate (arrow), lies next to an astrocyte. B: Control culture. sick, vacuolated oligodendrocytes lie beneath a layer of astrocytes.
Three oligodendrocytes (arrows) lie on layer of astrocytic processes. F: Sixty minutes PFP. A fragmented culture shows a layer of degen-
C: Control culture. A large flattened, healthy astrocyte with a single erating oligodendrocytes overlying a layer of minimally affected astro-
nucleus is shown. D: Sixty minutes PFP. An oligodendrocyte with a cytes. Magnification 750 in B; 1,000 in A,C–F.
plete lysis. Examples of such cells showed complete loss of forin for 1.5 hr and then stained with PI. For each field, PI
all membrane veils and were captured by both immuno- images were superimposed on corresponding phase con-
fluorescence and DiI staining. Some were found displaying trast micrograph and the cells counted unfixed. In cultures
PI positive nuclei associated with few GalC positive rem- exposed to perforin, the ratio of PI-positive cells ranged
nants of cytoplasmic fragments and processes (data not between 42 and 100% (mean 77%, SD 24), whereas the
shown), whereas others presented as arrays of cytoplasmic range in control cultures remained between 9 and 15%
fragments around an asymmetrically-enlarged and dis- (mean 11% , SD 2).
torted nucleus (Fig. 2D). This type of cellular fragmenta-
tion most probably represented end-stage osmotic lysis. Ultrastructural Features of Perforin-Induced
Damage
Kinetics of Perforin-Mediated Lysis By EM, the cytoplasm of normal oligodendrocytes in
Experiments were repeated using either 36, 48, 60 or vitro was more electron-dense than that of astrocytes,
72 HU of perforin. In all cases, the kinetics of perforin- possessed short segments of rough ER and contained
induced injury proceeded systematically though cell swell- microtubules but no intermediate filaments (Fig. 4A,B).
ing (15–30 min), membrane fenestration and expansion After perforin-induced injury, these cells became edema-
(30 –90 min), membrane fragmentation (30 –90 min), and tous and displayed extensive cytoplasmic vacuolation of
end-stage lysis (90 –150 min). As mentioned above, het- the cell body and processes within 30 min of exposure. On
erogeneity occurred in the extent of lysis achieved by occasion, oligodendrocytes featured an extremely large
individual cells under the same culture conditions, with some single cytoplasmic vacuole, which most probably evolved
cells becoming arrested at a stage before end-stage lysis. In from the confluence of several vacuoles during the process
most cells, however, death was complete within 3 hr. of osmotic lysis (Fig. 5A). Fragmentation of unit mem-
branes and the presence of myelin-like formations were
Ratio of Cell Killing by Perforin noted at the perimeter of such vacuoles (Fig. 5B). Another
The ratio of PI-positive cells was calculated by change common to the cytoplasm of affected oligoden-
counting by LM approximately 300 cells in each of seven drocytes was evident at the 0.5 hr time-point, when
randomly-selected low power fields. For this, cultures formation of villus-like profiles, rich in microtubules, was
consisting of a mixture of early differentiating oligoden- encountered (Fig. 4C). This appearance probably corre-
drocytes and astrocytes, were exposed to 72 HU of per- sponded to images seen by LM (Fig. 3D).
7. 386 Zeine et al.
Fig. 4. EM of control cells and early perforin-induced cytoplasmic 30,000. C: Thirty minutes PFP. The cytoplasm of this oligodendro-
injury. A: Control culture. A normal oligodendrocyte lies to the left of cyte protrudes as villus-like processes into the extracellular space. Note
an astrocyte. The cytoplasm of the oligodendrocyte is distinctly denser. the numerous microtubules with the cell body and the villus-like
Magnification 12,000. B: Detail of the oligodendrocyte cytoplasm in profiles (arrows). Magnification 75,000.
(A). Note the microtubules in cross section (arrows). Magnification
8. Oligodendrocyte Lysis by Perforin 387
Fig. 5. Ultrastructural patterns of membrane damage in oligodendro- 30,000. C: Sixty minutes PFP. End-stage lysis is seen in an oligo-
cytes exposed to PFP. A: Thirty minutes PFP. This oligodendrocyte dendrocyte that has lost its cell membrane, cytoplasmic integrity and
has developed a large vacuole around the perimeter of which myelin- intact nuclear membrane. Magnification 14,000. D: Detail of (C) to
like figures are located. Magnification 12,000. B: Detail from A to show beginning disruption of the nuclear membrane. Magnification
show the lamellar patterns of the myelin droplets. Magnification 30,000.
9. 388 Zeine et al.
Fig. 6. Sixty minutes PFP. Two
interdigitating, apparently healthy
astrocytes display an abundance of
intermediate filaments and ro-
settes of ribosomes. The nucleus
of the cell on the right is normal
and a hemidesmosome (typical of
subpial astrocytes) is indicated (ar-
row). Magnification 19,000.
Oligodendrocytes examined after exposure to per- of perforin exposure, cell membranes were lost and nu-
forin for 1 hr exhibited complete loss of the cell membrane clear envelopes began to degenerate (Fig. 5C). This is the
with only remnants of cytoplasm remaining around the first report demonstrating that SJL mouse oligodendro-
nucleus (Fig. 5C). In addition, evidence of degeneration of cytes are susceptible to perforin-induced osmotic lysis, a
the nuclear envelope was noted (Fig. 5D). Similar pathol- highly conserved, classical cell-death mechanism (Ojcius
ogy, albeit delayed and to a lesser degree, was seen in et al., 1991a; Liu et al., 1995b) and this occurred at a dose
astrocytes in the same culture system but on most occa- level similar to that which kills other eukaryotic cells.
sions, astrocytes were relatively resistant to damage and Interestingly, oligodendrocyte changes observed
maintained a normal ultrastructure well into the experi- here in vitro held much in common with appearances
mental period (Fig. 6). described in MS lesions, ultrastructural studies of which
DISCUSSION have consistently documented that oligodendrocyte pa-
thology exhibits features suggestive of immune osmotic
The mechanisms underlying oligodendrocyte deple- lysis or necrosis (Prineas, 1985; Prineas and McDonald,
tion from the MS plaque remain to be clarified although
cytolytic mechanisms have repeatedly been implicated 1997). In contrast, evidence for oligodendrocyte apoptosis
(Raine, 1997b). One candidate molecule in this process is in MS and EAE has been lacking or, at most, rare
the pore-forming membranolytic molecule, perforin, and (D’Souza et al., 1996; Bonetti et al., 1997; Bonetti and
in this report, we have studied the effects of perforin on Raine, 1997; Waldner et al., 1997). Perforin is a potent
oligodendrocytes cultured from a strain of mouse (SJL) mediator of osmotic lysis and perforin-mediated cytotox-
most frequently applied to studies on MS and a strain from icity is induced by killer T-cells (Lowin et al., 1994; Kagi
which oligodendrocytes have been difficult to grow in the et al., 1994). The presence of killer cells and perforin in
past. Our results have shown that during normal develop- demyelinative lesions of EAE and MS is well-known
ment in culture, oligodendrocytes extended processes and (Selmaj et al., 1991b; Held et al., 1993; Olive, 1996; Rajan
elaborated flattened myelin-like membranes, as described et al., 1996; Gasque et al., 1998; Zeine et al., 1999), and
previously for the murine system (Barry et al., 1996). After a dual role has been proposed for these cell types whereby
exposure to perforin, the integrity of the membrane veils injury to oligodendrocytes and neurons is detrimental
was compromised as microscopic fenestrae appeared and whereas injury to immune cells is protective to the CNS
expanded rapidly, transforming the flat membrane sheets (Battistini et al., 1995; Malipiero et al., 1997; Zhang et al.,
into a delicate lacework (Fig. 2B,C). The cell bodies 1997; Matsumoto et al., 1998; Murray et al., 1998; Zeine
swelled and the cytoplasm became vacuolated and dis- et al., 1999). Most likely, the balance between the effector
rupted into fragments rich in microtubules. Within 60 min and immunoregulatory arms of the immune response de-
10. Oligodendrocyte Lysis by Perforin 389
termines the magnitude of CNS damage inflicted by the Kinase inhibitors block the mobilization and orientation of
perforin-based cytotoxicity pathway. In a study on MOG- perforin-containing granules toward the contact zone
induced EAE in perforin knockout mice, oligodendrocyte (Wei et al., 1998); and calreticulin blocks perforin lytic
pathology was not discussed. activity by stabilizing target cell membranes (Fraser et al.,
Ultrastructurally, injured oligodendrocytes in MS le- 2000). Thus, the development of strategies to block per-
sions appear swollen, with vacuolated cytoplasm and wid- forin activity in vivo holds promise for rescuing oligoden-
ened, degenerating or ruptured membranes (Field and drocyte targets during acute episodes of inflammatory de-
Raine, 1964; Perier and Gregoire, 1965; Prineas, 1985;
` ` myelination.
Rodriguez et al., 1993; Rodriguez and Scheithauer, 1994; The observation that astrocytes were more resistant
Raine, 1997b; Raine et al., 1999). Previous studies have than oligodendrocytes to perforin was not surprising con-
shown that myelin in acute lesions is transformed into sidering their general resilience to disease. We and others
vesicular networks (Kirk, 1979; Lee et al., 1990; Rodri- have shown the expression of perforin within the cyto-
guez and Scheithauer, 1994; Genain et al., 1999; Raine et plasm of astrocytes in vivo and in culture (Gasque et al.,
al., 1999). In this study, some images of oligodendrocytes 1998; Zeine et al., 1999). Astrocytes are the only cells
and their membranes undergoing lysis by perforin-induced known to harbor free cytosolic perforin, and they must
membrane fragmentation were reminiscent of changes therefore have some protective mechanism to guard their
described in acute MS lesions (viz. Suzuki et al., 1969; Fig. own membranes against perforin lysis. Furthermore, as-
11, loc.cit.; Rodriguez and Scheithauer, 1994; Fig. 4A, trocytes may be a source of perforin in the CNS and may
loc.cit.; Raine, 1997b; Fig. 5, loc. cit.; Prineas and Mc- play an effector or regulatory role during inflammation.
Donald, 1997; Fig. 13.35, loc. cit.; Raine et al., 1999; Figs. Thus, we have shown that oligodendrocytes cultured
6 – 8, loc. cit.). from the CNS of SJL mice are selectively vulnerable to
The search for molecules that readily lyse oligoden- cytolytic damage by perforin, and that early-stage cell lysis
drocytes has been difficult. Perforin-based cytotoxicity is involves the formation of fenestrae in the cell membranes,
most akin to complement toxicity. Although the C9 com- probably as a result of the coalescence of numerous sub-
ponent of complement is deposited in MS and EAE le- microscopic perforin-induced pores and the influx of wa-
sions, some studies have shown that human oligodendro- ter into the cell. Subsequently, cells either disintegrated
cytes are not sensitive to complement damage in culture into cytoplasmic droplets, developed large cytoplasmic
(Zajicek et al., 1995). Combining antibodies with com- vacuoles, formed disrupted membranous networks or be-
plement results in a reduction in the survival of oligoden- came totally devoid of cell membrane - all these changes
drocytes in culture via a toxicity mechanism that can be primary to nuclear disintegration. As stated above, many of
inhibited by the addition of normal immunoglobulins these morphologic patterns have been described in MS
(Scolding et al., 1989; Scolding et al., 1990; Stangel et al., and EAE previously and are in support of oligodendrocyte
2000). It is most unlikely, however, that immunoglobulin damage in MS being effected by a non-apoptotic process.
would be protective in the case of perforin-mediated lysis. In ongoing studies on tissue from EAE and MS (Zeine et
We therefore reaffirm a unique role for perforin in irre- al., in preparation), we have documented the upregulation
versible oligodendrocyte injury relevant to the pathogen- of perforin by western blotting during acute exacerbations
esis of MS and extend previous findings by providing and downregulation during remission. Perforin was
present within infiltrating cytotoxic cells and astrocytes by
detailed timed images from oligodendrocytes undergoing immunohistochemistry, and the extent of remyelination
osmotic lysis. correlated with the absence of perforin. Thus, in the series
With regard to previous works on perforin-mediated of molecular events leading to the formation of the MS
lysis, our results are in agreement with the findings of lesion, it seems that a role for perforin may very well be
Jones et al. (1991) showing that “not all PI-positive cells found at the acute end of the spectrum.
proceeded to lysis.” Thus, the possibility of sub-lethal
attack by perforin on individual cells remains open and ACKNOWLEDGMENTS
raises interest in the mechanism of resistance to perforin We are indebted to Michael Cammer for expert
( Jiang et al., 1990; Ojcius et al., 1990, 1991b). Of ther- technical assistance with digital imaging at the Analytical
apeutic relevance would be the conferring of resistance to Imaging Facility of the Albert Einstein College of Medi-
selected target cells, e.g., oligodendrocytes, in EAE and cine; to Dr. Hin Hark Gan of New York University and
MS. Such a prospect awaits the isolation of ‘L-protectin,’ Howard Hughes Medical Institute for discussion; to Dr.
a membrane-bound glycosylated molecule that interferes Jorge Larocca of the Albert Einstein College of Medicine,
with perforin function in resistant target cell membranes Department of Neurology, for advice; and to Patricia
(Liu et al., 1995b). Currently, some experience has been Cobban-Bond for administrative assistance. This research
gained by blocking perforin-mediated lysis via four classes was supported in part by grants from the National Multiple
of chemical reagents in vitro. Calcium chelators inhibit Sclerosis Society of the USA (NMSS RG 1001-J-10
exocytosis and perforin binding to membranes (Liu et al., [CSR], NMSS RG 2971 [WC] and NMSS RG 2843
1995b); H -ATPase inhibitors raise the pH within cyto- [EB]), the American Heart Association (CCL), and by
toxic granules leading to accelerated degradation of per- HHS Grants NS 08952 (CSR), NS 11920 (CSR), and NS
forin (Kataoka et al., 1996; Zeine et al., 1998); MAP- 19943 (EB).
11. 390 Zeine et al.
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