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a 76-year - old female presented to the emergency department with the main complaint of 7 days of dyspnea and 1 day of dysarthria and dizziness. auscultation findings showed an irregular heart beat with a diastolic rumbling murmur in the apical region and crackles in both lower lung fields. transthoracic echocardiography showed a moderate degree of aortic regurgitation with left ventricular (lv) ejection fraction of 70.8% and a 1.631.31-cm complex, echogenic, round, mass - like lesion attached to the left atrial side of the interatrial septum. transesophageal echocardiography revealed the same mass finding with prominent spontaneous echo contrast in the left atrium and decreased emptying velocity of the left atrial appendage (fig. 2). the diagnostic coronary angiogram revealed a round, movable mass lesion in the left atrium with feeding arteries originating from the conus branch and atrioventricular nodal artery of the right coronary artery and no significant stenosis in either coronary artery (fig. 3). the patient was transferred to cardiac surgery and underwent removal of the mass and a maze operation. macroscopic findings were compatible with myxoma, and the microscopic findings revealed an acid mucopolysaccharide - rich stroma composed of a myxoid matrix and polygonal cells with scant eosinophilic cytoplasm scattered throughout the matrix (fig. 4). after surgery, the atrial fibrillation was abolished and an electrocardiogram showed a normal sinus rhythm. follow - up transthoracic echocardiography was performed after surgery and no remnant mass was observed in the left atrium (fig., the patient is admitted to outpatient department regularly and has taken aspirin and angiotensin receptor blocker steadily without specific problems. the presence of possible tumor vessels originating from coronary arteries may be helpful in the decision on an operative strategy for cardiac myxoma. about 52% of cardiac myxoma is visualized by coronary angiograms according to previous reports, but catheterization of the chamber from which the tumor arises carries the risk of tumor embolization. | a 76-year - old female present to the emergency department with dysarthria, dizziness, dyspnea. the patient had hypertension and atrial fibrillation. brain mri revealed right cerebellar infarction. transthoracic echocardiography showed a large round mass in the left atrium. transesophageal echocardiography showed large complex echogenic round mass lesion attached on left atrial side of interatrial septum. coronary angiogram revealed round movable mass lesion in left atrium with feeding arteries originated from right coronary artery. she underwent removal of mass and maze operation, and pathologic finding was compatible with myxoma. |
akap79/150 is a protein scaffold thought to position specific kinases (pka, pkc) and phosphastases (calcineurin) in appropriate synaptic domains so that their activities can regulate excitatory synaptic strength. using a viral - mediated molecular replacement strategy in rat hippocampal slices, we found that akap is required for nmda receptor - dependent ltd solely due to its interaction with calcineurin. |
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traditionally, the major mechanism underlying parkinson 's disease was thought to be reduced dopaminergic synaptic transmission in the nigrostriatal system. however, neurons may also communicate by extrasynaptic transmission, which was recently found to affect dopamine neurotransmitter delivery through diffusion in the extracellular matrix. fibronectin, a ubiquitous extracellular matrix component, participates in the formation and regulation of anisotropic diffusion (i.e., diffusion facilitated in a certain direction) of the extracellular matrix, and dynamically regulates neuronal functional activities, such as neuroactive substance diffusion and receptor activation, anti - neuroinflammation, and cell adhesion. studies over the past two decades have demonstrated that there exist two major modes of neuron - glial communication in the central nervous system : synaptic transmission (or wiring transmission) and extrasynaptic transmission. in synaptic transmission, synaptically evoked astrocytes produce elevated ca signals, which can trigger the release of gliotransmitters and play neuromodulatory and information - integration roles in neuronal activity, synaptic transmission and plasticity. extrasynaptic transmission involves secretion of a wide variety of bioactive substances by neurons and neuroglia that are released or leaked into the extracellular space. these substances mediate bidirectional communication between neurons and neuroglia through molecular diffusion, resulting in a sustained neuromodulatory action on neuronal activity., volume transmission and intercellular communication, all of which reflect the specific function of extracellular space. there are four types of extrasynaptic transmission in the central nervous system (figure 1) : including the intercellular short distances, the cerebrospinal fluid, the long distances around nerve fibers, and long distances around blood vessels. the latter two types of extrasynaptic transmission display the properties of faster and longer signal transmission that occur among brain nuclei, cerebral areas and even cerebral hemispheres, and involve the movement of various bioactive substances, such as ions, neuropeptides, neurohormones and metabolites, as well as pathological hematologic exudates such as cell adhesion molecules, growth factors and inflammatory molecules. thus, extrasynaptic transmission plays important modulatory roles in neuronal functional activities. in the central nervous system, fibronectin is produced and secreted by neuroglial cells and endothelial cells and is assembled into the extracellular matrix. thus, fibronectin is important for extrasynaptic transmission, and to some degree exerts neuroprotective effects, such as anti - neuroinflammation. however, the neuroprotective mechanism of fibronectin remains to be thoroughly analyzed to reveal its important clinical value and to enhance understanding of extrasynaptic transmission and the functional role of the neuron- glia network. in turn, it is hoped that this knowledge will unveil the molecular pathogenesis and new therapeutic approaches in neurodegenerative disorders such as parkinson 's disease. here, we summarize the molecular structures and roles of fibronectin and integrin receptors in extrasynaptic transmission, the neuroprotective roles of fibronectin and its potential significance in parkinson 's disease. schematic diagram of the principal types of cerebral extrasynaptic transmission (modified and adapted from references). the four types of extrasynaptic transmission in the central nervous system involve : short distances for simple diffusion, such as autocrine and paracrine transmission, cerebrospinal fluid (csf) for endocrine diffusion, long distances around nerve bundles for preferential diffusion, and long distances around blood vessels for preferential diffusion. the left section shows the functional tracks of extrasynaptic transmission or volume transmission, and the right section shows the functional tracks of synaptic transmission (neural wiring transmission) without neuroactive substance leakage into the extracellular space. fibronectin is a heterodimeric glycoprotein encoded by a single gene and is disulfide - bonded at its carboxyl terminal. each monomer contains three types of repeats (type i, ii or iii) and constitutes multiple domains. the middle domain contains an arg - leu - asp (rgd)-binding motif that can bind to integrin and non - integrin receptors. the carboxyl terminal domain containing a heparin binding site and a variable sequence has a higher expression level in the embryonic period that becomes lower in the elderly, and displays a neuroprotective effect. fibronectin has a wide variety of cell sources, such as astrocytes, epithelial cells, fibroblasts, and mesenchymal cells, and participates in cell adhesion, proliferation and differentiation, epithelial tissue repair, immune regulation, neural regeneration, and other physiological activities. integrins, the receptors for fibronectin, are a family of transmembrane glycoprotein receptors and their molecular structure comprises one and one subunit bound by non - covalent bonds. they can recognize and bind to fibronectin and other extracellular matrix proteins or other receptors, and exert dual functions of cell adhesion and signal transduction. at present, it is well - established that there are nineteen and eight subunits that can combine to form at least 25 different integrin receptors in mammals. the majority of subunits only bind to one subunit and sometimes to multiple subunits, such as 41, 47, 61, 64, v1, v3, v5, v6 and v8. both and subunits have a large extracellular region (important for binding to the rgd sequence in various integrin ligands such as matrix molecules), a transmembrane region and a small intracellular region (no catalytic effect). integrins are functionally divided into three main subfamilies : 1 integrins (i.e., very late antigen integrins), 2 integrins (i.e., leukocyte integrins), and v integrins, with 3 integrins (i.e., cell adhesion integrins) also given more attention recently. that is, the binding of an integrin to its ligand can activate intracellular signaling events, which in turn affect the affinity of ligand and integrin. in most cases, integrins can combine with neighboring receptors (i.e., counter receptors) to form integrin - receptor complexes, or receptor mosaics, on the cell surface that transactivate intracellular signaling and modulate biological effects. for example, in the cerebrovascular endothelial cells, fibronectin mediates the mitogen - activated protein kinase signaling pathway via 51 and v3 receptors and promotes cell survival and proliferation. moreover, fibronectin plays a neurotrophic and anti - inflammatory role in the brain and promotes the growth and survival of neurons. it has been confirmed that the fibronectin type iii (fibronectin 3) modules of the neural cell adhesion molecule are involved in the direct interaction between neural cell adhesion molecule and fibroblast growth factor receptor in dopaminergic, hippocampal and cortical neurons, and then activate the mitogen - activated protein kinase and phosphatidylinositol 3-kinase / protein kinase b (pi3k / akt) signal transduction pathways and induce neuronal differentiation and proliferation in vitro. the administration of synthetic fibronectin peptide v can increase the survival of dopaminergic neurons in neural grafts in vivo and ameliorate motor dysfunction in parkinson 's disease animals, suggesting the anti - apoptotic effect of fibronectin. fibronectin can also inhibit the development of mechanical allodynia by injection into the spinal dorsal column after spinal injury. in microglia, fibronectin can activate the pi3k - akt and mitogen - activated protein kinase / extracellular signal - regulated kinase signaling pathway, enhance the expression levels of neurotrophic factors, and attenuate the release of the pro - inflammatory factor interleukin 1, all of which contribute to neural repair and neuronal survival. traumatic brain injury induced significantly increased lesion volume and apoptotic cell death in fibronectin knockout mice, but intravenous injection of fibronectin before the injury reversed the neural deficits, indicating that fibronectin is neuroprotective against traumatic brain injury and a novel target for therapeutic interventions. fibronectin can also promote survival and migration of transplanted primary neural stem cells in a mouse model of traumatic brain injury, and act as a possible therapeutic tool for traumatic brain injury. taken together, findings from the studies on the neuroprotective role of fibronectin are expected to lead to the development of new therapeutic approaches for parkinson 's disease and other neurodegenerative diseases, and indicate that fibronectin could become an important pharmacological tool for the study of specific functional aspects of extrasynaptic transmission, including neuroprotection and neuromodulation. the three major subfamilies (1, 2 and v) of integrins are all able to recognize and bind to extracellular matrix proteins and counter receptors and trigger a great deal of intracellular structural and signaling changes. these changes include the assembly of multi - molecular complexes onto cytoplasmic integrin tails to engage and organize the cytoskeleton, as well as the activation of signaling pathways that modify function and gene expression. cross - talk between integrin - mediated signaling and growth factor - mediated signaling can occur at various levels. furthermore, integrin - fibronectin ligand interactions play roles in cell adhesion, and in combination with growth factor receptors, regulate functional activities of growth factors via receptor transactivation and intracellular signaling events. in the central nervous system, 1 and v are extensively expressed in several cell types such as neurons, glia and epithelial cells, while 2 is only expressed in microglia. 51 is the only integrin receptor containing the 5 subunit and binds to only one fibronectin ligand, which, along with 31 and 41, promotes growth and regulates function in neural cells and cerebrovascular endothelial cells. it has been demonstrated that 1 and v integrins can transactivate a variety of growth factor receptors and mimic the somatotrophic or neuroprotective effect of related growth factors, such as fibroblast growth factor, insulin - like growth factor (igf)-1 and glial cell line - derived neurotrophic factor. in the dopaminergic neurons of the substantia nigra that have a high level of constitutive igf-1 receptor (igf-1r) expression, 1 integrins (e.g., 3, 4, 51) can activate the igf-1r/ phosphatidylinositol 3-kinase / protein kinase b signaling pathway by the igf-1-independent transactivation of igf-1r, and enhance igf - mediated neurotrophic effects in degenerating dopaminergic neurons. for example, fibronectin is abundant in pancreatic tumors and engages igf - ir to inhibit cell death by stimulating formation of a complex between 3 integrin and protein - tyrosine phosphatase shp-2. formation of this complex prevents shp-2 from dephosphorylating igf - ir and results in sustained phosphorylation of igf - ir and downstream activation of akt kinase, and the inhibition of apoptosis through up - regulation of the anti - apoptotic factor bcl. 1 integrin expression is required for igf - ir - mediated prostate cancer cell proliferation and anchorage - independent growth, and can bring about the extended activation of igf-1r and mimic and amplify the biological effects of igf-1. therefore, fibronectin - induced neuroprotection could be fully mediated by igf - ir without involvement of igf-1, suggesting that fibronectin and igf - ir could be important targets for the development of pharmaceuticals that mediate pro - survival signals. fibronectin is a non - collagen component among extracellular matrix proteins that are synthesized by many cell types, such as astrocytes, endothelial cells, fibroblasts and myoblasts. extracellular matrix proteins are then assembled into three - dimensional fibrillar networks surrounding neural cells to form a pericellular microenvironment and to support biomolecular flow. however, this microenvironment is not homogeneous allowing for the directional facilitation of information flow down different dynamic pathways, i.e., intercellular channels, to regulate neuron - glial extrasynaptic transmission. during brain development or injury, fibronectin and other matrix proteins may support and promote differentiation and migration of neuronal progenitor cells, or directional outgrowth of neurites. in the adult brain, the fibrillar fibronectin guides and controls the flow of extracellular fluid and the diffusion of various substances, which are driven by energy gradients, such as concentration, temperature and pressure gradients, and have slower diffusion properties, less safety, less spatial constraint and wider diffusion ranges. the diffusion capability of extrasynaptic transmission is determined by three parameters : 1) extracellular space volume fraction (= extracellular space volume / total tissue volume), which can be reduced due to cerebral atrophy in the elderly, especially in neurodegenerative diseases, 2) tortuosity, reflecting the condition of diffusion barriers such as altered fibrillar fibronectin architecture and other extracellular matrix macromolecules, fine neural processes, charged molecules and degrading enzymes, 3) nonspecific cellular uptake (k), indicating the degree of cell swelling. these diffusion parameters differ in various brain regions, showing heterogeneous diffusion in the central nervous system, and are easily affected by physiological and pathological factors such as molecular rearrangement, glial remodeling, cell swelling and elderly extracellular space shrinkage. therefore, the expression level of fibronectin can directly influence the diffusion and permeability of various neuroactive substances, to some degree by directional facilitation through intercellular channels due to local changes in homeostasis. these preferential channels exert an important regulatory role on neurotransmitter diffusion, and intercellular short distance and peri - neurofiber long distance extrasynaptic transmission. thus, they might adversely underlie disease pathology and be used as a potential target for the treatment of neurodegenerative diseases such as parkinson 's disease. in geriatric patients, there are significant alterations in volume, tortuosity and anisotropy of brain extracellular space. these changes may seriously affect intercellular channel permeability and communication through the neuron - glia network, and bring about synapse - transmitter leakage and transmitter - receptor mismatches that give rise to abnormal accumulation and diffusion of neuroactive substances. this mechanism might contribute to the pathogenesis of parkinson 's disease or other neurodegenerative diseases, and provide a pharmacological target to ameliorate deficiencies in neurotransmission, cell migration and drug delivery and treatment. along with other components of the extracellular matrix, such as glycosaminoglycans and proteoglycans, fibronectin accumulates in the extracellular matrix to form poriferous perineuronal nets to regulate matrix organization. for example, fibronectin may specifically bind to growth factor receptors or be involved in clearance of degraded products and direct cell behaviors, such as receptor activation that transduces signals into cells, in addition to its supportive and adhesive roles. these effects of fibronectin could bring about the altered structural and functional properties of extracellular matrix (e.g., neurotransmitter storage, metabolite clearance, and diffusion parameters), and underlie the molecular mechanism of neurodegenerative disorders such as parkinson 's disease. the expression level of fibronectin determines the nature and condition of extracellular matrix, and is readily affected by multiple physiological and pathological factors (e.g., ageing and neuroinflammatory response), as well as genetic and epigenetic factors (e.g., transcription factors and post - translational modifications). meanwhile, fibronectin, like other extracellular matrix macromolecules, contributes to diffusion barriers in the extracellular space and influences neuroglial activation, diffusion of various factors or neurotransmitters, information transmission and neurotrophic microenvironment, in spite of neuronal and glial processes also disturbing the local extracellular matrix architecture of the central nervous system. briefly, binding of fibronectin to integrins (1, 2, and v) triggers intracellular structural alterations and signaling cascades, including integrin - mediated signaling which can crosstalk with growth factor - mediated signaling at various levels and mimic the functions of growth factors via receptor transactivation and intracellular signaling events. therefore, fibronectin could be applied to study neuroprotection in neurodegenerative diseases such as parkinson 's disease. in the parkinson 's disease brain, the transmission of dopamine in the nigrostriatal pathway is greatly abated or blocked for two reasons : 1) deficit of striatal dopamine in the parkinson 's disease brain because of decreased dopamine synthesis in degenerating nigral neurons, and 2) altered matrix content and increased diffusion barriers to extrasynaptic transmission along the nigrostriatal pathway. the latter is the main pathological change in the parkinson 's disease brain because the nigrostriatal dopamine pathway mainly operates via volume transmission ; that is, nigral dopamine reaches target cells mostly by diffusion along the dopamine concentration gradient of the extracellular space. although the downregulated expression of fibronectin in the elderly brain can compensative and reduce the diffusion barrier and partly ameliorate deficits in dopamine diffusion, the increased volume fraction is still not reversed. therefore, fibronectin could be administered as a neuroprotective drug to augment the fibronectin levels in plasma and brain, which would not only enhance survival of dopaminergic neurons but would also maintain a better extracellular matrix status for unrestricted diffusion and traffic of dopamine along the nigrostriatal pathway. in particular, under circumstances in which wiring transmission is blocked, the use of extrinsic fibronectin has an important compensatory effect on the striatal dopamine deficit and protects against the development of parkinson 's disease. moreover, it is reasonable to predict that the molecular status of plasma fibronectin could be used as an additional diagnostic biomarker for risk assessment of parkinson 's disease, and that the increase of fibronectin in the cerebrospinal fluid could be an important parameter used to diagnose certain neurodegenerative diseases such as amyotrophic lateral sclerosis and multiple sclerosis. the proneuronal and metabolic effects of fibronectin will be helpful in formulating new therapeutic and diagnostic strategies. fibronectin can bind to and activate both integrin receptors and igf-1r, thus triggering igf-1r / phosphatidylinositol 3-kinase / protein kinase b signaling and enhancing survival of dopamine neurons. we refer to this cascade (summarized in figure 2) as the fibronectin - integrin - growth factor receptor - signal transduction - gene and protein expression cascade, through which altered extrasynaptic transmission may modulate the functional outputs of cells to compensate for deficits in synaptic transmission. therefore, fibronectin could likely be used as an endogenous repair protein of extracellular matrix, and its clinical application ameliorate the poor brain extracellular environment and achieve some therapeutic effects in neurodegenerative diseases. the study of the functional manipulation of fibronectin in neuron - glial extrasynaptic transmission should be expanded to broaden our understanding of the complex fibronectin- and integrin - mediated signaling networks, and to determine a new and effective approach to diagnosis and treatment of certain neurodegenerative diseases. a proposed schematic map of the neuroprotective mechanism of fibronectin. fibronectin may mediate a receptor - receptor interaction between integrins and igf-1r in the cell surface membrane. molecular cross - talk likely occurs between the intracellular signaling cascades resulting from this interaction, to finally produce the neuroprotective effects. trk : tyrosine receptor kinase ; igf-1r : insulin - like growth factor-1 receptor ; mek : mitogen - activated protein kinase kinase ; erk : extracellular signal - regulated kinase ; pi-3k / akt : phosphatidylinositol 3-kinases / protein kinase b. | most hypotheses concerning the mechanisms underlying parkinson 's disease are based on altered synaptic transmission of the nigrostriatal system. however, extrasynaptic transmission was recently found to affect dopamine neurotransmitter delivery by anisotropic diffusion in the extracellular matrix, which is modulated by various extracellular matrix components such as fibronectin. the present study reviewed the neuroprotective effect of fibronectin in extrasynaptic transmission. fibronectin can regulate neuroactive substance diffusion and receptor activation, and exert anti- neuroinflammatory, adhesive and neuroprotective roles. fibronectin can bind to integrin and growth factor receptors to transactivate intracellular signaling events such as the phosphatidylinositol 3-kinase / protein kinase b pathway to regulate or amplify growth factor - like neuroprotective actions. fibronectin is assembled into a fibrillar network around cells to facilitate cell migration, molecule and ion diffusion, and even drug delivery and treatment. in addition, the present study analyzed the neuroprotective mechanism of fibronectin in the pathogenesis of parkinson 's disease, involving integrin and growth factor receptor interactions, and discussed the possible therapeutic and diagnostic significance of fibronectin in parkinson 's disease. |
reagents were received from sigma aldrich chemical co. (bangalore, karnataka, india) and used without further purification. solvents were purchased from commercial sources from hyderabad, telangana, indiabased companies and purified by reported protocols. h and c nmr spectra were recorded on bruker 300 mhz and 500 mhz machines and calibrated against tetramethylsilane (tms). mass spectrometric data were acquired by an electrospray ionization (esi) technique on a qtofmicro quadruple mass spectrometer. the stock solutions (110 m) of ndin and ndina were prepared in chcl3. an aliquot of it was transferred to various ratios of chcl3/mch in different volumetric flasks (final volume of 2 ml) and allowed to equilibrate for 2 h prior to the uv / vis absorption measurements. all experiments were performed upon excitation at 350 nm in a quartz cell with a 1 cm path length. the stock solutions were prepared in a similar manner as for the absorption study and employed for emission measurements. the stock solutions were prepared in chcl3 in a similar manner as for the uv / vis study. sem images of ndin and ndina were recorded on an fei nova nanosem (hillsboro, or usa) operating at high vacuum. for sem imaging, the samples of ndia and ndina were sputtercoated with gold for 10 s at 0.016 ma ar plasma after dropcasting the solutions on glass coverslip and solvent evaporation at rt. tem samples were prepared by the paper blotting method followed by solvent evaporation on a holey carboncoated copper grid. the liquid sample in chcl3/mch (5:95, v / v) was dropcasted and solvent was allowed to evaporate on the surface naturally. reagents were received from sigma aldrich chemical co. (bangalore, karnataka, india) and used without further purification. solvents were purchased from commercial sources from hyderabad, telangana, indiabased companies and purified by reported protocols. h and c nmr spectra were recorded on bruker 300 mhz and 500 mhz machines and calibrated against tetramethylsilane (tms). mass spectrometric data were acquired by an electrospray ionization (esi) technique on a qtofmicro quadruple mass spectrometer. the stock solutions (110 m) of ndin and ndina were prepared in chcl3. an aliquot of it was transferred to various ratios of chcl3/mch in different volumetric flasks (final volume of 2 ml) and allowed to equilibrate for 2 h prior to the uv / vis absorption measurements. all experiments were performed upon excitation at 350 nm in a quartz cell with a 1 cm path length. the stock solutions were prepared in a similar manner as for the absorption study and employed for emission measurements. the stock solutions were prepared in chcl3 in a similar manner as for the uv / vis study. sem images of ndin and ndina were recorded on an fei nova nanosem (hillsboro, or usa) operating at high vacuum. for sem imaging, the samples of ndia and ndina were sputtercoated with gold for 10 s at 0.016 ma ar plasma after dropcasting the solutions on glass coverslip and solvent evaporation at rt. tem samples were prepared by the paper blotting method followed by solvent evaporation on a holey carboncoated copper grid. the liquid sample in chcl3/mch (5:95, v / v) was dropcasted and solvent was allowed to evaporate on the surface naturally. as a service to our authors and readers, this journal provides supporting information supplied by the authors. such materials are peer reviewed and may be reorganized for online delivery, but are not copyedited or typeset. technical support issues arising from supporting information (other than missing files) should be addressed to the authors. | abstractin the present work, two new naphthalene diimide (ndi) amphiphiles, ndin and ndina, were successfully synthesized and employed to investigate their selfassembly and optical properties. for ndina, which contains an amide group, aggregationinduced emission enhancement (aiee) was demonstrated in the presence of various ratios of methylcyclohexane (mch) in chloroform, which led to the visual color changes. this new amidecontaining ndina amphiphile formed nanobelt structures in chloroform / mch (10:90, v / v) and microcuplike morphologies in chloroform / mch (5:95, v / v). the closure of these microcups led to the formation of vesicles and microcapsules. the structural morphologies gained from the solvophobic control of ndina were confirmed by various complementary techniques such as infrared spectroscopy, xray diffraction, and scanning and transmission electron microscopy. in the absence of the amide moiety in ndin, no selfassembly was observed, indicating the fundamental role of hbonding in the selfassociation process. |
medical researchers also realized that treating only small areas originally involved by lymphoma with radiation was not good enough, if radiation had to be used as the only treatment. a large area covering all lymph node areas in the upper or lower half of the body had to be treated. when the upper half of the body was being treated, the efrt field was called the mantle field. a high - dose radiation exposure on the thorax is mainly used in the context of adjuvant radiotherapy after conservative or radical breast surgery, adjuvant or exclusive radiotherapy of lung and esophageal cancer, and as a complement to systemic treatment in lymphoma. irradiation of the heart increases the risk of the so - called radiation - induced heart disease (rihd). the incidence of rihd is 1030% by 510 years posttreatment ; the prevalence of rihd, in the setting of the modern protocols of delivering adjuvant radiotherapy, reduction in doses, and field radiation size, is still poorly defined. we describe the case of a 66-year - old female with a history of hodgkin 's lymphoma (type 1a) treated with mantle radiotherapy in 1973. she was admitted for congestive heart failure in radiation - induced heart disease. a cardiac computed tomography angiography (64 dual - source, caredose, and electrocardiogram pulsing mindose) showed calcified ascending aorta and pericardium calcification narrowing the right ventricle, bronchiectasis and fibrosis of the lungs, and esophagus [figure 1a and b ]. (a) multidetector computed tomography short - axis view of the calcification in the ascending aorta and the nearest thickened pericardium, (b) multidetector computed tomography long - axis view of the thickened wall of the esophagus a cardiac magnetic resonance imaging (1.5 t) revealed normal left ventricle size (vts 17 ml, vtsi 10 ml / m), with ejection fraction of 0.87, stroke volume (sv) of 70 ml, sv index 39 ml / m, aortic regurgitation and stenosis with aortic valve peak velocity of 163 cm / s and a mild thickness of pericardium (3 mm) and esophagus [figure 2 ]. magnetic cardiac imaging (magnetic resonance imaging), axial image demonstrating circumferential thickening of the pericardium (> 3 mm), normal volume of the heart, and calcification of the ascending aorta a transthoracic echocardiography was performed and documented normal ef, aortic regurgitation and aortic stenosis with effective orifice area of 0.78 cmq [figure 3a and b ], calcified mitral annulus, signs of constrictive pericarditis (pericardial calcifications, augmented thickness, and doppler signs of constriction high filling pressure in the left and right ventricle, annulus paradoxes, and diastolic flow reversal in expiration in the suprahepatic veins), and of myocardial damage (low - tissue doppler velocities at the mitral annulus level [figure 3c ], significant pulmonary hypertension [figure 3d ]). therefore, we wanted to observe better heart and the signs of constrictive pericarditis, so we performed a transesophageal echocardiogram (tee). surprisingly, we were unable to visualize the cardiac structures because of high and completely acoustic impedance and interfaces [figure 4 ]. the acoustic shadowing due to the interface of two different structures with a high level of impedance showed the suboptimal image and no resolution of cardiac structures. the resultant images were echodense with the lack of signal in the sector beyond the structure [video 1 ]. (a) transthoracic echocardiography view of aortic regurgitation, (b) transthoracic echocardiography showing aortic valve stenosis, (c) transthoracic echocardiography demonstrating the myocardial damage by low - tissue doppler velocities at the mitral annulus level, (d) transthoracic echocardiography showing pulmonary hypertension transesophageal echocardiography failure : the completely impedance to visualize the cardiac structure radiation therapy uses high - energy rays (or particles) to destroy cancer cells. to treat hodgkin 's disease, a carefully focused beam of radiation is delivered from a machine outside of the body. if the hodgkin 's disease was in the upper body, radiation was given to the mantle field, which included lymph node areas in the neck, chest, and under the arms. the long - term sequelae of mantle therapy include, especially the lung and cardiac disease but also involve the vessels and the organs in the neck and thorax (such as thyroid, aorta, and esophagus). in our case report, the significant thickness of esophagus [figure 1b ] caused the acoustic impedance due to the inability of tee to view correctly and complete cardiac structure. any degree of vision or level of depth of the probe did not capture defined images. the short- and long - term sequelae of mantle field radiation were not predictable and quantifiable and included, especially heart and thoracic structure labeling radiation - induced heart disease. in the literature, the descriptions of unavailable tee in a patient with a history of radiation therapy have been few, all of them have pointed out to the rihd or other radiation - induced effects. our case report emphasized the failure of the tee, in particular, the impedance of radiation - induced thickness of the esophagus hence the unavailability to collect good quality images during the examination | the long - term sequelae of mantle therapy include, especially lung and cardiac disease but also involve the vessels and the organs in the neck and thorax (such as thyroid, aorta, and esophagus). we presented the case of 66-year - old female admitted for congestive heart failure in radiation - induced heart disease. the patient had undergone to massive radiotherapy 42 years ago for hodgkin 's disease (type 1a). transesophageal echocardiography was performed unsuccessfully with difficulty because of the rigidity and impedance of esophageal walls. our case is an extraordinary report of radiotherapy 's latency effect as a result of dramatic changes in the structure of mediastinum, in particular in the esophagus, causing unavailability of a transesophageal echocardiogram. |
it has been reported that serious problems during general anesthesia occur in 0.4% of patients. it may be difficult to detect anaphylactic reactions during anesthesia because various medications are administered in short periods of time, and transient interactions between drugs may take place. there have been many reports on anaphylactic reactions due to propofol or muscle relaxants during anesthesia induction. in contrast, cases on anaphylactic reactions before induction are rare, but in the absence of adequate monitoring and immediate treatment, can be fatal to the patients. we report life - threatening conditions in a patient due to a severe anaphylactic reaction during transfer to the operating room before induction of anesthesia. an 84-year - old woman, 148 cm in height and 61 kg in weight was scheduled for open reduction and internal fixation due to left calcaneous fracture with dislocation. she had been taking hypertension medication for five years but had no previous history of asthma, allergic rhinitis, or atopic dermatitis. however, one year ago she was treated with conservative management after experiencing dizziness and general weakness following injection of an unknown drug. she received no premedication other than tridol, which was administered for pain control a day before the surgery. her blood pressure, heart rate, and temperature were 130/80 mmhg, 68 beat / min, 36.8c, respectively. after a negative intradermal skin test (2 mg / ml in normal saline with cefbuperazone (tomiporan), 1 g of cefbuperazone (tomiporan) was injected before transfer to the operating room. fifteen minutes later in the surgical waiting room, the patient had slight difficulty in breathing but had an alert mental state. five minutes later, as the patient was being moved to the operation bed, the patient became incontinent of urine and unconsciousness. her blood pressure was 105/65 mmhg, heart rate 62 beat / min, and arterial oxygen saturation 75%. intubation was performed immediately without using muscle relaxants. on auscultation, wheezing was apparent in both lungs and airway pressure increased to 30 cmh2o after the ventilator was applied. sudden, profound hypotension (blood pressure 62/43 mmhg) and bradycardia (heart rate 45 beat / min) developed. the patient was promptly loaded with fluid along with two doses of ephedrine 20 mg. her blood pressure rose to 85/54 mmhg and then dropped to 50/22 mmhg a minute later. a central line was placed in the right internal jugular vein and an arterial cannula was inserted into the right femoral artery. arterial blood gas values were fio2 1.0, ph 7.288, po2 355 mmhg, be 6.1 meq, and arterial oxygen saturation 98.3%. since the patient received no special medications other than the intravenous antibiotic injection before arriving at the operation room, a clinical diagnosis of anaphylactic reaction was made and methyl prednisolone (solumedrol) 500 mg was intravenously injected. three doses of epinephrine 20 g were administered but there was no response : blood pressure 60/36 mmhg, heart rate 113 beat / min, and blood pressure 49/37 mmhg, heart rate 130 beat / min. after three additional doses of 200 g of epinephrine were administered, the patient s blood pressure and heart rate were then maintained at 110130/5060 mmhg and 80100 beat / min, respectively. forty minutes after resuscitation, her blood pressure was maintained at around 120/60 mmhg with epinephrine infusion and the patient was transferred to the intensive care unit where a mechanical ventilator was applied. after an hour at the intensive care unit, arterial blood gas values were fio2 1.0, ph 7.256, po2 346 mmhg, pco2 49.7 mmhg, be 5.5 meq, and arterial oxygen saturation 99.7%. after two hours in the intensive care unit, arterial blood gas value normalized : fio2 0.3, ph 7.345, po2 87.5 mmhg, pco2 42.3 mmhg, be 5.8 meq, and arterial oxygen saturation 97.1%. after three hours in intensive care unit, the patient s was alert, her mean arterial blood pressure was > 80 mmhg, and she showed no signs of airway obstruction or wheezing on auscultation. five hours later, the patient was transferred to the general ward and surgery was postponed. eight weeks later, a skin prick test confirmed a positive reaction (wheal 4 3.5, flare 7 6 mm) to cefbuperazone (tomiporan). anaphylactic reaction is an immediate life - threatening reaction mediated by ige and propagated by mediators such as histamine, tryptase, leukotrienes, platelet activating factor, nitric oxide, inflammatory prostaglandins, and bradykinin. it can lead to intravascular volume depletion, myocardial suppression and increased airway resistance, that results in sudden severe hypotension, hypoxemia, tissue hypoxia, and acidosis. the most common causes that trigger generalized reaction during anesthesia are neuromuscular blocking agents, latex, and antibiotics, with antibiotics reported as the cause in 1015% of all cases. -lactam antibiotics, such as penicillin, cephalosporin, carbapenem, and monobactam antibiotics, are involved in generalized reactions in 80% of these cases. however, recent wide spread use of cephalosporin antibiotics during perioperative period to prevent infections has led to an increase in the incidence of drug reactions due to cephalosporin antibiotics. cephalosporins are semi - synthetic antibiotics, derivatives of cephalosporin acremonium, and are composed of a -lactam ring connected to a 6-member dihydrothiazine ring. cephalosporin exerts its antibacterial effect by disrupting synthesis of the peptidoglycan layer in bacterial cell wall. this drug has broad spectrum activity and is administered as a first - line prophylactic drug to prevent infections in surgical procedures. generally, the first generation cephalosporins work more effectively on gram positive bacteria and have less activity on gram negative bacteria. with few exceptions, the most common side effects of cephalosporins are skin reactions such as maculopapular exanthema and urticaria, which are reported to occur in 13% of patients. only 0.00010.1% of the side effects is anaphylactic reaction, which is thus very rare. according to fisher., the most common clinical feature of anaphylactic reaction is cardiovascular collapse, occurring in their study in 389 out of 442 people, along with bronchospasm occurring in 161 people, of which 72 have having asthmatic bronchospasm. among cutaneous signs, there were 201 people with erythema, 55 with rash, and 37 with urticaria. in accordance with these statistics, our patient showed all the important clinical features such as cardiovascular collapse, asthmatic bronchospasm, and erythematous rash. furthermore, in patients with severe reactions, the first clinical features were a fall in blood pressure in 122 people, difficulty to inflate in 115, flush in 94, coughing in 28, rash in 20, desaturation in 15, and cyanosis is 12 out of 440 people. in our case, the patient had difficulty in inflating with wheezing immediately on arrival to the operation room, and considering the normal range of blood pressure with signs of bradycardia and oxygen desaturation (78%), difficulty in inflation was the first clinical feature. while it has been reported that anaphylactic reactions occur most commonly after the age of 40 in women and 50 in men, only 0.4% of all anaphylactic reactions occur in over 80 years of age. therefore, the severe anaphylactic reaction noted in our 84-year - old patient is very unusual. according to laxenaire., grade iii life - threatening reactions including cardiovascular collapse, tachycardia or bradycardia, arrhythmia, and severe bronchospasm occurred in 62.6% of the patients in their study. bronchospasm only occurred in 21.3% of grade ii reactions, whereas 75.4% of the patients with grade iii reactions had bronchospasm., it took 20 minutes for the symptoms to occur after antibiotic injection in the ward, which is still unlike most immediate reactions in which symptoms start to manifest promptly. initiative resuscitation with ephedrine and phenylephrine failed to restore the patient s blood pressure but epinephrine was effective. the normalization of blood pressure can be explained by a decrease in allergic reaction due to -2 agonists along with an increase in blood pressure to the effects of epinephrine. anaphylactic reactions that occur during induction or anesthesia can be treated immediately. however, if airway obstruction and severe hypotension occur during transfer, as in this case, they can be fatal. if anaphylaxis occurs during anesthesia, several causative agents can be considered : myocardial infarction, drug overdose, pulmonary embolus, irritant - induced bronchospasm, endotracheal tube malfunction, aspiration, hypoglycemia, and stroke. however, our patient had no premedication before transferring to the operating room other than the antibiotics injection. thus, we were able to make a fast diagnosis of anaphylactic reaction due to antibiotics. our patient was injected with antibiotics after a negative skin test with 2 mg / ml in normal saline concentration. according to romano, 2 mg / ml in normal saline concentration is nonirritant in a control group but very sensitive in subjects that have immediate reactions to cephalosporin. today, the highest concentration accepted for prick and intradermal testing in europe is 2 mg / ml for cephalosporins. on the other hand, other studies have used normal saline concentrations in the range of 0.5250 mg / ml for hypersensitivity diagnosis. this is less than the amount used in our case but the possibility of a false negative result can not be eliminated because the test volume was too small [1315 ]. one limitation of this study is that there was no ige test result to corroborate the clinical findings that the patient had undergone an anaphylactic reaction. also, considering her clinical features, course of treatment, and the positive skin prick test, a diagnosis of anaphylactic reaction due to cephalosporin should still be made. however, as demonstrated in the present case, if they occur 2030 minutes after injection, they can take place during transfer from the ward to the operating room. therefore, careful observation and monitoring is required when patients are being transferred after a prophylactic antibiotics injection. | although uncommon, anaphylactic reactions during surgery are very dangerous and can result in serious morbidity. various anesthetics can trigger anaphylactic reactions, and incidents with cephalosporin antibiotics are on the rise. in the case presented, an 84-year - old woman scheduled for calcaneus fracture surgery, was injected with cefbuperazone as a prophylactic antibiotic. on the way to the operating room, before induction of anesthesia, the patient lost consciousness and showed signs of hypoxemia, and anaphylactic reaction, which included hypotension, bronchospasm, and rash. five hours after immediate intubation and fluid resuscitation, the patient was extubated and transferred to the general ward. eight weeks later, the skin prick test confirmed a positive reaction to cefbuperazone. |
soft tissue sarcomas (stss) are a heterogeneous group of solid tumors that arise from embryonic mesenchymal cells and that have distinct clinical and pathological features. microscopically, arcinosarcomas are biphasic tumors comprising an intimate admixture of carcinomatous and sarcomatous components with an abrupt or gradual transition from one to the other. stss of the genitourinary (gu) tract are relatively rare, accounting for only 2.1% of all stss and 1% to 2% of all malignant gu tumors [1 - 3 ]. because of the rarity of urological stss, clinical data are limited and prognosis is often considered unpredictable. recently, several groups have reported their long - term experiences with gu stss and have investigated prognosis and prognostic factors [4 - 6 ]. although the consensus is that total surgical resection provides the patient the best chance for cure, there is no universal agreement concerning prognostic factors for gu stss. several analyses of prognostic factors influencing the overall survival (os) of patients with gu stss have revealed that tumor stage, grade, size, and anatomic site are important for patient survival. to the best of our knowledge, no prior study has investigated clinical outcomes and prognostic factors of gu stss in a korean patient population. in this study, we investigated prognostic factors of gu stss on the basis of 28 years of experience with these tumors at a single center in korea. between january 1982 and july 2009, 48 patients who were histologically diagnosed with a gu sts at our institute were included in this study. sarcomas of the gu tract that originated from the kidney, ureter, bladder, prostate, paratesticular region, and retroperitoneum were included. sarcomas that originated from female genital organs such as the uterus, ovary, vulva, or vagina were excluded from this study. demographic and clinical data were retrieved by reviewing patients ' charts, clinical data, operative notes, radiological reports, and pathological reports. the variables analyzed were patient age, sex, tumor histology, tumor size, primary organ, metastasis at diagnosis, and status of surgical resection. tumors were divided into two groups on the basis of size : one group comprised tumors smaller than 5 cm, and the other comprised tumors larger than 5 cm. local recurrence or metastasis was defined as the first recurrence of disease at the primary tumor site or distant site detected by a radiographic modality, such as computed tomography. univariate analysis of variables to determine prognostic factors was performed by using the log rank test. the results of the cox model analysis are reported as hazard ratios and 95% confidence intervals. the kaplan - meier estimate of the survival curve was used to summarize the data, whereas differences between patient groups were assessed by the log rank test. between january 1982 and july 2009, 48 patients who were histologically diagnosed with a gu sts at our institute were included in this study. sarcomas of the gu tract that originated from the kidney, ureter, bladder, prostate, paratesticular region, and retroperitoneum were included. sarcomas that originated from female genital organs such as the uterus, ovary, vulva, or vagina were excluded from this study. demographic and clinical data were retrieved by reviewing patients ' charts, clinical data, operative notes, radiological reports, and pathological reports. the variables analyzed were patient age, sex, tumor histology, tumor size, primary organ, metastasis at diagnosis, and status of surgical resection. tumors were divided into two groups on the basis of size : one group comprised tumors smaller than 5 cm, and the other comprised tumors larger than 5 cm. local recurrence or metastasis was defined as the first recurrence of disease at the primary tumor site or distant site detected by a radiographic modality, such as computed tomography. univariate analysis of variables to determine prognostic factors was performed by using the log rank test. the results of the cox model analysis are reported as hazard ratios and 95% confidence intervals. the kaplan - meier estimate of the survival curve was used to summarize the data, whereas differences between patient groups were assessed by the log rank test. during the study period, 48 patients with gu stss were treated at our institution. the mean age at diagnosis was 47.1 years (range, 3 to 80 years). the mean follow - up duration was 43.6 months (range, 6 to 167 months). the most common site was the retroperitoneum (n=16, 33.3%) followed by the kidney (n=12, 25%), prostate (n=10, 20.8%), bladder (n=7, 14.5%), ureter (n=1, 2%), and paratesticular region (n=1, 2%). the mean tumor size was 9.5 cm (range, 2.2 to 24 cm) and 43 patients (89.5%) had tumors larger than 5 cm. the histological subtypes of sarcoma were as follows : leiomyosarcoma (24, 50%), rhabdomyosarcoma (9, 18.7%), and liposarcoma (4, 8%). of the 48 patients, 33 (68.7%) underwent surgical resection and 15 (31.2%) were treated by chemotherapy, radiotherapy, or both. at the time of analysis, 20 patients (41.6%) had died of their sts, 2 patients (4.1%) had died of unrelated causes, and 26 patients (54.1%) were alive. the 5-year estimated survival rate of patients with a retroperitoneal sarcoma was 82%, while that of patients with a bladder sarcoma was 73%. the 5-year estimated survival rate for patients who presented with prostate sarcoma was 44%, while that for patients who presented with renal sarcoma was 39%. there were no significant differences in survival according to age (p=0.79), gender (p=0.84), tumor size (p=0.59), or histological subtype (p=0.69) according to the univariate analysis (table 2). however, a significant difference in survival according to metastasis at diagnosis was observed (fig. 2). patients who underwent surgical resection also showed significantly better overall survival (fig. patients with retroperitoneal sarcomas had more favorable survival outcomes than did patients with bladder, kidney, or prostate sarcomas (fig. these three factors (metastasis, surgical resection, and primary organ) were also significantly associated with improved overall survival in the multivariate analysis (table 2). during the study period, 48 patients with gu stss were treated at our institution. the mean age at diagnosis was 47.1 years (range, 3 to 80 years). the mean follow - up duration was 43.6 months (range, 6 to 167 months). the most common site was the retroperitoneum (n=16, 33.3%) followed by the kidney (n=12, 25%), prostate (n=10, 20.8%), bladder (n=7, 14.5%), ureter (n=1, 2%), and paratesticular region (n=1, 2%). the mean tumor size was 9.5 cm (range, 2.2 to 24 cm) and 43 patients (89.5%) had tumors larger than 5 cm. the histological subtypes of sarcoma were as follows : leiomyosarcoma (24, 50%), rhabdomyosarcoma (9, 18.7%), and liposarcoma (4, 8%). of the 48 patients, 33 (68.7%) underwent surgical resection and 15 (31.2%) were treated by chemotherapy, radiotherapy, or both. at the time of analysis, 20 patients (41.6%) had died of their sts, 2 patients (4.1%) had died of unrelated causes, and 26 patients (54.1%) were alive. the 5-year estimated survival rate of patients with a retroperitoneal sarcoma was 82%, while that of patients with a bladder sarcoma was 73%. the 5-year estimated survival rate for patients who presented with prostate sarcoma was 44%, while that for patients who presented with renal sarcoma was 39%. there were no significant differences in survival according to age (p=0.79), gender (p=0.84), tumor size (p=0.59), or histological subtype (p=0.69) according to the univariate analysis (table 2). however, a significant difference in survival according to metastasis at diagnosis was observed (fig. 2). patients who underwent surgical resection also showed significantly better overall survival (fig. patients with retroperitoneal sarcomas had more favorable survival outcomes than did patients with bladder, kidney, or prostate sarcomas (fig. these three factors (metastasis, surgical resection, and primary organ) were also significantly associated with improved overall survival in the multivariate analysis (table 2). less than 5% of stss arise in the gu tract and only 15% of stss arise within the retroperitoneum. the rarity of gu sts is a major obstacle to clinical research ; contemporary data in the medical literature are limited. mondaini reviewed sarcomas of different histological types in a multicenter study involving eight different hospitals in tuscany, italy. the mskcc group reported two consecutive series, one including 43 patients treated between 1982 and 1989 and another including 131 patients treated between 1977 and 2003. the latter study was an extension of the former study with a prolonged follow - up. owing to the rareness of stss, and therefore the absence of randomized controlled trials, no standard treatment options for this disease exist. this multimodal approach has replaced amputation as the primary surgical treatment of choice. in the present study the mean tumor size of 9.5 cm found in the present study is slightly smaller than that reported previously [9 - 11 ]. the 5-year os was 51.4%, similar to the results of previous studies that reported 5-year os rates of 50%. dotan reported that the presence of metastasis and rhabdomyosarcoma were unfavorable prognostic variables. in our study, the presence of metastasis at diagnosis was associated with survival in both univariate and multivariate analyses. lewis reported that the presence of unresectable disease and incomplete surgical resection were the most significant factors predictive of disease - specific death. in a study by van dalen of 143 patients treated in the netherlands, complete tumor resection was correlated with better overall survival in the multivariate analysis. however, dotan reported that complete resection was not a significant factor predictive of disease - specific survival in univariate and multivariate analysis in 102 patients with primary tumors only. these results suggest that any type of surgical resection can provide the best chance of survival in patients presenting with primary disease or with primary and metastatic disease. consistent with this, we found that the absence of surgical resection was an unfavorable prognostic variable for overall survival. therefore, we suggest that surgical resection may contribute to a favorable prognosis in patients with urological stss. the prognosis of retroperitoneal stss has been reported to be more favorable than that of the other gu stss. disease prognosis was the worst for stss of the kidney, whereas the prognosis was better for retroperitoneum, bladder, and prostate sarcomas, as reported previously. according to a previous study, the size of stss is an important prognostic variable, in contrast with our findings. this discrepancy may be because stss arising from the retroperitoneum can attain a large size owing to the flexibility of the retroperitoneum. however, retroperitoneal stss showed a better prognosis than bladder, prostate, and renal stss in our study. the histological subtype of the gu stss was not a prognostic factor for disease - specific survival in either the univariate or the multivariate analysis ; in other words, we did not observe an association between histological subtype and disease - specific survival. surgical resection, metastasis at diagnosis, and the primary organ were significant predictors of prognosis. the data presented in this study contribute to our understanding of urological stss and may help in the development of an optimal therapeutic strategy to treat stss. | purposethe purpose of this study was to elucidate prognostic factors for survival and clinical outcomes of rological soft tissue sarcomas (stss).materials and methodsthis was a retrospective review of the medical records of 48 patients with urological sts treated from january 1982 to july 2009. demographic and pathological characteristics were compared. patients ' demographics, clinico - pathological parameters, overall survival, and the factors expected to predict survival, such as sex, age at diagnosis, primary organ, surgical resection, metastasis, and mass size, were analyzed. we evaluated differences in survival on the basis of histological subtype by kaplan - meier analysis and multivariate cox proportional hazards regression.resultsthe study included 34 males (70.8%) and 14 females (29.1%). the mean age at diagnosis was 47.1 years (range, 3 to 80). the most common site was the retroperitoneum (n=16), followed by the kidney (n=12), prostate (n=10), bladder (n=7), ureter (n=1), and paratesticular region (n=1). nineteen patients (39.5%) had other organ metastases at diagnosis. the most common subtypes of sarcoma were leiomyosarcoma (50%), rhabdomyosarcoma (18.7%), and liposarcoma (8%). the remaining 11 cases had other histological subtypes (22.9%). mean tumor size was 9.5 cm (range, 2.2 to 24). thirty - three patients (68.7%) underwent surgical resection. the overall survival rate at 5 years was 51.4%. in the univariate and multivariate analysis, surgical resection, primary tumor site, and metastasis at diagnosis remained significant predictors of prognosis. patients with retroperitoneal sarcoma had a higher overall survival rate by 5 years compared with patients with other organ sarcoma.conclusionsthe overall survival rate at 5 years was 51.4%. surgical resection, primary tumor site, and metastasis at diagnosis remained significant predictors of prognosis. |
traumatic cerebral aneurysms with carotid cavernous fistula (ccf) are rare and difficult to treat by surgery. if left untreated mortality rate of 50% is reported in these cases. hence, the presence of these lesions should be diagnosed and treated at the earliest in order to prevent the untoward effects due to their poor prognosis. a 40-year - old man presented to the emergency room following a road traffic accident. computed tomography scan of the brain (plain) revealed frontal hematoma, intraventricular hemorrhage, and subarachnoid hemorrhage with depressed skull fracture. on the 4 day of hospitalization, he developed increasing proptosis and conjunctival congestion [figure 1a ]. magnetic resonance (mr) imaging showed hyperintense signals in the basal cisterns suggestive of subarachnoid hemorrhage on t1-weighted sequence. time of flight mr angiogram maximum intensity projection image showed abnormal communication between internal carotid artery and cavernous sinus. however, the precise location of the rent was not identified [figure 1b and c ]. digital subtraction angiogram of the left internal carotid artery showed intradural aneurysm (caused by the trauma), which was seen communicating inferiorly with cavernous sinus, basillar plexus, and petrosal sinus [figure 2a and b ]. balloon occlusion test was not tolerated and hence we chose to occlude the aneurysm and the fistulous communication (preserving the parent artery) over parent vessel occlusion. 40-year - old male accident victim with skull fracture presented with increasing left - sided proptosis which was diagnosed as carotid cavernous fistula due to rupture of intradural aneurysm. (b) t1-weighted magnetic resonance (mr) imaging done shows hyperintensity in the basal cisterns suggestive of subarachnoid hemorrhage (arrow). (c) time of flight mr angiogram shows the fistulous communication on the left side (arrow). digital subtraction angiograms of left internal carotid artery in the (a) left oblique and (b) right oblique views show intradural aneurysm (white arrow) and the fistulous communication into the cavernous sinus and petrosal sinus (black arrows in a). (c) road map image with stable position of the micro catheter achieved distally with gentle maneuver (arrow). under general anesthesia, 6 french guiding catheter was placed in the left internal carotid artery. a gentle steam shaping of the micro catheter tip was done to facilitate the navigation of the distal end of the micro catheter into the sinus [figure 2c ]. selective micro - catheter angiogram showed high flow through the aneurysm into the cavernous sinus and inferior petrosal sinus. after achieving stable position of the micro - catheter distally, thereafter, the aneurysm was completely packed with multiple coils and complete occlusion was achieved with no flow into the fistulous communication. post - coiling angiogram showed complete obliteration of the fistula and exclusion of the aneurysm from the circulation [figure 3a c ]. long - term follow - up to assess the coil compaction and recanalization is waiting to be done. post - coiling left internal carotid angiogram in (a) right oblique and (b) left oblique projections show complete occlusion of the aneurysm and the fistulous communication (arrows). (c) townes view (arrow) shows the patent internal carotid artery with good flow in all the distal branches and no remnant fistula. a recent history of trauma with skull base fracture and presentation of ccf with no clear aneurysm and vasospasm on angiogram confirms the diagnosis of the traumatic cerebral aneurysm. in the present case, the traumatic internal carotid artery injury caused pseudoaneurysm dilatation, which ruptured into the venous sinus and caused the presentation of ccf with an opthalmological manifestation. supraclinoid segment aneurysms presenting as ccf is unusual and very few reports of these intradural aneurysms are known. this entity is difficult to access surgically and the presence of the fragile wall of the aneurysm increases the risk of complications during surgery., described the use of coil occlusion in treating these aneurysms. today, the treatment of choice for direct ccf is balloon occlusion of the rent of the internal carotid artery. this treatment option could not be used in the present case, since the inflated balloon could cause rupture of the aneurysm and worsen the condition of the patient. parent vessel occlusion could not be done due to the distal aneurysm and also because of the absence of cross - flow from the right internal carotid artery. selective exclusion of the fistula and the aneurysm while preserving the parent artery is the treatment of choice in this type of pathology. due to high flow from the aneurysm into the fistula, initially this communication needs to be occluded, for which a stable positioning of the micro - catheter in the sinus is required. cho., in their study have reported a case of the traumatic supraclinoid aneurysm with ccf where aneurysm with fistula was partially occluded with coils and glue was used in the second session to cure the remnant fistula. zhao., reported a similar case which was treated by endovascular coils and onyx embolization. in their case, they first coiled the aneurysm in spite of which there was continuous opacification of the cavernous sinus. hence, they used onyx 34 to occlude the residual fistula in the same session. in our case, we initially placed the pre - shaped micro catheter distally into the communicating portion and completely packed the same with micro coils and then coiled the aneurysm after obliterating the fistula. hence, we decided not to use any liquid embolic agents and only coils were placed to obliterate both the fistula and the aneurysm. one should be extremely careful while negotiating the micro catheter through the traumatic aneurysm into the fistulous site to prevent the complication of hemorrhage due to the rupture of the aneurysm. endovascular treatment is the treatment of choice in such cases and should be considered as an emergency procedure due to the high mortality rates encountered in these cases. | carotid cavernous fistulae (ccf) are abnormal communication between cavernous segment of the internal carotid artery and cavernous sinus. these entities are usually encountered in 0.2 - 0.8% of patients with traumatic skull base fractures. traumatic cerebral aneurysms are rare and account for less than 1% of intracranial aneurysms. ccf due to ruptured intradural traumatic aneurysm is very rare and difficult to treat by surgical methods. we present one such case of a 40-year - old man with post - traumatic ccf due to a ruptured intradural aneurysm successfully treated with endovascular embolization. |
water channels of the aquaporin (aqp) family achieve the diffusive water permeation required for biological function and form tetramers. since the discovery of the first water channel, aqp1, more than 300 aqps have been identified in many organisms from bacteria to plants and mammals. aqp family members are classified into two subfamilies : an aqp group that is highly selective for water, and an aquaglyceroporin group that allows both water and other small neutral solutes, like glycerol and urea, to permeate the membrane. thirteen aqps (aqp0 to 12) have been identified in mammals and 5 mammalian aqps have been solved at atomic resolution. the first atomic structure of human aqp1 was determined by electron crystallography, which revealed the unique aqp fold with six transmembrane helices (termed h1h6) and two short pore helices (termed hb and he), together with the highly conserved asn - pro - ala (npa) motifs in the middle of the membrane. each aqp monomer has a channel pore, and the individual pores work independently within a functional tetramer. in the channel pathway, the narrowest region, which is located between the npa motif and the extracellular pore entrance, is called the ar / r (aromatic / arginine) constriction site. in the aqp group, the width of the ar / r site is the same as the diameter of a water molecule. aqp4 is a water selective channel that is predominantly expressed in the brain, but also expressed in other tissues. there are three functional splicing isoforms of aqp4 and their expression ratio determines the size of the orthogonal arrays that aqp4 forms in the endfeet of astrocytes. the long aqp4 isoform with a full n - terminus starting with met1 (aqp4m1) contains palmitoylated cysteines and thus sterically impedes the formation of arrays, whereas the short isoform starting with met23 (aqp4m23) easily forms arrays due to the absence of palmitoylation sites. a recently discovered functional isoform, aqp4e, has 41 residues extending from the n - terminus of aqp4m1. its expression level is quite low in rat brain, but it transports water and has no ability to form arrays, similar to aqp4m1. the function of aqp4 arrays remains elusive, but the water permeability of functional isoforms is quite similar. aqp4 has important roles in maintaining brain homeostasis as well as in several brain pathologies, including brain edema and neuromyelitis optica. most aqps are thought to be in a constitutively open conformation, but some water channels adopt a closed conformation that is regulated by various mechanisms. among them, the spinach plasma membrane aqp sopip2;1 is a functionally and structurally well - studied aqp whose gating is regulated to protect the plant against drought or flood. a plausible channel gating mechanism of sopip2;1 regulated by phosphorylation, ph or ca was proposed based on x - ray structural studies combined with molecular dynamics simulations. the conformational changes in loop d mainly contribute to the central part of its gating. in the closed state, loop d forms an antiparallel -sheet to cover the cytoplasmic pore entrance with the displacement of leu197. in contrast, in the open state loop d does not adopt a secondary structure that is displaced to move away from the pore, and the n terminus of helix 5 extends into the cytoplasmic side. the model proposes that closure of the pore is stimulated by the dephosphorylation of two conserved serine residues (ser115 or ser274) or by protonation of a conserved histidine residue (his193). no experimental evidence to date, however, has demonstrated the phosphorylation of either ser115 in sopip2;1 or a corresponding site in any other plant aqp. this together with other evidence contradicting the model [1921 ] suggests that more information is needed to fully elucidate the gating mechanism regulated by phosphorylation. the water permeability of mammalian aqp0 is affected by the ph, partially due to the protonation state of a his residue in loop a, and by ca in a calmodulin - dependent gating mechanism. these mechanisms help to regulate osmotic pressure through aqps in response to surrounding conditions and are tightly linked to the physiological functions of the cells that express the water channels. mercury, silver and gold suppress the activities of several aqps [2326 ], but these elements are not suitable for clinical application due to their toxicity and non - specificity for aqps. many recent studies describing the application of aqp inhibitors for human diseases have revealed the potential of aqps as therapeutic targets. acetazolamide (aza or azm), a well - known carbonic anhydrase inhibitor, reduces the water permeability of aqp1 in xenopus laevis oocytes. some groups reported, however, that aza does not affect the water permeability of aqp1 in erythrocytes and aqp4 in brain glial cells [3133 ]. our previous studies using proteoliposomes indicated that aza inhibits aqp4 activity, but has no effect on aqp1. the results of in vitro assays using proteoliposomes are more reliable and reproducible than those obtained in assays using living cells, such as oocytes and mammalian cells, which may explain the discrepancy in the findings obtained with different systems. to investigate the structural effect of aza binding, we determined the aqp4 structure in complex with aza by electron crystallography at 5 resolution, and further validated the binding site using a molecular docking simulation study. preparation of the constructs, and expression and purification procedures for rat aqp4m23 (raqp4m23) were performed as previously described. purified protein was mixed with escherichia coli total lipid extract (avanti) at a lipid - to - protein ratio of 1.0 (w / w). the mixture was dialyzed in a dialysis button for 3 days against 10 mm mes (ph 6.0), 100 mm nacl, 50 mm mgcl2, 2 mm dithiothreitol and 1% glycerol. during dialysis, the temperature was maintained at 20c on the first day, increased to 37c on the second day and decreased again to 20c on the third day. after harvesting, 2d crystals were soaked in the same dialysis buffer containing 1 mm aza (sigma - aldrich), which was solvated with 0.05% n, n - dimethylformamide, and maintained at 20c for 13 h. specimens for cryo - electron microscopy were prepared using the conventional single carbon film technique with molybdenum grids and a final trehalose concentration of 7% (w / v). double - layered 2d crystals tended to be more slippery between layers in the presence of aza than in the absence of aza. the preparation using the carbon sandwich technique drastically increased the failure rate of merging data to reconstruct the 3d structure. thus, we chose the conventional technique to collect the images, although we had to take a lot of images to obtain good ones without an image shift. electron micrographs were recorded with a jem3000sff electron microscope equipped with a liquid - helium stage and a field - emission electron source with an acceleration voltage of 300 kv. images of specimens were tilted at angles of 0, 20, 45 and 60, and recorded on kodak so-163 film (carestream health) at a nominal magnification of 40 000 with a 2-s exposure time, corresponding to a total electron dose of 20 electron. the images were computationally unbent and corrected for the contrast transfer function (ctf). the fourier transform of each image diffracted around 5 resolution along the tilt axis (fig. 1). images from crystals of the predominant crystal type (p4212) were merged and used to calculate a 3d density map at 5 resolution by applying a negative b - factor of 200 (figs. 2 and 3). the characteristics of our electron crystallographic structure of raqp4m23 are summarized in table 1. the cryo - em density map of raqp4m23 bound with aza has been deposited in the electron microscopy data bank (http://www.emdatabank.org/) with accession code emd-3214. table 1.electron crystallographic datatwo - dimensional crystal space groupp4212 lattice constantsa = b = 69.1, c = 160.0 (assumed), = 90.0number of images used approximate tilt angle () 06 2038 4550 6047 total141resolution limit in membrane plane ()5.0 normal to membrane plane ()5.7range of underfocus ()520043 400number of observed reflections16 595number of unique reflections1006overall weighted phase residuals24.8overall weighted r - factor0.480used reflections are better than iq7. (a) iq plots calculated from fourier transforms of images of frozen - hydrated 2d crystals of aqp4 bound to aza at tilt angles of 0, 45 and 60. circles with the label text in the upper right indicate resolutions of 20, 7, 5 and 4. (b) representative lattice lines (0, 3), (1, 5) and (2, 7) showing a good match between the experimentally observed reflection data and the calculated curves. the measured phases for lattice line (0, 3) were mostly 0 or 180, indicating agreement with the p4212 symmetry. 2.overviews of the map calculated from electron micrographs shown with a superimposed atomic model of aqp4 tetramer. figures are viewed parallel with the membrane (a), from the extracellular side (b) and cytoplasmic side (c). the density map represented by the gray surface is contoured at 1.2 and clearly shows six transmembrane helices in each monomer. each aqp4 monomer is shown as a ribbon model, and one of four channel pores in a tetramer is indicated by a yellow transparent circle. figures are viewed from the extracellular side (a), and cytoplasmic side (b). the density map represented by the gray surface is contoured at 1.2 and the unexplained density identified with the fitting atomic model is shown in yellow and is located near the extracellular pore entrance. one of the tetramers is shown as a stick model, and the others are shown as a ribbon model. electron crystallographic data used reflections are better than iq7. iq plots and lattice lines. (a) iq plots calculated from fourier transforms of images of frozen - hydrated 2d crystals of aqp4 bound to aza at tilt angles of 0, 45 and 60. circles with the label text in the upper right indicate resolutions of 20, 7, 5 and 4. (b) representative lattice lines (0, 3), (1, 5) and (2, 7) showing a good match between the experimentally observed reflection data and the calculated curves. the measured phases for lattice line (0, 3) were mostly 0 or 180, indicating agreement with the p4212 symmetry. overviews of the map calculated from electron micrographs shown with a superimposed atomic model of aqp4 tetramer. figures are viewed parallel with the membrane (a), from the extracellular side (b) and cytoplasmic side (c). the density map represented by the gray surface is contoured at 1.2 and clearly shows six transmembrane helices in each monomer. each aqp4 monomer is shown as a ribbon model, and one of four channel pores in a tetramer is indicated by a yellow transparent circle. figures are viewed from the extracellular side (a), and cytoplasmic side (b). the density map represented by the gray surface is contoured at 1.2 and the unexplained density identified with the fitting atomic model is shown in yellow and is located near the extracellular pore entrance. one of the tetramers is shown as a stick model, and the others are shown as a ribbon model. a model of raqp4m23 was constructed from the high - resolution structure of raqp4s180d (pdb : 2zz9) to replace ser180 with asp using coot, and then fitted to a density map using the fit in map function of chimera. the aza coordinate was downloaded from pubchem (cid : 1986). after roughly removing the geometry distortion of the ligand using discovery studio 4.5 (biovia), d.01 (gaussian, inc.) with the restricted hartree - fock model (rhf/6 - 31g(d)). the optimized coordinates and the model were used for a molecular docking simulation with autodock vina. the docking search area covered the whole extracellular cavity of aqp4 in a large box (30 30 30) centered at the guanidino group of the arg216 residue. because the program predicted similar binding sites with a good score, only the three best high - scoring conformers are represented in fig. 4 to elucidate the fitness of the ligand and the em density map. the position of the sliced section is represented by the broken line in (a). high energetically favorable conformations of aza are shown as three favorable stick models (green : the most favorable) and aqp4 is shown as a ribbon model. the position of the sliced section is represented by the broken line in (a). high energetically favorable conformations of aza are shown as three favorable stick models (green : the most favorable) and aqp4 is shown as a ribbon model. here we collected images of raqp4m23 2d crystals and used them to calculate a 3d reconstruction at 5 resolution (table 1 and fig. 2). almost all of the features of the density map were completely consistent with the previously determined atomic models of raqp4m23 and raqp4m23s180d, because the aqp4 atomic model fit nicely with the whole density map using chimera. at first glance, the 5- map did not reveal a large density that could obstruct the channel entrance formed by the loop or the terminal domain of aqp4 (fig. 2). six transmembrane helices were clearly resolved at this resolution, and a water pathway was observed from the extracellular to the cytoplasmic side as being emphasized by transparent yellow in a pore region of the monomer (fig. all densities except that near the extracellular pore entrance could be explained by the fitted atomic model showing that the water channel in aqp4 is likely to be in an open conformation. large conformational changes in aqp4 were not observed during aza - induced reduction of aqp4 water permeability. the closed conformation of plant pip2 induced by a divalent cation is accomplished by a large conformational change in loop d at the cytoplasmic side. the 5 map shows an interesting feature a density on the extracellular side that protrudes into the channel pore entrance at 1.2 (fig. this density likely represents aza, and the interaction of aza and aqp4 suggests a direct role of aza in the inhibition of the water permeability of aqp4. furthermore, the center of the protrusion density is more than 1.35, which corresponds to the density of the helical region in the map. such a significant feature is not observed in the density map at the cytoplasmic pore entrance, even when contoured at 0.8, which is close to the noise level. the clearly obstructing density at the limited resolution of our map leads us to propose that aza is directly involved in water inhibition at the extracellular side. at the current resolution, however, the location of the loop region is sometimes unreliable, and thus we can not exclude the possibility that the density represents a part of the extracellular loop of aqp4. the validity of the assignment conclusion must be confirmed by other methods. to further confirm the assignment of the protruding density at the extracellular pore entrance, we performed a molecular docking simulation between aza and aqp4. the predicted positions of the ligand located around the protruding density (fig. 4). we selected the three most energetically favorable conformations for which binding free energy ranged from 5.1 to 4.9 kcal mol. all selected conformations occupied an almost identical position, suggesting that the protruding density is a thiadiazol ring of aza (fig. the most favorable conformation of aza was associated with 10 residues, including thr56, gly146, val147, thr148, thr149, his151, ile205, gly209, ala210 and arg216 from h1, loop c, and he (sticks in fig. 4). in particular, the conserved arg216 in he comprises the ar / r constriction site and is thus crucial for allowing water molecules to permeate. the location of aza seems to obstruct the ar / r constriction site of the channel pore by clogging it at the extracellular side. our previous water permeation experiments consistently revealed that aza does not suppress the water permeability of aqp1, but clearly reduces aqp4 activity. considering the sequence alignment between aqp1 and aqp4, the conservation mapping to the aqp4 structure showed that most of the residues at the cytoplasmic side are more conserved than those at the extracellular side (fig. because loops c and he are located close to the water pore, changes in their interaction with aza could influence the water permeability of aqp4. together, these findings support the notion that water permeation through aqp4 is directly prevented by aza binding at the extracellular side. further structural and functional studies are needed to clarify the precise location of aza binding and the precise mechanism of the inhibition of water passage. figures are viewed from the extracellular side (a) and the cytoplasmic side (b). the highest energetically favorable conformation of aza is shown as a stick model and aqp4 is shown as a ribbon model. the mapping sequence conservation between rat aqp4 and human aqp1 is color - coded (maroon : identical, cyan : different). figures are viewed from the extracellular side (a) and the cytoplasmic side (b). the highest energetically favorable conformation of aza is shown as a stick model and aqp4 is shown as a ribbon model. the mapping sequence conservation between rat aqp4 and human aqp1 is color - coded (maroon : identical, cyan : different). scale bar represents 20. in summary, our analysis of the crystal structure of aqp4 bound with aza yields insights into the inhibitor binding conformation. our electron crystallographic reconstruction provides a first glimpse of the inhibited aqp4 structure in the membrane. the aza - binding location near the extracellular pore entrance visualized by the 5- map is in good agreement with the molecular docking simulation. this work was supported by grants - in - aid for scientific research (s), the japan new energy and industrial technology development organization (nedo) and the japan agency for medical research and development (amed) (to y.f.) ; and grants - in - aid for scientific research (c) (to k.t.). funding to pay the open access publication charges for the article was provided by grants - in - aid for scientific research (s). | acetazolamide (aza) reduces the water permeability of aquaporin-4, the predominant water channel in the brain. we determined the structure of aquaporin-4 in the presence of aza using electron crystallography. most of the features of the 5- density map were consistent with those of the previously determined atomic model. the map showed a protruding density from near the extracellular pore entrance, which most likely represents the bound aza. molecular docking simulations supported the location of the protrusion as the likely aza - binding site. these findings suggest that aza reduces water conduction by obstructing the pathway at the extracellular entrance without inducing a large conformational change in the protein. |
developmental disabilities (dd) are a chronic delay in development, including conditions such as cerebral palsy, intellectual development disorder, autism, and mixed type dd. children with dd have problems with physical, cognitive, social, sensory, and communication skills2. these unique problems have a negative impact on the lives of their family as well as of the children with dd1. among the problems faced by children with dd, impairment in cognitive function and visual perception causes problems in processing environmental stimuli, which makes it difficult for them to adapt to their environment and live in independently2. after entering elementary school, due to an increase in demanding activities of daily living, problems caused by impairments of cognitive function and visual perception become more evident3. as compared to prior to school entry, school - aged children are required to complete tasks within a limited time at school or home and are expected to exhibit sophistication and organization in their activities4. the literature indicates that there are intervention methods to improve cognitive function and visual perception in children with dd, such as attention training programs, sensory integration, and tabletop activities5, 6. these methods are remedial approaches to restore and facilitate the improvement of cognitive function and visual perception caused by dd7. although various conventional treatment methods for children with dd have been applied in clinical settings, the controversies over the most effective protocol continues8. in order to compensate for these problems, recently computer - based cognitive rehabilitation (cbcr) has been used in clinical settings8. several studies concluded that cbcr was an effective treatment for recovery of cognitive function and visual perception9. however, most such studies recruited adults or the elderly with stroke, traumatic brain injury, or dementia10. few studies have investigated the effects of cbcr on cognitive function and visual perception in children with dd. additionally, the existing cbcr programs have predominantly targeted adults, and there is a lack of cbcr programs for children8. in 2011, kim developed a cbcr program for children called cotras - c8. however, there is still insufficient evidence for the clinical effectiveness of this program. therefore, there is a need to investigate the effectiveness of the cotras - c using objective methods. accordingly, in this study, we conducted a randomized controlled trial to investigate the effectiveness of the cbcr program for children with reference to their cognitive function and visual perception. we screened the participants based on the inclusion criteria used in previous studies on cbcr for children8, 11. the inclusion criteria for participation were as follows : (i) dd diagnosed by a pediatrician or rehabilitation physician ; (ii) children with dd, exhibiting similar symptoms (children with limited cognitive function and visual perception) ; (iii) presence of dd diagnosed by performance on the korean - developmental test of visual perception-2 (k - dtvp-2) ; (iv) a below normal score on the kaufman assessment battery for children (k - abc) ; (v) ability to use the controller with the upper extremity without spasticity ; and (vi) ability to understand instructions within three repeated explanations. parents signed the informed consent form before commencement of the study, according to the code of ethics of the world medical association (declaration of helsinki, version 2004). the first was the k - dtvp-2, which comprises 8 sub - tests (eye - hand coordination, position in space, copying, figure - ground, spatial relations, visual closure, visual - motor speed, and form constancy). this test can be used to measure visual perception in children aged 4 to 8 years, and helps to confirm the extent of visual perception disability according to developmental stages12. it provides an index of motor - reduced visual perception (mrp) and visual motor integration (vmi) and combines the two indices to interpret the overall level of visual perception (general visual perception ; gvp)13. the second measure was the k - abc, designed for use with children aged 2.5 to 12.5 years. it assesses the intelligence quotient and achievement, and entails 2 subscales, the achievement scale (ach) and mental processing composite (mpc), and 16 sub - tests. the expected mean raw score is 100, with a standard deviation of 1514. the number of sub - tests implemented changes depending on the age of the children tested. therefore, in this study, we used the sub - tests, depending on the age of the subjects. all clinical measurements were administered before and after the intervention by a blinded occupational therapist with 4 years experience. after the baseline session, eligible participants were randomly allocated to either the control group (cg) or experimental group (eg) by a research assistant who was not involved in the intervention. during the intervention sessions, the eg participants received 20 sessions (2 days a week for 10 weeks) of cbcr with the cotras - c program (netblue co., ltd, korea). the cotras - c consists of about 4,000 game - based training task on skills including attention, memory, visuo - motor organization, coordination, and others. the cotras - c with a character design and voice suitable for children, makes the training easy by using the touch monitor and cotras - c controller. the difficulty level of all the tasks can be modified to suit the individual participants abilities. training data are automatically stored for accuracy analysis, which helps develop an appropriate treatment plan8, 11. in this study, the eg participants received the visual perception training that comprised spatial relations, spatial memory, concentration, eye - hand coordination, eye movement, and figure - ground perception. the duration of intervention for the cg participants was matched to that of the eg participants. the cg participants received a conventional cognitive rehabilitation that focused on visual perception, using pencil and paper activities. all participants received either the eg or cg intervention for 30 minutes per a session. the test and independent t - test were used to compare the differences in the general characteristics of the two groups. the independent t - test was used to compare the between group means and changes in values, while the paired t - test was used to test the differences in the continuous variables within the groups. the 29 participants who fulfilled the inclusion criteria were randomly allocated to the two groups as follows : 15 to the eg and 14 to the cg. all participants characteristics have been summarized in table 1table 1.subjects characteristicseg (n = 15)cg (n = 14)gendermale910female64age (months)69.73 8.3371.93 8.07educationpreschool1010school54main care - givermother118grandmother46mean standard deviation. cg : control group ; eg : experimental group after intervention, the participants in both the groups showed a significant increase in their scores on the k - dtvp-2, ach of the k - abc, and mpc of the k - abc (p < 0.001) (tables 2table 2.subjects k - dtvp-2 scores before and after the interventionk - dtvp-2 (gvp)pre - testpost - testeg (n = 15)66.80 (3.63) 79.87 (2.92)cg (n = 14)67.79 (2.78) 76.43 (3.90)p < 0.05, p < 0.001.mean (standard deviation). cg : control group ; eg : experimental group ; gvp : general visual perception ; k - dtvp-2 : korean - developmental test of visual perception-2 and 3table 3.subjects k - abc scores before and after the interventionsachmpcpre - testpost - testpre - testpost - testeg (n = 15)494.20 (25.46) 540.13 (23.91)68.60 (3.74) 75.56 (4.09)cg (n = 14)508.64 (17.99) 531.42 (16.99)70.25 (3.18) 74.42 (3.26)p < 0.05, p < 0.001.mean (standard deviation). ach : achievement scale ; cg : control group ; eg : experimental group ; mpc : mental processing composite). additionally, there were significant differences in the changes in the k - dtvp-2, ach of the k - abc, and mpc of the k - abc between the two groups (p < 0.001) (table 4table 4.comparison of change in k - dtvp-2 and k - abc scoreseg (n = 15)cg (n = 14)k - dtvp-2 (gvp)13.07 (1.44) 8.64 (3.15)k - abc ach45.93 (4.23) 22.79 (2.75) mpc6.90 (0.71) 4.18 (0.80)p < 0.05, p < 0.001.mean (standard deviation). ach : achievement scale ; cg : control group ; eg : experimental group ; gvp : general visual perception ; k - abc : kaufman assessment battery for children ; k - dtvp-2 : korean - developmental test of visual perception-2 ; mpc : mental processing composite). p < 0.05, p < 0.001. mean (standard deviation). cg : control group ; eg : experimental group ; gvp : general visual perception ; k - dtvp-2 : korean - developmental test of visual perception-2 p < 0.05, p < 0.001. ach : achievement scale ; cg : control group ; eg : experimental group ; mpc : mental processing composite p < 0.05, p < 0.001. mean (standard deviation). ach : achievement scale ; cg : control group ; eg : experimental group ; gvp : general visual perception ; k - abc : kaufman assessment battery for children ; k - dtvp-2 : korean - developmental test of visual perception-2 ; mpc : mental processing composite although studies on the effectiveness of the cbcr program for children are lacking8, 11, it is increasingly used in clinical settings because of its advantages15. therefore, the present study investigated the effectiveness of the cbcr program with reference to cognitive function and visual perception in children with dd. the results of this study indicated that the cbcr program for children may improve cognitive function and visual perception in children with dd. the clinical improvement in the k - dtvp-2 and k - abc scores was greater in the eg after intervention, as compared to that in cg participants, confirming that cbcr is a more effective intervention for children with dd as compared to conventional intervention. most cbcr programs used in korea were developed for adults and these effectiveness has been confirmed by several studies. however, in some parts of cbcr program for adults, the stimuli presented on the screen are not suitable for children and do not sustain interest in them. therefore, it is difficult to use such programs with children8. to resolve these problems, kim developed a cbcr program for children called cotras - c8. the effectiveness of cbcr with the cotras - c on cognitive function and visual perception have been proved in several studies8, 11. the use of a computer program helps in the cognitive development of children by facilitating their intellectual ability, stimulating curiosity, and allowing for creative thinking16. in 2014, park indicated that children with dd who received cbcr with the cotras - c showed increased k - dtvp-2 and k - abc scores, which is consistent with the results of the present study11. visual perception is the ability to interpret and understand the environment by processing information that is contained in visible light. visual perception is closely related to cognitive function, and therefore, an improvement in visual perception has been found to have a positive impact on cognitive function13, which was also confirmed in the results of the present study. this study conclude that cbcr with the cotras - c can be used as a rehabilitation approach to improve cognitive function and visual perception in children with dd, which suggests that the cotras - c may be used as an alternative to other existing cbcr programs. although the sample size employed in the present study was small, the findings of this study are significant because cbcr is a novel approach that broadens the scope of clinic based intervention to home - based ones. additionally, children living in a modern society have several opportunities to receive this treatment17. the present study did not examine the effectiveness of cbcr with the cotras - c on the activities of daily living of children with dd. therefore, in future, studies with a large sample size are needed to investigate the clinical effectiveness of the cotras - c as a rehabilitation program for activities of daily living as well as cognitive function and visual perception. | [purpose ] this study aimed to investigate the effects of a computer - based cognitive rehabilitation program for children with developmental disabilities. [subjects ] subjects included 29 children with developmental disabilities. [methods ] the subjects were randomly allocated to either the experimental group or control group. experimental group subjects received computer - based cognitive rehabilitation using the cotras - c while control group subjects received conventional cognitive rehabilitation. all subjects received 20 sessions (2 days a week for 10 weeks) of the experimental or control intervention for 30 minutes. to compare the two groups, the korean - developmental test of visual perception-2 and kaufman assessment battery for children were performed before and after the intervention. [results ] both groups showed statistically significant improvement in their scores after intervention. additionally, there were significant differences in the scores between the two groups. [conclusion ] the computer - based cognitive rehabilitation with cotras - c may be helpful in improving the recovery of cognitive function and visual perception in children with developmental disabilities. |
to determine the accuracy of the holladay 1, hoffer q, sanders - retzlaff - kraff (srk)/t, and srk ii iol power calculation formulas in patients with high myopia in a subset of indian population. this study was conducted at sadguru netra chikitsalaya, chitrakoot, madhya pradesh, india. all consecutive patients attending our out patient department (opd) for phacoemusification with axial length (al) more than 24.5 mm (range 24.75 - 32.35 mm) between may 2009-october 2009 (in all patients hydrophilic acrylic foldable within the bag iol was implanted). pre - existing astigmatism > 3.0 diopters (d)corneal scarkeratoconuscomplications significantly affecting the refractive status (vitreous loss with iol implanted in sulcus or anterior chamber, high wound induced astigmatism). pre - existing astigmatism > 3.0 diopters (d) complications significantly affecting the refractive status (vitreous loss with iol implanted in sulcus or anterior chamber, high wound induced astigmatism). in our study, keratometry measurements were retrieved from records and a scan applanation ultrasonography was done on echorule2 and analyzed. the implanted iol power was used to calculate the predicted postoperative refractive error by four commercially available iol formulas : srk ii, srk - t, holladay 1, and hoffer q. with each formula, the mean error (me) was calculated from the difference between the formula predicted refractive error and actual postoperative refractive error. the spherical equivalent was measured by a single trained optometrist using an autorefractor and subjective retinoscopy 1 - 2 months after cataract surgery. repeated measures anova tests were done after making bonferroni corrections to have pair - wise comparisons between the formulas and p 3.0 diopters (d)corneal scarkeratoconuscomplications significantly affecting the refractive status (vitreous loss with iol implanted in sulcus or anterior chamber, high wound induced astigmatism). pre - existing astigmatism > 3.0 diopters (d) complications significantly affecting the refractive status (vitreous loss with iol implanted in sulcus or anterior chamber, high wound induced astigmatism). in our study, keratometry measurements were retrieved from records and a scan applanation ultrasonography was done on echorule2 and analyzed. the implanted iol power was used to calculate the predicted postoperative refractive error by four commercially available iol formulas : srk ii, srk - t, holladay 1, and hoffer q. with each formula, the mean error (me) was calculated from the difference between the formula predicted refractive error and actual postoperative refractive error. the spherical equivalent was measured by a single trained optometrist using an autorefractor and subjective retinoscopy 1 - 2 months after cataract surgery. repeated measures anova tests were done after making bonferroni corrections to have pair - wise comparisons between the formulas and p 3.0 diopters (d)corneal scarkeratoconuscomplications significantly affecting the refractive status (vitreous loss with iol implanted in sulcus or anterior chamber, high wound induced astigmatism). pre - existing astigmatism > 3.0 diopters (d) complications significantly affecting the refractive status (vitreous loss with iol implanted in sulcus or anterior chamber, high wound induced astigmatism). in our study, keratometry measurements were retrieved from records and a scan applanation ultrasonography was done on echorule2 and analyzed. the implanted iol power was used to calculate the predicted postoperative refractive error by four commercially available iol formulas : srk ii, srk - t, holladay 1, and hoffer q. with each formula, the mean error (me) was calculated from the difference between the formula predicted refractive error and actual postoperative refractive error. the spherical equivalent was measured by a single trained optometrist using an autorefractor and subjective retinoscopy 1 - 2 months after cataract surgery. repeated measures anova tests were done after making bonferroni corrections to have pair - wise comparisons between the formulas and p 24.5 mm holladay 1 caused the smallest me, + 0.24 d. fifty three percent of patients had mean absolute error less than 1.00 d. the hoffer q and srk - t caused a little larger hyperopic shift with mes of + 0.58d and + 0.92 d. srk ii caused the largest hyperopic error + 1.23 d. [table 1 ]. pair - wise comparisons between different formulas shows that p value between different formulas is significant (p 28.0 mm they found holladay 1 and srk / t more accurate than hoffer q. narvaez and co - authors also reported better performance of these formulas when compared with srk - ii. in studies by bang and co - authors on al > 27.0 mm and wang and co - authors on al > 25.0 mm using iol master reported that haigis formula is the best. ghanem and el - sayed reported that in patients with high axial myopia, the performance of srk - t, hoffer - q, holladay-2, and haigis formulas are comparable in low plus powered iol implantation and haigis formula is the best formula when minus power iol is implanted. however, we did not find performance of srk - t formula better in indian myopic population. our recommendation is to avoid using the srk ii and srk / t formula for iol power calculation in eyes with axial myopia in indian population with al longer than 24.50 mm. in our study, all four formulas had a tendency to cause a postoperative hyperopic refraction. our study had some limitations : (a) it was a single center retrospective study and (b) surgeons, and time of the postoperative refraction were not standardized. to conclude, the results of our study provide useful information to aid the choice of iol power in patients with high axial myopia in indian population during our daily practice with holladay 1 being the most accurate and srk ii and srk / t to be avoided. | efficacy of intraocular lens power calculation formulas in a subset of indian myopic population. retrospectively reviewed 43 patients who underwent phacoemulsification with high axial length (al) (> 24.5 mm, range 24.75 - 32.35 mm). the power of the implanted intraocular lens (iol) was used to calculate the predicted post - operative refractive error by four formulas : sanders - retzlaff - kraff (srk ii), srk / t, holladay 1, and hoffer q. the predictive accuracy of the formulas was analyzed by comparing the difference between the actual and predicted postoperative refractive errors. repeated measures analysis of variance (anova) tests were done to have pair - wise comparisons between the formulas and p < 0.05 was considered significant. a subcategory of axial length 24.5 - 26.5 mm was also tested. holladay 1, hoffer q and srk / t formulas showed a slight tendency toward resultant hyperopia, with mean error of + 0.24 diopters (d), + 0.58 d, and + 0.92 d, respectively. the holladay 1 formula provided the best predictive result overall. |
chronic low back pain (lbp) has long been one of the most common causes of disability in adults and is a very frequent condition among early retirees in industrialized societies. degenerative disc disease (ddd) is the most frequent problem in patients with lbp. in 1988, modic. thereafter, the medical term modic changes (mc) has appeared in various studies on spinal degenerative diseases. the prevalence of mc among patients with ddd of the lumbar spine varies between 19% and 59%. mc of types 1 and 2 are more common than those of type 3 and mixed changes. mc are considered a magnetic resonance imaging (mri) parameter determining morphological changes in spinal degenerative diseases. mc are thought to occur because of environmental, genetic, hormonal, mechanical, and degenerative factors, as well as because of the interaction of several unknown factors. among the environmental factors, dietary habits and trends of ddd patients however, their significance in the monitoring and treatment of the disease remains unclear. it is the single most common cause of disability in individuals aged over 45 years and the second most common reason for primary care physician visits5,6,7. the risk factors associated with ddd include advanced age, socioeconomic status, torsional stress, smoking, obesity, heavy lifting, vibration, trauma, immobilization, psychosocial factors, gender, height, hereditary and genetic factors, and occupations like machine drivers, carpenters, and office workers8,9,10,11,12. mc have been described as being strongly associated with lbp13,14,15. according to modic., these changes visible on mri scans can be classified into three different types14, 15. type 1 changes are seen on t2-weighted mri as areas of high signal intensity and on t1-weighted mri as areas of low signal intensity extending from the vertebral endplates. histological examination of material harvested during surgery has shown that mc type 1 manifest as disruption and fissuring of the endplate with regions of degeneration, regeneration, and vascular granulation tissue14. type 2 are seen as areas of high signal intensity on both t1- and t2-weighted images, manifesting as disruption of the endplates with increased reactive bone and granulation tissue. type 3 mc presumably represent bone sclerosis and are visualized on mri as low - signal intensity areas on both t1- and t2-weighted images13,14,15,16. the aim of the present study is to investigate the effects of eating habits on the modic classification in patients with ddd. this randomized, prospective, controlled, single - blinded study was conducted at an outpatient clinic for physical medicine and rehabilitation of lbp patients. this department is part of the rheumatology outpatient clinic. via face - to - face interviews, this form recorded the patients demographics, mc, smoking and alcohol use, concomitant diseases, disease duration, and nutritional status. the questions related to tobacco and alcohol use, frequency of breakfast consumption, daily water consumption, salt consumption, and frequency of consumption of fast food, eggs, milk, yogurt, cheese, whole wheat bread, white bread, butter, and margarine. for each food choice, the frequency of consumption was rated as every meal, once a day, 3 days a week, 2 days a week, 1 day a week, once every 15 days, once every 1 month, and not sure. in addition to demographic characteristics (age, gender, weight, height, and body mass index [bmi ]), patients were also questioned about their occupation, main symptoms, and time of diagnosis. other exclusion criteria included other comorbidities, unavailable mri data, and inability to speak turkish. all the recruited subjects signed informed consent forms before participating in the study, and the approval of the local ethics committee was obtained. the erythrocyte sedimentation rate (esr ; mm / h) was measured using the westergren method, and the serum c - reactive protein (crp ; mg / dl) level was determined using nephelometry. all statistical analyses were performed using statistical package for social sciences for windows software version 16.0 (spss inc., the kolmogorov - smirnov test was used to confirm that the data were within the ranges of normal distribution in both groups. data were compared between groups using the independent - samples t - test. statistical significance was set at p < 0.05 with a 95% confidence interval. most were female (85%), and the mean age of the entire cohort was 43.65 11.36 years. the mean disease duration was 4.16 1.59 years, and mean bmi was 27.52 5.32. in terms of smoking and alcohol use, 15 patients (37.5%) the mean daily tea consumption was 1.65 1.27 cups, while the mean water consumption was 1.12 0.92 l. thirty percent of the patients did know exactly how much milk they consumed ; 20% said they consumed it on 2 days a week, while 20% stated that they never consumed it. the frequency of yogurt consumption was 3 days a week for 47.5% patients, frequency of cheese consumption was 3 days a week for 92.5% patients, and frequency of white bread consumption was 3 days a week for 62.5% patients. half the patients did not know how frequently they consumed whole wheat bread, while 30% reported eating it 3 days a week. the frequency of margarine consumption was unknown by 72.5% patients ; 30% did not know how often they ate butter, while 27.5% reported that they ate it once every 15 days. the frequency of egg consumption was 1 day per week for 30% patients and once every 15 days for 27.5%. then, 60% did not know how often they consumed fast food, and 20% reported that they did not consume fast food. the frequency of red meat consumption was 2 days a week for 35% patients and once every 15 days for 35% patients. the frequency of white meat consumption was once every 15 days for 35% and 1 day a week for 30% patients (table 1table 1.frequency of consumption of various foods in terms of number of daysdo not knowneverone day a monthonce every 15 daysonce a weektwice a week3 days a weekevery daymilk12 (30%)8 (20%)4 (10%)3 (7.5%)3 (7.5%)8 (20%)2 (5%)0 (0%)yogurt2 (5%)1 (2.5%)0 (0%)2 (5%)7 (17.5%)9 (22.5%)19 (47.5%)0 (0%)white bread5 (12.5%)3 (7.5%)1 (2.5%)0 (0%)1 (2.5%)0 (0%)25 (62.5%)5 (12.5%)whole wheat bread20 (50%)1 (2.5%)1 (2.5%)0 (0%)1 (2.5%)0 (0%)12 (30%)5 (12.5%)margarine29 (72.5%)4 (10%)2 (5%)2 (5%)3 (7.5%)0 (0%)0 (0%)0 (0%)butter12 (30%)1 (2.5%)0 (0%)11 (27.5%)9 (22.5%)7 (17.5%)0 (0%)0 (0%)fish1 (2.5%)9 (22.5%)15 (37.5%)10 (25%)4 (10%)1 (2.5%)0 (0%)0 (0%)eggs2 (5%)0 (0%)0 (0%)5 (12.5%)12 (30%)10 (25%)11 (22.5%)0 (0%)fast food24 (60%)8 (20%)3 (7.5%)2 (5%)2 (5%)1 (2.5%)0 (0%)0 (0%)cheese0 (0%)1 (2.5%)0 (0%)0 (0%)1 (2.5%)1 (2.5%)37 (92.5%)0 (0%)red meat0 (0%)1 (2.5%)2 (5%)14 (35%)9 (22.5%)14 (35%)0 (0%)0 (0%)white meat0 (0%)2 (5%)5 (12.5%)14 (35%)12 (30%)7 (17.5%)0 (0%)0 (0%)). the mean body fat percentage of the patients was 31.22% 9.58% and fat amount was 23.37 10.22 kg. the average esr was 24 8 mm / h, and mean crp level was 1.42 1.2 mg / dl. the cervical disc was affected in 11 patients, the lumbar disc in 18, and both lumbar cervical discs in 11 patients. the modic classifications according to the type of disc degeneration are given in table 2table 2.modic classification according to degenerated disc typeregioncervicallumbarboth cervical and lumbartotal modic type 18 (2.5%)8 (20%)4 (10%)20 (50%)modic type 22 (5%)8 (20%)5 (12.5%)15 (37.5%)modic type 31 (2.5%)2 (5%)2 (5%)5 (12.5%)total (n, %) 11 (27.5%)18 (45%)11 (27.5%)40 (100%). a weak negative correlation was noted between fish and egg consumption and the modic types (r = 0.361, p = 0.42 ; r = 0.428, p = 0.14, respectively). the main aim of this study was to investigate the relationship between the diet of ddd patients and the degree of disease as assessed from the modic classification. the most important feature of our study is that the extent or frequency of water, salt, fast food, eggs, milk, yogurt, cheese, whole wheat bread, white bread, butter, and margarine consumption was recorded. the consumption of cheese, yogurt, and white bread is significantly more than that of other food. although cheese and yogurt are rich in calcium, they are also abundant in fats., a weak negative correlation was found between fish and egg consumption and the modic classification. these food products contain essential amino acids along with omega-3 and omega-6 fatty acids. in most cases in the present study, disc degeneration was observed in 4th and 5th decades of life, a finding similar to those of other studies17, 18. the prevalence of modic type 1 changes in this study was 50%, which is comparable what is reported in some previous studies19, 20, and some studies have reported a prevalence ranging from 43 to 59%. mc did not seem to influence the clinical course of pain and function and were not prognostic factors for recovery. clinicians need to be reminded to treat patients with chronic lbp by using a biopsychosocial model of recovery21. the pathogenetic mechanisms underlying mc are not completely understood. a suggested cause is disc degeneration, which increases the shear force on the lumbar vertebral endplates, leading to microfractures. mc could be either edema - initialized via endplate microfractures or an inflammatory response caused by proinflammatory chemicals seeping from the nucleus pulposus through such microfractures22. the current study is based on the assumption that mc could be the singular cause of lbp in some patients. however, these changes are often detected along with other mri findings (i.e., degeneration, bulges, and herniation), which could also cause lbp. in the present study, patients were excluded if they had a competing somatic disease such as disc herniation with symptomatic root compression ; nevertheless, it is still very likely that the pain in some cases could have another pathological cause or a combination of causes. a study conducted in the general population showed that the clinical profile of individuals with both disc degeneration and mc was more pronounced than that of individuals with only disc degeneration, suggesting that mc are the crucial element connecting lbp and clinical findings. to our knowledge johansen. studied the relationship between vitamin d and mc and surprisingly found that mc were more common in individuals with normal levels of vitamin d than in those with low levels. findings suggest that the link between vitamin d and mc is perhaps related to inflammation, though further confirmatory studies are needed23. according to our theoretical framework, individuals with mc are expected to have low levels of vitamin d because of an increased susceptibility to inflammation and/or because microfractures occur in the vertebrae because of increased levels of parathyroid hormone24, 25. in conclusion, although the results of our study are promising, further studies with comparison groups and larger samples are needed before a cause - and - effect relationship can be proposed. | [purpose ] this study was conducted to examine the association between modic classification and the eating habits in patients with degenerative disc disease (ddd) and to determine the influence of nutrition on disease severity. [subjects and methods ] sixty patients with ddd visiting a low back pain outpatient clinic were enrolled. through face - to - face interviews, they completed questionnaires regarding their demographics, disease activity, smoking and alcohol use, concomitant diseases, disease duration, and nutritional status.exclusion criteria were age 65 years, other comorbidities, missing mri data, and inability to speak turkish. [results ] forty patients were finally included in the study. the frequency with which they consumed water, salt, fast food, eggs, milk, yogurt, cheese, whole wheat bread, white bread, butter, and margarine was recorded. a weak negative correlation was observed between the modic types and fish and egg consumption. [conclusion ] modic changes, which indicate the severity of ddd, seem to be correlated to patients dietary habits. however, studies with comparison groups and larger samples are needed to confirm our promising results before any cause - and - effect relationship can be proposed. |
hepatitis b virus (hbv) infection can cause infectious liver diseases, and about 400 million people throughout the world are chronically infected with this virus (1, 2), which has a high risk of progression to the development of liver failure, liver cirrhosis, and hepatocellular carcinoma (1). according to the similarity in the sequence of hbv, this virus is classified into eight genotypes and named using capital alphabet letters (a to h) (3). recently, two other genotypes (i and j) were proposed for hbv (4, 5). the replication of hbv involves a unique process, which is the production of covalently closed circular dna (cccdna) from the hbv genome through the repair of relaxed circular dna (rcdna) in the nuclei of hepatocytes. the cccdna acts as the template for viral rna transcription that serves as a viral pregenomic messenger rna (mrna), or as messenger rna coding for the envelope (s), polymerase, core, and x proteins (7). most of the antiviral agents have a profound effect on rcdna whereas low or no effect on cccdna (the episomal form of hbv) (8, 9). relapse of hbv replication after discontinuation of antiviral therapy is not uncommon and may be the result of the persistence of viral cccdna. it has been shown that monitoring of cccdna of hbv in liver biopsy specimens may be a valuable marker for virus eradication (10). although detection and quantification of cccdna in liver biopsy samples is the gold standard (11), it should be noted that liver biopsy is not always possible. therefore, the detection of cccdna should be performed on other samples, when a liver biopsy specimen is not available. recent studies have indicated that the hbv cccdna level is a marker of hbv replication in the hepatocytes of hbv - infected patients (12). hepatitis b surface antigen (hbsag) level is an important marker of infection with hbv and it can be used to diagnose, manage, and monitor patients. also, the level of hbsag in the plasma of hbv - infected patients indirectly reflects the number of infected hepatocytes (13). it has been shown that the plasma hbsag level was related to hbv dna replication (7, 11). the hepatitis b surface antigen level has been proposed as a marker of infected liver or the amount of hbv cccdna, which persists in hepatocytes (14). the present study aimed to detect hbv cccdna in plasma sample of patients with hbeag - negative chronic active hepatitis b and investigate the association between viral load and hbsag level with the presence of cccdna in plasma samples of the iranian treatment - naive patients with chronic hepatitis b infection. from april 2012 to may 2015, 106 patients with chronic hepatitis b infection referred to tehran hepatitis center and the hospitals that are related to iran university of medical sciences were enrolled in this cross - sectional study. the participants were eligible if they had a positive hbsag and a negative hbeag for at least 6 months and were naive to antiviral treatment. the patients were excluded if they had coinfection with hepatitis c virus (hcv) or human immunodeficiency virus (hiv). this study was approved by the local ethics committee of the gastrointestinal and liver disease research center (gildrc) of iran university of medical sciences, and all the patients provided written informed consent. a peripheral blood sample was collected from each patient in an edta - containing sterile tube and after separation of the plasma by centrifugation (5 minutes at 3000 rpm), the plasma samples were stored at -80c for further experiments. the level of hbsag was measured by the roche hbsag ii assay on the cobas e411 system (roche diagnostics gmbh, mannheim, germany), according to the manufacturer s recommendations. this assay is based on chemiluminescent immunoassay (clia), which uses microparticles coated with monoclonal anti - hbs for the quantitation of hbsag in plasma samples. this commercial clia has narrow dynamic range of quantification (0.05 - 130.00 iu / ml) ; therefore, samples that contain high levels of hbsag must be retested after being diluted. if a result is found below the lower range, the specimen has to be run undiluted. hepatitis b virus dna quantitation in the patients ' plasma samples (500 l) was performed using cobas taqman 48 (roche diagnostics, hacienda drive pleasanton, ca, usa) kit and high pure extraction was used according to the manufacturer s recommendation. this assay is a real - time polymerase chain reaction (rt - pcr) method based on dual - labeled hybridization probe targeting the hbv precore and core regions. the detection limit of the cobas taqman 48 is 6 > to 1 10 iu / ml. the viral dna was extracted from 200 l of plasma samples using qiaamp dna mini kit (qiagen gmbh, hilden, germany), according to the kit instruction. to increase the specificity of cccdna detection, plasmid - safe dnase (epicentre, madison, wi, usa) was used to eliminate rcdna, single - stranded dna (ssdna), and replicative double - stranded dna (dsdna) prior to rt - pcr, based on the kit instructions. the rt - pcr was performed for the detection of hbv cccdna in plasma sample using the rt - pcr instrument, rotor - gene q (qiagen, germany), as described previously (15). briefly, a pair of primers (the sense primer : 5-actcttggactcbcagcaatg-3 ; 1702 - 1722, and the antisense primer : 5-ctttatacgggtcaatgtcca-3 ; 1962 - 1942) that can specifically amplify a dna region from hbv cccdna (not viral genomic dna) were used (7, 15). for amplification of hbv cccdna, 5 pmol of the taqman probe (5-fam - ctttttcacctctgcctaatcatctcwtgttca - tamra-3 ; 1860 - 1892) and 10 pmol of each primer were used. the rt - pcr was performed using a 25-l mixture containing 5 l of the dna template and 12.5 l maxima probe qpcr master mix (fermentas gmbh, st. leon - rot). the cycling program of rt - pcr consisted of an initial denaturing step at 95c for 10 minutes, followed by 45 amplification cycles at 95c for 15 seconds and at 59c for 1 minute. extracted dna from liver biopsy specimens of three patients with hbv infection, who gave consent and underwent a liver biopsy for diagnostic purpose, were used as positive controls for detection of hbv cccdna. liver biopsy samples were divided into 2 parts : one used for histological diagnosis, and the other was submerged into rnalater (ambion inc., austin, tx) and stored at -20c. all statistical analyses were performed using spss software version 16.0 (spss 16.0 for windows ; spss inc., chicago, illinois, usa). to find the normality of the data, the kolmogorov - smirnov test was used. analysis of continuous variables was carried out using independent samples t - test or mann - whitney u test. the chi - square test or fisher exact test was performed to assess associations between categorical variables. from april 2012 to may 2015, 106 patients with chronic hepatitis b infection referred to tehran hepatitis center and the hospitals that are related to iran university of medical sciences were enrolled in this cross - sectional study. the participants were eligible if they had a positive hbsag and a negative hbeag for at least 6 months and were naive to antiviral treatment. the patients were excluded if they had coinfection with hepatitis c virus (hcv) or human immunodeficiency virus (hiv). this study was approved by the local ethics committee of the gastrointestinal and liver disease research center (gildrc) of iran university of medical sciences, and all the patients provided written informed consent. a peripheral blood sample was collected from each patient in an edta - containing sterile tube and after separation of the plasma by centrifugation (5 minutes at 3000 rpm), the plasma samples were stored at -80c for further experiments. the level of hbsag was measured by the roche hbsag ii assay on the cobas e411 system (roche diagnostics gmbh, mannheim, germany), according to the manufacturer s recommendations. this assay is based on chemiluminescent immunoassay (clia), which uses microparticles coated with monoclonal anti - hbs for the quantitation of hbsag in plasma samples. this commercial clia has narrow dynamic range of quantification (0.05 - 130.00 iu / ml) ; therefore, samples that contain high levels of hbsag must be retested after being diluted. if a result is found below the lower range, the specimen has to be run undiluted. hepatitis b virus dna quantitation in the patients ' plasma samples (500 l) was performed using cobas taqman 48 (roche diagnostics, hacienda drive pleasanton, ca, usa) kit and high pure extraction was used according to the manufacturer s recommendation. this assay is a real - time polymerase chain reaction (rt - pcr) method based on dual - labeled hybridization probe targeting the hbv precore and core regions. the detection limit of the cobas taqman 48 is 6 > to 1 10 iu / ml. the viral dna was extracted from 200 l of plasma samples using qiaamp dna mini kit (qiagen gmbh, hilden, germany), according to the kit instruction. to increase the specificity of cccdna detection, plasmid - safe dnase (epicentre, madison, wi, usa) was used to eliminate rcdna, single - stranded dna (ssdna), and replicative double - stranded dna (dsdna) prior to rt - pcr, based on the kit instructions. the rt - pcr was performed for the detection of hbv cccdna in plasma sample using the rt - pcr instrument, rotor - gene q (qiagen, germany), as described previously (15). briefly, a pair of primers (the sense primer : 5-actcttggactcbcagcaatg-3 ; 1702 - 1722, and the antisense primer : 5-ctttatacgggtcaatgtcca-3 ; 1962 - 1942) that can specifically amplify a dna region from hbv cccdna (not viral genomic dna) were used (7, 15). for amplification of hbv cccdna, 5 pmol of the taqman probe (5-fam - ctttttcacctctgcctaatcatctcwtgttca - tamra-3 ; 1860 - 1892) and 10 pmol of each primer were used. the rt - pcr was performed using a 25-l mixture containing 5 l of the dna template and 12.5 l maxima probe qpcr master mix (fermentas gmbh, st. the cycling program of rt - pcr consisted of an initial denaturing step at 95c for 10 minutes, followed by 45 amplification cycles at 95c for 15 seconds and at 59c for 1 minute. extracted dna from liver biopsy specimens of three patients with hbv infection, who gave consent and underwent a liver biopsy for diagnostic purpose, were used as positive controls for detection of hbv cccdna. liver biopsy samples were divided into 2 parts : one used for histological diagnosis, and the other was submerged into rnalater (ambion inc. all statistical analyses were performed using spss software version 16.0 (spss 16.0 for windows ; spss inc., chicago, illinois, usa). to find the normality of the data, the kolmogorov - smirnov test was used. analysis of continuous variables was carried out using independent samples t - test or mann - whitney u test. the chi - square test or fisher exact test was performed to assess associations between categorical variables. a total of 106 patients infected with chronic hbv were recruited in this cross - sectional study. the mean (sd) age of the study patients was 41.1 12.4 years (age range, 20 - 62 years). from a total of 106 participants, 67 cases (63.2%) were males. the demographic characteristics and laboratory parameters of the iranian hbeag - negative patients with chronic hepatitis b infection are listed in table 1, and the relationship between these parameters and the detection of cccdna are shown in table 2. the results of the kolmogorov - smirnov test showed that the distribution of hbv dna levels and hbsag titers was irregular and normal for age, aspartate aminotransferase (alt), alanine aminotransferase (ast), and alkaline phosphatase (alp). abbreviations : alp : alkaline phosphatase ; alt : alanine aminotransferase ; ast : aspartate aminotransferase ; cccdna : covalently closed circular dna ; hbv : hepatitis b virus ; hbsag : hepatitis b surface antigen. values are presented as no. median of hbv viral load : because did not accept normal distribution in hbv viral load. statistically significant. cccdna : covalently closed circular dna ; hbsag : hepatitis b surface antigen ; alt : alanine aminotransferase ; ast : aspartate aminotransferase ; alp : alkaline phosphatase. median of hbv viral load : because did not accept normal distribution in hbv viral load. there was no statistically significant difference between age, laboratory parameters (alt, ast, hbv viral load levels, and hbsag titer), and the sex of the patients ; however, there was a statistically significant difference between some laboratory parameters (alp level [p = 0.045 with student t - test ]), and the presence of cccdna in plasma sample (p = 0.039 with fisher exact test) and the gender of the precipitants (table 1). in the present study, there were 19 (17.9%) individuals with positive result for detection of cccdna in plasma samples. a significant difference was seen in the hbv viral load levels between individuals with and without cccdna in their plasma samples (p < 0.0001) and the presence of cccdna in the plasma sample and high baseline hbv viral load (60,000 iu / ml) (p < 0.0001 with the fisher exact test). there was a meaningful correlation between the hbsag titer and the presence of cccdna in the patients plasma samples (p < 0.0043). also, a statistically significant difference was observed between the presence of cccdna in patients plasma specimens and high baseline hbsag titer (3,000 iu / ml) (p = 0.041 with the fisher exact test). in the current study, there was a statistically significant difference between ast level and the presence of cccdna in patients plasma specimens (p = 0.001). the hbv dna levels and hbsag titers between individuals with and without cccdna in their plasma samples were shown in figure 1. hepatitis b virus infection manifests with a wide range of clinical symptoms ; therefore, there is a need for sensitive and reliable markers to improve the management of this infection. covalently closed circular dna of hbv is responsible for viral persistence in the liver (16). there is little information about hbv cccdna and its function in vivo (15). the hbv cccdna level is a useful indicator for assessing the status of hbv replication in the liver, but its clinical use is extremely restricted because of invasive procedures during sampling (17). there are some reports indicating that the reason of chronic hbv infection is cccdna (18). on the other hand, it has been demonstrated that cccdna is the major cause for hbv reactivation after cessation of anti - hbv treatment (15). in this study, a significant association was seen between the hbv dna level and the presence of cccdna in the patients plasma samples (p < 0.000), and also between the hbsag titer and the presence of cccdna (p = 0.0043) in the patients plasma samples. one important stage of the hbv life cycle is the production of hbv cccdna, which serves as a template for hbv replication and plays a crucial role in the persistence of this viral infection (19, 20). it is suggested that the quantitation of intrahepatic hbv cccdna is valuable and reliable for evaluating the efficiency of anti - hbv therapy (11, 21), and hbv recurrence after liver transplantation (22). it should be noted that this viral marker has not been applied in clinical practice widely (17). the main disadvantage of detection of the hbv cccdna before treatment response is the requirement for liver biopsies (16). however, it has been reported that the hbv cccdna can also be detected in the plasma of hbv - infected patients (12, 15). in the current study, a significant relationship was observed between the hbv viral load and the presence of cccdna in the patients plasma. this finding is consistent with that reported by a study from hong kong (16) in hbeag - negative patients. it has been reported that serum cccdna of hbv was detectable in 85.2% hbeag positive and 48.1% negative chronic hepatitis b participants (12), whereas the hbv cccdna was detected in plasma specimens in 19 (17.9%) out of the total 106 patients in the present study. there was no statistically significant difference between the sex of the study population and age and laboratory parameters (alt, ast, hbv viral load levels, and hbsag titer) in the plasma samples ; however, there was a statistically significant difference between the gender of the patients and some laboratory parameters (alp level, and the presence of cccdna). (23) showed that there was no relationship between the titer of hbv dna and ast (p = 0.054), while plasma hbv dna titer was correlated with alt (p = 0.042) in hbeag negative patients. 15) showed a positive correlation between the sera cccdna level and alt level in patients with hbv reactivation, but not in individuals sera without hbv reactivation. their results indicate that the occurrence of cccdna in the sera is an early signal of liver damage (15). hepatitis b surface antigen is a marker of hbv infection, and serological tests for its detection have guided its diagnosis (24). the present study showed a positive correlation between plasma quantitative hbsag and the presence of cccdna in hbeag - negative chronic hepatitis b participants (p = 0.041). this result is consistent with another study that was conducted among hbeag - positive chronic hepatitis b individuals (16), but is different from the findings reported by studies from greece (25) and hong kong (26). it was shown that plasma hbsag is a reflection of the amount of cccdna when the level of cccdna is high. however, the quantitation of hbv cccdna is decreased by the immune clearance, and the percentage of cccdna to hbsag production with integrated hbv dna, in the genome of hepatocytes, may be lower than that in individuals with hbeag - positive chronic hepatitis b patients (27). mechanisms that regulate the production of hbsag from the integrated hbv dna are not entirely clear. thus, it is obvious that the production of hbsag has no relationship with the quantity of the hbv template (cccdna) and the replicative activity of the hbv in hbeag - negative chronic hepatitis b individuals. therefore, it seems that the quantity of the plasma hbsag in participants with hbeag - negative is not a reliable indicator of the hbv replicative efficiency (16). in the current study, the hbv dna, and hbsag level detected in one session and may be repeating these tests will be more useful ; however, we did not repeat them due to finance limitation. in conclusion, the findings of the present study confirmed the concept that the plasma hbv viral load level and the quantitation of hbsag have association with the presence of hbv cccdna in the sera specimen. therefore, it seems that detection of cccdna in the plasma specimens is reliable and informative marker. | background : covalently closed circular dna (cccdna) of hepatitis b virus (hbv) is a marker of hbv replication in the liver of patients infected with hbv.objectives:this study aimed to investigate the association between the presence of cccdna in the plasma samples of iranian treatment - naive patients with chronic hepatitis b infection and hbv viral load and hbsag levels.patients and methods : from april 2012 to may 2015, 106 treatment - naive patients with chronic hepatitis b infection were enrolled in this cross - sectional study. the hbsag titer was measured by the roche hbsag ii assay on the cobas e411 system, and hbv dna quantitation was performed using the cobas taqman 48 kit. real - time polymerase chain reaction was performed for the detection of hbv cccdna.results:the mean (sd) age of the patients was 41.1 12.4 years (range, 20 - 62 years). from a total of 106 study participants, 67 (63.2%) were males. the hbv cccdna was detected in plasma specimens in 19 (17.9%) out of the total 106 patients, and a significant relationship was found between the presence of cccdna in plasma sample of males (23.9%) and females (7.7%) (p = 0.039). also, a significant correlation was found between the presence of cccdna in plasma sample of the patients and hbv viral load level (p < 0.0001) and hbsag titer (p = 0.0043).conclusions : this study showed that cccdna can be detected in the plasma specimen of 17.9% of iranian treatment - naive patients with chronic hepatitis b infection. therefore, designing prospective studies focusing on the detection of cccdna in these patients would provide more information. |
early childhood caries is a complex disease involving the maxillary primary incisors within a month after eruption and spreads rapidly to involve other primary teeth.1 it is a serious socio - behavioral and dental problem that afflicts infants and toddlers worldwide.2 the definitions of ecc in the published literature vary, making comparisons among studies difficult.1,3 for example, some of the definitions have included 1 or more incisors with decay,4 2 or more incisors with decay,5 and even 3 or 4 maxillary incisors with decay.6 therefore, in 2003, the american academy of pediatric dentistry (aapd) defined ecc as the presence of one or more decayed (noncavitated or cavitated), missing (due to caries), or filled tooth surfaces in any primary tooth in a child up to 71 months of age or younger. the academy also specifies that, in children younger than 3 years of age, any sign of smooth surface caries is indicative of severe early childhood caries (s - ecc). from ages 3 through 5, 1 or more cavitated, missing (due to caries) or filled smooth surfaces in primary maxillary anterior teeth or decayed, missing, or filled score of 4 (age 3), or 5 (age 4), or 6 (age 5) surfaces constitutes s - ecc.7 ecc is a multi - factorial disease.2 the factors include a susceptible host, fermentable carbohydrate diet, presence of dental plaque, high number of cariogenic micro - organisms such as mutans streptococci, lactobacillus and time.8 it is also thought that there may be unique risk factors for caries in infants and young children. ecc has been associated with demographic characteristics, oral hygiene practices, parental attitudes, educational status of mother, socio - economic status, temperament of the child, mouth breathing habit, siblings, pacifiers dipped in honey, children with chronic illness or special health care needs and other feeding habits, maternal nutrition, psychosocial issues, frequent use of medications and parenting practice.915 even ethnicity has been identified as a factor, as the prevalence of tooth decay in children remains high among ethnic minorities and various north american aboriginal population.16,17 ecc is a serious public health problem in very young children, and although it is not life threatening, if left untreated it may lead to pain, bacteremia, compromised chewing ability, and toxic overdose of analgesics (acetaminophen) administered during the early stages, followed by malocclusion in permanent dentition, phonetic problems, suboptimal health, lower self - esteem, and failure to thrive.18,19 also, it has been demonstrated that dental caries can gradually reduce a child s ability to gain weight, which may get reversed after complete oral rehabilitation.20 ecc has also been described as a social, political, behavioral, medical, psychological, economical and dental problem.21 it is considered as a social and political problem because it is endemic in disadvantaged children, regardless of race, ethnicity, or culture. these disadvantaged children suffer from diseases ; hunger, lack of education, family support, and parental employment.22 ecc can be a medical problem, because infants with ecc continue to grow at a slower pace compared to caries - free infants.23 children born after maternal complications during pregnancy or who have had traumatic births are at risk of developing ecc.24 moreover, children with severe ecc often require costly treatment with hospitalization under sedation or general anesthesia.25 preventive methods are not applied to many vulnerable children, who later develop serious dental problems. the prevalence of ecc is estimated to range from 1% to 12% in preschoolers of developed countries26 and from 50% to 80% in high - risk groups,15,17,21,24 including immigrants and aboriginal canadians. the prevalence of ecc and its association with feeding habits and oral hygiene practices in preschool children of kerala state which is a neighboring state of karnataka in southern india, was evaluated by jose and king. they also concluded that the preschool children in kerala, who were at high risk from developing caries lesions were those children with poor oral hygiene, who consumed snacks, who were given snacks as reward, and those who belonged to lower socio - economic status.2 however, because most studies on ecc have been conducted among specific ethnic, immigrant, and lower socio - economic communities, extrapolation of current risk assessment models to the general population is problematic.21 bangalore, being a metropolitan city within karnataka state, has inhabitants from different socioeconomic and cultural backgrounds. the city has leveraged its prowess in the information technology (it) industry to emerge as the leading it - bpo (information technology - business process outsourcing) destination in india. despite the seriousness of problems due to ecc, there has been a paucity of prevalence studies in bangalore, which may be due to the difficulty of access to this age group. the most tragic fact about ecc may be that measures, which could render the condition entirely preventable, have not been implemented due to the multi - factorial origin of this disease. hence, knowledge on prevalence and associated factors of ecc is necessary to develop targeted interventions for prevention of subsequent tooth decay, and to decrease the number of children that require emergency treatment. so, the aim of this study was as follows : to determine the prevalence of ecc in children aged between 8 and 48 months in urban bangalore, india.to determine possible associations of ecc with factors such as chronological age, birth weight, socio - economic status, educational status of the mother, feeding habits, and oral hygiene practices. to determine the prevalence of ecc in children aged between 8 and 48 months in urban bangalore, india. to determine possible associations of ecc with factors such as chronological age, birth weight, socio - economic status, educational status of the mother, feeding habits, and oral hygiene practices. this cross - sectional survey was conducted in bangalore city, which is the capital of karnataka state in india. with an estimated population of 5.8 million in 2001, bangalore is the 3 most populous city in india and the 28th most populous city in the world. all inhabitants of the city use the local tap which has a low - fluoride level of 0.7 ppm for domestic purposes. this survey consisted of a random sample of 1500 children, both male and female aged between 8 and 48 months, attending playschools and private hospitals in different parts of urban bangalore city, karnataka. in the present study, private hospitals were selected for the collection of data for children less than 2 years, and play homes and day care centers were selected for older children. the age group selected for this study was 8 48 months because by 8 months of age at least 2 central incisors erupt and it has also been established that mutans streptococci can be found in the mouth from as early as 6 months of age, even prior to tooth eruption.27 all children were included in the study after obtaining informed consent from the mother. not all individual data sets were complete and those with missing data sets were excluded from the modeling step. a pilot survey was conducted among 20 children, in order to pre - test the method of examination, data collection forms, and to train and familiarize the examiner with the survey. the preliminary investigation was conducted in the research institute among the group of children of the same age, which was considered as an analogous population. full agreement was reached on this result between the study examiner (pp) and two experienced professor (ss, ps) in the department of pediatric dentistry. clinical examination was performed by a sole examiner (pp) using disposable mouth mirrors for indirect vision of lingual areas of the teeth, and torch light. mouth mirrors were used for indirect vision of lingual areas of the teeth. the community periodontal index (cpi) probe was used to confirm visual evidence of caries on the occlusal, buccal and lingual surfaces. during the examination, the older children were seated on a chair and infant were examined with assistance of their mothers, by means of the knee - to - knee technique. a prior schedule for data collection was prepared and an average of 4050 children was examined per day. a dental surgeon was sitting close to the examiner so that the codes could be easily heard and recorded correctly. radiographs were not taken due to practical reasons. the who criteria (1997) for carious lesions were used to diagnose caries.28 the mothers of children attending play schools and nursing homes were informed of the nature of the investigation directly or through the teachers. they completed a dental health questionnaire which included a series of questions regarding the child s chronological age, birth weight, socio - economic status, educational status of mother, feeding habits and oral hygiene practices. the clinical and questionnaire data were analyzed using the statistical package for social sciences, raleigh, north carolina, usa (spss) version 12. the percentage of caries - affected and caries - free children within each variable category was compared using cross - tabulation procedure and the relative proportions within each group were analyzed using the chi - square test of association. in the above tests, p value of <.05 was considered as statistically significant. the mothers of children attending play schools and nursing homes were informed of the nature of the investigation directly or through the teachers. they completed a dental health questionnaire which included a series of questions regarding the child s chronological age, birth weight, socio - economic status, educational status of mother, feeding habits and oral hygiene practices. the clinical and questionnaire data were analyzed using the statistical package for social sciences, raleigh, north carolina, usa (spss) version 12. the percentage of caries - affected and caries - free children within each variable category was compared using cross - tabulation procedure and the relative proportions within each group were analyzed using the chi - square test of association. in the above tests, p value of <.05 was considered as statistically significant. the overall caries prevalence of 1500 children of urban bangalore whose ages ranged from 848-months was 27.5% (n = 413) with a mean deft of 0.854. the caries prevalence in different age groups showed an increase with age (table 1). in the present study, the number of children affected with ecc in relation to birth weight of children (table 2), socio - economic status (table 3), and mother s education level (table 4) was evaluated. low birth weight as defined by the who is weight at birth of less than 2.5 kg (5.5 lb). of the 1500 children studied, 499 (33.2%) children had low birth weight (< 2.5 kgs), and 27% of these children were affected with ecc. children with family annual income of less than rs 50,000 (us $ 1000) were included in group 1 and group 4 included children from higher family annual income of more than rs 200,000 (us $ 4000). the occurrence of ecc was found to be higher in children of low socio - economic status and uneducated mothers (table 3, 4). the effect of different feeding habits such as manner of feeding, on - demand breast feeding, bottle feeding at night, nutritional supplements given, in - between meal snacking, and use of pacifiers in the occurrence of ecc were analyzed (table 5). the 1500 children included in this study cleaned their teeth on their own or were assisted by parents. the methods of cleaning, frequency of cleaning, cleaning aids used, dentifrice used and initiation of tooth brushing were analyzed as shown in table 6. the oral health of preschoolers is an overlooked aspect of childhood health and well - being, especially in cases of ecc. these children constitute a population vulnerable to caries because of their dependence and inability to communicate with their parents. in southern india, dental caries prevalence in children below 6 years of age was comparatively low as reported by gupta in relation to other parts of the country. the mean deft found in karnataka (bangalore), andhra pradesh and kerala were 0.6, 1.63, and 2.1 respectively.30 another study was conducted to compare the prevalence and pattern of caries in 4 - 5-year - old children of urban bangalore and non - urban chickaballapur within karnataka state, india. the results showed caries prevalence of 66.3% with a mean deft of 2.9 in bangalore city whereas in chickballapur, the prevalence was 58.4% and the mean deft was 2.3.31 in the present study, the prevalence of ecc in urban bangalore within karnataka state was 27.5% with a mean deft of 0.854. this shows a decreased trend of caries prevalence in urban bangalore from 1987 to 2005. this level is higher than formerly reported in several european countries, but lower than the levels among several native north - american communities.32,33,34 the low prevalence observed in this study could be due to the increased use of fluoridated toothpastes available in the market, increased oral hygiene awareness as there are many oral health education programs organized by well - established dental institutions of bangalore. another possible reason could be to the rapid growth and development of bangalore city not only area with respect to the area but also in terms of employment opportunities and education status. this could lead to increased awareness among the general public especially mothers, primary care health providers and secondary caretakers towards the dental health of their children. determining the prevalence of caries in pre - school children is a difficult process as the children of this age group are not easily accessible, uncooperative, a detailed examination of the oral cavity can not be easily accomplished and no separate criteria has been developed for evaluating the extent and degree of caries in children below 3 years because of varied number of erupted teeth.35 similar to the results of the previous studies, our present study also showed that caries prevalence increased significantly with age.2,36,37 the children of group 7 (4448 months) demonstrated higher caries prevalence (37.2%) than the younger age groups. the finding that caries experience and the number of confirmed cases of ecc increased with increasing age was as expected, because there is an increasing number of erupted primary teeth which become exposed to the oral environment and cariogenic challenge. also, as children grow older there is a change in the dietary habits and hygiene practices.21 low birth weight and preterm births, predisposes to high levels of streptococcal colonization, in addition to favoring the development of enamel hypoplasia and salivary disorders.38 in the present study, 27% of low birth weight children were affected with ecc, which was not statistically significant. this is in accordance with the findings of another study by shulman.39 however, some other similar studies have found a significant association between low birth weight and ecc.40,41 the present study had a disproportionate sample size between low birth weight and normal birth weight children. therefore, further research is required to emphasize the correlation between low birth weight and caries prevalence in preschool children. the issues to be explored include the link between developmental enamel defects and caries, and the role of birth complications in the development of caries. low income affects the degree of education, health, values, life styles and access to health care information, thereby increasing susceptibility to caries. a statistically significant correlation was found between caries prevalence and low socio - economic status, measured in terms of the income. the present study showed that higher the income, lower the caries prevalence, which is in accordance with the observations of some previous studies.2,21,42 low socio - economic status may increase ecc risk in several ways. individuals from lower economic strata experience financial, social and material disadvantages that compromise their ability to care for themselves, obtain professional oral health care services and live in a healthy environment, all of which lead to reduced resistance to oral and other diseases.2 it has also been reported that children from low socioeconomic background have more fatalistic health beliefs, lower perceived need for and utilization of dental health care services and poor eating habits.21,43 as mothers are the primary caregivers of a child, low maternal education is related to higher caries prevalence in their children.2,21,44 in the study sample of 1500, 38% children whose mothers had no schooling were affected with caries. this was statistically significant when compared to those children whose mothers had received higher education. the results of this study are similar to other studies, which show a strong association between mother s education and presence of caries in their children. this may be attributed to the lack of information and education about the oral health care for children in uneducated mothers.2,21,37,44 however, recent research has shown a lack of any association between ecc and education level of the mother.45 the improper feeding patterns like bottle feeding beyond one year, prolonged or on - demand breast feeding and children put to bed with nursing bottle are responsible for an increase in the exposure of primary teeth to fermentable carbohydrates. this increase is likely to promote both an early colonization by oral mutans streptococci and an increase in the number of these microorganisms in the dental plaque and saliva, which increases the risk of developing caries.46 these bacteria are usually transmitted to the child by their mothers within the first 2 years of life which is known as the window of infectivity period in preschool children. the earlier these microorganisms are transmitted, the more severe will be the ecc in primary dentition.18 in the current study, 30% children were exclusively breast fed and 12.7% children exclusively bottle fed. this is in accordance with a study conducted in brazil by dini which stated that children who were exclusively breast fed had slightly higher caries prevalence.47 a recent study in the united states has indicated that infant breastfeeding and its duration, whether exclusive feeding or not, is not associated with any increased risk for ecc or s - ecc.48 a systematic review of the relationship between breastfeeding and ecc has thereby suggested that a definitive conclusion can not be drawn due to the inconsistent methodological approach in the research which makes it difficult to compare findings.49 this shows that the influence of infant feeding per se on ecc remains a complex and somewhat controversial issue. therefore, exclusive breastfeeding should be encouraged up to the sixth month, and maintained at least up to the second year, with flexibility in schedules or shifts, and complemented appropriately with weaning food.50 on - demand breast feeding was practiced by 554 mothers, of whom 164 (29.6%) children had caries. of the 755 children whose mothers did not practice on - demand breast feeding 202 (26.71%) children had caries. thus, there was significantly high caries prevalence in children whose mothers practiced on - demand breast feeding. ad libitum breastfeeding or breastfeeding for longer durations decreases plaque ph, thereby increasing the risk of ecc. therefore mothers should be informed of how they can reduce the probability of ecc by discouraging breast - feeding on demand, transitioning to use of a regular cup at 12 months of age, and cleaning the child s mouth regularly once the first primary tooth has erupted as suggested by american academy of pediatric dentistry guidelines.7 in the present study, of the 412 children who were bottle - fed at night, a significantly higher percentage (40.7%) of children had caries in comparison with the other group. this conclusion agrees with other studies that examined bottle feeding in detail and reported that the duration of bottle - feeding, particularly at night, is the most important determinant for ecc development rather than bottle - feeding itself.50,51,52 the reasons for this include decreased salivary flow and swallow reflex during sleep which allows liquid carbohydrate to remain in the mouth and pool around the teeth. this decreased rate at which carbohydrates are cleared from the oral cavity is a determinant in caries initiation. the anterior placement of the tongue protects the mandibular anterior teeth from the decay causing carbohydrate solutions.53 in the present study, 76.8% children consumed snacks between meals. a statistically significant correlation was found between caries prevalence and consumption of in - between meal snacks. children having in - between meal snacking habit had higher caries prevalence (29.2%). milk consumption has decreased whereas the consumption of soft drinks, juices, non - citric beverages, and carbohydrates has increased. these habits have been correlated with a higher prevalence of ecc.54,55 the sugar intake in the early stages of life may accelerate the accumulation of mutans streptococci in the infant s mouth which is a risk factor for caries. the mutans streptococci colonization is directly proportional with increased snacking habits leading to increase in caries prevalence among preschool children.56 the present study supports the earlier studies which state that frequent consumption of foods, snacks and drinks in - between meals increases the risk for caries.21,36 hence, nutritional recommendations of limiting the snacking time among children and encouraging regular meals is essential. nutritional supplements and dietary factors it has also been suggested that pre - natal and peri - natal malnutrition are often the causes of enamel hypoplasia, reduced salivary secretion and low buffering capacity.57,58 in addition, poor oral hygiene, insufficient exposure to fluoride, and general psychosocial stress is common in underprivileged children. these variables may impede the natural resistances to the disease cycle of bacterial invasion, demineralization and dental caries.1 it has been shown previously that nutritional supplementation is one of the effective strategies for the prevention and control of dental caries.58,59 in the present study, nutritional supplementation included vitamins, iron, calcium and fluorides. those children who consumed these supplements had significantly lower caries experience. the results of this study also suggest that, while evaluating prolonged nursing habits with bottle or breast - feeding as a contributory factor of ecc, other factors such as intake of cariogenic food and oral hygiene habits should also be considered. a study has shown that dipping a pacifier in sugar is associated with early colonization of mutans streptococci in pre - dentate infants, but a systematic review conducted by perssini did not show any consistent correlation between the use of pacifiers and the development of ecc regardless of dipping the pacifier in sugar or not dipping.27,60 the present study also did not show any significant relation between the use of pacifiers and ecc. many authors have shown that regular tooth brushing may counteract the effects of a cariogenic diet.15,33,61,62,63 therefore, variables such as tooth brushing under supervision, frequency of brushing, type of dentifrice, and tooth cleaning aids were analyzed in this study. previous studies and experience have shown that preschool children do not understand or have the manual dexterity to maintain good oral hygiene.64,36 hence, parental assistance and guidance is essential to reduce the risk of developing caries. tooth brushing by parents or caregivers has the potential of removing dental plaque more effectively, optimally saturating the oral environment with fluoride, thereby decreasing the risk of caries among their children.47 in the present study, prevalence of caries in those children who practiced tooth brushing by themselves was significantly higher than in those children who brushed under parental supervision. the result of this study is in accordance with another recent study which showed that children brushing without assistance by the mother were at high risk of developing ecc.36 in contrast, it has been observed in another similar study that adult supervision of children s tooth brushing was not associated with ecc.21 however, the importance of prolonged participation of parents in tooth cleaning of these pre - school children should be emphasized. when compared to those children who brushed or cleaned their teeth more than once a day, these children had significantly higher caries prevalence. this suggests that tooth brushing with a frequency of at least twice a day under parental guidance using a fluoridated toothpaste may spare the teeth from developing caries by removing dental plaque more effectively, there by optimally saturating the oral environment with fluoride, and decreasing the risk of caries among their children.65 parental assistance and daily frequency of tooth brushing have been shown to be major determinants of declining ecc experience in european countries.61,66 fluoride is presently the corner stone of dentifrice anti - caries therapy. tooth - brushing with fluoridated dentifrice has played a major role in the decline of caries worldwide.65 the results of the present study also showed that those children who used fluoridated dentifrice had significantly lower caries prevalence. the impact of the use of fluoridated dentifrice and the total fluoride intake in preschool children is a controversial issue. a concentration of 1450 ppm fluoride in toothpastes was shown to reduce caries in children but this increased the chances of fluorosis.67,68 the concentration of fluoride in toothpastes was not assessed as it varies from country to country depending on the government regulations. in india, most of the commercially available toothpastes are either nonfluoridated ayurvedic toothpaste (anchor, meswa) or fluoridated (colgate, pepsodent) with a fluoride content of more than 1000ppm. most of the parents were not familiar with the difference in toothpaste content and many did not know the effect of fluoride content in toothpastes on their child s teeth. in most cases, the parents used the same toothpaste, which they were using, for their children with perhaps lesser amount of paste. this may be due to lack of awareness among parents about the benefits and risks associated with use of fluoridated toothpastes in preschool children. therefore, it is time that parents are instructed to delay the use of fluoridated dentifrice until the child is older than 24 months and use non - fluoridated toothpaste in these young children. in older children above 2 years, a pea - sized amount of fluoridated toothpaste should be used. instructions should be given to the parents so that they make sure their children rinse and expectorate thoroughly after tooth brushing to avoid excessive ingestion of fluoride from toothpaste.69,70 at present tooth - brushing and other mechanical cleaning procedures are considered to be the most reliable means of maintaining oral hygiene. brushes are more effective in cleaning proximal, marginal and occlusal areas of the teeth. the soft brush is preferable for most uses in young children because of the likelihood of gingival tissue trauma and increased inter - proximal cleaning ability.63 the present study showed that 1132 (89.4%) of the children used baby brush to clean their teeth. these children had significantly lower caries prevalence, when compared to those children who cleaned their teeth using their finger. in a similar study, it was found that 60% of the children who used tooth brush to clean their teeth were free of caries.2 therefore, dental health behaviors such as use of fluoridated dentifrice with baby toothbrush significantly reduced ecc experience, possibly reflecting desirable parental dental knowledge and education. however, these preferred parental behaviors may be subject to a recall and response bias. some of the limitations in the present study were deriving detailed and accurate information from the parents regarding the feeding practices, contents of the feeding bottle, composition of snacks consumed and weaning. also, the information obtained from mothers regarding child rearing practices may not be totally reliable. data obtained may serve as a base line for planning and evaluation of community development and oral health promotion programs. although future community initiatives have the potential to increase community knowledge of ecc, the ultimate challenge will be sustaining long term behavioral change among parents. the burden of dental decay in this population reveals the need for effective preventive methods. thus, both medical providers and those working with pregnant women and the very young must play an integral role in the prevention and early detection of ecc. it is important for pediatricians, family physicians and other health service providers encountering expectant mothers and very young children to be cognizant of ecc and its ramifications, as they represent the first line of defense. ultimately community - based solutions should be explored and can be strengthened if used in conjunction with the existing and emerging strategies for promoting early childhood oral health and preventing dental decay. these strategies should include suppression of maternal reservoirs of mutans streptococci, health education of mothers to prevent vertical transmission of mutans streptococci, promoting the first dental visit by 12 months, providing anticipatory guidance, motivational interviewing (mi), using chemotherapeutics (e.g., fluoride varnish, silver diamine fluoride, povidone iodine[butadiene ], chlorhexidine), water fluoridation, promote use of fluoridated dentifrice, dietary counseling, self - examination for early signs of ecc, and other previously tested methods which are potentially simple, cheap, effective, and appropriately practical for this community.7,18,21,71 although future community initiatives have the potential to increase community knowledge of ecc, the ultimate challenge will be sustaining long - term behavioral change among parents, caregivers and the community at large leading to appropriate parenting practices. the reason for increased ecc risk could be cultural differences in child rearing, infant feeding practices, and oral health beliefs. this survey has identified certain risk factors for the presence of ecc in preschool children within an indian city. general dentists can use this information to target 15-year - old children and use the above preventive methods. public funded oral health programs need to target children from lower socio - economic status and develop effective strategies to promote breast feeding and discourage inappropriate bottle feeding. 21 mi of mothers is a recent approach which is shown to have a greater positive effect on preschool children s dental health than the traditional health education approach. this approach enhanced the preventive behavior of mothers of young children at high risk of developing caries.71 another important aspect of ecc which has not been explored is the role of care - takers of children attending day - care centers in cities. recently, mani have suggested that the care - takers of children attending day - care centers have an important role in promoting oral health among preschool children. during the recent years, there has been an increase in the number of working mothers in metropolitan cities ; therefore most of the children spend a considerable amount of time in day - care centers. these parents have very limited control over their child s activities in such day - care centers. this indicates that further studies on ecc should focus more attention on family dynamics, environmental factors, behavior and society in which the children grow up. determining the prevalence of caries in pre - school children is a difficult process as the children of this age group are not easily accessible, uncooperative, a detailed examination of the oral cavity can not be easily accomplished and no separate criteria has been developed for evaluating the extent and degree of caries in children below 3 years because of varied number of erupted teeth.35 similar to the results of the previous studies, our present study also showed that caries prevalence increased significantly with age.2,36,37 the children of group 7 (4448 months) demonstrated higher caries prevalence (37.2%) than the younger age groups. the finding that caries experience and the number of confirmed cases of ecc increased with increasing age was as expected, because there is an increasing number of erupted primary teeth which become exposed to the oral environment and cariogenic challenge. also, as children grow older there is a change in the dietary habits and hygiene practices.21 low birth weight and preterm births, predisposes to high levels of streptococcal colonization, in addition to favoring the development of enamel hypoplasia and salivary disorders.38 in the present study, 27% of low birth weight children were affected with ecc, which was not statistically significant. this is in accordance with the findings of another study by shulman.39 however, some other similar studies have found a significant association between low birth weight and ecc.40,41 the present study had a disproportionate sample size between low birth weight and normal birth weight children. therefore, further research is required to emphasize the correlation between low birth weight and caries prevalence in preschool children. the issues to be explored include the link between developmental enamel defects and caries, and the role of birth complications in the development of caries. social class may affect caries risk in many ways. low income affects the degree of education, health, values, life styles and access to health care information, thereby increasing susceptibility to caries. a statistically significant correlation was found between caries prevalence and low socio - economic status, measured in terms of the income. the present study showed that higher the income, lower the caries prevalence, which is in accordance with the observations of some previous studies.2,21,42 low socio - economic status may increase ecc risk in several ways. individuals from lower economic strata experience financial, social and material disadvantages that compromise their ability to care for themselves, obtain professional oral health care services and live in a healthy environment, all of which lead to reduced resistance to oral and other diseases.2 it has also been reported that children from low socioeconomic background have more fatalistic health beliefs, lower perceived need for and utilization of dental health care services and poor eating habits.21,43 as mothers are the primary caregivers of a child, low maternal education is related to higher caries prevalence in their children.2,21,44 in the study sample of 1500, 38% children whose mothers had no schooling were affected with caries. this was statistically significant when compared to those children whose mothers had received higher education. the results of this study are similar to other studies, which show a strong association between mother s education and presence of caries in their children. this may be attributed to the lack of information and education about the oral health care for children in uneducated mothers.2,21,37,44 however, recent research has shown a lack of any association between ecc and education level of the mother.45 the improper feeding patterns like bottle feeding beyond one year, prolonged or on - demand breast feeding and children put to bed with nursing bottle are responsible for an increase in the exposure of primary teeth to fermentable carbohydrates. this increase is likely to promote both an early colonization by oral mutans streptococci and an increase in the number of these microorganisms in the dental plaque and saliva, which increases the risk of developing caries.46 these bacteria are usually transmitted to the child by their mothers within the first 2 years of life which is known as the window of infectivity period in preschool children. the earlier these microorganisms are transmitted, the more severe will be the ecc in primary dentition.18 in the current study, 30% children were exclusively breast fed and 12.7% children exclusively bottle fed. this is in accordance with a study conducted in brazil by dini which stated that children who were exclusively breast fed had slightly higher caries prevalence.47 a recent study in the united states has indicated that infant breastfeeding and its duration, whether exclusive feeding or not, is not associated with any increased risk for ecc or s - ecc.48 a systematic review of the relationship between breastfeeding and ecc has thereby suggested that a definitive conclusion can not be drawn due to the inconsistent methodological approach in the research which makes it difficult to compare findings.49 this shows that the influence of infant feeding per se on ecc remains a complex and somewhat controversial issue. therefore, exclusive breastfeeding should be encouraged up to the sixth month, and maintained at least up to the second year, with flexibility in schedules or shifts, and complemented appropriately with weaning food.50 on - demand breast feeding was practiced by 554 mothers, of whom 164 (29.6%) children had caries. of the 755 children whose mothers did not practice on - demand breast feeding 202 (26.71%) children had caries. thus, there was significantly high caries prevalence in children whose mothers practiced on - demand breast feeding. ad libitum breastfeeding or breastfeeding for longer durations decreases plaque ph, thereby increasing the risk of ecc. therefore mothers should be informed of how they can reduce the probability of ecc by discouraging breast - feeding on demand, transitioning to use of a regular cup at 12 months of age, and cleaning the child s mouth regularly once the first primary tooth has erupted as suggested by american academy of pediatric dentistry guidelines.7 in the present study, of the 412 children who were bottle - fed at night, a significantly higher percentage (40.7%) of children had caries in comparison with the other group. this conclusion agrees with other studies that examined bottle feeding in detail and reported that the duration of bottle - feeding, particularly at night, is the most important determinant for ecc development rather than bottle - feeding itself.50,51,52 the reasons for this include decreased salivary flow and swallow reflex during sleep which allows liquid carbohydrate to remain in the mouth and pool around the teeth. this decreased rate at which carbohydrates are cleared from the oral cavity is a determinant in caries initiation. the anterior placement of the tongue protects the mandibular anterior teeth from the decay causing carbohydrate solutions.53 in the present study, 76.8% children consumed snacks between meals. a statistically significant correlation was found between caries prevalence and consumption of in - between meal snacks. children having in - between meal snacking habit had higher caries prevalence (29.2%). milk consumption has decreased whereas the consumption of soft drinks, juices, non - citric beverages, and carbohydrates has increased. these habits have been correlated with a higher prevalence of ecc.54,55 the sugar intake in the early stages of life may accelerate the accumulation of mutans streptococci in the infant s mouth which is a risk factor for caries. the mutans streptococci colonization is directly proportional with increased snacking habits leading to increase in caries prevalence among preschool children.56 the present study supports the earlier studies which state that frequent consumption of foods, snacks and drinks in - between meals increases the risk for caries.21,36 hence, nutritional recommendations of limiting the snacking time among children and encouraging regular meals is essential. nutritional supplements and dietary factors have a profound and lasting effect on developing and developed dentition. it has also been suggested that pre - natal and peri - natal malnutrition are often the causes of enamel hypoplasia, reduced salivary secretion and low buffering capacity.57,58 in addition, poor oral hygiene, insufficient exposure to fluoride, and general psychosocial stress is common in underprivileged children. these variables may impede the natural resistances to the disease cycle of bacterial invasion, demineralization and dental caries.1 it has been shown previously that nutritional supplementation is one of the effective strategies for the prevention and control of dental caries.58,59 in the present study, nutritional supplementation included vitamins, iron, calcium and fluorides. the results of this study also suggest that, while evaluating prolonged nursing habits with bottle or breast - feeding as a contributory factor of ecc, other factors such as intake of cariogenic food and oral hygiene habits should also be considered. a study has shown that dipping a pacifier in sugar is associated with early colonization of mutans streptococci in pre - dentate infants, but a systematic review conducted by perssini did not show any consistent correlation between the use of pacifiers and the development of ecc regardless of dipping the pacifier in sugar or not dipping.27,60 the present study also did not show any significant relation between the use of pacifiers and ecc. many authors have shown that regular tooth brushing may counteract the effects of a cariogenic diet.15,33,61,62,63 therefore, variables such as tooth brushing under supervision, frequency of brushing, type of dentifrice, and tooth cleaning aids were analyzed in this study. previous studies and experience have shown that preschool children do not understand or have the manual dexterity to maintain good oral hygiene.64,36 hence, parental assistance and guidance is essential to reduce the risk of developing caries. tooth brushing by parents or caregivers has the potential of removing dental plaque more effectively, optimally saturating the oral environment with fluoride, thereby decreasing the risk of caries among their children.47 in the present study, prevalence of caries in those children who practiced tooth brushing by themselves was significantly higher than in those children who brushed under parental supervision. the result of this study is in accordance with another recent study which showed that children brushing without assistance by the mother were at high risk of developing ecc.36 in contrast, it has been observed in another similar study that adult supervision of children s tooth brushing was not associated with ecc.21 however, the importance of prolonged participation of parents in tooth cleaning of these pre - school children should be emphasized. most children in the present study brushed or cleaned their teeth once a day. when compared to those children who brushed or cleaned their teeth more than once a day, these children had significantly higher caries prevalence. this suggests that tooth brushing with a frequency of at least twice a day under parental guidance using a fluoridated toothpaste may spare the teeth from developing caries by removing dental plaque more effectively, there by optimally saturating the oral environment with fluoride, and decreasing the risk of caries among their children.65 parental assistance and daily frequency of tooth brushing have been shown to be major determinants of declining ecc experience in european countries.61,66 fluoride is presently the corner stone of dentifrice anti - caries therapy. tooth - brushing with fluoridated dentifrice has played a major role in the decline of caries worldwide.65 the results of the present study also showed that those children who used fluoridated dentifrice had significantly lower caries prevalence. the impact of the use of fluoridated dentifrice and the total fluoride intake in preschool children is a controversial issue. a concentration of 1450 ppm fluoride in toothpastes was shown to reduce caries in children but this increased the chances of fluorosis.67,68 the concentration of fluoride in toothpastes was not assessed as it varies from country to country depending on the government regulations. in india, most of the commercially available toothpastes are either nonfluoridated ayurvedic toothpaste (anchor, meswa) or fluoridated (colgate, pepsodent) with a fluoride content of more than 1000ppm. most of the parents were not familiar with the difference in toothpaste content and many did not know the effect of fluoride content in toothpastes on their child s teeth. in most cases, the parents used the same toothpaste, which they were using, for their children with perhaps lesser amount of paste. this may be due to lack of awareness among parents about the benefits and risks associated with use of fluoridated toothpastes in preschool children. therefore, it is time that parents are instructed to delay the use of fluoridated dentifrice until the child is older than 24 months and use non - fluoridated toothpaste in these young children. in older children above 2 years, a pea - sized amount of fluoridated toothpaste should be used. instructions should be given to the parents so that they make sure their children rinse and expectorate thoroughly after tooth brushing to avoid excessive ingestion of fluoride from toothpaste.69,70 at present tooth - brushing and other mechanical cleaning procedures are considered to be the most reliable means of maintaining oral hygiene. brushes are more effective in cleaning proximal, marginal and occlusal areas of the teeth. the soft brush is preferable for most uses in young children because of the likelihood of gingival tissue trauma and increased inter - proximal cleaning ability.63 the present study showed that 1132 (89.4%) of the children used baby brush to clean their teeth. these children had significantly lower caries prevalence, when compared to those children who cleaned their teeth using their finger. in a similar study, it was found that 60% of the children who used tooth brush to clean their teeth were free of caries.2 therefore, dental health behaviors such as use of fluoridated dentifrice with baby toothbrush significantly reduced ecc experience, possibly reflecting desirable parental dental knowledge and education. however, these preferred parental behaviors may be subject to a recall and response bias. some of the limitations in the present study were deriving detailed and accurate information from the parents regarding the feeding practices, contents of the feeding bottle, composition of snacks consumed and weaning. also, the information obtained from mothers regarding child rearing practices may not be totally reliable. data obtained may serve as a base line for planning and evaluation of community development and oral health promotion programs. although future community initiatives have the potential to increase community knowledge of ecc, the ultimate challenge will be sustaining long term behavioral change among parents. the burden of dental decay in this population reveals the need for effective preventive methods. thus, both medical providers and those working with pregnant women and the very young must play an integral role in the prevention and early detection of ecc. it is important for pediatricians, family physicians and other health service providers encountering expectant mothers and very young children to be cognizant of ecc and its ramifications, as they represent the first line of defense. ultimately community - based solutions should be explored and can be strengthened if used in conjunction with the existing and emerging strategies for promoting early childhood oral health and preventing dental decay. these strategies should include suppression of maternal reservoirs of mutans streptococci, health education of mothers to prevent vertical transmission of mutans streptococci, promoting the first dental visit by 12 months, providing anticipatory guidance, motivational interviewing (mi), using chemotherapeutics (e.g., fluoride varnish, silver diamine fluoride, povidone iodine[butadiene ], chlorhexidine), water fluoridation, promote use of fluoridated dentifrice, dietary counseling, self - examination for early signs of ecc, and other previously tested methods which are potentially simple, cheap, effective, and appropriately practical for this community.7,18,21,71 although future community initiatives have the potential to increase community knowledge of ecc, the ultimate challenge will be sustaining long - term behavioral change among parents, caregivers and the community at large leading to appropriate parenting practices. the reason for increased ecc risk could be cultural differences in child rearing, infant feeding practices, and oral health beliefs. this survey has identified certain risk factors for the presence of ecc in preschool children within an indian city. general dentists can use this information to target 15-year - old children and use the above preventive methods. public funded oral health programs need to target children from lower socio - economic status and develop effective strategies to promote breast feeding and discourage inappropriate bottle feeding. 21 mi of mothers is a recent approach which is shown to have a greater positive effect on preschool children s dental health than the traditional health education approach. this approach enhanced the preventive behavior of mothers of young children at high risk of developing caries.71 another important aspect of ecc which has not been explored is the role of care - takers of children attending day - care centers in cities. recently, mani have suggested that the care - takers of children attending day - care centers have an important role in promoting oral health among preschool children. during the recent years, there has been an increase in the number of working mothers in metropolitan cities ; therefore most of the children spend a considerable amount of time in day - care centers. these parents have very limited control over their child s activities in such day - care centers. this indicates that further studies on ecc should focus more attention on family dynamics, environmental factors, behavior and society in which the children grow up. overall, the findings of this survey will form part of a baseline for the oral health assessment for children below 6 years of age in urban bangalore city, india. from the results of this study, it can be concluded that caries prevalence in pre - school children of urban bangalore was 27.5% with a mean deft of 0.854 and the risk factors for ecc included age, low maternal education, low socio - economic status, improper feeding and oral hygiene habits. there is evidence that suggests children with this condition remain at a high risk for future caries attack. the early identification of poor oral hygiene and improper feeding habits should be considered in preventive health promotion strategies in low socio - economic communities of bangalore city, india. | objectives : early childhood caries (ecc) is a devastating form of dental decay with multi - factorial origin. the aim of this cross - sectional study is to investigate the prevalence and related risk factors of ecc in preschool children of urban bangalore (india).methods : a random sample of 1,500 children aged between 8 and 48 months were selected from various parts of urban bangalore. the status of dental caries was recorded according to the world health organization (who) criteria. information regarding oral hygiene practices, feeding habits, socio - economic status, birth weight, and educational status of the mother was obtained through a structured questionnaire given to mothers of preschool children. the data was subjected to statistical analysis using the statistical package for social sciences (spss) version 12.results:the prevalence of ecc in preschool children was 27.5%, while the mean deft was 0.854. ecc increased significantly with age. children whose mothers had no schooling and those who belonged to low socioeconomic group showed higher caries prevalence. a significant increase in caries prevalence was found in children accustomed to the practice of on - demand breast feeding and bottle feeding at night. caries also increased significantly when snacks were consumed between meals. however, increased frequency of tooth - brushing, parental supervision, use of a baby toothbrush, and fluoridated dentifrice significantly decreased caries prevalence.conclusion:ecc is a serious public health problem in this population and measures to increase awareness should be undertaken. the target candidates for oral health promotion programs should include mothers, general dentists, pediatricians, nurses, primary care health workers, care - takers at day - care centers and gynecologists. |
dissolved organic carbon (doc) export from mangrove swamps accounts for 10% of the global terrestrial flux of doc to coastal oceans. given recent reports of high concentrations of mercury (hg) in mangrove tissues and mangrove marsh waters, and the known high affinity of hg species for doc in surface waters, there is concern that a large hg flux may accompany the doc flux from mangroves. in other aquatic environments, transport of hg species is intimately linked to the transport of doc because of strong complexation, particularly in tidal wetlands. elevated mercury levels have long been reported in coastal aquatic food webs from areas near the extensive mangrove swamps of southwest florida. in recent years, hg concentrations in some fish and wading birds in the everglades have declined, but concomitant declines have not been observed in florida bay species. these decoupled trends suggest that mitigation efforts may have diminished the terrestrial accumulation of hg but have not similarly benefited coastal food webs, perhaps because the hg source to coastal food webs is different than that of the uplands. methylmercury (mehg), the form of hg that bioaccumulates, has several potential sources in coastal waters : it may be produced in low - oxygen environments in terrestrial systems and then exported to coastal waters ; it may be produced in coastal wetlands such as mangroves and then tidally pumped into surrounding waters ; or it may be produced within estuarine and coastal sediments. although elevated tissue concentrations of hg and mehg are known to occur in mangroves and high concentrations of aqueous hg and mehg have been reported in mangrove - dominated environments, remarkably little is known about the magnitudes of hg and mehg export from mangrove swamps into coastal and estuarine waters. this study sought to characterize the export of doc and hg from the mesohaline estuarine mangrove swamps and fresher interior sawgrass marshes of the shark river estuary (everglades national park, florida, usa) to nearshore open coastal waters to test the hypothesis that mangroves can be significant sources of doc, hg, and mehg to coastal waters. estimating mass fluxes of carbon and mercury species in estuarine environments is challenging because of bidirectional flows, rapidly changing concentrations, and the multiple factors that affect tidal interactions with wetlands. we capitalized on the known strong association between doc and hg and applied a recently developed strategy that uses in situ continuous measurements of fluorescent dissolved organic material (fdom) as a proxy. we first established the relationship of fdom to doc, total hg and mehg concentrations by measuring fdom in situ while collecting discrete water samples throughout the estuary over a range of salinities and constituent concentrations during both wet and dry seasons. we also measured fdom continuously at a fixed site in the middle of the estuary for approximately two weeks of each season. regression models developed from the estuary - wide data were then applied to convert the seasonal midestuary fdom time series to time series of doc, total hg, and mehg concentrations, which were in turn used to calculate fluxes and yields. the premise of this study was that fluxes past the midestuary site would integrate processes operating over the range of nontidal to tidal domains, thereby providing insight into the production and transport of doc and mercury species. the concentration and flux time series data improve our understanding of processes controlling production and transport of doc, total hg (thg), and mehg from mangrove - dominated areas, assessments previously unattainable in dynamic estuarine systems. the southwest florida coast is a region of mangrove - dominated tidal inlets and rivers. the shark river (si figure s1) is one such river, located entirely within everglades national park. shark river slough drains an area of predominantly freshwater sawgrass marsh in its headwaters, and expansive mangrove swamp characterizes the estuary s lower reaches. tides are semidiurnal with a mean amplitude of approximately 1 m. the everglades experiences two distinct precipitation seasons : a may october rainy season and a november april dry season. regional average annual rainfall is approximately 1.4 m. our midestuary sampling site was near gunboat island (si figure s1), approximately 10 km inland from the coast and adjacent to a u.s. water depth at the site is 9 m. hg deposition and cycling in the region are strongly coupled to the pattern of precipitation. soil hg concentrations in the lower shark river are relatively high (0.15 to 0.30 mg kg). during a 2005 reconnaissance study, water samples were collected from a number of southwest florida coastal rivers (si table s1) to identify areas of high hg. for the follow - on flux study, additional samples were collected from the shark river estuary during september 2122, 2010 (wet season) and april 56, 2011 (dry season) (si figure s1, si table s2). surface water samples were collected by boat directly into 1 l polycarbonate containers. during the september sampling period, water was also collected at gunboat island from 0.5 m below the air sea interface, at 2 m depth, and at 1 m above the bottom. these samples were collected using a peristaltic pump, a 20 cm length of c - flex tubing, and three teflon tubes (1/4 in. samples collected for doc analysis were filtered within 2 h of collection through prerinsed gelman gelcap filters (nominal cutoff of 0.45 m) into precleaned, precombusted 1 l or 125 ml amber glass bottles. these samples were refrigerated and express - shipped on ice to the usgs laboratory in boulder, co. samples were analyzed for doc content by wet chemical oxidation and for uv absorbance by diode array spectrophotometry (see the supporting information for details). specific uv absorbance (suva254) was calculated as the uv absorbance at 254 nm divided by the doc concentration. samples collected specifically for total mercury (thg) and methylmercury (mehg) analyses were drawn directly into acid - washed 1 l pet bottles and then immediately filtered using an all - teflon filtration tower, fired quartz fiber filters (0.7 m pore size), and teflon sample bottles. all field sampling followed mercury - clean procedures and included the collection of numerous field blanks to monitor for possible contamination. filtered waters were immediately acidified to 1% (v / v) with concentrated hcl and were stored at ambient temperature out of direct sunlight. all samples were express - shipped to the usgs mercury research laboratory in madison wi. epa method 1631 and 1630, respectively, as implemented by the usgs mercury research laboratory (see the supporting information for details). concurrent with collection of each discrete water sample, fluorescent dissolved organic matter (fdom) fluorescence and other parameters were measured in situ with an open - faced optical sensor (wet labs eco) attached to a water quality sonde (ysi 6920). for continuous measurements, in situ instruments were deployed at the gunboat island site during september 2030, 2010, and april 419, 2011. a flow - through fdom fluorometer (wet labs wetstar ; excitation wavelength 370 nm, emission wavelength 450 nm) fitted with a pump (sea - bird electronics 5 t) and a water quality sonde (ysi 6920) was mounted approximately 1.5 m above the seabed to collect data at 15 min intervals. the fluorometer was purged for one minute, followed by 60 s of data collection. temperature, salinity, dissolved oxygen, ph, and turbidity were also measured during the same 60 s interval. the data were binned by averaging over the last 30 s of each data - collection period. midway through each field deployment, the instruments were briefly removed and cleaned ; the resulting data gaps were filled by interpolation. fluorescence is reported here as quinine sulfate equivalents (qse ; the concentration of quinine sulfate dehydrate in ppb that results in an equivalent instrument response). clean - water offsets, determined prior and subsequent to field deployment, were less than 1% of the measured in situ sample fluorescence values. water discharge data were obtained from the usgs gunboat island stream gage, which was equipped with a side - looking acoustic doppler velocimeter that measured cross - sectional velocities every 15 min. discharge measurements were calibrated using a moving boat equipped with a downward - looking acoustic doppler current profiler, and discharge was calculated according to published methods. concentrations of doc, filtered total mercury (fthg), and filtered methylmercury (fmehg) at gunboat island were derived from the continuous in situ fdom measurements. discrete - sample data were used to develop season - specific type i linear regression models to relate doc, fthg, and fmehg concentrations to fdom as described in supporting information section s2 ; no simple relationships were found to relate particle - associated hg concentrations to fdom or other parameters measured continuously in situ. the regression models (one set applicable where freshwater and midestuary waters mixed and another set applicable where midestuary and saline waters mixed) were applied to the continuous fdom data to obtain continuous time series estimates of doc, fthg, and fmehg concentrations. the freshwater estuary submodels were applied where fdom > 125 qse ; the estuary seawater submodels, where fdom 125 qse, corresponding to inflection points in salinity, suva254, fdom, and constituent values (figure 1). measured dissolved organic carbon (doc) and mercury species as a function of salinity (in practical salinity units, psu) and dissolved organic matter fluorescence (fdom ; in quinine sulfate units, qse) in discrete samples of the shark river estuary, sept. 2122, 2010, and april 56, 2011 : doc (top row), specific uv absorbance at 254 nm (suva ; second row), filter - passing total mercury (fthg ; third row), and filter - passing methylmercury (fmehg ; bottom row). the small gray circles in parts a h show the modeled constituent values at gunboat island for comparison. the large black circles in parts i p indicate fdom 26 ng l), including the everglades. concentrations of fthg and fmehg in samples collected near mangroves were higher than in samples collected from nearby channel waters, suggesting that mangrove areas were contributing large amounts of doc and mercury to estuary and coastal waters. the 20102011 shark river flux study was designed to assess the magnitude of this contribution. water samples and in situ measurements were collected during portions of both the wet and dry seasons to help gauge the range of fluxes that may occur and to provide insights into underlying and perhaps contrasting processes. during the september wet - season sampling period (figure 2a), the gunboat island site experienced much greater precipitation than during the april dry - season period (figure 2d). the midestuary median salinity was 9 psu in september, with a range of over 21 psu ; the median salinity in april was much higher (31 psu), with a much smaller range (9 psu). median water height during the september period (0.1 ft) was higher than during april (0.7 ft). as is discussed below, the salinity gradient, precipitation, and regional water height all have important implications for the estuarine export of doc, thg, and mehg. times series of tidal - stream gage data and modeled constituent concentrations and fluxes in shark river estuary at gunboat island during sept. 2122, 2010, and april 56, 2011 : salinity, water height, and precipitation (top row) ; modeled dissolved organic carbon (doc), filtered total mercury (fthg), and filtered methylmercury (fmehg) concentrations (middle row), and modeled cumulative doc, fthg, and fmehg fluxes (bottom row). sampling during both seasons encompassed freshwater sawgrass slough, mangrove - dominated coastal swampland, and the higher salinity open coastal waters of ponce de leon bay (si figure s1). doc concentrations were highest near the most inland, low - salinity sampling sites ; concentrations were lowest offshore in ponce de leon bay (figure 1a, si table s2). the doc : salinity concentration gradient was steeper during the april dry - season sampling period than during september (figure 1a, e). similar to doc, the highest fdom values during both seasons were from inland fresh waters, while the lowest values were found in outer ponce de leon bay (si table s2). the down - estuary fdom gradient was steeper in the dry season. during both seasons, fdom and doc concentrations were strongly positively correlated (figure 1i, m ; r = 0.98 in september ; r = 0.99 in april). in contrast, suva254, a compositional property of doc related to doc aromaticity and source, exhibited maximum values at midrange salinities during both sampling periods (figure 1b, f). fall suva254 values peaked at 5 psu salinity, when the gunboat island median salinity was 9 psu (figure 2a). spring suva254 values peaked at 26 psu, when the median salinity at gunboat island was 31 psu (figure 2d). the spatial distribution of fthg was similar to that of suva254 in both seasons (figure 1), with both quantities peaking near the region of 5 psu salinity in september (close to salinities at gunboat island) and near 20 psu salinity in april (also close to salinities at gunboat island). likewise, september fmehg values peaked near 5 psu salinity, in the mangrove zone, with the lowest concentrations found at offshore salinities (figure 1d). in april, however, the spatial distribution of fmehg more closely resembled that of doc, with the highest concentrations at inland sites and progressively lower values offshore (figure 1h). even so, the april relationship between fdom and fmehg showed an inflection point (figure 1p) near 25 psu salinity (figure 1h), indicating a change in doc composition from that previously associated with inland fmehg. interestingly, the spatial distributions of particulate thg (pthg) and particulate mehg (pmehg) also resembled those of suva254 (si table s2), suggesting a possible coupling between the particulate and dissolved hg phases. these values for the distribution of mercury between particulate and filtered phases with the majority occurring in the filter - passing fractions are similar to those previously reported for the everglades. the concurrent change in fthg, fmehg, fdom, and suva254 values at the salinities found near gunboat island in both seasons (figure 1) strongly suggests that the primary zone of production of doc, thg, and mehg is geographically fixed within the mangrove portion of the estuary, in the general vicinity of gunboat island. if doc and hg production and hg methylation occurred within a specific salinity zone of the estuary, the peak would shift seasonally away from gunboat island salinities. if the large majority of doc and hg were produced in the fresh waters of the sawgrass slough or in the outer estuary and this was where the majority of hg methylation occurred, there would not have been a midestuary maximum. further evidence that the mangroves supply the majority of doc, hg, and mehg is that midestuary high suva254 values can not be explained by invoking an appreciable contribution of freshwater - derived or outer estuary doc (figure 1). that the mangrove zone is the primary source of doc and hg supply to the coast is consistent with the findings of romigh., who reported high doc fluxes from shark river mangroves. they also agree with rumbold., who found little hg contribution from uplands and the highest thg and mehg concentrations in the transition zone from sawgrass to mangrove, and with the high thg and mehg concentrations found in our 2005 southwest florida reconnaissance study (si table s1). one implication of this finding is that, since little of the hg and mehg is derived from the sawgrass portion of the estuary, reducing hg loads to sawgrass marsh areas may have little effect on coastal hg fluxes from mangroves. modeled constituent concentrations in the shark river estuary at gunboat island in september and april generally tracked the water - height variations that marked the flood and ebb of the semidiurnal tides and the longer - period spring the highest modeled doc, fthg, and fmehg concentrations for each time series (figure 2b, e) most often occurred just after the lowest water of the ebb tide, near the lowest salinity portion of the tidal cycle. lower concentrations, the result of mixing with coastal waters, were seen coincident with the higher relative salinities that accompanied the flood tides. this difference in concentration between the ebbing and flooding waters drove the majority of flux in shark river estuary. material export from estuaries is driven by both net flow (the residual water flow after tidal exchange) and tidal pumping (net transport resulting from different constituent concentrations in ebbing and flooding waters). for the periods measured, net water discharge from the estuary accounted for less than 5% of the estimated constituent flux in september and less than 1% in april. most shark river doc, fthg, and fmehg export was driven by tidal pumping. daily fluxes were always positive (seaward), except for a few periods in the middle of the april deployment (figure 2c, f). for september, estimated daily fluxes from the shark river estuary averaged 9.1 (0.6) 10 mg d for doc, 1.5 (0.2) 10 ng d for fthg, and 1.7 (0.2) 10 ng d for fmehg. in april, the average estimated total daily fluxes were much smaller : 1.5 (0.02) 10 mg d for doc, 1.7 (0.1) 10 ng d for fthg, and 1.1 (0.04) 10 ng d for fmehg. despite finding that all constituents were related to fdom, estimated flux through time was different for each constituent (figure 2) for example, between september 25 and 27, average daily fluxes were lower than those seen during the first three days of the measurement period to a degree that varied depending on the constituent : approximately 70% lower for doc, 60% lower for fthg, and 35% lower for fmehg (figure 2c). although tidal pumping is the engine that drives shark river doc and hg exports, it appears that flux magnitudes are controlled by hydrologic and geomorphic properties that affect material exchanges between marsh / swamp surfaces and tidal channel waters. tidal exchange volumes were similar during the september and april deployments, but mercury fluxes were significantly larger in the fall than in the spring (9 times higher for fthg ; 17 times higher for fmehg). water levels were also elevated in september : median water height was 0.6 ft higher and median high water was approximately 0.2 ft higher (figure 2a, c). the greater water heights in fall would have inundated more mangrove - swamp surface area, thus making available more mangrove - derived material for ebb transport to channel waters. the effect of this enhanced supply can be seen in the contrast between the modeled concentration time series in september and april (figure 2b, e). in april, after the tide reversed, a smooth decline in concentration accompanied the incoming flood. in september, however, modeled constituent values did not decline smoothly with the incoming tide. instead, high concentrations often persisted into the subsequent flood stage, likely due to greater inundation during this period of higher water height and slow draining, the result of ebb it appears from these observations that even modest increases in regional sea level due to storms, wind, or rising global sea level have the potential to significantly alter constituent fluxes from mangroves. following the heavy rains of september 27 (figure 2a), for example, estimated total daily fluxes doubled for doc and fthg and increased by 75% for fmehg (figure 2c). note that elevated modeled constituent concentrations (figure 2b) did not occur until a full day after the precipitation fell. the heavy rains may have connected a supra - tidal mangrove plain (normally isolated from the shark river) to interior intertidal zones, supplying them with doc and hg. the modeled concentrations and fluxes of all constituents remained high (figure 2c). a similar lag effect in flux response to precipitation is seen following the rainfall event of april 4 (figure 2d, f). we sought to compare our flux estimates to previously published values and to assess whether exports from mangroves might represent a significant flux of thg and mehg to coastal waters. to do this, it was necessary to convert our flux values to yields (fluxes per unit area) and make a series of assumptions to generate annual values. we first converted the doc, fthg, and fmehg daily fluxes to annual ones by assuming that our deployment periods were representative of their respective seasons (wet and dry). we then converted these to yields by normalizing the fluxes to the area of influence (see methods). this calculation provided an estimated annual doc yield for the shark river estuary of 180 (12.6) g c m yr. this value is somewhat higher than the estimated annual doc yield reported by romigh. (56 g c m yr) for a small mangrove subdrainage in the shark river estuary but is well within the 44381 g c m yr range reported for mangrove swamps worldwide in a review by bouillon. the similarity between our results and other published values indicates that our fdom - based methods provided doc yield estimates of a reasonable magnitude. the estimated annual yield for fthg was 28 (4.5) g m yr : eight times the highest previously reported wetlands value and 20 to 100 times more than is generally reported for wetlands. the estimated yield for fmehg was 3.1 (0.4) g m yr : 10 to 100 times values previously reported from terrestrial wetlands for fmehg and 5 to 100 times previously published values from terrestrial wetlands for unfiltered mehg. the estimated shark river fmehg yields are more than five times those observed in the st. mary s river, a north florida blackwater river with a high doc concentration and flux. however, the mehg yields found here are similar to those measured in a tule - dominated tidal marsh in san francisco bay (2.5 mg m yr). our estimates, in combination with other recent studies of tidal wetlands, indicate that tidal flushing of marshes and swamps represents a potentially large and previously unrecognized source of thg and mehg to estuarine and coastal aquatic ecosystems. to assess whether fthg and fmehg exports from mangroves to the coastal ocean might be significant in comparison to other potential sources, we multiplied the shark river yields derived above by the 1400 km of fringing mangrove swamps in southwest florida. this exploratory calculation resulted in estimated annual exports in the range of 250 mt doc, 39 kg fthg, and 4.3 kg fmehg. to include particulate fractions, we increased the mercury fluxes according to the average ratios of particulate to filter - passing hg species found in this (si table s2) and other studies for the everglades. the resulting total (combined particulate and filtered) hg exports were thereby found to be approximately 55 kg for thg and 6.5 kg for mehg. we caution that these calculations are wholly heuristic in nature, made only to scale up the magnitudes determined in this study for the purpose of understanding the potential implications of our shark river findings within the context of what is currently known about coastal mercury supply in general. longer time series preferably multiyear time series and studies at multiple locations are needed to adequately constrain fluxes at larger scales. do fluxes of this magnitude represent a significant source to the coastal environment in comparison to other potential sources ? for thg, the primary source to southwest florida coastal waters is currently thought to be atmospheric deposition, with freshwater runoff accounting for only a small fraction. flux from mangroves comparing the potential mangrove flux to estimates of the combined wet and dry deposition of thg to the 5000 km of adjacent coastal waters suggests that mangroves could account for a loading equivalent to approximately 40% of atmospheric thg deposition.. found hg in waters exported from a mangrove marsh to be more reactive with respect to methylation than was hg in ambient estuary water. for mehg, the dominant source to coastal waters in southwest florida is presently thought to be local production in coastal sediments and diffusion into overlying waters ; the amount discharged from uplands and that attributed to wet and dry atmospheric deposition is small. benthic flux magnitudes of mehg from florida coastal sediments have not been reported, but if magnitudes are similar to fluxes observed elsewhere (1 pmol m d in chesapeake bay ; 30 pmol m d in san francisco bay), then potential mehg loading from mangroves to the 5000 km coastal - ocean area would be the equivalent of 2.5 to 20 times the flux from sediments, more than 80% of the total flux. this assessment, though preliminary in nature, clearly establishes that tidal mangrove systems have the potential to be a significant source of thg and mehg to tropical coastal waters, in addition to their previously demonstrated contributions of doc. our findings, in conjunction with previously published reports from tidal herbaceous wetlands, bolster the suggestion that tidal pumping of doc and hg from tidal wetlands may represent an important source to coastal environments. additional studies are needed to quantify fluxes from mangroves and other tidal systems and to resolve the fate of the material once it enters the nearshore environment. if the large shark river fluxes found in this study are generally representative of tidal mangrove systems, there may be global implications. recently reported particularly high concentrations of hg in tissues and litter fall of mangroves, suggesting that mangroves are particularly efficient at canopy capture of atmospheric hg. high rates of canopy capture in an environment conducive to methylation combined with the high fluxes found here would result in a system particularly efficient at extracting hg from the atmosphere and transporting it to the coastal oceans as mehg, potentially generating large coastal fluxes of mehg wherever mangroves are found. continuous in situ measurements of fdom can serve as a powerful tool for investigating these doc, thg, and mehg mass fluxes in many tidal systems. such measurements are critically needed to improve our understanding of hg cycling, advance predictive models and budgets of carbon and hg in coastal areas, and inform hg total maximum daily load regulation development. further, high - resolution time series of concentrations and fluxes provide valuable insights into the myriad biological, physical, hydrologic, and geomorphic processes affecting tidal exchange insights that should in turn improve estimates of carbon and mercury fluxes into coastal oceans. | the flux of dissolved organic carbon (doc) from mangrove swamps accounts for 10% of the global terrestrial flux of doc to coastal oceans. recent findings of high concentrations of mercury (hg) and methylmercury (mehg) in mangroves, in conjunction with the common co - occurrence of doc and hg species, have raised concerns that mercury fluxes may also be large. we used a novel approach to estimate export of doc, hg, and mehg to coastal waters from a mangrove - dominated estuary in everglades national park (florida, usa). using in situ measurements of fluorescent dissolved organic matter as a proxy for doc, filtered total hg, and filtered mehg, we estimated the doc yield to be 180 (12.6) g c m2 yr1, which is in the range of previously reported values. although hg and mehg yields from tidal mangrove swamps have not been previously measured, our estimated yields of hg species (28 4.5 g total hg m2 yr1 and 3.1 0.4 g methyl hg m2 yr1) were five times greater than is typically reported for terrestrial wetlands. these results indicate that in addition to the well documented contributions of doc, tidally driven export from mangroves represents a significant potential source of hg and mehg to nearby coastal waters. |
benign cystic lesions of the knee region in the form of fluid collections in synovial recesses, bursae, and ganglion cysts are common and often discovered incidentally in routine magnetic resonance imaging (mri) examinations. mr has proved to be superior compared to other imaging techniques in the detection of cystic lesions of the knee [1, 2 ] and add information of the anatomical relationship with adjacent structures and concurrent knee pathologies. the prevalence of cystic lesions has been described in both symptomatic and asymptomatic knees in previous studies [46 ] but only a few studies have examined the association between occupation - related kneeling and squatting and knee pathologies demonstrated by mri. based on mri, amin. showed that both heavy lifting and frequent kneeling and squatting increased the risk for knee cartilage degeneration, particularly at the patellofemoral (pf) joint, and we have in a previous study showed that occupational kneeling increases the risk of degenerative tears in the medial meniscus. however, to our knowledge, no previous mri studies have examined the relationship between occupational kneeling and the prevalence of intra- and periarticular cyst - like lesions of the knee joint. biomechanical studies have shown that tibiofemoral (tf) contact forces increase considerably during deep knee flexion and force joint fluid into the posterior part of the knee joint [912 ]. direct and indirect loading of the knee joint during kneeling work demands could, therefore, be a predisposing factor in the development of both intra- and periarticular disorders of the knee joint. many workers in the construction industry are exposed to knee - straining work activities but floor layers are particularly exposed. studies have shown that floor layers on average spend 4050% of the daily work time in kneeling work positions and that floor layers have a significantly increased prevalence of knee complaints [1316 ]. however, the high prevalence of knee complaints may not be explained by knee osteoarthritis (oa) and meniscal tears alone but could be attributable to other knee pathologies, for example, intra- and periarticular cystic - like lesions. the aim of this study was to examine the occurrence of mri - detected intra- and periarticular cyst - like lesions including bursitides of the knee joint in floor layers in comparison with a group of graphic designers without any occupationally related knee demands. we, furthermore, evaluated the association between mri findings and self - reported knee complaints and assessed the association of concomitant joint effusion, and cyst - like lesions potentially communicating with the knee joint, meniscal tears, and knee oa. a danish sample of 286 male floor layers and 370 male graphic designers was established from trade union rosters comprising members aged 3670 years in 2004 and with residence in copenhagen or aarhus, denmark. floor layers install different kinds of floorings and their work tasks involve prolonged kneeling and frequent change from kneeling to standing work positions. they work primarily with the layout of texts and advertisements using visual display units and their work did not include any knee demands. respondents constituted 253 (89%) floor layers and 290 (78%) graphic designers. the questionnaire provided data on anthropometrical characteristics, history of employment, knee complaints (ache, pain, or nuisance during the past 12 mo), knee injuries (fractures involving the knee, meniscal injuries, anterior (acl), or posterior (pcl) cruciate ligament lesions), and knee - straining sports activity defined as ever participated in football, handball, badminton, tennis, volleyball, ice hockey, or weight lifting. respondents were invited to participate in additional examinations. written informed consent was obtained from 156 floor layers and 152 graphic designers. among those, a random sample of 92 (copenhagen, n = 45 ; aarhus, n = 47) floor layers and 49 graphic designers (copenhagen) underwent an mri examination of both knees (total 282 knees). examinations were conducted at 2 mr centres in aarhus (centre i) and copenhagen (centre ii), respectively. mri was performed by a 1.5 tesla scanner (symphonyvision, siemens medical systems, erlangen, germany) at centre i and by a 1.5 tesla scanner (infinion, philips, best, the netherlands) at centre ii. the following mri sequences was obtained at centre i : sagittal proton density fat - saturated turbo spin echo (tr / te, 3300/15 ms) and sagittal and coronal t2-weighted (4000/86 ms) fat - saturated turbo spin - echo ; coronal t1-weighted (608/20 ms) spin - echo sequences and axial proton density fat - saturated turbo spin echo (3450/15 ms). the section thickness was 4 mm with an intersection gap of 0.4 mm ; field of view was 200 200 mm and matrix 512 in all sequences. at centre ii, the mri sequences included sagittal proton density fat - saturated turbo spin echo (tr / te, 2500/18 ms) and sagittal and coronal t2-weighted (4000/85 ms) fat - saturated turbo spin - echo ; coronal t1-weighted (400/13 ms) spin - echo and axial proton density fat - saturated turbo spin echo (2880/17 ms). the section thickness was 4 mm with an intersection gap of 0.4 mm ; field of view was 150 150 mm and matrix 512 in all sequences. the same experienced musculoskeletal radiologist (ne) evaluated each of the 282 mri examinations. due to differences in the appearance of mri at the two centres, blinding of occupational affiliation was incomplete for participants from centre i, who were all floor layers. the presence of fluid accumulations of the following bursae and synovial recesses were evaluated : semimembranosus - gastrocnemius (popliteal cyst figure 1), medial and lateral subgastrocnemius, prepatellar, superficial, and deep infrapatellar, medial (mcl), and lateral (lcl) collateral ligament, biceps femoris, iliotibial, anserine, and semimembranosus - gracilis bursitides (figure 2) and hoffa 's fat pad recesses (figure 3), [14, 17, 18 ]. other cyst - like lesions included meniscal cysts, ganglion cysts of the cruciate ligaments, and extracapsular synovial cysts (figure 4). two types of fluid collections along the popliteus tendon (pt) were distinguished : cystic lesions originating from the proximal tibiofibular joint and the pt recesses. insertional cysts [17, 20 ] were assessed at the cruciate and collateral ligaments. the presence or absence (0 = none, 1 = present) of cystic lesions was registered and the length of fluid collections in the pt recess was measured in mm from the upper demarcation line of the proximal tibiofibular joint and distally. knee joint effusion was registered (0 = none, 1 = present) as the presence of a localized fluid collection in the intercondylar area, in the medial or lateral recess, or as a generalized fluid collection that distended the suprapatellar recess. abnormalities in the mr signal intensities predicting meniscal tears were classified in grades 13 and radiographic knee oa were classified using a modified ahlbck scale of joint space narrowing (jsn). the prevalence of cyst - like lesions was computed among floor layers and graphic designers, respectively, and associations with occupation assessed by logistic regression and summarized by odds ratios (or) with 95% confidence intervals (cis). models were adjusted for age, body mass index (bmi), knee injuries, and knee - straining sports. using logistic regression, we additionally assessed the association between cyst - like lesions and knee complaints and furthermore associations between joint effusion and the presence of cystic lesions potentially communicating with the knee joint capsule, meniscal lesions (grade 3) and radiographic knee oa (jsn 25%). the adjusted or with 95% ci was computed, and independent variables incorporated in the model of adjusted results were occupation, age, bmi, knee injuries, and knee - straining sports. statistical analyses were performed using stata (statacorp lp, college station, tx, usa). mean age and trade seniority were higher among graphic designers compared to floor layers, and graphic designers also had a higher proportion of previous knee injuries and knee - straining sports experience than floor layers. the two study groups were comparable regarding average bmi and the proportion of knee complaints. fluid accumulations in various bursae were a common finding in both trade groups (table 2(a)). among 141 participants, 113 (80.1%) had mri findings consistent with bursal fluid collections around the knee joint. comparing the two trade groups, 85.8% of the floor layers were classified as having at least one bursal fluid collection in one or both knees compared to 69.3% among graphic designers (or 2.94, 95% ci 1.177.44). the prevalence differed in various sites of the knee joint and was generally higher among floor layers than graphic designers except for fluid collection in the prepatellar bursa. floor layers had a higher prevalence of fluid collections in the pt recess (figure 1) compared to graphic designers, and the average extents of pt recesses were also larger in floor layers (mean 23.7 mm, 95% ci 21.326.1) than in graphic designers (mean 20.1 mm, 95% ci 17.622.6 ; p < 0.05). an unexpected finding was cystic lesions within the lateral portion of the popliteus muscle (figure 5). these abnormalities were seen with and without communication to a not distended pt recess and occurred almost exclusively in floor layers. extracapsular synovial cysts extending in the proximal direction from capsular defects of the dorsal aspect of the femoral condyles (figure 4) and fluid collections in the mcl, lcl, biceps femoris, and anserine - semimembranosus - gracilis bursae (figure 2) were only registered in floor layers. although nonsignificant, floor layers also had a higher prevalence of medial parameniscal cysts and fluid - filled recesses in the hoffa fat pad (figure 3). the prevalence of cystic lesions in the anterior part of the knee (prepatellar, superficial and deep infrapatellar, and anserine bursae) was found in 16 of 184 (8.7%) knees in floor layers compared to 12 of 98 (12.2%) knees in graphic designers (or 0.68, 95% ci 0.291.66). cystic lesions in the posterior part of the knee ; semimembranosus - gastrocnemius (figure 1) and subgastrocnemius bursitides (medial and/or lateral), and extracapsular synovial cysts (figure 4), were a common finding but significantly more prevalent in floor layers and found in 149 of 184 (80.9%) knees compared to 60 of 98 (61.2%) knees in graphic designers (or 2.70, 95% ci 1.504.84). the association between knee joint effusions and cyst - like lesions potentially communicating with the knee joint capsule, meniscal tears, and radiographic knee oa are given in table 3. the prevalence of joint effusions was 36.9% (34/92) and 28.5% (14/49) in floor layers and graphic designers (p = 0.32), respectively. although nonsignificant, joint effusions were more frequently associated with fluid collections in the semimembranosus - gastrocnemius and the subgastrocnemius bursae than pt recesses and extracapsular synovial cysts. knee complaints were most frequently associated with acl insertional cysts, and more common among subjects with fluid collections in the periarticular bursae than subjects with fluid collections in the peripatellar bursae. the risk of knee complaints was low among subjects with cystic lesions around the medial (parameniscal cysts) and lateral (pt recesses) meniscus (table 4). in a sample of floor layers exposed to occupational kneeling, we found a significantly higher prevalence of cyst - like lesions in the posterior part of the knee joint compared to a reference group of graphic designers and our findings revealed surprisingly few floor layers with cystic lesions in the anterior part of the knee joint. the subgastrocnemius bursa is located above the knee joint and deep in relation to the medial gastrocnemius muscle where it communicates with the posterior joint capsule. it has been shown that this bursa commonly communicates with the semimembranosus - gastrocnemius bursa. in the present study, 66.7% (40/60) of those with a fluid collection in the semimembranosus - gastrocnemius bursa the prevalence of fluid collections in the semimembranosus - gastrocnemius (popliteal cyst) bursa on mri reported in the literature ranges from 5% to 38%. our results seem to be in agreement with these results, although we observed a higher prevalence among floor layers (46.7%) compared to graphic designers (34.7%). the pathogenesis of this disorder has been explained by a slit - like communication of the posterior joint capsule and the bursae. compared to the posterolateral corner of the knee joint that obtains reinforcement from the ligament of wrisberg, the pt, and the pcl, the posteromedial corner has only supplemental reinforcement from the posterior horn of the medial meniscus. this relative weakness of the posteromedial joint capsule may allow joint fluid to move from the knee joint into the bursa. in addition, studies have shown that pull from the semimembranosus tendon and the medial meniscus during knee flexion opens this slit - like communication. however, cadaveric studies have shown that roughly 50% of popliteal cysts do not communicate with the knee joint [24, 25 ]. bursal enlargement could be the result of multiple microtrauma of the bursa related to muscle contraction during repeated kneeling. another hypothesis is that kneeling, which has been shown to increase contact stress in the medial tf compartment significantly, may predispose to progressive thinning of the posteromedial joint capsule due to tearing forces emanating from the pull of the meniscus and the semimembranosus tendon [10, 24 ]. work tasks with repeated and prolonged kneeling could, therefore, explain the high prevalence of fluid collections in the semimembranosus - gastrocnemius bursa found in floor layers. excess fluid accumulation in the prepatellar bursa has traditionally been associated with direct external pressure due to repeated kneeling [13, 2628 ]. although few cases, we found a higher prevalence of excess fluid accumulation in the infrapatellar bursae compared to the prepatellar in floor layers. many of the floor layers ' work tasks are performed in knee angles above 90-degree flexion. in these work positions, most of the directly related stresses between the knee and underlay are located around the tibiae tubercle, and not between the patellae and the underlay. mechanical stress in this area could explain the higher prevalence of excess fluid accumulation in the infrapatellar and anserine bursae (figure 2) in floor layers. our results are in accordance with a previous ultrasound study by myllymki. that found a higher prevalence of fluid accumulation in the infrapatellar bursa compared to the prepatellar bursa among carpet - layers compared to a reference group of painters. ganglion cysts originating from the tibiofibular joint were not detected, but fluid collections along the pt recesses were a common finding and showed a higher prevalence in floor layers compared to graphic designers ; significantly more floor layers had lesions in both knees. apparently, there is some confusion in the literature distinguishing ganglion cysts originating from the proximal tibiofibular joint from pt recesses. these ganglion cysts have a characteristic anatomical location and mr appearance and are rare with a prevalence of 0.76%. also, pt recesses have characteristic mr appearances on axial and coronal mr slices (figure 1),. however, these recesses have in many publications been interpreted on sagittal mr images to originate from the proximal tibiofibular joint [3, 4, 17, 2931 ]. the prevalence of fluid collections along the pt varies between 0% and 7% in asymptomatic knees [4, 2931 ] and between 3% and 12% in symptomatic knees [2931 ]. the high prevalence of pt synovial recesses in this study sample may be explained by differences in the mr sequences used. to our knowledge, the mr appearances of multilobulated cystic lesions in the popliteus muscle (figure 5) found in 13 knees of floor layers and 2 control knees have not been described previously. in most of the cases, the images of these cystic lesions demonstrated continuity to pt recesses. apart from meniscal tears, and knee oa, we did not find significant relations between concomitant knee joint effusion and synovial recesses along the pt or other cyst - like lesions potentially communicating with the knee joint capsule. the presence of cyst - like lesions communicating with the knee joint may, therefore, exist in knees without pathologic joint effusion and not act as a marker of joint effusion, which also has been suggested in previous studies [29, 30 ]. we found a high prevalence of knee complaints in both study groups but no clear association with mri findings. these findings are in accordance with results from a neurosensory knee mapping study. by probing the knee joint without anaesthesia, it was shown that the area of cruciate ligaments insertion in particular was associated with pain. however, bursal tissue has a rich neural innervation of free nerve endings, and deformation of bursae may also be responsible for pain associated with bursal fluid accumulation. the association between cyst - like lesions and knee pain has, however, been conflicting in previous studies. although non - significant, our findings showed a higher association between knee complaints and fluid accumulations in periarticular bursae compared to accumulations in peripatellar bursae. this finding corresponds to earlier results of hill. who in an mri study found an equal distribution of peripatellar bursitis among subjects with and without knee pain, whereas periarticular bursitis was significantly more prevalent in subjects with knee pain. examined the association between intra- and periarticular cyst - like lesions and incident knee pain, and they found no significant associations. they suggested that such lesions may be a secondary phenomenon, which occurs in painful knees rather than a primary trigger of knee pain. current and previous findings indicate that not all cyst - like lesions may be clinically significant. however, they may in addition to knee oa and meniscal tears to some extent explain previous reports of significantly increased prevalence of knee complaints among floor layers [8, 13, 15, 22 ] first, the power of the study was limited due to the small study sample and the low response rate from questionnaires. second, results could be biased if the decision to participate in the study was differentially influenced by previous or current knee complaints. as discussed in a previous publication with the same study sample, our analysis revealed that graphic designers had a greater tendency to participate if knee symptoms were present [relative risk (rr) 2.7, 95% ci 1.74.3 ], than among floor layers (rr 1.2, 95% ci 0.91.6). this may have introduced bias and led to an underestimation of the difference between the two study groups. third, differential selection of workers toward different occupations depending on their health status may be inventible in occupations with high physical demands and a healthy - worker selection may have influenced results. however, such selection mechanisms would typically result in an underestimation of the investigated association. finally, we acknowledge that some of the mr findings may be changes discovered incidentally in routine mr imaging and that we do not know the clinical relevance of these findings. however, our results may be of interest due to the marked difference in the prevalence of findings in the two trade groups as previous studies have shown a high occurrence of unexplained knee complaints among workers exposed to frequent and prolonged kneeling. in summary, this was the first study to assess the relationship between occupational kneeling and intra- and periarticular cyst - like lesions of the knee joint. results revealed a significantly higher prevalence of cystic lesions in the posterior part of the knee joint in floor layers compared to a reference group of graphic designers and findings call attention to a possible association with occupational kneeling. | objective. to determine the risk of intra- and periarticular cyst - like lesions of the knee joint in occupational kneeling. methods. magnetic resonance imaging of both knees (n = 282) was conducted in 92 male floor layers and 49 male graphic designers (referents), with a mean age of 55.6 years (range 4270 years). the prevalence of cyst - like lesions was computed among floor layers and graphic designers, respectively, and associations with occupation summarized by odds ratio (or) with 95% confidence intervals (cis). using logistic regression, models were adjusted for age, body mass index, knee injuries, and knee - straining sports. results. floor layers had a significantly higher prevalence of cyst - like lesions in the posterior part of the knee joint compared to graphic designers (or 2.70, 95% ci 1.504.84). floor layers also had a higher prevalence of fluid collections in the popliteus tendon recess (or 2.17, 95% ci 0.994.77) and large cystic lesions of the popliteus muscle (or 3.83, 95% ci 0.7818.89). the prevalence of cystic lesions in the anterior part of the knee joint was low among floor layers (8.7%) and there was no significant difference between the two trade groups (p = 0.34). conclusions. occupational kneeling increases the risk of cyst - like lesions in the posterior part of the knee joint. |
the term groove pancreatitis was first used in 1973 by becker to describe a specific form of chronic pancreatitis that resulted in scarring, which extended into the anatomic space (groove) between the pancreatic head, duodenum, and common bile duct. more recently, preoperative diagnosis of groove pancreatitis has been emphasized to avoid unnecessary radical surgery. groove pancreatitis is not common, and only one case was recorded, but not published, in korea. a 46-year old man was admitted to our hospital with severe postprandial abdominal pain in the right upper quadrant. he complained of right upper abdominal pain that lasted for six months and weight loss of 12 kg over the same period. during the last few weeks iu / l), total bilirubin 0.5 mg / dl (normal range 0.21.2), alkaline phosphatase 69 iu / l), and alt 20 iu / l (normal range 544 iu / l). tumor markers were also within normal range : carcinoembryonic antigen 1.46 ng / ml (normal range < 5.0 iu / l) and carbohydrate antigen 19 - 9 3.17 u / ml (normal range < 39 iu / l). abdominal ultrasonography showed a non - homogeneous mass measuring 32 mm in diameter between the pancreatic head, descending duodenum, and common bile duct, as well as swelling of the pancreatic head (figure 1). endoscopy revealed duodenal wall thickening adjacent to the major papilla, with narrowing of the duodenal lumen. computed tomography (ct) revealed swelling of the pancreatic head and a non - homogeneously enhanced, low - density area between the pancreatic head, descending duodenum, and common bile duct (figure 2a). the head of the pancreas was enhanced on contrast ct scan and peripancreatic vessel encasement was not seen (figure 2b). mr images revealed a mass between the pancreatic head, duodenum, and common bile duct that was hypointense on t1-weighted images (figure 3a) and hypointense relative to the pancreatic head on t2-weighted images (figure 3b). on t1-weighted images, we observed the medial wall thickening of the descending duodenum, several small cysts in the groove and thickened duodenal wall on enhanced images (figure 3c, 3d). the patient has been under conservative treatment for 2 months and his severe abdominal pain has improved. although he continues to have mild postprandial discomfort, he refuses evaluation and treatment for his disease. groove pancreatitis is a rare form of chronic pancreatitis affecting the groove between the pancreatic head, the duodenum, and common bile duct. there are two forms of groove pancreatitis : one is pure groove pancreatitis that only affects the groove, the other is segmental groove pancreatitis that affects the groove and the parenchyma of pancreas. commonly reported symptoms include severe upper abdominal pain, recurrent postprandial vomiting, jaundice, diarrhea, and weight loss. although the pathogenesis of groove pancreatitis remains unclear, it is suspected to be associated with previous history of disease in the biliary system, peptic ulcers, gastric resections, true duodenal wall cysts, pancreatic head cysts, pancreatic heterotopia in the duodenum, disturbed pancreatic juice flow in the santorini duct in the absence of the minor papilla, and hypersecretion ancillary to alcohol abuse. in one study, 17 cases of groove pancreatitis were reviewed. the 17 patients had a median age of 51 years, and 16 of the 17 patients were alcoholics. symptoms of abdominal pain were observed in 6, vomiting in 4 and jaundice in 2 of the patients. the pure form differentiates duodenal cancer, cbd cancer, and acute pancreatitis with phlegmon. for the segmental form, while pancreatic cancer mass is seen as non - enhanced, in this disease, dynamic ct generally reveals an enhanced mass in the groove or the pancreatic head. other findings suggestive of groove pancreatitis include cysts in the duodenal wall and/or the groove, and duodenal stenosis accompanied by wall thickening. poor enhancement of scar lesions in groove pancreatitis may be due to delayed circulation caused by proliferation of fibrous tissues with attendant arterial constriction. the most characteristic finding on mri is a sheet - like mass between the head of the pancreas and the duodenum, associated with duodenal wall thickening. the mass is hypointense compared to pancreatic parenchyma on t1-weighted images, and iso- to slightly hyperintense on t2-weighted images. dynamic imaging typically shows delayed enhancement. in our patient, the symptoms were postprandial right upper quadrant abdominal pain, watery diarrhea, and weight loss. abdominal ct revealed a non - homogeneously enhanced mass lesion at the groove and pancreatic head. abdominal ct revealed a mass with duodenal wall thickening, but endoscopic biopsy specimens obtained from the mucosa of the second portion of the duodenum only showed inflammation. abdominal mri revealed a sheet - like mass at the groove, associated with duodenal wall thickening and cysts., we were able to diagnose segmental groove pancreatitis preoperatively. although groove pancreatitis is not rare, only a few cases have been reported due to a lack of awareness. all clinicians should be familiar with its clinical features and keep them in mind for the differential diagnosis of pancreatic masses and duodenal stenosis. | groove pancreatitis is a special form of chronic pancreatitis in which scarring is found mainly in the groove between the pancreatic head, duodenum, and common bile duct. preoperative differentiation between groove pancreatitis and pancreatic cancer is difficult. here we report one case of segmental groove pancreatitis diagnosed by clinical and radiological features. the patient was a 46-year old man with severe abdominal pain, weight loss, and a long history of alcohol abuse. computed tomography revealed swelling of the pancreatic head and a heterogeneously enhanced low - density lesion in the groove. mr images revealed a mass in the groove that had a low signal on t1-weighted images and a low signal relative to the pancreatic head on t2-weighted images. t1-weighted images on dynamic study showed the medial wall thickening of descending duodenum, several small cysts in the groove and thickened duodenal wall. the patient has been under conservative treatment for 2 months and his severe abdominal pain has improved. |
ion channels are membrane proteins that provide a pathway for the movement of ions into or out of all cells and are critical for all physiological processes. some channels discriminate poorly among various ions and some are quite selective, allowing passage of predominantly only one or very few types of ions. in certain subtypes of ion channels, that is, the availability of the pathway is controlled by voltage or a ligand or, in some cases, by both. over the last few decades, considerable effort has been directed toward identifying the part of the protein that forms the physical gate of the channel. much of this work has been focused on voltage - gated k (kv) channels, and a clear picture has evolved. the pioneering work of armstrong (1966, 1971) probing squid axon k channels with tetraethyl ammonium ions and other quaternary ammonium (qa) compounds led to the idea that the gate was located on the cytoplasmic side of the protein. a key finding was that these blocking ions have access to their binding site in the channel pore only from the intracellular end of the channel and only when the channel is open. larger ions including the long - chain qa analogue decyltriethylammonium, c10, and peptides based on the shaker inactivation ball peptide (bp) are also open - channel blockers and are trapped by or interfere with the closing of the activation gate, results that add further evidence that the physical location of the gate is toward the cytoplasmic end of kv channels (armstrong and hille, 1972 ; yeh and armstrong, 1978 ; zagotta., 1990 ; demo and yellen, 1991 ; toro., 1992 ; choi., 1993 ; holmgren., additional studies using the shaker kv channel as a model (holmgren., 1997 ; liu., 1997 ; del camino., 2000 ; del camino and yellen, 2001) identified parts of the sixth membrane - spanning region (s6) that could be chemically modified whether the channels were open or closed and other regions in s6, more toward the extracellular end, that could be modified only if the channels were open. a critical part of these studies was the finding that even small ions like cd and ag were excluded from these deeper structures by the channels gates. finally, support for this picture of a relatively large intracellular gate in kv channels has come from the solution of the crystal structures of several types of k channels (doyle. thus, the idea of an intracellular gate is well established for kv channels, but do other k channels work the same way ? some studies indicated that the large - conductance, voltage- and ca - activated k (bk) channel may also have a large intracellular gate because, as in kv channels, both c10 and the bp are open - channel blockers of these channels, and the bp interferes with bk channel closing (li and aldrich, 2004, 2006). however, there are several other findings with bk channels that raise questions about this conclusion. for example, in contrast to the open - channel block of bk channels by c10 and the bp, a relatively large qa compound, n-(4-[benzoyl]benyl)-n, n, n - tributylammonium) (bbtba), does not require open, conducting channels to block (wilkens and aldrich, 2006 ; tang., 2009). in addition, ba ions can not access their bk pore - blocking site from either the extracellular or intracellular solutions when the channel is closed (miller, 1987), whereas closed kv channels can be blocked by external but not by internal ba (armstrong and taylor, 1980). thus, there are significant differences between the behavior of bk and kv channels that raise doubts that the gating of bk channels occurs through an intracellular physical gate like that in kv channels. if bk channels do not open and close via movement of a large intracellular part of its protein, how is the flow of k ions regulated ? one hypothesis is that the selectivity filter itself controls ion flow through the channel (li and aldrich, 2006 ; piskorowski and aldrich, 2006 ; tang., 2009), and recent studies have provided evidence in support of this idea (chen and aldrich, 2011 ; zhou., 2011). if bk channel gating occurs with structural changes in the selectivity filter, it might be expected that permeant ions would alter channel gating. rb is very similar in size and hydration energy to k and is permeant in all k channels. replacing k with rb or adding rb to k solutions shifts the voltage dependence of bk channel activation (demo and yellen, 1992 ; piskorowski and aldrich, 2006). the pore - blocking ions cs and ba have a somewhat larger effect in that they destabilize the bk channel closed state by 2.3 and 1.5 kcal / mole, respectively (miller., 1987 ; demo and yellen, 1992). finally, replacing k with the permeant tl ion produces significantly larger effects on bk gating : a + 38-mv shift of the voltage dependence of channel activation (piskorowski and aldrich, 2006). however, even though tl permeates bk (and kv) channels, its complex electronic structure may make it a less than ideal probe of k channel activity. although the actions of rb, cs, and ba on bk channel gating are consistent with the expectations of selectivity filter gating, the rather small effects and the fact that rb and cs ions affect the gating of kv as well as bk channels (swenson and armstrong, 1981 ; clay and shlesinger, 1983) indicate that these results can not be considered as successful tests of this hypothesis. nevertheless, if the selectivity filter acts as the gate in bk but not kv channels, there should be some distinct effects of permeant ions on some aspect of bk but not kv channel gating. because the interaction of blocking ions with the gates of kv channels helped establish the intracellular location of the gate in these channels, a selective alteration of these interactions in bk channels by permeant ions would constitute a convincing test of the selectivity filter gating mechanism thus, we used small (tetrabutyl ammonium [tba ]) and large (bp) molecules shown previously to interact with the gates of kv channels as probes of bk channel gating with k or rb as the permeant ion. as reported previously (li and aldrich, 2004, 2006), we found that these two molecules exhibited open - channel block of bk channels with k as the permeant ion, and the bp interfered with channel closing. however, simply replacing rb for k as the permeant ion produced a profound difference : these pore - blocking ions had access to their binding sites independent of the activation state of bk channels, and the bp no longer interfered with the closing of the activation gate. moreover, this effect was specific for bk channels : these blocking ions retained their interactions with the gates of shaker kv channels, independent of the permeant ion. the bk channel construct used here was the mouse form of kca1.1 (mslo) (butler., 1993). the shaker k (shb) channel was the inactivation - deleted form with amino acids 646 removed (hoshi., 1990). channel proteins were expressed by rna injection into oocytes from frogs (xenopus laevis). xenopus ovarian lobes were obtained from nasco, and individual oocytes were isolated by standard procedures. channel currents were obtained with excised inside / out macropatches with an amplifier (axopatch 200b ; molecular devices). a holding potential of 80 mv was used for experiments on bk channels, and 100 mv was used for shb channels. in bk channel experiments with rb as the permeant ion, the pipette (extracellular) solution for bk channel experiments consisted of (in mm) : 140 x - glutamate, 2 mgcl2, and 10 hepes, ph 7.2, where x was either k or rb. most bk channel experiments were done with a bath (cytoplasmic) solution containing 100 m free ca (in mm) : 140 x - glutamate, 3 nta, 1.11 cacl2, and 10 hepes, ph 7.2, but a 10-m solution was also used : 140 x - glutamate, 5 hedta, 3.6 cacl2, and 10 hepes, ph 7.2. these solutions also contained 50 m crown ether (sigma - aldrich) to chelate any ba contamination of the ca salts. the cytoplasmic solution for shaker channel experiments was nominally ca free (in mm) : 140 x - glutamate, 1 egta, and 10 hepes, ph 7.2. tba - cl was obtained from sigma - aldrich, and the enhanced shaker bp (e12kd13k) (murrell - lagnado and aldrich, 1993) was prepared by biopeptide co. the accuracy of channel block at negative potentials where the open probability is small can be compromised by leak currents. to minimize such possible errors, only patches with a high seal resistance (low leak) were used, which required minimal analogue leak subtraction. in a few experiments in k solutions, we found comparable tba block of bk channels without and with a p/4 procedure (not depicted). we preferred not to routinely use p/4 leak subtraction because this procedure increases noise, necessitating the averaging of currents from several depolarizations, which is problematic for use - dependent blockers. the minimal contribution of leak to the analysis is indicated by the fact that channel current and activation level smoothly approached zero at negative potentials where leak currents would continue to increase. also, because rb permeates both shaker and bk channels less well than k (latorre and miller, 1983 ; eisenman., 1986 ; yool and schwarz, 1991), channel currents would be smaller in rb than in k solutions and, therefore, leak would represent a larger fraction of total current. such a situation would tend to minimize the measured block in rb solutions, but our results actually show more block of bk channels at negative potentials than was seen in k solutions. in addition, we found no correlation of block levels with current levels with either k or rb as the permeant ion. the slow kinetics of block by the bp can make it difficult to determine steady - state current levels (zagotta., 1990 ; li and aldrich, 2006 ; and see fig. 5). to minimize this problem in some experiments with few expressed channels, we averaged multiple current records. for these, we used a 3-hz cycle time that we determined did not produce accumulated use - dependent block (not depicted). we indicate in the figure legends which examples were derived from averaging multiple records. channel activation at a particular voltage was determined by the magnitude of the tail current after repolarization to the holding potential. the voltage dependence of channel activation was fit by the boltzmann equation amplitude/(1 + exp[z(vm vh)f / rt ]) and presented as normalized to the maximum value. because of the large patch to patch variability in the half - activation voltage (vh) of heterologously expressed slo channels (stefani., 1997 ;, 1999 ; orio and latorre, 2005) of shifting the bk channel activation curves from multiple cells so that the half - activation voltage from each cell coincides with the overall mean value and, through an interpolation procedure, computing a mean voltage activation relation. estimates of the channel deactivation kinetics were obtained from fits of a single - exponential time function to the tail currents after a depolarization that fully activated the channels. as noted in introduction, the shaker bp is an open - state blocker of bk channels (li and aldrich, 2006). the data presented in fig. 1 were collected to summarize the previously reported behavior of the bp on bk channels. the top insets show bk channel currents collected in 100 m ca during depolarizations to the indicated membrane voltages in the absence (black traces) and presence (red traces) of 2 um of the bp applied to the inner surface of bk channels in an inside - out patch. the peptide produced minimal block at negative potentials where the channel open probability is small and much larger block at more depolarized potentials where the channels are open. 1 shows the close correlation between channel activation () and the fraction of unblocked channels (). this close correlation is not the result of a voltage - dependent block by the bp that just happens to occur over the gating voltage range, because shifting channel activation by a reduction in intracellular ca produces an equal shift in the voltage dependence of block by the bp (li and aldrich, 2006). (top inset) time course of raw bk channel currents recorded in 100 m ca in the absence (black) and presence (red) of 2 m bp at the potentials indicated. (main) voltage dependence of bk channel activation () and fraction of channels not blocked by the bp () in 100 m ca. (inset) raw current recorded at + 60 mv from a holding potential of 80 mv in the absence (black) and presence (red) of 2 m bp. (boxed inset) magnified view of the currents after repolarization to 80 mv, with the peak current in the bp scaled to match that of the control record. another property reflecting the open - channel block by the bp can be seen in the time dependence of currents recorded in the presence of the bp, for example, the red traces at 10 and + 10 mv in the top insets and at + 60 mv in the main inset (fig. the channels are closed and unblocked and begin to open normally, and the current in the presence of the blocker superimposes with the control records (black traces). then, as the channels open, they become increasingly blocked. a final hallmark of this bulky peptide is that it interferes with bk channel closing (li and aldrich, 2006). this can be seen in the boxed inset, which is a magnified view of the tail currents after repolarization in the absence (fig. the peak amplitude of the tail current in the bp was scaled to match that of the control record. the presence of the bp substantially slowed the tail current ; the channels could not completely close until the peptide left its binding site in the permeation pathway. although we have not analyzed this process in detail, li and aldrich (2006) did and found evidence that a small fraction of blocked channels might not pass back through the open state before closing. nevertheless, their results and ours clearly show that the presence of the bp profoundly interferes with the ability of bk channels to close. as described in introduction, the qa compound c10 preferentially blocks open bk channels (li and aldrich, 2004), but less work has been done examining the properties of block by the smaller qa ion, tba, on bk channels. as shown in fig. the top insets show bk channel currents collected in 100 m ca during depolarizations to the indicated membrane voltages in the absence (black traces) and presence (red traces) of 10 mm of internally applied tba. as with the bp, tba produced little block of the channels at the most negative potentials where the channels have a low open probability, but there was a very large block at potentials where the channels are mostly open. 2 where the voltage dependence of the fractional open probability and the fraction of unblocked channels in 100 m ca are plotted as closed squares and closed circles, respectively. the reciprocal relationship between current inhibition and activation is a hallmark of open - channel block by this relatively small qa ion. (top insets) time course of raw bk channel currents recorded in 100 m ca in the absence (black) and presence (red) of 10 mm tba at the potentials indicated. (main) voltage dependence of bk channel activation in 100 m ca () and in 10 m ca (). also shown is the voltage dependence of the fraction of channels not blocked by 10 mm tba in 100 m ca () and in 10 m ca (). solid lines, fits of the boltzmann equation to the data ; dashed lines, spline fits to the data. shifting the voltage dependence of channel activation to more positive potentials by reducing the intracellular ca level to 10 m (fig. 2,) produced an equivalent shift in the voltage dependence of channel unblock (), preserving the tight relationship between channel activation and channel block. it is also apparent from the raw current traces that tba block was very fast, because the amount of block appears to be essentially independent of time during the depolarization. 2, we found that tba, unlike the bp, did not interfere with channel closing but slightly increased the rate of channel closing consistent with the findings of li and aldrich (2004). 1 summarized past published work with the bp (li and aldrich, 2006), showing that it is an open - state blocker of bk channels and that it interferes with bk channel closing. 2 illustrated that tba also preferentially blocked open bk channels. as with kv channels, these types of findings lead naturally to a picture of bk channels with a ca and voltage - controlled intracellular gate that prevents even the relatively small tba ion from blocking the channel in its closed conformation. in this view, the intracellular gate must first move to its open position before these ions can occupy their blocking sites in the intracellular end of the pore. the existence of such an intracellular gate can also account for how the large, slow bp interferes with channel closing. although this picture of an intracellular gate is consistent with these data, we noted in introduction that there are other findings with bk channels that call such a picture into question. if bk channel gating occurs with such an intracellular gate, changing the permeant ion from k to rb should have little or no effect on the relationship between channel opening and block by tba and the bp. therefore, we examined the actions of tba and bp on bk channels in rb solutions, and an example of tba block is illustrated in fig. (a ; insets) time course of raw bk channel currents recorded in 100 m ca in the absence (black) and presence (red) of 10 mm tba at the potentials indicated. the voltage level before depolarization was 100 mv, and after the depolarization it was 80 mv (main) steady - state current voltage relation before (), during (), and after () the application of 10 mm tba in rb solutions. (b) voltage dependence of bk channel activation () and fraction of channel not blocked by tba () in 100 m ca. 3 a contain raw bk channel currents recorded at the indicated voltages with rb as the permeant ion in the absence (black traces) and presence (red traces) of 10 mm tba. it is clear from these examples that tba produces a substantial block of bk channel currents, even at very negative potentials where the channels have a low open probability. the main part of fig. 3 a illustrates this same property over a larger voltage range and also shows that block by tba is fully reversible in rb solutions. the relationship between bk channel activation and block by tba in this patch 3 b. unlike the close correspondence between channel activation and tba block seen in k solutions (fig. 2), when rb is the permeant ion, block was almost completely independent of voltage. it is clear that bk channel block by tba in rb was almost independent of membrane voltage and, significantly, there was substantial block of the channels even at very negative potentials where the open probability was quite small. state - independent block of bk channels by tba in rb solutions. voltage dependence of bk channel activation () and fraction of channels not blocked by tba () in 100 m ca. data from six to nine experiments, except n = 3 at 90 mv. replacing k with rb as the permeant ion also had a profound influence on bk channel block by the bp, as illustrated in fig. 5. the left side of fig. 5 shows examples of bk channel currents in k solutions in the absence (black) and presence (red) of 2 m bp at the indicated potentials. as has been reported previously (li and aldrich, 2006) and can also be seen in fig. 1, the current with k as the permeant ion in the presence of the peptide perfectly follows the control current at the beginning of the depolarization. then only after the channels have been opened by the depolarization is the current inhibited. in contrast (fig. 5, right), there was no such time - dependent change of current in the presence of the bp in rb solutions : the current recorded with the bp is much smaller than control levels immediately upon depolarization and remains so throughout the pulse. raw currents at the indicated potentials in the absence (black) and presence (red) of 2 m bp recorded in 100 m ca in k (left ; calibration : 1.2 na, 50 msec) and rb (right ; calibration : 0.25 na, 50 msec) solutions. for the rb solutions, the voltage level before depolarization was 100 mv, and after the depolarization it was 80 mv. the data in fig. 6 demonstrate that the lack of time dependence of bp block in rb solutions seen in fig. 5 was because, with rb as the permeant ion, the bp had already blocked bk channels at the holding potential while they were closed. 6 a show bk channel currents recorded in rb solutions in the absence (black traces) and presence (red traces) of 2 m bp at the indicated potentials. it can be seen that the bp strongly blocked bk channels, even at negative potentials where the channel opening probability is low. the main part of fig. 6 a illustrates this same observation over a larger voltage range and also illustrates the reversible nature of bp block in rb solutions. fig. 6 b shows the relationship between channel activation and bp block in this example patch. it is clear that with rb as the permeant ion, the bp blocked closed channels essentially as well as it did open channels, in contrast to blocking only open channels when k was the permeant ion (fig. (a ; insets) time course of raw bk channel currents recorded in 100 m ca in the absence (black) and presence (red) of 2 m bp at the potentials indicated. (main) steady - state current voltage relation before (), during (), and after () application of 2 m bp in rb solutions. (b) voltage dependence of bk channel activation () and fraction of channels not blocked by bp () in 100 m ca. (inset) bk currents in response to repolarization to 80 mv from + 40 mv in the absence (black) and presence (red ; average of four records) of 2 m bp, with the peak current in the bp scaled to match that of the control record. b shows bk channel tail currents recorded in rb in the absence (black) and presence (red) of the bp, with the amplitude of the record in the presence of the bp scaled to match the control data. it is clear that the bp had no effect on the ability of the bk channels to close when rb was the permeant ion, again in sharp contrast to the strong interference that occurs in k solutions (fig. these differences in the behavior of the bp with rb as the permeant ion were consistent findings, as shown in fig. 7 establishes that, in rb solutions, the bp blocked bk channels essentially independently of whether they were open or closed. 7 shows bk channel closing time constants over a range of potentials in the absence () and presence () of the bp. there was little or no affect of the bp on the channel closing rate with rb as the permeant ion. voltage dependence of bk channel activation () and fraction of channels not blocked by 2 m bp () in 100 m ca. (inset) voltage dependence of the time constant of deactivation in the absence () and presence () of the bp. as described in introduction, the overall weight of evidence strongly supports the existence of an intracellular gate for kv channels. if bk channels are gated differently, for example within the selectivity filter, these two types of channels would likely differ in how permeant ions interact with state - dependent blocking ions. with k as the permeant ion, the bp is an open - channel blocker of shaker k channels and interferes with the channel gate closing (zagotta., we tested if this remained true with rb as the permeant ion, and the results are illustrated in fig. open - state block of shaker k channels by the bp in k and rb solutions. (top inset) currents recorded at 40 mv from a holding potential of 100 mv in the absence (black) and presence (red) of 2 m bp. (main) voltage dependence of shaker channel activation () and fraction of channels not blocked by 2 m bp (). (inset) shaker channel currents in response to repolarization to 100 mv (from + 20 mv) in the absence (black) and presence (red) of 2 m bp, with the peak current in the bp scaled to match that of the control record. (top inset) currents recorded at 40 mv from a holding potential of 100 mv in the absence (black) and presence (red) of 2 m bp. (main) voltage dependence of shaker channel activation () and fraction of channels not blocked by 0.5 m bp (). (inset) shaker channel currents in response to repolarization to 120 mv (from + 20 mv) in the absence (black) and presence (red) of 2 m bp, with the peak current in the bp scaled to match that of the control record. 8 a shows time - dependent block of shaker k channels in k solutions produced by 2 m bp during a depolarization to 40 mv (red trace), and the main part of fig. the inset contains tail currents recorded in the absence (black) and presence (red) of the bp, showing that the bp strongly interfered with channel closing in k solutions. 8 b shows that the open - channel block properties of shaker k channels did not change when rb replaced k as the permeant ion. the time - dependent block by the bp was preserved (fig. 8 b, top), and the voltage dependence of the fraction of channels not blocked by the bp () mirrored channel activation () in rb solutions just as in k solutions. the inset demonstrates that the bp continued to interfere with channel closing, even with rb as the permeant ion, all in sharp contrast with the behavior of bk channels. as summarized in introduction, there is overwhelming evidence that kv channels, including shaker channels, contain a cytoplasmic gate that occludes the ion permeation pathway to prevent ion flow when the channels are in their closed, nonconducting state. depolarization induces a conformational change that removes this intracellular restriction and allows permeant ion flow through the open channel. significant contributions to this picture of the kv channel gate have come from studies that showed that relatively small, intracellular qa compounds like tea and tba can reach their blocking sites in the pore only after the cytoplasmic gate opens upon channel activation (armstrong, 1966 ; armstrong and hille, 1972 ; french and shoukimas, 1981 ; choi., 1993 ; del camino., 2000 ; zhou., 2001). other important results confirming the intracellular location of the gate include the finding that large qa compounds and the bp are not only open - channel blockers of kv channels but also interfere with the channel s activation gate (demo and yellen, 1991 ; choi., 1993). as in kv channels, c10 and the bp can only block open bk channels, and the bp interferes with bk channel closing (li and aldrich, 2004, 2006), which suggests that the bk channel may also be gated at the intracellular end of the pore in these channels. this view would seem to be supported by our data with tba in k solutions showing that this small qa also required an open channel for block. however, there is strong evidence that a cytoplasmic gate does not guard the inner entrance to bk channels. a bulky tba derivative, bbtba, does not require open, conducting bk channels to block the channel pore (wilkens and aldrich, 2006 ; tang., 2009), and deep pore sites near the selectivity filter can be modified by large methanethiosulfonate derivatives, even when the channels are closed (zhou., 2011). thus, the location of the physical gate of bk channels must lie elsewhere than at the cytoplasmic end of the protein. if the physical gate of bk channels is not at the inner end of the pore as it is in kv channels, where could it be ? considering the limited extent of the pore, there are only a few possible locations, and the selectivity filter has been suggested to be the leading candidate (li and aldrich, 2006 ; piskorowski and aldrich, 2006 ; tang., 2009). the selectivity filter in bk channels is able to clearly distinguish between the very similar k and rb ions, so if the selectivity filter is the actual physical gate, it would be expected that there would be large differences between the gating of k and rb ions. as described in introduction, there are differences, but these are rather small and, furthermore, also occur in kv channels with their cytoplasmic gate. thus, there is no definitive test that the bk channel gates within the selectivity filter. however, a recent study provided sufficient evidence to keep selectivity filter gating in the running (chen and aldrich, 2011). in this study, the authors found a strong coupling between the protonation of the side chain of a deep pore amino acid and bk channel gating. these results indicate that channel gating causes a conformational change that alters the physical environment (polar or nonpolar) of the side chain of this amino acid that is at the cytoplasmic entrance to the selectivity filter. in spite of the small effect of permeant ions on bk channel gating, it still seems reasonable to expect that if the selectivity filter is the bk channel gate, there should be permeant ion as reviewed here, there are strong interactions between kv and bk channel gating and pore - blocking ions including tba and the bp, at least in k solutions. there are also strong interactions between permeant ions and pore - blocking ions in a variety of k channels (neyton and miller, 1988a, b ; spassova and lu, 1998 ; immke., 1999 ; immke and korn, 2000 ; guo and lu, 2001 ; thompson and begenisich, 2001, 2003), and there are differences in permeant ion occupancy of the k channel selectivity filter (doyle., 1998 ; morais - cabral., 2001 ; zhou and mackinnon, 2003). these properties of k channels lead us to consider that a sensitive test for selectivity filter gating in bk channels would be the role of permeant ions in the interaction between blocking ions like tba and the bp and channel gating. in this view, a positive test would have two results : (1) a permeant ion specific difference in how tba and the bp interact with the activation gate of bk channels, and (2) no such difference in shaker k channels. we found that tba and the bp were open - channel blockers with k as the permeant ion but blocked closed and open channels when rb replaced k as the permeant ion. in contrast, the state - dependent bp block of shaker channels was largely insensitive to the nature of the permeant ion. thus, we consider our findings to represent a positive test for selectivity filter gating in bk channels. our view is that a bk channel with a closed selectivity filter has a conformation that allows a k ion to bind near the tba- and bp - blocking sites in the pore. these ions can not bind to their blocking sites in the channel as long as k occupies its site, so they can not block a closed channel. the blockers and the k ion need not bind at exactly the same site ; they only need to compete for binding, either directly or electrostatically. in this view, when the bk channel opens, k ions will no longer significantly occupy its site in the selectivity filter, allowing tba and the bp to block the open channel. if rb ions do not significantly occupy the selectivity filter site, tba and the bp will block both closed and open channels when rb is the permeant ion. in this model, the interference of the bp with bk channel closing in k solutions is because the binding of k to the site contributes to the stability of the closed state. thus, because occupancy of the site by the bp and k ions is mutually exclusive, the channel can not be completely closed until bp vacates the site. bp exit from the channel takes much longer than normal deactivation, so closing will be slowed. a selectivity filter gate has been identified in cng channels (contreras and holmgren, 2006 ; contreras., 2008), which are nonselective ion channels that are similar in structure to k channels, especially in the selectivity filter region. cng channels are activated by the binding of cyclic nucleotides to the channel s large intracellular domain (kaupp and seifert, 2002), analogous to the binding of ca to the large cytoplasmic domain of bk channels. it may be that coupling the binding of an intracellular ligand to the opening of the channel pore is best accomplished with a selectivity filter gate rather than by the intracellular parts of the sixth membrane - spanning domain, as occurs in kv channels. in any case, our results demonstrate large differences in how blocking ions like tba and the bp interact with the gating in kv and bk channels. the simplest interpretation of our findings is that the activation gates in bk channels reside within the selectivity filter rather than in the intracellular end of the s6 domain, which forms the gating region of kv channels. | membrane voltage controls the passage of ions through voltage - gated k (kv) channels, and many studies have demonstrated that this is accomplished by a physical gate located at the cytoplasmic end of the pore. critical to this determination were the findings that quaternary ammonium ions and certain peptides have access to their internal pore - blocking sites only when the channel gates are open, and that large blocking ions interfere with channel closing. although an intracellular location for the physical gate of kv channels is well established, it is not clear if such a cytoplasmic gate exists in all k+ channels. some studies on large - conductance, voltage- and ca2 + -activated k+ (bk) channels suggest a cytoplasmic location for the gate, but other findings question this conclusion and, instead, support the concept that bk channels are gated by the pore selectivity filter. if the bk channel is gated by the selectivity filter, the interactions between the blocking ions and channel gating should be influenced by the permeant ion. thus, we tested tetrabutyl ammonium (tba) and the shaker ball peptide (bp) on bk channels with either k+ or rb+ as the permeant ion. when tested in k+ solutions, both tba and the bp acted as open - channel blockers of bk channels, and the bp interfered with channel closing. in contrast, when rb+ replaced k+ as the permeant ion, tba and the bp blocked both closed and open bk channels, and the bp no longer interfered with channel closing. we also tested the cytoplasmically gated shaker k channels and found the opposite behavior : the interactions of tba and the bp with these kv channels were independent of the permeant ion. our results add significantly to the evidence against a cytoplasmic gate in bk channels and represent a positive test for selectivity filter gating. |
the study was approved by the university malaya medical centre (ummc) medical ethics committee. | staphylococcus haemolyticus is one of the pathogens that harbor a high level of antibiotic resistance. here, we highlighted the potential determinants for multidrug resistance and virulence from the draft genome of staphylococcus haemolyticus strain c10a, isolated from a patient with chronic obstructive pulmonary disease exacerbation. |
cell transplantation into the damaged myocardium (cell therapy) has received extensive attention as a regenerative tool, and the accumulated evidence from both pre - clinical and clinical studies suggests that it has the potential to restore heart function. currently, cellular cardiomyoplasty is being actively explored as therapy for regenerating the damaged myocardium, using different cell types, mostly derived from bone marrow or skeletal muscle. the clinical trials have highlighted the need to improve survival, engraftment and differentiation of the transplanted cells. clearly a number of issues remain including the number of cells, the choice of an appropriate cell source that is able to provide all the main cell types of the heart and also the poor retention of the transplanted cells. in this review we will focus on cardiac tissue engineering (cte), with particular attention to the improvements that this methodology can offer compared to conventional cell injection therapy. during the last few years, cellular therapy for the diseased heart has shown encouraging results on cardiac function in animal models of heart ischemia, using a variety of different cells, even without clear cardiovascular differentiation of the transplanted cells. several years after the first human clinical applications using non - cardiac stem cells as a therapy for acute myocardial infarction, heart failure or refractory angina, it should be recognized that the results are mixed, with benefits ranging from absent to transient, but marginal at most. pre - clinical studies demonstrated promising results and also short - term effects in patients were observed ; however, in most trials long - term follow - up has shown that conventional pharmacological therapy often has the same late outcome as cellular therapy. cell therapy decreased the death rate of the endogenous myocytes, improved neoangiogenesis or positively affected ventricular remodelling in short - term follow - up, probably by secretion of paracrine factors. the small improvements in ventricular function, though, especially in the long - term follow - up, are probably because of the inability of the cells to actually form new cardiomyocytes. the first limitation of direct cell injection is the considerable cell death that occurs in the first 7 days after infarction because of inflammation and ischemia. zhang. found that high levels of cardiomyocyte death occurred within 4 days after implantation into injured hearts, and suggested ischemia as the main cause. similar data were reported in various animal models, thereby using different cell types and routes of administration. generally more than 50% of the cells die within the first days after delivery, and only 510% of surviving cells could be reported in a pig and a rat model. the ischemic environment induces cell death and needs vascularization because survival of transplanted cells is directly proportional to tissue perfusion. the lack of matrix, particularly important during collection and preparation of the cells for transplantation, can also induce cell death and is mediated by the anoikis signalling pathway. different strategies have been developed in order to enhance cell survival upon transplantation, and include the use of bioactive cytoprotective molecules to prevent cell death. studies with transgenic overexpression of akt, bcl-2 or hypoxia inducible factor-1, showed enhanced survival. in addition, in vitro pre - conditioning or simultaneous administration with insulin - like growth factor 1 (igf-1), vegf or free radical scavengers are all strategies that have demonstrated to increase the benefits of cell therapy. however, co - administration of bioactive molecules results in a temporary benefit. alternatively, genetic modification of transplantable cells, in order to enhance survival, has been tried, but remains risky for possible tumorigenic side effects. to this end, cte represents a possible solution to increase cell survival and cell retention in the heart. different bioactive molecules can be linked to a specific matrix in order to provide their controlled release. coupling pdgf - bb and igf-1 to self - assembling peptide nanofibres demonstrated a prolonged delivery of the growth factors and resulted in an improved cardiac function compared to a control group that received only a single bolus injection [5, 6 ]. interestingly, it was recently demonstrated that injection of rat cardiac progenitor cells (cpcs) together with igf-1 modified peptide nanofibres (nf - igf-1) enhanced cardiac function. the combination preserved left ventricular (lv) function and increased myocyte regeneration, as compared to saline, cells or nf - igf-1 only. although it is possible to improve cell survival by increasing resistance to death stimuli, another important problem in cardiomyoplasty is related to low cell retention in the heart. different studies demonstrated that a large number of cells are trapped in other organs, if injected systemically, or leaking from the site of myocardial injection [8, 9 ]. when cells are injected into an arrested heart by thiopental injection, cell retention is four times higher than injection into a beating heart. this indicates that cardiac contraction and/or myocardial perfusion are important in the early washout of cells. in addition, an increase in cell retention is obtained by fibrin glue application on the site of injection. survival and retention can also be improved when cells are transplanted together with a matrix such as liquid collagen or fibrin glue, resulting in improved cardiac function and remodelling. it is evident that the first step to optimize the repair of a damaged myocardium is to increase cell retention and survival. this aspect is particularly important if we consider the absolute number of cardiomyocytes that are lost after mi and that one of the main limitations of cellular therapy is the number of cells that are theoretically needed to replace them. however, an appropriate cell source is also fundamental. in order to obtain regeneration and to restore the damaged myocardium, cells should be used that are available in large numbers and capable of differentiating into cardiomyocytes and all the other cell types composing the heart. cardiac stem / progenitor cells seem a logical cell source to exploit for cardiac regeneration therapy. their presence in the heart, the frequent co - expression of early cardiac progenitor transcription factors and the capability for ex vivo and in vivo differentiation towards cardiac lineages, offer promise of enhanced cardiogenic potency compared to other cell sources. in ischemic heart disease both cardiomyocytes and the extracellular matrix are pathologically disrupted or modified. therefore, it could be important to exploit a combined procedure aiming at regenerating both myocardial cells and the extracellular matrix. with the increased knowledge in (stem) cell biology, cte is developing as a strategy to combine scaffold material and cells in order to provide a regenerative approach. the matrix should provide mechanical support to ventricular chamber integrity, in order to limit ventricular wall dilatation, and also provide a favourable environment for transplanted cells to enhance cell survival, proliferation and differentiation. the ideal matrix should : (i) be biodegradable, (ii) not induce any immune response by the host, (iii) support electro - mechanical properties of the heart and be replaced with newly synthesized ecm. recent advances in cell culture and cte have facilitated the development of suitable cell - engineered, biodegradable grafts. the optimal biomaterials and cell types, however, have not been identified. moreover, even a cell scaffold combination ideal for a defined pathology (e.g. post - infarct limited scar) might not be adequate for another one (e.g. end - stage hf because of dilated cardiomiopathy or congenital malformation). two main approaches of cte are performed : in vitro and in vivo cte. in vitro cte the bio - complex is then cultivated in vitro and applied on the epicardial surface. in the in vivo cte approach cells are injected with the matrix and formation of the biocomplex occurs at the site of injection. in vivo or in situ cte has recently emerged and involves injection of a mixture of biomaterials and cells. with this approach, this will improve survival and retention of the transplanted cells at the same time, and provide mechanical support to the damaged ventricle. the aim is to obtain regeneration and replacement of the damaged tissue in the natural milieu of the damaged myocardium. this approach is easy and feasible, but cell growth and differentiation can not be controlled as tightly as in the in vitro model. christman. transplanted myoblasts, fibrin glue or myoblasts in fibrin glue in rats at 1 week after mi and reported that, 24 hrs after injection, no differences in engrafted cells could be observed between cells transplanted with or without fibrin glue. however, after 4 weeks the percentage of engrafted cells was significantly higher with the combined treatment. both fibrin glue treatments displayed a small significant reduction of the scar size as compared with cell injection alone, probably because of improved neovascularization of the infarcted area. when fibrin glue was injected directly into the scar in chronic heart failure (e.g. 5 weeks after mi), the treatment improved fs, increased wall thickness and lv internal diameter) already at 2 days after injection. interestingly, 5 weeks after injection (10 weeks after mi), deterioration of fs was observed in both groups, although wall thickness was preserved in the fibrin - injected group. one of the advantages of injectable biopolymers is the possibility to deliver the hydrogel percutaneously via catheters, thereby avoiding large surgical procedures and the associated risks and costs. studied different catheter delivery methods with variable concentrations of fibrinogen and thrombin, and demonstrated that this approach was feasible for future clinical application. in addition, they demonstrated that, by using human msc full, cardiac - specific retention was increased after transplantation in a rat model of acute mi when cells were transplanted with fibrin compared to saline. this resulted in a decrease in the presence of mscs in liver and kidney, but not in the lungs. collagen can also be used in a liquid form thereby having the advantage that cells are better and more homogeneously distributed than in the preformed scaffold. the easy gelation at physiological temperature and the possibility to combine with other hydrogels (e.g. engineered heart tissue model [eht ]) are useful features of this protein. in addition to in vitro preparation tissue engineering, huang. injected liquid collagen in a rat model of reperfusion injury. they demonstrated a higher number of capillaries 5 weeks after matrix injection when compared to saline group, but similar to fibrin or matrigel usage. in a similar study, injection of collagen in contrast with the previous study, no matrix infiltrating cells or induction of vascularization were observed in the histological analysis. the application of alginate as a gel directly injected into the heart has been also reported. when alginate was injected directly in the myocardial wall of a 7-day - old mi, the alginate matrix significantly increased scar thickness, systolic and diastolic wall thickening, and cardiac function as compared to saline or cardiomyocyte injection alone. similar beneficial effects in scar thickness and remodelling were obtained also when alginate was injected in a 2-month - old infarct. used a vegf - a or pdgf - bb modified alginate scaffold and injected this into a mi model. the controlled release of the growth factors (alone or in combination) from the matrix was confirmed experimentally in vitro. although the addition of growth factors induced vessel formation and increased smooth muscle cell infiltration, no differences in left ventricle diastolic diameter and ejection fraction could be observed. another intriguing approach is the use of a - cellular matrix as a gel that can be injected into the heart. singelyn. recently reported the isolation of ecm from pig ventricle as a gel. this cardiac ecm is a viscous liquid solution up to room temperature, and will become a gel at 37c. preliminary studies demonstrated that the gel, when placed in a clinically compatible catheter, can be pushed through with minimal resistance, indicating the clinical applicability of the materials. in vitro cte cells are cultivated under strict culture conditions in order to enhance survival, proliferation and differentiation. the formed patch is then applied onto the epicardial surface of the heart in order to provide mechanical support to the damaged heart and enhance cardiac performance. the matrix and cultivation parameters play an important role to guide and organize seeded cells in order to control and obtain a myocardial patch that is as similar to native heart tissue as possible. the advantage of the in vitro approach is the possibility to control the shape and size of the construct, as well as organization and differentiation rate of the seeded cells. the main limitation of this approach is the need for nutrient diffusion that limits the thickness of the construct itself. therefore a vascularized tissue is required, together with mature contractile myocardial cells. the use of a perfusion - bioreactor can improve viability of the cultivated cells and/or enhance differentiation by applying mechanical stress. different types of materials have been used to cultivate different cell types and then applied on the damaged ventricular wall to evaluate heart regeneration. one of the first well - established 3d - myocardial in vitro models was created almost 10 years ago by zimmerman., and called eht. ehts consist of a mixture of liquid collagen, growth factor - reduced matrigel and neonatal rat cardiomyocytes. the mixture is then casted into a circular mould, statically cultivated for 7 days and then subjected to mechanical stretch for seven more days. when transplanted into the heart the constructs integrated fully in the host myocardium 4 weeks after in vivo implantation, having a diameter of about 450 m, and formed new capillaries connected with the recipient s vessels. engraftment and electrical coupling of the eht patches was demonstrated by the propagation of electrical responses in remote myocardium and improvement of myocardial function. mixed neonatal cardiomyocytes and fibrin gel in a small silicone tube, made a longitudinal slit and placed the tube around the femoral artery of a recipient rat, to generate an intrinsic supply. cardiac - like contractility properties and a positive chronotropic response to epinephrine and a positive inotropic response to ca. preformed porous dry collagen has been already used in clinical settings for 30 years as a haemostatic agent to repair tissue injuries and prevent haemorrhages. kofidis. utilized preformed collagen matrix for the first time to cultivate neonatal rat cardiomyocytes and presented a model of in vitro contractile cardiac tissue. when these constructs were attached to the epicardial surface of infarcted rat hearts, they induced neovascularization and reduced lv dilatation up to 3 weeks. we seeded human umbilical cord derived stem cells in the same collagen matrices and transplanted them into 7-day - old infarcts in a mouse model. the cell - matrix treatment prevented myocardial wall thinning, limited post - ischemic remodelling and displayed a lower end - diastolic volume and improved ejection fraction, compared to single treatments. so this approach seems to improve the efficiency of cell treatment at the time - points studied (1 and 6 weeks), corresponding to an improvement in an early post - infarct cardiac remodelling, vascularization and angiogenesis, but the data are not demonstrating real myocardial regeneration. the clinical relevance of this matrix was also tested in a small non - randomized clinical trial with patients having a chronic scar, an ef < 35% and indication of concomitant single off - pump coronary artery bypass graft surgery. ten patients received direct injection of bone marrow mononuclear cells and ten patients received cells seeded into a scaffold of 7 5 0.6 cm and sutured over the infarct and peri - infarct areas. after 1 year follow - up (10 3.5 months), no adverse events were reported, suggesting feasibility and safety of the treatment. in addition, improved deceleration time, decreased lv end - diastolic volume and reduced scar area thickness could be observed. although promising, further studies are warranted in order to better evaluate cardiac function improvement through this scaffold, because of the concomitant coronary artery bypass graft surgery mediated revascularization. further modifications of this pre - formed collagen matrix were studied by schussler. they coupled an rgd peptide to the collagen material in order to increase viability, contractile performance and differentiation of seeded cardiomyocytes. when compared to non - modified collagen, rgd modified scaffold increased viability of transplanted cardiomyocytes up to 1 month in vitro. in addition, the cells were better aligned and elongated, with the presence of cross striations, and contractile performance was increased threefold as compared with the non - modified scaffolds. as mentioned before, the choice of an appropriate cell source is fundamental in order to obtain improvement in cardiac function, not only by secretion of paracrine factors and by improving vasculogenesis, but also by regeneration of cardiomyocytes. to this end we cultivated cpcs in the form of cardiospheres, and cultivated them in a preformed commercially available collagen scaffold or in gelatine - based scaffolds [27, 28 ]. cpcs grown as cardiospheres represent per se a real cardiac microtissue, in which the more differentiated cells are located in the external layers whereas those proliferating and expressing stemness and angiogenic markers are in the core. moreover our previous observations [26, 29, 30 ] suggest that the csps microtissue might fulfil important criteria of tissue engineering technology for cardiac diseases, that is : long - term maintenance of differentiation capacity, in vitro and in vivo, multicellular type cultivation, potential for self - organization, polarization and microstructure formation between different cells, production of an extracellular matrix, vascularization, including induction of microvessels development, and connection to the host capillary system after implantation (after heterotopic injection), development of inter - tissue super - structures and compatibility with high - efficiency stable gene transfer technologies to engineer complementary cell phenotypes or provide therapeutic interventions (after both lenti / adeno / adeno - associated virus infection). thus even if csps alone appear to provide an optimal cardiogenic environment, preliminary data shows that csps can grow within and populate different kinds of scaffolds currently in use for tissue engineering. in particular csps seeded in collagen or gelatine - based scaffolds have shown prolonged viability during culture (up to 7 days or more) (fig. some differences in the spreading out and production of extracellular matrix were observed, with a more consistent proliferation in the commercial collagen scaffold. (a) csps pre - labelled with the fluorescent vital, seeded in collagen (a1 and a2) or gelatine (a3 and a4) matrix and cultivated for 1 week ; (b) scanning electron microscope analysis of csps in collagen and gelatine scaffolds ; (c) rt - pcr analysis of csps cultivated in the matrices. csps displayed very good integration with both matrices, as demonstrated by scanning electron microscope analysis (fig. in addition rt - pcr analysis for the expression of cardiac markers was performed, and demonstrated that after 7 days in culture csps seeded in collagen and gelatine scaffolds retained their cardiogenic commitment, as demonstrated by expression of nkx 2.5, gata-4, connexin-43 and tni (fig. our hypothesis is that autologous cardiac stem / progenitor cells, in combination with the optimal biodegradable biomaterial, can be used to build tissue - engineered cardiac patches, which could preserve survival, growth and differentiation potential of the embedded cells. moreover, this biocomplex could even serve as a cellular reservoir, allowing their slow migration along the epicardial surface to the damaged cardiac tissue, where they could exert a paracrine function, encouraging both local angiogenic / anti - apoptotic activity and resident progenitors recruitment and activation these cells, named cardiomyocytes progenitor cells appeared to be cpcs, restricted to the cardiac lineage that is able to differentiate into cardiomyocytes under precise conditions. preliminary data indicate that cardiomyocytes progenitor cells seeded in preformed collagen matrix are able to proliferate and to colonize the scaffold and could represent another cell source for cte. in vivo or in situ cte has recently emerged and involves injection of a mixture of biomaterials and cells. with this approach, this will improve survival and retention of the transplanted cells at the same time, and provide mechanical support to the damaged ventricle. the aim is to obtain regeneration and replacement of the damaged tissue in the natural milieu of the damaged myocardium. this approach is easy and feasible, but cell growth and differentiation can not be controlled as tightly as in the in vitro model. christman. transplanted myoblasts, fibrin glue or myoblasts in fibrin glue in rats at 1 week after mi and reported that, 24 hrs after injection, no differences in engrafted cells could be observed between cells transplanted with or without fibrin glue. however, after 4 weeks the percentage of engrafted cells was significantly higher with the combined treatment. both fibrin glue treatments displayed a small significant reduction of the scar size as compared with cell injection alone, probably because of improved neovascularization of the infarcted area. when fibrin glue was injected directly into the scar in chronic heart failure (e.g. 5 weeks after mi), the treatment improved fs, increased wall thickness and lv internal diameter) already at 2 days after injection. interestingly, 5 weeks after injection (10 weeks after mi), deterioration of fs was observed in both groups, although wall thickness was preserved in the fibrin - injected group. one of the advantages of injectable biopolymers is the possibility to deliver the hydrogel percutaneously via catheters, thereby avoiding large surgical procedures and the associated risks and costs. studied different catheter delivery methods with variable concentrations of fibrinogen and thrombin, and demonstrated that this approach was feasible for future clinical application. in addition, they demonstrated that, by using human msc full, cardiac - specific retention was increased after transplantation in a rat model of acute mi when cells were transplanted with fibrin compared to saline. this resulted in a decrease in the presence of mscs in liver and kidney, but not in the lungs. collagen can also be used in a liquid form thereby having the advantage that cells are better and more homogeneously distributed than in the preformed scaffold. the easy gelation at physiological temperature and the possibility to combine with other hydrogels (e.g. engineered heart tissue model [eht ]) are useful features of this protein. in addition to in vitro preparation tissue engineering, huang. injected liquid collagen in a rat model of reperfusion injury. they demonstrated a higher number of capillaries 5 weeks after matrix injection when compared to saline group, but similar to fibrin or matrigel usage. in a similar study, injection of collagen improved cardiac stroke volume and ejection fraction. however, in contrast with the previous study, no matrix infiltrating cells or induction of vascularization were observed in the histological analysis. the application of alginate as a gel directly injected into the heart has been also reported. when alginate was injected directly in the myocardial wall of a 7-day - old mi, the alginate matrix significantly increased scar thickness, systolic and diastolic wall thickening, and cardiac function as compared to saline or cardiomyocyte injection alone. similar beneficial effects in scar thickness and remodelling were obtained also when alginate was injected in a 2-month - old infarct. used a vegf - a or pdgf - bb modified alginate scaffold and injected this into a mi model. the controlled release of the growth factors (alone or in combination) from the matrix was confirmed experimentally in vitro. although the addition of growth factors induced vessel formation and increased smooth muscle cell infiltration, no differences in left ventricle diastolic diameter and ejection fraction could be observed. another intriguing approach is the use of a - cellular matrix as a gel that can be injected into the heart. singelyn. recently reported the isolation of ecm from pig ventricle as a gel. this cardiac ecm is a viscous liquid solution up to room temperature, and will become a gel at 37c. preliminary studies demonstrated that the gel, when placed in a clinically compatible catheter, can be pushed through with minimal resistance, indicating the clinical applicability of the materials. cells are cultivated under strict culture conditions in order to enhance survival, proliferation and differentiation. the formed patch is then applied onto the epicardial surface of the heart in order to provide mechanical support to the damaged heart and enhance cardiac performance. the matrix and cultivation parameters play an important role to guide and organize seeded cells in order to control and obtain a myocardial patch that is as similar to native heart tissue as possible. the advantage of the in vitro approach is the possibility to control the shape and size of the construct, as well as organization and differentiation rate of the seeded cells. the main limitation of this approach is the need for nutrient diffusion that limits the thickness of the construct itself. therefore a vascularized tissue is required, together with mature contractile myocardial cells. the use of a perfusion - bioreactor can improve viability of the cultivated cells and/or enhance differentiation by applying mechanical stress. different types of materials have been used to cultivate different cell types and then applied on the damaged ventricular wall to evaluate heart regeneration. one of the first well - established 3d - myocardial in vitro models was created almost 10 years ago by zimmerman., and called eht. ehts consist of a mixture of liquid collagen, growth factor - reduced matrigel and neonatal rat cardiomyocytes. the mixture is then casted into a circular mould, statically cultivated for 7 days and then subjected to mechanical stretch for seven more days. when transplanted into the heart the constructs integrated fully in the host myocardium 4 weeks after in vivo implantation, having a diameter of about 450 m, and formed new capillaries connected with the recipient s vessels. engraftment and electrical coupling of the eht patches was demonstrated by the propagation of electrical responses in remote myocardium and improvement of myocardial function. mixed neonatal cardiomyocytes and fibrin gel in a small silicone tube, made a longitudinal slit and placed the tube around the femoral artery of a recipient rat, to generate an intrinsic supply. cardiac - like contractility properties and a positive chronotropic response to epinephrine and a positive inotropic response to ca. preformed porous dry collagen has been already used in clinical settings for 30 years as a haemostatic agent to repair tissue injuries and prevent haemorrhages. utilized preformed collagen matrix for the first time to cultivate neonatal rat cardiomyocytes and presented a model of in vitro contractile cardiac tissue. when these constructs were attached to the epicardial surface of infarcted rat hearts, they induced neovascularization and reduced lv dilatation up to 3 weeks. we seeded human umbilical cord derived stem cells in the same collagen matrices and transplanted them into 7-day - old infarcts in a mouse model. the cell - matrix treatment prevented myocardial wall thinning, limited post - ischemic remodelling and displayed a lower end - diastolic volume and improved ejection fraction, compared to single treatments. so this approach seems to improve the efficiency of cell treatment at the time - points studied (1 and 6 weeks), corresponding to an improvement in an early post - infarct cardiac remodelling, vascularization and angiogenesis, but the data are not demonstrating real myocardial regeneration. the clinical relevance of this matrix was also tested in a small non - randomized clinical trial with patients having a chronic scar, an ef < 35% and indication of concomitant single off - pump coronary artery bypass graft surgery. ten patients received direct injection of bone marrow mononuclear cells and ten patients received cells seeded into a scaffold of 7 5 0.6 cm and sutured over the infarct and peri - infarct areas. after 1 year follow - up (10 3.5 months), no adverse events were reported, suggesting feasibility and safety of the treatment. in addition, improved deceleration time, decreased lv end - diastolic volume and reduced scar area thickness could be observed. although promising, further studies are warranted in order to better evaluate cardiac function improvement through this scaffold, because of the concomitant coronary artery bypass graft surgery mediated revascularization. further modifications of this pre - formed collagen matrix were studied by schussler. they coupled an rgd peptide to the collagen material in order to increase viability, contractile performance and differentiation of seeded cardiomyocytes. when compared to non - modified collagen, rgd modified scaffold increased viability of transplanted cardiomyocytes up to 1 month in vitro. in addition, the cells were better aligned and elongated, with the presence of cross striations, and contractile performance was increased threefold as compared with the non - modified scaffolds. as mentioned before, the choice of an appropriate cell source is fundamental in order to obtain improvement in cardiac function, not only by secretion of paracrine factors and by improving vasculogenesis, but also by regeneration of cardiomyocytes. to this end we cultivated cpcs in the form of cardiospheres, and cultivated them in a preformed commercially available collagen scaffold or in gelatine - based scaffolds [27, 28 ]. cpcs grown as cardiospheres represent per se a real cardiac microtissue, in which the more differentiated cells are located in the external layers whereas those proliferating and expressing stemness and angiogenic markers are in the core. moreover our previous observations [26, 29, 30 ] suggest that the csps microtissue might fulfil important criteria of tissue engineering technology for cardiac diseases, that is : long - term maintenance of differentiation capacity, in vitro and in vivo, multicellular type cultivation, potential for self - organization, polarization and microstructure formation between different cells, production of an extracellular matrix, vascularization, including induction of microvessels development, and connection to the host capillary system after implantation (after heterotopic injection), development of inter - tissue super - structures and compatibility with high - efficiency stable gene transfer technologies to engineer complementary cell phenotypes or provide therapeutic interventions (after both lenti / adeno / adeno - associated virus infection). thus even if csps alone appear to provide an optimal cardiogenic environment, preliminary data shows that csps can grow within and populate different kinds of scaffolds currently in use for tissue engineering. in particular csps seeded in collagen or gelatine - based scaffolds have shown prolonged viability during culture (up to 7 days or more) (fig. some differences in the spreading out and production of extracellular matrix were observed, with a more consistent proliferation in the commercial collagen scaffold. (a) csps pre - labelled with the fluorescent vital, seeded in collagen (a1 and a2) or gelatine (a3 and a4) matrix and cultivated for 1 week ; (b) scanning electron microscope analysis of csps in collagen and gelatine scaffolds ; (c) rt - pcr analysis of csps cultivated in the matrices. csps displayed very good integration with both matrices, as demonstrated by scanning electron microscope analysis (fig. in addition rt - pcr analysis for the expression of cardiac markers was performed, and demonstrated that after 7 days in culture csps seeded in collagen and gelatine scaffolds retained their cardiogenic commitment, as demonstrated by expression of nkx 2.5, gata-4, connexin-43 and tni (fig. our hypothesis is that autologous cardiac stem / progenitor cells, in combination with the optimal biodegradable biomaterial, can be used to build tissue - engineered cardiac patches, which could preserve survival, growth and differentiation potential of the embedded cells. moreover, this biocomplex could even serve as a cellular reservoir, allowing their slow migration along the epicardial surface to the damaged cardiac tissue, where they could exert a paracrine function, encouraging both local angiogenic / anti - apoptotic activity and resident progenitors recruitment and activation these cells, named cardiomyocytes progenitor cells appeared to be cpcs, restricted to the cardiac lineage that is able to differentiate into cardiomyocytes under precise conditions. preliminary data indicate that cardiomyocytes progenitor cells seeded in preformed collagen matrix are able to proliferate and to colonize the scaffold and could represent another cell source for cte. cte is a rapidly evolving field that is receiving increased attention as a promising approach to repair the damaged myocardium. cardiac cell therapy has been extensively studied in the last decade and different clinical trials were performed with several cell types. despite the sometimes conflicting results, feasibility and safety of cellular therapy was demonstrated, although the benefits, in terms of long - term improvement of cardiac function, are still doubtful. cte offers the advantage to shelter transplanted cells by providing a physiological environment and by protecting them from death stimuli, overcoming two of the main problems of direct cell injection. in addition, transplantation of cells together with a matrix provides mechanical support to the infarcted ventricular wall and can prevent post - infarction ventricular dilatation. the choice of the appropriate cell source and matrix plays a fundamental role in a successful tissue engineering application. for cardiac use, cardiomyocytes have been extensively studied as a possible cell source for heart regeneration. however, their use is not clinically applicable and other cell sources need to be considered. cpcs seem to be the most promising cell source because of their intrinsic capacity to proliferate, thereby providing suitable cell numbers, but most importantly for their natural commitment and differentiation potential to the cardiac lineages. | abstracttissue engineering is an increasingly expanding area of research in the cardiovascular field that involves engineering, chemistry, biology and medicine. cardiac tissue engineering (cte) aims to regenerate myocardial damage by combining cells, matrix, biological active molecules and physiological stimuli. the rationale behind cte applications is that in order to regenerate the ventricular wall after a myocardial infarction it is necessary to combine procedures that regenerate both cardiomyocytes and the extracellular matrix. the application of (stem) cells together with a matrix could represent an environment protected from the inflammatory and pro - apoptotic signals, a stemness / survival reservoir slowly releasing cells and factors promoting tissue regeneration and angiogenesis. this review will focus on the applications and advantages that cte application could offer compared to conventional cell therapy. |
dokonano analizy dokumentacji medycznej pacjentw z samoistn odm opucnow leczonych w klinice chirurgii dziecicej uniwersytetu medycznego w biaymstoku. w latach 20042014 hospitalizowano 22 dzieci z powodu samoistnej odmy opucnowej 18 chopcw i 4 dziewczynki, w wieku od 14 do 17 lat (rednio 16,6 miesica). w 11 przypadkach (52%) w leczeniu jednostronnej odmy opucnowej zastosowano jednorazowy drena jamy opucnowej, z dobrym skutkiem. w pozostaych 11 przypadkach wystpi nawrt odmy, w czasie od 3 tygodni do 2 lat. u pacjentw z odm nawrotow wykonano drena jamy opucnowej oraz tomografi komputerow (tk) klatki piersiowej. szeciu pacjentw z tej grupy, na podstawie obrazu tk, w ktrym stwierdzono obecno pcherzy rozedmowych, zakwalifikowano do torakoskopii (video - assisted thoracoscopic surgery vats). w trakcie vats wykonano zamknicie przetok oskrzelowo - opucnowych w obrbie pknitych pcherzy rozedmowych za pomoc ptli endoloop. u chopca, u ktrego nie stwierdzono efektu terapeutycznego po vats, wykonano minitorakotomi z dostpu pod pach za pomoc staplera usunito szczyt puca oraz wykonano pleurodez, z dobrym skutkiem. poznanie czynnikw ryzyka powstania nawrotowej samoistnej odmy opucnowej pozwala na kwalifikowanie chorych do odpowiednich, maoinwazyjnych procedur leczniczych vats. w przypadku niepowodzenia vats, minitorakotomia pachowa zapewnia powodzenie leczenia, zaoszczdzajc choremu blu pooperacyjnego i zapewniajc dobry efekt kosmetyczny. a primary spontaneous pneumothorax (psp) is defined as a spontaneously occurring pneumothorax in a person without clinically apparent underlying lung disease or trauma. the psp occurs at a frequency of 7.4 - 18 cases per 100,000 population per year in men and 1.2 - 6 cases per 100,000 population per year in women. the psp typically occurs in young adults (peak age incidence 20 - 30 years) and is uncommon in the paediatric population [2, 3 ]. therefore the research on psp has focused on management strategies in adults, and paediatric population data are lacking. the management of psp varies and depends on the experience and the speciality of the physician - in - charge. the aim of this study was to review paediatric spontaneous pneumothorax and describe diagnostic and therapeutic approaches and to review our institutional experience with spontaneous pneumothorax. the patients in this study consisted of 22 consecutive paediatric patients with confirmed psp, who were treated from 2004 to 2014 at the paediatric surgery clinic and were identified from an administrative database. we excluded patients with secondary pneumothorax due to external or surgical trauma, underlying lung disease (asthma, malignancy, infection, connective tissue disease, or congenital lung disease). demographic and clinical data were collected, including preoperative factors (age, height, weight), duration of hospital stay from the time of presentation to the ed to discharge, length / duration / type of preoperative management, type of procedure, recurrence of pneumothorax, failure rate of the procedure, length of postoperative air leak, narcotic requirement, and complications. the patients were predominantly male there were 18 boys and 4 girls (boys to girls ratio 4 : 1). the mean age of the patients was 16 years, 6 months month (range 14 - 17). the mean body mass index was 20.1 (ranging from 17 to 24). in all patients, the diagnosis of psp was confirmed by chest radiographs. in 11 patients (52%), chest tubes inserted on diagnosis of the pneumothorax were removed 48 hours after placement and resulted in resolution of the pneumothorax without need for additional intervention. one patient with persistent air leak after chest tube placement for more than seven days have had computed tomography (ct) of the chest, and after confirming of the existence of apical blebs was treated with video - assisted thoracoscopic surgery (vats) procedure with success. another 10 patients were hospitalised because of recurrence of psp, from three weeks to two years after the first episode of psp (mean 5 months 1 month). all of those patients were treated with thoracostomy and had ct of the chest performed. in five patients, ct of the chest revealed the apical blebs, and those patients prolonged postoperative air leak (over seven days) was observed in three patients (50%) ; those patients required reoperation during the same hospitalisation. one of those patients had resection of the apex of the lung with endo - gia stapler. another patient with recurrent psp, after two vats procedures that failed to visualise apical blebs, was treated with success using an open mini axillary thoracotomy and resection of apical blebs with endo - gia stapler. the tube was placed over a needle after instilling local anaesthetic in the fifth intercostal space at the midaxillary line. chest tubes were secured to the skin with suture, and a sterile dressing was applied after attaching the chest tube to a underwater seal collection system set at 10 cm h2o suction. a chest x - ray was repeated 4 - 6 hours after removal to evaluate for recurrent pneumothorax. three of the patients with pneumothorax required longer than 48 hours before chest tube removal but did not require additional intervention. another two patients were operated on because of persistent air leak after chest tube placement for over seven days. before the surgical intervention, a chest ct was routinely performed for patients with recurrent psp to localise the bullae and blebs, which identified apical blebs in 6 out of 11 patients (54.5%). indications for operation video - assisted thoracoscopic surgery (vats) were : persistent air leak after chest tube placement for more than seven days in one patient, recurrent ipsilateral pneumothorax in four patients, and occurrence of a contralateral pneumothorax in one patient with apical blebs confirmed in ct of the chest. valved trocars were used to allow co2 insufflation to assist with lung compression and to allow visualisation of the entire visceral pleura. video - assisted thoracoscopic surgery procedures were performed under a 5-mm thoracoscopic view using a standard three - port approach. a 5-mm trocar was introduced through an intercostal space in the midaxillary line for insertion of a 30 telescope. if the patient already had a chest tube, this was removed and the chest tube tract was used for insertion of a second 5-mm trocar for insertion of operating instruments. a third trocar was inserted in the anterior axillary line, 5 mm or 12 mm trocar, for insertion of an endoloop or an endo - gia stapler if blebectomy was to be performed. in the absence of evident bullae, 250 ml of saline solution was instilled into the pleural cavity and the lung was ventilated to identify the air leak. at the time of the initial vats procedure, apical blebs were identified in four out of five patients. if blebs were visualised, bleb resection was performed using endoloops or an endoscopic stapling device (figs. the lung was inspected for bleeding and, after pulmonary reinsufflation, checked for air leaks. pleural adhesions after pleural abrasion visible during the second video - assisted thoracoscopic surgery (vats) in a 17-yearold patient apical bleb in a 16-year - old patient resection of apical bleb using an endoloop in a 16-year - old patient line of endo - gia stapler resection of an apex of left lung because of apical blebs in a 17-year - old patient mechanical pleurodesis was achieved using a cautery scratch. at the end of the procedure, the chest tubes were typically removed when the air leak ceased and postoperative chest radiographs showed no residual pneumothorax. on average, postoperative pain medications, which included perfalgan, dolargan, or morphine, were given intravenously or intramuscularly for 1 - 2 days, after which the patients were given oral analgesics. the mean interval between the initial hospitalisation and recurrence was 5 months 1 month (range from 3 weeks to 2 years). recurrent pneumothorax was evacuated by closed thoracostomy with success in four patients (40%). after a second vats procedure in three patients, we did not observe recurrent psp in two of these patients. one patient with recurrent psp, after two vats procedures failed to visualise apical blebs, was treated successfully with an open mini axillary thoracotomy and resection of apical blebs with endo - gia stapler (fig. 1 and 4). the mean follow - up period was 4 years 3 months 1 month (range 6 months 10 years). there were no postoperative deaths, and none of the patients required monitoring in the intensive care unit. data are presented as the mean standard deviation or frequencies and percentages, as appropriate. comparisons were made using mann - whitney u test and, as appropriate, and statistical significance was considered when p < 0.05. patients with primary spontaneous pneumothorax are generally young and healthy. most cases of primary spontaneous pneumothorax (psp) occur in healthy adolescents and in young adults in the absence of prior lung disease. the psp is a relatively rare disease entity in children. according to margolis., who performed a retrospective study of 156 patients with psp, the median age of presentation was 19 years, and 69% of patients were male. the goal in the management of psp is to re - expand the lung and prevent recurrence with minimal morbidity. rupture of blebs and bullae may be responsible for the occurrence of most cases of psp. the formation of bullae is most likely to be multifactorial, including physical characteristics, smoking, anatomic abnormalities of the bronchial tree, sex differences, genetic factors, and growth [5, 7 ]. among our patients there were no smokers, but they generally had asthenic features, and the mean body mass index (bmi) was 20.1. previous studies have suggested that growth during adolescence causes a rapid increase in the vertical dimension of the thorax compared to the horizontal dimension. this rapid increase causes an increase in negative pressure at the apex of the lung, which may lead to formation of bullae and may cause psp upon rupturing [5, 7 ]. the most recent guidelines from the american college of chest physicians consensus statement (2001) recommend that patients with small pneumothorax should be observed in the emergency department for 3 - 6 hours and discharged home if repeat chest radiograph shows no progression of pneumothorax [8, 9 ]. the average rate of recurrent pneumothorax after the first episode of psp with conservative treatment was reported at 30%, and it seems to be higher in children, ranging from 50 to 60% [5, 1012 ]. the british thoracic society recommends needle aspiration as the initial treatment of choice, but the american college of chest physicians consensus prefers insertion of small - bore catheters (14 fr) or chest tubes (16 - 22 fr). needle aspiration is safe and less painful and leads to a shorter hospital stay than chest tube insertion. however, needle aspiration is only successful in approximately 60% of patients, and more than half of these patients needed to be hospitalised. chan and lam in their study found an even lower success rate after simple needle aspiration of psp (50.5%), but only 15% success rate if the pneumothorax was larger than 40%. nonetheless, pain scores were greater in the tube thoracostomy group. in our department we initially treat patients with psp with chest tube insertion, and in many cases only continuous suction allows complete evacuation of air from the pleural space and full expansion of the lung. some authors suggest that life - threatening haemothorax might be related to the use of the anterior approach. there are reports of pulmonary artery injury and cardiac tamponade after decompression of the pneumothorax performed in the second intercostal space, midclavicular line [8, 16 ]. bearing in mind those complications we use the fifth intercostal space in the anterior or mid - axillary line for chest tube drainage, and we have not observed thoracostomy complications in our series of patients. pulmonary fibrosis, a history of smoking, asthenic habitus, and younger age have been reported to be independent risk factors for recurrences after the first episode of psp [5, 17 ]. recurrence rates after a first episode of psp treated with chest drainage vary from 16 to 52%. after a first recurrence, the likelihood of subsequent recurrences seems to increase progressively up to 62% for a second and 83% for a third recurrence, which is why an operative intervention is indicated in such cases [1, 18 ]. some authors advocate surgery also for patients with air - leak persisting beyond 14 days. in our department, patients with psp are qualified for surgical treatment when air - leak persists beyond seven days. prior to that, we perform ct of the chest to identify blebs these patients will benefit from surgical intervention and resection of bullae. also, the american college of chest physicians (accp) state that patients should be operated on at the second recurrence, except for those at risk scuba divers, pilots etc., or those presenting with persistent air leak. in the recent years vats video - assisted thoracoscopic surgery was first reported in 1989 for treatment of secondary spontaneous pneumothorax in children with cystic fibrosis. video - assisted thoracoscopic surgery is safe and effective, with low morbidity and minimal invasiveness, and has been performed with good results in children with psp [5, 7, 11 ]. the recurrence rate of psp in the group of children after vats has been reported as 4.9 - 11.8% [5, 7, 11 ]. these recurrence rates are higher than those of adults 1.7 - 8.3% [22, 23 ]. the higher risk of recurrence in children is not related to surgical failure, but it is often associated with the formation of new bullae. it has been reported that in children who develop recurrent psp, half will have recurrence on the contralateral side. the use of ct scanning is advocated to identify contralateral blebs or bullae [20, 24 ]. we also observed a significant decrease in recurrences with increasing experience, which is consistent with the observations of other authors. the thoracoscopic approach is a relatively new technique, and there is an impact of the learning curve. the aetiology of postoperative recurrence includes undiscovered bullae, inadequate resection margins that fail to include healthy lung tissue, coexisting severe emphysema, and newly grown bullae [5, 26 ]. several authors have recently reported their experiences with fibrin glue reinforcement of staple lines during thoracoscopic treatment of pulmonary bleb disease in adults [27, 28 ]. comparing the results of chest tube drainage and vats for recurrent psp, patients undergoing vats or limited thoracotomy for psp have recurrence rates of 13% and 0%, respectively, after surgical intervention with the resection of blebs with the lung tissue, we advocate our patients to avoid activities that put additional strain on the lungs, including scuba diving, strenuous exercise, and playing wind musical instruments. a strong and adequate pleural adhesion is indicated to prevent recurrence of the pneumothorax. regarding the optimal method for pleurodesis reports in adult patients have advocated blebectomy plus mechanical pleurodesis or chemical pleurodesis [22, 23 ]. pleurectomy has a very low incidence of recurrence, ranging from 0 to 1%, by open thoracotomy, transaxillary minithoracotomy, or vats. unfortunately, pleurectomy may lead to increased morbidity, related to postoperative haemorrhage complications [30, 31 ]. also, pleurectomy may make secondary thoracotomy extremely difficult, and performance of an apical pleurectomy alone will not prevent recurrence in the lower part of the chest. talc poudrage is rarely performed, even though available data show that both vats bullectomy plus pleurodesis and medical talc poudrage without bullae treatment are equally effective. talc poudrage has two main drawbacks : the risk of postoperative pleural infection ; and the creation of tight adhesions that are difficult to free, so another surgical intervention becomes necessary in the future. in view of those facts, pleural abrasion is widely accepted as a reasonable compromise and is successfully performed in our department. the last resort treatment for children who have failed vats is an open thoracotomy with direct surgical treatment of the subpleural bleb or cyst. several studies comparing vats to open surgery have documented lower recurrence rates after thoracotomy than after vats [3234 ]. open thoracotomy is more effective in preventing recurrence but greatly increases discomfort and morbidity. in view of diminishing morbidity, better cosmetic result, and shortening of the hospital stay, in our department we prefer an axillary mini - thoracotomy approach, even though the view offered by an axillary mini - thoracotomy is so limited that it makes it almost impossible to examine the lower lobe. in our series we had to perform thoracotomy only in one patient. although our study is limited by the small number of patients, we conclude that most patients resolve their spontaneous pneumothorax and air leak with tube thoracostomy alone. for those patients in whom chest tube drainage is not effective, and for those with recurrent psp, early vats and bullectomy combined with pleural abrasion is the most efficient intervention, obviating long periods of ineffective chest tube drainage. | introductionprimary spontaneous pneumothorax (psp) occurs at a frequency of 7.4 - 18 cases per 100 000 population per year. the psp typically occurs in young adults and is uncommon in children. the aim of this study was to review our institutional experience with psp in children.material and methodstwenty - two paediatric patients with confirmed psp, treated from 2004 to 2014 at the paediatric surgery clinic. there were 18 boys and 4 girls. the mean age was 16 years, 6 months 1 month (range 14 - 17). the mean body mass index (bmi) was 20.1 (ranging from 17 to 24).resultsthe recurrence rate of psp was 48%. the mean interval of the recurrence was 5 months 1 month (range from 3 weeks to 2 years). recurrent pneumothorax was evacuated by thoracostomy with success in four patients. the first video - assisted thoracoscopic surgery (vats) procedure had a failure rate of 50%. after second vats procedure, we did not observe recurrent psp in two patients. one patient with recurrent psp, after two vats procedures, was treated with success, with an open mini axillary thoracotomy. the mean follow - up period was 4 years 3 months 1 month (range 6 months 10 years). we have not noted any intraoperative complications.conclusionsalthough our study is limited by the small number of patients, we conclude that most patients resolve their spontaneous pneumothorax and air leak with tube thoracostomy alone. for those patients in whom chest tube drainage is not effective, and for those with recurrent psp, early vats and bullectomy combined with pleural abrasion is the most efficient intervention. |
obstructive sleep apnea (osa) syndrome represents a serious health hazard and is recognized as an independent risk factor for adverse cardiovascular outcomes such as hypertension, arrhythmias, stroke, and coronary heart disease. sympathetic activation, oxidative stress, and systemic inflammation have been shown to be the main intermediary mechanisms linking intermittent hypoxia (ih), the marker of osa, with deleterious cardiovascular and metabolic consequences leading to enhanced cardiovascular morbidity and mortality [2, 3 ]. prior to the occurrence of cardiovascular events, sleep apnea is associated with several subclinical cardiovascular alterations including nocturnal hypertension and early atherosclerosis that are related to both vasculature remodeling (such as increased intima - media thickness, arterial plaque formation, and arterial stiffness [58 ]) and endothelial dysfunction. osa patients exhibit altered endothelial function with desensitization of the alpha and beta 2-adrenergic receptors, altered no - dependent vasodilatation, and hypersensitivity to vasoconstriction (induced by angiotensin ii (ang ii)) [1012 ]. alterations in endothelial function precede the development of morphological atherosclerotic changes and subsequent clinical complications. digital pulse amplitude augmentation in response to hyperemia (endopat) is one of the validated methods for measuring endothelial function. endopat has the advantage of being easy to perform compared to other endothelial function assessment techniques. a main advantage of the system is that the contralateral arm serves as an internal control that can be used to correct for any systemic drift in vascular tone during the test. there is demonstration that peripheral arterial tone (pat) values allow quantifying cardiovascular risk and predicting late adverse cardiovascular events. in osa patients, reversing early disorders in the cardiovascular system before the occurrence of major clinical events, such as myocardial infarction or stroke, may be a means of reducing cardiovascular risk. continuous positive airway pressure (cpap) the first line therapy for osa has been suggested in small size randomized controlled trials (rcts) as being able to reverse some of these subclinical alterations as well as endothelial dysfunction. however, cpap acceptance is poor in some subgroups of osa patients and recent large rcts demonstrate that cpap alone is not enough to reduce cardiometabolic risk in osa patients. thus a crucial issue is to develop alternative or combined treatments to address early, or delay, deleterious osa - related cardiovascular consequences. statins were initially introduced for the prevention of cardiovascular risk because of their lowering lipid effects. during the last decade, numerous in vivo and in vitro studies have described pleiotropic effects of statins, independent of their lipid - lowering properties. some of the reported pleiotropic effects of statins may impact intermediate mechanisms underlying cardiovascular risk in osa patients. simvastatin treatment is able to reduce sympathetic tone and normalize autonomic function in chronic heart failure (chf) rabbits by inhibiting central ang ii mechanisms and therefore the superoxide pathway. statins are also able to reduce ih - induced hypertension to improve carotid compliance and to reduce cardiac infarction hypersensitivity on ih exposed rats. these beneficial vascular effects have also been reported in normolipidemic patients with isolated systolic hypertension where statins reduce large artery stiffness and blood pressure. statins are also known to stabilize atherosclerosis plaques, induce inhibition of vascular smooth muscle cell proliferation as well as platelet aggregation, and reduce vascular inflammation [2426 ]. statins through their pleiotropic properties that impact intermediary mechanisms might modify cardiovascular outcomes in osa. the aim of this study was thus to determine the effect of 3 months of atorvastatin treatment on endothelial function, blood pressure, and early signs of atherosclerosis in osa patients, through a randomized double - blind placebo - controlled trial. the study was conducted in accordance with applicable good clinical practice requirements in europe, french law, ich e6 recommendations, and ethical principles of the helsinki declaration (south africa 1996 and edinburgh 2000). the study was approved by an independent ethics committee (comit de protection des personnes, grenoble, france, irb0005578) and registered on the clinicaltrials.gov site (nct00669695). written informed consent was obtained from all included patients. an external data quality control was performed systematically for some criteria (such as informed consent, complications, and adverse events) and by a random selection of 10% of the case report forms for other criteria. this multicenter, randomized, double - blind, parallel group study compared atorvastatin 40 mg / day versus placebo over 12 weeks. the primary endpoint was change in endothelial function from baseline to 12 weeks, measured by pat. other endpoints included office blood pressure (bp), early carotid atherosclerosis (intima - media thickness (imt) and carotid diameters), arterial stiffness measured by pulse wave velocity (pwv), and metabolic and inflammatory parameters. patients were recruited from sleep laboratories of 3 university hospitals (grenoble and angers, france and geneva, switzerland). only subjects diagnosed with osa (apnea - hypopnea index (ahi) > 30/h) aged over 18 years and who gave written informed consent were eligible. patients presenting any of the following criteria were not included : history of stroke, coronary heart disease, chronic respiratory failure, hypothyroidism, already on statin treatment, multiple antihypertensive medications, pregnant or lactating women, alcohol consumption > 3 units / day, treatment by itraconazole, ketoconazole, protease inhibitor, fibrates, antivitamin k, diltiazem, verapamil, erythromycin, clarithromycin, or cyclosporine. the determination of the sample size was based on published data on the beneficial effect of an oral appliance using the same endpoint, showing that pat improved from 1.77 0.4 at baseline to 2.0 0.4 after treatment. a change of approximately 0.23 in this variable was anticipated after 3 months of atorvastatin treatment. no study to date has demonstrated the range of improvement in pat values after an intervention that is predictive of reduced morbidity or mortality. the sample size calculated to obtain significant differences with 80% statistical power and an alpha error of 0.05 showed that 57 patients per group were necessary. an interim analysis initially planned was scheduled after the inclusion of 25 patients per group. patients underwent an overnight polysomnography. after waking up and while still in a fasting state the epworth sleepiness scale was completed and arterial blood gases analysis was performed in order to exclude obesity hypoventilation syndrome. patients randomly allocated to the statin group received 40 mg / day atorvastatin (tahor, pfizer laboratories, france) during 12 weeks. patients randomly allocated to the control group received placebo (lactose, lc2 laboratories, france) similarly administered. in order to maintain the double blind status, atorvastatin tablets were encapsulated in capsules identical to the lactose placebo capsules (by lc2 laboratories, france). twelve weeks after the baseline visit, the same parameters were measured to compare the effect of the statin treatment with placebo. the primary endpoint was the effect of atorvastatin treatment on endothelial function between baseline and 12 weeks, as measured by pat. the secondary objectives of the study were to determine the effect of atorvastatin treatment on bp, imt and carotid diameters, arterial stiffness (pwv evaluation), and metabolic parameters. overnight sleep studies were scored manually according to standard criteria and an ahi was calculated from the number of apneas and hypopneas per hour according to international guidelines. clinical bp was measured using a mercury sphygmomanometer on three occasions, in line with european society of hypertension - european society of cardiology guidelines. mean arterial bp (mabp) was calculated as dbp + 1/3(sbp - dbp). after bp measurements, endothelial function was assessed by reactive hyperaemia using the finger plethysmographic methodology (pat) with the endopat device (itamar medical ltd, caesarea, israel) as previously described. pat index was calculated as the natural logarithm of the average amplitude of pat signal 90 to 120 seconds after deflation divided by average amplitude of the pat signal during the 210 seconds prior to cuff inflation. carotid - to - femoral pulse wave velocity (pwv) was used for the arterial stiffness evaluation. to determine the carotid - to - femoral pwv, two pulse transducers were fixed on the skin over the right common carotid and femoral arteries. the time delay was measured with a complior device, between the troughs of simultaneously recorded pulse waves and averaged over 10 consecutive cycles. the carotid - femoral pwv was calculated as the distance between the arterial sites divided by the time delay. b - mode ultrasonography was performed using an hp sonos 2500 (hewlett packard) machine with a sectorial 7.5 mhz probe. the method used to determine the mean common carotid imt and luminal diameter has been previously described. both common carotid arteries were studied consecutively in the long axis with a probe incidence allowing good quality images. the imt was defined as the distance separating the most internal parts of these lines and the luminal diameter by the distance between the blood - intima interfaces on the anterior and posterior walls. the images were recorded in end - diastole and then analyzed by specific validated software (timc laboratory, chu grenoble, france). imt and diameter measurements were carried out on areas free of plaques and then averaged. the imt and luminal diameter values were the mean values for the two common carotid arteries. carotid ultrasonography was performed by two operators who were blinded to the other study data. the analysis of carotid parameters using the specific software was performed by the same operator throughout the entire study. after peripheral blood sampling, plasma glucose and serum triglyceride levels were measured automatically (modular 700, roche, meylan, france). serum insulin was measured using a radio - immunometric sandwich assay (cis bio international, gif - sur - yvette, france). the high - sensitivity c - reactive protein (hs - crp) level was measured using automated immunonephelometry (behring nephelometer ii analyzer, dade behring, germany). urinary leukotriene e4 (lte4, a validated marker of proinflammatory cysteinyl leukotriene production) and 11-dehydro - thromboxane b2 (11-dhtxb2) were quantified using liquid chromatography - tandem mass spectrometry. statistical analysis was performed by using ncss 97 software (kaysville, utah, usa). the data were analyzed in intention to treat, which includes all patients who signed the informed consent form. missing baseline data were replaced by the median of each group and missing data at 12 weeks were replaced by the median of the opposite group (maximum bias method). baseline data were compared by a student or a mann - whitney test for continuous data (depending on the validity of the normality of distributions) and by a chi - square test for categorical data. for the analysis of data evolution between baseline and 12 weeks, a repeated measure two - way analysis of variance (anova) all p values were two - tailed and a p value < 0.05 was considered significant. fifty - one patients were included and randomized (n = 25 in the statin group, n = 26 in the placebo group) between 16 may 2008 and 1 june 2012 (36 patients in grenoble between 16 may 2008 and 20 january 2012, 11 patients in angers between 13 may 2011 and 1 june 2012, and 4 patients in geneva between 16 three patients (2 from the statin group and 1 from the placebo group) presented adverse effects such as myalgia or digestive disorders. three patients withdrew : 1 from the statin group and 2 from the placebo group (see flow - chart presented in figure 1). key demographics for the study population included age 54 11 years, 21.6% female, bmi 28.5 4.5 kg / m. there were no significant difference regarding baseline demographic data between the groups and no differences in baseline bp and sleep apnea characteristics. baseline hdl cholesterol levels were significantly higher in the statin group (table 1). after 12 weeks of treatment, adherence was not significantly different between the two groups (94.7 0.07% in the statin group versus 96.6 0.05% in the placebo group). after 12 weeks, there was no improvement in endothelial function when the statin intervention group was compared with the placebo group. the mean difference in pat measurements between the groups was 0.008 (0.29 ; 0.28), p = 0.979 (table 2). this intermediary analysis, initially planned in the study protocol, revealed no positive effect of treatment on the primary endpoint. moreover, as a simulated calculation with 3 times the number of included patients also showed no positive effect of treatment, the study was then stopped based on futility for primary outcome pat measured endothelial function. sbp significantly decreased after 12 weeks of atorvastatin treatment with a mean difference between groups of 6.34 mmhg (12.68 ; 0.01), p = 0.050 (table 3 and figure 2). after 12 weeks, there was no effect of statin treatment in reducing arterial stiffness compared with the placebo group. the mean difference in pwv measurements between the groups was 0.54 m / s (0.45 ; 1.52), p = 0.189 (table 2). after 12 weeks of treatment both carotid imt and left and right luminal carotid diameters remained unchanged in both groups (table 2). total and ldl cholesterol levels significantly improved after 12 weeks of atorvastatin treatment (p < 0.0001), whereas hdl cholesterol was unchanged compared to the placebo group. moreover, in both groups, glycemia, insulinemia, the homa index reflecting insulin resistance, and the glycated hemoglobin a1c level did not change (table 4). hs - crp, lte4, and 11-dhtxb2 were unchanged after 12 weeks of treatment (table 5). this multicenter, randomized, double - blind, and parallel group study in osa patients was the first to investigate the effect of statin treatment on osa - related cardiovascular outcomes. pat reflects changes in digital microvessel dilatation which is only partly dependent on nitric oxide. it has been demonstrated that pat and flow - mediated dilatation (fmd) measurements have significant but differing relations with cardiovascular and metabolic risk factors. in fact, fmd and pat measure different aspects of vascular biology and provide distinct information regarding vascular function in conduit versus smaller digital vessels. using pat, we showed that 3 months of atorvastatin neither improved endothelial function nor reduced early signs of atherosclerosis or arterial stiffness. in this relatively healthy population of osa patients, we did not confirm the beneficial effect on vascular compliance that we have previously reported with statins in rats exposed to intermittent hypoxia. this result is in accordance with another human study showing that, despite improvement in the lipid profile, 6 weeks of atorvastatin treatment (40 mg / day) failed to improve endothelial dysfunction in the first - degree relatives of patients with premature coronary artery disease. the atorvastatin dosage used here (40 mg / day) may have potentially been too low to improve endothelial function and atherosclerosis markers in osa patients. indeed, a higher atorvastatin dosage (80 mg / day) was shown to improve endothelial function assessed by flow - mediated dilation and to reduce large artery stiffness in normolipidemic hypertensive patients. an alternative explanation might be that we did not include patients with comorbidities such that a majority of our osa patients did not exhibit sufficient endothelial dysfunction or severe stiffening at baseline to detect an effect. however and importantly, we showed that this statin dosage is able to lower systolic office blood pressure in osa patients. this observation is in accordance with previous results from our group showing that in rodents statin treatment reduces ih - induced blood pressure elevation. however, outside the area of sleep apnea, such a beneficial effect has also been reported in normolipidemic patients with isolated systolic hypertension [23, 37 ]. in vascular smooth muscle cells statins statins have also been shown, in the chf rabbit, to reduce sympathetic tone, inhibiting central ang ii mechanisms and therefore the superoxide pathway. there is growing evidence that nad(p)h oxidase - derived reactive oxygen species induced by ang ii play an important role in the central regulation of autonomic activity and cardiovascular function in various pathological states. as previously shown in ih exposed rats, the hypertensive effect and cardiac infarction hypersensitivity induced by ih were abolished by antioxidant treatments (such as tempol and melatonin) which were able to normalize dhe level and nadph expression. all these mechanisms might potentially be involved in the blood pressure lowering effect of statins observed here in osa patients. in this study, the impact of statin in reducing blood pressure (around 6 mmhg mean difference) is clinically relevant. this is particularly true in view of the limited impact of cpap treatment in reducing bp [40, 41 ]. indeed, among patients treated for hypertension, even 1 to 2 mmhg mean differences in office blood pressure are already associated with reduced odds of stroke and major cardiovascular events [4244 ]. law. have underlined the importance of lowering blood pressure in everyone over a certain age, rather than measuring it in everyone and treating it in some. larger reductions in blood pressure are known to produce larger reductions in the risk of all major cardiovascular events. finally, we also showed that statin treatment improved the lipid profile in normolipidemic osa patients, in accordance with previous studies on normolipidemic hypertensive patients [23, 37 ]. we observed here that 3 months of statin treatment induced a decrease of 2 mmol / l in total cholesterol and of 1.68 mmol / l in ldl cholesterol that could significantly reduce cardiovascular risk. mmol / l under statins is able to reduce the risk of ischaemic heart disease events by about 60% and stroke by 17%. recent large rcts demonstrate that cpap alone is not sufficient to address cardiometabolic risk in osa patients. thus, a combination of statin treatment with cpap therapy could be useful to better control blood pressure in osa patients and to reduce associated cardiovascular morbidity and mortality. indeed, in view of the high risk of cardiovascular disease (cvd) in patients with osa, the use of statins in this group of patients, irrespective of their baseline cholesterol levels, should be encouraged. while absolute risk assessment is essential when considering primary prevention for cvd, an uncritical application of the framingham risk equation may result in the underuse of statins in patients with osa. thus, when the framingham risk tool is used to manage statin treatment for primary cvd prevention in osa patients, a lower cvd risk threshold than that recommended by current guidelines may need to be set. evidence from large registries and long - term prospective trials is now required to determine the potential synergic beneficial effects and the rate of new cardiovascular events in patients with osa receiving combined statin and cpap therapy. finally, combined statin and cpap therapy should be put in a realistic perspective compared to the undisputed effects of weight loss and/or exercise not only on blood pressure but also on the cardiometabolic consequences of sleep apnea [20, 47, 48 ]. | rationale. accumulated evidence implicates sympathetic activation as inducing oxidative stress and systemic inflammation, which in turn lead to hypertension, endothelial dysfunction, and atherosclerosis in obstructive sleep apnea (osa). statins through their pleiotropic properties may modify inflammation, lipid profile, and cardiovascular outcomes in osa. methods. this multicenter, randomized, double - blind study compared the effects of atorvastatin 40 mg / day versus placebo over 12 weeks on endothelial function (the primary endpoint) measured by peripheral arterial tone (pat). secondary endpoints included office blood pressure (bp), early carotid atherosclerosis, arterial stiffness measured by pulse wave velocity (pwv), and metabolic parameters. results. 51 severe osa patients were randomized. key demographics for the study population were age 54 11 years, 21.6% female, and bmi 28.5 4.5 kg / m2. in intention to treat analysis, mean pat difference between atorvastatin and placebo groups was 0.008 (0.29 ; 0.28), p = 0.979. total and ldl cholesterol significantly improved with atorvastatin. systolic bp significantly decreased with atorvastatin (mean difference : 6.34 mmhg (12.68 ; 0.01), p = 0.050) whereas carotid atherosclerosis and pwv were unchanged compared to the placebo group. conclusion. in osa patients, 3 months of atorvastatin neither improved endothelial function nor reduced early signs of atherosclerosis although it lowered blood pressure and improved lipid profile. this trial is registered with nct00669695. |
concomitanced with craniofacial dysostosis, short arms, and other anomalies, while silver described a syndrome of congenital hemihypertrophy, short stature, and elevated urinary gonadotrophins. silver syndrome is a genetically heterogeneous disorder, with a very wide variation in phenotype. it is also the first human disorder related with epigenetic mutation affecting two different chromosomes. although the incidence of russell silver syndrome is largely unknown, it is estimated to be 1 in 50,000100,000 births. described the major features of russell silver syndrome : low birth weight (2 standard deviation [sd ]), poor postnatal growth 2 sd, and preservation of occipitofrontal circumference and asymmetry. here, we present a rare case of russell silver syndrome associated with low conus medullaris. silver syndrome, however, tubbs. reported a case of russell silver syndrome with tethered cord. this is believed to be the first and only reported case of russell silver syndrome associated with tethered spinal cord until now. we report a rare case of a 2-year - old male child, a product of nonconsanguineous marriage, who was clinically diagnosed as russell silver syndrome associated with low conus medullaris. the patient presented with characteristic features of russell silver syndrome, such as intrauterine growth retardation with subsequent marked postnatal growth impairment, body asymmetry, and triangular face. because of preeclampsia, the mother underwent a cesarean delivery at 32 weeks of gestation. at birth, his weight was 1140 g, length was 34 cm, and had a head circumference of 28 cm. the patient was well appearing, but short and thin, with triangular facies, broad forehead, and low - set prominent ears. asymmetry of the face with left eyelid ptosis, micrognathia, high - arched palate and downturned mouth corners, and hemihypertrophy on the right side, involving the lower limb and cryptorchidism, were also noted [figure 1 ]. his height was 71 cm and weight was 6450 g, which were low as per his age, and his head circumference was 45 cm. the phenotypic appearance of the child there were no cutaneous findings, such as caf - au - lait macules or occult spinal dysraphism. all the basic investigations were within the normal limit, including the level of the hormones. magnetic resonance imaging (mri) was obtained and it revealed a conus medullaris at the inferior border of the l3 vertebral body and l5-s1 posterior vertebral fusion defect [figure 2 ]. however, urodynamic study showed overactive detrusor and detrusor - sphincter dyssynergia which could indicate the presence of tethered cord syndrome. the patient was taken into follow - up with suspicion of tethered cord syndrome. spinal magnetic resonance imaging conus medullaris at the inferior border of the l3 vertebral body there was a delay in the early motor milestones owing to the decreased muscle bulk. according to denver ii developmental screening test, which was performed at the age of 14 months, language development was 14 months degree, fine motor development was 11.5 months degree, gross motor development was 10 months degree, and personal social development was 13 months degree. additional informed consent was obtained from the patient 's legal guardian for whom identifying information is included in this article. russell silver syndrome is a genetically heterogeneous disorder with each case having varying symptomatology. the degree of heterogeneity of the syndrome is emphasized by the large number of different potential genetic bases which have been put forward as causal. maternal uniparental disomy of chromosome 7 (mat - upd(7)) was for many years the only recognized genetic abnormality in patients with russell silver syndrome, accounting for approximately 10% of all cases. the phenotype of children with russell silver syndrome and mupd(7) is thought to differ from other russell silver cases with less distinct facial characteristics, less micrognathia, and no downturned corners of the mouth reported. it is also characterized by clinical features such as hemiatrophy, asymmetry, triangular face, high forehead, preserved head circumference, prominent low - set ears, small jaw, clinodactyly, camptodactyly, skeletal asymmetry, hypospadias, spinal deformity - scoliosis and/or kyphosis. urogenital anomalies, growth hormone deficiency, congenital heart disease, and cleft palate or limb defects have also been reported. there is no pathognomonic radiological feature for russell silver syndrome, but delayed bone age, clinodactyly, phalangeal hypoplasia, ivory epiphyses, and second metacarpal pseudoepiphysis have been reported as suggestive features. the case reported by tubbs. describes the occurrence of russell silver syndrome with tethered cord in a 20-year - old male. the patient also had a thoracic - progressing levoscoliosis. in the mri, conus medullaris was seen at the superior border of the l3 vertebral body with no fatty filum. one of the suggestions made by the authors was the possibility that an undiagnosed spinal cord tethering might be the culprit of some of the scoliosis cases that are associated with russell silver syndrome. yamaguchi. performed a study that is considered to be the largest report in the english literature on spinal deformity in russell the study determined the prevalence of scoliosis and kyphosis in the general population of persons with russell silver syndrome and it included 163 patients. the researchers considered that patients with russell silver syndrome have a high prevalence of spinal deformity. of the 163 respondents, 14% reported scoliosis, 3.1% reported kyphosis, and 3.8% reported both kyphosis and scoliosis, with an average age of diagnosis of 8 years. haslam. and arai. reported abnormal excretory urograms, unilateral chronic pyelonephritis, unilateral ureteropelvic obstruction, severe vesicoureteral reflux, and horseshoe kidney. these clinical features occur frequently in vater syndrome which has a known propensity for tethered cord syndrome. we did not diagnosed any structural anomaly of the urinary system in our patient, but urodynamic study showed overactive detrusor and detrusor - sphincter dyssynergia. anticholinergic therapy was started. with at least 40% of the cases with an unknown etiology, russell silver syndrome has a range of phenotype from mild to severe, with heterogeneous genetic features and a heterogeneous group of conditions. tethered cord syndrome is not a frequent component of russell silver syndrome. there could be an association between tethered cord syndrome and scoliosis, which is seen in russell silver syndrome, but there are not enough cases described in literature to prove this correlation. however, it is important to consider tethered cord syndrome in patients with russell silver syndrome, to avoid scoliosis and other long - term complications. the authors certify that they have obtained all appropriate patient consent forms. in the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal. the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity can not be guaranteed. the authors certify that they have obtained all appropriate patient consent forms. in the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal. the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity can not be guaranteed. | russell silver syndrome is a rare heterogeneous disorder mainly characterized by intrauterine and postnatal growth retardation, craniofacial disproportion, clinodactyly, variation in urogenital development, and skeletal asymmetry. it is rare to come across tethered cord - associated russell silver syndrome. we report a rare case of russell silver syndrome associated with low conus medullaris in a 2-year - old patient with demonstrative phenotype. magnetic resonance imaging indicated a low conus medullaris at the inferior border of the l3 vertebral body. urodynamic study revealed detrusor - sphincter dyssynergia and detrusor overactivity. a decision to follow - up the patient was made because of the suspicion of tethered cord syndrome. even though tethered cord syndrome is not a common finding in russell silver syndrome, it is important to consider tethered cord syndrome to avoid scoliosis and other long - term complications. |
strabismus is an ocular alignment disorder characterized by a horizontal, vertical, and/or torsional deviation of one eye relative to the other, which may result in compromised binocular vision and amblyopia.1,2 strabismus is classified roughly into comitant and incomitant types. according to a study conducted in central china, strabismus was prevalent in 108 (5.0%) out of the 2,363 eligible students.3 clinically, strabismus also exhibits stereopsis impairment. at present, surgery is the major method of strabismus correction.4 optimal eye movements and alignment require a functional oculomotor system. initially, it was reported that eom dystrophy can lead to squint,5 and subsequently, duane s retraction syndrome was found to be closely related to muscular malnutrition.6 moreover, abnormal positions of the eoms can also lead to strabismus.7 additionally, dysfunction of the eom pulleys had been shown to be involved in incomitant strabismus.8,9 strabismus can affect the binocular visual function, resulting in impaired visual fusion in strabismus patients.10 functional magnetic resonance imaging (fmri) has been widely used to study strabismus. a previous report described reduced gray matter volume in the occipital eye field (oef) and parietal eye field (pef) and an increase in the gray matter volume in the frontal eye field (fef) of adults with strabismus.11 the fef is an area of the frontal cortex that can trigger eye movements.12 another group demonstrated reduced functional connections between the v1 and v2 areas in macaque monkeys with strabismic amblyopia.13 it has been shown that the mean diffusivity of the occipital tracts was increased in patients with strabismic amblyopia.14 the regional homogeneity (reho) method, a resting - state fmri (rs - fmri) measurement method, is widely used to determine the local synchronization of spontaneous fmri signals, and it provides important information on the activities of the brain.15,16 the reho method has been successfully deployed to investigate pathological mechanisms of neural diseases such as optic neuritis,17 sleep disorders,18 and parkinson s disease.19 however, it has not yet been used to explore the pathophysiological changes in comitant strabismus (cs). to our knowledge, our study is the first to evaluate regional spontaneous brain activity in subjects with cs and demonstrate its relationship with behavioral performances. ten men and ten women with cs were recruited from the ophthalmology department of the first affiliated hospital of nanchang university hospital. the diagnostic criteria for congenital strabismus included the following : 1) strabismus from birth ; 2) stereopsis defects (no visual fusion) ; 3) binocular uncorrected or corrected visual acuity (va) being equal ; and 4) with alternated cover. the angles of the strabismus group were equal, and the large squint angle range was 5060 delta. subjects were excluded if they had any one of the following conditions : 1) acquired strabismus, incomitant strabismus, concealed oblique ; 2) conditions of eye diseases, trauma, or optic neuropathy (eg, infection, inflammation, ischemic disease, compression damage, eye surgery, intraocular placeholder lesions) ; 3) psychiatric disorders, diabetes, cardiovascular disease, cerebral infarction disease ; and 4) drug or alcohol addiction. twenty healthy controls (hcs ; ten men, ten women) with similar age range, sex ratio, and education status were also recruited for this study. all hcs met the following requirements : 1) normal brain parenchyma on cranial mri ; 2) no ocular disease with uncorrected or corrected va > 1.0 ; 3) no psychiatric disease ; and 4) accessible to the mri scanning (eg, no cardiac pacemaker or implanted metal devices). this study was authorized and approved by the first affiliated hospital of nanchang ethics committee. all research methods followed the declaration of helsinki and conformed to the principles of medical ethics. all subjects participated voluntarily and were informed of the purposes, methods, and potential risks before signing an informed consent form. mri scanning was performed with a 3-t mr scanner (trio, siemens, germany). t1-weighted images were acquired with a three - dimensional spoiled gradient - recalled sequence in an axial orientation. we obtained 176 high - resolution images taking 5 minutes (repetition time = 1,900 ms, echo time = 2.26 ms, thickness = 1.0 mm, gap = 0.5 mm, acquisition matrix = 256256, field of view = 250250 mm, flip angle = 9) and 240 functional images covering the whole brain taking 10 minutes (repetition time = 2,000 ms, echo time = 30 ms, thickness = 4.0 mm, gap = 1.2 mm, acquisition matrix = 6464, flip angle = 90, field of view = 220220 mm, 30 axial slices with gradient - recalled echo - planar imaging pulse sequence). the 240 functional images were analyzed as described previously.17 briefly, the data were filtered by mricro (nottingham university, nottingham, uk) and preprocessed using spm8 (the mathworks, inc., natick, ma, us) and dparsfa (institute of psychology, cas., then, the data were processed with slice timing, head motion correction, spatial normalization, and smoothening with a gaussian kernel of 666 mm full - width at half - maximum (fwhm). after spatially normalized to the montreal neurological institute (mni) space using the standard echoplanar image template and resampled at a resolution of 333 mm, the fmri images were detrended and bandpass - filtered (0.010.08 hz) to reduce the effects of low - frequency drift and physiological high - frequency respiratory and cardiac noise. based on kendall s coefficient of concordance (kcc), reho computation was performed with the rest software (center for cognition and brain disorders, hznu., hangzhou, zhejiang, people s republic of china), as previously described.17 to investigate the group differences in the reho values between patients with cs and hcs, fmri data were fitted with a general linear model (glm) with the spm8 toolkit. p 2.3. two - sample student s t - test was used for continuous data for behavioral performances. all statistical analyses were done with the ibm spss statistics 20.0 software (ibm corporation, armonk, ny, usa). brain regions with different between - group reho values were classified as regions of interest (rois) with the rest software. for each roi, finally, correlation analysis was performed to investigate the relationship between the mean reho value in each of those different areas and the related behavioral performances. all the clinical data of the patients with cs were collected, including the course of the disease and the best - corrected va. ten men and ten women with cs were recruited from the ophthalmology department of the first affiliated hospital of nanchang university hospital. the diagnostic criteria for congenital strabismus included the following : 1) strabismus from birth ; 2) stereopsis defects (no visual fusion) ; 3) binocular uncorrected or corrected visual acuity (va) being equal ; and 4) with alternated cover. the angles of the strabismus group were equal, and the large squint angle range was 5060 delta. subjects were excluded if they had any one of the following conditions : 1) acquired strabismus, incomitant strabismus, concealed oblique ; 2) conditions of eye diseases, trauma, or optic neuropathy (eg, infection, inflammation, ischemic disease, compression damage, eye surgery, intraocular placeholder lesions) ; 3) psychiatric disorders, diabetes, cardiovascular disease, cerebral infarction disease ; and 4) drug or alcohol addiction. twenty healthy controls (hcs ; ten men, ten women) with similar age range, sex ratio, and education status were also recruited for this study. all hcs met the following requirements : 1) normal brain parenchyma on cranial mri ; 2) no ocular disease with uncorrected or corrected va > 1.0 ; 3) no psychiatric disease ; and 4) accessible to the mri scanning (eg, no cardiac pacemaker or implanted metal devices). this study was authorized and approved by the first affiliated hospital of nanchang ethics committee. all research methods followed the declaration of helsinki and conformed to the principles of medical ethics. all subjects participated voluntarily and were informed of the purposes, methods, and potential risks before signing an informed consent form. mri scanning was performed with a 3-t mr scanner (trio, siemens, germany). t1-weighted images were acquired with a three - dimensional spoiled gradient - recalled sequence in an axial orientation. we obtained 176 high - resolution images taking 5 minutes (repetition time = 1,900 ms, echo time = 2.26 ms, thickness = 1.0 mm, gap = 0.5 mm, acquisition matrix = 256256, field of view = 250250 mm, flip angle = 9) and 240 functional images covering the whole brain taking 10 minutes (repetition time = 2,000 ms, echo time = 30 ms, thickness = 4.0 mm, gap = 1.2 mm, acquisition matrix = 6464, flip angle = 90, field of view = 220220 mm, 30 axial slices with gradient - recalled echo - planar imaging pulse sequence). the 240 functional images were analyzed as described previously.17 briefly, the data were filtered by mricro (nottingham university, nottingham, uk) and preprocessed using spm8 (the mathworks, inc., natick, ma, us) and dparsfa (institute of psychology, cas., then, the data were processed with slice timing, head motion correction, spatial normalization, and smoothening with a gaussian kernel of 666 mm full - width at half - maximum (fwhm). after spatially normalized to the montreal neurological institute (mni) space using the standard echoplanar image template and resampled at a resolution of 333 mm, the fmri images were detrended and bandpass - filtered (0.010.08 hz) to reduce the effects of low - frequency drift and physiological high - frequency respiratory and cardiac noise. based on kendall s coefficient of concordance (kcc), reho computation was performed with the rest software (center for cognition and brain disorders, hznu., hangzhou, zhejiang, people s republic of china), as previously described.17 to investigate the group differences in the reho values between patients with cs and hcs, fmri data were fitted with a general linear model (glm) with the spm8 toolkit. p 2.3. two - sample student s t - test was used for continuous data for behavioral performances. all statistical analyses were done with the ibm spss statistics 20.0 software (ibm corporation, armonk, ny, usa). brain regions with different between - group reho values were classified as regions of interest (rois) with the rest software. for each roi, finally, correlation analysis was performed to investigate the relationship between the mean reho value in each of those different areas and the related behavioral performances. brain regions with different between - group reho values were classified as regions of interest (rois) with the rest software. for each roi, finally, correlation analysis was performed to investigate the relationship between the mean reho value in each of those different areas and the related behavioral performances. all the clinical data of the patients with cs were collected, including the course of the disease and the best - corrected va. as shown in table 1, there were no obvious differences in weight (p=0.453), age (p=0.705), best - corrected va right (p=0.316), and best - corrected va left (p=0.705) between the patients with cs and the hcs. compared to hcs, patients with cs had significantly increased reho values in the right inferior temporal cortex (ritc)/right fusiform gyrus (rfg)/right cerebellum anterior lobe (rcal), right lingual gyrus (rlg), and bilateral cingulate gyrus (bcg) (figure 1 [red areas ] and table 1). specifically, the mean reho values of altered reho regions between the cs group and hcs are shown in figure 2 and table 2. the differences of reho values in altered reho regions between the cs and hc groups may serve as diagnostic markers for strabismus. to test this possibility, we extracted the mean reho values of different brain regions and performed the receiver operating characteristic (roc) curve analysis. in our study, the values of the area under the curve (auc) for the ritc / rfg / rcal (figure 3a) and the cluster of bcg (figure 3b) were 0.938 and 0.940, respectively. as shown in table 1, there were no obvious differences in weight (p=0.453), age (p=0.705), best - corrected va right (p=0.316), and best - corrected va left (p=0.705) between the patients with cs and the hcs. compared to hcs, patients with cs had significantly increased reho values in the right inferior temporal cortex (ritc)/right fusiform gyrus (rfg)/right cerebellum anterior lobe (rcal), right lingual gyrus (rlg), and bilateral cingulate gyrus (bcg) (figure 1 [red areas ] and table 1). specifically, the mean reho values of altered reho regions between the cs group and hcs are shown in figure 2 and table 2. the differences of reho values in altered reho regions between the cs and hc groups may serve as diagnostic markers for strabismus. to test this possibility, we extracted the mean reho values of different brain regions and performed the receiver operating characteristic (roc) curve analysis. in our study, the values of the area under the curve (auc) for the ritc / rfg / rcal (figure 3a) and the cluster of bcg (figure 3b) were 0.938 and 0.940, respectively. to our knowledge, this is the first study to evaluate the effect of cs on resting - state brain activity using the reho technique. compared to hcs, patients with cs showed significantly increased reho values in the ritc / rfg / rcal, rlg, and bcg. the human inferior temporal cortex consists of the inferior temporal gyrus, the middle temporal gyrus, and the fusiform gyrus. the inferior temporal cortex is not only responsible for visual shape selectivity but also involved in the classification of visual information.20,21 previous studies have demonstrated that the inferior temporal gyrus responds selectively to 3d structures defined by binocular disparity.22,23 yan observed that patients with comitant extropia showed decreased white matter volumes in the right inferior temporal gyrus.24 in agreement with this finding, we found significantly increased reho values in the ritc in patients with cs. another complication often occurring in cs patients is the stereovision impairment.25 the reho values in the ritc were higher in cs patients, which may explain the compensation of stereovision in these patients. the cerebellum is involved in balance and motor control, as well as the execution of accurate eye movements.26 consistently, one group reported that the cerebellum is involved in the execution of eye and hand movements.27 a previous study has shown that cerebellar vermis activation is related to visually guided saccades.28 joshi and das29 found that the posterior interposed nucleus (pin) in the cerebellum plays an important role in conjugate eye movements in strabismic monkeys. in support of these findings, we also observed higher reho values in the cerebellum anterior lobe of patients with cs, indicating a stronger cerebellar activity than hcs. this suggests that functional reorganization may be occurring in the cerebellum anterior lobe to compensate for the conjugate eye movement abnormalities. the lingual gyrus and fusiform gyrus are related to vision processing.30 a previous study reported that the lingual gyrus was a stable brain region in the visual system network and that the area involved in color and visual motion (area v4) is located in the lingual and fusiform gyri of the prestriate cortex.31,32 visual snow is also related to the dysfunction of lingual gyrus.33 the fusiform gyrus, a fusiform face area (ffa), is a hub for face processing.34 yang reported that the lingual visual cortex was highly activated in patients with infantile esotropia, suggesting a potential role in optical fusion. in our study, we also found that the reho values in the rlg and fusiform gyrus were significantly higher, which may be related to the compensation of stereovision in cs. the cingulate gyrus is an integral part of the limbic system, which is involved with emotion formation, depression, and pain.3638 the limbic system is closely related to memory and emotion.39 in the present study, we also found that patients with cs had higher reho values in areas in the bcg, which may suggest that strabismus has a significant changes in the limbic system. besides, the higher reho values in these areas may be related to compensation by the limbic system. we found that patients with cs had abnormal spontaneous activity in specific regions of the brain, which may be related to the compensation of stereovision in cs. however, there are some limitations to our study, such as the relatively small sample size. moreover, for some subjects, the scan time was too long, so body movement could have affected the reho findings. moreover, future research should distinguish between different types of cs to more accurately assess brain function activity changes. | objectiveto investigate the underlying regional homogeneity (reho) of brain - activity abnormalities in patients with comitant strabismus (cs) and their relationship with behavioral performance.methodstwenty patients with cs (ten men and ten women) and 20 (ten men and ten women) age-, sex-, and education - matched healthy controls (hcs) underwent resting - state functional magnetic resonance imaging scans. the reho method was used to assess local features of spontaneous brain activities. patients with cs were distinguished from hcs by receiver operating characteristic curve. correlation analysis was performed to explore the relationship between the observed mean reho values of the different brain areas and behavioral performance.resultscompared to hcs, the patients with cs showed significantly increased reho values in the right inferior temporal cortex / fusiform gyrus / cerebellum anterior lobe, right lingual gyrus, and bilateral cingulate gyrus. we did not find any relationship between the observed mean reho values of the different brain areas and behavioral performance.conclusioncs causes dysfunction in many brain regions, which may explain the fusion compensation in cs. |
surgical intervention in epilepsy is a widely accepted, effective therapy in patients with pharmacoresistant partial epilepsy. seizure semiology correlates with the seizure origin, spread, and release phenomenon and can help in the estimation of the anatomical brain localization. finger snapping has been noted as a common act for centuries and is employed in securing someone else 's attention. the sound of the snap is created by forcing air out between the fingers with an audible crack. automatisms involving the distal segments of the upper or lower limbs are frequent during seizures and, when unilateral, often indicate ipsilateral seizure onset, mostly in the temporal lobe or in the orbitofrontal region,. finger snapping could be interpreted as a temporal lobe automatism, but the fierceness of the finger - snapping movement associated with tonic abduction of the upper limb could indicate a focus in the supplementary motor area (sma),. we describe two patients in whom invasive intracranial eeg evaluation was performed to localize the seizure onset for surgical intervention. both patients showed ictal snapping of the right hand fingers, which has not previously been described in literature. a 34-year - old right - handed man was seen at the montreal neurological hospital for investigation of refractory seizures. his seizures began at the age of seven, two years after a right - sided head trauma. early eegs revealed an active epileptiform anomaly over the right midfrontal region. over the years, he showed fluctuation in frequency and severity of seizures, which eventually remained refractory. 1.5-tesla mri showed no structural abnormalities. during admission for video - eeg, typical clinical seizures occurred predominantly during sleep, without aura, and were defined as tonic posturing with fencing, extension of the right arm, snapping of the right hand fingers, and some pedaling movement. electroencephalography telemetry showed several seizures not only with bilateral background activity changes, such as generalized epileptiform anomalies, but also with focal attenuation over the right frontal or temporal regions. for nine days, intracranial stereo - eeg exploration with 11-contact depth electrodes was performed, exploring both frontal lobes and the right temporal lobe (fig. 1). active interictal epileptiform discharges were seen in the right hippocampal formation, and an independent, less active interictal epileptiform anomaly was found in the mesial aspect of the right orbitofrontal lobe. twenty - five seizures were recorded, mostly nocturnal, with a semiology typical for this patient 's seizure pattern : they were usually brief, lasting 22 to 40 s, consisting of tonic posturing with fencing, extension of the right arm and elevation and flexion of the left arm, deviation of the head to the right, and discreet pedaling with both legs. right hand finger snapping was observed in 10/25 (40%) seizures during the second half of the ictal phase. electrographical onsets were bilateral and frontal with often right - sided predominance and maximum involvement of the sma. the finger snapping coincided with the discharge spreading to the right temporal lobe to both mesial hippocampal and more superficial temporal channels. a typical seizure was electrically evoked when the right mesial orbitofrontal area was stimulated (biphasic square wave stimulus, 60 hz, 4 ma, 3 s), with finger snapping occurring 12 s after onset and while a rhythmic discharge was seen in the right temporal lobe (supplementary video 1). the patient first underwent a restricted resection of the right anterior orbitofrontal region and, one year later, a more extensive anterior frontal resection with no changes in seizure frequency or in semiology. a 15-year - old right - handed girl was seen at the university medical center utrecht for investigation of pharmacoresistant seizures. magnetic resonance imaging showed a left mesial parietal focal cortical dysplasia. during admission for video - eeg, typical clinical seizures consisted of gazing and grunting, twitching of the upper body, head, right arm, and ictal eeg showed irregular slow wave activity over the left laterofrontal and frontotemporal regions and diffuse spread in the left hemisphere. intracranial subdural grids were placed over the mesial aspect of both the frontoparietal region and the convexity of the left centroparietotemporal neocortex (fig. 1). active interictal multifocal epileptiform discharges occurred near the mesial parietal lesion, more laterally over the parietotemporal transition area and the inferotemporal surface. twenty - two typical seizures were recorded, and 3/22 (13%) were with typical finger snapping which started few seconds after seizure onset. these three attacks consisted of gazing and grunting, twitching of the head, and a right hand finger snapping. ictal corticography showed gamma activity over the mesial aspect of the parietal lobe and caudal to the lesion prior to clinical symptoms. the discharge then spread to the parietotemporal transition area and the inferotemporal surface (supplementary video 1). this patient eventually underwent resection, and the lesion turned out to be focal cortical dysplasia (2b). resection was restrained by eloquent functions of the brain tissue, postoperative mri showed rests of the cortical dysplasia, and postsurgical seizures remained unchanged. a 34-year - old right - handed man was seen at the montreal neurological hospital for investigation of refractory seizures. his seizures began at the age of seven, two years after a right - sided head trauma. early eegs revealed an active epileptiform anomaly over the right midfrontal region. over the years, he showed fluctuation in frequency and severity of seizures, which eventually remained refractory. 1.5-tesla mri showed no structural abnormalities. during admission for video - eeg, typical clinical seizures occurred predominantly during sleep, without aura, and were defined as tonic posturing with fencing, extension of the right arm, snapping of the right hand fingers, and some pedaling movement. electroencephalography telemetry showed several seizures not only with bilateral background activity changes, such as generalized epileptiform anomalies, but also with focal attenuation over the right frontal or temporal regions. for nine days, intracranial stereo - eeg exploration with 11-contact depth electrodes was performed, exploring both frontal lobes and the right temporal lobe (fig. 1). active interictal epileptiform discharges were seen in the right hippocampal formation, and an independent, less active interictal epileptiform anomaly was found in the mesial aspect of the right orbitofrontal lobe. twenty - five seizures were recorded, mostly nocturnal, with a semiology typical for this patient 's seizure pattern : they were usually brief, lasting 22 to 40 s, consisting of tonic posturing with fencing, extension of the right arm and elevation and flexion of the left arm, deviation of the head to the right, and discreet pedaling with both legs. right hand finger snapping was observed in 10/25 (40%) seizures during the second half of the ictal phase. electrographical onsets were bilateral and frontal with often right - sided predominance and maximum involvement of the sma. the finger snapping coincided with the discharge spreading to the right temporal lobe to both mesial hippocampal and more superficial temporal channels. a typical seizure was electrically evoked when the right mesial orbitofrontal area was stimulated (biphasic square wave stimulus, 60 hz, 4 ma, 3 s), with finger snapping occurring 12 s after onset and while a rhythmic discharge was seen in the right temporal lobe (supplementary video 1). the patient first underwent a restricted resection of the right anterior orbitofrontal region and, one year later, a more extensive anterior frontal resection with no changes in seizure frequency or in semiology. a 15-year - old right - handed girl was seen at the university medical center utrecht for investigation of pharmacoresistant seizures. magnetic resonance imaging showed a left mesial parietal focal cortical dysplasia. during admission for video - eeg, typical clinical seizures consisted of gazing and grunting, twitching of the upper body, head, right arm, and ictal eeg showed irregular slow wave activity over the left laterofrontal and frontotemporal regions and diffuse spread in the left hemisphere. intracranial subdural grids were placed over the mesial aspect of both the frontoparietal region and the convexity of the left centroparietotemporal neocortex (fig. 1). active interictal multifocal epileptiform discharges occurred near the mesial parietal lesion, more laterally over the parietotemporal transition area and the inferotemporal surface. twenty - two typical seizures were recorded, and 3/22 (13%) were with typical finger snapping which started few seconds after seizure onset. these three attacks consisted of gazing and grunting, twitching of the head, and a right hand finger snapping. ictal corticography showed gamma activity over the mesial aspect of the parietal lobe and caudal to the lesion prior to clinical symptoms. the discharge then spread to the parietotemporal transition area and the inferotemporal surface (supplementary video 1). this patient eventually underwent resection, and the lesion turned out to be focal cortical dysplasia (2b). resection was restrained by eloquent functions of the brain tissue, postoperative mri showed rests of the cortical dysplasia, and postsurgical seizures remained unchanged., patients extended the right arm and snapped the right hand fingers. in one patient when this patient was requested to snap his fingers on purpose, he could not snap as loudly. in both patients, the sma was involved early in the seizure, with spread to the orbitofrontal and parietal areas. the eeg activity at the start of the finger snapping during these seizures coincided with the spread of discharge into the temporal lobe, which suggests that finger snapping represents another upper limb automatism generated or modulated by the temporal lobe. the localizing precision is, however, limited as snapping occurred only during seizure spread. in the first patient, the predominant activity was ipsilateral to the finger snapping, but the contralateral temporal lobe was not implanted. the temporal seizure activity seemed contralateral, but, in this case, the ipsilateral side was not explored. however, video - eeg showed only contralateral activity. within the temporal lobe, the spread of activity seemed to differ between patients which could result from different recording methods, depth versus corticography. the sampling of the implantations imposes a limitation to our hypothesis. in both patients, epilepsy surgery of the mesiofrontal and parietal cortex the semiology was unchanged, which fits with our hypothesis of the involvement of the sma and temporal lobes. investigation of ictal behavior through video - eeg monitoring has led to the recognition of a number of typical clinical signs, including oral and limb automatisms such as lip smacking, swallowing, chewing, and exploratory movements in temporal lobe epilepsy,. finger snapping has not been described as a typical ictal sign or following electrocortical stimulation. it might be a type of rhythmic ictal nonclonic hand motions which are associated with contralateral temporal lobe seizures. a number of studies specifically compared semiologic features of frontal lobe seizures with those of temporal lobe seizures,,,. leg movements such as pedaling and kicking, tonic posturing, and tonic clonic movements seem to occur more frequently in frontal lobe seizures, while oral, exploratory, and hand automatisms are seen more frequently in temporal lobe seizures,. automatisms have been quantitatively analyzed by yang., and they suggest limb movements during temporal lobe epilepsy to be predominantly distal, while, in frontal lobe epilepsy, proximal movements are predominant. ictal finger snapping might be interpreted as a combination of two common features of epileptic seizures. the start of snapping in fact follows a tonic abduction of the distal part of the right arm with high muscle tension in the fingers, which is seen in supplementary motor seizures,. the subsequent repetitive snapping of fingers can be interpreted as an automatism of the right hand, originating from the involvement of the temporal lobe,. this is supported by the evolution of the movement in patient 2 in whom the finger - snapping movement seems to start as a slow rhythmic pill - rolling movement, gets stronger, and eventually becomes finger snapping. this could emphasize that finger snapping could be atypical unilateral motor automatisms but, in a way, similar to classical temporal automatisms. in two cases of ictal finger snapping, the ictal onset zone was near the supplementary motor area, with spread to the temporal lobe. we hypothesize that this semiology is a combination of muscle tension of the hand from supplementary motor area activation followed by an automatism of snapping originating from temporal lobe involvement. the following are the supplementary data related to this article.supplementary video 1supplementary video sequences patient 1.seizure 1 in this seizure, rhythmic activity is seen bilaterally in the sma, spreading into the orbitofrontal area. during the finger - snapping movements, the seizure consists of sudden arousal with deviation of the head to the right followed by tonic posture of the right arm and snapping of the right hand fingers. the patient rolls onto his right side and moves to a sitting position.seizure 2 in this seizure, the onset is bilateral with a low - voltage fast discharge seen diffusely in the right frontal lobe structures. this is followed by a rhythmic polyspike discharge seen exclusively in the right temporal lobe. postictally, there is a marked attenuation and slowing of background activity predominating in the right and in the frontal lobe. clinically, it consists of a sudden arousal without aura associated with breathing change. there is tonic posturing of the arms and body, tonic deviation of the head to the right, and snapping of the right hand fingers.supplementary video sequences patient 2.seizure 1 in this seizure, gamma activity is seen in the posterior interhemispheric area. spike wave complexes arise in the parietal grid, and, during the finger - snapping movements, there is an increase in spike wave complexes over the inferotemporal area. clinically, the seizure starts with grimacing, a sudden deviation of the head to the left, and pulling off the blankets. the patient, now looking at her right hand, moves her right arm up and starts snapping the right hand fingers.seizure 2 in this seizure, gamma activity is first seen in the anterior interhemispheric area, spreading towards the posterior interhemispheric electrodes mixed with synchronous spike waves at 1 hz in the parietal, temporal, and later frontal grids. during the finger - snapping movements, there is a transition into 8-hz spike wave activity in the anterior interhemispheric and parietal grids. clinically, the seizure starts with deviation of the head to the left and movement to an upward position with the upper body. the patient moves her right arm to the backside and starts snapping the right hand fingers, while her head still deviates to the left.supplementary material.supplementary figure 2, eeg patient 1depth eeg of patient 1 at time of seizure onset. lo : left orbitofrontal electrodes, lc : left cingulate, ls : left sma, ra : right amygdala, rh : right hippocampus, ro : right orbitofrontal, rs : right sma. the uppermost channel of the group of channels from one electrode is the deepest contact of that electrode.supplementary figure 3, eeg patient 2intracranial subdural eeg of patient 2 at time of seizure onset. iha : interhemispheric strip anterior (1 8), ihp : interhemispheric strip posterior (1 8). the parietal grid is an 8 8 grid, and the temporal grid is a 2 8 grid. the uppermost channel of the group of channels of a strip electrode is the contact furthest away from the craniotomy of that electrode. the parietal grid shows the channels in order from left to right and then from top to bottom., rhythmic activity is seen bilaterally in the sma, spreading into the orbitofrontal area. during the finger - snapping movements, the seizure consists of sudden arousal with deviation of the head to the right followed by tonic posture of the right arm and snapping of the right hand fingers. the patient rolls onto his right side and moves to a sitting position. in this seizure, the onset is bilateral with a low - voltage fast discharge seen diffusely in the right frontal lobe structures. this is followed by a rhythmic polyspike discharge seen exclusively in the right temporal lobe. postictally, there is a marked attenuation and slowing of background activity predominating in the right and in the frontal lobe. there is tonic posturing of the arms and body, tonic deviation of the head to the right, and snapping of the right hand fingers. spike wave complexes arise in the parietal grid, and, during the finger - snapping movements, there is an increase in spike wave complexes over the inferotemporal area. clinically, the seizure starts with grimacing, a sudden deviation of the head to the left, and pulling off the blankets. the patient, now looking at her right hand, moves her right arm up and starts snapping the right hand fingers. in this seizure, gamma activity is first seen in the anterior interhemispheric area, spreading towards the posterior interhemispheric electrodes mixed with synchronous spike waves at 1 hz in the parietal, temporal, and later frontal grids. during the finger - snapping movements, there is a transition into 8-hz spike wave activity in the anterior interhemispheric and parietal grids. clinically, the seizure starts with deviation of the head to the left and movement to an upward position with the upper body. the patient moves her right arm to the backside and starts snapping the right hand fingers, while her head still deviates to the left. lo : left orbitofrontal electrodes, lc : left cingulate, ls : left sma, ra : right amygdala, rh : right hippocampus, ro : right orbitofrontal, rs : right sma. the uppermost channel of the group of channels from one electrode is the deepest contact of that electrode. iha : interhemispheric strip anterior (1 8), ihp : interhemispheric strip posterior (1 8). f : frontal (2 4). the parietal grid is an 8 8 grid, and the temporal grid is a 2 8 grid. the uppermost channel of the group of channels of a strip electrode is the contact furthest away from the craniotomy of that electrode. the parietal grid shows the channels in order from left to right and then from top to bottom. | we describe two patients who showed snapping of the right hand fingers during invasive intracranial eeg evaluation for epilepsy surgery. we correlated the eeg changes with the finger - snapping movements in both patients to determine the underlying pathophysiology of this phenomenon. at the time of finger snapping, eeg spread from the supplementary motor area towards the temporal region was seen, suggesting involvement of these sites. |
although oral phosphodiesterase (pde)-5 inhibitors are generally an effective and well - tolerated treatment modality for patients with erectile dysfunction (ed), pde-5 therapy can not cure the underlying disorder in the corpus cavernosum and has limitations such as reduced efficacy in patients with diabetes or radical prostatectomy, lack of spontaneity of the sexual act, and a contraindication in those who take nitrates. gene therapy was introduced in the field of ed in the late 1990s with various therapeutic genes. however, most of these studies remain at a preclinical level, even though the preclinical results are promising. despite the well - known safety of plasmid dna as a gene delivery vector in humans viral vectors exert high gene transfer efficiency but have many drawbacks including systemic toxicity, induction of host immune responses, and possible integration into the host genome. these obstacles have led to the development of new gene delivery methods, such as electroporation-, ultrasound-, and polymer - mediated gene transfer techniques. electroporation technology has emerged as a simple, nontoxic, safe, and effective method for delivering foreign genes into target cells or tissues. a transient and reversible increase in cell membrane permeability with local delivery of electrical pulses to target cells by using electrodes is a major mechanism for electroporation - mediated enhanced gene delivery. in preclinical experiments, in vivo electroporation has been shown to significantly enhance gene transfer efficiency while maintaining the safety properties of plasmid dna. the phase i clinical trial of electroporation - mediated delivery of interleukin-12 plasmid with dose escalation in patients with metastatic melanoma showed the technique to be safe, effective, and reproducible. a dna vaccine against the hepatitis b virus delivered by in vivo electroporation was also shown to be effective in patients with chronic hepatitis b. in the ed field, however, electroporation - mediated gene delivery is in its infancy. electroporation has been applied to deliver adenovirus encoding neuronal nitric oxide synthase into penis in vivo. those authors used a single electroporation parameter (100 v, 8 pulses with a duration of 40 ms, and 1 second between pulses). it was reported that electroporation of skeletal muscle induces myofiber damage and a transient decrease in contractile function. because the electroporation threshold may differ between cell types and tissues, this threshold should be determined for specific cells or tissues. parameters of the electrical pulses used for electroporation also affect the initial steps of plasmid gene translocation through the plasma membrane by regulating membrane permeability and are the key factor in determining transfection efficiency. however, optimal electroporation conditions for delivering plasmid dna into the corpus cavernosum and the adverse effects of electroporation on the erectile tissue have not yet been documented in detail. in the present study, we determined electroporation - mediated transfection efficiency by using reporter genes in different stimulation settings in normal or diabetic mice in vivo. we used a surface - type plate platinum electrode rather than a needle - type electrode to deliver electrical pulses into the erectile tissue because the plate electrode may reduce electroporation - related pain in the clinical situation. pcmv - luc was purchased from promega (madison, wi, usa) and was introduced into escherichia coli strain jm109 (promega) and purified by use of qiafilter plasmids giga kits (qiagen, valencia, ca, usa). the concentration of plasmid dna was determined by using 1 (od260)=50 g of dna. specific pathogen - free c57bl/6 mice were purchased from orient bio (seongnam, korea) and bred in our pathogen - free animal facility. the experiments were approved by the institutional animal care and use subcommittee of our university. the mice were anesthetized with ketamine (100 mg / kg) and xylazine (5 mg / kg) intramuscularly and placed on a thermoregulated surgical table. with the use of sterile technique, the pcmv - luc (100 g/40 l) was injected into the midportion of the corpus cavernosum. immediately after plasmid dna injection, square wave - pulse electroporation (ecm 830, harvard apparatus, holliston, ma, usa) was delivered to the injection sites by using two parallel 3-mm plate platinum electrodes (tweezertrodes, harvard apparatus). to determine the optimal in vivo electroporation - mediated gene delivery condition into the corpus cavernosum, different electroporation conditions were set at 5 - 50 v, 8 - 16 pulses with a duration of 40 - 100 ms, and 1 second between pulses. we compressed the penis at the base with an elastic band immediately before injection, and the compression was maintained for 30 minutes after injection to minimize blood drainage via the dorsal veins. the incision was closed with 6 - 0 vicryl (polyglactin 910) sutures. at 3 days after injection, the penis was harvested and gene expression was measured by luciferase assay. on the basis of these initial results, we also examined the efficacy of electroporation - mediated gene delivery into the penis in diabetic mice, in which diabetes was induced in 2-month - old male mice by intraperitoneal injections of streptozotocin (50 mg / kg) for 5 days consecutively as previously described. at 8 weeks after the induction of diabetes, pcmv - luc (100 g/40 l) was injected into the corpus cavernosum of diabetic mice. fasting and postprandial blood glucose levels were determined with an accu - check blood glucose meter (roche diagnostic, mannheim, germany) before the mice were sacrificed. for the in vivo luciferase assay, the tissues were homogenized in 200 l of 1 reporter lysis buffer (promega). after incubation for 30 minutes at room temperature, the homogenates were centrifuged at 11,000 rpm for 10 minutes. the protein concentrations of the extracts were determined by using a pro - measure kit (intron biotechnology inc., seongnam, korea). an amount of 100 l of the luciferase assay reagents (promega) was dispensed into luminometer tubes. then 50 l of the lysates were added to a luminometer tube containing the luciferase assay reagent, and the tubes were briefly mixed by vortexing. the luminometer was programmed to perform a 3-second measurement delay followed by a 10-second measurement read for luciferase activity. luciferase activity was measured in terms of relative light units (rlus) by using a luminometer (turner biosystems, sunnyvale, ca, usa). the final values of luciferase were reported in terms of rlu / mg total protein. to determine the undesired consequences of electroporation, a midportion of each penile segment was harvested and immediately fixed in 10% formalin phosphate - buffered solution before paraffin embedding. intracavernous pressure (icp) and systemic blood pressure were measured during electrical stimulation of the cavernous nerve as previously described. stimulation parameters were 5 v at a frequency of 12 hz, a pulse width of 1 ms, and a duration of 1 minute. during tumescence, the total icp was determined by the area under the curve from the beginning of cavernous nerve stimulation to a point 20 seconds after stimulus termination. systemic blood pressure was measured by using a noninvasive tail - cuff system (visitech systems, apex, nc, usa). the ratios of maximal icp and total icp (area under the curve) to mean systolic blood pressure (msbp) were calculated to adjust for variations in systemic blood pressure. the group comparisons of parametric data were made by one - way analysis of variance followed by newman - keuls post hoc tests. we used mann - whitney u tests or kruskal - wallis tests for nonparametric data. pcmv - luc was purchased from promega (madison, wi, usa) and was introduced into escherichia coli strain jm109 (promega) and purified by use of qiafilter plasmids giga kits (qiagen, valencia, ca, usa). the concentration of plasmid dna was determined by using 1 (od260)=50 g of dna. specific pathogen - free c57bl/6 mice were purchased from orient bio (seongnam, korea) and bred in our pathogen - free animal facility. the experiments were approved by the institutional animal care and use subcommittee of our university. the mice were anesthetized with ketamine (100 mg / kg) and xylazine (5 mg / kg) intramuscularly and placed on a thermoregulated surgical table. with the use of sterile technique, the pcmv - luc (100 g/40 l) was injected into the midportion of the corpus cavernosum. immediately after plasmid dna injection, square wave - pulse electroporation (ecm 830, harvard apparatus, holliston, ma, usa) was delivered to the injection sites by using two parallel 3-mm plate platinum electrodes (tweezertrodes, harvard apparatus). to determine the optimal in vivo electroporation - mediated gene delivery condition into the corpus cavernosum, different electroporation conditions were set at 5 - 50 v, 8 - 16 pulses with a duration of 40 - 100 ms, and 1 second between pulses. we compressed the penis at the base with an elastic band immediately before injection, and the compression was maintained for 30 minutes after injection to minimize blood drainage via the dorsal veins. the incision was closed with 6 - 0 vicryl (polyglactin 910) sutures. at 3 days after injection, the penis was harvested and gene expression was measured by luciferase assay. on the basis of these initial results, we also examined the efficacy of electroporation - mediated gene delivery into the penis in diabetic mice, in which diabetes was induced in 2-month - old male mice by intraperitoneal injections of streptozotocin (50 mg / kg) for 5 days consecutively as previously described. at 8 weeks after the induction of diabetes, pcmv - luc (100 g/40 l) was injected into the corpus cavernosum of diabetic mice. fasting and postprandial blood glucose levels were determined with an accu - check blood glucose meter (roche diagnostic, mannheim, germany) before the mice were sacrificed. for the in vivo luciferase assay, the corpus cavernosum tissues were quick - frozen in liquid nitrogen. the tissues were homogenized in 200 l of 1 reporter lysis buffer (promega). after incubation for 30 minutes at room temperature, the homogenates were centrifuged at 11,000 rpm for 10 minutes. the protein concentrations of the extracts were determined by using a pro - measure kit (intron biotechnology inc., seongnam, korea). an amount of 100 l of the luciferase assay reagents (promega) was dispensed into luminometer tubes. then 50 l of the lysates were added to a luminometer tube containing the luciferase assay reagent, and the tubes were briefly mixed by vortexing. the luminometer was programmed to perform a 3-second measurement delay followed by a 10-second measurement read for luciferase activity. luciferase activity was measured in terms of relative light units (rlus) by using a luminometer (turner biosystems, sunnyvale, ca, usa). the final values of luciferase were reported in terms of rlu / mg total protein. a midportion of each penile segment was harvested and immediately fixed in 10% formalin phosphate - buffered solution before paraffin embedding. intracavernous pressure (icp) and systemic blood pressure were measured during electrical stimulation of the cavernous nerve as previously described. stimulation parameters were 5 v at a frequency of 12 hz, a pulse width of 1 ms, and a duration of 1 minute. during tumescence, the total icp was determined by the area under the curve from the beginning of cavernous nerve stimulation to a point 20 seconds after stimulus termination. systemic blood pressure was measured by using a noninvasive tail - cuff system (visitech systems, apex, nc, usa). the ratios of maximal icp and total icp (area under the curve) to mean systolic blood pressure (msbp) were calculated to adjust for variations in systemic blood pressure. the group comparisons of parametric data were made by one - way analysis of variance followed by newman - keuls post hoc tests. we used mann - whitney u tests or kruskal - wallis tests for nonparametric data. immediately after injection of pcmv - luc (100 g/40 l) into the corpus cavernosum of normal mice, each animal received electroporation of the injection sites with eight 40-ms pulses at voltages of 5, 10, 30, or 50 v. electroporation profoundly induced gene expression in the corpus cavernosum tissue of normal mice in a voltage - dependent manner. at voltages of 30 and 50 v, gene expression was significantly higher in the normal mice that received electroporation than in those treated with pcmv - luc alone. the highest gene expression was noted in the mice stimulated at 50 v (fig. 1). we further determined transfection efficiency at different electroporation settings : eight 40-ms pulses, sixteen 40-ms pulses, eight 100-ms pulses, and sixteen 100-ms pulses at a voltage of 30 v. we observed significantly higher gene expression in the mice stimulated with sixteen 40-ms pulses and eight 100-ms pulses than in those stimulated with eight 40-ms pulses (fig. 2). undesired consequences of electroporation were determined by gross inspection of the stimulated area and by h&e staining. we found an extensive electrical burn scar in the penis of mice that received electroporation with eight 40-ms pulses at a voltage of 50 v. some degree of burn scarring was noted in the penis of mice stimulated with sixteen 40-ms pulses, eight 100-ms pulses, and sixteen 100-ms pulses at a voltage of 30 v. no detectable burn scarring was noted in mice stimulated with eight 40-ms pulses at a voltage of 20 or 30 v (fig. 3). thus, electroporation - mediated target organ damage may occur in proportion to the intensity, duration, and frequency of the electrical pulse. h&e staining revealed an area of extensive fibrosis in the corpus cavernosum of normal mice that received electroporation at 50 v (fig. these findings suggest that eight 40-ms pulses at a voltage of 30 v is the optimal in vivo electroporation condition into the corpus cavernosum, which induces good transfection efficiency with no adverse events related to electroporation. we also examined electroporation - mediated transfection efficiency in streptozotocin - induced diabetic mice. at 8 weeks after the induction of diabetes, pcmv - luc (100 g/40 l) was injected into the corpus cavernosum of diabetic mice. the animals then received electroporation at the injection sites with eight 40-ms pulses at a voltage of 30 v. similar to the results in normal mice, electroporation significantly increased gene expression in the corpus cavernosum of diabetic mice compared with that in mice treated with pcmv - luc alone (fig. fasting and postprandial blood glucose concentrations were significantly higher in the diabetic mice than in the controls (fasting glucose, 396.549 mg / dl vs. 106.313.9 mg / dl ; postprandial glucose, 550.814.0 mg / dl vs. 136.35.45 mg / dl, p<0.01, respectively). the ratios of maximal icp and total icp to msbp were significantly lower in the diabetic mice than in the controls (maximal icp / msbp, 0.290.03 vs. 0.560.01 ; total icp / msbp, 14.41.5 vs. 31.20.8 ; p<0.01, respectively). immediately after injection of pcmv - luc (100 g/40 l) into the corpus cavernosum of normal mice, each animal received electroporation of the injection sites with eight 40-ms pulses at voltages of 5, 10, 30, or 50 v. electroporation profoundly induced gene expression in the corpus cavernosum tissue of normal mice in a voltage - dependent manner. at voltages of 30 and 50 v, gene expression was significantly higher in the normal mice that received electroporation than in those treated with pcmv - luc alone. the highest gene expression was noted in the mice stimulated at 50 v (fig. 1). we further determined transfection efficiency at different electroporation settings : eight 40-ms pulses, sixteen 40-ms pulses, eight 100-ms pulses, and sixteen 100-ms pulses at a voltage of 30 v. we observed significantly higher gene expression in the mice stimulated with sixteen 40-ms pulses and eight 100-ms pulses than in those stimulated with eight 40-ms pulses (fig. 2). undesired consequences of electroporation were determined by gross inspection of the stimulated area and by h&e staining. we found an extensive electrical burn scar in the penis of mice that received electroporation with eight 40-ms pulses at a voltage of 50 v. some degree of burn scarring was noted in the penis of mice stimulated with sixteen 40-ms pulses, eight 100-ms pulses, and sixteen 100-ms pulses at a voltage of 30 v. no detectable burn scarring was noted in mice stimulated with eight 40-ms pulses at a voltage of 20 or 30 v (fig. 3). thus, electroporation - mediated target organ damage may occur in proportion to the intensity, duration, and frequency of the electrical pulse. h&e staining revealed an area of extensive fibrosis in the corpus cavernosum of normal mice that received electroporation at 50 v (fig. these findings suggest that eight 40-ms pulses at a voltage of 30 v is the optimal in vivo electroporation condition into the corpus cavernosum, which induces good transfection efficiency with no adverse events related to electroporation. we also examined electroporation - mediated transfection efficiency in streptozotocin - induced diabetic mice. at 8 weeks after the induction of diabetes, pcmv - luc (100 g/40 l) was injected into the corpus cavernosum of diabetic mice. the animals then received electroporation at the injection sites with eight 40-ms pulses at a voltage of 30 v. similar to the results in normal mice, electroporation significantly increased gene expression in the corpus cavernosum of diabetic mice compared with that in mice treated with pcmv - luc alone (fig. fasting and postprandial blood glucose concentrations were significantly higher in the diabetic mice than in the controls (fasting glucose, 396.549 mg / dl vs. 106.313.9 mg / dl ; postprandial glucose, 550.814.0 mg / dl vs. 136.35.45 mg / dl, p<0.01, respectively). the ratios of maximal icp and total icp to msbp were significantly lower in the diabetic mice than in the controls (maximal icp / msbp, 0.290.03 vs. 0.560.01 ; total icp / msbp, 14.41.5 vs. 31.20.8 ; p<0.01, respectively). the main observation of the present study was that in vivo electroporation profoundly enhanced the gene transfer efficiency of plasmid dna in the corpus cavernosum in proportion to the intensity of the electrical pulse. however, we also observed damage to the erectile tissue as a result of the electroporation : the higher the intensity, duration, and frequency of the electrical pulse, the higher the possibility of target organ damage. some authors have shown that short - duration high - voltage pulses result in efficient dna transfer, whereas others have suggested that longer intermediate - voltage pulses result in more efficient gene transfer. the results of our study make clear that both voltage and duration of the electrical pulse affect the transfection efficiency. however, we must consider the tissue damage produced by electroporation in experimental settings when we apply this technique. although the highest gene expression was noted at a voltage of 50 v with eight 40-ms pulses, we also observed critical burn scarring and necrosis of the penis at this stimulation parameter. furthermore, an increase in frequency or duration of the electrical pulse also induced damage to the penis even at the same voltage (30 v). specifically, we observed some degree of burn scarring in the penis of mice stimulated at sixteen 40-ms or eight 100-ms pulses but not at eight 40-ms pulses. in a phase i trial of electroporation - mediated delivery of interleukin-12 plasmid in patients with metastatic melanoma, there were no serious adverse events related to electroporation. the authors reported that topical application of 1% lidocaine was effective for minimizing pain. besides the parameters of the electrical pulses used for electroporation, it is also important to determine which type of electrode is ideal for electroporation - mediated gene delivery into the corpus cavernosum. in the present study, we applied a surface - type plate platinum electrode to penile skin to deliver electrical pulses into the underlying cavernous tissue. further studies are needed to test whether direct application of electrical pulses into the corpus cavernosum by use of a needle - type electrode would result in different transfection efficiency or a different safety profile. functional and structural derangements in cavernous vasculature are one of the most important mechanisms involved in the pathogenesis of ed from vascular risk factors. recently, much attention has focused on therapeutic angiogenesis to recover cavernous angiopathy and to restore erectile function by using a variety of proangiogenic genes at a preclinical level. a previous study reported that in vivo electroporation - mediated delivery of hypoxia - inducible factor-1 plasmid dna significantly improves perfusion through increased capillary density and collateral vessel formation in the hind limb of mice compared with mice that received plasmid dna alone. electroporation - mediated gene delivery of proangiogenic factors, such as fibroblast growth factor-2, vascular endothelial growth factor, and hepatocyte growth factor, have also demonstrated efficacy in a mouse model of hind limb ischemia, which further reinforces the usefulness of electroporation in gene delivery for therapeutic angiogenesis. therefore, it is worthwhile to apply the electroporation technique in the field of ed with a target of therapeutic angiogenesis. further studies are necessary to determine whether electroporation - mediated delivery of therapeutic plasmid genes can achieve better clinical outcomes than does conventional plasmid or virus - mediated gene therapy. a lack of study showing the long - term time course of gene transfection efficiency or electroporation - mediated adverse events with the use of a therapeutic gene is a limitation of this study. with regard to determining specific aspects of an electroporation protocol, including transfection efficiency and adverse tissue damage, our study may provide important practical information for electroporation - mediated gene therapy in the field of ed. electroporation - mediated gene therapy lacks the efficacy concerns or biohazard considerations implicit in plasmid or viral vectors. moreover, our protocol will be useful in future refinement of electroporation - mediated gene delivery protocols for the corpus cavernosum. we have established the optimal electroporation conditions for maximizing gene transfer into the corpus cavernosum of mice while avoiding damage to the erectile tissue. our results suggest that electroporation with eight 40-ms pulses at a voltage of 30 v is not injurious to the penis and is an optimal in vivo electroporation parameter in the corpus cavernosum of mice. the electroporation - mediated gene delivery technique will be a valuable tool for gene therapy in the field of ed. | purposeelectroporation is known to enhance the efficiency of gene transfer through a transient increase in cell membrane permeability. the aim of this study was to determine the optimal conditions for in vivo electroporation - mediated gene delivery into mouse corpus cavernosum.materials and methodsdiabetes was induced in c57bl/6 mice by intraperitoneal injections of streptozotocin. after intracavernous injection of pcmv - luc (100 g/40 l), different electroporation settings (5 - 50 v, 8 - 16 pulses with a duration of 40 - 100 ms) were applied to the penis to establish the optimal conditions for electroporation. gene expression was evaluated by luciferase assay. we also assessed the undesired consequences of electroporation by visual inspection and hematoxylin - eosin staining of penile tissue.resultselectroporation profoundly induced gene expression in the corpus cavernosum tissue of normal mice in a voltage - dependent manner. we observed electrical burn scars in the penis of normal mice who received electroporation with eight 40-ms pulses at a voltage of 50 v and sixteen 40-ms pulses, eight 100-ms pulses, and sixteen 100-ms pulses at a voltage of 30 v. no detectable burn scars were noted in normal mice stimulated with eight 40-ms pulses at a voltage of 30 v. electroporation also significantly induced gene expression in diabetic mice stimulated with 40-ms pulse at a voltage of 30 v without injury to the penis.conclusionswe have established the optimal electroporation conditions for maximizing gene transfer into the corpus cavernosum of mice while avoiding damage to the erectile tissue. the electroporation - mediated gene delivery technique will be a valuable tool for gene therapy in the field of erectile dysfunction. |
accessory scrotum is the rarest developmental anomaly of scrotum. because it is usually associated with perineal lipomatous mass or anomalies in the anorectal and genitourinary system, early diagnosis and proper treatment are important to improve the quality of patients ' lives., we report a case of perineal accessory scrotum with a lipomatous hamartoma in a 46-year - old man. it is a good opportunity to know the natural course of both accessory scrotum and lipomatous hamartoma in an adult patient. a 46-year - old male patient was admitted to dongguk university gyeongju hospital because of a perineal mass. the patient was born with the mass but he did n't undergo any treatment. there was no history of rapid growth or other complications. he was a hepatitis b virus carrier and he had a 20-year history of rheumatoid arthritis. physical examination revealed a round, pedunculated mass arising from the left lateral aspect of the anus. the mass was soft, nontender and movable, measuring 5 cm 4 cm. there was a 3.5 cm 1.5 cm sized elongated skin - tag - like protuberance on the left lateral portion of the main mass. the rugated skin of the protuberance was contiguous with the covering skin of the main mass. there were no abnormalities in the anorectal and genitourinary system. under the impression of a lipoma, we completely excised the mass. on section, the main mass was a lump of ill - defined yellow adipose tissue partly divided by whitish fibrous septa. the elongated small protuberance was composed of whitish soft tissue and had a rubbery texture (fig. microscopically, the main mass consisted of predominant mature adipose tissue that was neither encapsulated nor well circumscribed from surrounding tissue (fig. small bundles of bland smooth muscle fibers, mature neural tissue and blood vessels were scattered within the adipose tissue mass (fig. the main mass was diagnosed as a hamartoma with predominant adipose tissue (lipomatous hamartoma). the skin - tag - like protuberance was composed of numerous, irregularly oriented smooth muscle bundles, resembling the tunica dartos (fig. there are four representative developmental anomalies of the scrotum ; penoscrotal transposition, bifid scrotum, ectopic scrotum, and accessory scrotum. among them, accessory scrotum is extremely rare. more than 40 cases have been reported in the english - language literature [2 - 6 ]. accessory scrotum is a kind of ectopic scrotal tissue but never containing a testicle within it. most of them are diagnosed in early childhood, especially in boys, and always arise in the perineum posterior to a normally developed original scrotum. accessory scrotum may occur as an isolated anomaly but some could be associated with perineal lipoma / hamartoma or anomalies in the anorectal and genitourinary system. in a review of 23 cases of accessory scrotum, 19 cases (83%) six out of 19 cases (32%) had anomalies such as anorectal malformations or penoscrotal transposition. even though there was no perineal mass, accessory scrotum was associated with genitourinary and anorectal anomalies including vacterl (vertebral, anal, cardiac, tracheoesophageal, renal and limb anomalies) association. although accessory scrotum itself is a benign lesion, thorough physical examination and imaging studies to detect coexistent other anomalies is necessary. prenatal early diagnosis can be made by an ultrasound examination, if we are aware of these rare congenital anomalies. when accessory scrotum is accompanied with a perineal lipomatous mass, accessory scrotum is usually attached to a lipomatous mass as a pedunculated skin tag or an incompletely separated protruding second nodule. close anatomic relationship as well as frequent coexistence of two lesions suggest the possibility that the lipomatous mass is involved in the pathogenesis of accessory scrotum. we support the hypothesis of sule. emphasizing the role of mesenchymal tissue within the perineal mass in the formation of accessory scrotum. embryologically, scrotal tissue is derived from the labioscrotal swellings that are lateral to the genital tubercle. after 12 weeks of gestation, both labioscrotal swellings migrate inferomedially and fuse to form the scrotum. in our opinion, aberrantly developed fat tissue in the perineum may disrupt only minor cellular population in the inferior portion of a labioscrotal swelling. but, continuous growth of intervening fat tissue forms a tumor - like mass and pushes the disrupted precursor scrotal cells. however, we are not sure how some accessory scrotum is developed without perineal mass. in these cases, perineal lipomatous masses are interesting in pathologic aspect as well as clinical presentation. they have been diagnosed as lipoma or hamartoma. many authors named this lesion lipoma but detailed microscopic features and biologic nature were seldom described in the previous reports. in the present case, we noted some scattered mature neural tissue and smooth muscle bundles within the lipomatous mass. hamartoma is a benign tumor - like malformation and is composed of abnormally proliferated mature cells and tissue that normally present in the affected regions. hamartoma is usually asymptomatic but enlarged hamartoma can lead to complications such as mechanical obstruction, pressure effect, hemorrhage and fracture. especially, clinicians and pathologists should take more interest when it is encountered in adult patients, because malignant transformation of hamartoma has been reported, albeit rarely. in the present case, the hamartoma did n't cause any complications except discomfort. here, we present a 46-year - old man who was born with a perineal mass that consisted of accessory scrotum and lipomatous hamartoma. this case will be helpful for evaluation of perineal mass and the possibility of accessory scrotum should be kept in mind. the clinicopathologic features of accessory scrotum and lipomatous hamartoma in our adult patient were similar to those of children 's cases. | accessory scrotum is a rare congenital anomaly that is often associated with perineal tumor or other developmental abnormalities. because most cases are diagnosed and treated in early childhood, little is known about the natural biologic course of this entity and associated lesions through time. we present a case of accessory scrotum associated with lipomatous hamartoma in a 46-year - old man who was born with a perineal mass. we evaluate the clinicopathologic features and discuss the pathogenesis with a review of the literature. |
approximately 20%-30% of patients are diagnosed with metastatic disease, and 70%-80% of patients are presented with localized or locally advanced disease at the time of diagnosis, which is potentially curable by radical surgical resection alone. among patients who undergo radical resection for localized disease, an understanding of the pathogenesis of rcc at the molecular level, and randomized clinical trials, have established the standard role of the orally administered vascular endothelial growth factor receptor and platelet derived growth factor receptor inhibitor sunitinib for the treatment of advanced rcc. clinical trials are the standard way to evaluate the efficacy of new investigational therapies [5 - 7 ]. in various types of cancer (e.g., melanoma, ovarian cancer, lung cancer), data suggests that there exist a trial effect, ' whereby clinical trials participants may have an improved outcome simply by participating in a clinical trial itself [5 - 10 ]. positive effects of clinical trials participation may include improved routine care and treatment delivery, quality control, and close observation, leading to better patient compliance and clinician practice. the effect of clinical trial participation on the outcome of treatment in metastatic rcc is poorly defined. this knowledge would be important, as treatment options and clinical trials with targeted therapies for patients with metastatic rcc have significantly expanded in recent years. thus, we aimed to study the effect of clinical trial participation on the outcome of metastatic rcc patients treated with sunitinib. we reviewed the records of patients (unselected cohort, international multicenter database) with evidence of metastatic rcc, who were treated with sunitinib between february 1, 2004, and december 31, 2013, in six centers across two different countries : the united states (sidney kimmel comprehensive cancer center at johns hopkins, baltimore, md) and israel (institute of oncology, meir medical center, kfar saba ; department of oncology, asaf harofe medical center, zerifin ; department of oncology, rambam medical center, haifa ; department of oncology, sheba medical center, tel hashomer ; department of oncology, soroka university medical center, beer - sheva). patients treated with sunitinib within clinical trials were identified and individually matched by clinicopathologic factors to patients treated with sunitinib as standard therapy out of a clinical trial. patient data were retrospectively and personally collected by the investigator d.k. from electronic medical records and paper charts, including the following baseline clinical characteristics and known prognostic factors [12 - 19 ] : age, sex, pretreatment smoking status (active versus past / never), histology (clear cell versus non clear cell), past nephrectomy, the time interval from initial diagnosis to sunitinib treatment initiation, prior systemic therapies, number of metastases sites, presence of lung / liver / bone metastases, eastern cooperative oncology group (ecog) performance status, hemoglobin level, corrected (for albumin) calcium level, pre - treatment neutrophil to lymphocyte ratio (nlr), sunitinib induced hypertension, and sunitinib dose reduction or treatment interruption. data on the concomitant use of medications, including angiotensin system inhibitors (angiotensin converting enzyme inhibitors and angiotensin ii receptor blockers) and bisphosphonates, was gathered from patients electronic medical records and paper charts, pharmacy records, and by contacting patients and other treating physicians as needed. the outcome data was last updated on december 31, 2013, including the objective response rate, progression free survival (pfs), and overall survival (os). patients who did not progress or die by december 31, 2013 were censored in pfs analysis or os analysis, respectively. sunitinib was administered orally, usually at a starting dose of 50 mg once daily, in 6-week cycles consisting of 4 weeks of treatment followed by 2 weeks without treatment. on treatment dose reduction or treatment interruption were done for the management of adverse events, depending on their type and severity, in accordance to the standard guidelines. treatment was continued until evidence of disease progression on scans, unacceptable adverse events, or death. patient follow - up generally consisted of regular physical examinations and laboratory assessments (hematologic and serum chemical measurements), every 4 - 6 weeks, and imaging studies performed every 12 - 18 weeks. for the evaluation of response, the response evaluation criteria in solid tumors (recist) ver. 1.1 was applied. in patients with only bone metastases, only complete response, stable disease, or progressive disease were noted, not partial response. the response was assessed by independent radiologists and treating physicians (while the patients were on treatment in each center, as part of standard patient follow - up). pfs was defined as the time from the initiation of sunitinib treatment until evidence of disease progression on scans or death of any cause. overall survival was defined as the time from the initiation of sunitinib treatment to death of any cause. patients in the groups of clinical trials participants and non - participants were individually matched by age, sex, rcc histology, prior nephrectomy, prior systemic therapy, sunitinib induced hypertension, sunitinib dose reduction / treatment interruption, the use of angiotensin system inhibitors, heng risk, and pretreatment nlr. clinicopathologic characteristics and response rate were compared between clinical trials participants and non - participants, by chi - square test or fisher 's exact test for nominal data, and two - sample t test (or mann - whitney non - parametric test) for continuous measures. in all tests, cox proportional hazards regression model was used for comparison of pfs and os between the two groups. furthermore, to determine if participation in a clinical trial is independently associated with treatment outcome, a univariate analysis (unadjusted) of association between each clinicopathologic factor and clinical outcome was performed for the entire patient cohort, using logistic regression for response rate and cox regression model for survival outcomes (pfs and os). factors with significant association in the univariate analysis were included in multivariate cox proportional hazards regression model to determine their independent effects. pfs and os times (probability and median) were estimated from kaplan - meier curve. the research was carried out in accordance with the approval by the institutional review board committee of our institutions. we reviewed the records of patients (unselected cohort, international multicenter database) with evidence of metastatic rcc, who were treated with sunitinib between february 1, 2004, and december 31, 2013, in six centers across two different countries : the united states (sidney kimmel comprehensive cancer center at johns hopkins, baltimore, md) and israel (institute of oncology, meir medical center, kfar saba ; department of oncology, asaf harofe medical center, zerifin ; department of oncology, rambam medical center, haifa ; department of oncology, sheba medical center, tel hashomer ; department of oncology, soroka university medical center, beer - sheva). patients treated with sunitinib within clinical trials were identified and individually matched by clinicopathologic factors to patients treated with sunitinib as standard therapy out of a clinical trial. patient data were retrospectively and personally collected by the investigator d.k. from electronic medical records and paper charts, including the following baseline clinical characteristics and known prognostic factors [12 - 19 ] : age, sex, pretreatment smoking status (active versus past / never), histology (clear cell versus non clear cell), past nephrectomy, the time interval from initial diagnosis to sunitinib treatment initiation, prior systemic therapies, number of metastases sites, presence of lung / liver / bone metastases, eastern cooperative oncology group (ecog) performance status, hemoglobin level, corrected (for albumin) calcium level, pre - treatment neutrophil to lymphocyte ratio (nlr), sunitinib induced hypertension, and sunitinib dose reduction or treatment interruption. data on the concomitant use of medications, including angiotensin system inhibitors (angiotensin converting enzyme inhibitors and angiotensin ii receptor blockers) and bisphosphonates, was gathered from patients electronic medical records and paper charts, pharmacy records, and by contacting patients and other treating physicians as needed. the outcome data was last updated on december 31, 2013, including the objective response rate, progression free survival (pfs), and overall survival (os). patients who did not progress or die by december 31, 2013 were censored in pfs analysis or os analysis, respectively. sunitinib was administered orally, usually at a starting dose of 50 mg once daily, in 6-week cycles consisting of 4 weeks of treatment followed by 2 weeks without treatment. on treatment dose reduction or treatment interruption were done for the management of adverse events, depending on their type and severity, in accordance to the standard guidelines. treatment was continued until evidence of disease progression on scans, unacceptable adverse events, or death. patient follow - up generally consisted of regular physical examinations and laboratory assessments (hematologic and serum chemical measurements), every 4 - 6 weeks, and imaging studies performed every 12 - 18 weeks. for the evaluation of response, the response evaluation criteria in solid tumors (recist) ver. 1.1 was applied. in patients with only bone metastases, only complete response, stable disease, or progressive disease were noted, not partial response. the response was assessed by independent radiologists and treating physicians (while the patients were on treatment in each center, as part of standard patient follow - up). pfs was defined as the time from the initiation of sunitinib treatment until evidence of disease progression on scans or death of any cause. overall survival was defined as the time from the initiation of sunitinib treatment to death of any cause. patients in the groups of clinical trials participants and non - participants were individually matched by age, sex, rcc histology, prior nephrectomy, prior systemic therapy, sunitinib induced hypertension, sunitinib dose reduction / treatment interruption, the use of angiotensin system inhibitors, heng risk, and pretreatment nlr. clinicopathologic characteristics and response rate were compared between clinical trials participants and non - participants, by chi - square test or fisher 's exact test for nominal data, and two - sample t test (or mann - whitney non - parametric test) for continuous measures. in all tests, cox proportional hazards regression model was used for comparison of pfs and os between the two groups. furthermore, to determine if participation in a clinical trial is independently associated with treatment outcome, a univariate analysis (unadjusted) of association between each clinicopathologic factor and clinical outcome was performed for the entire patient cohort, using logistic regression for response rate and cox regression model for survival outcomes (pfs and os). factors with significant association in the univariate analysis were included in multivariate cox proportional hazards regression model to determine their independent effects. pfs and os times (probability and median) were estimated from kaplan - meier curve. the research was carried out in accordance with the approval by the institutional review board committee of our institutions. forty - nine patients treated with sunitinib within clinical trials (nct00083889, nct00130897, nct00444314) were identified and individually matched, via clinicopathologic factors, to 49 patients treated with sunitinib as the standard therapy out of a clinical trial. clinicopathologic factors used to individually match patients were age (median, 64 years), sex (male, 67%), histology (clear cell, 86% ; n=42), past nephrectomy (92%, n=45), prior systemic therapy (16%, n=8 ; including interferone n=5 in clinical trials participants and n=6 in non - participants, or il-2 n=3 in participants and n=2 in non - participants), sunitinib induced hypertension (45%, n=27), sunitinib dose reduction / treatment interruption (41%, n=20), use of angiotensin system inhibitors (37%, n=18), heng risk stratification (favorable 25%, n=12 ; intermediate 59%, n=29 ; poor 16%, n=8), and pre - treatment nlr (3 in 55%, n=27). the groups were balanced with regard to the presence of lung (p=0.62)/liver (p=0.27)/bone (p=0.67) metastases, number of metastatic sites (p=0.8), smoking status (p=0.97), presence of sarcomatoid component in tumor histology (p=0.84), and subsequent lines of therapy (p=0.7 and p=0.85 for subsequent second and third lines of therapy). in clinical trial participants, subsequent second line of therapy included sorafenib (n=13), bevacizumab (n=6), and everolimus (n=5) ; moreover, subsequent third line of therapy included everolimus (n=2), bevacizumab (n=1), and sunitinib rechallenge (n=1). in clinical trial non - participants, subsequent second line of therapy included temsirolimus (n=7), everolimus (n=5), sorafenib (n=4), pazopanib (n=3), and bevacizumab (n=2) ; and subsequent third line of therapy included pazopanib (n=2), and axitinib (n=1). in clinical trial participants versus non - participants, objective response was partial response / stable disease 80% (n=39) versus 74% (n=36), and progressive disease at first imaging evaluation within the first 3 months 20% (n=10) versus 26% (n=13) (p=0.63 ; odds ratio, 1.2) (table 2). 1) was 10 versus 11 months (hazard ratio [hr ], 0.96 ; p=0.84), and median os (fig. 2) 23 versus 24 months (hr, 0.97 ; p=0.89) (table 2). smoking status (hr, 3.35 ; p 3 (hr, 2.5 for > 3 vs. 3 ; p 3 (hr, 1.8 for > 3 vs. 3 ; p=0.009) were individually associated with os. status of clinical trial participation (yes vs. no) was not associated with pfs (hr, 1.15 ; p=0.48). factors independently associated with pfs were smoking status (hr, 2.18 ; p=0.009, for active vs. never / past smokers), non - clear cell histology (hr, 3 vs. clear cell histology ; p=0.008), use of angiotensin system inhibitors (hr, 0.56 for yes vs. no ; p=0.03), and pre - treatment nlr > 3 (hr, 1.95 for > 3 vs. 3 ; p=0.015). factors that were independently associated with os were smoking status (hr, 2.9 ; p=0.001, for active vs never / past smokers), non - clear cell histology (hr, 2.1 vs. clear cell histology ; p=0.032), and pre - treatment nlr > 3 (hr, 1.4 for > 3 vs. 3 ; p=0.02). forty - nine patients treated with sunitinib within clinical trials (nct00083889, nct00130897, nct00444314) were identified and individually matched, via clinicopathologic factors, to 49 patients treated with sunitinib as the standard therapy out of a clinical trial. clinicopathologic factors used to individually match patients were age (median, 64 years), sex (male, 67%), histology (clear cell, 86% ; n=42), past nephrectomy (92%, n=45), prior systemic therapy (16%, n=8 ; including interferone n=5 in clinical trials participants and n=6 in non - participants, or il-2 n=3 in participants and n=2 in non - participants), sunitinib induced hypertension (45%, n=27), sunitinib dose reduction / treatment interruption (41%, n=20), use of angiotensin system inhibitors (37%, n=18), heng risk stratification (favorable 25%, n=12 ; intermediate 59%, n=29 ; poor 16%, n=8), and pre - treatment nlr (3 in 55%, n=27). the groups were balanced with regard to the presence of lung (p=0.62)/liver (p=0.27)/bone (p=0.67) metastases, number of metastatic sites (p=0.8), smoking status (p=0.97), presence of sarcomatoid component in tumor histology (p=0.84), and subsequent lines of therapy (p=0.7 and p=0.85 for subsequent second and third lines of therapy). in clinical trial participants, subsequent second line of therapy included sorafenib (n=13), bevacizumab (n=6), and everolimus (n=5) ; moreover, subsequent third line of therapy included everolimus (n=2), bevacizumab (n=1), and sunitinib rechallenge (n=1). in clinical trial non - participants, subsequent second line of therapy included temsirolimus (n=7), everolimus (n=5), sorafenib (n=4), pazopanib (n=3), and bevacizumab (n=2) ; and subsequent third line of therapy included pazopanib (n=2), and axitinib (n=1). in clinical trial participants versus non - participants, objective response was partial response / stable disease 80% (n=39) versus 74% (n=36), and progressive disease at first imaging evaluation within the first 3 months 20% (n=10) versus 26% (n=13) (p=0.63 ; odds ratio, 1.2) (table 2). 1) was 10 versus 11 months (hazard ratio [hr ], 0.96 ; p=0.84), and median os (fig. 2) 23 versus 24 months (hr, 0.97 ; p=0.89) (table 2). smoking status (hr, 3.35 ; p 3 (hr, 2.5 for > 3 vs. 3 ; p 3 (hr, 1.8 for > 3 vs. 3 ; p=0.009) were individually associated with os. status of clinical trial participation (yes vs. no) was not associated with pfs (hr, 1.15 ; p=0.48). factors independently associated with pfs were smoking status (hr, 2.18 ; p=0.009, for active vs. never / past smokers), non - clear cell histology (hr, 3 vs. clear cell histology ; p=0.008), use of angiotensin system inhibitors (hr, 0.56 for yes vs. no ; p=0.03), and pre - treatment nlr > 3 (hr, 1.95 for > 3 vs. 3 ; p=0.015). factors that were independently associated with os were smoking status (hr, 2.9 ; p=0.001, for active vs never / past smokers), non - clear cell histology (hr, 2.1 vs. clear cell histology ; p=0.032), and pre - treatment nlr > 3 (hr, 1.4 for > 3 vs. 3 ; p=0.02). in the present study, we did not find a clinical trial effect in patients with metastatic rcc treated with sunitinib. the outcome of clinical trial participants was similar to that of matched patients treated out of a trial, in terms of objective response, pfs, and os. furthermore, in a univariate - multivariate analysis of the entire patient cohort (trial participants plus non - participants), the status of clinical trial participation was not associated with treatment outcome. a positive effect of clinical trial participation on treatment outcome, may be explained by multiple factors, including a protocol effect (the way therapy is given), care effect (incidental aspects of care), hawthorne effect (changes in health care practitioners and patients behavior due to the knowledge that they are under monitoring), placebo effect, treatment (per clinical trial) in high - volume centers (per clinical trial) versus non - academic institutions (i.e., participation in clinical trials may represent a quality characteristic of medical centers. thus, healthcare systems that are active in clinical trials may deliver better outcomes for patients than healthcare systems that are inactive in trials) [9 - 11,20 ]. another potential association between clinical trial participation and outcome may also arise if clinicians who tend to recruit to trials also tend to be more competent clinicians. however, some studies found only a weak evidence to suggest that clinical trials participation leads to a positive effect on the outcome of participants [8,9,20 - 22 ]. furthermore, other studies found a trial effect only when the investigational treatment arm of a clinical trial was more efficacious than the standard treatment arm. moreover, improvement in outcomes of patients participating in clinical trials, as reported by some studies, might be related to bias from methodological difficulties as data collection, i.e., retrospective analysis, patient selection (e.g., exclusion of patients with coexisting medical conditions), a better follow - up of patients treated per clinical trials than out of trials, and failure to publish studies reporting negative trial effects. various reasons might be put forward to explain the present study findings, of similar outcomes between clinical trial participants and non - participants. it is possible that institutional standardization of care (e.g., regular follow - up by a dedicated nurse who specialized in the management of sunitinib toxicity), may provide the same benefit as treatment, thus eliminating the potential for the participation effects. second, our patients were treated with an oral targeted agent. in most studies reporting clinical trial effect, patients were treated with intravenous chemotherapy and radiation therapy. to the best of our knowledge, the effect of clinical trial participation on the outcome of modern targeted therapies is poorly defined. finally, some data suggests that when stratified by tumor type, the beneficial effect of trial enrollment may be seen only in selected types, such as breast, lung and colon cancer, and may not be seen in other types, such as melanoma, pancreatic and hepatobiliary cancers. first, this is a multicenter retrospective study that represents an unselected heterogeneous cohort of patients that were treated with sunitinib, including all histologic variants of rcc, and patients who were treatment nave and those with a history of prior therapy. moreover, our data do not exclude the possibility of a trial effect at other institutions. nonetheless, the outcome of the present study patient population (i.e., median pfs of 10 - 11 months, and median os of 23 - 24 months) is similar to the previously published data on patients with metastatic rcc that are treated with sunitinib. second, we are unable to exclude the possibility that unequal distribution of unidentified clinicopathologic parameters in our patient cohort may have biased the observed results. thus, our study might have been underpowered to show a difference in the outcome. other clinicopathologic factors, including status of clinical trial participation, that were not found to be significantly associated with disease progression in the present study might have been important in a larger patient cohort. fourth, whether our findings are specific to sunitinib or generalizable to other tyrosine kinase inhibitors is not known. despite these limitations, our clinical observation on the status of clinical trial participation does not impact the outcome of sunitinib treatment in metastatic rcc an may contribute to treatment decisions, patient selection, and clinical trials design. given the above evidence, discussion between clinicians and mrcc patients about participation in a clinical trial should focus on the improvement of future therapy, and not the direct benefit of research participation. physicians should not imply a survival benefit when counseling metastatic rcc patients about entering clinical trials. further prospective studies may be warranted, to test and confirm our hypothesis generating observation in larger patient cohorts, to define the association between clinical trial participation and outcome in different subgroups of patients (e.g., according to risk by prognostic models, clear cell versus non - clear cell histology, and first line versus advanced line treatment). we investigated the effect of clinical trial participation on outcome of patients with mrcc who were treated with sunitinib. after accounting for other clinicopathologic factors, we were unable to show that participation in a clinical trial may in itself provide an outcome advantage to these patients. the present study may improve confidence that the trial treatment effects will translate to the real - world setting. discussion between clinicians and mrcc patients regarding participation in a clinical trial should focus on the improvement of future therapy rather than the direct outcome of research participation. | purposestudies suggested the existence of a trial effect ', in which for a given treatment, participation in a clinical trial is associated with a better outcome. sunitinib is a standard treatment for metastatic renal cell carcinoma (mrcc). we aimed to study the effect of clinical trial participation on the outcome of mrcc patients treated with sunitinib, which at present, is poorly defined.materials and methodsthe records of mrcc patients treated with sunitinib between 2004 - 2013 in 7 centers across 2 countries were reviewed. we compared the response rate (rr), progression free survival (pfs), and overall survival (os), between clinical trial participants (n=49) and a matched cohort of non - participants (n=49) who received standard therapy. each clinical trial participant was individually matched with a non - participant by clinicopathologic factors. pfs and os were determined by cox regression.resultsthe groups were matched by age (median 64), gender (male 67%), heng risk (favorable 25%, intermediate 59%, poor 16%), prior nephrectomy (92%), rcc histology (clear cell 86%), pre - treatment nlr (> 3 in 55%, n=27), sunitinib induced hypertension (45%), and sunitinib dose reduction / treatment interruption (41%). in clinical trial participants versus non - participants, rr was partial response / stable disease 80% (n=39) versus 74% (n=36), and progressive disease 20% (n=10) versus 26% (n=13) (p=0.63, or 1.2). the median pfs was 10 versus 11 months (hr=0.96, p=0.84), and the median os 23 versus 24 months (hr=0.97, p=0.89).conclusionsin mrcc patients treated with sunitinib, the outcome of clinical trial participants was similar to that of non - participants who received standard therapy. |
chemokines are small cytokines known for their ability to induce migration of cells such as lymphocytes, dendritic cells (dc), macrophages, and stem cells. based on the cellular context and the site of expression, chemokines can be divided into inflammatory chemokines, that are synthesized and promote recruitment of cells during inflammation and homeostatic chemokines, that are constitutively expressed in specific tissues where they regulate leukocyte homing [1, 2 ]. some chemokines participate both in immune defense during inflammation and in physiological trafficking of resting leukocytes [1, 2 ]. moreover, some inflammatory chemokines are crucial components of tumor microenvironment and have a pivotal role in tumor progression, enhancing cancer cell migration to distant organs. chemokine scaffold, that is, a conserved protein structure, dependent on two disulfide bonds linking cysteine residues. based on the relative position of their cysteine residues located in the n - terminal region, chemokines can be divided into four subfamilies, cxc, cc, c, and cx3c [1, 2 ]. cxc chemokines can be further subdivided depending on the presence or absence of an elr (glu, leu, and arg) amino acid motif. elr cxc chemokines attract neutrophils and possess angiogenic properties, whereas elr cxc chemokines are angiostatic and attract t and b lymphocytes as well as natural killer (nk) cells. cc chemokines promote the migration of monocytes, dc, lymphocytes, eosinophils, and basophils. lymphotactin / xcl1 and fractalkine / cx3cl1 are the only members of the c and cx3c chemokine families, respectively. lymphotactin attracts t and b lymphocytes and nk cells, whereas fractalkine attracts predominantly t and b lymphocytes, nk cells, and monocytes [1, 2 ]. chemokines mediate their functions through binding to seven transmembrane g - protein - coupled receptors defined as cxcr, ccr, cr, or cx3cr [1, 2 ]. furthermore, some chemokines bind to multiple receptors and some receptors recognize more than one chemokine. cx3cl1 consists of a chemokine domain linked to a transmembrane domain via an extended mucin - rich stalk of an extracellular domain. the chemokine is synthesized as membrane - anchored form and may be cleaved in the soluble form by different metalloprotease. [5, 6 ]. the membrane - anchored cx3cl1 form functions as an adhesion molecule promoting retention of leucocytes to endothelial cells under physiological flow conditions. the soluble cx3cl1 form is released following constitutive shedding operated by a disintegrin and metalloprotease (adam)10, whereas shedding under inflammatory conditions is mediated primarily by adam17 [8, 9 ]. soluble cx3cl1 resembles a conventional chemokine exhibiting efficient chemotactic activity for human monocytes, nk cells, t cells, dendritic cells and, as demonstrated by our group, for a subset of germinal center b cells [5, 11 ]. cx3cl1 expression has been reported in many cell types of hematopoietic or nonhematopoietic origin, such as endothelial and epithelial cells, lymphocytes, neurons, microglial cells, and osteoblasts. cx3cl1-driven chemotaxis and adhesion are mediated by cx3cr1 that is expressed on different cell types such as nk cells, cd14 monocytes, cytotoxic effector t cells, b cells, neurons, microglia, smooth muscle cells, and tumor cells [11, 1315 ]. cx3cl1 is involved in leukocyte recruitment associated with numerous inflammatory disorders and in tumorigenesis process in which the chemokine show pro- and antitumoral properties. the different roles of cx3cl1 make it an attractive candidate for the development of therapeutic strategies. this review will summarize the multiple roles of the cx3cl1/cx3cr1 axis in the pathogenesis of inflammation and cancer. chemokines and adhesion molecules provide signals for trafficking, adhesion, and migration of leukocytes at sites of injury and inflammation. in this context, cx3cl1 promotes the accumulation of immune cells that express cx3cr1, generating a vascular gateway for cytotoxic effector cells and being detrimental in several inflammatory diseases [6, 17, 18 ]. increased levels of soluble cx3cl1 have been detected in serum, bronchoalveolar lavage fluids, and supernatants from airway smooth muscle cells, lung endothelium, and airway epithelium of allergic asthma and rhinitis patients. both high secretion of cx3cl1 and upregulation of cx3cr1 function by nave and memory cd4 t cells play a critical role in the recruitment of inflammatory cells after allergen stimulation [19, 20 ]. it has been demonstrated that transfer of cd4 t cells from wild type mice into cx3cr1 deficient mice restores the clinical features of asthma, highlighting the therapeutic potential of the cx3cl1/cx3cr1 axis. rheumatoid arthritis (ra) is a chronic joint disease characterized by massive infiltration of inflammatory cells into multiple joints, leading to hyperplasia of synovium and destruction of cartilage and bone. previous studies have defined the role of cx3cl1 in pathogenesis of ra and other chronic diseases such as polymyositis and dermatomyositis [2326 ]. cx3cl1 has been detected on fibroblast - like synoviocytes and endothelial cells in ra synovium where it contributes to the accumulation of cx3cr1 t cells, macrophages, and dendritic cells. following interaction of cx3cl1 with its receptor, these latter inflammatory cells adhere to endothelial cells, migrate into the synovium, and secrete cytokines [2326 ]. in a murine model of collagen - induced arthritis (cia), inhibition of cx3cl1 attenuated clinical symptoms, ameliorated histopathological features, and reduced joint infiltration of inflammatory cells. similarly to human ra, polymyositis and dermatomyositis are characterized by chronic muscle inflammation with infiltration of cx3cr1 cytotoxic t cells and macrophages, recruited by cx3cl1 expressing vascular endothelial cells and other inflammatory cells. in experimental autoimmune myositis, the treatment with anti - cx3cl1 antibody reduced the numbers of muscle infiltrating cells and ameliorated histological inflammatory lesions pointing to the potential therapeutic role of cx3cl1 inhibition and/or cx3cl1/cx3cr1 block in the treatment of ra and inflammatory myopathies. atherosclerosis is a disease affecting arterial blood vessels as the result of fatty materials accumulation such as cholesterol. monocytes - derived foam cells are the hallmark in both early and advanced atherosclerotic lesions and evidence indicates that chemokines play important roles in directing migration of these cells from the blood to the vessel wall [29, 30 ]. in this respect, high levels of cx3cl1 have been found in vascular smooth muscle cells (vsmcs) of coronary atherosclerotic plaques, whereas mononuclear cells and vascular endothelium express cx3cr1 [31, 32 ]. the cx3cl1 membrane - bound form promotes cell - cell interactions, whereas the soluble form, cleaved by the cysteine protease cathepsin s and expressed by vsmcs, regulates the adhesion and capture of circulating monocytes to the sites of atherogenesis. genetic deletion of cx3cl1 or its cognate receptor dramatically reduces monocyte recruitment in the artery wall and the subsequent development of lesions in murine models of atherosclerosis, suggesting that the chemokine / receptor axis represents an attractive therapeutic target for clinical trials in cardiovascular disease. recent studies in mice show that treatment with a cx3cr1 antagonist induces potent inhibition of atherosclerotic lesions. an important role for the cx3cl1/cx3cr1 axis has also been demonstrated in renal diseases such as glomerulonephritis, tubulointerstitial nephritis, pyelonephritis, and renal allograft rejection. upregulation of cx3cl1, expressed preferentially on the apical membrane of renal tubular epithelial cells, has been reported in glomerulonephritis where cx3cl1 acts as a chemoattractant and adhesion molecule [35, 36 ]. indeed the apical cx3cl1, firmly anchored to the membrane, facilitates recruitment and retention of the majority of cx3cr1 leukocytes that infiltrate the kidney during glomerulonephritis or other nephropathies [35, 36 ]. both cx3cl1 and cx3cr1 are upregulated in patients with chronic liver disease and associated with the severity of liver fibrosis. an increased expression of adam10 and adam17 by hepatic stellate cells (hsc) has been demonstrated in these patients. shedding of cx3cl1 by the two metalloproteases facilitates the recruitment and adhesion of cx3cr1 inflammatory cells and the paracrine stimulation of hsc in liver disease [37, 38 ]. the cx3cl1/cx3cr1 axis plays also important roles in inflammatory bowel diseases. in patients with crohn 's disease, there is a significant increase of cx3cl1 mrna expression in inflamed lesions compared to noninflamed colonic mucosa suggesting a relevant impact of this chemokine in intestinal inflammation. experiments in mice demonstrate that intestinal lamina propria (lp) macrophages express high levels of cx3cr1. thus, deletion of cx3cr1 resulted in a significant reduction of macrophage recruitment to lp with a decreased translocation of bacteria to mesenteric lymph nodes and their ability to take up pathogens. these findings point to cx3cr1 as a specific marker for lp macrophages and a critical component in maintaining lp macrophage homeostasis. in recent years, increasing evidence for an inflammatory component in age - related macular degeneration has been demonstrated. under physiological conditions, cx3cl1 is constitutively expressed in retina and retinal pigment epithelium, whereas microglia cells express cx3cr1. dysfunction in cx3cl1/cx3cr1 signaling leads to accumulation in the subretinal space of both inflammatory macrophages and microglia cells that participate to the development of retinal degeneration. recently, two common single - nucleotide polymorphisms (snps) located in the open reading frame of the human cx3cr1 gene have been described, namely t280 m and v249i. a methionine replaces a threonine residue whereas a valine is replaced by an isoleucine in the v249i variant. the frequencies of these two variants are significantly associated to a lower risk of inflammatory diseases such as atherosclerosis, coronary artery disease, susceptibility to human immunodeficiency virus infection, age - related macular degeneration, and crohn 's disease [4449 ]. cx3cl1 is abundantly expressed by neurons in the central nervous system (cns), where it regulates the communication between neurons, glia, and microglia cells, sustaining the normal microglial activity through interaction with its receptor. cx3cr1 is also highly expressed by microglia, astrocytes, and hippocampal neurons in the cns [50, 51 ]. thus, cx3cl1 serves as a neuronal regulatory protein controlling microglia activation under physiological conditions. depending on type of cns injury, the cx3cl1/cx3cr1 alzheimer 's disease (ad) is a progressive neurodegenerative disorder characterized by alteration of neurons and loss of memory and cognitive function. the pathological hallmarks of ad are massive deposits of amyloid beta (a) in the brain leading to microglia activation and neuroinflammation. proinflammatory chemokines, cytokines, and neurotoxins contribute to neuronal degeneration observed in ad. in mice overexpressing amyloid precursor protein, cx3cr1 depletion results in a reduction of a deposition and amelioration of the disease [52, 53 ]. in contrast, other experimental ad models show that cx3cr1 deficiency enhances microglia activation and worsens memory and cognitive functions [54, 55 ] suggesting that this discrepancy may be related to the different mouse models used in these studies. depletion of dopaminergic neurons in the substantia nigra characterizes parkinson 's disease (pd). recent evidence suggests that microglia activation is a key component contributing to this dopaminergic degeneration [56, 57 ]. high cx3cl1 levels correlate with the progression and severity of the disease as demonstrated in cx3cl1 or cx3cr1 deficient mice which show an increased brain neurodegeneration [58, 59 ]. in a mouse model of pd, the two forms of endogenous cx3cl1 have been detected with different effects on disease progression. the soluble form exhibits neuroprotective capability since it decreases dopaminergic neuron loss, reduces impairment of motor coordination, and ameliorates microglial activation and proinflammatory cytokine release. in contrast, the membrane - bound form is not neuroprotective but mediates proinflammatory functions. further studies are necessary to expand the knowledge on the role of both forms in in vivo models of neuroinflammation and neurodegeneration. human immunodeficiency type i (hiv) infection causes hiv - associated neurocognitive disorders (hand) including hiv - dementia. the above - discussed balance between neuroprotective and neurotoxic roles of cx3cl1 is also observed in hiv dementia. on the one hand, cx3cl1 has a clear protective effect since its upregulation and secretion inhibit apoptosis of hippocampal neurons induced by neurotoxic viral proteins [61, 62 ]. on the other hand, cx3cl1 is involved in neuronal damage through its activity on microglia cells that secrete proinflammatory cytokines. moreover, microglia and macrophages expressing cx3cr1 are in turn activated and recruited to the neuronal injury sites, thus amplifying the inflammatory response [51, 63 ]. the role of cx3cl1/cx3cr1 axis in the different neurological disease is summarized in table 1. the expression of cx3cr1 in nk cells and some t cells subsets has provided the rationale to exploit the role of the chemokine / receptor in cancer immunotherapy setting the stage for potential therapeutic approaches. the dual function of cx3cl1 as chemoattractant for leukocytes and adhesion molecule for tumor cells that express the receptor may explain the discrepancies reported in the literature regarding the tumorigenesis process. recently, expression and function of cx3cl1 and cx3cr1 have been demonstrated in glioma tumors irrespectively of their histotype and clinical severity. gliomas are the most common brain tumor in humans, characterized by high invasion rate and diffuse infiltration of the cns. gliomas include heterogeneous tumors classified according to the pathological characteristics as astrocytomas, oligodendrogliomas, and glioblastoma [64, 65 ]. cx3cl1 negatively regulates glioma cell invasiveness by promoting tumor cell aggregation when expressed as transmembrane protein. the induction of cell to cell contact by cx3cl1 prevented detachment of tumor cells from the tumor aggregate that is required for the invasion process [64, 65 ]. treatment of glioma cells with recombinant transforming growth factor (tgf)-beta 1 reduced cx3cl1 expression at mrna level, facilitating glioma cell detachment and dispersion. moreover, erreni and colleagues demonstrated that glioblastoma cancer stem cells and progenitor cells express both cx3cl1 and cx3cr1, indicating that this axis operates early in tumorigenesis process. neuroblastoma (nb) is the most common extracranial tumor of childhood that originates from the neural crest and presents with metastases at diagnosis in about half of patients. this tumor regresses spontaneously in infant, whereas children older than one year have poor prognosis. the bone marrow is the preferred site of nb metastases, but also bone, liver, and skin are frequently involved [67, 68 ]. previous studies have demonstrated that several human nb cell lines express functional cx3cr1 and cx3cl1. soluble cx3cl1 stimulates nb cells that express cx3cr1 to transmigrate through cx3cr1/cx3cl1 human bone - marrow endothelium, suggesting a prometastatic effect of the chemokine / receptor axis. in other studies, delivery of the cx3cl1 gene into nb cell lines induces an effective antineuroblastoma immune response mediated by nk cells and t lymphocytes. this chemokine gene therapy is amplified by anti - gd2 antibody / il-2 fusion protein and may provide a promising approach for neuroblastoma. tumors of nonneuronal origin, for example, prostate, pancreas, and breast cancer, show overexpression of cx3cr1 that regulates adhesion and migration of tumor cells to metastatic sites. in prostate cancer, shulby and colleagues demonstrated in vitro that cx3cl1 and its receptor direct prostate cancer cells to the bone marrow and guide their preferential migration towards human osteoblasts. bone marrow endothelial cells express the membrane form of cx3cl1, that is cleaved by osteoblasts and released as soluble molecule able to attract prostate cancer cells. not only osteoblasts but also mesenchymal stromal cells secrete soluble cx3cl1 in the acellular fraction of bone marrow. this generates a gradient that attracts cx3cr1-bearing cells from the blood to the bone marrow. these findings support the rationale for the use of cx3cr1, cx3cl1, and metalloproteases responsible for its cleavage as potential therapeutic targets for prostate cancer. pancreatic ductal adenocarcinoma (pdac) represents a highly aggressive tumor characterized by rapid progression and chemoresistance. the peculiarity of this tumor is tropism for local peripheral nerves that is a major cause of local tumor recurrence. tumor cells from pdac patients strongly expressed cx3cr1 that mediates migration to cx3cl1 constitutively expressed by neural cells. the high frequency of cx3cr1 and the marked perineural invasion in padc patients supported the concept that cx3cr1 may have an important role in the spreading of pancreatic cancer cells along peripheral nerves and in predicting early tumor relapse after surgery. to confirm this hypothesis, marchesi and colleagues demonstrated that a tumor infiltrated in peripheral nerves was present only in mice injected with cx3cr1-transfected padc cells. in conclusion, the cx3cr1/cx3cl1 axis could represent a potential therapeutic approach using antagonists to cx3cr1 capable to inhibit tumor neurotropism in padc. in epithelial ovarian carcinoma (eoc) cx3cl1 is produced by epithelial cells and promotes malignant cell proliferation. cx3cr1 is virtually absent in normal ovarian surface epithelium and accumulates during the course of tumorigenesis process [76, 77 ]. the interaction between cx3cl1 and cx3cr1 facilitates cell migration and cell adhesion between eoc cells and peritoneal mesothelial cells, contributing to eoc cell proliferation. metastasis is the main cause of morbidity and mortality associated with this tumor [78, 79 ]. several experimental models have analyzed the influence of the cx3cr1/cx3cl1 axis in the biology of breast cancer and the possible therapeutic targeting of cx3cl1. normal and malignant breast tissues express cx3cr1 but its overexpression increases the ability of tumor cells to migrate to skeleton and brain where bone stromal cells and neurons release soluble cx3cl1 [79, 80 ]. in this view, cx3cl1 transgenic mice inoculated with cancer cells show a strong reduction of skeletal dissemination compared to wild - type animals. in addition high levels of cx3cl1 correlate with good prognosis and are identified as prognostic factors for disease survival. these antitumoral effects are due to immunological mechanisms since the chemokine enhances the recruitment of cd8 t cells, cd1a, dcs, and nk cells, inducing both innate and adaptive immunity. therefore, cx3cl1 expression could be considered an essential biomarker for predicting prognosis and identify patients eligible for immunomodulating therapy in breast cancer. cx3cl1 is considered a prognostic biomarker also for patients with colorectal cancer and hepatocellular carcinoma. colorectal cancer (crc) is the second cause of cancer death since one half of all patients develop metastasis especially in the liver and lung. high cx3cl1 expression has been found to be a marker of better prognosis in crc. in murine models soluble cx3cl1, produced by colon cancer cells, drastically reduced their metastatic potential and growth in the target organs through immune mechanisms. the antitumor effects of cx3cl1 are mostly related to attraction of cytotoxic effector t lymphocytes and nk cells. expression of both cx3cl1 and cx3cr1 influences the clinical features and prognosis in patients with hcc since high expression of the chemokine / receptor axis correlates with better prognosis and tumor differentiation. in a murine model, tang and colleagues showed that transfer of the cx3cl1 gene into tumor cells elicited tumor - specific cytotoxic t cells and increased production of il-2 and ifn- in tumor tissue leading to inhibition of hcc growth. in this respect, cx3cl1 may be considered a chemokine suitable for immunoprevention or gene therapy in colorectal cancer, hepatocellular carcinoma, and, more recently, in gastric adenocarcinoma where its expression correlates with induction of both innate and adaptive immunity. in conclusion, two different mechanisms operate in cancer, leading to protumoral or antitumoral effects of cx3cl1. on the one hand, cx3cl1 stimulates a strong immune response with the recruitment of nk cells and tumor - specific t cells. on the other hand, cx3cl1 plays a pivotal role in tumor angiogenesis, as well as in adhesion and migration of cancer cells, reinforcing their ability to spread and metastasize. we have previously demonstrated that cx3cl1 is expressed on human nave, germinal center (gc), and memory b cells, the major b cell subsets from secondary lymphoid tissues. in these sites, soluble cx3cl1 produced by t follicular helper and follicular dendritic cells was found to control both the trafficking of human cx3cr1 centrocytes present in the gc light zone and their in vivo survival and differentiation. recently, a novel role for cx3cr1/cx3cl1 axis has been also delineated for b cell malignancies. our group demonstrated the involvement of cx3cr1 in the crosstalk between neoplastic b cells and tumor microenvironment of patients with chronic lymphocytic leukemia (b - cll). b - cll cells were found to coexpress cx3cr1 and membrane - anchored cx3cl1 on the cell surface and constitutively release the soluble form of the chemokine. only a fraction of b - cll samples was found to be attracted in vitro by cx3cl1. leukemic b cells upregulated cxcr4 upon incubation with cx3cl1 and this was paralleled by increased chemotaxis to cxcl12. nurse - like cells generated from cll patient blood coexpressed cx3cr1 and cx3cl1, but only a small proportion of them migrated in vitro to cx3cl1 that is not secreted by nurse - like cells. based upon these findings, we have proposed a model whereby the cx3cr1/cx3cl1 axis may contribute to interactions between cll cells and tumor microenvironment by increasing cxcl12-mediated attraction of leukemic cells to nlc and promoting directly adhesion of cll cells to nlc. in studies by other groups, cx3cr1 expression was investigated in different types of b cell lymphomas by reverse transcriptase - polymerase chain reaction (rt - pcr), immunohistochemistry, and flow cytometry. b cell lymphomas include about 80% of the malignant lymphomas and comprise different subtypes. in particular, diffuse large b cell lymphoma (dlbcl) is the most frequent subtype, representing 30%35% of all non - hodgkin lymphomas (nhl). dlbcl has predominant centroblastic morphology, is highly proliferating and invades the gc subverting the physiological microenvironment. follicular lymphoma (fl) has centrocytic and centroblastic components in different ratios depending on tumor grade and, compared to dlbcl, shows lower proliferative activity and slower invasion of gc by tumor cells. mantle cell lymphoma (mcl) is a relatively rare type of nhl that displays an aggressive course with a continuous relapse pattern. marginal zone lymphomas (mzls) are indolent b cell lymphomas with variable symptoms related to lymphoma location. in particular, extranodal mzls of mucosa - associated lymphoid tissue (malt) arise in gastrointestinal tract but may affect every organ in the body. malt lymphoma infiltrates b cell follicles in the peyer 's patch marginal zone, spreading in the surrounding tissue, and show the same cytological features and immunophenotype as mzls. dlbcl, fl, mcl, and malt lymphoma were found to express cx3cr1 at mrna and protein levels suggesting a functional role for this receptor in the interaction between lymphoma cells and tumor microenvironment [94, 95 ]. cx3cr1 expression in human fl, mcl, and mzl has been confirmed by our group (table 2) (unpublished data). in addition, we have demonstrated for the first time that freshly isolated malignant b cells express the membrane - bound form of cx3cl1 (table 2) (unpublished data). to investigate the functional activity of cx3cr1 in contrast, the chemokine did not induce locomotion of fl and mzl cells (table 2) (unpublished data). these preliminary results provide evidence that lymphoma b cells show a different migratory behavior in response to cx3cl1 compared to their normal counterparts. thus, fl cells, which are of gc origin, did not migrate in vitro to soluble cx3cl1 that, in contrast, was found by our group to be chemotactic for normal gc b cells. in contrast, a minority of mcl cell fractions migrated to soluble cx3cl1 whereas their normal counterparts, that is, nave b cells, did not. the cx3cl1/cx3cr1 axis plays an important function in the pathophysiology of several inflammatory, infectious, and neurological processes and in various forms of cancers. as apparent from this review, the cx3cl1/cx3cr1 axis promotes inflammation and tumor growth in the majority of disease models discussed. cx3cl1 shedding may be blocked using chemical antagonists of adam10/adam 17 or cathepsin s, although this approach is far from being selective. | fractalkine / cx3cl1, the only member of the cx3c chemokine family, exists as a membrane - anchored molecule as well as in soluble form, each mediating different biological activities. it is constitutively expressed in many hematopoietic and nonhematopoietic tissues such as endothelial and epithelial cells, lymphocytes, neurons, microglial osteoblasts. the biological activities of cx3cl1 are mediated by cx3cr1, that is expressed on different cell types such as nk cells, cd14 + monocytes, cytotoxic effector t cells, b cells, neurons, microglia, smooth muscle cells, and tumor cells. the cx3cl1/cx3cr1 axis is involved in the pathogenesis of several inflammatory cancer including various b cell malignancies. in tumors the interaction between cancer cells and cellular microenvironment creates a context that may promote tumor growth, increase tumor survival, and facilitate metastasis. therefore the role of the cx3cl1/cx3cr1 has attracted interest as to the development of potential therapeutic approaches. here we review the different effects of the cx3cl1/cx3cr1 axis in several inflammatory and neurodegenerative diseases and in cancer, with emphasis on human b cell lymphomas. |
although contact angle hysteresis phenomenon has been well known for several decades, its nature is not fully explained yet. numerous papers have been published in which this phenomenon was investigated both experimentally and theoretically, and it would be impossible to cite all of them. the appearance of contact angle hysteresis was originally attributed to the surface roughness, its chemical heterogeneity and/or surface active impurities present in the liquid. some general characteristic features of the hysteresis, among others, are discussed in adamson and gast s monograph. as is well known, practically on all real solid surfaces contact angle hysteresis is observed, and one may claim that all real surfaces possess some roughness and/or chemical imperfections of the surface structure on the molecular scale. the microscopic contact angle can vary across such surfaces, and the experimental macroscopic advancing and receding contact angles measured using a few microlitre droplet settled on the surface are averaged values of the microscopic angles. various aspects concerning relations between microscopic and macroscopic contact angles, also in relation to contact angle hysteresis, were published by decker.. amount of roughness (at the most ngstrom level) of the contact line does not correlate with the amount of hysteresis, resulting from dramatic changes in the advancing and receding contact angles that occurred after uv / ozone treatment. gaydos and neumann deduced that minimum patch size of the heterogeneous surface to produce contact angle hysteresis was about 1 m. the effect of surface roughness on the hysteresis may depend not only on the roughness heights but also the roughness topology. on the other hand, krumpfer and mccarthy stated that because the advancing and receding events are not generally the reverse of one another this leads to the expectation that most surfaces should exhibit contact angle hysteresis even if they are not dirty, rough or chemically heterogeneous. indeed, even on smooth and low surface energy solids like teflon [7, 8 ], self - assembled hexadecyltrichlorosilane monolayers deposited on glass slide or silicon, or polystyrene with fluorocarbon substituents, a considerable hysteresis of water and other liquids was observed. lately, krumpfer and mccarthy discussed contact angle hysteresis appearing on hydrophobic and superhydrophobic surfaces concluding that it is due to receding contact line pinning, and the amount of hysteresis depends on the sign of curvature of the tops of posts. simultaneously, the authors describe procedures for obtaining smooth surfaces with the hysteresis ranging within 0.51 which can be obtained, for example, by covering the silicon surface with poly(dimethylsiloxane) through the process of silanization. different origins of hysteresis, which appears even on molecularly smooth solid surfaces, have been recently presented by starov and velarde. they considered thermodynamic equilibrium of the system : solid surface / liquid drop / gas and the presence of derjaguin pressure behind the three - phase contact line. they concluded that although in some systems contact angle hysteresis was due to surface roughness and its mechanical or chemical heterogeneity, thehysteresis could be found even on homogeneous perfectly flat surfaces as a consequence of the peculiar shape of the derjaguin isotherm in the partial wetting case. moreover, they also found that static advancing contact angle is not affected by the solid surface roughness if its height is less than 1030 nm. discussed the so - called intrinsic hysteresis appearing during measurement of static contact angle in which an adsorbed film at the three - phase contact line is considered. earlier chibowski [12, 13 ] assumed the presence of the liquid film behind the drop after the three - phase line has retreated and derived an equation for calculation of the apparent surface free energy from the contact angle hysteresis. it should be mentioned that in most published papers, the contact angle hysteresis mostly deals with water sessile drops on different solid substrates, from partially hydrophilic up to superhydrophobic, and relatively small number of papers have been published where contact angle hysteresis of liquids other than water is reported [1416 ]. therefore, the purpose of this study was to investigate contact angle hysteresis of water, formamide and diiodomethane on phospholipid 1,2-dipalmitoyl - sn - glycero-3-phosphocholine (dppc) layers deposited on three different solid supports, i.e. glass, mica and poly(methyl methacrylate) (pmma). it seemed to us interesting to learn whether and how much the kind of solid support, covered with the same amount of dppc monolayers, affected the contact angle and its hysteresis. another question was whether on the same surface the amount of hysteresis was different depending on the liquid used. for the bare solid surfaces and surfaces with two statistical dppc monolayers deposited the afm images were recorded. dppc (semi - synthetic, 99 %) was purchased from sigma and used without further purification. the probe liquids employed for contact angle measurements were : water from milli - q plus system (resistivity, 18.2 m cm), formamide (ucb co., belgium, > 99 %) and diiodomethane (poch s.a, gliwice, poland, p.a.). before deposition of dppc, the surfaces of microscope glass slides (20 26 mm), mica (continental trade, warsaw, poland) plates (8 16 mm) and pmma (plastic - group, lublin, poland) plates (20 20 mm) were carefully prepared. the details of surfaces pretreatment were published earlier. the successive dppc layers were obtained by pouring from a microsyringe appropriate volume of its aqueous solution, or in the case of pmma methanolic solution, on the solid support surfaces. it was calculated taking 0.57 nm area for a dppc molecule and the geometric surface of the solid plate. the consecutive layer was deposited on the previous one by depositing the same volume of the solution on the surface covered with dried dppc layer(s). the contact angles were measured at room temperature (20 1 c) using a gbx contact angle meter (france) equipped with a video - camera system and computer software for the contact angle calculation from the shape of the sessile droplet. then, a 1-l volume was sucked into the syringe from the droplet, and receding contact angle was read out. the readings were taken both on the left and right sides of the 2d droplet profile for all three test liquids and on each statistical monolayer of dppc deposited on the solid supports. three series of the contact angle measurements were conducted on each support using the three liquids. in each series and for each liquid, the contact angles were measured for two to three droplets. thus, the arithmetic mean value of the contact angle of a particular liquid was calculated from about 1218 readings. the topography of bare solid surfaces used as the supports and those with two statistical dppc monolayers deposited was imaged with an atomic force microscope nanoscope iii (veeco, usa) equipped with standard silicon or silicon nitride tips. the afm images were analysed using wsxm program (version develop 8.0 scanning probe microscope software). dppc (semi - synthetic, 99 %) was purchased from sigma and used without further purification. the probe liquids employed for contact angle measurements were : water from milli - q plus system (resistivity, 18.2 m cm), formamide (ucb co., belgium, > 99 %) and diiodomethane (poch s.a, gliwice, poland, p.a.). before deposition of dppc, the surfaces of microscope glass slides (20 26 mm), mica (continental trade, warsaw, poland) plates (8 16 mm) and pmma (plastic - group, lublin, poland) plates (20 20 mm) were carefully prepared. the successive dppc layers were obtained by pouring from a microsyringe appropriate volume of its aqueous solution, or in the case of pmma methanolic solution, on the solid support surfaces. it was calculated taking 0.57 nm area for a dppc molecule and the geometric surface of the solid plate. the consecutive layer was deposited on the previous one by depositing the same volume of the solution on the surface covered with dried dppc layer(s). the contact angles were measured at room temperature (20 1 c) using a gbx contact angle meter (france) equipped with a video - camera system and computer software for the contact angle calculation from the shape of the sessile droplet. then, a 1-l volume was sucked into the syringe from the droplet, and receding contact angle was read out. the readings were taken both on the left and right sides of the 2d droplet profile for all three test liquids and on each statistical monolayer of dppc deposited on the solid supports. three series of the contact angle measurements were conducted on each support using the three liquids. in each series and for each liquid, the contact angles were measured for two to three droplets. thus, the arithmetic mean value of the contact angle of a particular liquid was calculated from about 1218 readings. the topography of bare solid surfaces used as the supports and those with two statistical dppc monolayers deposited was imaged with an atomic force microscope nanoscope iii (veeco, usa) equipped with standard silicon or silicon nitride tips. the afm images were analysed using wsxm program (version develop 8.0 scanning probe microscope software). the 3d afm images of the bare solid substrates and their roughnesses are shown in fig. 1. the average roughness of its surface is 0.57 nm. also, average surface roughnesses of glass and pmma are small, 1.78 and 3.89 nm, respectively. these values are much below those (1030 nm) discussed by starov and velarde that would affect static advancing contact angle. therefore, the contact angle hysteresis, if appears on these surfaces, can be considered as resulting from different origin than the surfaces roughness. 2 are presented 3d afm images of the two statistical dppc monolayers deposited on the solid surfaces shown in fig. 1. again, the average roughness of the layers deposited on mica is the smallest one, and that on glass is the same as that of bare glass surface. also on pmma surface, the roughness is not much larger than that on its bare surface (compare figs. 1 and 2). in the case of the dppc layers, the distribution of the roughness is broader than that of the appropriate bare support surface. anyway, it can be concluded that the layers roughness should not be a direct cause of the contact angle hysteresis if it appears.fig. 13d afm images of the bare surfaces of glass (a), pmma (b) and mica (c), the roughness along the marked line, and the roughness of total surface shown from top to bottom, respectively. 23d afm images of two statistical dppc monolayers deposited on the surfaces of glass (a), pmma (b) and mica (c), the layer roughness along the marked line, and roughness of the total surfaces from top to bottom are shown, respectively. also, rms and average height values, h a, are given 3d afm images of the bare surfaces of glass (a), pmma (b) and mica (c), the roughness along the marked line, and the roughness of total surface shown from top to bottom, respectively. also, rms and average height values, h a, are given 3d afm images of two statistical dppc monolayers deposited on the surfaces of glass (a), pmma (b) and mica (c), the layer roughness along the marked line, and roughness of the total surfaces from top to bottom are shown, respectively. also, rms and average height values, h a, are given the surface tension and its components of the probe liquids : water, formamide and diiodomethane, their boiling point, vapour pressure, the molecule volume and the area per molecule are listed in table 1. the two liquids, water and formamide, are polar and diiodomethane is apolar liquid. the liquids evidently differ in their vapour pressure at room temperature and the molecular size (volume), as well as in the nature and strength of the interactions.table 1surface tension and its components of the probe liquids used for contact angle measurements in milli - newton per meter, and some other parameters characterizing the liquidsprobe liquid l llw l+ l lab boiling point cvapour pressure at 20 c mmhgvolume per molecule, nm area per molecule, nm water, h2o72.821.825.525.551.010017.50.0300.059formamide, hconh2 58.039.02.2839.619.02100.080.0660.197 diiodomethane, ch2i2 50.850.80001820.820.1340.215 l liquid surface tension, l liquid lfshitz van der waals component, l liquid electron acceptor component, l liquid electron donor component calculated assuming spherical shape of the molecule surface tension and its components of the probe liquids used for contact angle measurements in milli - newton per meter, and some other parameters characterizing the liquids l liquid surface tension, l liquid lfshitz van der waals component, l liquid electron acceptor component, l liquid electron donor component calculated assuming spherical shape of the molecule before discussing the contact angles presented in this paper, some problems dealing with the interpretation of experimentally measured contact angles should be briefly mentioned. a detailed discussion on this issue can be found in a paper published by marmur, although he has introduced many forms of contact angles, like : geometric, ideal, young, actual, apparent, advancing, receding, most stable and dynamic, and one may become a bit confused as to what contact angle is actually measured in a given solid / liquid drop / gas system. on the other hand, a careful reading of this paper helps understanding the problems encountered in the interpretation of the experimental contact angles. anyway, the experimental contact angle is always a macroscopic quantity, and its value can be an average of many microscopic contact angles that may appear around the droplet perimeter on a rough solid surface. however, as was mentioned above, it is the case of a solid surface having roughness of micrometer size [3, 10, 20 ]. nevertheless, in practical liquid drop / solid surface systems, the macroscopic contact angle is an apparentcontact angle. if the surface is micrometrically rough, then the ratio of the droplet size to the roughness size should be two to three orders of magnitude larger in order to measure meaningful apparent contact angles. in the systems investigated in this paper, the roughness of the surfaces is only a few nanometers ; therefore, the droplet of 3 l volume completely fulfills the condition of the above - mentioned ratio, and because during depositing the droplet from automatic deposition system on the solid surface its volume is slowly increased up to 3 l, we consider thus measured contact angle as an apparent advancing contact angle, which is probably close to the maximum advancing contact angle. then, after sucking 1 l volume of the liquid from the settled droplet into the syringe, the three - phase contact line has retreated, and the measured contact angle is the apparent receding contact angle, which again can be considered as that close to the minimum receding contact angle. it is believed that behind the droplet a liquid film is present [10, 11, 13, 15, 21 ]. both thus measured contact angles are equilibrium ones, but they are not young s contact angles.in consequence, the observed hysteresis of a given probe liquid contact angle can be considered as the difference between the apparent contact angle of the droplet surrounded by bare solid surface and the apparent receding contact angle of the droplet behind which some film of the liquid is present. the advancing and receding contact angles of these liquids on the dppc layers deposited on glass, mica and pmma are plotted in figs. 3, 4 and 5. the first general conclusion that can be drawn by analysing these results is that while on strongly polar solid substrates (glass and mica), the largest contact angles (both advancing and receding) were measured for diiodomethane droplets (figs. 3 and 4), and on weakly polar pmma substrate, the contact angles of diiodomethane are the lowest among these three probe liquids (fig. 5). it can be also easily noticed that generally the diiodomethane contact angle hysteresis is the smallest. the second general observation is that the reproducibility of the measured contact angles is relatively good, especially that of the advancing ones. it is a well - known fact that reproducibility of receding contact angle is generally worse than that of advancing, both measured on the same surface. the third observation is that larger changes in the contact angles occur with the increasing number of dppc layers deposited on higher energy surfaces of glass and mica than on those deposited on weakly polar pmma, whose surface possesses only some electron donor interactions (s = 1020 mj / m) [2123 ]. finally, the greatest changes in the probe liquids contact angles occur on first two to three statistical monolayers when compared to those on the bare substrate surface.fig. 3advancing and receding contact angles of water, formamide and diiodomethane on dppc monolayers deposited on glassfig. 4advancing and receding contact angles of water, formamide and diiodomethane on dppc monolayers deposited on micafig. 5advancing and receding contact angles of water, formamide and diiodomethane on dppc monolayers deposited on pmma advancing and receding contact angles of water, formamide and diiodomethane on dppc monolayers deposited on glass advancing and receding contact angles of water, formamide and diiodomethane on dppc monolayers deposited on mica advancing and receding contact angles of water, formamide and diiodomethane on dppc monolayers deposited on pmma to better depict the effect of the substrate surface properties on the contact angle hysteresis, in figs. 6, 7, and 8 are plotted the contact angle hysteresis values for water, formamide and diiodomethane on the dppc layers deposited on glass, mica and pmma. it increases sharply with increasing number of monolayers on glass (from 7 to 17), much less on the monolayers deposited on mica (from 7 to 11), and it does not change much on the layers on pmma (68), except that on one statistical dppc monolayer (11), where it is larger than on glass and mica. these results evidently show that contact angle hysteresis of highly polar water depends on the solid substrate surface properties, even though up to five dppc monolayers were deposited. it may be postulated that this must be due to differences in the layers structures and dppc molecules orientation, which to some extent is confirmed by afm images too (fig. 2). moreover, some restructuring of the dppc layer structure upon prolonged contact with water can also occur [24, 25 ]. however, this is not so evident in the case of formamide contact angles measured on the same dppc layers, which is also a polar liquid whose hysteresis results are shown in fig. generally the hysteresis increases with the dppc layer thickness on these three solid substrates, and it practically does not change on three to five layers on mica (1011). however, no clear relationship can be found with the kind of substrate. in fig. 8 are plotted the contact angle hysteresis values of apolar diiodomethane droplets on the dppc layers. the hysteresis changes for this liquid show a different trend than those for water and diiodomethane. on the layers deposited on glass and mica, the hysteresis decreases with increasing layers thickness, and it increases on the layers present on pmma surface. in fact, the diiodomethane contact angle hysteresis in all cases is below 9 ranges between 2 and 9.fig. 6contact angle hysteresis of water on dppc monolayers deposited on glass, mica and pmmafig. 7contact angle hysteresis of formamide on dppc monolayers deposited on glass, mica and pmmafig. 8contact angle hysteresis of diiodomethane on dppc monolayers deposited on glass, mica and pmma contact angle hysteresis of water on dppc monolayers deposited on glass, mica and pmma contact angle hysteresis of formamide on dppc monolayers deposited on glass, mica and pmma contact angle hysteresis of diiodomethane on dppc monolayers deposited on glass, mica and pmma to summarize the above discussed results, it is evident that contact angle hysteresis on the same dppc film, but deposited on different solid substrates, differs significantly for highly polar water and practically apolar diiodomethane. on the other hand, this is not the case for the contact angle hysteresis of formamide (fig. as the heights of the roughness are not the principal cause of the observed hysteresis, it must be due to some differences in structures of the layers, and the contact angle hysteresis of a given probe liquid depends not only on the liquid nature but also on the dppc molecule / solid surface interactions which reflect in the orientation of the dppc molecules, i.e. with their polar head or hydrophobic tails outward. on strongly polar glass and mica surfaces, more hydrophobic tails should be directed outward than in the case of weakly polar pmma surface, on which they may be less ordered. the afm images (fig. 2) and the contact angles (figs. 3, 4 and 5) show that the dppc layers might not be tightly packed and uniform, if deposited in the above described way, and the packing, organization and orientation of the molecules in the film are those of a gel state. hence, water can penetrate the film and interact also with the bare support surface and, therefore, the contact angles are relatively low on the films deposited on glass and mica surfaces, and much higher on the films on pmma surface. the increase in contact angle hysteresis also points that the probe liquid droplet penetrates deeper into the phospholipid layer structure during the receding of the three - phase contact line. hence, the differences in the values of contact angles and their hystereses reflect different strengths of the solid / liquid interactions. however, the nature of the liquid molecule is of great importance too. especially large differences in the contact angle hysteresis are observed on the two to five statistical monolayer - thick layers deposited on different substrates (figs. water is the smallest molecule among these probe liquids (table 1), and according to van oss., it shows strong electron donor l and electron acceptor l+ interactions, while formamide possesses only strong l parameter. the access of the probe liquid molecules to the polar head and apolar chains of dppc molecules is reflected in the measured contact angles. this access seems to be more important in the case of receding contact angles measured after the three - phase contact line of the droplet has retreated, and the liquid may or may not penetrate into the dppc layers, and thus, a liquid film is left behind the drop. some additional information can be obtained from the calculated surface free energies using the contact angle hysteresis approach, eq. (1) [12, 13].1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \gamma_{\text{s}}^{\text{tot } } = \frac{{{\gamma_{\text{l}}}{{\left ({ 1 + \cos { \theta_a } } \right)}^2}}}{{\left ({ 2 + \cos { \theta_{\text{r } } } + \cos { \theta_{\text{a } } } } \right) } } $ $ \end{document } thus, calculated values are apparent ones, as in fact macroscopic contact angles are too, and this model has been discussed elsewhere [12, 13 ]. despite the values are apparent ones, they provide information about the surface free energy changes taking place with increasing thickness of the layers and depending on the nature of the liquid used, i.e. polar or apolar. thus, calculated values from contact angle hysteresis of water and diiodomethane for dppc layers deposited on the three solid supports are plotted in fig. 9. in the case of layers deposited on glass and pmma, the apparent surface free energy values determined from diiodomethane contact angles are practically constant on all five layers, and they differ only slightly from those of bare solids, respectively, despite some changes in the advancing and receding contact angles and their hysteresis that occurs (figs. 3, 5, 6 and 8). however, the surface free energy calculated from diiodomethane contact angles for dppc layers deposited on mica decreases significantly, about 9 mj / m on the two to five monolayer - thick layers (fig. this means that much stronger dispersion interactions are present on the bare mica surface than on dppc monolayers, where the dispersion energy of the hydrocarbon tails seems to be weaker. thus, the apparent surface free energy values determined from water contact angles hysteresis are higher than those determined from diiodomethane contact angles (fig. the smallest changes appear in the case of dppc layers deposited on pmma whose surface shows relatively weak electron donor s and no electron acceptor s+ interactions [2123 ]. this suggests that polar heads of dppc molecules are less accessible than on the two other substrates. the dppc layers energy changes on glass and mica determined from water contact angles run almost parallel to each other, and they are a little bigger on glass. the greatest energy decrease occurs on first two monolayers, and then the changes are small. thus, the interactions of polar water molecules are weaker on the layers than on the bare surface of glass and mica, on which probably stronger hydrogen bonding can be formed.fig. 9apparent surface free energy of dppc monolayers deposited on glass, mica and pmma calculated from contact angle hysteresis of water and diiodomethane (ch2i2) apparent surface free energy of dppc monolayers deposited on glass, mica and pmma calculated from contact angle hysteresis of water and diiodomethane (ch2i2) contact angle hysteresis depends on both the solid and liquid properties. on the studied dppc layers deposited on three different solid supports, the contact angle hysteresis is larger for polar liquids (water and formamide) than apolar diiodomethane. the surface tension and its components of the probe liquids are different, as well as size of their molecules and the vapour pressure at room temperature. the hysteresis is larger on the dppc layers deposited on the surfaces having strong polar interactions, i.e. glass and mica, than on weakly polar pmma (the differences in hysteresis amount appear even on five statistical monolayers of dppc). for water and formamide, the hysteresis increases with the layer thickness increase, but for diiodomethane, it increases only on the layers deposited on pmma and decreases on the layers deposited on glass and mica. such behaviour clearly indicates that interrelation between solid support and liquid properties is decisive for the contact angle and its hysteresis on the same kind of layer, here dppc. it is believed that in the studied systems, the contact angle hysteresis does not result from the surface (film) roughness, which is only a few nanometers high, but that it is due to the liquid film presence behind the drop (derjaguin pressure) in the receded state of the droplet. additional information can be obtained by calculation of the apparent surface free energy from the contact angle hysteresis, where both the advancing and receding contact angles are taken into account. thus, the obtained results give better insight into liquid / solid surface interactions which depend both on the liquid and the surface properties. | advancing and receding contact angles of water, formamide and diiodomethane were measured on 1,2-dipalmitoyl - sn - glycero-3-phosphocholine (dppc) layers deposited on three different solid supports glass, mica and poly(methyl methacrylate). up to five statistical monolayers were deposited on the surfaces by spreading dppc solution. it was found that even on five statistical dppc monolayers, the hysteresis of a given liquid depends on the kind of solid support. also on the same solid support the contact angle hysteresis is different for each probe liquid used. the afm images show that the heights of roughness of the dppc films can not be the primary cause of the observed hysteresis because the heights are too small to cause the observed hystereses. it is believed that the hysteresis is due to the liquid film present right behind the three - phase solid surface / liquid drop / gas (vapour) contact line and the presence of derjaguin pressure. the value of contact angle hysteresis depends on both the solid surface and liquid properties as well as on intermolecular interactions between them. |
hypothyroidism is a multiorgan endocrine disorder which results in metabolic dysfunction involving brain, nerves, heart, and muscular systems. neurological abnormalities of the central and peripheral nervous system occur in conjunction with systemic abnormalities in hypothyroid and are present in 30%80% of cases but are noted only incidentally. moreover, some signs and symptoms of neurological manifestations such as somnolence, impaired memory and concentration, neuropathy, and depression may sometimes contribute significantly to disability. peripheral nervous system abnormalities have been studied in hypothyroid patients by variety of techniques. on the contrary, quantification of central nervous system (cns) dysfunction hypothyroidism has been reported to affect both electroencephalogram and visual evoked potential (vep) in response to flash stimulation, causing particularly the delay in conduction. recent studies have even established the reversibility of delay in vep sensory conduction in hypothyroid patients following the appropriate thyroxin replacement using electrodiagnostic techniques. clinical hypothyroidism in middle age is also associated with decrease in cognitive functions, especially memory. cognitive tests of patients with moderate to severe hypothyroidism indicate difficulties in performing calculations, recent memory loss, reduced attention span, and slow reaction time (rt). these are not attributable to attention deficit but to specific retrieval deficits after excluding interfering factors related to the perception of disease. hypothyroid patients showed prolongation of latencies only in the early event - related potential components, with speeding of sensory and cognitive processing after treatment. the insidious and steady history of hypothyroidism in adults indicates that it is a process which starts early but probably goes unnoticed. subclinical hypothyroid (sch) is asymptomatic and frequently a laboratory diagnosis stage with normal levels of free triiodothyronine (ft3) and t4 but a mildly increased serum thyroid - stimulating hormone (tsh) levels (4.25 iu / ml). it has been useful to identify individuals at high risk of development to overt hypothyroid. few recent studies have reported potential risk of development of neuropsychiatric dysfunction especially memory and event - related latency in sch, but the association with serum tsh remains largely unexplored. as electrophysiological study could possibly be useful in documenting the subtle abnormalities, this study was planned to assess the vep and cognitive function of memory and rt in sch subjects and find their association, if any, with the levels of serum tsh. a cross - sectional analytical study on adult sch subjects was conducted in the department of physiology, himalayan institute of medical sciences (hims), srhu, dehradun, after approval from the institutional ethical committee. the study volunteers were selected by method of probability sampling. using the mean values of p100 latency (vep) in hypothyroid cases (96.9 6.0 mm) and euthyroid controls (90.03 2.7 mm), sample size of 36 was calculated by formula for mean difference, two - sided test with alpha error of 0.05 and power of 90%. an equal number of age- and sex - matched euthyroid controls were also recruited (n = 36). equally literate, clinically healthy adults diagnosed with sch in the age group of 2040 years were recruited from the medical opd of hims hospital after they fulfilled the inclusion criteria : diagnosed as sch as per levels of serum tsh > 5 iu / ml up to 15 iu / ml and normal values of serum ft3 and free thyroxine (ft4). diagnosis was based on the confirmatory biochemical parameters (serum ft3, ft4, and tsh) in our hospital. the normal ranges of serum ft3, ft4, and tsh were 2.04.2 pg / ml ; 0.61.7 ng / dl, and 0.344.24 iu / ml. equal numbers of clinically healthy age- and sex - matched euthyroid controls were recruited from the attendants of patients and residents in and around srhu campus. common exclusion criteria were used to exclude the participants having diabetes mellitus, hypertension, bipolar disorder, tuberculosis, anemia, recent delivery (9 months), pregnancy, obesity (body mass index [bmi ] 30 kg / m), multiple endocrine syndromes, neuromuscular disorder, severe myopia, cataract, glaucoma, and maculopathy by detailed clinical history and systemic examinations including eye. following investigations were done : fasting blood sugar, x - ray chest, electrocardiogram, and hemoglobin estimation. patients with any clinical symptoms or signs referable to cns dysfunction, smokers, alcoholics and those taking drugs affecting thyroid status (lithium, nonsteroidal anti - inflammatory drugs) or drugs acting on cns were also excluded from the study. structured case reporting form was used to collect relevant clinical history, demographic, and anthropometric data. neuropack octopus (nihon kohden japan) was used to measure the latency, and amplitude of vep wave form. ab clock test (abct) and digit spanning test were used for cognitive impairment assessment. bio impedance machine was used to measure bmi, % body fat, and visceral fat. fixed metered scale and weighing machine (krups) were used to measure height and weight. volunteers were asked to report to the physiology department in morning hours on all working days. a standard case reporting form was administered by the investigator at the point of entry to collect information on demographic and anthropometric characteristics such as height, weight, bmi, personal medical history of past and present illness, and family history with detailed history of chronic medication, visual history related to diseases affecting vision, addiction, and smoking. a reversal flash pattern of checks with rate of 2 per se cond was used and values were recorded by computer - based program. the subjects were properly instructed and motivated to provide full cooperation. they were instructed to have their scalp oil free and to avoid beverages and strenuous exercise on the day of recording. they were seated comfortably in an air conditioned and sound proof dark, quiet room and instructed to fixate their one eye (other being closed with a patch) on the central red spot of the tv monitor, kept at one - meter distance. before commencement of the test, the recordings were done in the forenoon from 10 am to 12 noon after a light breakfast in the sitting posture. the skin of the scalp, the electrode disc, and electrical impedance were checked for proper functioning. the subjects were allowed to wear the usual spectacles (if any) during the test. the black and white checks (15 mm l5 mm size) subtending an angle of 32 min of an arc, were generated on the monitor through electronic generator of the evoked potential recorder. the electrical activity had low cut of 2 hz and high cut of 0.3 khz filters. the evoked responses were recorded and averaged after giving a trial of 256 checker board stimuli. the visual evoked potential - positive (vep - p) complexes were analyzed in terms of absolute peak latencies and pi amplitude. latencies were used to analyze the differences between cases and controls. experimental procedure a cognitive function involving visuospatial skills was tested by abct, and working memory was tested by digit spanning test. investigator by the gesture with his / her finger asked the subject to draw with pencil, a circle of modest size on the given blank sheet of paper. when the circle was drawn, subject was asked to put in the numbers as in the face of a clock and set the hands of clock to show ten past eleven. scoring template was placed over the completed clock with the template 's 12 oclock line placed over the subject 's 12. subject was asked to listen to some numbers at the rate of one number per second carefully and later asked to repeat the digits in monotone without any variation in pitch of voice. both the forward and backward scores were added together and compared with standard chart for final score. ninety - five percentile standard score value of 124 was set as cutoff for memory difficulties. medicraft 653 mp) with a sensitive clock (measures time up to 1/10 ms ; accuracy : + one digit). apparatus is equipped with four light stimuli (red, blue, green, and yellow). after familiarizing oneself with the procedure, apparatus and demonstration, three practice trials were given to every volunteer before final recording. the readings of vrt were recorded in a quiet adequately illuminated room with subject sitting comfortably in the chair at a constant distance from instrument. random visual signals were presented, and subjects were instructed to respond to the flashing of the light immediately by pressing switch - off knob on the panel. three readings were recorded, and minimum rt was taken as the final rt for that sensory modality of that subject. data management and statistical analysis spss (ibm spss statistics for windows, version 19.0. quantitative data were represented as mean and sd and qualitative data as proportions and percentages. differences in means of quantitative variables (e.g., vep latency, amplitude, rt) in the groups were tested by unpaired t - test and qualitative variables (memory scores) by mann whitney u - test. correlation of the serum tsh with the continuous variables, for example, vep latency and rt were assessed by pearson product moment correlation coefficient and with the qualitative variables such as memory scores by spearman correlation. the study volunteers were selected by method of probability sampling. using the mean values of p100 latency (vep) in hypothyroid cases (96.9 6.0 mm) and euthyroid controls (90.03 2.7 mm), sample size of 36 was calculated by formula for mean difference, two - sided test with alpha error of 0.05 and power of 90%. an equal number of age- and sex - matched euthyroid controls were also recruited (n = 36). equally literate, clinically healthy adults diagnosed with sch in the age group of 2040 years were recruited from the medical opd of hims hospital after they fulfilled the inclusion criteria : diagnosed as sch as per levels of serum tsh > 5 iu / ml up to 15 iu / ml and normal values of serum ft3 and free thyroxine (ft4). diagnosis was based on the confirmatory biochemical parameters (serum ft3, ft4, and tsh) in our hospital. the normal ranges of serum ft3, ft4, and tsh were 2.04.2 pg / ml ; 0.61.7 ng / dl, and 0.344.24 iu / ml. equal numbers of clinically healthy age- and sex - matched euthyroid controls were recruited from the attendants of patients and residents in and around srhu campus. common exclusion criteria were used to exclude the participants having diabetes mellitus, hypertension, bipolar disorder, tuberculosis, anemia, recent delivery (9 months), pregnancy, obesity (body mass index [bmi ] 30 kg / m), multiple endocrine syndromes, neuromuscular disorder, severe myopia, cataract, glaucoma, and maculopathy by detailed clinical history and systemic examinations including eye. following investigations were done : fasting blood sugar, x - ray chest, electrocardiogram, and hemoglobin estimation. patients with any clinical symptoms or signs referable to cns dysfunction, smokers, alcoholics and those taking drugs affecting thyroid status (lithium, nonsteroidal anti - inflammatory drugs) or drugs acting on cns were also excluded from the study. structured case reporting form was used to collect relevant clinical history, demographic, and anthropometric data. neuropack octopus (nihon kohden japan) ab clock test (abct) and digit spanning test were used for cognitive impairment assessment. bio impedance machine was used to measure bmi, % body fat, and visceral fat. fixed metered scale and weighing machine (krups) were used to measure height and weight. volunteers were asked to report to the physiology department in morning hours on all working days. a standard case reporting form was administered by the investigator at the point of entry to collect information on demographic and anthropometric characteristics such as height, weight, bmi, personal medical history of past and present illness, and family history with detailed history of chronic medication, visual history related to diseases affecting vision, addiction, and smoking. the scalp electrodes were placed relative to bony landmarks according to octopus system. a reversal flash pattern of checks with rate of 2 per se cond was used and values were recorded by computer - based program. they were instructed to have their scalp oil free and to avoid beverages and strenuous exercise on the day of recording. they were seated comfortably in an air conditioned and sound proof dark, quiet room and instructed to fixate their one eye (other being closed with a patch) on the central red spot of the tv monitor, kept at one - meter distance. before commencement of the test, the recordings were done in the forenoon from 10 am to 12 noon after a light breakfast in the sitting posture. the skin of the scalp, the electrode disc, and electrical impedance were checked for proper functioning. the subjects were allowed to wear the usual spectacles (if any) during the test. the black and white checks (15 mm l5 mm size) subtending an angle of 32 min of an arc, were generated on the monitor through electronic generator of the evoked potential recorder. the electrical activity had low cut of 2 hz and high cut of 0.3 khz filters. the evoked responses were recorded and averaged after giving a trial of 256 checker board stimuli. the visual evoked potential - positive (vep - p) complexes were analyzed in terms of absolute peak latencies and pi amplitude. latencies were used to analyze the differences between cases and controls. experimental procedure a cognitive function involving visuospatial skills was tested by abct, and working memory was tested by digit spanning test. investigator by the gesture with his / her finger asked the subject to draw with pencil, a circle of modest size on the given blank sheet of paper. when the circle was drawn, subject was asked to put in the numbers as in the face of a clock and set the hands of clock to show ten past eleven. scoring template was placed over the completed clock with the template 's 12 oclock line placed over the subject 's 12. subject was asked to listen to some numbers at the rate of one number per second carefully and later asked to repeat the digits in monotone without any variation in pitch of voice. both the forward and backward scores were added together and compared with standard chart for final score. ninety - five percentile standard score value of 124 was set as cutoff for memory difficulties. medicraft 653 mp) with a sensitive clock (measures time up to 1/10 ms ; accuracy : + one digit). apparatus is equipped with four light stimuli (red, blue, green, and yellow). after familiarizing oneself with the procedure, apparatus and demonstration, three practice trials were given to every volunteer before final recording. the readings of vrt were recorded in a quiet adequately illuminated room with subject sitting comfortably in the chair at a constant distance from instrument. random visual signals were presented, and subjects were instructed to respond to the flashing of the light immediately by pressing switch - off knob on the panel. three readings were recorded, and minimum rt was taken as the final rt for that sensory modality of that subject. data management and statistical analysis spss (ibm spss statistics for windows, version 19.0. quantitative data were represented as mean and sd and qualitative data as proportions and percentages. differences in means of quantitative variables (e.g., vep latency, amplitude, rt) in the groups were tested by unpaired t - test and qualitative variables (memory scores) by mann correlation of the serum tsh with the continuous variables, for example, vep latency and rt were assessed by pearson product moment correlation coefficient and with the qualitative variables such as memory scores by spearman correlation. the scalp electrodes were placed relative to bony landmarks according to octopus system. a reversal flash pattern of checks with rate of 2 per se cond was used and values were recorded by computer - based program. the subjects were properly instructed and motivated to provide full cooperation. they were instructed to have their scalp oil free and to avoid beverages and strenuous exercise on the day of recording. they were seated comfortably in an air conditioned and sound proof dark, quiet room and instructed to fixate their one eye (other being closed with a patch) on the central red spot of the tv monitor, kept at one - meter distance. before commencement of the test, the recordings were done in the forenoon from 10 am to 12 noon after a light breakfast in the sitting posture. the skin of the scalp, the electrode disc, and electrical impedance were checked for proper functioning. the subjects were allowed to wear the usual spectacles (if any) during the test. the black and white checks (15 mm l5 mm size) subtending an angle of 32 min of an arc, were generated on the monitor through electronic generator of the evoked potential recorder. the electrical activity had low cut of 2 hz and high cut of 0.3 khz filters. the evoked responses were recorded and averaged after giving a trial of 256 checker board stimuli. the visual evoked potential - positive (vep - p) complexes were analyzed in terms of absolute peak latencies and pi amplitude. experimental procedure a cognitive function involving visuospatial skills was tested by abct, and working memory was tested by digit spanning test. investigator by the gesture with his / her finger asked the subject to draw with pencil, a circle of modest size on the given blank sheet of paper. when the circle was drawn, subject was asked to put in the numbers as in the face of a clock and set the hands of clock to show ten past eleven. it was prompted at each stage. put in the numbers.put the time as ten past eleven. scoring template was placed over the completed clock with the template 's 12 oclock line placed over the subject 's 12. subject was asked to listen to some numbers at the rate of one number per second carefully and later asked to repeat the digits in monotone without any variation in pitch of voice. both the forward and backward scores were added together and compared with standard chart for final score. ninety - five percentile standard score value of 124 was set as cutoff for memory difficulties. experimental procedure rt was recorded by visual multiple choice rt apparatus (model no. medicraft 653 mp) with a sensitive clock (measures time up to 1/10 ms ; accuracy : + one digit). apparatus is equipped with four light stimuli (red, blue, green, and yellow). after familiarizing oneself with the procedure, apparatus and demonstration, three practice trials were given to every volunteer before final recording. the readings of vrt were recorded in a quiet adequately illuminated room with subject sitting comfortably in the chair at a constant distance from instrument. random visual signals were presented, and subjects were instructed to respond to the flashing of the light immediately by pressing switch - off knob on the panel. three readings were recorded, and minimum rt was taken as the final rt for that sensory modality of that subject. data management and statistical analysis spss (ibm spss statistics for windows, version 19.0. quantitative data were represented as mean and sd and qualitative data as proportions and percentages. differences in means of quantitative variables (e.g., vep latency, amplitude, rt) in the groups were tested by unpaired t - test and qualitative variables (memory scores) by mann correlation of the serum tsh with the continuous variables, for example, vep latency and rt were assessed by pearson product moment correlation coefficient and with the qualitative variables such as memory scores by spearman correlation. thirty - six adult cases (2040 years of age) diagnosed as having sch were studied for evoked potentials and cognitive functions. the cases and controls had comparable height, weight, and obesity markers - bmi, visceral fat, and % body fat. the number and proportion of females were considerably more than males, i.e., 31 (86.1%). a significant difference was seen between serum tsh levels of sch cases and euthyroid controls (p 0.001). no significant differences regarding ft3 and ft4 levels were observed between sch cases and controls [table 1 ]. demographic and biochemical parameters among euthyroid controls and subclinical hypothyroid cases cns affection was observed by comparing the p100 and n135 latency of veps for both cases and controls. no significant differences were seen regarding p100 latencies in both the eyes among cases and controls (p > 0.05 each) although the duration of latency in sch cases was more than the euthyroid controls. n135 latency period was higher in sch cases as compared to controls in the right eye (p = 0.035) but not in the left eye (p = 0.169) [table 2 ]. monocular latencies of n75, p100, and n135 visual evoked potential waves among euthyroid controls and subclinical hypothyroid cases significantly, lower standard digit spanning score was observed in sch cases as compared to euthyroid controls (p < 0.001). normal (124 standard score) value of digit spanning test was observed among 8 controls as compared to only 1 case. below normal (< 124 standard score) value was observed in higher number of cases than controls 35 out of 36 as compared to control. sch cases were significantly associated with below normal standard digit spanning score (< 124) in our study (p = 0.027) [table 3 ]. no significant difference was observed in visuospatial domain by the abct between euthyroid control and sch cases. digit spanning and ab clock test score among euthyroid controls and subclinical hypothyroid cases rt to visual stimuli for all the primary colors of the visible spectra showed a slower response time in sch cases in comparison to controls, and the differences were statistically highly significant (p < 0.01 each) [table 4 ]. visual reaction time for different wavelengths of light among euthyroid controls and subclinical hypothyroid cases serum tsh had significant negative correlation with digit spanning score (r = 0.40, p = 0.001). although the serum tsh levels had negative correlation with latency in generation of n75 (r = 0.10, p = 0.57), p100 (r = 0.053, p = 0.78), and n135, (r = 0.11, p = 0.43) potential waves, the correlation was nonsignificant. neurological alterations in hypothyroidism have shown that cns is more vulnerable to the effects of hypothyroidism than the peripheral nervous system. therefore, electrophysiological neurological studies can be performed in hypothyroid subjects early in the course of thyroid deficiency to detect nervous system involvement. evoked potentials such as veps provide a reliable and objective measure of function in related sensory system and tracts. the normal response to pattern stimulation is a triphasic response with a prominent positive wave (p100) with a peak latency of 84105 (mean : 96 4) ms which is suggestive of cns activity. the p100 waveform is generated in the striate and peristriate occipital cortex due to the activation of the primary visual cortex and also due to the discharge of the thalamocortical fibers. the latency depends on salutatory conduction due to myelination, whereas the amplitude of wave primarily on number of functioning axons in the nerve. slow conduction or increased latency usually implies defects in myelination and loss of amplitude due to axonal dysfunction. the p100 is a prominent peak that shows relatively little variation between the subjects, minimal within inter - ocular difference, and minimal variation with repeated measurements over time. therefore, the present study focused more on the p100 latency values among the study groups, and its correlation with the levels of serum tsh. the mean n75 values in controls and cases for both the eyes were not significantly different. the mean p100 latency for the monocular vision from both the right and left eye was marginally higher in cases as compared to controls but was statistically nonsignificant. newly diagnosed hypothyroid patients also observed nonsignificant difference between overt hypothyroid and sch, but a marginally higher latency of p100 was observed in hypothyroid cases (mean : 97 6 ms) as compared to controls (96 4 ms). significantly prolonged latency in p100 was observed in several studies in hypothyroid cases in both the eyes which reduced following treatment with triiodothyronine. also not all cases of hypothyroidism present with prolonged latency in p100 as observed by khedr. in 52% of age- and sex - matched hypothyroid cases. the increase in p100 latency may vary with number of checks in the pvep in hypothyroid cases as observed by osterweil., who found increased p100 latency in only 20 checks of pvep and not in 50 checks and attributed it to central retinal dysfunction. the above studies have observed changes in latency of p100 (vep) in long - standing hypothyroidism or untreated patients with obvious neurological involvement. it may be possible that sch duration may not be long enough in our study and changes in myelination it is an important parameter representing the integration of sensory, motor, and coordination system of the body. in our study, there was a significant increase in vrt for all the spectra of light in sch patients as compared to the control group. our findings in sch cases are similar to the findings of del ser quijano. shah and nahar in their study found a significant increase in both audio and vrt in hypo as well as hyperthyroid patients. vrt was significantly increased in hypothyroid patients even in middle age (4050 years) as reported by vedavathi. the long rt in hypothyroid and sch patients can be explained because of generalized decrease in metabolic rates affecting the sensory receptors, neural pathways, and skeletal muscles. measurement of vrt is a simple procedure and can act as a sensitive indicator of thyroid dysfunction in sch, in the absence of any other clinical pathology. cognitive assessment using digit spanning test for working memory in sch cases observed a median score of 79 (interquartile range [iqr ] = 9), which was lower than the median score in euthyroid controls (median = 96, iqr = 16.5). complementing our study,. also found that sch cases performed significantly worse in the verbal, visual memory, and global cognitive performance but improved on thyroxin replacements. similar findings were observed by sunita. across the young and elderly sch otherwise normal population in both forward and backward digit test and by martino and strejilevich in bipolar disorder subjects where the sch showed a poorer performance with cognitive function of verbal memory and attention. a recent longitudinal study in the adults has observed that a decline in the digit spanning backward test (reflecting working memory) and clock command test of visuospatial and construction was linked to the above range of tsh levels in sch subjects. sch was associated with lower standardized score 120 (< 95 percentile score of 124) of digit spanning test in our study (p = 0.02) but failed to find any association with visuospatial abct. it can be said that working memory is slightly less in cases as compared to controls, which could be because of slow processing due to modulation of calorigenesis and oxygen consumption leading to decrease in metabolic rates. it may also be explained by slow neural conduction and transduction at the sensory receptors, due to myelination and decreased voltage - gated sodium current density resulting in decreased slope of action potential as observed in overt hypothyroid cases. thyroid deficient subjects have decline in working memory and rt to stimuli which is an evidence of involvement of cns much earlier in stage and which may increase with the duration of elevated tsh. we have tried to rule out the subtle neurological risk factors in sch subjects along with defect in visual equity before the vep recording, yet we could not establish long latency in p100 in them since the duration of symptoms before diagnosis was not considered. periodic evaluation of neurological tests may help in earlier management of symptoms before the person becomes clinically hypothyroid. a follow - up study of vep on sch subjects is needed to provide evidence for the effect of high tsh on sensory conduction in cns. decline in working memory and rt to visual stimuli is an evidence of the involvement of cns in sch. | background : central nervous system (cns) involvement is insidious and may occur early in subclinical hypothyroid (sch) state which can be picked up by electrophysiological study. this study aims to record visual evoked potential (vep), event - related latency and cognitive functions, and find their association with the levels of serum thyroid - stimulating hormone (tsh) in patients with sch.materials and methods : in this cross - sectional study, 36 adult sch patients and an equal number of age- and sex - matched euthyroid controls were included. pattern reversal vep, visual reaction time (rt), digit spanning test, and ab clock test (abct) were done in both sch cases and euthyroid controls. the observed values were analyzed for comparison of mean values between the groups and correlation of recorded variables with the levels of serum tsh.results:sch cases showed a higher p100 (vep) latency in both the right (103.2 12.3 vs. 102.7 6.8 ms) and left eye (101.1 9.1 vs. 96.2 10.7 ms) as compared to controls, but the difference was statistically insignificant. a significant delay in rt was observed on visible spectra of light in sch cases (p < 0.001). digit spanning score (forward and backward) in sch cases was significantly lower than controls (p < 0.001), and a lower standardized score (< 124 or < 95th percentile) was significantly associated with sch state (p = 0.027). no significant difference was observed in visuospatial domain by abct between both the groups although the median score was lower in sch cases. only digit spanning score showed a significant negative correlation with tsh levels (r = 0.4 ; p = 0.001).conclusion : decline in working memory and rt to visual stimuli is an evidence of the involvement of cns in sch. prolonged latency in vep may depend on the duration of sch. |
trauma is among the top five causes of death in the world, showing different incidence rates from one country to another.1, 2 treatment and management of trauma involving craniofacial region still represents a challenge, due to the presence of important anatomical structures. this part of the body includes, in fact, not only vital organs, but also sensorial and motor systems as visual, auditory, olfactory and speech, composed by both numerous organs located in maxillofacial bones. moreover, maxillofacial region is the most relevant area concerning the psychological and social aspects. facial physiognomy, in fact, is used for both self and interpersonal identification, and social relationships ; any damage to this region may cause an imbalance in mental health context. the different rates of resistance and elasticity, associated to the position within the district and the shape of the bone itself, are related to the highest incidence of fractures in the mandibular and nasal bones. there are several causes of maxillofacial fractures, such as motor vehicle accidents, car crashes, sport activities, fights, etc. unintentional injuries are responsible for almost 90% of all pediatric deaths, and the head region constitutes the most affected area in pediatric patients.4, 5, 6 the analysis of gender and age distribution showed that the highest prevalence of craniofacial trauma involved young male victims. this can be related to sociocultural behavior, like the need to experience risk, beyond the abuse of alcohol or drugs.4, 5, 6, 7, 8 other reasons, like the major physical activity in heavy works or sports, performed often by young men, can explain the strong correlation ; though, the reason why trauma represents one of the first death causes in young population may be also correlated to the low rate of slow progressive diseases, like cancer, atherosclerosis or cardiovascular diseases that usually require years to show the first clinical symptoms. for the mentioned reason and the early occurring age, craniofacial trauma can be associated with severe temporary or permanent sequels, including physical, emotional, social, and economic damage as well, represented by medical care expenses for a long - term period.4, 8 although several papers have been published regarding maxillofacial trauma, the topic is still debated and discussed frequently in the literature. among various solutions proposed, recombinant human bone morphogenetic protein-2 (rhbmp-2) represents a new but already well described therapeutic option, which has showed promising results. autologous blood - derived products that have recently become commercially available do not contain bmp and have not been shown to be capable of inducing newly formed bone in the standard rat subcutaneous implant model of osteoinductivity. bmps are members of transforming growth factors - beta (tgf - beta) group and play an important role in embryonic development including brain and bone formation. at present, nearly 20 different bmps have been identified, but only bmps-2, -4, -6, and -7 have been shown to have significant osteoinductive properties. rhbmp-2 has been shown to successfully reconstruct defects ranging from isolated areas of the jaw to the entire restoration of defects. an advantage of rhbmp-2 is that it does not require a donor site, with de novo bone formation and no need for a bone graft. it has also been observed how rhbmp-2, respect native bmp, is more effective in the bone fracture healing, to shorten the consolidation phase. for these reasons, patients can return to normal life earlier, and the hospital stay is reduced. an adjunctive benefit of rhbmp-2 is represented by its ability to help soft tissue healing as well as in maxillofacial region. the effectiveness of rhbmp-2 when utilized for reconstructing mandibular continuity defects has previously been described. boyne in a randomized controlled trial demonstrated de novo organ tissue growth in humans from a recombinant human protein ; authors underlined how rhbmp-2/acs safely induced adequate bone for the placement and functional loading of endosseous dental implants in patients requiring staged maxillary sinus floor augmentation. in another study published in 2007, the bony regeneration of premaxillary clefts in humans using rhbmp-2 in a collagen sponge carrier was evaluated ; authors concluded that clefts of the anterior maxilla can have complete osseous regeneration induced by rhbmp-2 as an effective alternative to conventional anterior iliac particulate marrow cancellous bone grafts. rhbmp-2 also showed great results when applied in reconstruction of large defects occurring to mandible after tumor resection and osteonecrosis treatment.14, 15 while bmp could be an effective aid for the treatment of craniofacial trauma in adults, further considerations must be taken when the patient is still in a growing age. fda established that rhbmp-2 is contraindicated in patients who (1) are pregnant, (2) may be allergic to any of the materials contained in the devices, (3) have an infection near the area of the surgical incision, (4) have had a tumor removed from the area of the implantation site or currently have a tumor in that area, or (5) are skeletally immature. the general use of bmp-2 in immature patients is not recommended, due to the possible insurgence of side effects and the limited experience. guidelines for age, weight and level dependent dosage of rhbmp-2 in the pediatric patients are not present in literature yet. although extensive data on rhbmp-2 use in immature patients are lacking, several studies described the off - label use of rhbmp-2 to achieve spinal fusion in children with compromised bone healing.16, 17, 18 moreover, concerning the maxillofacial district, a case of a 14 teenage female who had complete regeneration of a mandibular defect with an off - label use of rhbmp-2 has been reported. even if boyne has shown that pediatric patients can be able to achieve a complete osseous regeneration without bone grafting after a large continuity mandibular resections, it is hard to imagine the same degree of bone regeneration without the aid of rhbmp-2. furthermore, the surgical treatment itself sometimes may interfere with the growth of pediatric patients. the treatment method applied is often modified according to the child 's age and development. bone morphogenic proteins possess good osteoinductive properties that enhance healing and are used in the treatment of adult patients with recalcitrant non - unions and spinal fusion procedures successfully to facilitate union / fusion. manufacturers of commercially available rhbmp-2 have stated that it is contraindicated for use in the pediatric population because they have not been able to provide data that establish the safety and efficiency of bmp-2 in children below 18 years of age. the possibility of selecting a conservative treatment versus an open surgical approach is quite debated. the application and the development of bmp-2 for maxillofacial trauma can play an important role for the management of large maxillofacial defect due to traumatic event. the choice to treat craniofacial fractures in a closed approach may cause severe facial deformities, which are extremely difficult to correct in the future. the surgeon should analyze the risks of impacting skeletal growth versus obtaining acceptable stability and reduction for healing. the avoidance of the sequela of nonoperative treatment and its effects should be avoided for the severe fractures if at all possible. surgeon must evaluate facial growth, development of the paranasal sinuses and the dental status. the surgeon must consider the general situation of the patient, evaluating maintenance of the airway, balance of fluid and electrolyte levels and adequate nutritional intake during treatment. there has been some discussion regarding the use of resorbable plates and screws in pediatric facial fractures. some authors recommend the use of resorbable plates and screws for growing patients whereas others do not.21, 22 rhbmp-2 has been used in orthopedic trauma surgery to treat tibia fractures. govender, in a clinical trial, used rhbmp-2 associated with unreamed nails in open tibia fractures for the first time. during bone formation, bmp-2 plays a fundamental role in a complicated cascade of events that involves a number of several stimulatory factors and cells types. even if bmp-2 exhibits direct action in recruiting cells to the area and stimulating cell differentiation, the natural biology of bone formation remains intact. those growth factors are therefore thought to initiate, stimulate and increase the normal bone formation cascade. they showed statistically significant reduction of secondary interventions, which was the main parameter taken in consideration in this study. moreover, secondary clinical outcomes like hardware failures, fracture healing time, infection wound healing were significantly better when the rhbmp-2 was utilized. aro instead observed that the application of a dose of 1.5 mg / ml rhbmp-2, in association to a reamed nail fixation, for the treatment of open tibia fractures does not significantly shorten the healing time. they also found similar infection rates in the control (11%) compared to the rhbmp-2 group (19%). recently, alt evaluating both clinical and economic aspects, investigated the effectiveness of rhbmp-2 for tibia fracture treatment again. they highlighted how the use of rhbmp-2 reduces secondary interventions in patients with grade iii open tibia fractures treated with an unreamed nail. furthermore, they confirmed the ability of rhbmp-2 to shorten healing time (even if the difference observed was not significant) and sequentially, to impact the economic aspect, like reduction of health costs and productivity loss. even if application of rhbmp-2 sometimes showed unclear outcomes, undoubtedly, it has still the potential to bring some advantages in complex clinical situation and trauma. currently the existing problems related to rhbmp-2 application in oral maxillofacial surgery and trauma are well documented. several tests were performed to determine the effects of rhbmp-2 on fertility reproduction and perinatal toxicity. all those researches demonstrated how the treatment of gravid rabbits with those growth factors did not result in maternal toxicity or gross fetal abnormalities.26, 27, 28 moreover, toxicology studies using intravenous administration of rhbmp-2 have demonstrated no toxic effects at doses much higher than those recorded in the clinical application for the bone trauma management. some journal articles have reported the presence of bmp-2 in a number of human neoplasms. however, those growth factors have not been suggested to play a role in the primary neoplastic process.29, 30 rhbmp-2 showed the potential to be an effective therapeutic option for treatment of many trauma and craniofacial bone defects. future studies, investigating the long - term effects of rhbmp-2 in large samples, may help to provide scientific evidence in trauma treatment. however, more clinical multi central trials need to be realized in order to be effective those potential therapeutic device. the loma linda oral and maxillofacial surgery department is today a referring center for the use of growth factor in oral and maxillofacial surgery. boyne, loma linda university professor emeritus, on 1996 published a pilot study performed on adult male macaca fascicularis (rhesus) monkeys in order to observe the effect of two dose ranges of rhbmp-2 on bone regeneration following bilateral hemimandibulectomies. the result of this work indicates that rhbmp-2 can bring about osseous regeneration of critical sized hemimandibulectomy defects in rhesus monkeys. ten years later, after several animal research and after the fda release, bmp-2 has been currently used in the management of craniomaxillofacial critical size defect. from 2007 those growth factors have been used for the management of the cleft palate in young patients. clefts of the anterior maxilla can have complete osseous regeneration induced by rhbmp-2 as an effective alternative to conventional anterior iliac particulate marrow cancellous bone grafts. in my opinion, and accordingly with urist, bmp-2 gives the surgeon the possibility of having the control of osteogenesis. however even if the cost is still high, the application of the growth factors represents the future for avoiding large bone grafting procedure reducing the discomfort of the patient. future studies about the ideal carrier should be performed in order to better manage the action and the release of those powerful proteins. | in recent years, recombinant human bone morphogenetic protein-2 (rhbmp-2) has been introduced as a therapeutic option in the treatment of several congenital and acquired craniofacial defects. although there have been promising clinical results, the international literature still lacks complete guidelines, including limits and indications for the use of rhbmp-2. the possible indications for rhbmp-2 in patients undergoing facial trauma are discussed in this article. |
the size of engineered nanomaterials makes many novel and innovative products, as evident by the increasing number of commercially available nanotechnology products. thus, there is a huge concern surrounding the potential toxicity of these nanomaterials and there is a need to sufficiently test such materials. the goal here is to understand and control risk, and both toxicity testing and physicochemical characterisation should be conducted. although our current understanding of risk associated with nanomaterials is limited, attempts have been made in order to assess this systematically. recently, aschberger they have indicated the risk expected from metal and metal oxide nanomaterials, which was particularly relevant in the case of algae and daphnia. they have attributed the risk from such materials their exposure to both the particles and corresponding dissolved ions. there is a general consensus within the nanoecotoxicological community that physicochemical characterisation of nanomaterials in complex media is not a trivial matter, and so the reliability of such measurements is vital if we are to understand and control the risk imposed by nanomaterials [2, 3 ]. in recent years, the oecd initiative has adopted a holistic approach to this problem and that physicochemical characterisation should be carried out with as many parameters as possible, to include redox potential. the prospect (ecotoxicology test protocols for representative nanomaterials in support of the oecd sponsorship programme) project is the uk 's contribution to this oecd initiative, and the uk is responsible for two types of nanomaterials : cerium oxide (ceo2) and zinc oxide (zno). out of all seventeen oecd parameters identified, redox potential is the most ambiguous in its definition, what this parameter means and how it is measured, in a nanoecotoxicological context. integral to any ecotoxicological investigation is the ability to measure the redox conditions of a given system as indicated by the redox potential. in nature, redox reactions are an important part of phenomena such as mineral weathering, bacterial respiration, and degradation of pollutants. in soil chemistry, for example, the redox potential value can estimate whether the soil is aerobic or anaerobic, and whether chemical compounds such as fe oxides or nitrate have been chemically reduced or are present in their oxidised form. in natural waters, redox reactions include the oxidation of organic matter and various reduction reactions such as the reduction of oxygen to water, nitrate to elementary nitrogen dioxide, iron (iii) to fe (ii), sulphate to sulphide, and carbon dioxide to methane. in terms of nanomaterial toxicity, redox potential is a parameter that has been associated with inducing oxidative stress. recently, burello and worth, in their prediction of a given nanomaterial to induce oxidative stress, have developed a theoretical framework that combines measurements of nanoparticle particle size and redox potential. the need to accurately measure redox potential is evident, in particular we need to understand the extent that nanomaterials can influence the natural redox phenomena should these materials be released into the environment. redox potential is a measure of a system 's affinity for electrons, and the measurement of redox potential will only have meaning when there are reduced and oxidised species, called the redox couple, in the liquid media. the redox couple undergoes a redox reaction, in which the reduction (gain of electrons) of one redox species is accompanied by the oxidation (loss of electrons) of another. the movement of electrons, governed by kinetics (e.g., transport limitations of the redox species to the electrode), creates an electric potential. the potential measured is determined by the ratio of activities of oxidised and reduced species, as defined by the nernst equation ; this is a thermodynamic property. the redox potential can be directly measured using a potentiometer (high impedance voltmeter) with an oxidation - reduction potential (orp) electrode. this is the recommended technique under the current oecd guidelines for the testing of nanomaterials (nms) ; essentially it is a measurement of potential difference (in mv) across a two - electrode system, that is, an inert platinum (pt) electrode and silver - silver chloride (ag / agcl) reference electrode. have recently adopted this approach to measure the redox potential of nanomaterial dispersions, that is, cerium oxide dispersed in synthetic freshwater algal medium. there are theoretical and practical difficulties associated with the measurement of redox potential and these, although not well recognised, have already been discussed for many years. firstly, redox potential is based on the concepts of equilibrium thermodynamics, and as such it can only be adequately measured at equilibrium. a reliable redox potential measurement requires that equilibrium be established not only at the electrode, but also among the various redox couples in solution. secondly, most redox potential measurements represent mixed potentials, and certain redox species may not contribute significantly towards the redox potential value ; that is, not all will react sufficiently fast enough at the electrode and therefore will not contribute towards stable and reliable redox potential measurements. if the particles themselves act as a redox species, then there are various factors that may prevent them contributing towards the final orp value, including sedimentation events, diffusion limitations, and the barrier of electron exchange at the pt electrode. if this is true, then redox potentials of nanomaterial dispersions are likely to be solely dominated by dissolved redox species in the media, rather than contributions arising from the particles themselves. in this study although the orp probe has been conveniently used in the past by scientists to directly measure redox potential, it is essential that the reliability of such data should be questioned when measuring nanomaterial dispersions. there is the risk that researchers may treat such a tool as a black box and thus may not be fully aware of the inherent limitations in the use of such a tool in these measurements. in this study, the orp values of six nanomaterial (either zno or ceo2 dispersed in one of the four liquid media) dispersions will be measured using an orp probe. the orp values of the nanomaterial dispersions will be compared relative to each other and to the corresponding media blank, to identify if there is any evidence of redox contributions as a result of the particles themselves. if there are redox contributions from the particles themselves, then this is likely to happen when dispersions are stable, as this would allow sufficient time for the particles to interact with the pt electrode. consequently as part of the investigation, the properties associated with the different nanomaterials will be characterised, parameters of interest to include particle size, zetapotential, and dispersion stability (as reported by the so - called half life values). zeta - potential is a well - known parameter that characterizes the electric properties of solid surface in contact with liquid and is a way to probe surface charge. the magnitude of this value is related to dispersion stability, that is, the higher the value, the better the dispersion stability. the concept of half - life has been put forward in the oecd guidelines as the measurand to indicate dispersion stability through time that is, the larger the half - life value ; the longer it takes for the concentration to reduce by half and thus the more stable the dispersion. lastly, the corresponding scanning electron microscopy (sem) data, that is, the primary particle size (mean feret diameter) and the corresponding standard deviation, will also be reported. all experiments were performed in a temperature - controlled laboratory, and for the redox potential measurements the temperature of the dispersions were monitored using a temperature probe (reported value of ~20c) to ensure that any change in the readings was not attributed to temperature changes in the dispersions. the nms supplied from the prospect programme were of two types, either ceo2 or zno, and are as follows : nanograin ceo2 (from umicore belgium), nanosun zno (from micronisers, australia), micron zno (from sigma aldrich, uk), z - cote zno (from basf, germany), micron ceo2 (from sigma aldrich, uk), ceria dry ceo2 (from antaria, australia). the particles were used as received and did not contain any added surface stabilisers. di water (resistivity of 18 mohm) from a millipore, milliq system was used to prepare all aqueous solutions and suspensions. for the purpose of zetapotential measurements, di water with 5 mm sodium chloride (sigma aldrich, uk) was employed in addition to deionised water ; the nacl here served as background electrolyte for the measurement of zetapotential. (chemical compositions) used for making up the ecotox media were obtained from the university of exeter, one of our collaborators in the prospect project. three types of ecotox relevant media were prepared accordingly and for long - term storage, the ecotox solutions were autoclaved and kept refrigerated until needed. seawater, in which 25 g per l of tropic marine sea salt (tropical and marine limited) was made up, resulting in ph ~8.8. this was prepared by firstly dissolving appropriate salts (196 mg cacl22h2o, 82 mg mgso47h2o, 65 mg nahco3, 0.002 mg na2seo3, as obtained by appropriate dilutions of a 2 mg / ml stock solution) in 1 l of di water. upon continued stirring, further di water was added so that conductivity of the solution was between ~360 and 480 s / cm. first, salts (11.76 g cacl22h2o, 4.93 g mgso47h2o, 2.59 g nahco3, 0.23 g kcl) were dissolved separately in 1 l of di water to make four separate stock solutions. second, 25 ml of each salt stock solution was aliquot into a clean bottle and diluted in di water (made up to 1 l volume). third, 200 ml of the stock solution from step 2 was aliquoted and further diluted with di water (made up to 1 l volume). nanomaterials were dispersed using the protocol as previously reported (tantra, jing, gohil 2010) (http://www.nanotechia-prospect.org/publications/basic). briefly, this involved weighing the nanoparticle powder into small, clean vials using an analytical mass balance. dispersion was carried out by adding the appropriate liquid media (fish, daphnia, seawater, or di water) dropwise (5 drops from a pasteur pipette) and mixing using a spatula so as to produce a thick paste before adding 15 ml of liquid media and stirring gently, using the same spatula. the formation of a thick paste as a first step was necessary to allow the efficient displacement of powder - air interface with the powder - liquid interface. the subsequent deagglomeration step was carried out using an ultrasonic probe (130 watt ultrasonic processors) ; this was done by inserting the ultrasonic probe tip (6 mm ti) half way down the 15 ml volume of dispersed nanoparticles, and sonication was carried out with 90% amplitude for 20 s. after sonication, the nanoparticle suspension was diluted using the appropriate liquid media, in order to make up to 1 l total volume. a glass rod was used to gently mix the final dispersion, to ensure homogeneity. the dispersions (in the four different media) were stored in separate precleaned 1 l media bottles and left undisturbed. analyses of redox potentials, half - lives, and zetapotentials were conducted on the day immediately after the dispersions were made. sem images were obtained using a supra 40 field emission scanning electron microscope from carl zeiss (welwyn garden city, hertfordshire, uk), in which the optimal spatial resolution of the microscope is a few nanometres. in - lens detector images were acquired at an accelerating voltage of 15 kv, a working distance of 3 mm, and a tilt angle 0. the sem was calibrated using a sira grid calibration set (sira, chislehurst, kent, uk). these are metal replicas of cross - ruled gratings of area 60 mm with 19.7 lines / mm for low magnification and 2160 lines / mm for high magnification calibrations, accurate to 0.2%. for analysis of the as received nanoparticle powders, a sample of each powder was sprinkled over a sem carbon adhesive disc ; one side of the carbon disc was placed securely on a metal stub, whilst the other side was exposed to the nanoparticle powder. excess powder was removed by gently tapping the stub on its side until a light coating of powder on the surface became apparent. an adequate magnification was chosen for image acquisition for example, for the estimation of primary particle mean diameter, the shape and limits of the primary particles should become apparent. sem micrographs were analysed by manually tracing contours of primary particles onto a transparency sheet. the transparency sheet was scanned for further image analysis using image j software, which automatically calculated particle diameter dimensions. redox potentials were measured using an orp oakton waterproof orp testr, purchased from cole - parmer, uk. this, in effect, measures the potential difference across two electrodes (a pt electrode against a double junction ag / agcl reference electrode). the orp instrument manufacturer has specified a resolution of 1 mv, with an accuracy of 2 mv. prior to use, the electrode was preconditioned in clean tap water for 30 minutes before a final rinse with distilled water. when making measurements, the electrode was carefully placed in a vial containing the nanomaterial dispersion sample ; there must be sufficient liquid sample to cover the sensing element. the electrode was carefully stirred a little and then placed in a fixed position, slightly above the bottom of the container. the signal output was allowed to settle for 5 minutes before a reading, the field potential, was noted. at this point, the signal was stable and there was no further change observed within the next few minutes. after measurement, the electrode was cleaned with tap water and rinsed with distilled water, after which further measurements could be made. when not in use, the electrode was stored in oakton electrode storage solution. the redox potential orp electrode was calibrated against ysi zobell orp calibration solution (purchased from cole - palmer). this reagent was made available in dry form and was reconstituted with 125 ml of di water prior to use, after which the solution has ~6-month expiry date. this standard solution was used to verify the performance of the electrode at the beginning and end of the study. for ag / agcl reference, the redox potential value for zobell solution was quoted to be 231 10 mv (depending on temperature) ; at ~20c, this value was ~237 mv. redox potential measurements were carried out on freshly dispersed nanomaterial in the four chosen media, as detailed above. all field potential values recorded were subjected to an additive correction factor of + 206 mv. this was necessary so that the final value was reported as if the reference electrode was a standard hydrogen reference electrode (she) instead of the ag / agcl, as previously documented. the conversion from ag / agcl to she is typically on the order of 200 to 220 mv, and voltage correction is temperature dependent and also varies slightly with the concentration of kcl (~3.5 m) in the electrode filling solution. turbidity was measured using an hf scientific - micro100 ri turbidity meter (cole - palmer, uk) ; this meter has an infrared light source that meets the international standard iso 7027 for turbidity measurements. the meter was calibrated on standards, which are based on amco - aepa-1 microspheres ; these standards are traceable to standard formazin suspension. standard values of 1000, 10 and 0.02 ntu were used to calibrate the meter. prior to use, the meter was allowed to warm up for 30 minutes. sample cuvettes (hf scientific (usa)) were used to hold the samples. note that glass thickness may vary from cuvette to cuvette and within the same cuvette. hence, individual vials were indexed ; indexing of the cuvette entails finding the point of the cuvette that light passes through that gives the lowest reading and, once indexed, the holder can be marked accordingly. prior to their use, cuvettes were cleaned, in accordance with manufacturer 's instructions. this involved washing the interior and exterior of the cuvette with a detergent (2% hellmanex in di water) ; it was then rinsed several times in distilled water before finally rinsing in di water. the cuvette was further rinsed with the sample two times before filling (30 ml) and analysed. the cuvette was placed into the meter and signal allowed to settle before taking readings. turbidity readings were taken at regular time intervals. when not in use, the vials (containing the dispersions) were stored in the dark. electrophoretic measurements were obtained using a zetasizer nano zs (malvern instruments, uk) equipped with a 633 nm laser. the reference standard (dts1230, zetapotential standard from malvern) prior to their use, these cells were thoroughly cleaned with ethanol and deionised water, as recommended by the instrument vendor. for analysis, the individual cell was filled with the appropriate sample and flushed before refilling ; measurement was carried out on the second filling. malvern instrument 's dispersion technology software (version 4.0) was used for data analysis, and zetapotential values were estimated from the measured electrophoretic mobility data using the smoluchowski equation, as documented in a previous publication. table 1 shows the redox potential results associated with dispersions of the prospect nanomaterials in four different media. the table also contains the redox potential values of the corresponding blank media, that is, liquid media with no nanomaterial. the values in brackets show the difference of orp readings upon addition of the nanomaterials with respect to the corresponding blank media. results show that orp values are dominated by the type of liquid media used for dispersions. nanomaterial dispersions in seawater resulted in a much smaller orp values in comparison with dispersions made in other media. in addition, this is also the case with the orp values of the corresponding blank media, that is, blank seawater having the smallest orp value, of 384 mv, compared to the rest of the blank liquid media (redox potential values all above 400 mv). the orp values reported here is not specific to a single chemical species and thus represent an aggregate oxidization - reduction of all species that can react at the pt working electrode. the fact that the type of liquid media itself seems to have some contribution to the final orp values is not surprising, as dissolved redox species in the liquid can easily interact with the pt electrode of the orp probe. in fact, such orp measurements are often employed as an accurate gauge of water quality and for the monitoring of dissolved species in the water. seawater in particular is shown to be more reducing in nature, that is, due to higher concentration of reducing agents (such as no2) in such media, if compared to the other liquid media. furthermore, we expect a much - reduced level of oxidising agent such as dissolved oxygen in such a high saline solution, as the more saline the water can be the less oxygen the water can hold. if there is a reduction in oxidising agents, then this also has an effect of lowering the orp value. the orp readings reported here were taken three times with very little variation among the replicates, that is, not more than 2 mv. however, the second and third replicates were acquired soon after acquiring the 1st replicate, by taking the orp probe out of the dispersion and reimmersing it back into the dispersion. the variations in the replicates here thus represent variations of the instrument 's accuracy ; they will not represent any variations that might be due to other factors such as differences in dispersion quality. table 1 also shows the change in orp values (values reported in brackets) upon addition of the nanomaterials relative to the corresponding blank media. results show that, in most cases, there is a change of less than ~10 mv associated upon addition of the nanomaterials. there are only three cases in which the orp value change is greater than 15 mv : nanograin ceo2 in fish medium, micron ceo2 in di water, and ceria dry ceo2 in fish media. currently, we offer no explanation as to why there is a much larger change in orp values in these three cases, apart from potential variations in dispersion quality, for example, due to potential redox contaminants associated with the different samples received. in addition, no real differentiation can be made between zno and ceo2 particles, with z - cote zno (basf, germany) having the same 9 mv change as the ceria dry ceo2 (antaria, australia), when both are dispersed in di water. the other physicochemical characterization associated with the nanomaterials are shown in tables 2, 3, and 4 below, which correspond to zetapotential, turbidity, and sem particle size measurements, respectively. results show that zetapotential values of nanomaterials when dispersed in seawater can not be successfully measured (due to high conductivity) and thus displayed as n / a in table 2. such unsuccessful measurements were reported in the corresponding quality report at the end of the measurement. in general, results indicate high zetapotential values for nanomaterials that are dispersed either in di water or di water + 5 mm nacl. results of the di water are similar to the corresponding di water + nacl case ; the addition of nacl into the di water was carried out so as to have greater confidence in the di water results, as the measurement of zetapotential usually involves the presence of inert background electrolyte. overall, results show that nanomaterials are most stable when dispersed in di water (or di water + nacl) and least stable when in an ecotox media. this is true apart for the case of micron ceo2 showing that it is least stable in di water, that is, 7 mv when compared to other ecotox media such as fish medium, that is, 22 mv. currently, no explanation is available for this behaviour, and dispersion stability was further measured by using the concept of half - life values (as previously discussed in the introduction, the larger the half - life, the more stable the dispersion). results in table 3 show that, as with zetapotential measurements, results in di water are generally more stable (with the largest associated with z - cote zno of 4038 minutes) than when in ecotox media. however, unlike the zetapotential values, micron ceo2 also shows the same trend, that is, most stable in di water than when in an ecotox media. the reason for this discrepancy lies in the fact that dispersion stability was measured in two different ways : through the measurement of interparticle force (zetapotential) or through analyzing the stability via sedimentation measurements (turbidity with time). the former measurement is solely governed by the electric properties of the solid surface in contact with liquid, which will subsequently contribute towards sedimentation rate ; the latter measurement is not only determined by the zetapotential value but also by other factors, for example, particle size (in which the larger particles are expected to sediment at a much faster rate). the sem results (table 4) show the mean (feret) primary particle sizes and the corresponding standard deviations (to reflect on the polydispersity of the primary particle size). results presented in table 4 show that the mean primary particle sizes of the samples range from ~30 nm to ~890 nm, the largest being the micron zno and micron ceo2 from sigma aldrich. the sd values show the large degree of polydispersity associated with samples received : high polydispersity associated with sigma aldrich samples, less so with nanosun zno, microniser. the sem micrographs indicated that all particles tested here were highly aggregated together into agglomerates of irregular shape. if there is any particle contribution towards the final orp values, then, out of the two types of nanomaterials tested, we expected ceo2 to have a bigger contribution compared to zno. this is on the basis that ceo2 particle can act as a redox couple of ce(iv)/ce(iii), which is not the case for zno. if ceo2 particles had contributed to the final orp value, then we expect this to occur with nanograin ceo2 (having the smallest particle size of ~30 nm, as shown in table 4) and when dispersed in di water, as this resulted in a highly stable dispersion (noted by its high dispersion stability value of 2676 min and high zeta potential value of 33 mv, as shown in tables 3 and 2, resp.). a highly stable dispersion will mean sufficient time to allow particles to diffuse to the pt electrode, thus interacting with the pt electrode in order to contribute towards the final orp reading. hence, we expected the redox potential to be affected most by the nanograin ceo2 in di water and clearly this was not the case. as shown in table 2, nanograin ceo2 in di water only resulted in an orp value change of 11 mv compared to a change of 21 mv when the same particles were dispersed in fish medium. overall, results suggest that the particles have minor effects on the final orp readings. the study investigated the redox potential measurements, using orp probe electrode, of different zno and ceo2 dispersions, in various liquid media. the variations in the orp readings for the different dispersions could not be regarded as being highly significant and were mainly governed by the type of liquid media that the nanomaterials were dispersed in. this is not surprising, as orp values are dominated by the amount of dissolved chemical species in the liquid media. dispersions in seawater resulted in the lowest orp values, suggesting that the media is reducing in nature. this was attributed to a much higher concentration of reducing agents such as sulphites or a reduction in the concentration of dissolved oxygen under a high salinity environment. the study shows that there was little contribution from the particles themselves towards the final orp reading, with no significant differentiation between ceo2 and zno. as it is clear that redox potential measurements using an orp electrode will not indicate a particle 's contribution towards the final redox potential value, the work has highlighted the need to have better tools for such measurements. there are several alternatives to using the orp probe, including x - ray photoelectron spectroscopy (xps) and electron paramagnetic resonance (epr) and electron energy loss spectroscopy (eels). however, these technologies rely on the indirect measurement of redox potential and the accuracy of the values reported may come into question. | redox potential has been identified by the organisation for economic co - operation and development (oecd) as one of the parameters that should be investigated for the testing of manufactured nanomaterials. there is still some ambiguity concerning this parameter, i.e., as to what and how to measure, particularly when in a nanoecotoxicological context. in this study the redox potentials of six nanomaterials (either zinc oxide (zno) or cerium oxide (ceo2)) dispersions were measured using an oxidation - reduction potential (orp) electrode probe. the particles under testing differed in terms of their particle size and dispersion stability in deionised water and in various ecotox media. the orp values of the various dispersions and how they fluctuate relative to each other are discussed. results show that the orp values are mainly governed by the type of liquid media employed, with little contributions from the nanoparticles. seawater was shown to have reduced the orp value, which was attributed to an increase in the concentration of reducing agents such as sulphites or the reduction of dissolved oxygen concentration. the lack of redox potential value contribution from the particles themselves is thought to be due to insufficient interaction of the particles at the pt electrode of the orp probe. |
during percutaneuos coronary intervention (pci) procedures, several complications can occur, such as dissections, thrombi, ulcers or plaque, may appear as angiographic hazy or uncertain images. a proper diagnosis is important to guide the correct management in the catheterization laboratory and to improve patient outcomes. angiography has important limitations to characterize those entities that, with contrast, can be diagnosed accurately thanks to intravascular imaging.1) among the intracoronary imaging techniques available, intravascular ultrasound (ivus) and optical coherence tomography (oct) are the most commonly used for research. both techniques provide great value for characterization of coronary lesions and for guidance of pci. ivus is very useful to measure the vessel wall and to evaluate plaque burden, whereas the oct (with almost microscopic resolution) is an excellent method for characterizing luminal and intimal lesions.2 - 4) herein, we present a case that illustrates the ability of oct for accurate diagnosis of an intracoronary post - angioplasty that had an uncertain hazy image, and its usefulness as a guide for therapeutic strategy. a 73-year - old female patient underwent coronary angiography after being admitted with a non - st elevation myocardial infarction. we determined that the patient had 2 diseased coronary vessels, with the culprit lesion in the right coronary artery (rca) and a severe lesion in the left anterior descending artery (lad) (fig. 1). we treated the rca lesion with implantation with a bare metal stent (bms). ten hours before the second procedure, the patient was given a sub - cutaneous dose of 1 mg / kg enoxaparin. following the current recommendation guidelines,5) we gave the patient a 0.3 mg / kg enoxaparine dose by intravenous administration immediately before the angioplasty procedure ; we decided against giving the patient unfractionated heparin. we performed a predilatation procedure using a compliant balloon angioplasty in the lad without angiographic complications, and after that we implanted a bms of 2.523 mm. in the control post - pci angiography because of the uncertain interpretation by angiography, we decided to perform oct in order to ascertain the mechanism of the angiographic hazy image (dissection, thrombus, plaque prolapse). we used a c7-xr, saint jude oct frequency domain system with a dragon fly catheter (st. the oct study showed an intra - stent protruding signal rich mass with irregular borders and low - backscattering, a finding compatible with a white thrombus. the stent was fully expanded but showed some strut malapposition at the proximal border (fig. the final result was significant reduction of the thrombus and correction of the stent malapposition (fig. the patient was discharged 3 days after the procedure and did not experience any additional clinical events. at 4 months acute stent thrombosis may occur in patients, with or without acute coronary syndrome.4)6 - 12) rinaldi.6) identified 4 independent predictors of stent thrombosis during pci : higher baseline platelet count, acute myocardial infarction indication, use of a coil or self - expanding stent, and preprocedural thrombus. in this particular case, a great thrombus burden was found during the first acute pci performed in the rca, but no angiographic thrombotic images that were found suggested lad thrombosis prior to pci. we had not performed an initial oct, so a possible previous thrombus could be present, but had remained undetected by angiography. however, we consider that a high - risk patient, the stent malapposition and possibly an inadequate anticoagulation could explain the acute stent thrombosis. an alternate explanation, such as prolapsed thrombus, could not be dismissed without a previous oct. an incorrect diagnostic leads to an incorrect choice in therapeutics and potential damage to the patient. the choice of ivus or oct in order to obtain a specific diagnosis of an intravascular lesion depends in most of the cases on the availability and or experience in the laboratory. the oct has a spatial resolution of about 10 - 15 m with a penetration depth of 2 to 3 mm. the frequency - domain oct modality, allows for high - resolution imaging, with pullbacks of 50 mm in less than 3 seconds, with minimum use of contrast and without coronary occlusion requirement.2)3) oct allows the characterization of histologic composition of the lesion and a precise identification of the luminal border, with high sensitivity to detect tiny dissections, plaque prolapse or intraluminal thrombus (even to distinguish white and red thrombus).2 - 4)7 - 12) in contrast, ivus has a spatial resolution much lower, around 130 m, and does not allow the accurate characterization of some intravascular images.4)8)9) comparing both intravascular image techniques and after performing pci in their study, kawamori.8) could identify thrombus in 15% with oct, and 5% with ivus. in the setting of pci for acute coronary syndrome kubo.9) could detect thrombus in all patients with oct, but only in 33% of patients with ivus. in this case, discrimination between dissections, plaque prolapsed, or thrombus was crucial to the choice of treatment. a dissection would have required a new stent implantation, and it would certainly have contraindicated thrombus aspiration. optical coherence tomography is a straightforward and useful method to assess misleading angiographic lesions, probably with higher accuracy than ivus when scanning intravascular or intra - stent images, and may be useful during pci procedures when angiography is uncertain. | although its use in daily practice is not common, optical coherence tomography (oct) is a powerful research tool in invasive cardiology. this report describes a hazy angiography image after percutaneous coronary intervention that has been assessed using oct. based on the results of the oct, the patient underwent an elective coronary angioplasty with standard anticoagulation. after implantation of the stent, an intracoronary hazy image was seen on angiography. the use of oct permitted a correct diagnosis and a successful treatment. this paper provides a discussion of the advantages and disadvantages of oct, and a comparison with intravascular ultrasound. |
epilepsy is one of the common neurological disorders, which require immediate medical attention and long - term therapy. the incidence is approximately 0.30.5% in different world populations with a prevalence rate of five to 10 per thousand people. the overall aim of treating epilepsy should be complete control of seizures, without causing any untoward reaction due to the medication. patients perceptions often take account of other parameters such as effects of epilepsy on daily activities and functions. epilepsy can be associated with profound physical, psychological, and social consequences ; and its impact on a person 's quality of life (qol) can be greater than that of chronic conditions. people with epilepsy have been shown to report a poorer qol because they are more likely to have poor self - esteem and a high level of anxiety and depression. in some patients, the social stigma and impact on qol can pose a greater challenge than the clinical severity. health - related quality of life (hrqol) is recognized as an important outcome in epilepsy treatment. research assessing the qol associated with successful treatment of epilepsy is far behind that of other chronic conditions such as cancer, diabetes, and cardiovascular disease. very few studies have been carried out on qolie-31 in india and research in this area will identify factors affecting qol and may lead to strategies that improve the management of patients with epilepsy. this study was therefore conducted to determine the level of health - related qol of patients of epilepsy in a tertiary care teaching hospital. this was a cross - sectional, questionnaire - based study conducted in a tertiary care teaching hospital from march to october 2013 after approval from the institutional ethics committee. respondents were adults aged at least 18-year - old with a diagnosis of epilepsy for at least a year. they were explained the nature and purpose of the study and necessary consent were obtained. patients with associated psychotic disorders, severe mental retardation, strokes, head injuries, brain tumors, and patients who have had recent brain surgery were excluded. questionnaires were developed to collect sociodemographic data (age, sex, employment status, educational level) and clinical aspects of epilepsy (seizure frequency, duration of epilepsy, and medication). seizures frequency was defined as the number of seizures occurring in the last year prior to the interview. the quality of life in epilepsy (qolie-31) was used for collecting data on health - related qol with the permission of the research and development (rand) corporation. it consists of seven subscales, which are seizure worry, emotional well - being, energy / fatigue, cognitive functioning, medication effects, social functioning, overall qol, and one item of overall health. the responses were used likert rating scales, which were later transformed into linear scales that ranged between 0 and 100. descriptive statistics were expressed as mean standard deviation (sd) and percentage as appropriate. the unpaired t - test or one - way analysis of variance (anova) the correlation coefficient was used to measure the relationship between seizure frequency and subscale and overall score. descriptive statistics were expressed as mean standard deviation (sd) and percentage as appropriate. the unpaired t - test or one - way analysis of variance (anova) the correlation coefficient was used to measure the relationship between seizure frequency and subscale and overall score. descriptive statistics were expressed as mean standard deviation (sd) and percentage as appropriate. the unpaired t - test or one - way analysis of variance (anova) the correlation coefficient was used to measure the relationship between seizure frequency and subscale and overall score. totally, 60 patients of epilepsy (irrespective of the type of epilepsy) were included in the study consisting of 35 men and 25 women. the range of seizures frequency in the past 1-year was 14 with a mean of 2.367, and mean duration of epilepsy was 6.9 years [table 1 ]. demographic characteristics of patients with epilepsy the mean total score of qolie-31 was 64.61 [table 2 ]. the highest mean score was the emotional well - being effects, 70.53 and the lowest was seizure worry subscale, 57.55. one - way anova showed significant differences in the mean score of sub - scales of qolie-31 (p < 0.001). the total score of seizure worry were significantly lower as compared to all other subscales in qolie-31 (p < 0.001). there was no significant difference in a total score among all other subscales when compared to each other in a post - hoc analysis. total score of qolie-31 sub scales in epilepsy patients there were no significant differences in the mean of total qol scores between groups for sociodemographic and clinical characteristics except for drug therapy [table 3 ]. there was a significant difference in the total score of qolie-31 within the monotherapy and polytherapy group. differences of qol score for sociodemographic characteristics and drug therapy in epilepsy patients comparison of various subscale score and overall score between different monotherapy groups was done using one - way anova [table 4 ]. there was the statistical significant difference in score of cognitive effect and medication effect between the three monotherapy groups. a post - hoc analysis showed that statistical significant difference was seen between phenytoin and carbamazepine group and valproate and carbamazepine group in case of cognitive effect. similarly, for a medication effect score was significantly better in carbamazepine group as compared to valproate group. improving the qol in a person with a seizure disorder is an essential component of the management of such patients. the mean total score of qolie-31 in our study, was almost similar to a study conducted in india but higher than studies conducted in australia (52.9), africa (52.1). a study in malaysia has reported a higher mean total score of qolie-31 (68.9). even though the majority of the studies had used qolie-31 questionnaire (different translations), different study methodologies with different inclusion and exclusion criteria would have accounted for the different scores. the pattern of scores of qolie-31 subscales of our study was partially similar to the studies conducted in africa and malaysia. in our study, the emotional well - being subscale was highest, and seizure worry the lowest. the difference in pattern may be due to the reason that different countries have dissimilarities of beliefs, culture, and socioeconomic factors which in turn can affect qol measures, thus findings from other countries, may not be relevant to the local situation. there was no significant difference in the qol scores and sociodemographic characteristics such as education and employment status. in our study, unemployment did not have any impact on qol score because most of them fit independent category that is either students or housewives. however, other studies have reported that unemployment are often related to the state of seizure control, the age of onset and duration of illness, the type of medication, severity and frequency of seizures. in our study in general, the literature says that people with frequent seizures had significantly poorer hrqol than those with infrequent or no seizures., reported that seizures frequency was the most important clinical predictor of psycho - social dysfunction and emotional maladjustment. longer duration of epilepsy has been reported as a predictor for poor qol due to greater complications and disabilities. the probable reason may be small sample size in the present study. in our study, this may be due to the fact that patients on polytherapy have more severe and complicated disease. however, in contrast to this finding, other studies have reported that there was no association between qolie-31 and type of drug therapy. among patients receiving monotherapy, furthermore, scores for medication effect were better in carbamazepine group as compared to valproate group indicating that the patients in the valproate group were more worried about the side effects of the drug. this may be due to better adverse effect profile of carbamazepine as compared to other drugs. it is evident from our study that there are many factors that influence qol of people with epilepsy. among them patients who were on monotherapy had a better qol mainly because of the lesser side effects. adding clinical counseling and other interventions to address the physical, mental, psychological, social, and emotional aspects of health well - being is likely to achieve better health outcomes for epilepsy patients. raising awareness in society regarding the existence of effective therapy through public educational campaigns might help in eliminating the stigma of epilepsy and may improve qol of epilepsy patients. | objectives : health - related quality of life (qol) is an important outcome in epilepsy treatment. very few studies have been carried out on the quality of life in epilepsy (qolie-31) in india. the present study aimed to determine the level of health - related qolie-31 in patients of epilepsy.materials and methods : this was a cross - sectional, questionnaire - based study conducted in a tertiary care teaching hospital. respondents were adults aged at least 18-year - old with a diagnosis of epilepsy. qolie-31 was used for collecting data on health - related qol. the unpaired t - test or one - way analysis of variance was used to compare means of qol scores between groups.results:totally, 60 patients of epilepsy were included in the study. the mean (standard deviation) total score of qolie-31 was 64.61. a score of cognitive and medication effect were significantly better in carbamazepine group as compared to valproate group.conclusions:patients on monotherapy had a better qol as compared to patients receiving polytherapy. |
sheehan 's syndrome (ss) is defined as pituitary hormone deficiency due to ischemic infarction of the pituitary gland as a result of massive postpartum uterine hemorrhage. although the onset of ss can involve acute severe panhypopituitarism in some patients, the majority of ss patients are diagnosed with a clinically subtle partial pituitary deficiency and therefore their diagnoses and treatments are delayed for many years. the prevalence of ss is not clearly known, presumably due to the great number of nondiagnosed patients. it is a rarely encountered disorder in developed countries due to good obstetric care [3, 4 ]. however, its prevalence is estimated to be still high in developing countries in which many deliveries take place at home. zargar. estimated the prevalence of ss to be 3.1% among women in india, and about two - thirds of those women had given birth at home. as the etiopathogenesis of ss is not clear, disorders of coagulation have been investigated in some studies. it has been reported that disseminated intravascular coagulation (dic) can cause postpartum hypopituitarism [7, 8 ]. in addition, cakir. found protein s deficiency in 2 out of 12 patients with ss. importantly, in another study, gokalp. investigated inherited hypercoagulation as a risk factor of ss. they found that frequency of mthfr (methylenetetrahydrofolate reductase) c677 t and mthfr a1298c polymorphisms was significantly higher among their 38 patients with ss compared to the healthy control group. in addition, factor ii (g20210a), factor v (g1691a), and pai-1 4g/5 g mutations were also more common among the ss patients, but no significant differences were observed. our aim in this study was to investigate gene polymorphisms of mthfr c677 t and a1298c, factor ii (g20210a), factor v (g1691a), and pai-1 (plasminogen activator inhibitor-1) 4g/5 g that are associated with inherited hypercoagulation and tnf- (tumor necrosis factor - alpha) 308 g > a that is associated with apoptosis of pituitary cells due to autoimmunity. knowing the frequencies of these polymorphisms among ss patients will be helpful in understanding their roles in the etiopathogenesis of ss. fifty - three patients who were previously diagnosed with ss and 43 healthy women were enrolled in this study which was conducted between 2011 and 2013. the blood samples of patients with ss who were followed up by the endocrinology department of erciyes university medical school were collected following 12 hours of fasting. the healthy female volunteers in the control group were chosen from hospital staff and their relatives. in addition to the demographic information, the medical history and drug use of the participants were examined in detail. the most important inclusion criterion for the ss group was having the exact diagnosis of ss. therefore, it was emphasized that all of the following criteria for diagnosis were met : (a) at least one pituitary hormone deficiency found in basal levels or via dynamic tests if required ; (b) massive postpartum uterine hemorrhage history at last delivery ; (c) agalactia and amenorrhea after the last delivery ; (d) exclusion of all other causes of pituitary deficiency ; (e) observation of partial or complete empty sella on magnetic resonance imaging (mri). exclusion criteria for ss patients were having comorbidity or being on other treatments than glucocorticoid and thyroid hormone replacement therapies which were adequately performed. in order to avoid the effects of replacement therapies of growth hormone (gh) and gonadal steroids on genotyping studies, such treatments were stopped 3 months before the blood collection for the genetic analyses. in addition, women in the control group did not have any histories of disease and were not on any drug therapies. in addition, the insulin tolerance test or glucagon stimulation test was performed to identify growth hormone (gh) deficiency and secondary adrenal failure. basal hormone levels including free t4 (normal : 0.881.72 ng / dl), thyroid stimulating hormone (tsh ; normal : 0.575.6 miu / ml), adrenocorticotropic hormone (acth ; normal : 046 pg / ml), cortisol (normal : 923 g / dl), prolactin (prl ; normal for postmenopausal women : 2.429.8, and premenopausal women : 3.329.8 ng / ml), follicle stimulating hormone (fsh ; normal for postmenopausal women : 23.9119.1, and premenopausal women : 2.09.8 miu / ml), luteinizing hormone (lh ; normal for postmenopausal women : 16.354.8, and premenopausal women : 0.717.3 miu / ml), estradiol (e2 ; normal for postmenopausal women : 14.444.5, and premenopausal women : 18.9246.7 pg / ml), and insulin - like growth factor-1 (igf-1 ; reference intervals varied by age) were measured in the hormone laboratories of erciyes university medical school. methods of assays and commercial kits were as follows : gh : immunoradiometric assay (irma), immunotech sas, france ; igf-1 : irma, immunotech sas, france ; acth : irma, cisbio bioassays, france ; cortisol : radioimmunoassay (ria), immunotech s.r.o, czech republic ; prl, tsh, ft4, fsh, lh, estradiol : immunoassay, siemens advia centaur xp - usa. 2 ml venous blood samples were collected in edta - containing tubes for dna analyses. total genomic dna was extracted by standard methods and dna samples were stored at 20c until the analyses of polymorphisms. genotyping studies were conducted in erciyes university genome and stem cell center (genkok). a total of 20 l pcr mixture with 5 l sample dna was analyzed by using a lightcycler faststart dna master hybprobe to perform the genotypings of mthfr c677 t and a1298c, factor ii (prothrombin) g20210a, and factor v g1691a according to manufacturer 's instructions (roche diagnostics, germany). in addition, roche lightcycler 480 software was used for detecting different genotypes of these polymorphisms. genotypes of the tnf--308 g / a and pai-1 4g/5 g polymorphisms were detected by analyses of polymerase chain reaction (pcr) and restriction fragment length polymorphism (rflp) results. the genomic region of interest was amplified by using pcr for 30 cycles with a denaturation temperature of 94c for 3 min, 94c for 30 sec, 60c for 30 sec, and 72c for 30 sec. amplified 98 base pair (bp) products were digested overnight with bsl i at 55c and subjected to 4% agarose gel electrophoresis. the pcr procedure was performed in a total volume of 50 l containing 5 l genomic dna, 10x pcr buffer, dntps (2.5 mm), mgcl2 (1.5 mm), taq dna polymerase (1 u / ml), 5aggcaataggttttgagggccat-3 forward primers, and 5tcctccctgctccgattccg-3 reverse primers for tnf-. the cycling conditions consisted of denaturation at 95c for 5 min, followed by 30 consecutive cycles of 95c for 1 min, 60c for 1 min, and 72c for 1 min, with a final elongation at 72c for 5 min. the 107 bp pcr products were separated by electrophoresis on a 2% agarose gel and visualized with uv illumination and ethidium bromide staining. after amplification, the pcr products were digested overnight at 37c with nco i restriction enzyme and analyzed by 3% agarose gel electrophoresis. binomial variables were analyzed using pearson 's chi - square test or fisher 's exact test. moreover, the odds ratios (or) were calculated with 95% confidence intervals (ci) using logistic regression analysis. comparisons of genotype frequencies were made between patients and controls, and p values less than 0.05 were considered as statistically significant. panhypopituitarism was detected in 38 (71.7%) patients with ss, while partial hypopituitarism was detected in 15 (28.3%) patients. importantly, all patients had gonadotropin and gh deficiencies, but deficiencies of acth, tsh, and prolactin were not found in all of the patients (table 1). when comparing groups, it was revealed that there was no significant difference between the mean age of the 53 cases with ss and the 43 healthy controls, 63.2 12.5 and 60.3 9.3 years, respectively. in addition, no difference was detected in terms of mean body mass indexes (bmis). kg / m in the ss group and 29.2 3.3 kg / m in healthy women. moreover, the ss and healthy control groups did not significantly differ according to polymorphism ratesof factor ii (g20210a), factor v (g1691a), mthfr (c677 t and a1298c), pai-1 (4g/5 g), and tnf- (308 g > a) genes, except for pai-1 (4g/5 g). the pai-1 (4g/5 g) mutation was detected in 26 (60.5%) of the 43 control cases and in 20 (37.7%) of the 53 ss cases. the physiological enlargement of the pituitary gland during pregnancy plays a significant role in onset of ss, because severe bleeding does not lead to pituitary deficiency in women unless they are pregnant. even though the pathogenesis of ss has not yet been fully clarified, the basis of its pathology has been identified as infarction and ischemic necrosis that develops due to the interruption of arterial blood flow in the anterior pituitary gland. however, the cause of the interruption in the blood flow is not clear. considered potential mechanisms are arterial thrombosis similar to that seen in stroke, development of arterial spasm as a result of severe hypotension that is due to massive uterine bleeding, or compression of pituitary vessels due to relatively small sella turcica volume associated with enlargement of the pituitary during pregnancy. furthermore, autoantibodies detected in many patients against the pituitary gland have been suggested as a contributing factor in the etiopathogenesis of ss. in the etiopathogenesis of ss, our study findings did not yield a significant difference between the control and ss patient groups in terms of mutations of blood composition anomalies that lead to inherited hypercoagulation. in other words, in the ss group none of the genes had higher polymorphism rates than those in the control group. there are numerous other acquired (prolonged immobilization, pregnancy, oral contraceptive pills, advanced age, obesity, cigarette use, hypertension, etc.) or genetic (protein c or s deficiency, antithrombin - iii deficiency, platelet gpiib / iiia hpa - ib mutation, elevated levels of factors vii, viii, ix, and xii, von willebrand disease, fibrinogen, etc.) factors that are known to cause hypercoagulation. an important limitation of our study is that not all of the parameters associated with hypercoagulation were investigated. therefore, it is hard to conclude, based only on our study findings, that inherited hypercoagulation is not involved in ss. although, the mutation rates of mthfr c677 t and a1298c genes show social differences, homozygous mutations are estimated to be 10%, and heterozygous mutations are about 40% [16, 17 ]. approximately, 15% of the population is reported to carry both heterozygous genetic mutations together, due to their close relations. although the rates of mthfr gene polymorphisms were not higher in the ss group than in the control group in our study, more important issues are whether the ss patients had vitamin b6, vitamin b12, and/or folate deficiency and thus a disorder in mthfr gene expression during their last pregnancies. the heterozygous mutations in factor ii (prothrombin) g20210a are encountered at a 2 - 3% rate among caucasians. plasma factor - ii levels (prothrombin) increase as a result of mutations. in that case, venous thrombosis risk among individuals carrying this mutation is increased by 2 - 3 times. one study reported that coronary artery risk is increased by 1.31 times in individuals with prothrombin g20210a mutation. while varying across different populations, the incidence rate of factor v leiden heterozygous mutation is approximately 5%, but its homozygous mutation is rare. as a result of the mutation in the factor v gene (g1691a), activated this, in turn, results in a deterioration of the bleeding and coagulation balance in favor of coagulation. this mutation is reported to increase thrombosis risk fivefold when heterozygous, and by 1080-fold when homozygous [21, 22 ]. factor v leiden has also been shown to increase complications such as miscarriage, preeclampsia, and abruptio placentae by a minimum of 2 - 3 times. plasminogen activator inhibitor-1 (pai-1) inhibits tissue plasminogen activator (tpa) and urokinase which are proteins leading to plasminogen activation and fibrinolysis. the estimated frequences of pai-1 4g/4 g, 4g/5 g and 5g/5 g are, respectively, about 35%, 50%, and 15% [23, 24 ]. polymorphisms of pai-1 4g/4 g and 4g/5 g are reported to cause an increase in pai-1 levels and thus risk of arterial thrombosis rather than of venous thrombosis [25, 26 ]. pregnant women who carry the homozygous pai-1 4g/4 g mutation are also known to have an increased risk of miscarriage. however, there are other studies reporting that patients with ischemic stroke and coronary artery disease do not have increased pai-1 4g/5 g polymorphism [28, 29 ]. interestingly, our results showed that the 4g/5 g polymorphism rate was 60.5% among healthy women, while it was 37.7% among ss patients. this finding may suggest that the 4g/5 g polymorphism does not always lead to thrombophilia, unless accompanied by some acquired factors. tnf- (tumor necrosis factor - alpha) is a cytokine that plays an important role in the regulations of immune functions, cell proliferation and differentiation, apoptosis due to autoimmunity, coagulation, adipocyte, lipid and glucose metabolism. the 308 g / a polymorphism of tnf- which leads to elevated tnf- levels is known to have an important impact on the development of autoimmunity diseases, metabolic syndromes, cancers, and psychiatric disorders [30, 31 ]. autoimmunity is a potential risk factor in the development of ss and 35% of patients have been shown to have anti - pituitary autoantibodies. these antibodies are presumably generated in reaction to the necrotic pituitary gland tissue found after ischemic infarction and seem to be responsible for the chronic but progressive course of ss. on another note, increased estrogen levels during pregnancy make adenohypophysis cells, particularly the lactotroph cells, more vulnerable to apoptosis due to autoimmunity as a result of elevated expression of the tnf- gene in rats. this condition may also be associated with the vulnerability of an enlarged pituitary to ischemic necrosis at the end of pregnancy. estrogen levels gradually increase during pregnancy and peak close to completion of the full term. in this regard, we hypothesized that autoimmunity caused by tnf- 308 g / a mutation can be a contributing factor in the etiopathogenesis of ss. however, no increase in the rate of tnf- 308 g / a polymorphism was observed among the ss patients when compared to the control group. as demonstrated in our study, the disorders suggested for ss etiopathogenesis and the associations among them are highly complicated. however, the results of our study showed no increase in factor ii, factor v, mthfr, pai-1, and tnf- gene polymorphism rates among ss patients compared to the control group. hence, genetic factors other than these gene polymorphisms should be researched in the etiopathogenesis of ss. clarifying ss etiopathogenesis via further studies will be particularly beneficial in identifying women susceptible to ss prior to disease development. | sheehan 's syndrome (ss) is defined as pituitary hormone deficiency due to ischemic infarction of the pituitary gland as a result of massive postpartum uterine hemorrhage. herein, we aimed to investigate the roles of factor ii (g20210a), factor v (g1691a), mthfr (c677 t and a1298c), pai-1 4g/5 g, and tnf- (308 g > a) gene polymorphisms in the etiopathogenesis of ss. venous blood samples were obtained from 53 cases with ss and 43 healthy women. standard methods were used to extract the genomic dnas. factor ii (g20210a), factor v (g1691a), and mthfr (c677 t and a1298c) polymorphisms were identified by real - time pcr. pai-1 4g/5 g and tnf- (308 g > a) gene polymorphisms were detected with polymerase chain reaction (pcr) and restriction fragment length polymorphism (rflp) methods. according to statistical analysis, none of the polymorphisms were found to be significantly higher in the ss group compared to the control group. hence, we suggest that genetic factors other than factor ii, factor v, mthfr, pai-1, and tnf- gene polymorphisms should be researched in the etiopathogenesis of ss. |
aric is an ongoing, prospective, observational study of the natural history of cv risk factors and atherosclerotic diseases. detailed study rationale, design, and procedures have been previously published.11 the original cohort included 15 792 participants recruited between 1987 and 1989 using probability sampling of middle - aged (45 to 64 years old) men and women from 4 communities in the united states (forsyth county, nc ; jackson, ms ; suburban minneapolis, mn ; and washington county, md). enrollment of african americans varied by field center (jackson 100% ; forsyth county 12% ; minneapolis < 1% ; washington county < 1%). subsequent follow - up visits occurred at 3-year intervals up to 1998, with annual telephone interviews conducted between visits and to the present. institutional review boards from each site approved the study, and informed consent was obtained from all participants. the fourth aric exam occurred between 1996 and 1998, was attended by 11 656 participants, and is the basis for this analysis given that plasma n - terminal pro b - type natriuretic peptide (ntprobnp) levels were measured on samples obtained at this visit. we excluded 2519 participants for the following reasons : missing plasma nt - probnp levels (n=405) ; self - reported race other than african american or caucasian (n=31) ; prevalent or missing status regarding prevalent cvd (defined as coronary artery disease, heart failure, atrial fibrillation / flutter, or stroke ; n=1989), or missing baseline characteristics of interest (n=94). ntprobnp levels were measured using an electrochemiluminescent assay on a cobas e411 analyzer (roche diagnostics, indianapolis, in), as previously described.12 the measurement range was 5 to 35 000 pg / ml, with a value of 2.5 pg / ml assigned to participants with levels below the limit of detection. the coefficient of variation was 3.5% to 4.7%.12 established definitions for htn, obesity, dm, and smoking status, as previously described in aric, were utilized.13 annual household income was determined by participant self - report at visit 1 (19871989). htn was defined as systolic (sbp) or diastolic blood pressure (dbp) 140 or 90 mm hg, respectively, or use of antihypertensive medications, which were classified according to the medispan therapeutic classification system.14 body mass index (bmi) was calculated from measured height and weight, with obesity defined as a bmi 30 kg / m. creatinine and fasting glucose were measured according to standardized protocols ; estimated glomerular filtration rate (egfr) was calculated with the chronic kidney disease epidemiology collaboration (ckd - epi) equation with chronic kidney disease (ckd) defined as an egfr < 60 ml / min per 1.73 m.15 prevalent dm was defined as fasting glucose 126 mg / dl or nonfasting glucose 200 mg / dl, self - report of physician diagnosis of diabetes, or use of antidiabetic medications. among participants without dm, the homeostatic model assessment method was used to quantify insulin resistance (homa - ir) as fasting glucose (mg / dl)insulin/405.16 urine microlbuminuria was measured and categorized as previously described, with microalbuminuria defined as a urine albumin - to - creatinine ratio of 30 to 300 mg / g.17 genotyping methods for estimating genetic ancestry among african americans in the aric study have been previously described.18,19 briefly, genotyping was performed on stored dna from visit 1 using the illumina beadlab platform at the center for inherited heart disease research (johns hopkins university, baltimore, md). single - nucleotide polymorphisms (snps) with frequencies that significantly differed between african and caucasian ancestral populations were defined as ancestry informative markers (aims). among african americans, who represent an admixed population, percentage european ancestry (pea). race - specific frequencies of each snp among the aims were estimated from west african and european samples to provide a bayesian prior for ancestral allele frequencies. pea was available in 1656 (86%) of african americans in the final study population for this analysis. summary statistics for covariates were calculated as counts (percentages), and medians (interquartile ranges ; iqrs) for categorical and continuous data, respectively. comparisons between african americans and caucasians were made by chi - square () or wilcoxon rank - sum tests, as appropriate. a cumulative distribution plot was derived in order to compare natural log - transformed ntprobnp levels according to race. three sensitivity analyses were performed to compare ntprobnp levels between african americans and caucasians when restricted to : (1) participants with detectable ntprobnp levels (5.0 pg / ml) ; (2) healthy participants, defined as no prevalent cvd, htn, dm, ckd, past or current smoking, or use of antihypertensive medications, aspirin, or lipid - lowering therapy and bmi < 25 kg / m ; and (3) participants from forsyth county, north carolina, which was the only aric field center with substantial numbers of both african americans and caucasians. multivariable linear regression was used to assess the associations between race and ntprobnp. given the non - normal distribution (right - skewed) of ntprobnp, values were natural log transformed before entry into regression models. the multiplicative effect (percent difference) on log ntprobnp levels was estimated by exponentiating the beta coefficients (95% confidence interval [ci ]) for each covariate. for example, african - american race is associated with 40% lower log ntprobnp levels than caucasians because e=0.60. multivariable logistic regression was used to calculate the adjusted odds of nondetectable ntprobnp levels according to race. covariates included in multivariable models included race, age, gender, bmi, heart rate, sbp and dbp, fasting glucose, egfr, annual household income, htn, antihypertensive medication, dm, smoking status, and microalbuminuria. in additional analyses, homa - ir was added into the multivariable logistic and linear regression models to account for the association between insulin resistance and plasma ntprobnp levels. multivariable linear regression analyses were also repeated among african americans, with inclusion of pea as a covariate. before entry into regression models, all analyses were performed using stata software (11.2 ; stata corp, college station, tx). aric is an ongoing, prospective, observational study of the natural history of cv risk factors and atherosclerotic diseases. detailed study rationale, design, and procedures have been previously published.11 the original cohort included 15 792 participants recruited between 1987 and 1989 using probability sampling of middle - aged (45 to 64 years old) men and women from 4 communities in the united states (forsyth county, nc ; jackson, ms ; suburban minneapolis, mn ; and washington county, md). enrollment of african americans varied by field center (jackson 100% ; forsyth county 12% ; minneapolis < 1% ; washington county < 1%). subsequent follow - up visits occurred at 3-year intervals up to 1998, with annual telephone interviews conducted between visits and to the present. institutional review boards from each site approved the study, and informed consent was obtained from all participants. the fourth aric exam occurred between 1996 and 1998, was attended by 11 656 participants, and is the basis for this analysis given that plasma n - terminal pro b - type natriuretic peptide (ntprobnp) levels were measured on samples obtained at this visit. we excluded 2519 participants for the following reasons : missing plasma nt - probnp levels (n=405) ; self - reported race other than african american or caucasian (n=31) ; prevalent or missing status regarding prevalent cvd (defined as coronary artery disease, heart failure, atrial fibrillation / flutter, or stroke ; n=1989), or missing baseline characteristics of interest (n=94). ntprobnp levels were measured using an electrochemiluminescent assay on a cobas e411 analyzer (roche diagnostics, indianapolis, in), as previously described.12 the measurement range was 5 to 35 000 pg / ml, with a value of 2.5 pg / ml assigned to participants with levels below the limit of detection. established definitions for htn, obesity, dm, and smoking status, as previously described in aric, were utilized.13 annual household income was determined by participant self - report at visit 1 (19871989). htn was defined as systolic (sbp) or diastolic blood pressure (dbp) 140 or 90 mm hg, respectively, or use of antihypertensive medications, which were classified according to the medispan therapeutic classification system.14 body mass index (bmi) was calculated from measured height and weight, with obesity defined as a bmi 30 kg / m. creatinine and fasting glucose were measured according to standardized protocols ; estimated glomerular filtration rate (egfr) was calculated with the chronic kidney disease epidemiology collaboration (ckd - epi) equation with chronic kidney disease (ckd) defined as an egfr < 60 ml / min per 1.73 m.15 prevalent dm was defined as fasting glucose 126 mg / dl or nonfasting glucose 200 mg / dl, self - report of physician diagnosis of diabetes, or use of antidiabetic medications. among participants without dm, the homeostatic model assessment method was used to quantify insulin resistance (homa - ir) as fasting glucose (mg / dl)insulin/405.16 urine microlbuminuria was measured and categorized as previously described, with microalbuminuria defined as a urine albumin - to - creatinine ratio of 30 to 300 mg / g.17 genotyping methods for estimating genetic ancestry among african americans in the aric study have been previously described.18,19 briefly, genotyping was performed on stored dna from visit 1 using the illumina beadlab platform at the center for inherited heart disease research (johns hopkins university, baltimore, md). single - nucleotide polymorphisms (snps) with frequencies that significantly differed between african and caucasian ancestral populations were defined as ancestry informative markers (aims). among african americans, who represent an admixed population, percentage european ancestry (pea) race - specific frequencies of each snp among the aims were estimated from west african and european samples to provide a bayesian prior for ancestral allele frequencies. pea was available in 1656 (86%) of african americans in the final study population for this analysis. summary statistics for covariates were calculated as counts (percentages), and medians (interquartile ranges ; iqrs) for categorical and continuous data, respectively. comparisons between african americans and caucasians were made by chi - square () or wilcoxon rank - sum tests, as appropriate. a cumulative distribution plot was derived in order to compare natural log - transformed ntprobnp levels according to race. three sensitivity analyses were performed to compare ntprobnp levels between african americans and caucasians when restricted to : (1) participants with detectable ntprobnp levels (5.0 pg / ml) ; (2) healthy participants, defined as no prevalent cvd, htn, dm, ckd, past or current smoking, or use of antihypertensive medications, aspirin, or lipid - lowering therapy and bmi < 25 kg / m ; and (3) participants from forsyth county, north carolina, which was the only aric field center with substantial numbers of both african americans and caucasians. multivariable linear regression was used to assess the associations between race and ntprobnp. given the non - normal distribution (right - skewed) of ntprobnp, values were natural log transformed before entry into regression models. the multiplicative effect (percent difference) on log ntprobnp levels was estimated by exponentiating the beta coefficients (95% confidence interval [ci ]) for each covariate. for example, african - american race is associated with 40% lower log ntprobnp levels than caucasians because e=0.60. multivariable logistic regression was used to calculate the adjusted odds of nondetectable ntprobnp levels according to race. covariates included in multivariable models included race, age, gender, bmi, heart rate, sbp and dbp, fasting glucose, egfr, annual household income, htn, antihypertensive medication, dm, smoking status, and microalbuminuria. in additional analyses, homa - ir was added into the multivariable logistic and linear regression models to account for the association between insulin resistance and plasma ntprobnp levels. multivariable linear regression analyses were also repeated among african americans, with inclusion of pea as a covariate. before entry into regression models, all analyses were performed using stata software (11.2 ; stata corp, college station, tx). compared to caucasians, african americans were more likely to be female, hypertensive, and have microalbuminuria ; however, ntprobnp levels in african americans (median, 43 pg / ml ; iqr, 18, 88) were significantly lower than in caucasians (median, 68 pg / ml ; iqr, 36, 124 ; p<0.001 ; table 1 ; figure 1). ntprobnp levels below the limit of detection were also more frequent among african americans (9%) than caucasians (2% ; p<0.001). significantly lower ntprobnp levels in african americans were also found when analyses were restricted to (1) participants with detectable levels of ntprobnp, (2) healthy participants, or (3) participants at the forsyth county, north carolina, field center (data not shown). baseline characteristics of african - american and caucasian participants without prevalent cardiovascular disease at aric visit 4 (19961998) data presented as percentage or median (interquartile range) for categorical and continuous data, respectively. ace indicates angiotensin converting enzyme ; arb, angiotensin receptor blocker ; aric, atherosclerosis risk in communities ; bmi, body mass index ; bpm, beats per minute ; ca, calcium ; dbp, diastolic blood pressure ; egfr, estimate glomerular filtration rate ; homa - ir, homeostatic model for assessment of insulin resistance ; ntprobnp, n terminal pro b type natriuretic peptide ; sbp, systolic blood pressure ; uacr, urine albumin creatinine ratio. natural log ntprobnp values are lower in african americans, as compared to caucasians (p<0.001). for example, the frequency of natural log ntprobnp levels 4 (ntprobnp=54.6 pg / ml) is 60% in african americans, compared to 40% of caucasians. the lower limit of the natural log of ntprobnp is truncated at 0.92 based upon the lowest value of ntprobnp of 2.5 pg / ml. aric indicates atherosclerosis risk in communities ; ntprobnp, n - terminal pro b - type natriuretic peptide. in multivariable linear regression analysis, adjusted log ntprobnp levels were significantly lower (40% ; 95% ci, 43, 36) in african americans than caucasians, independent of other factors associated with ntprobnp levels (figure 2). covariates also associated with lower ntprobnp levels included male gender, higher bmi, heart rate, and dbp, as well as higher glucose and egfr. in contrast, covariates associated with higher levels of ntprobnp included increasing age, lower household income, beta - blocker use, current smoking, higher sbp, and presence of microalbuminuria. with inclusion of homa - ir into the multivariable linear regression model, increased insulin resistance was also significantly associated with lower ntprobnp levels (14% ; 95% ci, 16, 11), although this did not attenuate the relationship between african - american race and lower plasma ntprobnp levels (37% ; 95% ci, 41, 33). in subgroup analyses, ntprobnp levels were consistently lower in african americans, as compared to caucasians (figure 2). forest plot of percent difference in log ntprobnp levels in african americans, compared to caucasians, overall and across subgroups of aric participants. african - american race is significantly associated with lower ntprobnp levels, as compared to caucasians, in the overall study population and within subgroups. values shown are from multivariable adjusted linear regression analyses (see methods for covariates). for the insulin - resistant subgroups, aric indicates atherosclerosis risk in communities ; homa - ir, homeostatic model for assessment of insulin resistance ; ntprobnp, n - terminal pro b - type natriuretic peptide. multivariable logistic regression was performed to examine factors associated with nondetectable plasma nt - probnp levels (table 2). after multivariable adjustment, african - american race was associated with significantly increased odds of having nondetectable levels (multivariable adjusted odds ratio [or ], 5.74 ; 95% ci, 4.22, 7.80). in an additional analysis with inclusion of homa - ir in the model, african - american race remained significantly associated with increased odds of nondetectable ntprobnp (or, 5.51 ; 95% ci, 3.91, 7.77). characteristics significantly associated with nondetectable plasma ntprobnp among aric participants nondetectable plasma defined as ntprobnp < 5 pg / ml. data presented as percentage or median (interquartile range) for categorical and continuous data, respectively. aric indicates atherosclerosis risk in communities ; bmi, body mass index ; bpm, beats per minute ; dbp, diastolic blood pressure ; egfr, estimate glomerular filtration rate ; ntprobnp, n terminal pro b type natriuretic peptide ; or, odds ratio. or adjusted for all covariates listed plus age, annual household income, history of hypertension, systolic blood pressure, antihypertensive medication class, diabetes mellitus, smoking status, and the presence of microalbuminuria. ors for continuous variables reflect odds per 1 sd (bmi=5.5 kg / m ; heart rate=9.5 bpm ; dbp=10 mm hg ; fasting glucose=31 mg / dl ; egfr=16.5 ml / min per 1.73 m). among the 1656 african americans in whom pea data were available, median pea was 15% (iqr, 11, 23). in multivariable linear regression models, a 1 sd (10%) increase in pea was associated with 7% (95% ci, 1, 13) higher adjusted ntprobnp levels (p=0.025 ; figure 3). the relationship between genetically determined percent european ancestry and plasma ntprobnp levels among self - reported african americans in aric. among self - reported african americans, with increasing proportion of european ancestry aric indicates atherosclerosis risk in communities ; ntprobnp, n - terminal pro b - type natriuretic peptide. compared to caucasians, african americans were more likely to be female, hypertensive, and have microalbuminuria ; however, ntprobnp levels in african americans (median, 43 pg / ml ; iqr, 18, 88) were significantly lower than in caucasians (median, 68 pg / ml ; iqr, 36, 124 ; p<0.001 ; table 1 ; figure 1). ntprobnp levels below the limit of detection were also more frequent among african americans (9%) than caucasians (2% ; p<0.001). significantly lower ntprobnp levels in african americans were also found when analyses were restricted to (1) participants with detectable levels of ntprobnp, (2) healthy participants, or (3) participants at the forsyth county, north carolina, field center (data not shown). baseline characteristics of african - american and caucasian participants without prevalent cardiovascular disease at aric visit 4 (19961998) data presented as percentage or median (interquartile range) for categorical and continuous data, respectively. ace indicates angiotensin converting enzyme ; arb, angiotensin receptor blocker ; aric, atherosclerosis risk in communities ; bmi, body mass index ; bpm, beats per minute ; ca, calcium ; dbp, diastolic blood pressure ; egfr, estimate glomerular filtration rate ; homa - ir, homeostatic model for assessment of insulin resistance ; ntprobnp, n terminal pro b type natriuretic peptide ; sbp, systolic blood pressure ; uacr, urine albumin creatinine ratio. natural log ntprobnp values are lower in african americans, as compared to caucasians (p<0.001). for example, the frequency of natural log ntprobnp levels 4 (ntprobnp=54.6 pg / ml) is 60% in african americans, compared to 40% of caucasians. the lower limit of the natural log of ntprobnp is truncated at 0.92 based upon the lowest value of ntprobnp of 2.5 pg / ml. aric indicates atherosclerosis risk in communities ; ntprobnp, n - terminal pro b - type natriuretic peptide. in multivariable linear regression analysis, adjusted log ntprobnp levels were significantly lower (40% ; 95% ci, 43, 36) in african americans than caucasians, independent of other factors associated with ntprobnp levels (figure 2). covariates also associated with lower ntprobnp levels included male gender, higher bmi, heart rate, and dbp, as well as higher glucose and egfr. in contrast, covariates associated with higher levels of ntprobnp included increasing age, lower household income, beta - blocker use, current smoking, higher sbp, and presence of microalbuminuria. with inclusion of homa - ir into the multivariable linear regression model, increased insulin resistance was also significantly associated with lower ntprobnp levels (14% ; 95% ci, 16, 11), although this did not attenuate the relationship between african - american race and lower plasma ntprobnp levels (37% ; 95% ci, 41, 33). in subgroup analyses, ntprobnp levels were consistently lower in african americans, as compared to caucasians (figure 2). forest plot of percent difference in log ntprobnp levels in african americans, compared to caucasians, overall and across subgroups of aric participants. african - american race is significantly associated with lower ntprobnp levels, as compared to caucasians, in the overall study population and within subgroups. values shown are from multivariable adjusted linear regression analyses (see methods for covariates). for the insulin - resistant subgroups, aric indicates atherosclerosis risk in communities ; homa - ir, homeostatic model for assessment of insulin resistance ; ntprobnp, n - terminal pro b - type natriuretic peptide. multivariable logistic regression was performed to examine factors associated with nondetectable plasma nt - probnp levels (table 2). after multivariable adjustment, african - american race was associated with significantly increased odds of having nondetectable levels (multivariable adjusted odds ratio [or ], 5.74 ; 95% ci, 4.22, 7.80). in an additional analysis with inclusion of homa - ir in the model, african - american race remained significantly associated with increased odds of nondetectable ntprobnp (or, 5.51 ; 95% ci, 3.91, 7.77). characteristics significantly associated with nondetectable plasma ntprobnp among aric participants nondetectable plasma defined as ntprobnp < 5 pg / ml. data presented as percentage or median (interquartile range) for categorical and continuous data, respectively. aric indicates atherosclerosis risk in communities ; bmi, body mass index ; bpm, beats per minute ; dbp, diastolic blood pressure ; egfr, estimate glomerular filtration rate ; ntprobnp, n terminal pro b type natriuretic peptide ; or, odds ratio. or adjusted for all covariates listed plus age, annual household income, history of hypertension, systolic blood pressure, antihypertensive medication class, diabetes mellitus, smoking status, and the presence of microalbuminuria. ors for continuous variables reflect odds per 1 sd (bmi=5.5 kg / m ; heart rate=9.5 bpm ; dbp=10 mm hg ; fasting glucose=31 mg / dl ; egfr=16.5 ml / min per 1.73 m). among the 1656 african americans in whom pea data were available, median pea was 15% (iqr, 11, 23). in multivariable linear regression models, a 1 sd (10%) increase in pea was associated with 7% (95% ci, 1, 13) higher adjusted ntprobnp levels (p=0.025 ; figure 3). the relationship between genetically determined percent european ancestry and plasma ntprobnp levels among self - reported african americans in aric. among self - reported african americans, with increasing proportion of european ancestry, there is a significant increase in plasma ntprobnp levels. aric indicates atherosclerosis risk in communities ; ntprobnp, n - terminal pro b - type natriuretic peptide. our principal finding is that african americans have substantially lower plasma ntprobnp levels, compared to caucasians. the lower levels of plasma ntprobnp in african - american individuals were observed across clinically relevant subgroups and appeared to be independent of other factors associated with low levels of ntprobnp, including obesity and insulin resistance. pea among african americans also correlated with ntprobnp levels, which not only supports the observation of racial differences in np levels, but also suggests a genetic basis for lower np levels between individuals of west - african and european ancestry. given that previous experimental, genetic, and clinical studies support that reductions in np secretion are associated with salt - sensitive htn and incident dm,2,4 our finding of lower levels of circulating np levels in african - american individuals may, in part, explain the increased susceptibility to htn and dm. furthermore, the relative reduction in ntprobnp level in african americans versus caucasians observed in our study (40%) is comparable to that reported with obesity (20% to 40%),20 suggesting physiological and clinical significance. previous studies of hospitalized patients suggest that african americans may have lower np levels than caucasians.2,79 however, these studies largely evaluated persons presenting to the emergency department, such that the coexistence of prevalent cvd and risk factors may have confounded the associations between race and np levels.7,9,21 in contrast, the aric study afforded the opportunity to examine the impact of race on np levels in a community - based cohort of more than 9000 individuals free of prevalent cvd. this sample size also allowed adjustment for confounding factors, and we found that african americans had lower plasma ntprobnp levels than caucasians, despite greater frequencies of female gender, htn, microalbuminuria, and lower socioeconomic status among african americans, each of which correlated with higher, rather than lower, levels of np. moreover, the finding of lower levels of plasma ntprobnp in african americans was independent of other factors previously reported to be associated with low levels of ntprobnp, including obesity, insulin resistance, and dm. the significant and independent association between pea and ntprobnp levels among african americans also suggests a genetic basis for lower np levels in african americans. african americans have a higher prevalence of htn, left ventricular hypertrophy, and microalbuminuria, conditions that should raise np levels.6,2224 consequently, the finding in aric that african - american individuals have lower np levels, compared to white individuals, is counterintuitive and suggests that nonhemodynamic factors may contribute. the association between pea and ntprobnp levels among african americans indicates an underlying genetic component. one biologically plausible way in which np levels may be decreased is through impaired synthesis or reduced release from cardiomyocytes. indeed, previous studies indicate that variations in processes related to the synthesis of ntprobnp, such as genetic polymorphisms in the nppb gene that influence transcription, translation, and/or post - translational processing, may contribute to lower ntprobnp levels, although there are limited data on racial differences in the genetics of the np system.25 alternatively, non - nppb gene variations that affect np production and processing may be involved as well. for example, corin is a protein that is partly responsible for cleavage of nps into the active carboxy - terminus hormone and inactive amino - terminus propeptide.26 variants in the corin gene are more common among african americans, and experimental mouse models overexpressing the corin variant observed in african americans demonstrate increased salt sensitivity, htn, and hypertrophy, thereby recapitulating the clinical phenotype.4,27 however, variation in the corin gene may not fully explain the observed lower levels of ntprobnp in aric given that the assay used detects both the 76-amino - acid ntprobnp as well as the full 108-amino - acid prohormone (probnp),28 suggesting that np levels in african americans may, in part, be regulated upstream of corin. future studies quantifying circulating probnp, ntprobnp, and bnp levels may help clarify the role of corin mutations in african americans, but are beyond the scope of this study. clearance of ntprobnp is not mediated through the same processes that lead to clearance of the active hormone, bnp.29 therefore, it is also possible that the lower levels of ntprobnp observed among african americans may be related to enhanced clearance in african americans, compared to caucasians. bnp levels were not measured in aric ; however, ntprobnp and bnp levels have previously been demonstrated to be highly correlated,30,31 suggesting that lower ntprobnp levels also reflect lower bnp levels, and that differences in np between races may be determined upstream of clearance in np regulation pathways. however, further studies are needed to elucidate and clarify the relative importance of synthesis, release, and clearance mechanisms to the lower np levels observed in african americans. strengths of the present investigation include the large sample size, routine measurement of plasma ntprobnp in a community - dwelling population, standard ascertainment of clinical characteristics, use of multivariable adjusted analyses, and consistency of the finding of lower plasma ntprobnp levels across several sensitivity and subgroup analyses. plasma ntprobnp levels in ambulatory community - dwelling participants may be below the limit of detection ; however, in a sensitivity analysis restricted to individuals with detectable levels, plasma ntprobnp remained significantly lower in african americans, as compared to caucasians. plasma bnp was not measured in aric ; therefore, it is possible that active np hormone levels may not differ according to race. however, ntprobnp and bnp levels are highly correlated, even within a range of values well below thresholds diagnostic of heart failure.3032 atrial natriuretic peptide (anp) was not measured in aric, although other studies have indicated that mid - regional pro - anp levels may also be lower in african americans, compared to caucasians.9 we did not assess cardiac structure and function with echocardiography, given that these measures were not obtained during aric visit 4. however, compared to caucasians, african americans tend to have greater wall thickness,24 which is typically associated with higher np levels and therefore should bias the result toward the null ; however, we found that plasma ntprobnp levels were significantly lower in african americans, lending further validity to these results. though we adjusted for multiple factors that may contribute to ntprobnp levels, there may be residual confounding. in the estimation of pea, which is based upon samples from west africa and europe further studies are needed to elucidate specific genetic loci related to np levels in african americans. finally, in this cross - sectional study, we can not determine a causal association between low plasma np levels and htn, obesity, and/or dm. however, it has previously been demonstrated in aric that low plasma np levels are associated with increased risk for dm.2,3 african americans have a higher prevalence of htn, left ventricular hypertrophy, and microalbuminuria, conditions that should raise np levels.6,2224 consequently, the finding in aric that african - american individuals have lower np levels, compared to white individuals, is counterintuitive and suggests that nonhemodynamic factors may contribute. the association between pea and ntprobnp levels among african americans indicates an underlying genetic component. one biologically plausible way in which np levels may be decreased is through impaired synthesis or reduced release from cardiomyocytes. indeed, previous studies indicate that variations in processes related to the synthesis of ntprobnp, such as genetic polymorphisms in the nppb gene that influence transcription, translation, and/or post - translational processing, may contribute to lower ntprobnp levels, although there are limited data on racial differences in the genetics of the np system.25 alternatively, non - nppb gene variations that affect np production and processing may be involved as well. for example, corin is a protein that is partly responsible for cleavage of nps into the active carboxy - terminus hormone and inactive amino - terminus propeptide.26 variants in the corin gene are more common among african americans, and experimental mouse models overexpressing the corin variant observed in african americans demonstrate increased salt sensitivity, htn, and hypertrophy, thereby recapitulating the clinical phenotype.4,27 however, variation in the corin gene may not fully explain the observed lower levels of ntprobnp in aric given that the assay used detects both the 76-amino - acid ntprobnp as well as the full 108-amino - acid prohormone (probnp),28 suggesting that np levels in african americans may, in part, be regulated upstream of corin. future studies quantifying circulating probnp, ntprobnp, and bnp levels may help clarify the role of corin mutations in african americans, but are beyond the scope of this study. clearance of ntprobnp is not mediated through the same processes that lead to clearance of the active hormone, bnp.29 therefore, it is also possible that the lower levels of ntprobnp observed among african americans may be related to enhanced clearance in african americans, compared to caucasians. bnp levels were not measured in aric ; however, ntprobnp and bnp levels have previously been demonstrated to be highly correlated,30,31 suggesting that lower ntprobnp levels also reflect lower bnp levels, and that differences in np between races may be determined upstream of clearance in np regulation pathways. however, further studies are needed to elucidate and clarify the relative importance of synthesis, release, and clearance mechanisms to the lower np levels observed in african americans. strengths of the present investigation include the large sample size, routine measurement of plasma ntprobnp in a community - dwelling population, standard ascertainment of clinical characteristics, use of multivariable adjusted analyses, and consistency of the finding of lower plasma ntprobnp levels across several sensitivity and subgroup analyses. plasma ntprobnp levels in ambulatory community - dwelling participants may be below the limit of detection ; however, in a sensitivity analysis restricted to individuals with detectable levels, plasma ntprobnp remained significantly lower in african americans, as compared to caucasians. plasma bnp was not measured in aric ; therefore, it is possible that active np hormone levels may not differ according to race. however, ntprobnp and bnp levels are highly correlated, even within a range of values well below thresholds diagnostic of heart failure.3032 atrial natriuretic peptide (anp) was not measured in aric, although other studies have indicated that mid - regional pro - anp levels may also be lower in african americans, compared to caucasians.9 we did not assess cardiac structure and function with echocardiography, given that these measures were not obtained during aric visit 4. however, compared to caucasians, african americans tend to have greater wall thickness,24 which is typically associated with higher np levels and therefore should bias the result toward the null ; however, we found that plasma ntprobnp levels were significantly lower in african americans, lending further validity to these results. though we adjusted for multiple factors that may contribute to ntprobnp levels, there may be residual confounding. in the estimation of pea, which is based upon samples from west africa and europe further studies are needed to elucidate specific genetic loci related to np levels in african americans. finally, in this cross - sectional study, we can not determine a causal association between low plasma np levels and htn, obesity, and/or dm. however, it has previously been demonstrated in aric that low plasma np levels are associated with increased risk for dm.2,3 the lower ntprobnp levels observed in african americans, compared to caucasians, may help to explain racial disparities in cardiometabolic risk given that lower np levels may increase the risk of htn, obesity, and dm, conditions more prevalent among african americans. however, further studies are needed to confirm the causal role of nps and clarify the mechanisms leading to lower levels of np, of which our results suggest that genetic variation likely contributes. ultimately, augmentation of the np system in individuals with a relative np deficiency may prove to be an efficacious strategy for the prevention of cardiometabolic risk. the atherosclerosis risk in communities study is carried out as a collaborative study supported by national heart, lung, and blood institute (nhlbi) contracts (hhsn268201100005c, hhsn268201100006c, hhsn268201100007c, hhsn268201100008c, hhsn268201100009c, hhsn268201100010c, hhsn268201100011c, and hhsn268201100012c). its contents are solely the responsibility of the authors and do not necessarily represent official views of the ncats or the national institutes of health. | backgroundnatriuretic peptides promote natriuresis, diuresis, and vasodilation. experimental deficiency of natriuretic peptides leads to hypertension (htn) and cardiac hypertrophy, conditions more common among african americans. hospital - based studies suggest that african americans may have reduced circulating natriuretic peptides, as compared to caucasians, but definitive data from community - based cohorts are lacking.methods and resultswe examined plasma n - terminal pro b - type natriuretic peptide (ntprobnp) levels according to race in 9137 atherosclerosis risk in communities (aric) study participants (22% african american) without prevalent cardiovascular disease at visit 4 (19961998). multivariable linear and logistic regression analyses were performed adjusting for clinical covariates. among african americans, percent european ancestry was determined from genetic ancestry informative markers and then examined in relation to ntprobnp levels in multivariable linear regression analysis. ntprobnp levels were significantly lower in african americans (median, 43 pg / ml ; interquartile range [iqr ], 18, 88) than caucasians (median, 68 pg / ml ; iqr, 36, 124 ; p<0.0001). in multivariable models, adjusted log ntprobnp levels were 40% lower (95% confidence interval [ci ], 43, 36) in african americans, compared to caucasians, which was consistent across subgroups of age, gender, htn, diabetes, insulin resistance, and obesity. african - american race was also significantly associated with having nondetectable ntprobnp (adjusted or, 5.74 ; 95% ci, 4.22, 7.80). in multivariable analyses in african americans, a 10% increase in genetic european ancestry was associated with a 7% (95% ci, 1, 13) increase in adjusted log ntprobnp.conclusionsafrican americans have lower levels of plasma ntprobnp than caucasians, which may be partially owing to genetic variation. low natriuretic peptide levels in african americans may contribute to the greater risk for htn and its sequalae in this population. |
proteolytic lysosomal enzyme activity may be responsible for detrimental effects on the corneal endothelium.1,2 glycolytic lysosomal enzymes have been studied less extensively and their role in endothelial cell loss during corneal preservation is still incompletely understood.3 the use of adjuvant corticosteroid in the preservation medium has demonstrated reduced levels of glycolytic lysosomal enzymes.4,5 however, the lysosomal activity found during preservation for routine corneal transplantation did not correlate with graft success.6 the mean corneal storage time in this study was 73 hours.6 the question of whether lysosomal activity at longer preservation times (14 days) may be more substantial and could be reduced through the use of corticosteroids has not been completely addressed. the second question to be addressed is the magnitude of the source of the glycolytic lysosomal enzymatic activity. if, as expected, it is from the cellular elements of the cornea, then removal of the corneal epithelium should have a substantial effect on the measured glycolytic enzymatic activity. nine of the eyes were selected for mechanical deepithelialization prior to removal of the corneoscleral rim in order to protect the corneal endothelium. they were stored for 14 days at 4c. at the conclusion of the preservation, 5 ml of the solution was frozen in dry ice and transported frozen for -glucuronidase activity. the hydrocortisone solution was prepared in the following manner by the admixture pharmacy department at the ohio state university hospitals. water - soluble hydrocortisone powder (sigma h0396) was dissolved aseptically in the optisol to achieve a final concentration of 10 m in nine containers and 10 m in nine containers. at the conclusion of the preservation period, 5 ml of the optisol solution was removed from the chamber and immediately frozen with dry ice and transported to the mayo medical laboratories for measurement of the -glucuronidase activity using the csf protocol. the substrate, phenolphthalein mono--glucuronic acid, is hydrolyzed by the enzyme at ph 4.5 for 18 hours. nine of the eyes were selected for mechanical deepithelialization prior to removal of the corneoscleral rim in order to protect the corneal endothelium. they were stored for 14 days at 4c. at the conclusion of the preservation, 5 ml of the solution was frozen in dry ice and transported frozen for -glucuronidase activity. the hydrocortisone solution was prepared in the following manner by the admixture pharmacy department at the ohio state university hospitals. water - soluble hydrocortisone powder (sigma h0396) was dissolved aseptically in the optisol to achieve a final concentration of 10 m in nine containers and 10 m in nine containers. at the conclusion of the preservation period, 5 ml of the optisol solution was removed from the chamber and immediately frozen with dry ice and transported to the mayo medical laboratories for measurement of the -glucuronidase activity using the csf protocol. the substrate, phenolphthalein mono--glucuronic acid, is hydrolyzed by the enzyme at ph 4.5 for 18 hours. the activity for the epi no hct (control) group (mean : 4.302 ; sem : 0.586) and the epi 10 m hct group (mean : 4.472 ; sem 435) were found to be not significantly different (tukey test, confidence level of 95%). the dep - no hct group (mean : 2.178 ; sem : 0.271) and the epi 10 m hct group (mean : 2.072 ; sem : 0.437) was not found to be significantly different (tukey test, confidence level of 95%). 10 m hct groups were each found to be significantly different than the dpi no hct and the epi 10 m hct groups (tukey test, confidence level of 95%). endothelial cell damage may occur from proteolytic lysosomal enzymes.1,2 the addition of corticosteroid to the preservation solution has been shown to reduce lysosomal enzyme activity and to have a protective effect on the corneal endothelium.4,5 lysosomal enzyme levels have not been found to correlate as a predictor of graft outcome in an analysis of lysosomal enzyme levels from a solution of preserved corneas used for penetrating keratoplasty with relatively short periods of preservation (73 hours).6 this study indicates that -glucuronidase activity for prolonged corneal preservation (14 days, 4c) may be reduced in two ways. the second method is by adding water - soluble hydrocortisone to achieve a concentration of 10 m. while the lysosomal enzyme activity was not found to correlate with graft success it may be that reducing lysosomal activity by these methods will allow longer preservation times and viable endothelium.6 a study compared a hydrocortisone supplemented solution (h - sol) to optisol with 12 day preservation and found similar endothelial cell loss for each group. lysosomal enzyme activity was not measured in this study and there are other differences other than supplemental steroid in the two solutions used in this study.7 in summary, we found substantial reduction in -glucuronidase activity with the removal of the corneal epithelium and with the additional of 10 m hydrocortisone. whether this will correlate with improved corneal endothelium and the ability to preserve corneas longer is as yet unknown. | purpose : to analyze the levels of -glucuronidase during prolonged (14 day) corneal preservation with epithelialized (epi) and deepithelialized (dep) corneas and the effect of supplemental hydrocortisone (hct) on these levels.methods:thirty-six freshly excised bovine corneas were preserved in optisol solution (4c) for 14 days with the following conditions epi / no supplemental hct, dep no supplemental hct, epi/108 m hct, and epi 104 m hct. -glucuronidase activity levels were measured at the end of this period.results:-glucuronidase levels (nmol / ml / h) for each group were found to be : epi no hct : 4.302, sem 0.586 ; dep no hct : 2.178, sem 0.271 ; epi 108 m hct : 4.472, sem 0.435 ; epi 104 m hct : 2.072, sem 0.437. the epi no hct and epi 108 m hct were not significantly different as were the dep no hct and epi 104 m hct groups. the epi no hct group and the epi 108 m hct group were independently significantly different from the dep no hct and the epi 104 m hct groups.conclusions:the corneal epithelium contributes significantly to the formation of the lysosomal enzyme, -glucuronidase, during preservation. the addition of 104 m hct decreases the production of -glucuronidase during corneal preservation in this model. |
hypertension is a global public health problem, accounting for substantial morbidity and mortality through heart disease, stroke, blindness, and renal failure.1,2 globally, it accounts for at least 45% of heart disease, and 51% of deaths resulting from stroke.1 hypertension was once considered rare in sub - saharan africa (ssa), but it has become currently a widespread problem, with immense socioeconomic importance.3 in addition, there is evidence that the prevalence of hypertension and cardiovascular diseases is increasing rapidly in ssa.4,5 mass migration of rural africans to urban areas and rapid changes in lifestyle and risk factors account for this rising prevalence.3 the epidemiological picture of hypertension in ssa, compared against other regions, is grimmer, with the region having the highest prevalence, of 46%, compared against 30% in high - income countries.6,7 this high prevalence is expected to be reflected in increased prevalence of complications of hypertension in the region. it has also been projected that this prevalence will continue to increase at an alarming rate, with ssa bearing the brunt of the disease burden because of ignorance, poverty, inadequate health care provision, and erosion of traditional lifestyles.3,8,9 this will impose considerable strain on scarce health resources, and further impoverish the region. therefore, the thrust of public health policies within the region should be primary prevention of hypertension.10 success of these primary prevention programs will be dependent on accurate estimates of the prevalence of hypertension in our communities. this will enable precise quantification of the burden imposed by hypertension on our national and regional resources. epidemiological transition is at various stages in different places, and the true situation in ssa is largely unknown,11 especially in the rural areas.12 therefore, there is an important need for better epidemiological data, and hypertension - related outcome trials in ssa.3 data on the prevalence of cardiovascular risk factors, such as hypertension, are scarce in ssa.13 in addition, limited information exists about the prevalence of cardiometabolic risk factors and the burden of cardiovascular disease in the adult nigerian population, especially in the rural setting.14 moreover, few population - based studies have been conducted recently in nigeria on the prevalence and correlates of hypertension among the populace, in both urban and rural communities.12,13,1521 therefore, this study sought to determine the prevalence of hypertension in adult populations living in three rural communities of ife north local government area (lga) of osun state, south west nigeria. this will further help to define the pattern of hypertension in this rural population, and will also add to national and global databases. one thousand adults of age 1590 years, from three rural towns of ife north were recruited into this cross - sectional study over a 6-month period, using a multistage proportional stratified random sampling technique.22 the minimum sample size was determined using the formula for calculating sample size in the population;23 the population of the ife north lga was 153,274, according to the 2006 population census.24 the participants data were collected after community mobilization and consent. the permission of traditional rulers of each community was sought, and informed consent was obtained from each participant. the procedure followed the world health organization guidelines for conducting community surveys.25 three major towns, ipetumodu, edunabon, and moro, were randomly selected from the seven major towns of ife north local government. each of these towns, or communities, is divided further into census enumeration areas (eas) for demographic purposes ; ipetumodu has 800 eas, edunabon has 340 eas, and moro has 130 eas, according to the ife north local government local government office of the national population commission. ten percent of eas were randomly selected from each town, in proportion to the number of eas in each town. this translated to 80 eas for ipetumodu, 34 eas for edunabon, and 13 eas for moro. thereafter, households were randomly selected from each ea, where the selected household had at least one eligible respondent. six households per ea were randomly selected in ipetumodu, 10 households per ea in edunabon, and 23 households per ea in moro. in each household, at least one eligible and consenting respondent, of age 15 years or older, was interviewed. ethical approval was granted by the ethics and research committee of the obafemi awolowo university teaching hospital, ile - ife. all sociodemographic data were obtained using a standard questionnaire, and anthropometric variables and blood pressure (bp) were measured.16,17 after about 10 minutes of quiet sitting, three readings of blood pressure (bp) were taken at intervals of 35 minutes using an electronic bp monitor (omron healthcare inc, vernon hills, il, usa), after calibration with a mercury sphygmomanometer, according to recommendations of the nigerian hypertension society.26,27 the mean of three bp readings was used for analysis, following the manufacturer s guide. in 1997, the federal ministry of health and social services, lagos, released the final report of a national survey on noncommunicable diseases in nigeria, which included the prevalence of hypertension in both the rural and urban areas.27,28 in this survey, hypertension was defined as mean systolic blood pressure (sbp) and diastolic blood pressure (dbp) of 160/95 mmhg, using the 1978 definition of the world health organization;16,29 this was the basis of the cutoff point used in this study. hypertensive patients on regular drug therapy for hypertension were also included in this study.16 based on the seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure (jncvii) guidelines30 and the world health organization and international society of hypertension guidelines (who / ish),31 a second cutoff point for hypertension, of 140/90 mmhg, was adopted in this study, for comparison with other studies that used this criterion. using the jncvii classifications of hypertension,16,30 participants were categorized as follows for sbp : normal : 80 mmhg ; prehypertension : 8089 mmhg ; stage 1 hypertension : 9099 mmhg ; and stage 2 hypertension 100 mmhg. a subject with both sbp and dbp of 140/90 mmhg is hypertensive by jncvii30 and who / ish 1999 criteria.31 with isolated systolic hypertension (ish), sbp is 140 mmhg, while dbp is 80 mmhg ; prehypertension : 8089 mmhg ; stage 1 hypertension : 9099 mmhg ; and stage 2 hypertension 100 mmhg. a subject with both sbp and dbp of 140/90 mmhg is hypertensive by jncvii30 and who / ish 1999 criteria.31 with isolated systolic hypertension (ish), sbp is 140 mmhg, while dbp is 0.05). the mean dbp was 77.511.7 mmhg for males, and 76.412.5 mmhg for females (p>0.05). other descriptive variables except age were significantly different between sexes, as shown in table 2 (p 160/95 mmhg as a cutoff) in 2003, and the estimates of 8%10% among rural dwellers in nigeria in 1997.27,28 the 26.4% prevalence of hypertension that we obtained in these three rural communities is lower than recent estimates of hypertension prevalence, of 32.9% and 36.6%, in a semiurban community in ghana,10 and in nigeria,16 respectively, using the 140/90 mmhg definition. this finding corroborates earlier studies, which have observed lower bp and prevalence of hypertension in rural populations, compared against urban and semiurban populations in africa.3,9,3336 we found that the prevalence of hypertension was higher in men than in women, as also shown in previous studies.3336 in addition, sbp increased with age up to 6170 years, while dbp increased up to 4150 years old. earlier reports had also indicated increased hypertension with age.28,37,38 the pattern of hypertension in this study showed a prevalence of ish (13.1%) higher than that of idh (7.7%), similar to earlier reports.37,38 our results also showed that there were significant positive correlations between some anthropometric indicators of obesity (wc, hc, and bmi) and bp. this finding supports earlier investigations, which have reported significant positive correlations between anthropometric factors, such as bmi, and sbp and dbp.16,34,36 some nigerian authors have recently reported high prevalence of hypertension in some rural communities, as observed in this study. oladapo found a 20.8% prevalence of hypertension, while onwubere,12 asekun - olarinmoye,18 okeahialam,11 onwuchekwa,19 andy,20 and ahaneku gi reported prevalence of 46.4%, 13.16%, 20.9%, 18.3%, 23.6%, and 44.5%, respectively, in their recent studies. these various studies on hypertension prevalence in nigeria reported a wide range of prevalence rates using the bp cutoff point of 140/90 mmhg. the prevalence rates range between 13.16%46.4% ; the reason for this wide disparity is due partly to methodological differences in these studies. those who reported higher prevalence rates, such as onwubere (46.4%) and ahaneku (44.5%), conducted their studies mainly among elderly and middle - aged adults, in populations of the eastern part of nigeria. the other studies, from the rest of nigeria, reported prevalence rates of 13.16%26.4% ; in some of these studies, the study populations included adolescents and young adults. in addition, asekun - olarinmoye,18 oladapo,14 and our study report 13.16%, 20.8%, and 26.4% prevalence, respectively, in south west nigeria. onwuchekwa and andy found 18.3% and 23.6% prevalence, respectively, in the south south nigeria. these studies have shown a trend of increasing hypertension in nigeria ; hence, the need to increase efforts aimed at prevention and treatment of hypertension. screening for hypertension should be intensified, since screening activities are an important component of any prevention and control program.4 this is because they allow for detection of previously - unaware hypertensive individuals, and providing them with early treatment to prevent cardiovascular disease.4 further studies are necessary to assess the impacts of socioeconomic status and lifestyle in relation to hypertension in this region. the prevalence of hypertension in the three rural communities was 26.4%, indicating a trend towards increasing prevalence of hypertension in this rural population. in addition, there were significant positive correlations between anthropometric indicators of obesity and blood pressure in adult residents of these communities. | backgroundthe prevalence of hypertension is increasing rapidly in sub - saharan africa, but data are limited on hypertension prevalence. in addition, few population - based studies have been conducted recently in nigeria on the prevalence and correlates of hypertension in both urban and rural communities. therefore, we determined the prevalence of hypertension in adults in the three rural communities of ipetumodu, edunabon, and moro, in south west nigeria.materials and methodsone thousand adults between 15 and 90 years of age were recruited into this cross - sectional study, over a 6-month period, using a multistage proportional stratified random sampling technique. sociodemographic data and anthropometric variables were obtained, and resting blood pressure (bp) was measured using an electronic sphygmomanometer. diagnosis of hypertension was based on the jnc vii guidelines, the who / ish 1999 guidelines, and the bp threshold of 160/95 mmhg.resultsfour hundred and eighty - six men (48.6%) men and 514 women (51.4%) participated in the study. their mean age, weight, height, and body mass index were 32.314.7 years, 6213 kg, 1.50.1 m, and 23.02 kg / m2, respectively. the prevalence of hypertension, based on the 140/90 mmhg definition, was 26.4% (male : 27.3% ; female : 25.4%). the prevalence of hypertension, based on the 160/95 mmhg definition, was 11.8% (male : 13.5% ; female : 10.1%). there were significant positive correlations between bp and some anthropometric indicators of obesity.conclusionthe prevalence of hypertension in the three rural communities was 26.4%, indicating a trend towards increasing prevalence of hypertension. there was also a significant positive correlation between anthropometric indicators of obesity and bp in this population. |
the identification of a stem cell in a tissue is a major challenge of pivotal importance. being able to detect the stem cell population allows for powerful approaches to study cell differentiation dynamics by, for example, lineage tracing (barker., 2007, busch., 2015). additionally, it provides a first step toward ex vivo propagation of primary stem cells in organoid cultures (lancaster., 2013, sato., 2009), important for applications in regenerative medicine. moreover, stem cell populations relevant for disease progression, such as cancer stem cells, are promising targets for therapeutic intervention. stem cells are typically rare, which makes their discovery by traditional population - based assays very difficult. for example, it took decades of dedicated research to define the population of hematopoietic stem cells (hscs) (eaves, 2015), but it remains an open question how much heterogeneity exists within this subpopulation of bone marrow cells (wilson., 2015). similarly, the discovery of intestinal stem cells (van der flier and clevers, 2009) took years of work, and heterogeneity within this compartment remains under debate (buczacki., 2013). the recent availability of single - cell mrna sequencing methods allows profiling of healthy and diseased tissues with single - cell resolution (grn., 2015,, 2015, patel., 2014, paul., 2015, the transcriptome of a cell can be interpreted as a fingerprint, revealing its identity. however, biological gene expression noise (eldar and elowitz, 2010, raj and van oudenaarden, 2008) and technical noise because of amplification of minute amounts of mrna from a single cell (brennecke., 2013, 2014) affects the readout and makes it a challenge to discriminate cell types based on their transcriptome. by sequencing large numbers of randomly sampled single cells from a tissue these inventories can be screened for cell types of particular interest, such as stem cells. an obvious strategy for the identification of the stem cell is the derivation of a lineage tree from single - cell sequencing data. however, transcriptomes of randomly sampled cells only represent a snapshot of the system, and temporal differentiation dynamics can not be directly derived. however, if the system of interest comprises all differentiation stages, such as the intestinal epithelium or the bone marrow, then attempts can be made to infer a lineage tree by assembling single - cell transcriptomes in a pseudo - temporal order. existing approaches assume a continuous temporal change of transcript levels to assemble differentiation trajectories (bendall., 2014, here we present a method to identify rare and abundant cell types of a system and use these cell type classifications to guide the inference of a lineage tree. we investigate the general properties characterizing the position of a cell type within the lineage tree and identify the number of branches and the transcriptome uniformity of a cell type as features correlating with the degree of pluripotency. we show that our approach successfully recovers the identity of the stem cell in the intestine and in the bone marrow, two systems with a well described stem cell population. we then use our method to predict multipotent cell populations in the adult human pancreas. to develop a robust approach for the inference of differentiation trajectories, we used a previously published dataset from a lineage tracing experiment comprising the progeny of lgr5-positive mouse intestinal stem cells (grn., 2015). this system is ideal for testing the inference of differentiation dynamics because the lineage tree is already well characterized (figure 1a). the continuously self - renewing intestinal epithelium is arranged in crypts and villi, with a small number of lgr5 + stem cells, also known as crypt base columnar cells (cbcs), residing near the crypt bottom. these cbcs give rise to rapidly proliferating transit - amplifying (ta) cells that migrate upward along the crypt - villus axis and develop into the terminally differentiated cell types (barker, 2014, van der flier and clevers, 2009). although absorptive enterocytes constitute the most abundant cell type, the secretory lineage comprises rare cells, such as mucus - producing goblet cells, hormone - secreting enteroendocrine cells, and antimicrobial paneth cells. labeled cells were collected 5 days after label induction using an lgr5-creert2 construct and a rosa26-yfp reporter with a loxp - flanked transcriptional roadblock (figure 1b). we first improved the robustness of the initial clustering step of our previously developed raceid algorithm (grn., 2015) by replacing the k - means clustering with k - medoids clustering (figure s1). second, we noticed that the previously used gap statistic (tibshirani., 2001) was not ideal for determining the cluster number. although increasing the number of clusters in many cases leads to a growing gap statistic, the decrease of the within - cluster dispersion (tibshirani.,, we thus determine the cluster number by identifying the saturation point of the within - cluster dispersion. together, these two changes lead to a more robust initial clustering of raceid2 (experimental procedures ; figure s1). for the intestinal lineage tracing data (experimental procedures), raceid2 recovered a larger group of lgr5 + stem cells (cluster 2) and early progeny (clusters 1 and 8) as well as the major mature cell types ; i.e., enterocytes (cluster 3), goblet (clusters 4 and 19), paneth (clusters 5 and 6), and enteroendocrine cells (cluster 7) (figures 1c and 1d). these cell types could be unambiguously assigned based on the cluster - specific upregulation of marker genes inferred by raceid2 (table s1). one of the major challenges for the inference of differentiation pathways in a system with multiple cell lineages is the determination of branching points. to overcome this problem, we predefined the topology of the lineage tree by allowing differentiation trajectories linking each pair of clusters. a putative differentiation trajectory links the medoids of two clusters, and the ensemble of all inter - cluster links defines the possible topology of the lineage tree. to minimize the effect of technical noise and, at the same time, the computational burden, we first reduce the dimensionality of the input space requiring maximal conservation of all point - to - point distances. in a second step, we assign each cell to its most likely position on a single inter - cluster link. to find this position, the vector connecting the medoid of a cluster to one of its cells is projected onto the links between the medoid of this and all remaining clusters, and the cell is assigned to the link with the longest projection after normalizing the length of each link to one. the projection also defines the most likely position of the cell on the link (figure 2a), reflecting its differentiation state (experimental procedures). if this strategy is applied to the intestinal data, then only a subset of links is populated (figure 2b). to determine links that are more highly populated than expected by chance and are therefore candidates for actual differentiation trajectories, we computed an enrichment p value based on comparison with a background distribution with randomized cell positions (figure 2b ; figure s2a). furthermore, we reasoned that the coverage of a link by cells indicates how likely it is that this link represents an actual differentiation trajectory and not only biased perturbations driving the transcriptome of a given cluster preferentially toward the transcriptome of another cluster without leading to actual differentiation events. we defined a link score as one minus the maximum difference between the positions of each pair of neighboring cells on the link after normalizing the length of each link to one (figure s2b). if this score is close to one, then the link is densely covered with cells with only small gaps in between. if the link score is close to zero, the cell density is only concentrated near the cluster centers connected by this link. the computationally inferred intestinal lineage tree is consistent with the known lineage tree (figure 1a). secretory cell types (clusters 4, 5, 6, and 7) populate individual branches emanating from the central lgr5 + cluster, and absorptive enterocytes (cluster 3) differentiate from the same group via a more abundant group of ta cells (cluster 1). we compared the inferred lineage tree to the tree predicted by monocle (trapnell., 2014), a recent method for the derivation of branched lineage trees that does not rely on a predefined tree topology, and found that monocle could not resolve the different branches of secretory cells (figure s2). more precisely, we try to identify, from the lineage tree, the cell population with the highest degree of multipotency. we noticed that different cell types showed a variable number of populated links to other clusters. the link score is reflected by the thickness of the line in our graphical representation (figure 2b). we also show links with a low link score because they are informative about the associated cell state. for example, a cell type with many low - scoring links can fluctuate toward a diversity of fate biases, whereas cell types with only a few links are much more canalized. these two scenarios reflect a more promiscuous transcriptome, such as expected for stem cells, versus a more confined transcriptome, as expected for a mature cell type. in our data, cluster 2, which contains cells positive for lgr5 and other established stem cell markers (ascl2 and clca4) (figure 2c), was the most highly connected cluster. another putative property of stem cells is the tendency to exhibit a more uniform composition of the transcriptome in comparison with differentiated cells. mature cell types frequently express a small number of genes at very high levels, crucial for cell type - specific functions. the transcriptome of paneth cells, for instance, is dominated by high numbers of lysozymes and other host defense genes. the uniformity of the transcriptome is reflected by shannon s entropy (shannon, 1948), and this concept has previously been applied to study cellular differentiation (anavy., 2014, banerji., 2013, piras., 2014) (experimental procedures). we anticipate that the transcriptome of a multipotent cell type is more uniform in each individual cell. in addition, multiple state biases could coexist within this population that can give rise to diverse mature cell types upon external stimuli, or stochastically, leading to high entropy (banerji., 2013, ridden., 2015). for the intestinal lineage tracing data, both paneth and goblet cells had clearly reduced entropy compared with lgr5-positive cells, whereas the entropy of enterocytes and enteroendocrine cells was comparable with stem cells (figure 2d). we found that, for all analyzed datasets (see below), the number of links discriminates better between multipotent and differentiated cells when rescaled by the entropy. therefore, the simplest score that performs well in discriminating multipotent cells from the remaining cell types was a product of the median entropy (after subtracting the minimal entropy observed in the system) and the number of links of a cluster (experimental procedures). this score exhibits a clear maximum for cluster 2 comprising the lgr5 + stem cells (figure 2d). we named our algorithm stemid for the lineage tree inference and the derivation of this score. next we wanted to test whether stemid could identify lgr5 + cells in a larger and more complex dataset comprising intestinal cells of various independent experiments conducted in our lab. in this dataset, we combined 3 weeks and 8 weeks of lgr5 lineage tracing data. a subset of those was enriched in secretory cells by fluorescence - activated cell sorting (facs) on cd24 (van es., we also sorted non - traced cd24 + control cells (experimental procedures ; figure s3). raceid2 revealed the known intestinal cell types within this dataset based on cluster - specific expression of known cell type marker genes and subdivided these into stages of differentiation or maturation (figures 3a and 3b ; figure s3a). a full list of differentially expressed genes for each cluster is given in table s2. for example, intestinal stem cells in cluster 7, marked by high expression of lgr5 and clca4 (figure 3b), were connected directly to all secretory branches, whereas ta cells (cluster 5) primarily give rise to enterocytes (cluster 10) (figure 3c ; figures s3c and s3d). interestingly, we observed two distinct differentiation trajectories for paneth cells (clusters 13 and 14), one via a dll1-positive common precursor of paneth and goblet cells (cluster 1) and another one directly connecting stem cells (cluster 7) or ta cells in cluster 5, marked by upregulation of the cell - cycle gene pcna, directly to the mature paneth cell clusters. both the dll1-dependent (van es., 2012) and the direct route (farin., 2014, sawada., 1991), which was observed after ablation of paneth cells, have been described. we were not able to recapitulate this finding with a minimum spanning tree - based alternative approach (figure s3e). we then computed the stemid score and found that the lgr5+/clca4 + cells (cluster 7) exhibit the highest score (figure 3d). the second highest score was observed for cluster 21, which represents a common progenitor to paneth and goblet cells. the ta cells in cluster 5, which our lineage inference identifies as progenitors with an enterocyte fate bias, acquire the third - highest stemid score. noticeably, paneth cells in cluster 13 and mature goblet cells in cluster 2 show the same connectivity as the stem and progenitor cells in clusters 7, 5, and 21, but rescaling by entropy helps correctly assign a mature state to these cells (figure s3f). in conclusion, stemid could identify intestinal stem cells and distinguish progenitor populations from more mature intestinal cell types. to test the performance of stemid in a different biological system, we applied the algorithm to single - cell sequencing data of mouse bone marrow cells. these cells were selected based on physical interactions between doublets or larger groups of cells and are thus not sampled randomly from all cell types in the bone marrow. this dataset was complemented with kitsca-1lincd48cd150 hscs (kiel., 2005) cell types identified by raceid2 were dominated by the myeloid lineage and comprised hscs, erythroblasts, megakaryocytes, two groups of granulocytes (neutrophils and eosinophils), macrophages, a small group of b lymphocytes, and several clusters representing progenitor stages of the myeloid lineage (figures 4a and 4b ; figure s6a). a full list of differentially expressed genes for each cluster is shown in table s3. cluster 1 comprises almost exclusively sorted hscs (figure s4b). the inferred lineage tree (figure 4c ; figures s6c and s6d) indicates that hscs differentiate into multipotent progenitor cells (cluster 5) but are also directly linked to mature lineages. hscs and multipotent progenitors are both linked to megakaryocytes (cluster 4), eosinophils (clusters 10 and 29), macrophages (cluster 28), and two branches covering a spectrum of progenitor and mature states of the neutrophil (clusters 11, 3, 2, 14, 12, and 22) and erythroid lineage (clusters 9, 8, 7, 6, and 13), respectively. the b lymphocytes are only directly linked to the hscs, suggesting that cluster 5 represents a myeloid progenitor population, and no lymphoid progenitors were present in our sample. the inferred lineage tree is therefore consistent with the existence of a common myeloid progenitor population giving rise to erythrocytes, megakaryocytes, granulocytes, and macrophages (orkin and zon, 2008). stemid determines the highest score for cluster 1 and, therefore, correctly recovers hscs among all cell types in the mixture (figure 4d ; figure s6). the second - highest score discriminates the multipotent myeloid progenitors (cluster 5) from the remaining cell types, and the third - highest score was assigned to the earliest progenitor of the erythroblast lineage. therefore, the level of multipotency also correlates with the stemid score of bone marrow - derived cells. the high connectivity of cluster 1 provides evidence for early fate biases already in hscs. moreover, the high entropy of hscs reflects a more uniform transcriptome in individual cells of this population. the entropy distribution across all cells in this cluster is shifted in comparison with all other groups (figure 5a). in general, the inter - cluster variability substantially exceeds the intra - cluster variability. the narrow entropy distribution of cluster 1 also rules out a strong dependence on the cell cycle. however, we also observed that 54 of the 276 hscs (20%) show distinct fate biases, revealed by low expression of lineage - specific marker genes (figure 5b), a finding that is consistent with a recent report based on lineage tracing (peri., 2015). because the sensitivity of single - cell sequencing is limited, this number is almost certainly an underestimation. we note that most hscs (112 of 276) are assigned to the link with the multipotent progenitor (cluster 5). we can not address whether the observed fate bias persists during differentiation or whether stochastic switching between distinct cell fates occurs during differentiation. our observation is also consistent with a recent single - cell transcriptome analysis showing an unexpected heterogeneity of myeloid progenitor cell populations and suggests the existence of an early cell fate bias (paul., 2015). we observe very similar sets of marker genes, as found in this study, but our lineage inference permits an analysis of the temporal dynamics of gene expression. as an example, we extracted all cells from the neutrophil branch (clusters 1, 11, 3, 2, and 12) in pseudo - temporal order derived from the projection coordinates and clustered temporal expression profiles by using self - organizing maps (experimental procedures). a z - score of gene expression values along this trajectory reveals that the raceid2 clusters represent sets of cells with common modules of co - expressed genes and that gene expression within these modules changes smoothly over time (figure 5c). although ribosomal protein - encoding genes and other components of the translational machinery slowly decline during differentiation, other genes are transiently switched on in progenitor populations (e.g., elane) or immature neutrophils (e.g., ngp) or only upregulated in mature cells (e.g., retnlg). finally, we note that the identification of the hsc population by stemid is robust to changing the contribution of this population to the mixed sample. for example, when only ten hscs are randomly selected and all others are discarded from the dataset, stemid still assigns the highest score to the small hsc cluster (data not shown). in summary, stemid could successfully identify the stem cell type in a complex mixture of cells isolated from bone marrow. the inferred lineage tree recovered known trajectories but suggested an early cell fate bias present already in hscs. after having demonstrated that stemid can robustly identify the stem cell population in two distinct biological systems, we applied the algorithm to predict multipotent cell populations in a less characterized system : the human pancreas. the pancreas consists of acinar cells that produce the digestive enzymes, ductal cells secreting bicarbonate to neutralize stomach acidity, and hormone - producing endocrine cells that regulate hormone metabolism (jennings., 2015). it is unclear which multipotent cells maintain pancreatic homeostasis and can give rise to different mature cell types during regeneration upon injury. although early studies have suggested that, in humans, these cell populations could reside within the exocrine compartment or that dedifferentiation of exocrine cells could give rise to endocrine cells (bonner - weir. 2015), the identity of multipotent cell populations is still unclear (jiang and morahan, 2014). we sequenced pancreatic cells from human donors (experimental procedures), and application of raceid2 revealed all major cell types, including different subpopulations of acinar and ductal cells ; hormone - producing,,, and pancreatic polypeptide producing (pp) cells ; and stellate cells (figures 6a and 6b ; figures s5a and s5b). a full list of differentially expressed genes for each cluster is shown in table s4. in particular, we discovered novel subpopulations of ductal cells. in one of these groups (cluster 14), the cell surface glycoprotein ceacam6 was significantly upregulated (p 0.7 is 40% for k - means versus 73% for k - medoids. the corresponding fractions are 58% versus 83% for the bone marrow data and 40% versus 90% for the pancreas data. to infer differentiation trajectories, we assign every cell onto a specific link between its cluster of origin and another cluster based on the longest projection of the vector connecting the cluster center with the cell position onto these links. this adequately reflects how much a cell has moved from the most representative cell state in the same cluster (the medoid) toward another cell identity (or vice versa). if significantly more cells reside on a link than expected by chance, this provides strong evidence that cells of the cluster of origin exhibit a pronounced transcriptome bias toward another cell fate. in addition, if a continuum of cell states covers a given link, as evidenced by a high link score, then this link represents a strong candidate for an actual differentiation trajectory. significant links with reduced link scores, on the other hand, indicate plasticity of the connected cell types in a sense that the transcriptome of a cell type can, to some extent, fluctuate toward another fate. the quality of our lineage inference is supported by the recovery of known differentiation trajectories in the intestinal epithelium and the bone marrow. remarkably, we recovered a rare alternative differentiation pathway where lgr5 + cells differentiate directly into paneth cells without intermediate dll1 + progenitors (farin. we could also show, for the intestinal and the bone marrow data, that stemid infers a lineage tree with substantially higher resolution in comparison with methods published previously (haghverdi., 2015, trapnell., 2014 ; figure s6). the derived lineage tree for the bone marrow suggested that, in contrast to the classical view of dichotomous differentiation via a hierarchy of increasingly restricted progenitor populations (giebel and punzel, 2008), a cell fate bias already exists at stages as early as the hsc stage (figure 5b). this observation is consistent with a recent single - cell transcriptome analysis revealing heterogeneity of the common myeloid progenitor cell population, indicating early fate bias (paul., 2015). moreover, direct generation of progenitors restricted to the myeloid fate from mouse hscs has been described in the past (yamamoto., 2013), and the existence of unipotent cells within human hscs (notta., 2016) and classically defined mouse multipotent progenitor populations was shown recently (peri., 2015). for both model systems, the stemid score, which quantifies very general properties of a cell type (i.e., the number of links and the entropy of the transcriptome), ranks raceid2-predicted cell types by their level of multipotency. lgr5 + cbcs and sorted hscs acquire the highest score among all cell types of the intestine and bone marrow, respectively, demonstrating the performance of our algorithm. we could further demonstrate the performance of stemid on two previously published datasets (figure s7) for cells from developing lung epithelium (treutlein., 2014) and differentiating human radial glial cells (pollen., 2015). potential problems for the stemid algorithm arise in the absence of intermediate progenitors or the occurrence of unrelated cell types. in the absence of intermediate progenitors, stemid infers a link to a more multipotent population. for example, it is known that a spectrum of progenitors will reside on this trajectory, and, as we have observed for the other lineages, an early fate bias toward lymphocytes could exist in hscs. in the absence of intermediate progenitors, a link to a more multipotent population reflects all information on the lineage relationship that can be extracted from the data. if the stem cell itself is missing from the sample, stemid will identify the cell type with the highest level of multipotency. the presence of unrelated cell types in the mixture could lead to false positive links. however, because the feature space is high - dimensional, it is likely that none of the links between an unrelated cell type and the remaining lineage tree will be significantly populated. we also argue that links of mature cell types to related progenitor or stem cell populations were identified with high specificity (oftentimes only a single link in line with previous findings was detected). this makes the occurrence of significant links between unrelated cell types unlikely. finally, we used stemid to screen human adult pancreatic cells for multipotent cell populations. it is unclear which adult pancreatic cell types can give rise to the different mature pancreatic lineages during normal tissue turnover or regeneration. although initial evidence suggested that multipotent cells within the ductal compartment could differentiate into endocrine cells both in humans and mice (jiang and morahan, 2014), subsequent lineage - tracing experiments produced contradictory results. although mouse lineage tracing of carbonic anhydrase ii (ca2)-positive ductal cells revealed that these cells give rise to cells upon injury (bonner - weir., 2008), lineage tracing of sox9-, muc1-, or hnf1-positive cells could not confirm this finding (furuyama., 2011, kopinke and murtaugh, 2010, kopp., 2011, solar., 2009). using stemid, we were able to predict distinct sub - populations of ductal cells with varying differentiation potential. although ductal cells marked by high levels of ceacam6 are predicted to differentiate into,, and pp cells, another sub - population expressing high levels of the ferritin complex primarily appears to give rise to cells and acinar cells. we note that the latter sub - population does not express any of the markers used in previous lineage - tracing experiments, but we caution that expression of these genes might be too low to be reliably detected by single - cell mrna sequencing. we further remark that cell differentiation in the adult pancreas might not be conserved between human and mouse. we provide the well documented r source code for raceid2 and the stemid algorithm at https://github.com/dgrun/stemid. we hope that stemid will be useful for a better understanding of differentiation dynamics in a variety of systems. for lineage - tracing experiments, we injected 0.4 mg tamoxifen into 3-month - old lgr5-creert2 c57bl6/j mice bred to rosa26lsl - yfp reporter mice. crypts were isolated from mice as described previously (sato., 2009). pancreatic cadaveric tissue was procured from a multiorgan donor program and only used when the pancreas could not be used for clinical pancreas or islet transplantation, according to national laws, and when research consent was present. human islet isolations were performed in the islet isolation facility of the leiden university medical center according to a modified protocol originally described by ricordi. pancreatic tissue samples were fixed overnight in 4% formaldehyde (klinipath), stored in 70% ethanol, and subsequently embedded in paraffin. after deparaffinization and rehydration in xylene and ethanol, respectively, antigen retrieval was performed in citric buffer for 20 min. primary antibodies were rabbit anti - ftl (ab69090), mouse anti - glucagon (ab10988), and guinea pig anti - insulin (ab7842). alexa fluor - conjugated secondary antibodies against rabbit, mouse, and guinea pig immunoglobulin g (igg) (life technologies ; a11008, a10037, and a21450) were used at a dilution of 1:200. nuclear counterstaining was done by embedding with dapi vectashield (vector laboratories, h-1500). we used c57bl/6 female or male mice from 23 to 52 weeks bred in our facility. experimental procedures were approved by the dier experimenten commissie of the royal netherlands academy of arts and sciences and performed according to the guidelines. bone marrow was isolated from femur and tibia by flushing hank s balanced salt solution (hbss, invitrogen) without calcium or magnesium, supplemented with 1% heat - inactivated fetal calf serum (fcs) (sigma). the protocol was carried out as described previously (grn., 2015). read mapping and quantification were done as described previously (grn., 2015). the algorithm and follow - up analyses are described in full detail in the supplemental experimental procedures. d.g. and a.v.o. conceived the study. d.g. developed the algorithm and performed all computational analyses. single - cell sequencing of pancreatic cells and antibody staining were performed by m.j.m with the help of g.d. single - cell sequencing of intestinal cells was performed by k.w. with the help of a.l. | summaryadult mitotic tissues like the intestine, skin, and blood undergo constant turnover throughout the life of an organism. knowing the identity of the stem cell is crucial to understanding tissue homeostasis and its aberrations upon disease. here we present a computational method for the derivation of a lineage tree from single - cell transcriptome data. by exploiting the tree topology and the transcriptome composition, we establish stemid, an algorithm for identifying stem cells among all detectable cell types within a population. we demonstrate that stemid recovers two known adult stem cell populations, lgr5 + cells in the small intestine and hematopoietic stem cells in the bone marrow. we apply stemid to predict candidate multipotent cell populations in the human pancreas, a tissue with largely uncharacterized turnover dynamics. we hope that stemid will accelerate the search for novel stem cells by providing concrete markers for biological follow - up and validation. |
stigma is a broad term that includes direct discrimination and systemic discrimination (link. the stigma of schizophrenia has been studied extensively in many cultures and has been shown to have a variety of negative impact on finance, quality of life, (thornicroft. healthcare professionals are not immune to these biases and negative attitudes have been found at greater rates in non - psychiatric settings than in psychiatric settings (bjorkman. most studies of discrimination against those with schizophrenia assessed the attitudes of the general public in different cultures (angermeyer and matschinger 2005 ; anglin. fewer studies have examined the expectation and subjective experience of discrimination by those with schizophrenia (dickerson. 2002 ; ertugrul and ulug 2004 ; thornicroft. 2009 ; yanos. 2008) and most of these utilized small samples with a few exceptions (lee. 2005 ; thornicroft. one of the largest surveys of this kind in the united states was conducted by otto wahl using a sample of 1,301 individuals with mental illness recruited from the national alliance on mental illness (nami) but less than 20% of this sample identified as having a schizophrenia spectrum disorder. the results for this group are not separated out. given the observed differences in attitudes towards people with schizophrenia versus those with depression (nordt. 2006), it is unclear whether these findings can be generalized to individuals with schizophrenia. the wahl study is unusual in that it identified how commonly discrimination, prejudice, or support is experienced in specific social situations. such data is essential to optimally target interventions. a framework that separately considers the experience or expectation of discrimination in different social contexts may also support or challenge proposals that stigma 2001), that it is related to unrealistically elevated fear of violence (link. 1999a), that it is related to perceptions of treatability or that it is not related to beliefs about etiology (angermeyer and matschinger 2005). 2005) and the international study of stigma by the indigo study group (thornicroft. 2009) both provide large diagnosis - specific study of the experience of stigma by individuals with schizophrenia. the consistency of methodology in the indigo study allows for a rare comparison of stigma in different countries but despite the scale of the study, the authors acknowledge that they were not able to investigate in any detail the complex features of stigma and discrimination that might apply in culture or context specific settings. this article reports on the first large - scale study examining to whom americans with schizophrenia disclose their diagnosis, whether they experience positive, negative or neutral reactions to such disclosures and some specific consequences of prior disclosures. the national alliance on mental illness (nami) commissioned harris interactive to conduct an online survey of people living with schizophrenia, recruited via nami e - mail lists and the nami website. the survey questions were developed by a panel that included the authors in consultation with harris interactive. this survey was restricted to individuals over the age of 18 who self - identified as having schizophrenia, schizoaffective disorder or another schizophrenia spectrum disorder. those results are available online (http://www.nami.org/content/navigationmenu/schizophreniasurvey/analysis_living_with_schizophrenia.htm) and will be analyzed in a separate article. this study will focus on the sections of the survey that describe sources of support as well as positive and negative experiences of individuals who self - identify as having schizophrenia. descriptive statistics such as percent frequencies for categorical data and means and standard deviations for continuous data were computed. as most data were not normally distributed, spearman rank correlations were computed to investigate the interrelationships between measures. step - wise regression techniques, both linear and logistic, were used to model factors of interest with inclusion / exclusion criteria of p < 0.15. the significance level was set at p < 0.05. the current post - hoc analysis of the data from harris interactive was based on a de - identified dataset. this protocol was therefore deemed exempt from review by the cedars - sinai medical center irb. the initial harris interactive study was made possible with funds from astrazeneca, solvay, and wyeth. this post - hoc analysis did not require any additional funding and the authors do not have any material conflicts of interest to report. a total of 258 people self - reporting a diagnosis of schizophrenia or schizoaffective disorder qualified and completed the survey. only 38% of the sample was employed.table 1sample characteristicsgender male (%) 45 female (%) 55age 1824 (%) 8 2534 (%) 20 3544 (%) 28 4554 (%) 28 5564 (%) 14 65 + (%) 1 mean41.8 sd11.5race caucasian (%) 78 african - american (%) 4 hispanic (%) 7 other (%) 9education hs or less (%) 17 some college (%) 46 college or more (%) 37employment employed (%) 38 unemployed (%) 41 retired (%) 11 student (%) 17 homemaker (%) 11income less than $ 35 k (%) 65 $ 35 k$74,999 (%) 18 $ 75 k$99,999 (%) 5 $ 100 k or more (%) 5 decline to answer (%) 8current mh poor / fair (%) 58status good (%) 24 very good / excellent (%) 17 decline to answer (%) 8 sample characteristics individuals with schizophrenia varied in how open they reported they were about their diagnosis depending on their relationship with the other person (see table 2). all openness scores were rated by participants on a scale of 1 (not at all open in disclosing diagnosis) to 4 (completely open regarding diagnosis). the response rate for each type of relationship varied as many subjects did not have particular types of relationships. response rates were lowest for relationships with spouse / significant others and with children with only about half of the respondents indicating that they had such relationships. the highest mean openness scores were for the categories of doctors and spouses / significant others with mean scores of 3.6 and 3.3, respectively where 3 corresponds to quite a bit open about one s diagnosis of schizophrenia and 4 corresponds to completely open about their diagnosis. neighbors had the lowest mean openness score of 1.7 where 2 would correspond to somewhat open about one s schizophrenia diagnosis and 1 correspond to not at all open about one s schizophrenia diagnosis.table 2openness scores by type of relationshipmean scoresdresponse raten%parents3.31.023993extended family2.61.125197spouse / significant other3.41.015359children2.31.314657friends2.71.025498coworkers2.11.117869employer2.21.217769place of worship2.01.117769neighbors1.70.924896doctors3.60.725699law enforcement2.01.118371overall mean score2.60.7258 openness scores by type of relationship to consider the potential availability of support from a particular type of relationship, the percentage of all respondents who identify being at least somewhat open about their diagnosis was calculated for each relationship type (see fig. 1). most individuals with schizophrenia were not open about their diagnosis with children, police / correction officers and persons at their place of worship. only 25% of women reporting being at least somewhat open with police / correction officers compared to 50% of men.fig. 1percentage of subjects who report being at least somewhat open about their schizophrenia diagnosis with specific individuals or groups percentage of subjects who report being at least somewhat open about their schizophrenia diagnosis with specific individuals or groups although this survey did not directly ask about levels of social isolation, the number of different types of relationships for each respondent was calculated as a proxy. the mean number of types of relationships for respondents to this survey was 8.8 relationships with a standard deviation of 1.8. across all relationships, an average openness score was computed for each participant, again on a scale of 1 (not at all open) to 4 (completely open). in examining overall openness scores, we found a negative correlation with the number of types of relationships a respondent had (rs = 0.340, p < 0.001) and positive correlation with the rating of current mental heath status (rs = 0.302, p < 0.001). the results of the step - wise linear regression analysis of an individual s average openness score are presented in table 3. in the unadjusted analysis, only the self - reported current mental health status and the number of types of relationships significantly predicted overall level of openness. in the adjusted analysis, these two predictors remained significant after controlling for gender, race, marital status, physical health, income, and employment status. however, these two factors individually do not account for a large portion of the variability in mean openness scores, with each having a low partial correlation coefficient. other unmeasured factors might be more influential on openness given that the adjusted analysis model had a low overall r = 0.222.table 3results of step - wise linear regression in determining predictors of openness scorevariableprobability value (p)coefficientsepartial correlation (r)unadjusted analysisadjusted analysisage0.7038age of onset0.3638years treated0.3026female0.33280.12090.13050.08380.0102caucasian0.27650.06360.19200.10300.0115receiving treatment0.7420family member diagnosed0.1740education0.6937current mental health good0.07700.01150.25850.10150.0076 very good<0.0001<0.00010.72040.13580.1018physical health (very good)0.49160.07910.21280.12070.0127employment status0.42150.12860.14600.09570.0085marital status0.12380.02420.22830.10060.0138income < $ 25 k0.2195 # of relationships (response rate)<0.0001<0.00010.17300.02880.1051 results of step - wise linear regression in determining predictors of openness score for all different types of relationships, some individuals with schizophrenia report being treated better, some worse and some report being treated no differently (see fig. 2). more individuals with schizophrenia report being treated worse by police / correctional officers than any other group and more report being treated better by parents than any other group. although relatively few people are open about their schizophrenia at their place of worship, among those who are open only 18% report being treated worse compared to 22% who report being treated better and a majority who report being treated no differently.fig. 2percentage of subjects who report being treated better worse or no better by specific individuals or groups after disclosing their diagnosis percentage of subjects who report being treated better worse or no better by specific individuals or groups after disclosing their diagnosis to further investigate the treatment experience of our subjects by doctors, the sample responding to that question were dichotomized (treated worse versus treated the same or better) and logistic regression modeling was used to identify characteristics of the subjects who perceived being treated worse by their doctor after revealing their diagnosis (see table 4). a total of 250 subjects reported revealing their diagnosis to their doctor and 61 of these reported being treated worse. in the unadjusted analysis, only gender was a statistically significant predictor of perceiving worse treatment by doctors. in the adjusted analysis, gender, employment status, and age of onset were statistically significant factors predicting perceived worse treatment. individuals with an earlier age of disease onset were less likely to perceived worse treatment, as were those who were employed. however, women were 2.5 times more likely than men to perceive worse treatment from their physicians after disclosing their diagnosis of schizophrenia.table 4results of logistic regression in determining predictors of perceiving worse treatment by doctors after disclosing diagnosisvariableprobability value (p)odds ratio(95% ci)unadjusted analysisadjusted analysisage0.3065age of onset0.05370.02200.959(0.924, 0.994)years treated0.3631female0.04550.00872.476(1.257, 4.878)caucasian0.6680receiving treatment0.4634family member diagnosed0.8447education0.5832current mental health (very good)0.1736physical health0.4728employment status0.36060.04800.494(0.245, 0.994)marital status0.2178income0.6910level of openness (completely)0.1238 results of logistic regression in determining predictors of perceiving worse treatment by doctors after disclosing diagnosis individuals with schizophrenia describe a variety of specific positive and negative experiences around their diagnosis with positive experiences (ex., encouraging their recovery, taking an interest in their condition / disease) among the most common reactions (fig. 3). however, a large majority report experiencing some negative reactions in this survey : 85% reported being treated as if they lack intelligence, 80% reported hearing negative comments and 71% reported that someone was afraid to be left alone around them. more than a third reported that they never or rarely experienced others taking an interest in their condition, 91% reported that someone has avoided the topic of their illness at least once, and more than half reported that someone they relied upon became more distant since their diagnosis.fig. 3percent or subjects who experienced specific positive and negative reactions since being diagnosed percent or subjects who experienced specific positive and negative reactions since being diagnosed a larger percentage of subjects report being able to rely on their mental health provider than family or friends (fig. however, about half of all respondents report that their medical conditions are not taken as seriously when doctors know of their schizophrenia. more women than men report difficulties getting their medical conditions taken seriously and report that it is more difficult to get access to physical health care when their diagnosis of schizophrenia is known, consistent with the findings that women are more likely to report being treated worse after disclosing a diagnosis of schizophrenia.fig. 4percentage of individuals with schizophrenia who agree or strongly agree with the following statements percentage of individuals with schizophrenia who agree or strongly agree with the following statements taken together the findings of this survey suggest that many people with schizophrenia or schizoaffective disorder are well aware of social stigma and that their experience of such stigma varies in different social settings. this is consistent with findings from an earlier us study of serious mental illnesses (wahl 1999) and from schizophrenia studies that include data from other countries (thornicroft. 2009). while this survey does not directly ask for their emotional responses to the limited supports and negative reactions, it is clear that stigma impacts their lives in ways that most people would experience as profoundly painful. this pattern could be explained if individuals believe that their diagnosis will be better received when they are not perceived as currently impaired by schizophrenia. however, this correlation also suggests that those who are currently suffering are more isolated. the correlation between a greater variety of relationship types and lower overall openness can be explained if the social skills that increase the range of socialization also increase awareness of stigma. a larger percentage of women reported being at least somewhat open about their diagnosis with their spouse or significant other while a larger percentage of men reported being open with their employers and with police / correctional officers. it is unclear whether this difference is due to gender differences in the rates of intimate relationships, employment or contact with the justice system. these gender differences as well as the overall pattern of diagnosis disclosure provide valuable information to help focus the scenarios that are practiced within psychiatric rehabilitation, cognitive - behavioral treatment and social skills training for individuals with schizophrenia. although recent studies have demonstrated that spirituality and religion can play a profound role for large fractions of individuals with schizophrenia (mohr. 2006), in our sample, only 39% disclose their schizophrenia diagnosis in their places of worship. taken together with the relatively positive response experienced by those who do disclose their diagnosis in this setting (only 18% report being treated worse), our findings suggest that places of worship may be an underutilized avenue of support. however, this opportunity should be viewed within the context of a recent study that suggests that only about 22% of americans attend worship services each week (hadaway and marler 2005). it is unclear whether individuals with schizophrenia have more or less contact with their place of worship than the general public, but given the social isolation that is often associated with this disease, it is possible that the 39% of respondents who disclose their diagnosis in places of worship may represent a large majority of all respondents who attend services regularly or have significant social contact with others in that setting. the relatively low rates of being treated worse in places of worship (18%) compares favorably to healthcare settings where 24% report being treated worse. a disturbingly large percentage of respondents report that they have greater difficulty getting treatment for their medical problems and that doctors who know their schizophrenia diagnosis are perceived as regarding their medical problems less seriously. this finding suggests that biased treatment may play a role in the 60% of premature deaths in people with schizophrenia that are due to medical conditions as described in a recent review (nasmhpd 2006). in that report, seven factors are identified as causes for this greater mortality, including factors related to medications, systems issues and the effects of psychiatric symptoms. our study suggests that another factor, specific to an awareness of the diagnosis may affect the quality of medical care provided to those with schizophrenia. 2006) and that other specialties may have greater rates of stigmatizing beliefs (bjorkman. 2008 ; chin and balon 2006). taken together with the results of this study, stigma by healthcare providers must be strongly suspected of playing a role in the premature mortality of individuals with schizophrenia. because healthcare professionals are taught that schizophrenia is a disease, our findings of healthcare bias supports a prior study that questions the effectiveness of anti - stigma campaigns based on education about biological etiologies (angermeyer and matschinger 2005). however, since healthcare professionals are also routinely exposed (at least briefly) to individuals with schizophrenia during training, this finding also challenges a study that suggests stigma is more directly related to a lack of familiarity (corrigan. the experience of stigma in healthcare settings also provides an interesting perspective on the proposal that stigma is based on conceptions of treatability (angermeyer and matschinger 2005) or dangerousness (angermeyer and matschinger 2005 ; link. individuals in healthcare settings should have better than average knowledge about the actual data on treatability and violence associated with schizophrenia. future research should assess whether healthcare providers are, in fact, knowledgeable about treatability and the risk of violence, and whether variation in perceived treatability and perceived risk accounts for the perceived prejudicial medical treatment reported in this study. if stigma is dependent on beliefs about treatability and risk of violence and if our findings are verified by subsequent studies, the prejudicial treatment in healthcare settings bodes poorly for public education campaigns as it is suggests that in order to combat stigma we would need to provide more education than is currently provided to professional healthcare workers. this study also suggests the importance of gender in the doctor - patient relationship for individuals with schizophrenia. women were statistically more likely to report being treated worse by doctors who know of their schizophrenia diagnosis and a larger percentage of women reported that their medical problems were taken less seriously and that it was harder to get access to physical health care when their diagnosis of schizophrenia was known. prior studies suggest that women may receive less aggressive medical care compared to men (gan. 2000 ; wexler. 2005) and this study suggests that women with schizophrenia may be a doubly vulnerable population. more broadly, the very different groups in this study suggest that a unitary approach to stigma may be an oversimplification. more than twice as many subjects report getting treated worse by police / correctional officers than by people at their place of worship. thus, interventions in law enforcement settings such as crisis intervention teams may have a far greater impact on the negative experiences of individuals with schizophrenia than interventions with other groups. by contrast, in places of worship, education may shift from a focus on tolerance to a focus on how to create a welcoming environment where individuals with schizophrenia may be more inclined to share their personal struggles with this disease. caucasians are overrepresented in our sample and prior studies suggest that minorities may differ from caucasians in the dynamics of family caregiving (magaa. future studies could benefit from greater attention to the experience of ethnic and racial minorities. more importantly, this sample all self - identified as having schizophrenia or schizoaffective disorder and therefore may not be representative of the many individuals who do not have this level of insight. the sample was recruited through nami, an organization that provides a broad range of education and which engages in advocacy. thus, individuals may be sensitized to the issues of stigma through nami s education programs. also, individuals with more negative experiences around their disease may be more prone to be involved with nami. the convenience sample obtained through an online survey is also skewed towards those with access to a computer and those who are comfortable using this technology. however, it is important to recognize that there are limitations to any other single source of data collection on this subject. if this survey was conducted through live interviews, individuals with more negative symptoms and individuals with greater shame around their disease may be less inclined to participate. in addition, live interviews of individuals in treatment centers, clubhouses or residential settings would each skew the sample in different ways (ex., towards individuals who are getting active treatment, towards individuals who are more socially engaged or towards individuals that are less able to live independently). if this survey was done via paper and the mail, the increment in motivation necessary to return such surveys would risk a bias towards individuals with more profound negative experiences and less profound negative symptoms. in the experience of the authors through their work on a variety of projects with nami, our members with serious mental illness are often more able and willing to use the internet than many of our other members. finally, because this is a secondary analysis of a dataset, the findings should be further tested using this study to generate primary hypotheses. | stigma against those with schizophrenia has demonstrated deleterious effects. however, less is known about the experience of individuals who disclose this diagnosis and how such disclosures differ by social situations. this study examines diagnosis disclosure in different contexts. a convenience sample of 258 adults with schizophrenia recruited via the internet and e - mail lists completed an online survey. subjects were more open about their diagnosis with doctors, parents and friends than with employers or police. those who report very good current mental health or who had fewer types of relationships were more open overall. although reactions to disclosure varied, many report worse treatment by police and better treatment by parents after disclosure. many also experienced worse treatment for medical problems after disclosing their schizophrenia diagnosis. these results support targeted anti - stigma interventions. it also suggests that stigma must be understood through individual experience in specific contexts rather than as a unitary experience. |
pulmonary arterial hypertension (pah) is a group of diseases characterized by progressive increase in pulmonary vascular resistance (pvr) (mean pulmonary arterial pressure [pap ] > 25 mm hg at rest or > 30 mm hg with exercise, as confirmed by right heart catheterization), leading to right ventricular failure and premature death. elevated pvr may result from multiple mechanisms, including vasoconstriction, proliferative and obstructive remodeling of pulmonary vessel wall, inflammation, and thrombosis. pah is a rare disease with an incidence of only 5.9 cases per 1 million, and primary pah is an aggressive disease with a median survival of 2.8 years. dasatinib is an oral multi - target tyrosine kinase inhibitor of bcr - abl, platelet - derived growth factor receptor (pdgfr), src, discoidin domain receptor, and c - kit and has a ~300-fold greater potency in abl inhibition compared to imatinib in in vitro studies. in 2010, dasatinib was approved by the food and drug administration for treatment of chronic myeloid leukemia (cml) as a frontline therapy and as a second - line treatment after imatinib failure. however, previous case reports have identified several concerns over the development of pah in dasatinib - treated cml patients [3 - 8 ]. here, we describe two cml patients who developed pah while receiving dasatinib treatment, but were improved shortly after discontinuation of dasatinib. a 43-year - old male patient without comorbidity was diagnosed with chronic phase (cp) cml in 1999. he had been treated with imatinib until hematological progression ; this patient was subsequently treated with a daily regimen of 140 mg dasatinib beginning from july 15, 2005. after several adjusted doses were administered because of repeated thrombocytopenia, the final dasatinib dose was restored in june 2010 to 140 mg daily and was maintained without additional adverse events (aes). on april 15, 2011, 69 months after initial dasatinib administration, the patient presented with new york heart association (nyha) functional class ii dyspnea. a chest radiograph revealed that the patient had grade 2 pleural effusion (pe). furosemide (40 mg / day) and prednisolone (30 mg / day) were administered for 3 weeks. although pe improved with this treatment, the patient s dyspnea persisted to the same degree. a transthoracic doppler echocardiogram revealed a highly increased right ventricular systolic pressure (rvsp) of 92 mm hg, an enlarged right heart cavity (right atrial area of 37.8 cm2, basal right ventricular end - diastolic diameter of 44.5 mm), and a small amount of pericardial effusion. the left heart chambers and left ventricular ejection fraction were normal. the systolic pap was estimated at 90 mm hg, and right ventricular function was impaired, as assessed by a reduced tricuspid annular plane systolic excursion of 15.6 mm. other potential causes of pah (such as congenital heart disease, pulmonary embolism, and parenchymal lung disease) were ruled out by chest contrast - enhanced 3-dimensional computed tomography. b - type natriuretic peptide (bnp) levels were elevated to 353.33 pg / ml. no causative medication had been taken. on may 9, 2011, dasatinib was discontinued, and a regimen of sildenafil, a calcium channel blocker, and diuretics was initiated. one month later, subjective symptoms improved to nyha functional class i. follow - up echocardiography revealed an improved rvsp of 79 mm hg and an estimated systolic pap of 80 mm hg. re - evaluation revealed a significant improvement, with rvsp decreased to 57 mm hg and pe completely absent 2 months after dasatinib discontinuation. on july 4, 2011, the patient started ponatinib at a dose of 45 mg once daily, and this treatment has been continued without any aes. the changes in clinical course, cardiopulmonary parameters and treatment history are depicted in fig. he was initially treated with interferon - alpha and later received a daily regimen of 400 mg imatinib that was dose - adjusted over time because of aes. because no cytogenetic response was achieved on imatinib, dasatinib was initiated at a dose of 140 mg daily from december 2, 2005 and was initially well tolerated. however, in february 2008, the patient complained of intermittent nyha class ii dyspnea on effort, and his chest radiograph revealed right pe ; with sustained dasatinib treatment, the symptom progressed to nyha class iii on february 24, 2009. at that time, dasatinib was discontinued, and treatment with low dose sildenafil (25 mg / day) was initiated. however, a chest computed tomography scan revealed mild pulmonary congestion, and a transthoracic doppler echocardiogram showed an increased rvsp of 71 mm hg and a small amount of pericardial effusion. laboratory tests for human immunodeficiency virus (hiv) and rheumatic diseases, including antinuclear antibodies, antibodies to double - stranded dna, and anti - scl-70 antibodies, were negative. a careful review of concurrent medications or toxins suspected to play an essential role in the development of pah revealed no causative evidence. after switching to nilotinib (800 mg / day) in may 2009, the patient s subjective dyspnea improved, and his rvsp decreased to 55 mm hg. bnp levels returned to 28 pg / ml, and the 6-minute - walk test value improved from 480 m to 615 m. the patient intermittently discontinued nilotinib because of repeated hyperbilirubinemias and was transferred to our hospital in july 2009. eighteen months later, he lost cytogenetic response with an e292v mutation ; therefore, a daily dose of 100 mg dasatinib was reintroduced in december 2010. although the patient previously experienced dasatinib - associated pah, we chose to administer dasatinib in order to overcome the patient s mutation - related resistance. the dose of dasatinib (40 - 60 mg / day) was then adjusted according to the severity of dyspnea, and a major cytogenetic response on dasatinib was maintained. after dasatinib administration, the patient again experienced a sustained nyha functional class ii dyspnea with serial elevation of left ventricular systolic pressure observed in a follow - up echocardiogram (fig. on april 11, 2011, a daily dose of 45 mg ponatinib was prescribed without improvement in pah while maintaining a complete cytogenetic response and a major molecular response. in august 2012, the patient 's subjective dyspnea was improved to nyha functional class i. the changes in clinical course, cardiopulmonary parameters and treatment history are depicted in fig. a 43-year - old male patient without comorbidity was diagnosed with chronic phase (cp) cml in 1999. he had been treated with imatinib until hematological progression ; this patient was subsequently treated with a daily regimen of 140 mg dasatinib beginning from july 15, 2005. after several adjusted doses were administered because of repeated thrombocytopenia, the final dasatinib dose was restored in june 2010 to 140 mg daily and was maintained without additional adverse events (aes). on april 15, 2011, 69 months after initial dasatinib administration, the patient presented with new york heart association (nyha) functional class ii dyspnea. a chest radiograph revealed that the patient had grade 2 pleural effusion (pe). furosemide (40 mg / day) and prednisolone (30 mg / day) were administered for 3 weeks. although pe improved with this treatment, the patient s dyspnea persisted to the same degree. a transthoracic doppler echocardiogram revealed a highly increased right ventricular systolic pressure (rvsp) of 92 mm hg, an enlarged right heart cavity (right atrial area of 37.8 cm2, basal right ventricular end - diastolic diameter of 44.5 mm), and a small amount of pericardial effusion. the left heart chambers and left ventricular ejection fraction were normal. the systolic pap was estimated at 90 mm hg, and right ventricular function was impaired, as assessed by a reduced tricuspid annular plane systolic excursion of 15.6 mm. other potential causes of pah (such as congenital heart disease, pulmonary embolism, and parenchymal lung disease) were ruled out by chest contrast - enhanced 3-dimensional computed tomography. b - type natriuretic peptide (bnp) levels were elevated to 353.33 pg / ml. no causative medication had been taken. on may 9, 2011, dasatinib was discontinued, and a regimen of sildenafil, a calcium channel blocker, and diuretics was initiated. one month later, subjective symptoms improved to nyha functional class i. follow - up echocardiography revealed an improved rvsp of 79 mm hg and an estimated systolic pap of 80 mm hg. re - evaluation revealed a significant improvement, with rvsp decreased to 57 mm hg and pe completely absent 2 months after dasatinib discontinuation. on july 4, 2011, the patient started ponatinib at a dose of 45 mg once daily, and this treatment has been continued without any aes. the changes in clinical course, cardiopulmonary parameters and treatment history are depicted in fig. he was initially treated with interferon - alpha and later received a daily regimen of 400 mg imatinib that was dose - adjusted over time because of aes. because no cytogenetic response was achieved on imatinib, dasatinib was initiated at a dose of 140 mg daily from december 2, 2005 and was initially well tolerated. however, in february 2008, the patient complained of intermittent nyha class ii dyspnea on effort, and his chest radiograph revealed right pe ; with sustained dasatinib treatment, the symptom progressed to nyha class iii on february 24, 2009. at that time, dasatinib was discontinued, and treatment with low dose sildenafil (25 mg / day) was initiated. however, a chest computed tomography scan revealed mild pulmonary congestion, and a transthoracic doppler echocardiogram showed an increased rvsp of 71 mm hg and a small amount of pericardial effusion. laboratory tests for human immunodeficiency virus (hiv) and rheumatic diseases, including antinuclear antibodies, antibodies to double - stranded dna, and anti - scl-70 antibodies, were negative. a careful review of concurrent medications or toxins suspected to play an essential role in the development of pah revealed no causative evidence. after switching to nilotinib (800 mg / day) in may 2009, the patient s subjective dyspnea improved, and his rvsp decreased to 55 mm hg. bnp levels returned to 28 pg / ml, and the 6-minute - walk test value improved from 480 m to 615 m. the patient intermittently discontinued nilotinib because of repeated hyperbilirubinemias and was transferred to our hospital in july 2009. eighteen months later, he lost cytogenetic response with an e292v mutation ; therefore, a daily dose of 100 mg dasatinib was reintroduced in december 2010. although the patient previously experienced dasatinib - associated pah, we chose to administer dasatinib in order to overcome the patient s mutation - related resistance. the dose of dasatinib (40 - 60 mg / day) was then adjusted according to the severity of dyspnea, and a major cytogenetic response on dasatinib was maintained. after dasatinib administration, the patient again experienced a sustained nyha functional class ii dyspnea with serial elevation of left ventricular systolic pressure observed in a follow - up echocardiogram (fig. on april 11, 2011, a daily dose of 45 mg ponatinib was prescribed without improvement in pah while maintaining a complete cytogenetic response and a major molecular response. in august 2012, the patient 's subjective dyspnea was improved to nyha functional class i. the changes in clinical course, cardiopulmonary parameters and treatment history are depicted in fig. the cause of pah can be classified as idiopathic, hereditary, or associated with congenital heart disease, connective tissue disease, hiv infection, portal hypertension, drugs and toxins, and hemoglobinopathies. although right heart catheterization was not performed to establish a diagnosis of pah in our two patients, they had an estimated systolic pap of over 30 mm hg in echocardiogram. these patients did not have any possible causes of pah such as family history of pah, congenital heart disease, infection, rheumatologic disease or hiv. pah in our patients continued to worsen during administration of dasatinib but became normalized after switching to another tyrosine kinase inhibitor. according to french reports, the lowest estimated incidence of pah occurring in patients exposed to dasatinib was 13 of 2,900 (0.45%). preclinical findings have suggested that an imbalance in pdgfr expression contributes to excessive smooth muscle proliferation observed in ovine perinatal pulmonary hypertension. another study found that platelet - derived growth factor (pdgf) and pdgfr mrna are overexpressed in pulmonary arterial smooth muscle cells and endothelial cells from the pulmonary arteries of patients displaying severe pah ; in addition, immunostaining of these tissues confirms high protein levels of pdgf / pdgfr. the ability to inhibit pdgfr signaling suggests that both imatinib and dasatinib have similar effects on pulmonary vascular remodeling ; furthermore, imatinib was effective for the treatment of pah through blockade of pdgfr phosphorylation in several studies. therefore, a unique mechanism of dasatinib - associated pah may exist independent of effects on pdgf / pdgfr signaling. the main difference in the kinase profile between dasatinib and imatinib is the inhibition of src family kinases. preclinical findings suggest that src kinase activity is related to hypoxic vasoconstriction in the rat pulmonary artery. therefore, src activity may be one of the main mechanisms of dasatinibrelated pah. the patients presented in this report and other patients in some previously published cases have several common characteristics (table 2). first, dasatinib had been given for a long period of time (19 - 52 months) before clinical manifestations of pah developed. the occurrence of pah at a late onset suggests a chronic pathological mechanism with an insidious, as opposed to acute, inflammatory, or cardiac etiology. in our case, the first patient developed dyspnea after approximately 69 months on dasatinib, and the second patient developed dyspnea after 38 months in the first instance and almost immediately after starting dasatinib in the second instance. for the second patient, during the second administration of dasatinib, we observed a short time interval between the initiation of dasatinib and diagnosis of pah. given that this was a re - trial of dasatinib and his baseline rvsp was already elevated to 55 mm hg at the time of restarting dasatinib, the short interval between initiation of dasatinib and manifestation of pah symptoms can be explained. second, these patients demonstrated rapid symptomatic and hemodynamic improvement (within several days to 4 months) after discontinuation of dasatinib. this reversibility is remarkable because normalization of pulmonary hemodynamics is not usually achieved in pah with usual medical treatments. third, a majority of patients have had a history of pe with pericardial effusion. it was previously reported that pe occurred in 17%-54% of patients on dasatinib, which suggests a similar mechanism of development for dasatinib - associated pah and pe. given these common characteristics, we suggest that dasatinib is the primary cause of pah in our two cases. in conclusion, long - term treatment with dasatinib may contribute to pah, which may improve spontaneously after discontinuation. routine cardiopulmonary evaluation before and during treatment with dasatinib will be needed in patients with clinical manifestations. | we describe two cases of pulmonary arterial hypertension (pah) that occurred under dasatinib treatment and were resolved after dasatinib discontinuation. two patients with chronic phase chronic myeloid leukemia (cml) were switched to dasatinib therapy because of hematological progress while receiving imatinib. these patients had new york heart association (nyha) functional class ii dyspnea with elevated right ventricular systolic pressure (rvsp), which progressed under dasatinib treatment. after dasatinib treatment was discontinued, subjective symptoms were improved to nyha functional class i and the follow - up transthoracic doppler echocardiography showed improved rvsp. treatment with an alternate tyrosine kinase inhibitor was initiated and had been continued without development of dyspnea or elevation of rvsp. this report suggests that dasatinib can cause the reversible pah, therefore, routine cardiopulmonary evaluation before and during treatment with dasatinib may be needed in cml patients with clinical manifestations. |
pertussis, also known as whooping cough, is an acute respiratory tract infection caused by bordetella pertussis. although the incidence rate of pertussis has significantly decreased following the wide use of pertussis vaccine, b. pertussis infection is continuously occurring as a small outbreak even in several developed countries with high vaccination coverage (1 - 4). there has been the resurgence of reported pertussis cases primarily in infants younger than 1 yr old, especially less than 3 months of age, in adolescents and young adults worldwide (5, 6). the possible causes of increased incidence in adolescents and adults include waning of vaccine immunity, adaptation of circulating b. pertussis strains, development of diagnostic methods, and active surveillance due to increased awareness of pertussis (7 - 9). household contact with infected adolescents and adults also becomes the major source of pertussis infection in infants who are not fully immunized, and this problematic circulation may consequently threaten overall public health (10, 11). the epidemiological characteristics of pertussis can vary depending on the definition of case, diagnostic method of confirmed case, national reporting system, network organization for epidemiological investigation, and the local vaccination schedule. national immunization program (nip) of korea consists of three primary series of diphtheria - tetanus - acellular pertussis vaccine (dtap) at 2, 4, and 6 months, followed by a first booster at 15 - 18 months and a second booster between 4 and 6 yr of age. recently, a tetanus toxoid, reduced diphtheria and acellular pertussis (tdap) booster vaccine in adolescents aged 11 - 12 yr is added to korean nip in 2012. the vaccination rate of primary dtap continues to be approximately 94% (12, 13), and there is a mandatory notification system in korea. an annual average of about 11.5 cases of pertussis has been reported to the korea center for disease control and prevention (kcdc), however, the reported cases of pertussis have increased since the 2000s. in addition, the kcdc reported that the incidence of pertussis was markedly increased (more than about 5.5 times) in 2009, compared to the previous (14). also, the incidence of pertussis in 2011 was more than two times compared with the incidence in 2009 (15). we may predict the substantial outbreak in the future in our country. given the importance of accurately determining the epidemiological features of infant pertussis and the lack of reliable existing data in korea, this study was conducted to describe the clinical characteristics of laboratory confirmed cases less than 1 yr of age and to evaluate the relative importance of family members on infants who are vulnerable to b. pertussis transmission. a prospective, multicenter, observational study was conducted between january 2009 and september 2011. we evaluated all infants clinically suspected of pertussis infection because of a cough lasting at least 2 weeks with at least one of the following symptoms : paroxysmal coughing ; inspiratory whooping ; post - tussive vomiting or apnea without other known cause. we obtained information on current respiratory manifestations, radiologic findings and immunization status for each infant. diagnostic approaches for pertussis nasopharyngeal aspirates (npa), or swab samples if aspirates were not possible, and blood samples were collected within 2 days at admission. laboratory tests were performed at the vaccine bio institute (vbi) of the catholic university of korea. it is determined as the criteria for laboratory - confirmed pertussis case when the subjects is applicable to one of the following criteria : 1) positive result of b. pertussis on culture of nasopharyngeal aspirates (npa) or swab ; this sample was collected and cultured on regan - lowe culture medium at 37 for more than 1 week ; 2) positive result of b. pertussis in pcr or real - time pcr (rt - pcr) of npa or swab ; pcr was done by the method glare. reported (16), and rt - pcr was done by modified method of reischl u. colleagues manual (17) ; 3) positive serology which defined as pertussis toxin (pt) antibody in a single serum sample that was higher than cut - off value (24 eu / ml) of enzyme - linked immunosorbent assay (elisa) kit (ibl, hamburg, germany) or a 4-fold increased change in anti - pt antibody between acute - phase and convalescent - phase serum. after the laboratory confirmation on pertussis, the parent or legal guardian for registration of index case was contacted as soon as possible, and all infants who are eligible for inclusion criteria were registered to the study immediately upon receipt of the consent form. to all family members if cough started at least 7 days before the onset of symptoms in the index case, it was requested to participate in the study as household contacts. and they were registered for the study immediately upon receipt of the consent form, and interviewed using the standard questionnaire to collect demographic and clinical data. also, for all long - term household contacts, it was asked to visit for the study to collect respiratory samples (for culture, pcr, rt - pcr identification of b. pertussis) and/or serum samples (for elisa). student 's t - test and fisher 's exact test were applied for comparing categorical variables between the groups. data were analyzed using spss statistical software, version 15.0 for windows (chicago, il, usa). this study was approved by the institutional review boards (irb) of seoul st. mary 's hospital (irb approval number : xc09tim - i0081k) and of other 2 hospitals. a prospective, multicenter, observational study was conducted between january 2009 and september 2011. we evaluated all infants clinically suspected of pertussis infection because of a cough lasting at least 2 weeks with at least one of the following symptoms : paroxysmal coughing ; inspiratory whooping ; post - tussive vomiting or apnea without other known cause. we obtained information on current respiratory manifestations, radiologic findings and immunization status for each infant. diagnostic approaches for pertussis nasopharyngeal aspirates (npa), or swab samples if aspirates were not possible, and blood samples were collected within 2 days at admission. laboratory tests were performed at the vaccine bio institute (vbi) of the catholic university of korea. it is determined as the criteria for laboratory - confirmed pertussis case when the subjects is applicable to one of the following criteria : 1) positive result of b. pertussis on culture of nasopharyngeal aspirates (npa) or swab ; this sample was collected and cultured on regan - lowe culture medium at 37 for more than 1 week ; 2) positive result of b. pertussis in pcr or real - time pcr (rt - pcr) of npa or swab ; pcr was done by the method glare. reported (16), and rt - pcr was done by modified method of reischl u. colleagues manual (17) ; 3) positive serology which defined as pertussis toxin (pt) antibody in a single serum sample that was higher than cut - off value (24 eu / ml) of enzyme - linked immunosorbent assay (elisa) kit (ibl, hamburg, germany) or a 4-fold increased change in anti - pt antibody between acute - phase and convalescent - phase serum. after the laboratory confirmation on pertussis, the parent or legal guardian for registration of index case was contacted as soon as possible, and all infants who are eligible for inclusion criteria were registered to the study immediately upon receipt of the consent form. to all family members if cough started at least 7 days before the onset of symptoms in the index case, it was requested to participate in the study as household contacts. and they were registered for the study immediately upon receipt of the consent form, and interviewed using the standard questionnaire to collect demographic and clinical data. also, for all long - term household contacts, it was asked to visit for the study to collect respiratory samples (for culture, pcr, rt - pcr identification of b. pertussis) and/or serum samples (for elisa). student 's t - test and fisher 's exact test were applied for comparing categorical variables between the groups. data were analyzed using spss statistical software, version 15.0 for windows (chicago, il, usa). this study was approved by the institutional review boards (irb) of seoul st. mary 's hospital (irb approval number : xc09tim - i0081k) and of other 2 hospitals. of a total of 65 clinically suspected cases, 21 infants (32.3%) were enrolled to be the confirmed cases of b. pertussis infection in this study. the age range was from 22 to 198 days old with a mean age of 2.5 months old. nine patients (42.9%, average age : 47.4 days) had not received any dtap vaccinations, 9 (42.9%, average age : 77.2 days) received 1 dose, 2 (9.5%, average age : 104.5 days) received 2 doses, and 1 (4.8%, age : 198 days) received 3 doses of dtap (table 1). pertussis occurred mostly in spring, summer and early fall, peaking in april, june, and september. although paroxysmal cough manifested for more than 1 week in all cases, whooping cough was present in only 3 cases (14.3%). there were 4 cases (19.0%) of pneumonic infiltration, 3 cases (14.3%) of hyperinflation on radiologic examinations. four patients with abnormal chest radiographic finding were not immunized with dtap vaccination, and remaining 3 patients were received with 1 dose of dtap vaccination. two patients had concomitant infections of rhinovirus and respiratory syncytial virus, respectively, and severity was not increased in these patients. absolute lymphocytosis was noted in 12 patients (57.1%). of note, absolute lymphocytosis was found more commonly in patients (8/9, 88.9%, average age : 45.8 days) who did not receive dtap vaccination than patients (4/12, 33.3%, average age : 88.0 days) who did receive dtap vaccination (p = 0.024, table 2). however, the mean length of hospitalization for patients without dtap vaccination was 15.4 6.6 days, which was significantly longer than the 8.8 3.8 days of patients with dtap vaccination (p = 0.009, table 2). two patients were admitted to the intensive care unit (icu) ; both of them did not receive dtap vaccination, and one of these patients treated with a mechanical ventilator. there were no deaths due to pertussis during the study period (table 1). on the result of diagnostic methods, pcr had the highest sensitivity showing positivity for all 21 cases (100%), however, confirmed cases with culture were 9 (42.9%) which showed the lowest sensitivity. although rt - pcr and serological test were not performed in all patients, high sensitivity was observed in all patients done with these tests. total of 72 family members and long - term guardians of index infant cases were enrolled in this study for confirming household transmission. the composition of those members were as followings ; 42 parents, 20 siblings, 10 relatives. seventy members (97.2%) showed respiratory symptoms, and 42 members (60%) with respiratory symptoms had a history of antibiotics use. of these 72 family members, 38 members (38/72, 52.8%) were confirmed with laboratory criteria of pertussis as followings ; all 38 members were positive by pcr, 31 members (who received elisa assay) were all positive serology, 12 members were positive culture. finally, a total of 18 potential family sources (18/21, 85.7%) were identified for confirmed cases, of which 20 members (20/38, 52.6%, 15 family) were parents and 8 (8/38, 21.1%, 6 family) were siblings. other 10 members (6 family ; 6 grandparents, 4 aunts) accounted for 26.3% (10/38). in particular, mothers provided the highest proportion (13/38, 34.2%) of presumed sources of infection (fig. a resurgence of reported pertussis has been documented in a number of countries with high vaccine coverage since the 1990s (1, 4, 18). dtap vaccination coverage in korea is very high, with 94% of the children completing the primary series of pertussis immunization. despite the nationwide vaccination program with high coverage understanding the epidemiological and clinical features of pertussis in community is important because it remains a major cause of morbidity and mortality worldwide. in this study, we have made the demographic data from confirmed pertussis in an attempt to compare the clinical outcomes according to dtap immunization. generally, pertussis mostly occurred in young infants under 6 months of age who did not complete the primary series of dtap vaccination. depending on the immunization status of our patients, there were significant differences in clinical outcome similar to other studies (11, 19, 20). the duration of hospital stay was significantly longer ; and the severe cases admitted to icu were found in patients without pertussis vaccination as compared to those with vaccination. the absolute lymphocytosis in laboratory findings was shown primarily in patients without the vaccination history. even though all patients had paroxysmal cough for more than 1 week, whooping cough was present in only 14.3% of the patients. because the presenting symptoms may be nonspecific, it is important for clinicians to suspect pertussis when there is prolonged cough or absolute lymphocytosis, especially in young infants with no or incomplete dtap vaccination. the highest numbers of pertussis cases was in april, june, and september, which have relatively warm temperatures in korea. few patients occurred in winter as other respiratory pathogens are commonly considered in the differential diagnosis. similar studies from other countries have also shown that the reported pertussis case mostly occurred in a summer months with high temperatures (21, 22). since the number of patients is too small, it is unclear to know the seasonality of pertussis based only upon this result. in addition, various factors such as environmental factors, school opening, and climate can affect the seasonal characteristics of pertussis epidemiology. however, because clinical presentations of pertussis often resemble influenza - like symptoms and those of other respiratory diseases such as mycoplasma pneumoniae and adenovirus, this lack of awareness and nonspecific clinical characteristics may be often associated with underdiagnosed condition of pertussis. culture of nasopharyngeal secretions is essential for diagnosis, however, the result showed the lowest sensitivity (42.9%) in our patients. moreover, this result requires a long incubation period up to 10 to 14 days, after which the patient can be delayed in critical treatment and infect other persons with contact. although rt - pcr and serological test were not performed in all patients, these methods also have higher sensitivity than culture. serologic testing can help the diagnosis of patients with atypical symptoms, for whom clinical samples are collected at later time from the onset of disease. this study is, to our knowledge, the first to document the presumed importance of household transmission in korea. in 18 families (85.7%) of our patients where a source of infection was established, parents (52.6%), grandparents and aunts (26.3%), or siblings (21.1%) in particular, mothers provided the highest proportions of presumed sources of infection. in our study, the majority of household members showed respiratory symptoms such as prolonged cough and recent history of antibiotics treatment for respiratory infections. however, none of them did know that pertussis might cause severe or chronic cough in adolescents and adults.. there has been an increase in the reported number of pertussiss cases in many countries, primarily among adolescents and adults, which is special concern because adolescents and young adults are a recognized reservoir of infection for neonates and infants, who are at high risk for pertussis - related morbidity and mortality (23 - 26). several household studies have noted the predominant role of parents in the transmission of pertussis to susceptible infants (27, 28). for this reason, the usa advisory committee on immunization practices (acip) has recommended the cocooning strategy that tdap vaccine be administered to all caregivers of infants less than 1 yr, in an effort to protect young infants against pertussis since 2006 (29). likewise, the cocooning strategy is important in korea, in keeping with high morbidity and mortality in young infants without immunization (30). despite the introduction of tdap vaccine in our country, vaccine use is only beginning, perhaps because of an incomplete understanding of the burden of pertussis among community population and the lack of national policy and education concerned with the necessity of tdap vaccination. the immunization policy in korea recently introduced the booster dose of tdap vaccine as part of routine immunization in adolescent. therefore, the change in the epidemiology of pertussis should be noted carefully. in conclusion, pertussis is still present in korea and remains a major morbidity in young infants. most infants were too young to have received the primary series of dtap vaccination before infection. household members were identified as a potential source of pertussis infection, and they should be encouraged to receive booster immunization with tdap to minimize pertussis transmission to this vulnerable group on infants. nationwide pertussis reporting is also urgently required to better understand disease transmission patterns, for early recognition and prevention of outbreak and for the evaluation of vaccination policy. universal recommendation for pertussis booster vaccination at 11 to 12 yr is expected to decrease the transmission of pertussis in households in korea. | a recent resurgence of pertussis has raised public health concerns even in developed countries with high vaccination coverage. the aim of this study was to describe the clinical characteristics of infant pertussis, and to determine the relative importance of household transmission in korea. the multicenter study was prospectively conducted from january 2009 to september 2011. we identified the demographic and clinical data from these patients and performed the diagnostic tests for pertussis in their household contacts. twenty - one patients with confirmed pertussis were included in the analysis. all infections occurred in infants younger than 6 months of age (mean age, 2.5 months) who had not completed the primary dtap vaccination except for one patient. infants without immunization history had a significant higher lymphocytosis and longer duration of hospital stay compared to those with immunization. all were diagnosed with pcr (100%), however, culture tests showed the lowest sensitivity (42.9%). presumed source of infection in household contacts was documented in 85.7%, mainly parents (52.6%). pertussis had a major morbidity in young infants who were not fully immunized. household members were responsible for pertussis transmission of infants in whom a source could be identified. the control of pertussis through booster vaccination with tdap in family who is taking care of young infants is necessary in korea. |
the role of low - density lipoprotein cholesterol (ldl - c) in the pathophysiology of atherosclerosis has been unequivocally established, and the use of ldl - lowering agents, especially statins, has led to a significant reduction of cardiovascular events in both primary1 and secondary2 prevention. however, a significant cardiovascular risk remains even after the achievement of optimal ldl concentrations, and among the major statin trials, the maximum relative risk reduction has not exceeded 47%.3,4 on the other hand, epidemiological data have clearly demonstrated a strong inverse relationship between high - density lipoprotein cholesterol (hdl - c) levels and the risk of cardiovascular events, which is independent of the ldl - c levels and remains relevant even when ldl - c levels are below 70 mg / dl.46 furthermore, the absolute benefits of ldl lowering are greater in patients with low hdl - c concentrations.4,7 thus, extensive research is being carried out to identify new hdl - raising drugs in the hope that this would lead to further reduction of cardiovascular risk, and cholesteryl ester transfer protein (cetp) inhibitors are being actively studied for that purpose. cetp is a hydrophobic glycoprotein that is secreted mainly from the liver, predominantly derived from kupffer cells,8 and circulates in the plasma, bound mainly to hdl. in individuals with essentially normal lipid levels, cetp concentration is ~14 g / ml,9,10 while the concentration may be 7080% higher among those with hyperlipidemia.10,11 the role of cetp is to promote the transfer of cholesteryl esters from hdl to very low - density lipoprotein and ldl.12 therefore, cetp inhibition raises hdl - c levels and decreases ldl - c levels (fig. 1). cetp inhibition became an attractive antiatherogenic target when rodents lacking plasma cetp activity were found to have elevated hdl levels and resistance to diet - induced atherosclerosis.10,12 subsequently, patients with cetp gene mutations were also found to have elevated hdl levels and decreased incidence of coronary heart disease (chd).13,14, torcetrapib has been shown to inhibit the development of atherosclerosis.15 furthermore, in human studies, torcetrapib produced a dose - dependent increase of hdl - c up to 106% and a reduction in ldl - c up to 42%.16,17 thus, the investigation of lipid level management to understand its impact in atherosclerotic events (illuminate) trial was conducted to test the hypothesis that torcetrapib would decrease cardiovascular events in high - risk populations.18 in this study, 15,067 patients at high cardiovascular risk were randomized to receive atorvastatin plus 60 mg of torcetrapib daily versus atorvastatin plus placebo. in patients who received torcetrapib, there was an increase of 72.1% in hdl - c and a decrease of 24.9% in ldl - c, as compared with baseline (p 20% per 10 years).41 in the anacetrapib group, as compared with placebo, there was a 138.1% increase in hdl - c levels and a 39.8% reduction in ldl - c levels. in addition, anacetrapib, as compared with placebo, increased apo - a1 levels by 44.7% and decreased apo - b levels by 21.0%. furthermore, treatment with anacetrapib was associated with a 31.7% reduction in non - hdl cholesterol, a 36.4% reduction in lipoprotein(a) levels, and a 6.8% reduction in triglyceride levels, beyond the changes seen in the placebo group.41 there were no appreciable differences between the anacetrapib group and the placebo group in the percentage of patients with adverse events that were thought to be related to the study drug or that led to its discontinuation. there were also no significant differences between the two groups in the mean change in systolic or diastolic blood pressure or in the percentage of patients with a reported increase in blood pressure. moreover, there were no significant between - group differences in the serum levels of potassium, chloride, bicarbonate, or aldosterone, thus proving that anacetrapib was devoid of off - target hyperaldosteronism.41 a two - year extension to the define trial showed that an additional two years of treatment with anacetrapib was well tolerated with durable lipid - modifying effects on hdl - c and ldl - c.42 of note, anacetrapib has been shown to increase apo - b clearance in mildly hypercholesterolemic subjects and this effect may also be responsible for the observed anacetrapib - induced ldl - c lowering.43 in an animal model, anacetrapib has been shown to dose - dependently reduce atherosclerosis and improvelesion stability.44 the effects of anacetrapib on cardiovascular outcomes are currently being evaluated in the ongoing evaluation of the effects of anacetrapib through lipid modification (reveal) this is a phase iii trial designed to determine whether treatment with anacetrapib could reduce the risk of a composite end point (coronary death, myocardial infarction, or coronary revascularization) in patients with circulatory problems who have their ldl - c optimally treated with a statin. it has randomized 30,624 subjects to anacetrapib 100 mg daily or matching placebo with a predicted median follow - up of four years. the study will include men and women 50 years of age with a history of myocardial infarction, cerebrovascular atherosclerotic disease, peripheral arterial disease, or diabetes mellitus with other evidence of symptomatic chd. evacetrapib is a novel, potent, and selective cetp inhibitor, which has been shown to elevate hdl - c without inducing aldosterone or increasing blood pressure.45 as monotherapy, evacetrapib produced dose - dependent increases in hdl - c from baseline of 53.6%128.8% and decreases in ldl - c of 13.6%35.9%.46 in combination with statin therapy, evacetrapib, 100 mg daily, compared with statin monotherapy, produced increases in hdl - c of 78.5%88.5% and decreases in ldl - c of 11.2%13.9%.46 in a meta - analysis of 14 randomized treatment arms over a mean of two months, evacetrapib significantly increased hdl - c by an average of 86% and reduced ldl - c by an average of 21.11% without any effect on the level of triglycerides.47 meta - regression suggested a dose - dependent effect of evacetrapib on hdl - c and ldl - c, but not triglyceride levels. this meta - analysis also suggested equivalent rates of adverse events in subjects receiving evacetrapib and placebo.47 in a recent study, evacetrapib, alone or in combination with statins, was found to increase cholesterol efflux capacity (cec), including the abca1-specific cec, and also increase pre - beta-1 hdl.48 these effects made evacetrapib a promising target for the potential reduction of cardiovascular risk, given the recent evidence that cec has a strong inverse correlation with incident cardiovascular events,49 although the hypothesis that a therapeutic increase of cholesterol efflux improves cardiovascular outcome remains to be proven in clinical studies. the process of the abca1-specific cec is shown and described in figure 2. addition of evacetrapib to atorvastatin compared to placebo, high intensity atorvastatin, and atorvastatin with ezetimibe to evaluate ldl - c lowering in patients with primary hyperlipidemia (accentuate) this study had enrolled 396 hypercholesterolemic patients with atherosclerotic cardiovascular disease and/or diabetes mellitus, and its primary outcome measure was the percent change in ldl - c from baseline to three months. in addition, the effects of evacetrapib on cardiovascular outcomes were being evaluated in the a study of evacetrapib in high - risk vascular disease (accelerate) this was a trial designed to determine whether treatment with evacetrapib could reduce the risk of a composite end point (cardiovascular death, myocardial infarction, stroke, coronary revascularization, or hospitalization for unstable angina) in clinically stable patients with high - risk vascular disease (history of acute coronary syndrome, cerebrovascular atherosclerotic disease, peripheral arterial disease, or diabetes mellitus with documented cad). the study had enrolled 12,095 patients and had a predicted median follow - up of four years. however, on october 12, 2015, eli lilly and company and the accelerate study s academic leadership have accepted the recommendation of the independent data monitoring committee to terminate the trial due to insufficient efficacy. lilly discontinued the development of evacetrapib for the treatment of high - risk atherosclerotic cardiovascular disease and concluded all the other studies in the program. the study was not stopped for safety findings, but the independent data - monitoring committee based its recommendation on data from periodic data reviews, which suggested that there was a low probability that the study would achieve its primary end point based on results to date [https://investor.lilly.com/releasedetail.cfm?releaseid = 936130 ]. more recently, another novel cetp inhibitor, ta-8995, was studied in a multicenter, randomized, phase 2, double - blind, placebo - controlled, parallel - group study (tulip trial) in patients with mild dyslipidemia, as monotherapy and in combination with statin therapy.50 administration of ta-8995 caused a dose - dependent increase of hdl - c levels by up to 179.1% and of apo - a1 levels by up to 63.4%, whereas it reduced the ldl - c levels by 45.3% and the apo - b levels by 33.7%. in addition, the levels of lipoprotein(a) were reduced by up to 36.9%. in combination with statins, furthermore, treatment with the 10 mg dose of ta-8995 resulted in a 36.7% increase in serum - mediated cholesterol efflux.50 in this study, there was no effect of ta-8995 (whether given as monotherapy or in combination with a statin) noted on any laboratory safety parameter, including serum aldosterone, salivary cortisol, high sensitivity crp, or endothelin 1. in addition, there was no effect of ta-8995 on serum electrolyte concentrations, blood pressure, or insulin resistance.50 cetp inhibitors comprise a new class of agents that significantly increase hdl - c and also have an ldl - lowering effect. however, the first trials with the cetp inhibitors torcetrapib and dalcetrapib failed to show a reduction in cardiovascular events, and another trial with the cetp inhibitor evacetrapib was recently stopped, because the independent data - monitoring committee suggested that there was a low probability that the study would achieve its primary end point. nevertheless, newer cetp inhibitors with more favorable effects on lipids and devoid of any detrimental off - target characteristics, such as anacetrapib, are presently studied in ongoing clinical trials. the results of these outcome trials are expected to provide a final answer to the question whether cetp inhibition might actually be effective for the reduction of cardiovascular risk or the cetp inhibition chapter will need to come to an end. | the atheroprotective role of high - density lipoprotein cholesterol (hdl - c) in cardiovascular disease has been unequivocally established, and epidemiological data have clearly demonstrated a strong inverse relationship between hdl - c levels and the risk of cardiovascular events, which is independent of the low - density lipoprotein cholesterol (ldl - c) levels. thus, it would be logical to hypothesize that raising hdl - c might potentially lead to a reduction of cardiovascular risk. cholesteryl ester transfer protein (cetp) promotes the transfer of cholesteryl esters from hdl to very low - density lipoprotein and ldl. therefore, cetp inhibition raises hdl - c levels and decreases ldl - c levels. the first trials with cetp inhibitors failed to show a reduction in cardiovascular events. however, newer cetp inhibitors with more favorable effects on lipids are presently being tested in clinical trials with the hope that their use may lead to a reduction in cardiovascular risk. this review aims to provide the current evidence regarding cetp inhibition, as well as the clinical and scientific data pertaining to the new cetp inhibitors in development. |
1.8 million people died in 2009 due to aids - related causes, and an estimated 2.6 million people were newly infected with hiv in world. india carries the third largest number of hiv infected patients in the world after south africa and nigeria. hiv infection causes depletion of cd4 cells in peripheral blood and lymphoid tissue causing cd8 cell dysfunction. quantitation of cd4 helper lymphocytes is thus essential in the staging and monitoring of patients infected with hiv. throughout the course of disease, the total t - cell levels remain fairly constant despite a fall in cd4 cell count due to compensatory rise in cd8 cells.. therefore the ratio of cd4 cell to cd8 cells is a significant measure which is of greater magnitude as compared to absolute cd4 cell count of disease progression in hiv - infected subjects. plasma hiv-1 rna levels have been shown to be a strong predictor of rapid progression to aids after seroconversion that is independent of cd4 counts. high viral load is currently considered to be one of the main indicators of hiv - induced immune deterioration. since the onset of hiv / aids epidemic, the oral cavity has played a key role in helping to define the natural history of hiv / aids. few studies have evaluated the relationship of cd4/cd8 ratio with oral manifestations [58 ]. high cd8 lymphocytosis and low cd4/cd8 ratio have also been associated with oral candidiasis (oc) [5, 7 ]. studies from western countries have also shown association of oral manifestations with higher hiv viral load [912 ]. however till date no asian study has attempted to assess the relationship between oral manifestations with cd4/cd8 ratio and hiv viral load. the possible reasons for absence of studies related to hiv viral load conducted in asia could be financial and resource constraints. in the present study, a group of hiv patients from south indian population were analyzed to identify possible association of oral manifestations with cd4/cd8 count ratio and hiv viral load and to evaluate the diagnostic utility of correlating main oral manifestations for low cd4/cd8 ratios (0.60). hiv viral load was also documented from 30 patients among the study sample of 103 patients and was subsequently constellated in 2 groups (20,000 copies / ml). oral manifestations were diagnosed according to presumptive criteria of eec - clearinghouse classification (1993). a single examiner trained in oral medicine (g.s.) examined and recorded all oromucosal lesions. statistical analysis was done using spss software version 11. associations between subject variable and each type of oral lesion were analyzed using chi - square test. odds ratio and 95% confidence interval were used in logistic regression analysis for association between oral manifestations and cd4/cd8 ratio within group i. mann - whitney u test was used for differences between mean values of group of patients. the probability that the patient has cd4/cd8 ratio 0.30 when a specific oral manifestation is absent. the demographic distribution of 103 patients (age range 2468 years ; mean age 37.1 years ; 70 males, 33 females) consisted of all south indians. social demographics, their oral habits, usage of antiretroviral therapy and laboratory parameters of the study cohort can be observed in table 1. among the study population 101 patients (98.3%) had cd4/cd8 ratio less than 1 (normal range 0.031.61) which is suggestive of an advanced immunosuppression in this study (p value 20,000 copies / ml was seen in patients below cd4/cd8 ratio 0.60). erythematous candidiasis (ec) (49.3%), melanotic hyperpigmentation (40.6%), and xerostomia (37.7%) were common oral findings within group i, whereas within group ii melanotic hyperpigmentation (28.6%) followed by ec (17.9%) were more frequent oral manifestations. overall, ec (38.8%) was most common finding followed by melanotic hyperpigmentation (35.9%), xerostomia (32.0%), oral hairy leukoplakia (ohl) (17.5%), and linear gingival erythema (lge) (table 3). current mean cd4/cd8 ratios for groups with and without oral manifestations are given in table 4. the mean absolute cd4 count for the study cohort with presence of any oral manifestation was 196.18 cells / mm, and mean cd4/cd8 ratio for the study cohort was 0.24 (s.d 0.22). the lowest mean cd4/cd8 ratio of 0.16 among individual oral manifestations was found for pseudomembranous candidiasis (pc) (mean absolute cd4 count126.78 cells / mm). mann - whitney u test revealed a statistically significant difference between mean values of group when any oral manifestation was present (p 20,000 copies / ml and remaining had hiv viral load 20,000 copies / ml had some type of oral manifestation (statistically significant ; p.05). smoking may produce particulate matter, noxious chemicals, and heat that can modify more local factors. oral manifestations were equally prevalent in alcoholics (81.3% ; 39/48) as well as nonalcoholics (80% ; 44/55). no statistical significance was also observed for individual oral manifestations with gender, smoking and alcohol (p >.05). since myriad factors can affect t - cell subsets, changes in cd4 t cells and cd8 t cells reflected through cd4/cd8 ratio should be investigated over a time frame, preferably a 36 month period to assess the immune status of patient. the clinical significance of cd4/cd8 ratio and hiv viral load are well established for hiv disease progression, but as to date, no consensus has been reached in scientific literature as to what comprises the standard grouping for cd4/cd8 ratios and hiv viral load. the prevalence of ec was 38.8% (table 3) that was comparable to studies conducted by campo. (40%). among candidal infections the most common finding was ec (38.8%) followed by pc (8.7%), angular cheilitis (ac) (5.8%), and hyperplastic candidiasis (1.9%). prognostic significance for both ec and pc has been reported to be similar by two longitudinal studies by dodd. fonseca. 2000 correlated oral manifestations with cd4/cd8 ratio in 51 hiv positive children and concluded that patients with cd4/cd8 ratio 20,000 copies / ml) (table 6). campo., margiotta., and laurenco and figueiredo had also found a positive correlation between high hiv viral loads > 10,000 copies / ml and oral candidiasis. in our study no correlation could be found between ohl and high viral loads. this was in contrast to other studies where ohl was associated to high hiv viral load [9, 24, 30 ]. patton. found that patients with ohl were twofold more likely to have a higher viral load than people without oral lesions, whereas moura. had suggested ohl as a clinical marker for hiv viral load > 3000 copies/l. our finding of negative correlation for ohl should be explored in future studies whether there is any difference in relationship between ohl and high viral loads in developing and developed nations. the prevalence of oral manifestations of those patients on antiretroviral therapy (art) was comparatively lesser as compared to patients not on art (78.6% versus 82.0% ; p =.669), though difference was not statistically significant (table 7). this finding was in concurrent with other studies [3234 ]. among all oral manifestations, this finding is similar to other studies conducted in india [15, 34 ]. various reasons that have been attributed to increased oral pigmentation in patients with art are increased release of msh caused by deregulation of cytokines in hiv disease, smoking, addison 's disease, and use of melanocyte stimulating drugs like art. ohl can also be considered as a measure of efficacy of art [12, 16 ]. there was a regressive, but not statistically significant, effect of art on ohl. various studies have documented a reduced prevalence of ohl in patients who are taking art [16, 31, 35 ]. the protease inhibitors, a class of art, are also known to exert anticandidal activity by inhibiting secreted aspartyl proteinases [saps ] secreted by candida species. saps are the major virulence factors for candida species and their inhibition would mean a reduced virulence and hence decreased prevalence of oral candidiasis that has been reported in various studies [36, 37 ]. the absence of protease inhibitors in our study sample could be the probable reason why we could not observe a dramatic reduction in ec and pc. this finding was similar to other studies conducted in asia [1416, 34 ]. the possible reason could be a functionally incomplete reconstitution that might lead to development of human papilloma virus-(hpv-) induced lesions like warts, condylomas, and focal epithelial hyperplasia. the probable enhanced antiviral effect of art on hpv related to genetics as well as the virulence factors could be the reasons for absence of warts in asian countries. the presence of oral manifestations in patients on art may indicate advanced immune suppression or failure of the therapy [34, 36 ]. among subsample of hiv viral load population (n 30), 15 patients (50%) were on art and they had hiv viral load.05). a greater sample size of patients on art could be directed towards future research for its association with oral manifestations. unfortunately, no effective vaccine has been found so far, and there is little hope that it will be developed in near future which can combat the virus or at least stimulate the host immune system. in this study more studies are needed to carry out association between oral manifestations and cd4/cd8 ratio, which is a better laboratory marker than absolute cd4 count for immune suppression. so far, studies have been conducted only in western countries with almost scarcity of literature in developing world. studies similar to ours need to be done with cohorts comprising of samples of resource - constrained nations, which can be compared with those from developed nations to evaluate if there are differences. future studies could be directed towards association between oral manifestations and hiv viral load, a better marker of long - term immune deterioration. a greater sample study size for hiv viral load in developing nations will be required to confirm the findings in our study. people infected with hiv need to be educated, inspired, and empowered with knowledge so that they can protect themselves from the impact of aids. to ensure a better care and quality of life to these patients linkages with social sector programmes for accessing social support to the patients, recreational and skill development support, transport subsidy to ensure art care, and integrating nutritional support within treatment regimens are certain strategies that can be implemented. by regular intraoral examinations at frequent intervals dentists can play a small but significant role of ensuring an effective integrated delivery of health services for hiv - infected patients. this can occur only if there is joint action / strategy between health care providers and various hiv - related government as well as nongovernment organizations. the possibility of an oral lesion index to predict hiv in resource poor countries with no access to cd4 count or viral load was suggested recently. however to incorporate an oral lesion index, certain regional parameters and research studies that employ rigorous research methodologies with emphasis on hiv viral load and cd4/cd8 ratio need to be done. there is a need to compare the results of various cross - sectional studies, and more longitudinal studies are required in developing nations to formulate an oral lesion index that can help as a marker for hiv - related immune suppression. ec had good predictive values (84.5% and 85%) for group i cd4/cd8 ratio 20,000 copies / ml. there was reduced prevalence of oral manifestations in patients on art. more studies need to be carried out, especially in asia, for correlation of oral manifestations with hiv viral load and cd4/cd8 ratio. | the objective of the present research was to determine the prevalence of oral manifestations in an hiv infected population from south india and evaluate their association with hiv viral load and cd4/cd8 ratio. intraoral examination of 103 patients, whose cd4/cd8 ratio was available, were conducted. hiv viral loads were available for thirty patients only. the prevalence of oral manifestations was 80.6% (83/103). the most common oromucosal lesion was erythematous candidiasis (ec) (38.8%) followed by melanotic hyperpigmentation (35.9%). patients having any oral manifestation had a mean cd4/cd8 ratio of 0.24. ec had positive predictive value of 85.0% for cd4/cd8 ratio 20,000 copies / ml) (p 20,000 copies / ml). |
perhaps one of the most important concepts in inorganic chemistry is the nature of the bonding interactions between a metal center and its ligands. the covalency, or charge donation from the ligands to the metal, of these bonds influences the chemistry of transition metal complexes, including their reactivity, redox potentials, and magnetic exchange. ligand bonds is of fundamental importance when attempting to rationalize the properties and reactivity of inorganic complexes. the influence of bonding on the metal orbitals occurs through two distinct mechanisms. the first is via the direct dilution of the metal d orbitals due to mixing with the ligand orbitals and is termed symmetry restricted covalency due to its symmetry - dependent nature. the second, more subtle, mechanism is a distortion of the metal d orbital radial wave functions due to bonding (central field covalency). accordingly, in a molecular orbital (mo) picture, the impact of symmetry restricted covalency on the ground state of an inorganic complex can be described as a linear combination of metal and ligand orbitals according to eq 1, where (1) represents the amount of ligand character mixed into the metal d manifold.1 several experimental techniques have been developed to assess the covalency of metal ligand bonds, including ground state methods such as analysis of hyperfine and superhyperfine couplings obtained from epr and excited state techniques like visible absorption and x - ray absorption spectroscopies (specifically the metal l - edge and ligand k - edge). as discussed in the cited references, these methods have provided significant insights into the nature of metal ligand bonding and have greatly improved our understanding of many inorganic systems. it should be noted, however, that challenges and limitations exist for all of these methods. the extraction of covalency from epr requires an epr active compound with resolvable ligand superhyperfine coupling. obtaining covalency information from absorption measurements depends on the presence of suitable resolved spectral features and accurate intensities. additional experimental challenges are presented by the ultrahigh vacuum conditions needed for transition metal l - edges and the k - edges of light atom ligands (c, n, o). due in part to these limitations, the extraction of reliable covalency values is often quite challenging if not impossible, so additional methods for obtaining this information are valuable. the xes process begins with ionization of a 1s core electron from the metal using high energy incident x - rays ; the photons emitted during the radiative decay of electrons from higher - lying states are then analyzed, allowing xes to probe the occupied states of a metal compound. in this way, xes provides information that is complementary to that obtained from xas and that is sensitive to the bonding interactions of a complex. further, as a hard x - ray technique that probes core orbitals, xes is inherently element selective and applicable to a wide range of sample states and environments. the first row transition metal k xes spectrum can be divided into two regions : the intense k mainline (composed of the k1,3 and k lines) at low energy and the valence - to - core region at higher energies (figure 1). the valence - to - core transitions have been shown to arise from orbitals of dominantly ligand ns and np character with a small amount of metal np mixing that provides a dipole allowed mechanism for the observed intensity. as such, the valence emission features are sensitive to the identity and electronic structure of the bound ligands. furthermore, the valence - to - core region can be effectively modeled using a straightforward frozen - orbital one - electron scheme based on density functional theory (dft), as previously detailed. the coupling of experiment to computations in this manner has enabled valence - to - core xes to become a powerful probe of the environment around a metal center, allowing, for example, the identification of a central carbide in femoco, the detection of bridging oxos in the mn4cao5 cluster of photosystem ii, and the assessment of the electronic structure of hydrogenase model compounds. development of the k mainline, on the other hand, has received relatively less attention in the recent literature, especially with respect to molecular systems. assigned as metal 3p to 1s transitions, the mainline has long been known to be sensitive to the spin state at the metal center, with dramatic differences observed (e.g., decrease in k intensity and decrease in the splitting between the k and k1,3) as the nominal spin at the metal is reduced. as intense, dipole - allowed transitions, the mainlines have been used to assess changes in metal spin and oxidation state. these changes can be understood in a multiplet framework and are well established to be largely due to modulations of the exchange integrals between the 3p hole and the valence 3d electrons in the final state ; a schematic of the various states involved for a 3d metal is provided in figure 2(38) with extension to other d counts similarly possible. however, to date, the analysis of k mainlines has had limited application beyond their use as simple fingerprints for spin state. we note, however, that cramer and co - workers previously invoked covalency to rationalize the reduced k intensity seen for rubredoxin as compared to fecl4 and similar observations were made by gamblin and urch as well as glatzel and bergmann. these studies thus provided the first hints that the k mainlines were not simply isolated probes of spin state, although, to our knowledge, the contributions of covalency to the k mainline spectra have never been systematically investigated. pictorial depiction of the transitions giving rise to the k mainlines. in brief, 1s ionization from a totally symmetric s ground state (1s3p3d) gives rise to s and s intermediate states (1s3p3d) that are split only by the small 1s3d exchange and, thus, are nearly degenerate. enumerating the possible final states in the absence of spin orbit coupling, one p and three p final states (1s3p3d) are accessible ; the parent 3d terms are shown in parentheses. the intensity of the formally allowed transition to the (p)p state is calculated to be very small and does not contribute significantly to the spectral shape. as the splitting between the k mainline features is governed largely by the 3p3d exchange integrals, they too should be modulated by the metal ligand covalency. by taking the mo description of covalency expressed in eq 1 together with the knowledge that the exchange integrals between the metal 3p core orbitals and the ligand orbitals are expected to be much smaller than the one center d3p|d3p integrals, expressions of the type d3p|d3p reduce to eq 2, where clearly has a direct influence on the observed splitting (m and n are rational numbers that depend on the actual orbitals involved and g1 and g3 are the slater exchange integrals between the 3p and 3d electrons)2 herein, we experimentally observe significant differences in the k mainline spectra for a series of nominally high spin, d fe(iii) compounds. to explain these effects, we derive analytical expressions for the k mainline splitting for d systems that define the k1,3 these expressions nicely describe the observations made between metals of different d counts and demonstrate the necessity of invoking covalency to explain the trends seen between the high spin ferric compounds studied. the ligand field expressions are, however, very general in nature, so we also employ computations to obtain a more quantitative understanding of these spectra. crystal field multiplet calculations are well - established as being able to simulate k mainlines, so we begin by reproducing the experimentally observed effects using this methodology and confirm the mainline dependence on the p these calculations offer the ability to independently tune the spin orbit coupling, ligand field, and the coulomb and exchange integrals and allow us to deconvolute the effects of each of these parameters. although the ability to separately tune these parameters is of much utility in isolating individual contributions, the empirical nature of these modifications limits the information that may be extracted. thus, in order to obtain deeper insights, we have developed a protocol for the calculation of k mainlines using restricted active space configuration interaction (ras ci) calculations as implemented in orca. these calculations are a significant improvement over the multiplet methodology as they explicitly and nonempirically include covalency, spin orbit coupling, and ligand field effects. despite failing to properly include dynamic correlation, these calculations provide valuable insight into the chemical factors that affect k mainlines and, together with the ligand field expressions, establish a theoretical framework to assess the contributions of symmetry restricted covalency to these spectra. importantly, for an initial test set of four high spin ferric compounds, the general trends in the calculated spectra reproduce experiment and the observed changes are shown to correlate with the ligand character mixed into the metal d orbitals. this correlation between the k mainline splitting and calculated metal ligand covalency is then extended to a wider range of experimental data for three additional high spin fe(iii) compounds that were previously reported, demonstrating that the splitting between the k1,3 and k may be used generally as a quantitative probe of metal ligand covalency. the covalent modulation of the k mainline splitting is of the same order of magnitude as the changes induced by a two - electron modulation of the d count (at the atomic limit) ; hence, these results also serve as a cautionary note when employing mainlines as isolated fingerprints for spin state. the implications of these results for the broader application of k mainlines as a probe of transition metal active site electronic structure are discussed. anhydrous fef3, fecl3, and febr3 were obtained from aldrich and used without further purification. (et4n)[fe(sar)4 ] (ar = 2,3,5,6-tetramethylphenyl) was prepared by modification of the method outlined in ref (43). namely, lithium thiolate was prepared by in situ deprotonation of the thiol with a lithium ethoxide solution, the latter obtained by treatment of lithium hexamethyldisilazide with excess ethanol. due to the air sensitivity or hygroscopic nature of these compounds, samples for xes measurements were prepared by grinding to a fine powder, packing into 1 mm aluminum spacers without dilution, and sealing with 38 m kapton tape. all xes spectra were collected at ssrl beamline 62 (3 gev, 350 ma) or chess c - line (5.3 gev, 200 ma). for ssrl data, the incident beam energy was set to 8 kev using a si(111) liquid nitrogen cooled monochromator and was calibrated using a fe foil. focusing mirrors were used to achieve a 150 200 m beam at the sample, providing 10 photons / s. if necessary to prevent sample damage or detector saturation, aluminum filters were inserted before the sample to attenuate the incident beam. energy resolution of the xes spectra was achieved using a crystal array spectrometer employing five spherically bent ge(620) crystals (100 mm diameter, 1 m radius of curvature) aligned on intersecting rowland circles, as described previously. samples were maintained at < 20 k in an oxford cfi208 continuous flow liquid helium cryostat and were positioned at 45 with respect to the incident beam. a he filled flight path was used between the sample and spectrometer to reduce signal attenuation and emitted x - rays were detected using an energy resolving si drift detector with a 3 mm vertical slit. spectra were collected over the energy range of 7013 to 7123 ev with steps of 0.2 ev (70137079 ev) and 0.15 ev (70797123 ev). collection of data at chess was done using a setup very similar to that at ssrl. in brief, the incident beam was set to 9 kev using upstream multilayers (90 ev band - pass) and focused to a 1 3 mm spot providing 1 10 photons / s. the sample was maintained below 30 k using a displex cryostat and an array of five spherically bent ge(620) crystals was used for energy selection. a silicon drift detector with a 3 mm vertical slit was used to detect emitted x - rays and data were collected over the range of 7017 to 7121 ev with 0.36 ev steps (70177082 ev) and 0.24 ev (70827121 ev). for all spectra, the signal was normalized with respect to the incident flux measured in an upstream ion chamber. the spectrometer energy was calibrated using scans of fe2o3 and reference energies of 7044.67, 7060.62, 7092.38, and 7107.42 ev. damage scans were performed on each sample to determine acceptable exposure times per spot. if needed, data were collected from multiple spots on a sample to avoid radiation damage. all scans that showed no evidence of damage were averaged using pymca and the area under the spectrum from 70177120 ev was set to 1000. averaged mainline spectra were fit using blueprint xas version 1.2. reported values are obtained from the positions of the fit components corresponding to the k and k1,3 peaks and are the average from at least 18 good fits. in addition to the fits for the fef3, fecl3, febr3, and (et4n)[fe(sar)4 ] measured for the present study, fits were also obtained for the previously reported fe(acac)3, (tpfp)fecl, and fecl4 data. representative fits and tabulated numerical data for all compounds are provided in figure s1 and table s2 in the supporting information. crystal field multiplet calculations were carried out using the model implemented by thole, the atomic theory developed by cowan, and the crystal field interactions developed by butler. spectra were calculated for 3p to 1s emission from fe d ions and were energy shifted by 7055.1 ev to match experimental spectra. except when specified otherwise, lorentzian broadenings of 1.60 ev (70587100 ev) and 5.10 (70207058 ev) as well as a global gaussian broadening of 1.20 ev were used to simulate lifetime and instrumental broadenings. ci can be broken down into two steps : an initial dft calculation to generate quasi - restricted orbitals (qros) followed by the ras the initial dft calculations were performed using the bp86 functional, the zeroth - order regular approximation (zora) for relativistic effects following the model potential implementation of van wllen, the scalar - relativistically recontracted def2-tzvp basis set, and the conductor - like screening model (cosmo) in an infinite dielectric. geometry optimizations were performed using this same level of theory, beginning with crystal structure coordinates. qros were visualized using chimera 1.5.3 and were used to perform orbital coefficient analysis with moanalyzer ; they were also used as an input for the following ras ci and, when applicable, casscf calculations. covalent mixings and thus serve well as an input into the ras ci calculations. these calculations are explained in detail in the supporting information, so only a brief description will be presented here. in short, the ras ci calculations are performed to calculate the mainline transitions between the photoionized 1s3p3d intermediate state and all accessible 1s3p3d final states while taking into account the full multiplet structure of this region., one containing the core 1s and 3p orbitals and the other containing the valence 3d orbitals. these orbitals are frozen and then one electron is removed from the core, allowing calculation of all possible septet and quintet states (which are themselves allowed to mix via spin transitions between the desired intermediate states and final states can then be calculated, generating computed mainline spectra. for sample inputs and further explanation, as noted in the introduction, the sensitivity of k mainlines to the spin state of 3d transition metal centers is well established and has been previously explained theoretically. less explored are the differences seen within a given spin / oxidation state, though these are generally thought to be small, leading to the common assumption that the k mainline serves as a fingerprint for spin state. however, as shown in figure 3 for a series of high - spin ferric complexes, even compounds of the same oxidation state and spin state can show large differences in the k mainline spectra. namely, the energy splitting between the k and k1,3 for these compounds varies from 13.6 to 18.2 ev, a difference of nearly 5 ev (table 1). k mainline spectra for a series of ferric compounds that demonstrate significant differences in mainline appearance despite all compounds being high spin fe(iii). previous studies and the present analysis (vide infra) have demonstrated that k mainline spectra are relatively insensitive to spin orbit, coulombic repulsion, and ligand field effects (supporting information figures s2s4) but instead are dominated by intra - atomic exchange. this thus implies that the splitting between the two dominant k mainline features must primarily correlate with the number of unpaired electrons on the metal which is, in turn, simply a function of the d configuration. however, the actual splitting requires a detailed inspection of the multiplets that arise in the intermediate and final states. because this is a nontrivial procedure, we provide a comprehensive collection of the theoretical expressions that correlate the k mainline splitting to the d configuration (table 2) and detail the derivation of these equations in the supporting information (section 11). in the derivation, it is assumed that in the final state only the exchange couplings between the unpaired 3p electron and the unpaired d electrons in their electronic ground state contribute to the splitting. this obviously is a strong simplification, which, however, makes it possible to reach some general conclusions. in the equations, covalency is accounted for in terms of the stevens orbital reduction factors t and e (for the t2 g and eg orbitals, respectively) ; these factors correspond to the quantity in eq 2. with these equations in hand, it is illuminating to plot the values of e in terms of 4g1 + 42g3 (a measure of p d exchange) for the free ion case of t = e = 1 (figure 4) to visualize the dependence of e on the 3d count. for d configurations where both high and low spin ground states are available, respectively ; except for the low spin ground states, these expressions are also valid for tetrahedral complexes after removal of the indices g and u. dependence of the k mainline energy splitting on the number of 3d electrons is shown. when high and low spin ground states are possible, these are indicated with red and blue markers, respectively. although figure 4 nicely rationalizes in a very general way many of the observed oxidation and spin state dependencies of metal k mainlines, there are still factors that will influence the effective values of 4g1 + 42g3, and thus possibly modulate the shapes of the mainlines, which must be assessed. namely, these factors include the identity of the metal, its oxidation state, and the interaction of the metal with ligands. for higher metal oxidation states or later transition metals, the radial wave functions will be more contracted leading to higher intrinsic values of 4g1 + 42g3. in addition, the effects of symmetry restricted and central field covalency on the metal must be also considered. to estimate the magnitude of the contribution that changes in effective oxidation state have on the exchange energy, 4g1 + 42g3, we performed hartree fock and ab initio ci calculations (details below and in the computations section) of the electronic multiplets and slater condon parameters for transition metal ions in various oxidation states (figure 5). as expected, these values increase by, at most, 10% with increasing oxidation state, reflecting a contraction of the radial wave functions upon oxidation. notably, this change even between different formal oxidation states is less than what is observed experimentally in the series of high spin ferric compounds (25% decrease). effect of varying oxidation state (electron count) on the effective exchange energy (4g1 + 42g3) (per spins pair) from hartree fock limit calculations using the computer code by r. d. cowan (see supporting information for computational results and list of values of 4g1 + 42g3). as the oxidation state - dependent modification of the p d exchange integrals does not appear sufficiently large to account for the changes observed in the high spin ferric mainlines, we were left with, as has been suggested previously, metal ligand covalency as the operative factor. we thus fully explored the role of covalency from both a crystal field multiplet and ras ci perspective and then use the new insights obtained to return to these ligand field expressions at the end. this analysis further develops the information content of k mainlines beyond the common fingerprint interpretation. a detailed description of our calculations for free atoms and ions is contained in the supporting information (section 12), where we also provide a comprehensive summary of dipole selection rules for all multiplets that are involved in the mainline calculations for any d configuration. together with the extensive tabular material in section 12 of the supporting information, covalency estimates can even be performed without any recourse to ab initio calculations. simulation of k mainlines has traditionally been accomplished with a crystal field multiplet approach that has yielded significant insights into the behavior of mainlines. within such a quasi - atomic approach, various parameters can be modified to effect spectral change : (1) the 10 dq value parametrizing the ligand field splitting, (2) the slater condon electronic repulsion parameters fdd2 and fdd4 that describe the electronic repulsion within the d shell, (3) the parameters fpd2 and g1/g3 that describe the 3p/3d coulomb and exchange integrals respectively, and (4) the 3p and 3d spin orbit coupling constants. the influence of many of these parameters has been investigated previously, though in order to fully calibrate our new ras ci approach, a systematic study of these effects for d fe(iii) can be found in the supporting information (section 9). as expected on the basis of the theoretical analysis in table 2 and exemplified for fe below, of these parameters, only g1 and g3 have a significant influence on the calculated spectral shapes (figure 6). scaling the p d exchange integrals allows for the entire range of observed splittings to be obtained, confirming previous assignments. variation of 3p/3d or 10 dq (consistent with cramer and co - workers, supporting information figures s2 and s4) within reasonable limits leads to essentially no discernible changes, whereas modifying fpd and fdd over wide ranges has only small impacts on the calculated spectra (supporting information figure s3). calculated atomic multiplet spectra on a ferric ion showing the effect of reduced g1 and g3 values. to demonstrate that the experimental k1,3 k energy splittings could be reproduced with these calculations, the slater integrals were all scaled by an amount necessary for the calculation to correctly match experiment (supporting information figure s5). a scalar energy shift and broadening d exchange parameters increase as expected from fef3 to fe(sar)4 (supporting information table s1) and are generally in agreement with the covalency values obtained from dft calculations (table 3) ; by varying the values of the d d and p d repulsion parameters we estimate the uncertainty in the gpd values to be 5%. we note, however, that equally good simulations of the experimental data could be obtained with different sets of parameters and that the solution space becomes even larger upon consideration of charge transfer and lower symmetry systems. a less empirical methodology to investigate the effect of covalency on the mainline spectra is thus desirable. to further support the assignment that the changes in the k mainlines are due to differences in metal ligand covalency, dft calculations were employed. because dft generally leads to a fairly realistic description of covalency (with a slight tendency toward overestimation), it is instructive to compare the calculated metal ligand covalencies obtained from an analysis of the qros to the experimental energy splittings for the high - spin ferric compounds (figure 7). although the covalency for these complexes is anisotropic (e.g., different in each d orbital, table 3), given the experimental resolution it is appropriate to take the average covalency as a measure of the reduction of the p d exchange (defined by taking the average of the lwdin d populations for the metal d based qros). figure 7 clearly demonstrates that a correlation exists between the experimentally observed mainline splitting and the calculated average covalencies. although this correlation is satisfying and certainly captures the essential physics of the problem, it can not be made any more quantitative because dft is unable to explicitly calculate the multiplets that account for the k mainlines. k energy splittings and the sums of the qro coefficients from the metal - based d orbitals. as explained in the computations section and in the supporting information, it is straightforward to set up a restricted active space configuration interaction protocol in which all intermediate and final states that enter the mainline calculation are explicitly represented. thus, provided one uses as inputs for these calculations the dft qro orbitals that show the correct covalent mixings with the ligand orbitals, one might hope for a near - quantitative reproduction of the experimental spectra. as shown in figures 8 and 9 and table 4, the calculated k mainline spectral shapes and energy splittings correlate very well with experiment (figures 8 and 9) and reproduce all of the major trends. importantly, the magnitude of the change across the series of compounds is in excellent agreement with what is observed experimentally (4.37 ev versus 4.54 ev). furthermore, the calculated mainline splittings correlate extremely well with the mo coefficients on the d - based orbitals found in table 3 (supporting information figure s6). there is a systematic error in the absolute transition energies that we attribute to the combined effects of basis set incompleteness, scalar relativistic effects, and missing dynamic correlation. however, as shown by the success of related methods for the calculation of x - ray absorption and emission spectra, this error is not relevant for chemistry as it is highly systematic and can be eliminated through calibration. the calculated splittings are significantly larger than the experimentally measured ones, despite the fact that properly covalently diluted molecular orbitals have been employed and all integrals have been calculated correctly. the reason for this behavior is simply that equations of the form of eq 2 are grossly oversimplified and, possibly counter widespread belief, calculating the naked (unscreened) electron electron interaction over covalently diluted mos is simply not enough to obtain accurate results. what is missing are the effects of dynamic correlation which go a long way in providing a screening of the electron electron interaction and this could be achieved, for example, by second - order multireference perturbation theory (raspt2), as in the related work by odelius. however, this method is not available to us at the present stage of development. alternatively, these effects are also highly systematic and hence can be accounted for, with considerable computational advantages, through very modest parametrization as shown by the success of the dft / cis and dft / rocis methods. efforts along these lines are underway in our research group. with the results of ras ligand covalency, we last turn to quantitatively applying this relationship to other ferric systems. by applying this same methodology to three previously reported high spin fe(iii) compounds, we see the obtained relationship is generally applicable (figure 10) ; this result is qualitatively the same if experimental intensity - weighted average energies are used instead of fit peak positions (supporting information figure s7). of course, the covalency numbers obtained from an analysis of orbital coefficients are artificial and will vary depending on the level of theory used for the calculations ; hence, calibration to an accepted value is necessary to establish a quantitative correlation between the k mainline splitting and covalency. as many possibilities exist for reference values, all of which will give slightly different correlations, we leave this exercise to the reader and simply demonstrate that, according to the measure of covalency employed here, the observed trend is applicable across a broad range of compounds. correlation between experimental k mainline splitting and qro - derived covalency values for an expanded series of ferric compounds. lastly, it is worthwhile to calibrate the size of the effect shown in figure 10 with the expected changes associated with varying d count. by using the qro calculated t and e values (table 3) in the d ligand field expression from table 2, we can observe the effect of covalency compared directly to that from d count (figure 11). the inclusion of this covalent reduction reduces the splitting to what would be expected for a d ion (for highly covalent complexes) or d ion (for the highly ionic fluoride). further, for a d electron configuration, this is also complicated by the fact that d and d configurations should have identical splitting in the free ion limit. these results indicate that extreme caution must be exercised when attempting to relate an absolute k mainline splitting to a given d configuration or metal spin state. correlations of this type will certainly be possible but will only be valid over a very restricted range of possible chemical variations. effect of covalency on the k mainlines is demonstrated alongside the effect of d count. the four compounds from the high spin ferric series reported here are represented by white circles. in this work, we have investigated in detail the multiplets that contribute to the initial, intermediate, and final states of k mainline xes spectra and derived general equations governing the energy splitting between the k1,3 and k mainline features for any d count metal. these equations reinforce previous work demonstrating that the splitting of the mainline is primarily due to the 3p-3d exchange integrals (4g1 + 42g3), with high spin states giving rise to relatively intense k features and large energy separations between the two peaks. this free - ion picture of the mainline shape being governed by the number of unpaired electrons on the metal adequately rationalized early observations and is the level of interpretation that has dominated the literature ever since. although this correlation is often true, it breaks down when applied to complexes that are not highly ionic. indeed, from the data presented here especially febr3 and fe(sar)4, vida supra and elsewhere (nibr2, rubredoxin, nitrogenase mofe protein), it is clear that in many cases the mainlines deviate substantially from what would be expected based upon the known nominal metal oxidation and spin states. in these cases, applying the standard interpretation of k mainlines will lead to incorrect conclusions about the metal electronic structure. from the equations in table 2, we have demonstrated that the value of e is dominated by the effective value of the quantity 4g1 + 42g3, which depends upon a number of factors, including the metal identity and oxidation state, with higher values found for late metals and higher oxidation states. further, it is also modified by the stevens orbital reduction factors that account for symmetry restricted metal ligand covalency, reducing the predicted mainline splittings from what would be expected for a free ion (figure 11). of these factors, only the covalent dilutions are subject to appreciable variation for compounds with a given metal and oxidation state, implicating covalency as the source of the observed modulations of the mainline splittings. using the series of high - spin ferric compounds as an example, we have proposed a ras ci based protocol for calculating k mainlines, which eliminates much of the empiricism associated with the established crystal field multiplet - based approach. these relatively simple quantum chemical calculations correlate very well with the experimental data and reproduce all important effects. in terms of a very favorable ratio of cost to performance, we chose to take advantage of the fact that density functional theory typically delivers molecular orbitals that have realistic metal ligand mixing ratios (e.g., covalent dilutions) and use these orbitals in the ras ci calculations, which properly account for all multiplet and spin orbit effects. the missing dynamic correlation contributions in these calculations lead to calculated splittings that are overestimated with respect to experiment, though the correlation between theory and experiment is excellent. additionally, because the qros used for these calculations have reasonable covalent dilutions, they were also used to demonstrate that a quantitative relationship between k mainline splitting and covalency may be obtained. doing so provides an intuitive picture that nicely rationalizes the observed effects in chemically meaningful terms, though it should be kept in mind that orbitals are not physical observables and that one is arguing in terms of static one - electron pictures that become invalid in the case of dominant multiplet effects or strong electronic relaxation. furthermore, when approaching such estimates of covalency, it must be clearly understood that they are based on a specific physical model of the electronic structure of a given complex (single determinant mo theory, typically spin - restricted) together with a series of approximations that allow for a relationship of that electronic structure to actual spectroscopic observables (typical assumptions include frozen orbitals, various one - center approximations, and the neglect of metal ligand overlap). clearly, many of these assumptions are severe and it should not come as a surprise when estimates obtained by different techniques differ. importantly, the compositions of individual orbitals do not qualify as physical observables and hence any such procedure is not physically rigorous. it is, however, difficult to deny the usefulness of the underlying intuitively appealing pictures that greatly help to rationalize trends among series of related molecules. it is useful at this point to compare the determination of covalency from k mainlines to existing experimental methods (e.g., epr superhyperfine couplings, metal l - edges, and ligand k - edges). analysis of superhyperfine couplings in epr spectra offers a measure of the spin delocalized onto the ligands in the electronic ground state, thus providing a way to quantify the mixing of metal and ligand orbitals. because the superhyperfine couplings are dependent on the identity of the ligands present, these measurements provide ligand - specific covalency values. obvious requirements include complexes that are epr active and ligands with nuclear moments, restricting the range of compounds for which this type of analysis may be performed. metal l - edge and ligand k - edge xas monitor transitions from core orbitals (metal 2p or ligand 1s, respectively) to unoccupied valence orbitals with appreciable metal 3d or ligand np character. both of these techniques rely upon the measurement of accurate normalized intensities in order to infer covalency. as the observed intensity is a product of the absorber character in the acceptor mo (i.e.,) and the radial transition moment dipole integral, these methods rely on the proper factorization of these quantities. hence, the abstraction of a covalency number from metal or ligand xas is inherently indirect. it is also important to note that the intrinsic transition dipole moments for a given metal or ligand are known to vary significantly with oxidation state, effective charge, and nature of the donor / acceptor interaction, thus introducing significant uncertainties. it should be noted that theoretical approaches can greatly aid in the determination of these values. however, this requires accurate intensities, which can present a significant experimental challenge and in cases of nearby / overlapping edges becomes prohibitive. thus, though covalency measured by all of these methods has provided great insights into a number of systems, additional methodologies are clearly desirable. in contrast, the determination of covalencies from k mainlines relies upon the covalent reduction of the 3p3d exchange integral as manifested in the energy splitting between the k and k1,3 features. in a simple picture this is perhaps similar to the measurement of metal l - edges in that the metal character that is lost to the ligands is being probed ; hence, k mainlines also provide a measure of the average metal ligand covalency. in contrast, though, the analysis of k mainlines does not reference oscillator strengths, so the absolute intensities of the xes features are not important and only accurate relative energies are needed. after accounting for all uncertainties in data acquisition and processing, the splitting of the mainline can be determined to within 0.5 ev for a typical high spin complex, which corresponds to a covalency determination to within a few percent using the calibration in figure 10, a level of precision that compares favorably to existing methods. in addition to having a reasonable sensitivity to covalency relative to other techniques, k xes also offers numerous experimental benefits. first, it can be applied to any transition metal and is not dependent on the presence of magnetic coupling between the metal and ligands. as a hard x - ray spectroscopy it can readily be applied to a wide range of sample environments including measurements on dilute solutions and in extreme pressure cells that may be inaccessible with other techniques. this advantage is particularly clear in comparison to first row transition metal l - edges and ligand k - edges (of c, n, o), which require highly damaging low energy x - rays and uhv conditions that limit in situ applications. ligand covalency with promise to shed light onto many systems that would otherwise be experimentally inaccessible. lastly, as can clearly be seen in figure 11, the reduction in mainline splitting due to covalency can be as large as the reduction expected from a change in formal d - count. thus, any attempt to relate a given mainline splitting to a specific oxidation / spin state must be undertaken with extreme caution. cases certainly exist where such an analysis is possible comparisons between metal oxides of differing oxidation states, for example though these situations are likely the exception rather than the rule. rather than reduce the utility of k mainlines, however, the theoretical developments contained herein expand the information content that may be extracted from k mainlines and further the ability to quantitatively interpret these data. in this work, we have demonstrated that the mainlines of metal k xes spectra are a sensitive probe of the covalency of metal complexes in addition to carrying spin - state information. the effect of covalency was established and explored using both crystal field multiplet and straightforward ras ci calculations. ci approach, in combination with a detailed analysis of the multiplets that contribute to k mainline in general d configurations, yielded new insights into the chemical factors governing mainlines. it is now possible to obtain an experimental estimate of covalency analogous to that provided by epr, metal l - edges, or ligand k - edges from k mainlines. finally, these results indicate that caution must be used when attempting to obtain spin state information from k mainlines due to the competing and possibly overwhelming effect that covalency has on mainline shape and energy. | the mainline feature in metal k x - ray emission spectroscopy (xes) has long been recognized as an experimental marker for the spin state of the metal center. however, even within a series of metal compounds with the same nominal oxidation and spin state, significant changes are observed that can not be explained on the basis of overall spin. in this work, the origin of these effects is explored, both experimentally and theoretically, in order to develop the chemical information content of k mainline xes. ligand field expressions are derived that describe the behavior of k mainlines for first row transition metals with any dn count, allowing for a detailed analysis of the factors governing mainline shape. further, due to limitations associated with existing computational approaches, we have developed a new methodology for calculating k mainlines using restricted active space configuration interaction (ras ci) calculations. this approach eliminates the need for empirical parameters and provides a powerful tool for investigating the effects that chemical environment exerts on the mainline spectra. on the basis of a detailed analysis of the intermediate and final states involved in these transitions, we confirm the known sensitivity of k mainlines to metal spin state via the 3p3d exchange coupling. further, a quantitative relationship between the splitting of the k mainline features and the metal ligand covalency is established. thus, this study furthers the quantitative electronic structural information that can be extracted from k mainline spectroscopy. |
it was an observational, prospective, cross - sectional study conducted in patients with bronchial asthma. the study was carried out from november 2013 to march 2015 in the department of pulmonary medicine in a tertiary care hospital. a written informed consent was taken, and patients were enrolled in the study as per the inclusion criteria. patients presenting with acute exacerbation of bronchial asthma with peak expiratory flow (pef) at randomization 75% of their known best (in the past 12 months) or in the absence of this information, 75% of their predicted pef (predicted normal)patients of both genders in the age group of 18 years and abovepatients on inhaled corticosteroids and using metered dose inhalers (mdis) for a period of at least 3 months (in addition to other drugs). patients presenting with acute exacerbation of bronchial asthma with peak expiratory flow (pef) at randomization 75% of their known best (in the past 12 months) or in the absence of this information, 75% of their predicted pef (predicted normal) patients of both genders in the age group of 18 years and above patients on inhaled corticosteroids and using metered dose inhalers (mdis) for a period of at least 3 months (in addition to other drugs). after enrollment, demographic data of patients, drug history, and information regarding inhaler device were recorded. five of them included symptoms present at night, early in the morning, history of respiratory difficulty, wheezing, and limitation of activities. questions on the frequency of usage of rescue therapy and on forced expiratory volume at 1 s (fev1) completed the questionnaire. there were 15 questions in the questionnaire divided into four domains, which included assessment of activity limitation, symptoms of asthma, exacerbation due to environmental stimuli, and emotional disturbances due to the disease. the responses in asthma control and qol were assessed for the questions as per the symptoms in the preceding 3 months. the data were analyzed using spss software version 16 (spss south asia pvt. ltd. pearson 's chi - square test was used to test the association between demographic and clinical characteristics across the variables regarding asthma inhaler technique. multiple logistic models were used to identify the independent risk factors that were associated with the improper use of inhaler devices. the odds ratio with 95% confidence intervals patients presenting with acute exacerbation of bronchial asthma with peak expiratory flow (pef) at randomization 75% of their known best (in the past 12 months) or in the absence of this information, 75% of their predicted pef (predicted normal)patients of both genders in the age group of 18 years and abovepatients on inhaled corticosteroids and using metered dose inhalers (mdis) for a period of at least 3 months (in addition to other drugs). patients presenting with acute exacerbation of bronchial asthma with peak expiratory flow (pef) at randomization 75% of their known best (in the past 12 months) or in the absence of this information, 75% of their predicted pef (predicted normal) patients of both genders in the age group of 18 years and above patients on inhaled corticosteroids and using metered dose inhalers (mdis) for a period of at least 3 months (in addition to other drugs). after enrollment, demographic data of patients, drug history, and information regarding inhaler device were recorded. the patients were asked to use inhaler in the presence of an investigator. as per the checklist five of them included symptoms present at night, early in the morning, history of respiratory difficulty, wheezing, and limitation of activities. questions on the frequency of usage of rescue therapy and on forced expiratory volume at 1 s (fev1) completed the questionnaire. there were 15 questions in the questionnaire divided into four domains, which included assessment of activity limitation, symptoms of asthma, exacerbation due to environmental stimuli, and emotional disturbances due to the disease. the responses in asthma control and qol were assessed for the questions as per the symptoms in the preceding 3 months. the data were analyzed using spss software version 16 (spss south asia pvt. ltd., bengaluru, karnataka, india). pearson 's chi - square test was used to test the association between demographic and clinical characteristics across the variables regarding asthma inhaler technique. multiple logistic models were used to identify the independent risk factors that were associated with the improper use of inhaler devices. the odds ratio with 95% confidence intervals the mean age of patients was 51.54 14.6 years and 57.3% of them were females. nearly 22.7% of the patients were uneducated and 17.9% and 17% of the patients were smokers and alcoholics, respectively. the drugs prescribed and comorbidities seen in the study population are shown in table 2. clinical characteristics of the study patients drugs prescribed and comorbidities in the study population the median duration of inhaler use was 24 months. of the total 330 patients using inhaler, 190 (57.5%) were using a spacer. patients who had received education on inhaler technique constituted 87% of the total study population and 80.3% were following the prescription of mdi as directed by their physician. improper technique for the use of inhaler was defined as < 75% of the correct steps done for each device. a total of 209 patients (63.4%) performed the steps properly. from the 12 steps for the assessment of mdi with spacer, step 11 was incorrectly performed by 72.6% of the individuals, that is, breathing normally for at least 3060 s postinhaler use. this was followed by step 4 (44.2%) and step 5 (43.2%) that included breathing out fully before inhalation and breathing out fully away from spacer, respectively, as shown in figure 1. on assessing patients on mdi without spacer, step 11 and step 4 were incorrectly performed by majority of the patients. assessment of steps of using metered dose inhaler with spacer based on the scoring system, the number of patients was categorized into poorly controlled, partially controlled, and well - controlled asthma as shown in figure 2. asthma control questionnaire score of the study patients (n = 330) all patients with well - controlled asthma had proper technique of inhalation. patients with < 70% of predicted fev1 performed the steps improperly when compared to patients with predicted fev1 70% as shown in table 3. relationship of inhaler technique with asthma control and predicted forced expiratory volume percentage in 1 s regarding qol, scores below 4 indicate a poor qol. all domains of qol were above 70% with the highest score in emotional domain and the lowest in environmental domain. patients with improper technique of inhaler device had lower qol scores with respect to all the domains in comparison with those having proper technique (p < 0.001). the improper use of asthma inhaler devices was observed in 121 (36.6%) patients. patients with age 40 years had a higher risk of improper inhalation technique compared to patients < 40 years of age (p = 0.041). gender, education, occupation, comorbidities, and other habits did not show any significant association with improper inhaler technique. patients who visited the outpatient department (opd) and those who were admitted in hospital in the past 3 months had a higher risk of improper technique when compared to patients with no opd visits or hospital admissions (p = 0.03). patients who had received education on inhaler use (p = 0.001) and who were following regular prescription of inhaler use (p = 0.04) performed the steps properly in comparison to those who did not receive education and were not following prescription, respectively. after controlling other risk factors, multivariable adjustment revealed that patients who lack education on inhaler device (p < 0.05) were more likely to use the asthma device improperly compared to those who received education. in addition, patients with poorly controlled asthma (p < 0.001) had a faulty technique and did not use the inhaler properly. improper technique for the use of inhaler was defined as < 75% of the correct steps done for each device. a total of 209 patients (63.4%) performed the steps properly. from the 12 steps for the assessment of mdi with spacer, step 11 was incorrectly performed by 72.6% of the individuals, that is, breathing normally for at least 3060 s postinhaler use. this was followed by step 4 (44.2%) and step 5 (43.2%) that included breathing out fully before inhalation and breathing out fully away from spacer, respectively, as shown in figure 1. on assessing patients on mdi without spacer, step 11 and step 4 based on the scoring system, the number of patients was categorized into poorly controlled, partially controlled, and well - controlled asthma as shown in figure 2. asthma control questionnaire score of the study patients (n = 330) all patients with well - controlled asthma had proper technique of inhalation. patients with < 70% of predicted fev1 performed the steps improperly when compared to patients with predicted fev1 70% as shown in table 3. relationship of inhaler technique with asthma control and predicted forced expiratory volume percentage in 1 s regarding qol, scores below 4 indicate a poor qol. all domains of qol were above 70% with the highest score in emotional domain and the lowest in environmental domain. patients with improper technique of inhaler device had lower qol scores with respect to all the domains in comparison with those having proper technique (p < 0.001). the improper use of asthma inhaler devices was observed in 121 (36.6%) patients. patients with age 40 years had a higher risk of improper inhalation technique compared to patients < 40 years of age (p = 0.041). gender, education, occupation, comorbidities, and other habits did not show any significant association with improper inhaler technique. patients who visited the outpatient department (opd) and those who were admitted in hospital in the past 3 months had a higher risk of improper technique when compared to patients with no opd visits or hospital admissions (p = 0.03). patients who had received education on inhaler use (p = 0.001) and who were following regular prescription of inhaler use (p = 0.04) performed the steps properly in comparison to those who did not receive education and were not following prescription, respectively. after controlling other risk factors, multivariable adjustment revealed that patients who lack education on inhaler device (p < 0.05) were more likely to use the asthma device improperly compared to those who received education. in addition, patients with poorly controlled asthma (p < 0.001) had a faulty technique and did not use the inhaler properly. the anti - asthmatic medications are administered through inhaler devices and their effectiveness in clinical practice can be affected by many factors. the amount of drug reaching the target organ is important for improved efficacy in the treatment of asthma. this in turn depends on the type of inhaler device, technique of inhalation, and patient 's compliance to inhalers. the common causes of uncontrolled asthma include incorrect inhaler technique in up to 80% of the patients and at least 50% of the patients show poor adherence. proper technique of inhaler use improves disease outcome and qol. of the total 330 patients, there was a female preponderance and most of the patients had been diagnosed with asthma for more than 1 year. all the patients were prescribed steroids, and a large number of patients (83%) were prescribed beta-2 agonists followed by antihistamines. allergic rhinitis, hypertension, and diabetes were the common comorbidities seen in the patients. various studies reveal that 8.6%, 14%, and 16.7% of the patients were using mdi devices with spacers. after two sessions of training, 48.5% of the patients failed to demonstrate the correct technique of inhaler use and therefore it becomes obligatory to improve the standard of care in patients with asthma. assessment of individual steps in inhaler technique revealed that 72.6% of the patients on mdi with spacer and 67.1% of the patients on mdi without spacer did not perform the step of breathing normally for at least 3060 s postinhaler use. this was the most common step performed incorrectly in the study where a gap provided between the two actuations required the patients to breathe normally so as to prevent oropharyngeal deposition and allow the drug particles to reach the lower respiratory tract. this common error of not waiting for at least 30 s between inhalations has been reported by other authors. in addition, it is considered as one among the seven essential steps of mdi technique. hence, a need to emphasize on this step for proper use of inhaler device is essential. another step performed incorrectly by patients was breathing out fully before inserting the mouth piece or breathing out fully away from the spacer in patients on mdi with spacer. in patients using mdi with spacer, the step of breathing out fully was incorrectly performed by 77% of the participants and breathing out away from the device was incorrectly performed in 83% of the participants. about 50% of the patients do not perform the step of proper exhalation before inhalation. lack of proper exhalation prior to inhalation was one among the three most common errors in 25% of the inhaler users. holding breathe for at least 10 s postinhalation, i.e., step 9 was done incorrectly in 42.6% of the patients. a study on patients using mdi without spacers found the maximum number of patients who exhaled incorrectly (65.88%) followed by breath holding (45.88%). in another study, authors put forth that the major incorrect steps were not exhaling properly before inhalation (62%), not holding breathe correctly (57%), and not correctly shaking the inhaler (55%) for pressurized mdi. less than half (41.3%) of the patients adhered to the step of breath holding for 10 s. an interesting finding from this study and previous studies suggests that the breath holding for 10 s is the step performed incorrectly or missed by most of the patients using mdi without spacer. this can be overcome by using mdi with spacer which is easier to use, does not need the breath - holding period, and is not affected by hand breath incoordination. according to the gina guidelines, the goal of asthma management is to achieve clinical control. an important finding of this study was that patients with poorly controlled asthma (92.6%) compared to those with partially controlled asthma (7.4%) had a significant association with technique and are more likely to use asthma device improperly (p < 0.0001). few other studies had similar findings where inhaler misuse was associated with an increased risk of poor asthma control. inhaler technique was also compared to ventilation capacity to know whether low fev1% of predicted scores was associated with difficulty in using inhaler devices. on analysis, 86.8% of the patients with low fev1% of predicted scores were found to perform the steps improperly. low fev1% of predicted scores indicates obstructive defect and thereby poor disease control, leading to difficulty in performing the right technique. however, in another study, low fev1 was not associated with faulty inhaler technique. studies assessing asthma control have found at least 50% of patients with poorly controlled asthma. another cross - sectional study constituted 24.6% of patients with controlled asthma, 46.3% with partially controlled asthma, and 29.1% with uncontrolled asthma. asthma qol measured by aqlq correlates well with emotional and environmental aspects of the disease along with symptom and activity limitations. mean aqlq score was 5.6 1.3 and cutoff score was set at 5.4 in comparison to the present study where the mean aqlq score was 3.8 0.57 and cutoff value was at 4. asthma adversely affects physical, psychological, and social domains of qol. in the present study, environmental domain had a lower score of 71.93% followed by symptom domain (74.46%) and activity domain (77.04%). a study showed an improvement in all the domains of qol, except environmental exposure. exposure to various stimuli such as cigarette smoke, dust, strong smells, air pollution, or weather changes affects environmental domain. though studies claim that stress and negative emotions exacerbate the symptoms of asthma, it was not seen in the present study due to proper counseling and reassurance by the physicians, and most probably, it can also be due to patients sharing their experiences with fellow patients suffering from the same disease. patients in the age group 40 years (81.8%) performed the step improperly when compared to patients in the age group of < 40 years (18.2%). similar findings were seen in a study, where maximum number of patients with incorrect technique belonged to the age group of 4160 years. education and regular reinforcement in all patients and more so in elderly is required to ensure a precise technique. a study comparing inhaler technique in adult and pediatric patients with asthmatics suggested that the former should be given supplemental instructions so that they take their medications properly. uncontrolled asthma remains a frequent cause of emergency department (ed) visits and hospital admissions. the improper use of inhaler can cause uncontrolled asthma and frequent ed visit. on analysis, a significant association (p = 0.031) was seen, where of the total patients performing technique correctly, 65% had no hospital visits, 20.6% visited opd, and only 14.4% had admission in the past 3 months. patients using inhaler devices inappropriately were at greater odds of visiting the ed in hospitals, compared to those with appropriate technique. a large body of evidence suggests that inhaler technique can be improved by education from a health professional or other person trained in correct technique. in our study, patients (91.9%) who received education were at a lower risk of making errors (p = 0.001). studies suggest that as many as 25% of the patients with asthma had never received verbal inhaler technique instructions. only an estimated 11% of the patients receive follow - up assessment and education on their inhaler technique. step - by - step instructions with repeated demonstrations of inhaler use and assessment of participant comprehension, i.e., teach - to - goal (ttg) strategy can be a beneficial step toward this. a hospital - based intervention through ttg results in fewer health - related events in asthma. patients following regular prescriptions of mdi performed the technique perfectly in the present study and there was a statistically significant (p = 0.040) difference as compared to those not following the prescriptions. in a study where 233 patients were compliant with treatment, technical error was seen only in 15.8% of the patients. multiple logistic regression analysis of variables that was significantly associated with improper inhaler technique revealed poorly controlled asthma and lack of education of inhaler device as significant predictors of improper inhalation technique (p < 0.001). another recent study using regression model states that uncontrolled asthma and lack of education on asthma are associated with improper technique. thus, the present study puts forward the various factors associated with improper inhaler use which can reduce the delivery of required dose to the lungs. this in turn can attenuate the effectiveness of medication and can be a cause of therapeutic failure. one of the limitations of the study was that as it is a cross - sectional study, temporal sequence between the patients ' performance of the inhaler technique and the level of asthma control could not be established. furthermore, as this study has been done in a single tertiary care hospital where patients are educated about the correct technique, it does not represent the inhaler practice at the national level. hence, a multicentric study to assess inhaler technique and different aspects of asthma management is the requisite. breathing normally between two actuations, breathing out fully before inserting the mouth piece, or breathing out fully away from the spacer in patients on mdi with spacer were the erroneous steps in the use of inhaler. poor control of asthma and insufficient information about inhaler are strong predictors for faulty technique of inhaler use. improper technique in higher age groups makes it obligatory to stress on proper inhaler technique in this subset of patients. | introduction : there is a need to assess erroneous steps in the use of inhaler devices in people who have asthma. the objectives of this study were to assess the inhaler technique in patients who have asthma, the factors affecting improper technique, and the association of inhaler use with asthma control, hospital visits, and quality of life (qol) of patients who have asthma.methods:it was an observational, prospective, cross - sectional study conducted on patients with bronchial asthma. patients were enrolled in the study ; their history was recorded and they were asked to use inhaler in the presence of an investigator and the technique was scored. asthma control and qol of patients were assessed using asthma control questionnaire and mini asthma qol questionnaire.results:a total of 330 patients completed the study. nearly 36.6% of the patients performed the steps incorrectly. breathing normally for 3060 min postinhaler use was the most common step done incorrectly. patients with poorly controlled asthma (p < 0.001) and those with predicted forced expiratory volume at 1 s < 70% performed the steps erroneously (p < 0.001).conclusion : all patients, particularly those above 40 years, should be given proper instructions regarding inhaler use to obtain therapeutic advantage. |
eukaryotic gene expression is controlled at multiple levels, and splicing of mrna is an important regulatory step in the production of functional proteins. during mrna splicing, portions of the rna, introns, are removed by the spliceosome complex, and the remaining protein - coding rnas, exons, are joined together. alternative splicing, in which different combinations of exons are included in the final protein - coding mrna, is responsible for the diversity of protein - coding mrnas that can be derived from a single open reading frame [14 ]. the location of these splice junctions is generally defined by a g - t dna sequence signal at the splice donor and an a - g dna sequence signal at the splice acceptor [1, 5 ]. it is likely that more subtle sequence features in addition to the local sequence context of these splice sequences play a role in both constitutive splicing and alternative splicing. transcription and splicing appear to be mechanistically coupled [614 ], and the precise rules governing recognition and regulation of constitutive and alternative splicing are poorly understood. several recent papers have focused on the role that the packaging of the template dna into chromatin plays in the splicing process [6, 1523 ]. the packaging of eukaryotic dna into chromatin is expected to affect all dna templated processes. the fundamental subunit of chromatin is the nucleosome, 150 base pairs (bp) of dna wrapped around a histone octamer. the position and density of nucleosomes play key regulatory roles and are controlled both by chromatin regulatory complexes and by features intrinsic to the dna sequence [2426 ]. there is limited information on how these two determinants of nucleosome occupancy coordinate to regulate responses, such as transcription in mammalian cells. nucleosome forming and nucleosome inhibitory properties were derived from first principles more than three decades ago. more recently, maps of nucleosome distribution have enabled the development of computational models that use dna sequence features to predict the nucleosome forming or inhibitory potential of dna. position specific scoring matrices were developed by aligning strong nucleosome forming sequences and identifying the positions of enriched dinucleotide composition. machine learning tools [24, 28 ], such as support vector machines (svms), have been used to predict the intrinsic nucleosome occupancy likelihood (nol) for any dna sequence. the support vector machine used in these studies is discriminative, rather than generative, and uses dna sequences representing the strongest and the weakest affinity for forming nucleosomes. in these studies, dna sequences protected from mnase digestion genomic loci with the highest and the lowest nucleosome occupancy signals were used to train an svm. the resulting algorithm can be used to classify the nucleosome forming potential of any genomic sequence. a comparative assessment of available nucleosome occupancy prediction algorithms revealed that the svm trained on human chromatin worked well on related species with relatively large, complex genomes. these seminal discoveries affirmed an important role for intrinsic dna sequence features influencing chromatin organization and revealed the utility of combined genomics and computational approaches for chromatin research. these exonic nucleosomes have been proposed to act as speed bumps that allow for cotranscriptional splicing [32, 33 ]. additionally, posttranslational modifications of the nucleosomal histones at these exonic boundaries have been shown to affect splice site usage [18, 19, 34 ]. this exonic nucleosomal occupancy is conserved throughout metazoan evolution, indicating a role for genomic sequence in this process. we have previously described a computational model of nucleosomal occupancy trained on dna sequence content. a large fraction of nucleosome positions can be accurately predicted based on dna sequence, indicating a significant dna sequence component to nucleosome positioning [25, 26 ]. nucleosome positioning signals at exon boundaries are conserved throughout evolution, and we reasoned that the analysis of predicted dna - encoded nucleosomal occupancy would improve characterization of regulatory features associated with intron - exon boundaries. we used predictions of nucleosome occupancy to characterize intron / exon boundaries in five metazoan species and identified a cryptic sequence - based dna property that is specific to a subset of fundamental metabolic genes. our results suggest that, by defining regulated positions for nucleosomes, dna features other than the consensus splice site sequence play a role in splicing. the dna sequences for the current builds of all organisms (human, hg19 ; rat, rn4 ; zebrafish, danrer7 ; fly, dm3 ; worm, ce10 ; yeast, saccer3) in this analysis were downloaded from the ucsc genome bioinformatics website (http://hgdownload.cse.ucsc.edu/downloads.html). annotations for gene structure (transcription start site, strand, and boundaries between introns and exons) were acquired from the refgene tables of the ucsc genome browser public mysql database with the exception of yeast for which the sgdgene table was used. nucleosome occupancy likelihood (nol) scores were generated using the support vector machine (svm) model derived from the ozsolak a375 dataset (described in). the svm scores a 50 bp window of dna sequence and uses a 1 bp step sliding window for sequences longer than 50 bp. the resulting score indicates the likelihood that the associated 50-mer is nucleosome forming (positive value) or nucleosome inhibitory (negative value). gene ontology analysis was completed with the gorilla software using genes of interest in the target set and the list of all genes in the associated genome as the background set. for calculations of significance across genomic statistics in enriched ontological categories, outliers were defined as those values less than the first quartile minus the interquartile range (iqr) or greater than the third quartile plus the iqr. p values were then calculated using a random sample of the same size from the whole genome and all values from the set of interest to perform a two sample t - test with a p value of < 0.05 being considered significant. we were first interested in discovering if there was any intrinsic nucleosome occupancy information in the regions flanking intron - exon boundaries. we reasoned that if chromatin structure plays a role in cotranscriptional splicing, then a robust location to store that chromatin structural information would be within the dna sequence itself. to this end, we retrieved all intron - exon boundaries from the refseq annotation of the human genome. we calculated nucleosome occupancy likelihood (nol) scores for sequences spanning 1000 bp centered on intron - exon boundaries. nol scores were calculated for each 50-mer and the resulting value was assigned to the center nucleotide position. nol scores were plotted to reveal overarching trends of predicted nucleosome positions at intron - exon boundaries. consistent with previous observations, we detected characteristic nucleosome positions at these boundaries (figure 1(a)). sequence analysis of the regions surrounding these boundaries showed clear consensus splice donor and splice acceptor sequences (figure 1(a)). closer inspection of the plots shown in figure 1(a) revealed a sharp dip in average nol scores for the introns both upstream and downstream of exons. this sharp dip, identified by the nol, could only result from a set of sequence features that reduce the nucleosome forming potential at that location. we were next interested in understanding the characteristics of the sequences that contributed to this sharp dip. we hypothesized that the dip in the nol scores may represent a functional dna - encoded chromatin - regulatory structural element, as this is what the nol plots measure. to investigate this possibility, we aligned the entire dataset to this putative regulatory feature by centering each region on the minimum found in the 100 base pairs centered on the boundary (figure 1(b)). the percentage representation of each base at all positions was calculated and graphically represented to identify a previously undetected consensus sequence defining intron - exon boundaries. the sequence composition of these shifted subsets reflected equal representation of all four dna bases and therefore did not reveal any clear consensus sequence feature (figure 1(b), sequence diagram). this result suggests that a more cryptic combination of sequence features reflecting some physical property of dna may be defining this low scoring element. different biophysical properties emerge with different dna sequence combinations (e.g., dna wedge angles). we were, therefore, interested in determining if the dips upstream and downstream conferred by the dna sequence of the exon might identify a cryptic characteristic of intron boundary architecture. we calculated the location of the minimum nol score for each boundary window and then represented these data as a histogram of distance of the minima from the intron - exon boundary (figure 2(a)). we observed a striking overrepresentation 26 nucleotides (nt) from the annotated intron - exon boundary, upstream of the exon (u 26). we also found a similar feature + 26 nt from the exon - intron boundary, downstream of the exon (d + 26). the discovery of the u 26 and d + 26 features prompted us to investigate how many and what types of genes include these features. in order to understand the numbers and types of genes associated with the u 26 and d + 26 feature, we selected the exons represented in each of these groups, u 26 and d + 26, for further analysis. there are 9578 genes represented in the u 26 group and 7360 genes represented in the d + 26 group, representing 24.1% and 18.5% of all open reading frames tested, respectively. we next wanted to know if the u 26 and d + 26 features were found in the same sets or different sets of genes. 3369 genes, or 19%, overlap between the u 26 and d + 26 groups (figure 2(b)). as positioned nucleosomes flanking exons are phylogenetically conserved, we were next interested in determining whether the prominent u 26 and the d + 26 signature are conserved in other metazoan species. conservation of these features would suggest an important role for the u 26 and the d + 26 causing it to be maintained by evolution. we identified the nucleotide position of the minimum at the boundary between intron and exon for rat, zebrafish, fly, and worm. a conspicuous u 26 and d + 26 signature exists for all of these species (figures 2(c), 2(e), 2(g), and 2(i)). as with the human example this result led us to hypothesize that the signature was associated with a particular gene set that is conserved across each of the species tested. we next wanted to identify the feature that is present in groups of genes with related function. in order to test whether the u 26 or the d + 26 signatures identified groups of genes that share a common function, we searched for ontological enrichment. the u 26 and the d + 26 signatures both showed enrichment for overlapping groups of gene function (table 1). these groups include atpase activity, atp binding, helicase activity, and motor activity. with little exception, these enrichments persist throughout all metazoan species tested (table 1). these results led us to hypothesize that these groups of genes contain genomic characteristics that differ from the set of all genes. in order to test whether genes found in the enriched functional categories containing the u 26 and the d + 26 signature had genomic characteristics that varied significantly from the rest of the genome, we compared exon size, intron size, and number of exons for each function category to the same values calculated for the genome as a whole (figure 3). the exon sizes of genes included in each of the categories did not differ substantially from the exon sizes encoded by each genome (supplementary figure 1, available online at http://dx.doi.org/10.1155/2015/167578). intron sizes of the genes in the ontological function categories were significantly shorter when compared to the respective whole genomes with few exceptions (rat : motor activity, zebrafish : atp binding, and worm : atp binding). across all organisms, with the exception of atpase activity and helicase activity in rat, all ontological categories across all organisms exhibited significantly higher numbers of exons per gene. for example, the human genome has a median of seven exons per gene while the subset of human motor activity genes has approximately five times the number of exons with a median of 35 exons per gene. as our experiments, to this point, had been purely based on in silico experiments and genome sequence data, we wanted to see how our results compared to published in vivo data. we next were most interested in testing if the u 26 or the d + 26 signatures are observed in vivo. we used the nucleosome maps generated by the encode consortium for the two cell lines, gm12878 and k562. we retrieved all nucleosome measurements for intron - exon boundaries for each of the two cell lines from http://genome.ucsc.edu/ and identified the location of the minimum within the central 100 base pairs surrounding intron - exon boundaries as described previously using the nol scores. approximately 2.5% of genes contained intron - exon boundaries exhibiting the d 26 and u + 26 characteristic (table 2). we wanted to know whether this in vivo signature was associated with the shared common function consistent with those identified from the nol predictions. three of the four categories identified by the nol score signal, atp binding, atpase activity, and motor activity, were also significantly enriched in the in vivo data. these findings indicate that sequence - based nucleosome forming signals at the boundaries between introns and exons may play a role in the regulation of these genes. the upstream intron contains a region between the branchpoint sequence and the 3 splice site that is generally depleted of ag dinucleotides. this region is generally within 40 nucleotides of the ag splice site and encompasses the u 26 feature. we were interested in determining if the loci containing the u 26 feature were enriched or depleted for any dinucleotide occurrences relative to the rest of the genome. we calculated dinucleotide frequency for the ten dinucleotides for the loci containing the u 26 feature and compared that to the dinucleotide frequency for an equal number of other intron - exon boundaries in the genome (figure 4). we found that the intronic region off these genes is actually slightly more depleted of ag dinucleotides than other intron - exon boundaries. we were next interested in determining if the remaining dinucleotides showed differential intron and exon content. recent work has shown that differential g / c content plays a role in the intron exon definition and splice site selection [14, 38 ]. cc, cg, and gc dinucleotides were depleted in introns of the loci containing the u 26 feature. likewise, aa, at, and ta dinucleotides were enriched in the introns of the loci containing the u 26 feature compared to equivalent regions in the rest of the genome (figure 4). loci containing the u 26 feature have a lower overall intronic g / c content. the exonic region, however, shows the opposite trend with decreased a / t and increased g / c content. the cc dinucleotide feature most strongly defines this set of loci and is depleted from introns and enriched in exons. this would suggest that dinucleotide content plays a larger role in the definition of intron - exon boundary than was previously anticipated. we have identified a set of conserved genes sharing a common function using a nucleosome positioning signature. we have further characterized the set of genes as having increased numbers of exons while having average number and length of introns. thus, we have identified a set of conserved genes with common function and distinguishing features that suggest shared regulation. our observations and the classification of a particular subset of genes could not have been accomplished through alignment of nucleotide content. the nol scores point toward a physical property of dna related to the ability of a particular dna sequence to form a nucleosome. the organization and architecture of dna around the nucleosome may likely play a role in the mechanism of pre - mrna splicing. | the regulation of metazoan gene expression occurs in part by pre - mrna splicing into mature rnas. signals affecting the efficiency and specificity with which introns are removed have not been completely elucidated. splicing likely occurs cotranscriptionally, with chromatin structure playing a key regulatory role. we calculated dna encoded nucleosome occupancy likelihood (nol) scores at the boundaries between introns and exons across five metazoan species. we found that (i) nol scores reveal a sequence - based feature at the introns on both sides of the intron - exon boundary ; (ii) this feature is not part of any recognizable consensus sequence ; (iii) this feature is conserved throughout metazoa ; (iv) this feature is enriched in genes sharing similar functions : atpase activity, atp binding, helicase activity, and motor activity ; (v) genes with these functions exhibit different genomic characteristics ; (vi) in vivo nucleosome positioning data confirm ontological enrichment at this feature ; and (vii) genes with this feature exhibit unique dinucleotide distributions at the intron - exon boundary. the nol scores point toward a physical property of dna that may play a role in the mechanism of pre - mrna splicing. these results provide a foundation for identification of a new set of regulatory dna elements involved in splicing regulation. |
cysts related to the mullerian system usually occur in paraovarian (1) or paratubal regions (2). an eleven year old girl presented to the emergency department with a twenty four history of abdominal pain, fever and nausea. on presentation she was clinically dehydrated, pyrexial, and the lower abdomen was full with tenderness in the right iliac fossa and hypogastrium. inflammatory markers showed elevated c - reactive protein of 331 mg / ml (normal < 10 mg / ml) and a total white cell count of 29.7 x 10/l (range 5- 13.0) with a neutrophilia of 27.0 x 10/l (range : 1.5- 8.0). (fig 1&2) ultrasound image of longitudinal view of multicystic pelvic mass ultrasound image of coronal view of multicystic mass following resuscitation she underwent laparotomy on gram stain only a few white blood cells were seen and fluid culture did not identify an organism. the histopathological analysis revealed both cysts to be lined focally by ciliated and non - ciliated epithelium forming plicae. however, for the major part, the cysts were lined by acute inflammatory exudate. in the paratubal and paraovarian regions the majority of the cysts are of mullerian origin and can account for up to 76% of all the cysts (3). they are more common in adults and only occur rarely in adolescents (4%) (3). dilatation of these cysts could be anticipated after menarche, due to the secretory activity of the lining epithelium under the influence of the hormones (3). large cysts could cause symptoms such as nausea, pain and vomiting (4). some undergo torsion (5,6) with severe pain and other may also cause torsion of the fallopian tubes (2). in large adult series pelvic pain and a neoplastic potential has been identified (6,7) with the incidence varying from 1.69% to 2 %. no case of inflamed paratubal cysts appears in the english literature. we can only postulate that contributory factors in our case could have included low grade infection due to previous meningitis and vp shunts. it shows a wide range of findings (8), but the majority are simple cysts. multilocular cysts, as in our case are very rare accounting for only 4% of the cases (8). occasionally a normal ovary abutting a cyst gives a clue to the origin of these cysts but accurate pre op diagnosis remains rare. mri may give more detailed three dimensional information but essentially features are the same as an ultrasound. kishimota suggested that most paraovarian cysts were homogenous, near the ipsilateral round ligament and uterus (9). laparoscopy has been used successfully in paediatric practice to deal with large or complicated paraovarian cysts (4). the indication for open surgery is malignancy or suspicion of malignancy, dense adhesions or large cysts (6), as was the case in our patient. in our patient laparoscopic surgery would have been impossible due to the obliteration of the peritoneal cavity. in summary we report a very rare case of bilateral inflamed fimbrial cysts which were identified and excised during laparotomy for an acute abdomen. | paramesonephric duct remnants are an infrequent cause of abdominal symptoms in childhood. preoperative diagnosis is often difficult and diagnosis is usually made at surgery. we report a rare presentation of an acute abdomen in a child with bilateral inflamed fimbrial cysts. ultrasound revealed the presence of a multicystic lesion behind bladder. it was only at laparotomy the diagnosis of bilateral inflamed fimbrial cysts was establsihed. these were excised and the child made an uneventful post operative recovery. |
gastrointestinal (gi) autoimmune diseases are characterized by the uninhibited immune response in the gi tract. these diseases are characterized by an increased number of mononuclear cells in the intestinal epithelium, which can lead to destruction of intestinal crypts. some common gi autoimmune diseases are crohn 's disease and ulcerative colitis, commonly known as inflammatory bowel disease (ibd). recently published data show that the incidence of ibd is the highest in canada, and direct costs alone in the healthcare system amount to $ 1.8 billion. as such researchers have yet to answer what prompts the imbalance between tolerance and immune response leading to ibd. lately, ox40-ox40l interactions have come to light as potential treatment targets. ox40l is part of the tumor necrosis family, expressed on antigen presenting cells (apcs) (dendritic cells, macrophages, and b - cells), vascular endothelial cells, mast cells, natural killer cells, and on some t - cells. the interaction of ox40 and ox40l plays a crucial role in clonal expansion of antigen - specific t - cells. ox40l is also important in determining the amount of memory t - cells remaining after the immune response.[57 ] ox40l - ox40 interaction has been implicated in various inflammatory autoimmune diseases through many different animal models. however, it is important to recognize that ox40l - ox40 interactions are important for normal immune response as well. activation of ox40l produces anti - apoptotic proteins that aid in the survival and proliferation of t - memory cells. co - stimulatory signals delivered through ox40 have been implicated in selectively promoting the differentiation of t - helper (th)-2 cells, and help prolong antigen - specific proliferative response. ox40-deficient mice are shown to have impaired t - cell proliferation, even though the initial nave t - cell response remains unimpaired. these mice have also exhibited lower levels of anti - apoptotic protein : bcl-2 and bcl - xl. this implies that ox40 's role is mainly in sustaining the immune response, and that ox40 indeed has a non - pathological role as well. however, in normal mice, ox40-positive cells are observed only in lymphoid tissues, including peyer 's patches of the gut. in pathological condition, as demonstrated by mice with hapten - induced colitis or interleukin (il)-2 knockout mice with spontaneous colitis, ox40-positive cells are found in the lamina propria. when ox40 takes on a pathogenic role, it reactivates pre - existing self - reactive t - cells through constitutive activity.[51316 ] ox40 also starts to repress regulatory t - cells (t regs), which is responsible for toning down the immune response, contributing to the balance between tolerance and immunity. experiments conducted have shown that when anergic self - reactive t - cells are introduced in a host along with ox40 agonist, the self - reactive t - cells are activated from their anergic state, resulting in autoimmunity. this statement is further supported by the administration of anti - ox40l monoclonal antibody, which reduced the clinical and histopathological disease. in vivo treatment with anti - ox40l antibodies decreased t - cell inflltration in the colon and suppressed the production of primary inflammatory cytokines : interferon (ifn)-, il-2, and tumor necrosis factor (tnf)- in the lamina propria. combining this treatment with anti - tnf- antibodies further improved the therapeutic effect by further reducing ifn-, il-2, and tnf- production. in addition to animal models, many association studies have been done with ibd patients that have shown higher expression of ox40. ox40 signals are known to be transmitted through tnf receptor associated factors (trafs). traf2 and traf5 activate the nuclear factor kappa - b (nf-b) signalling pathway through ib kinase pathway though this has yet to be proven. recent studies have shown that traf2 is critical to ox40 signalling pathway, as traf2-deficient mice did not have the same degree of effector and memory t - cell generation as the wild - type. the role of traf3 and traf5 is still unclear and needs to be investigated further. ox40 also induces the expression of anti - apoptotic bcl-2 family members : bcl-2, bcl - xl, and bfl-1, which directly correlates with activity of nf-b1 as well. this gives strong proof that nf-b pathway as known to researcher now is involved in ox40 signalling pathway. overall, the bcl-2 family ensures that the cd4 t - cells do not undergo apoptosis after the immediate immune response, proven by studies showing that bcl-2-deficient mice failed to maintain high levels of cd4 t - cells 4 - 8 days after activation. ox40 signalling also maintains the active form of protein kinase b. protein kinase b leads to the expression of survivin, which is a member of the inhibitor of apoptosis (iap) family and regulates the division of t - cells. pi3k connects this pathway to several cyclin - dependant kinases, such as cyclin a, which are involved in cell cycle progression. through a pathway, which may be linked to pi3k, ox40 also increases the influx of ca in cd4 t - cells, leading to dephosphorylation and nuclear entry of transcription factors known as nuclear factor of activated t - cells (nfats), which regulate the production of cell cytokines. ox40 activation also downregulates cytotoxic t - lymphocyte antigen 4 (ctla-4), foxp3, and interleukin (il)-4. the decreased levels of these molecules, which are inhibitory in nature, lead to a strong response from t - cells helping them survive and proliferate. it is believed that ox40 interaction also leads to elongation of mrna half - life, thus allowing more cytokines to be expressed. considering the role that ox40 plays in the inflammatory immune response, it has the potential to treat some debilitating diseases. however, when using ox40 or ox40l as a possible treatment target, it is important to realize that at this point, researchers do not know what makes ox40 signalling one answer, though not complete, is provided by tslp, which is produced by mucosal epithelial cells or skin cells in response to allergens. however, unlike regular dcs, these tslp - activated dcs express ox40l, which leads to the differentiation of nave t - cells into inflammatory th2 cells, and these do not produce il-10, but produce tnf- instead. papers indicate that both tslp and ox40 can induce inflammatory diseases in recombination activating gene-2 (rag-2)deficient mice, indicating that the two molecules may share a common signalling pathway. since tslp - activated dcs will produce inflammatory response, tslp can be viewed as a target for treatment that will implicitly remove inflammatory ox40 signalling. considering the above, even though ox40 and ox40l may seem to be a tempting target for curing all kinds of inflammatory disease, it is important to remember that ox40 has a normal role in the human body as well. ox40-deficient mice did not show any severe side effects ; however, there were defects in t - cell proliferation and cellular immunity. these side effects can not be risked when considering possible clinical applications of the ox40l - ox40 axis. another question that arises is how ox40 or tslp can be knocked down in humans. furthermore, there are many things that are still unclear in the ox40l - ox40 signalling pathway. for example, the extent of involvement of nf-b and pi3k, which are molecules important for many other cell functions as well. another thing that is unclear is whether it is ox40 's inhibition or ox40l 's inhibition that will lead to desired results. a study found that using ox40l antibodies did not inhibit ibd, whereas using ox40 antibodies did. overall, changing ox40l - ox40 interactions may be tempting ; however, the target of this change needs to be investigated. ox40l - ox40 interaction has been implicated in various inflammatory autoimmune diseases through many different animal models. however, it is important to recognize that ox40l - ox40 interactions are important for normal immune response as well. activation of ox40l produces anti - apoptotic proteins that aid in the survival and proliferation of t - memory cells. co - stimulatory signals delivered through ox40 have been implicated in selectively promoting the differentiation of t - helper (th)-2 cells, and help prolong antigen - specific proliferative response. ox40-deficient mice are shown to have impaired t - cell proliferation, even though the initial nave t - cell response remains unimpaired. these mice have also exhibited lower levels of anti - apoptotic protein : bcl-2 and bcl - xl. this implies that ox40 's role is mainly in sustaining the immune response, and that ox40 indeed has a non - pathological role as well. however, in normal mice, ox40-positive cells are observed only in lymphoid tissues, including peyer 's patches of the gut. in pathological condition, as demonstrated by mice with hapten - induced colitis or interleukin (il)-2 knockout mice with spontaneous colitis, ox40-positive cells are found in the lamina propria. when ox40 takes on a pathogenic role, it reactivates pre - existing self - reactive t - cells through constitutive activity.[51316 ] ox40 also starts to repress regulatory t - cells (t regs), which is responsible for toning down the immune response, contributing to the balance between tolerance and immunity. experiments conducted have shown that when anergic self - reactive t - cells are introduced in a host along with ox40 agonist, the self - reactive t - cells are activated from their anergic state, resulting in autoimmunity. this statement is further supported by the administration of anti - ox40l monoclonal antibody, which reduced the clinical and histopathological disease. in vivo treatment with anti - ox40l antibodies decreased t - cell inflltration in the colon and suppressed the production of primary inflammatory cytokines : interferon (ifn)-, il-2, and tumor necrosis factor (tnf)- in the lamina propria. combining this treatment with anti - tnf- antibodies further improved the therapeutic effect by further reducing ifn-, il-2, and tnf- production. in addition to animal models, many association studies have been done with ibd patients that have shown higher expression of ox40. ox40 signals are known to be transmitted through tnf receptor associated factors (trafs). traf2 and traf5 activate the nuclear factor kappa - b (nf-b) signalling pathway through ib kinase pathway though this has yet to be proven. recent studies have shown that traf2 is critical to ox40 signalling pathway, as traf2-deficient mice did not have the same degree of effector and memory t - cell generation as the wild - type. the role of traf3 and traf5 is still unclear and needs to be investigated further. ox40 also induces the expression of anti - apoptotic bcl-2 family members : bcl-2, bcl - xl, and bfl-1, which directly correlates with activity of nf-b1 as well. this gives strong proof that nf-b pathway as known to researcher now is involved in ox40 signalling pathway. overall, the bcl-2 family ensures that the cd4 t - cells do not undergo apoptosis after the immediate immune response, proven by studies showing that bcl-2-deficient mice failed to maintain high levels of cd4 t - cells 4 - 8 days after activation. ox40 signalling also maintains the active form of protein kinase b. protein kinase b leads to the expression of survivin, which is a member of the inhibitor of apoptosis (iap) family and regulates the division of t - cells. pi3k connects this pathway to several cyclin - dependant kinases, such as cyclin a, which are involved in cell cycle progression. through a pathway, which may be linked to pi3k, ox40 also increases the influx of ca in cd4 t - cells, leading to dephosphorylation and nuclear entry of transcription factors known as nuclear factor of activated t - cells (nfats), which regulate the production of cell cytokines. ox40 activation also downregulates cytotoxic t - lymphocyte antigen 4 (ctla-4), foxp3, and interleukin (il)-4. the decreased levels of these molecules, which are inhibitory in nature, lead to a strong response from t - cells helping them survive and proliferate. it is believed that ox40 interaction also leads to elongation of mrna half - life, thus allowing more cytokines to be expressed. considering the role that ox40 plays in the inflammatory immune response, it has the potential to treat some debilitating diseases. however, when using ox40 or ox40l as a possible treatment target, it is important to realize that at this point, researchers do not know what makes ox40 signalling one answer, though not complete, is provided by tslp, which is produced by mucosal epithelial cells or skin cells in response to allergens. however, unlike regular dcs, these tslp - activated dcs express ox40l, which leads to the differentiation of nave t - cells into inflammatory th2 cells, and these do not produce il-10, but produce tnf- instead. papers indicate that both tslp and ox40 can induce inflammatory diseases in recombination activating gene-2 (rag-2)deficient mice, indicating that the two molecules may share a common signalling pathway. since tslp - activated dcs will produce inflammatory response, tslp can be viewed as a target for treatment that will implicitly remove inflammatory ox40 signalling. considering the above, even though ox40 and ox40l may seem to be a tempting target for curing all kinds of inflammatory disease, it is important to remember that ox40 has a normal role in the human body as well. ox40-deficient mice did not show any severe side effects ; however, there were defects in t - cell proliferation and cellular immunity. these side effects can not be risked when considering possible clinical applications of the ox40l - ox40 axis. another question that arises is how ox40 or tslp can be knocked down in humans. furthermore, there are many things that are still unclear in the ox40l - ox40 signalling pathway. for example, the extent of involvement of nf-b and pi3k, which are molecules important for many other cell functions as well. tampering with their signalling cascade can lead to other unforeseen side effects. another thing that is unclear is whether it is ox40 's inhibition or ox40l 's inhibition that will lead to desired results. a study found that using ox40l antibodies did not inhibit ibd, whereas using ox40 antibodies did. overall, changing ox40l - ox40 interactions may be tempting ; however, the target of this change needs to be investigated. ox40 signalling is pro - inflammatory, priming th2 to produce tnf-. however, even though the side effects in the ox40 deficient mice are little, there is no guarantee that side effects will not be present in humans. hence, changing ox40l - ox40 signalling may result in a myriad of side effects through the complicated cellular pathways, demanding that caution be taken before doing any clinical trials. | gastrointestinal (gi) autoimmune diseases have a high incidence in developed countries, such as canada and the us. some common gi autoimmune diseases include ulcerative colitis and crohn 's disease. these conditions are not only unpleasant for the patient, but also present a heavy burden on the healthcare system. ox40l, a member of the tumor necrosis family, has been identified as a key player in the pathological inflammatory response, which characterizes gi autoimmune diseases. ox40l is expressed in many cell types, including antigen presenting cells (apcs), t - cells, vascular endothelial cells, mast cells, and natural killer cells. the importance of ox40l - ox40 interactions in inflammatory autoimmune diseases is becoming more evident through different animal models, ranging from nematode models to mouse models. this literature review attempts to summarize the current literature regarding the role of ox40l - ox40 interactions in gi autoimmune inflammatory diseases and comment on its potential for treatment. various databases, including ovid medline and pubmed were used to retrieve articles regarding the role of ox40l - ox40 interactions in the pathogenesis of autoimmune diseases. these articles were then reviewed and summarized in a comprehensive manner. ox40l - ox40 interactions have a strong potential for becoming a treatment target ; however, there are still many gaps in the present knowledge, which need to be addressed before more definitive treatments can emerge. it is also suggested that upstream events leading to ox40l activation, such as thymic stromal lymphopoietin (tslp)-activated dendritic cells, be explored as treatment targets as well. ox40l - ox40 interaction is a possible venue for treatment of gi diseases ; however, the underlying mechanisms of actions and the downstream effects of ox40l knock down need to be investigated. |
sepsis is one of the most common infectious conditions during the neonatal period, and it is still a significant cause of morbidity and mortality, despite the outstanding development of neonatology in recent years. described as systemic inflammatory response (sirs) associated with a suspected or proven infection, the sepsis is an infectious disease of varied etiology, which determines degrees of inflammatory and metabolic responses [2, 3 ]. tumor necrosis factor (tnf), interleukin-1 (il-1), interleukin-6 (il-6), and interleukin-8 (il-8) are proinflammatory cytokines, whereas interleukin-10 (il-10) and transforming growth factor - beta (tgf-) are known as anti - inflammatory cytokines, both produced rapidly in the setting of neonatal sepsis. in the past years, several authors have supported the use of cytokines in the diagnosis of both early and late sepsis. previous results from our study, in which cytokines were measured in the plasma and in the umbilical cord blood at birth, support the idea that increased levels of either proinflammatory cytokines (tnf-) or anti - inflammatory cytokines (il-10) in neonates at birth change throughout the infectious process and there is also a positive and significant correlation between the levels of these cytokines. in the present review, we will cover the actual role of cytokines in the pathogenesis of neonatal sepsis and its value as a diagnostic criterion. neonatal sepsis is a systemic infection that occurs in newborns up to 28 days of age and it is a major cause of morbidity and mortality in newborns. according to world health organization, in 2010, 3.7 million newborns died before reaching 28 days of age in the united states, and 37% were due to infectious causes. as proposed by the international pediatric sepsis consensus conference in 2002, specific definitions for pediatric sirs and sepsis present important differences related to clinical signs and laboratory biomarkers specific to adults. the major differences between adults and children are that the diagnosis of pediatric sirs requires lower values for heart rate, leukocyte count, and systolic blood pressure and upper values for heart rate, respiration rate, or leukocyte count. in sepsis, to confirm the diagnosis, it is necessary the presence of bacterial infection suspected or confirmed by culture or other methods. taken together, some clinical findings can also help the diagnosis process such as : petechiae and purpura (in the setting of hemodynamic instability) ; fever, cough, and hypoxemia (in the setting of leukocytosis and pulmonary infiltrates). in adults, sepsis is defined as a complex clinical syndrome of severe systemic inflammatory response syndrome (sirs) with multiple physiological and immunological abnormalities, which is usually associated with bacterial or fungal infections [2, 810 ]. sepsis pathogenesis is associated with hemodynamic changes, disturbances of microcirculation and cellular changes that cause imbalance between blood flow and metabolic tissue requirements, leading to multiple - organ dysfunctions, which is responsible for the severe and often fatal form of the disease. neonatal sepsis and sepsis in adults are very different conditions, with implications for the epidemiology and pathophysiology and even in clinical management. in addition, differences between neonates and adults directly impact the involvement of cytokines during development of sepsis and the use of assessment of these mediators in clinical routine [1214 ]. although our focus here is neonatal sepsis, as far as possible we compare results of studies involving adults from those obtained from studies involving neonates. sepsis development can be initiated through recognition of one or more components of invading organism, including structural elements such as gram - negative endotoxins or secreted exotoxins that stimulate the local and systemic release of endogenous inflammatory mediators. among the inflammatory mediators are cytokines such as tnf-, ifn-, il-1, il-6, and il-8, which act favoring the migration and activation of immune cells [15, 16 ]. stimulation of polymorphonuclear leukocytes, histiocytes, platelets, and endothelial cells leads to the production of biologically active mediators, including platelet activating factor (paf), arachidonic acid metabolites, histamine, bradykinin, complement proteins, vasoactive peptides, and oxide nitric (no). the production and release of these proinflammatory mediators can induce a systemic inflammatory response characteristic of the initial phase of sepsis [17, 18 ]. for a long time, it was believed that sepsis was caused by an exacerbated inflammatory response generated from innate immune response triggered by bacterial infections. however, researchers eventually described the presence and importance of compensatory anti - inflammatory response syndrome, cars, which often occurs after hyperinflammatory phase, especially in patients who develop severe sepsis. in neonates, severe sepsis is characterized by the persistence and prevalence of proinflammatory mediators up to the third day after diagnosis, whereas these groups of cytokines are prevalent in sepsis, with good clinical evolution, just on the day of diagnosis. the study of inflammatory mediators and cytokines as biomarkers of neonatal sepsis are really important for syndrome diagnosis. recent research has pointed to the role of damage - associated molecular patterns (damps), which are intracellular proteins released in response to cell injury, such as high mobility group box protein 1 (hmgb-1). damps act as endogenous danger signals to activate and amplify the function of receptors such as receptor for advanced glycation end products (rage), hence perpetuating the inflammatory response. this may be particularly important for neonatal sepsis, because the maternal - fetal barrier is compromised by the inflammatory response, leading to translocation of damps into the fetus. the study of proinflammatory cytokines il-6, tnf-, il-1, and il-8 and anti - inflammatory cytokines il-10 and tgf- as reliable biomarkers of neonatal infection has been shown to be potentially useful for early sepsis diagnosis and to predict the severity of disease at early stages of the infection [2326 ]. cytokines are relatively small molecules with short serum half - life (from minutes to a few hours) and play a central role in immune response in neonates with sepsis. during sepsis, cytokine levels may be observed in picograms per milliliter of plasma or in nanograms or even micrograms per milliliter. in the 1990s, sepsis was believed to be associated with an exacerbated release of mainly proinflammatory cytokines, such as tumor necrosis factor (tnf-), interleukin (il-1, il-6, and il-12), interferon- (ifn-), and macrophage migration inhibitory factor (mif). the expression however, recent research on the pathophysiologic mechanisms underlying sepsis indicates that the profound proinflammatory response is counteracted by certain anti - inflammatory cytokines, including il-10, transforming growth factor (tgf-), and il-4, which attempt to restore immunological balance [18, 29, 30 ]. il-6 is a cytokine that shows early response to infection, preceding the increase in c - reactive protein and followed by tnf- release. it is synthesized by mononuclear phagocytes, endothelial cells, fibroblasts, and the decidua, chorion, amnion, and trophoblast cells soon after stimulation by microbial products [31, 32 ]. il-6 acts as a signal in the activation of t cells, and it induces the secretion of antibodies by b cells and the differentiation of cytotoxic t cells. it stimulates the release of other cytokines, particularly tnf- and il-1. il-6 is an early marker in the diagnosis of neonatal sepsis, increasing several hours before the increase in c - reactive protein. the sensitivity of these tests together can reach values close to 100%, hence the importance of these markers. it is capable of interfering with the production of c - reactive protein, so it can be detected earlier than c - reactive protein during bacterial infection. the c - reactive protein has both anti - inflammatory and proinflammatory effects during infection, since it mediates the elimination of pathogens, despite also inhibiting the interaction between endothelial cells and leukocytes. as the secretion starts 46 hours after stimulation and it peaks at 36 hours after infection, it is often used in the diagnosis of infection. biomarker to have a very short half - life, approximately 100 minutes in patients with meningococcal infection ; additionally, the circulating levels decrease or return to basal levels 24 hours after appropriate treatment in late - onset neonatal sepsis. several studies have reported sensitivity for detection of il-6 in 75 to 90% of circulating serum in the first 24 hours of infection, with a marked reduction in the diagnostic effectiveness 48 hours after the onset of symptoms and suspected sepsis [23, 25, 37, 38 ]. moreover, il-6 has been correlated with maternal chorioamnionitis and used in the initial diagnosis of early neonatal sepsis when detected at high levels in umbilical cord blood. similar results were found by our research group when analyzing liver of perinatal deaths diagnosed with fetal inflammatory response syndrome (firs) using immunoperoxidase method ; we observed that il-6 and c - reactive protein were overexpressed. when detected in the umbilical cord blood of newborns at term and without risk factors, il-6 does not have significant clinical utility in differentiating infected and healthy newborns. on the other hand, in newborns with premature rupture of membranes, il-6 showed high sensitivity and specificity in predicting funisitis and positive cord blood culture. the difference in il-6 levels in cord blood and blood of the newborn infant at birth is due to the kinetics of il-6. therefore, sample collection time is an important factor for the detection of high levels of il-6 in neonates. release of tnf- may occur approximately 30 minutes after lps injection, and the circulating levels reach their peak in approximately one and a half hour, with an estimated half - life of about 70 minutes [30, 32 ]. tumor necrosis factor (tnf-) is the prime mediator of septic shock in neonates and widespread tissue injury, and it regulates the secretion of il-1 [37, 38 ]. high levels of tnf- appear to be related to the severity of the disease, although some studies in adults do not confirm this relationship [44, 45 ]. the peak plasma concentration of tnf- is reached after an hour of experimental endotoxemia, with near - zero levels for three hours. there are evidences that tnf- is found free in plasma concomitant with the appearance of signs and symptoms of bacterial infection. systemic release of tnf- can cause vasodilation and increased vascular permeability leading to systemic edema, with decreased blood volume and hypoproteinemia that can progress to shock. there was stimulus for leukocyte and platelet adhesion, with clots formation in small vessels and consumption of coagulation proteins that may lead to disseminated intravascular coagulation. this condition may also progress to multiple - organ failure and death in early - onset neonatal [47, 48 ]. in addition, tnf- and il-1 were identified as crucial cytokines to development of fever and, therefore, belong to a group of pyrogenic cytokines [30, 49 ]. tnf- induces increased adherence of neutrophils and integrins to endothelial tissues and upregulates the endothelial expression of procoagulant proteins. moreover, together with il-1, tnf- was one of the first mediators identified in inflammatory sites. synergically, tnf- and il-1 amplify the inflammatory signals by activating macrophages to produce proinflammatory cytokines such as il-6 and il-8, as well as lipid mediators and reactive oxygen species leading to sepsis - induced organ dysfunction. tnf- acts through two receptors, tnfr1 (tnf receptor-1) and tnfr2 (tnf receptor-2), resulting in immune cell activation and release of several immunoregulatory mediators. the role of tnf- as a marker for the prediction of early - onset neonatal sepsis has been suggested. additionally, when used in combination with il-6, tnf- may achieve up to 98.5% sensitivity. on the other hand, santana and colleagues demonstrated that tnf- was not significantly different between sick and healthy neonates. discrepancy between the studies could be due to the fact that the kinetics of tnf- production is not fully understood in early life. furthermore, due to the short half - life of tnf- and its interaction with soluble receptor, the detection becomes difficult. thus, tnf- as a reliable biomarker for sepsis is compromised. in contrast, some studies, especially by pickler and colleagues, reported that high levels of tnf-, il-6, and il-1 are often associated with profiles of sepsis in neonates. these authors also confirmed the low sensitivity and specificity of tnf- to differentiate sepsis in infected newborns. experimental studies have shown that the injection of tnf causes a syndrome which is indistinguishable from septic shock. it was observed that the infusion of recombinant tnf- in humans results in sirs [5557 ]. it is believed that because this role in the inflammatory process results in severe clinical conditions of sepsis, tnf- may have a direct correlation with the severity of sepsis and the mortality rate during the development of sepsis in newborns at risk for infections. il-1 is a proinflammatory cytokine released by timely activated macrophages similar to those of tnf-. it signals through two distinct receptors, called il-1 receptor type i (il-1r1) and il-1r2, and has many effects on immune cells [30, 59 ]. in the inflammatory response cascade, namely, il-1 can be produced by the central nervous system, particularly in the hypothalamus, and also can be induced by infectious agents (bacterial endotoxins, viruses, fungi, and parasite antigens) and c5a complement, usually in one hour, reaching peak levels in 510 hours [61, 62 ]. during sepsis, il-1 seems to induce fever, coagulation, and hematopoiesis, promoting the extravasation of inflammatory cells. therefore, it has been noticed that il-1 is significantly increased in most patients with sepsis, and it has been associated with the severity of sepsis [63, 64 ]. moreover, persistently elevated levels of this cytokine have been correlated with the development of multiple - organ failure and with a worse prognosis in adults [45, 64 ]. interleukin-1 has been described as a marker of neonatal sepsis, although its diagnostic efficacy is lower than that of il-6 and tnf-. the diagnostic value of il-6, tnf-, and il-1 is limited by the time of blood sample collection, which should be as early as possible if neonatal sepsis is suspected, since these cytokines have very short half - life [46, 65 ]. therefore, analysis of a panel of cytokines, and not only the isolated measurement of a single cytokine, is required in order to characterize the inflammatory response in sepsis. il-8 belongs to the class of proinflammatory chemokines and it is produced by the placental cells, fetal monocytes / macrophages, and cells. il-8 is a chemokine which follows a course of time similar to that of il-6. this characteristic limits, to a great extent, the role of il-6 and il-8 as clinically useful biomarkers for all stages of sepsis, although they may be useful early in the disease prior to treatment in neonates. recently, the association of high levels of il-8 in the presence of retinopathy was determined in seventy - four very low birth weight preterm infants with clinical criteria of early infection whose cytokines were obtained during the first three days of life. the cut - off points for il-6 > 357 pg / ml, il-8 > 216 pg / ml, and tnf- > 245 pg / ml were significantly associated with the development of retinopathy. placental cells, fetal monocytes / macrophages, and endothelial cells are able to produce il-8 after infectious process originated in the uterus. il-8 levels increase about 90 minutes after infection and peak at about 120 minutes in septic neonates, whereas its circulating concentration decreases significantly 48 hours after birth, likewise the kinetics of il-6. only preterm infants less than 32 weeks of gestation may have increased levels of il-8 due to gestational age. data by dembinski and colleagues showed that il-8 levels were undetectable in the umbilical cord blood of healthy newborns. in the past years, il-8 has been extensively investigated as a predictive biomarker of early - onset neonatal sepsis that is corroborated by dllner and colleagues, who observed increased il-8 levels in the umbilical cord blood of infected preterm neonates. the disadvantage of il-8 measurement in comparison with il-6 levels is the limit of detection. the il-6 serum detection limit is > 0.7 pg / ml, whereas the serum detection limit of il-8 is > 10 pg / ml. anti - inflammatory cytokines, such as il-10 and tgf-, are important inflammatory mediators, since they play a major role in preventing excess proinflammatory response during sepsis. il-10 is produced by different types of immune system cells such as monocytes, macrophages, t and b lymphocytes, and nk cells. this cytokine suppresses the production of proinflammatory mediators including tnf-, il-1, il-6, ifn-, and gm - csf in cells of the immune system. high levels of il-10 have been correlated with poor prognosis of sepsis in adults, shown to be a useful predictor of severity in septic shock and death [76, 77 ]. however, it was shown that the appropriate response of il-10 may have a protective effect on sirs and that high il-6/il-10 ratio was found in patients with a worse prognosis. similarly, a high il-10/tnf ratio has also been associated with severe late - onset neonatal sepsis [26, 75 ]. in an experimental model, it was shown that the administration of recombinant murine il-10 protects from lethal endotoxemia, even when il-10 was injected 30 minutes after lps administration. in contrast, the immunoneutralization of il-10 led to increased levels of tnf and il-6 in circulating mice, and it also reversed the ability of il-10 to protect mice from lethal endotoxin. despite these clear protective effects of il-10 in lps - induced diseases, il-10 actions are not always beneficial. the effects of il-10 appear to depend on the time of administration in case of neutralizations. furthermore, the authors reported that il-10-deficient mice showed an earlier onset of lethality after experimental sepsis induced by cecal ligation and puncture as compared with wild type mice. tgf-, as well as il-10, is a member of the growth factor family and it is an important anti - inflammatory cytokine. tgf- was shown to play a role in tissue repair and fibrosis, as well as in sepsis - induced immunosuppression. in vitro, tgf- suppresses the release of proinflammatory mediators such as il-1 and tnf- from monocytes and macrophages. tgf- also inhibits t - lymphocyte functions, such as il-2, and the secretion of t cell proliferation, as well as promoting the development of t regulatory cells. studies involving evaluation of in vitro assays, experimental models of sepsis, and human clinical evaluations support the anti - inflammatory actions of tgf-. these experiments have shown that treatment with tgf- blocked endotoxin - induced hypotension, probably inhibiting the hypotensive effects of no and improved survival in a rat model of salmonella endotoxin - induced septic shock [72, 88 ]. another study reported that adult patients at the onset of sepsis presented high levels of tgf- even though these levels were not correlated with severity or prognosis of disease. recent data have demonstrated that tgf- reverses the depression of cardiac myocyte contraction, which is induced by cytokines such as tnf- and il-1 and by the serum of patients with septic shock. hence, it is suggested that tgf- may have cardioprotective effects in sepsis - induced cardiac injury. recently, il-7, which is a hematopoietic growth factor, has been reported to have antiapoptotic roles, essential for lymphocyte survival and growth [91, 92 ]. in addition to its antiapoptotic properties, it induces proliferation of cd4 and cd8 t cells. one of the characteristic features of sepsis is the profound loss of t cells in various lymphoid organs. during experimental sepsis, il-7 decreases cell apoptosis through the expression of antiapoptotic bcl-2 gene [94, 95 ]. another study showed that septic mice treated with recombinant il-7 (rhil-7) increase the local and systemic production of neutrophils and il-17, thus recruiting more neutrophils to the site of infection. nevertheless, further studies are necessary to understand the real role of il-7 in the pathogenesis of neonatal sepsis. another cytokine which has been studied and seems to be really involved in the pathogenesis of sepsis is il-22. in a small group of hospitals within a single health center it is believed that il-22 that is produced during sepsis may contribute to host defense and to stabilizing mucosal barrier functions under systemic infection conditions. however, the adverse effects of il-22 are also described in a model of polymicrobial peritonitis, in which the levels of il-22 and its receptor in the spleen and kidney were very high. the biological activity of il-22 is modulated by its antagonist, il-22 pb. treatment of mice with il-22 pb before sepsis led to increased accumulation of neutrophils and mononuclear phagocytes, as well as a reduction in bacterial burden at the site of infection. thus, the beneficial effects of il-22 are mediated by promoting tissue protection, whereas ill effects are connected with exacerbated inflammation. this dual role of il-22 implies that it participates in the pathophysiology of experimental sepsis. il-33 can induce t helper cells, mast cells, eosinophils, and basophils to produce th2 cytokines. il-33 mediates its biological effects through interaction with its receptors and with associated proteins abundantly expressed on the surface of th2 cells and mast cells. il-33 also functions as a chemotactic mediator for th2 cells [100, 101 ]. in mast cells, il-33 triggers the production and release of proinflammatory cytokines, promotes maturation, and induces degranulation. furthermore, il-33 amplifies the polarization of alternatively activated macrophages and it enhances tlr4-mediated cytokine production by macrophages. st2 exists in different splice variants, which results in a cell membrane - bound form and in a soluble form. the soluble form, sst2, is generated by alternative splicing and does not induce signaling, thus acting as a receptor for il-33. high levels of sst2 have been associated with the pathogenesis of sepsis, so sst2 may be a potential marker of poor prognosis [95, 105 ]. il-33 has beneficial effects in experimental sepsis, enhancing the accumulation of neutrophils through the upregulation of cxcr2 via grk2-dependent pathways at the site of infection and reducing the inflammatory response in systemic sites. however, it has not yet been determined whether the administration of il-33 actually represents a therapeutic strategy. the proinflammatory cytokine il-17a is mainly produced by th17 cells and is involved in the mediation of proinflammatory responses, hence triggering the production of many other cytokines such as il-1, il-6, and tnf-. it has recently been shown that increased il-17a levels have adverse effects during experimental sepsis in clp - induced sepsis models. whereas the blocking of il-17a was associated with reduced levels of bacteremia, proinflammatory cytokines and an increased survival rates of animals. it causes an increased production of il-4 itself and of other anti - inflammatory cytokines, suppressing the secretion of monocyte - derived proinflammatory cytokines. experimental studies have shown that il-4 increases the survival rates of mice exposed to lethal lps doses. similarly, in humans, mrna expression of il-4 was associated with survival of patients with severe sepsis. nonetheless, il-4 plasma levels of septic patients on the day of hospital admission were not different between the patients who survived and those who did not survive sepsis. it was recently suggested that polymorphisms in the il-4 gene promoters may affect the balance of th1 and th2 response and, consequently, predispose trauma patients to develop sepsis. although there are several studies suggesting that il-4 plays an important role in the pathogenesis of sepsis, its real role in the course of the disease remains unknown. procalcitonin (pct) is the hormone calcitonin, which is normally produced by the c cells of the thyroid gland, leading to massive release of pct into the bloodstream depending on the severity of sepsis. assicot and colleagues were the first to describe pct as a potential biomarker of sepsis and infection. the authors showed a more favorable pct kinetic profile than the profile of c - reactive protein and cytokines. pct circulating levels decrease within about 24 hours, when the infection is sufficiently treated. decreasing pct levels are, thus, associated with improved survival, whereas increased or persistently increasing pct levels are predictive of an unfavorable outcome [113, 114 ]. discrimination between infectious and noninfectious conditions for pct and decreasing pct levels in properly treated patients raised the hypothesis that pct levels can help in determining the antimicrobial therapy. pct serum concentrations may be increased in medullary thyroid carcinoma in the absence of bacterial infections and in conditions such as severe trauma, surgery, or postcardiac arrest, heat shock, stress birth, and various types of immunotherapies and some autoimmune diseases. however, as any other biomarker, pct levels must be evaluated within the clinical context of the patient. understanding the neonatal sepsis pathogenesis still remains a challenge, given its complexity and the inherent immunological characteristics of the newborn. cytokines seem to be one of the major mediators involved in the outcome of this entity. the imbalance between proinflammatory and anti - inflammatory cytokines appears to be related to both the severity and prognosis of neonatal sepsis. therefore, the use of cytokines as biomarkers of neonatal sepsis seems plausible and necessary, since the early diagnosis of neonatal sepsis directly influences the therapy and prognosis. | neonatal sepsis is a major cause of morbidity and mortality and its signs and symptoms are nonspecific, which makes the diagnosis difficult. the routinely used laboratory tests are not effective methods of analysis, as they are extremely nonspecific and often cause inappropriate use of antibiotics. sepsis is the result of an infection associated with a systemic inflammatory response with production and release of a wide range of inflammatory mediators. cytokines are potent inflammatory mediators and their serum levels are increased during infections, so changes from other inflammatory effector molecules may occur. although proinflammatory and anti - inflammatory cytokines have been identified as probable markers of neonatal infection, in order to characterize the inflammatory response during sepsis, it is necessary to analyze a panel of cytokines and not only the measurement of individual cytokines. measurements of inflammatory mediators bring new options for diagnosing and following up neonatal sepsis, thus enabling early treatment and, as a result, increased neonatal survival. by taking into account the magnitude of neonatal sepsis, the aim of this review is to address the role of cytokines in the pathogenesis of neonatal sepsis and its value as a diagnostic criterion. |
prostate cancer is the most common cancer and the second leading cause of cancer death in men. androgen deprivation therapy is the treatment of choice in patients with advanced metastatic disease, although progression typically occurs within 12 years of initial response [2, 3 ]. secondary hormonal therapy is an option in some patients ; however, most neoplasms ultimately progress to a situation of androgen - independent growth, namely hormone - resistant prostate cancer (hrpc), androgen - independent prostate cancer or, recently, castration - resistant prostate cancer. clinical manifestations include rising psa (prostate serum antigen) concentration, bone metastases, substantive pain, and soft - tissue / lymph node metastases. some forms of hrpc treatment are systemic radiation therapy, chemotherapy, immunotherapy and estrogen therapy. however, outcomes in hrpc are very poor and therefore novel agents are needed to improve the treatment of this condition. many approaches, such as drugs that target specific pathways involved in cell signaling, proliferation, apoptosis, immune modulation and angiogenesis, are currently under investigation to improve survival benefit. lenalidomide is a thalidomide analog and also known as a member of the so - called immunomodulatory drugs (imids) with immunomodulatory and antiangiogenic properties. in preclinical studies, lenalidomide demonstrated antitumoral activity, inhibiting hypoxia - induced factor-1 expression by endothelial cells and epithelial tumor cells, including prostate cells, and an increase in pc-3 prostate cancer cell apoptosis with monotherapy or in combination with docetaxel. using in vitro and in vivo models of prostate cancer, lenalidomide inhibited growth factor - induced invasion and produced synergistic effects in combination with docetaxel that slowed tumor progression. the few published reports about the use of lenalidomide alone or in combination in hrpc patients [8, 9, 10, 11, 12, 13, 14, 15, 16, 17 ] show clear evidence of its clinical antitumoral activity in this disease. in this paper, we demonstrate that lenalidomide not only has antitumoral activity but also has no associated hematologic toxicity. in this paper, we discuss the case of a 65-year - old male patient diagnosed in august 1999, by transurethral resection of the prostate, with a gleason 6/10 prostate adenocarcinoma. his past medical history included renal clear cell adenocarcinoma of both kidneys and constipation treated with lactulose. the serum psa at the time of diagnosis was elevated to 19.7 ng / ml (normal range 0.04.0). radiotherapy and combined androgen blockade (cab) therapy was initiated, comprising lutein - releasing hormone analog (zoladex) and bicalutamide 50 mg daily (table 1). the patient remained asymptomatic with a low psa level for 5 years (psa nadir 0.1 ng / ml). in january 2008, biochemical and bone progression was diagnosed with an increased psa of 12 ng / ml ; bone metastases were detected by bone and ct scans. however, 3 months later, his psa rose again to 37 ng / ml. in april 2008, the patient received a third hormonal therapy with cyproterone acetate for 2 months, but biochemical progression was detected with a new increase of psa to 64 ng / ml. subsequently, docetaxel - prednisone - based chemotherapy was started, but the patient 's psa level rose again after an initial drop (64123857 ng / ml) ; bone and ct scans showed bone - only metastases. in october, second - line chemotherapy with oxaliplatin and capecitabine on a compassionate - use basis was administered, but after 3 cycles of treatment, psa level was 233 ng / ml. in november 2008, this therapy was replaced by treatment with fulvestrant for compassionate use and, after the first month, psa level returned to 32 ng / ml. however, in march 2009, his psa value was higher than 300 ng / ml. sunitinib was proposed for compassionate use. after being treated for 5 months with sunitinib (50 mg daily on days 14, given every 42 days), in august 2009, the patient began treatment with vinblastine - adriamycin - estramustine and ketoconazole, which was discontinued after the third cycle given the clinical deterioration associated with more intense pain (initial ps 1 ; final ps 2) and increased levels of serum psa (1,146 ng / ml). in october, a new line of treatment with lenalidomide 25 mg / day for compassionate use together with analgesic agents was administered in november 2009. concomitant analgesic medication for bone pain consisted of ibuprofen 600 mg/8 h, morphine sulfate 10 mg for rescue and fentanyl patches that were discontinued in january 2010. the patient responded well to lenalidomide therapy, psa level markedly declined to 391 ng / ml and no hematological or cutaneous adverse events were documented ; similar hemoglobin levels were maintained before and after lenalidomide treatment (10.411.7 g / dl). the patient regained the ability to walk over 7 km daily, maintaining his lifestyle, and he improved his general performance status until april 2010, when fentanyl patches were reintroduced and his psa level rose to 422 ng / ml. in may 2010, an increased psa level of 893 ng / ml was accompanied by clinical worsening due to the evolution of the disease, and therapy was suspended. after that, estracyt (estramustine) 140 mg/12 h was started but no response was obtained ; psa level in july 2010 was > 5,000 ng / ml, and bone and ct scans revealed lymph node, liver and lung metastases. the progressive deterioration of the patient 's health led to hospital admission and death in october 2010. proliferation, angiogenesis and evasion of immune surveillance are important pathways by which hrpc progresses. based on antiangiogenic and antitumoral properties of imids, it has been described that lenalidomide inhibits tumor necrosis factor- production, diminishes levels of vegf and basic fibroblast growth factor, stimulates t cells and hinders angiogenesis. the antitumoral activity of lenalidomide is approximately 5,000 times more potent than thalidomide in animal models, and, furthermore, it has the additional advantage of being associated with a better toxicity profile. in fact, preliminary studies suggest that lenalidomide may have clinical activity in patients with metastatic castration - resistant prostate cancer [16, 17 ]. nevertheless, published data about the use of lenalidomide in hrpc are rare so far, particularly in chemotherapy pre - treated patients. all of the data have shown clear evidence of antitumor activity of lenalidomide alone [13, 14 ] or in combination with docetaxel [9, 12 ], paclitaxel [10, 15 ], ketoconazole or gm - csf in metastatic hrpc patients. responses described in these studies are promising with regard to survival advantage. according to our experience, the patient described here, pre - treated with chemotherapy, showed favorable response to lenalidomide therapy for 5 months improving his general health status and quality of life, even discontinuing fentanyl administration for 4 months. it is significant to note that psa levels declined, and disease response and pain reduction during lenalidomide administration was not accompanied by any toxicity. hematological tests did not show alteration in hemoglobin levels, and the patient did not experience cutaneous adverse effects. this observation provides an additional benefit and suggests that lenalidomide may be an attractive drug because of its antineoplastic activity and low side effect profile. the evolution of the disease with the appearance of metastases led to worsening of the clinical situation of the patient. the prognosis of prostate cancer is mainly determined by the presence or absence of metastases. in our patient, the development of bone, lymph node, liver and lung metastases led to progressive deterioration, which is a common complication due to widespread disease expansion. other treatments received by the patient mainly included subsequent lines of hormonal therapy, radiotherapy and chemotherapy with antineoplastic agents standard treatments in the management of hrpc. although reductions in psa levels were achieved for a limited period of time, successive changes of therapy were required because of relapse and disease progression. given the need for new agents that do not bear a potentially high cost in terms of side effects in the management of hrpc patients, lenalidomide is proposed as an interesting option. in conclusion, the present case report presents new evidence of the potential of lenalidomide to provide clinical benefits to hrpc patients because of its antitumoral activity and lack of toxicity ; the use of lenalidomide is supported in this patient population for delaying disease progression. | hormone - resistant prostate cancer (hrpc) occurs when prostate cancer is no longer responsive to hormone therapy. treatment options are limited, and there is a clear necessity for therapies that improve outcome. preclinical and clinical evidence supports the role of the immunomodulatory agent lenalidomide in hrpc. in this paper, we report that lenalidomide showed antitumoral activity in a patient with hrpc and bone metastases pre - treated with chemotherapy, decreased the psa level and improved the patient 's health status for the first 5 months. it is important to emphasize that it was not associated with hematologic toxicity. |
adult mesenchymal stem cells (mscs) represent an innovative tool for cell - based therapy of degenerative disorders, chronic inflammatory, and autoimmune diseases or allograft rejection. the understanding of the mechanisms that mediate and/or modulate the therapeutic potency of mscs is important from both a physiological and a clinical point of view. in this context, one key interest is to better understand the modulation of msc biology by toll - like receptors (tlrs) as they have been linked to the perpetuation of chronic inflammatory responses (chron 's disease, rheumatoid arthritis) through the recognition of conserved pathogen - derived components or endogenous ligands (also known as danger signals) that mscs will likely encounter in the sites of injury. here mscs have been isolated from multiple tissues of mesodermal origin, such as bone marrow (bm - mscs), adipose tissue (ad - mscs), umbilical cord blood and peripheral blood. mscs can be easily isolated by adhesion to plastic and expanded in vitro in serum containing media with no additional requirements for growth factors or cytokines. in culture thus, to date they are defined according to the criteria of the international society for cellular therapy by being negative for hematopoetic and endothelial markers such as cd11b, cd14, cd31, cd34, and cd45, and positive for a variety of many other markers, including hla class i, cd105, cd73, cd29, and cd90. therefore, mscs can be identified in vitro by their ability to differentiate into mesenchymal - type cells (adipocytes, osteoblasts, and chondrocytes) but also neurons, endothelial cells, astrocytes, and epithelial cells when cultured in the appropriate conditions [26 ]. the expression of human leukocyte antigen (hla) molecules class i (also called mayor histo - compatibility class i = mhc i) on all cells on the body allows the immune system to distinguish self from nonself. in the absence of immune suppression or tolerogenic mechanisms, cells expressing hla molecules stimulate t cells directly only if they possess appropriate costimulatory molecules cd80 (b7 - 1), cd86 (b7 - 2) or cd40-. allogeneic cells can also activate t cells through an indirect pathway where their hla antigens are presented by professional antigen presenting cells (apc). aside from hla class a remarkable unique feature of mscs is that they are considered to be immunoprivileged as they express low levels of cell - surface hla class i molecules whereas hla class ii, cd40, cd80, and cd86 are not detectable on the cell surface. stimulation with interferon (ifn)- has been shown to increase both class i and class ii molecules, however, mscs do not express costimulatory molecules cd80 (b7 - 1), cd86 (b7 - 2) or cd40, even after ifn- stimulation. mscs not only fail to induce activation of cd4 + cells but also escape lysis by cd8 + cytotoxic lymphocytes. even whole lymphocytes stimulated in vitro to target peripheral blood lymphocytes (pbls) derived from a specific donor, will lyse lymphocytes from that individual but not mscs derived from the same donor. further analyses indicate that mscs induce an abortive activation programme in fully differentiated cd8 + t cells so that major effector functions are not activated. a third important feature of mscs is that they are immunosuppressive and inhibit activation, proliferation, and function of immune cells, including t cells, b cells, nk cells and antigen - presenting cells (apcs) [6, 1324 ]. despite ample research in recent years, mostly in bm - mscs, the specific molecular and cellular mechanisms involved in the immunoregulatory activity of mscs remain controversial. there is evidence that the capability to modulate immune responses relies on both cell contact - dependent mechanisms and soluble factors secreted by mscs in response to cytokines released by activated immune cells. mscs may inhibit lymphocyte proliferation by a mechanism that requires, at least in part, the release of soluble factors such as hepatocyte growth factor (hgf), prostanglandin - e2 (pge2), transforming growth factor (tgf)-1, indoleamine 2,3-dioxygenase (ido), nitric oxide, and interleukin (il)-10 [14, 16, 18, 21, 22, 2529 ]. on the other hand, other studies have shown that bm - mscs may modulate t - cell phenotype resulting in the generation of cells with regulatory activity [14, 15, 19, 3034 ]. the biological characteristics mentioned above make mscs an interesting tool for cellular therapy and regeneration. this is supported by a number of studies in animal models of inflammatory diseases demonstrating an efficient protection against allograft rejection, graft - versus - host disease, experimental autoimmune encephalomyelitis, collagen - induced arthritis, sepsis and autoimmune myocarditis [13, 28, 3138 ]. in fact, mscs are being used in several clinical trials with a focus on their immunomodulatory capacities (http://clinicaltrials.gov/search/term=stem+cells?term=stem+cells). in this regard, cellerix is currently conducting a phase iii clinical trial using human ad - mscs to treat complex peri - anal fistula in crohn patients after having successfully completed phase i and ii trial with high efficacy rates [39, 40 ]. importantly, it is believed that the therapeutic potency, safety, and efficacy of treatment of inflammatory diseases with mscs reside to a large extent in their immunologically privileged phenotype and in their immunosuppressive capacity. interestingly, tlr activation has been implicated in the pathology of a number of inflammatory diseases including rheumatoid arthritis or inflammatory bowel disease (ibd), since they can either initiate or perpetuate the chronic inflammation due to the continue exposure to tlr ligands [41, 42 ]. therefore, the use of mscs in cell therapy for the treatment of inflammatory diseases deserves further investigation regarding the potential effects of tlr signaling on mscs biology and the potential implications in the immunogenicity and immunosuppressive capacity, which are of special relevance in terms of therapeutic potency. expression of tlrs at the rna and protein levels have been studied by rt - pcr, flow cytometry, and immunofluorescence. so far, consistent results demonstrate high mrna expression of tlr 1, 2, 3, 4, 5, and 6 in ad - mscs and bm - mscs from both human and mice, while inconsistent results have been reported on the expression of tlr 7 to 10 in the same studies. at the protein level, expression of tlr2, tlr3, tlr4, tlr7, and tlr9 has been reported by flow cytometry and immunofluorescence [23, 2831, 43 ]. have studied whether hypoxia can affect the expression of tlrs in human ad - mscs, as these cells may be used as a therapeutic approach in ischemic tissues. they demonstrated that exposure to hypoxic conditions significantly increase mrna of tlr1, 2, 5, 9, and 10. moreover, lipopolysaccharide (lps) challenge downregulates tlr2 and tlr4 expression in msc - derived osteoprogenitors. on the other hand, transduction of mscs with baculoviruses (a dna viral vector) upregulates expression of tlr3 and triggers tlr3 signaling pathway. as reviewed extensively somewhere else in this issue, tlr activation trigger myd88 dependent and independent downstream signalling cascades leading to the nuclear translocation of nf-b and other transcription factors and the activation of a number of genes (see figure 1) [4752 ]. it has been demonstrated that when ad - mscs or bm - mscs were stimulated with ligands specific for different tlrs the nuclear factor - kappa b (nf-b), mitogen - activated protein (map) kinases (mapks), pi3k signalling pathways were activated with a subsequent induction of several genes and cytokines, mainly cxcl-10, il-6 and il-8. however, differences in the induction of genes in response to tlr activation have been reported. for instance, in contrast to our observations in ad - msc, hwa cho., and tomchuck. have reported the induction of tumor necrosis factor (tnf)- and il-1 by lps and polyinosinic : polycytidylic acid (poly ic) in bm - msc and ad - msc, respectively [44, 53 ]. in response to tlr stimulation human ad - mscs induce the expression of manganese superoxide dismutase (mnsod), a key protective protein against oxidative stress in the mitochondria. it has been reported that induction of mnsod protects cells from oxidative stress leading to increased survival. in the settings of an inflammatory response, immune cells release vast amounts of reactive oxygen species which results in the generation of an oxidative milieu. based on these data we speculated that increased expression of mnsod by mscs in response to tlr ligand exposure would provide them with improved engraftment or survival at injured or inflamed sites, leading to enhanced therapeutic effects. this hypothesis is further supported by recent results showing that tlr4 activation protects mscs from oxidative stress - induced apoptosis. in fact, lps preconditioning of mouse bm - mscs can, when compared to unconditioned mscs, improve their survival and engraftment of mscs and increase the release of vascular endothelial growth factor (vegf) in a model of rat acute myocardial infarction leading to an enhanced therapeutic effect (improved cardiac function, reduced apoptosis of myocardium, reduced fibrosis and elevated vascular density after myocardial infarction) (see table 1). as indicated above, one of the main features of mscs is the potential to differentiate to several cell types of mesenchymal origin. some groups have reported the effects of tlr activation on msc differentiation with contradictory results. we found no effect on adipogenic differentiation but detected that poly i : c and lps increased osteogenic differentiation in human ad - mscs. however hwa cho. reported increased osteogenic differentiation of human ad - msc by lps and peptidoglycan (pgn) activation, whereas cpg oligodeoxynucleotides (cpg - odn) impaired it. the increased osteogenic differentiation in the presence of lps or pgn was accompanied by increased erk activation. these authors reported reduced adipogenic differentiation when pgn was present. on the other hand, whereas mo. reported increased osteogenic differentiation of human bm - mscs after prolonged lps activation, liotta. found no effect of tlr activation on adipogenic, osteogenic or condrogenic differentiation of human bm - mscs. reported that tlr2 activation by pam3cys reduced mouse bm - msc differentiation into the three mesodermal lineages. interestingly, they also found that myeloid - differentiation primary - response protein 88 (myd88)-deficient bm - mscs, when cultured in the appropriate differentiation media without additional stimulation with tlr ligands effectively differentiated into adipocytes but failed to differentiate into osteocytes and chondrocytes, indicating that this pathway may be involved in msc mutipotency. these discrepancies could be due to differences in culture conditions, between bone marrow and adipose - derived mscs, and between mouse and human cells (see table 1). the effect of tlr activation in the proliferation of mscs has been studied by several groups. stimulation with lps or pam3cys promoted proliferation of mouse bm - mscs [55, 58 ], but stimulation with lps, poly i : c, lipoteichoic acid (lta) and pgn showed not significant effects on human ad - mscs and bm - mscs [23, 44, 45 ]. however, hwa cho. reported that stimulation of ad - mscs with cpg - odns leads to a g1 arrest wich results in inhibition of proliferation (see table 1). migration to the appropriate site of injury is considered to play an important role in the therapeutic efficacy of mscs. in this context, demonstrated that tlr3 activation drives the migration of human bm - mscs using transwell and boyden chamber migration assays suggesting that this tlr signalling pathway may be manipulated to increase the biodistribution of infused mscs at the injured sites. moreover, lps, odns, ll-37 (an antimicrobial peptide), fibronectin fragment iii 1c (fn iii1c) and flagellin resulted in moderate to limited induced migration. on the other hand, pevsner - fischer. found that tlr2 activation impaired mouse bm - msc migration using wound healing nevertheless, further investigation will be required to better understand the potential role of tlr signalling in migration and biodistribution of mscs in vivo, which is of great clinical relevance. the potential use of allogeneic mscs relies on the special capacity of these cells to escape to the immune recognition. it is well established that tlr activation can modulate expression of costimulatory molecules in immune cells. therefore, it is of special interest from a therapeutic point of view to determine whether exposure of mscs to tlr ligands may induce the expression of hla - i, hla - ii, and costimulatory molecules (cd40, cd80, cd86) leading to an augmented immunogenic phenotype. we analyzed the expression of hla - i, hla - ii, cd80, and cd86 in human ad - mscs by flow cytometry 72 hours after stimulation with lps, poly i : c, and pgn. we found that lps, poly i : c, and pgn did not alter the expression of hla - ii, cd80, and cd86. poly i : c was the only tlr ligand capable to induce hla - i to some extend. furthermore, costimulation of human ad - mscs with ifn- (a well - known inducer of hla - i and hla - ii expression in mscs) in combination with either lps, poly i : c or pgn did not alter the inf--mediated induction of hla - i and hla - ii. similar results have also been reported in human bm - mscs [43, 57 ]. these results indicate that tlr activation does not significantly affect the immunogenic properties of human mscs (see table 1). these results are of great relevance regarding the use of allogeneic msc - based cell therapies. bm - msc and ad - mscs have been shown to possess the capacity to inhibit proliferation of immune cells upon mitogenic or allogeneic activation. as mentioned above, this immunosuppressive capacity of all mscs can become a key factor for their therapeutic use and potency. the mechanisms underlying the immunosuppression potential of mscs are not fully understood, but seem to require both cell - to - cell contact - dependent mechanisms and the release of soluble immune modulators (ido, pge2, tgf-1, nitric oxide, etc.) upon activation in response to immune cells. interestingly, some of these immune modulators are downstream of signalling pathways triggered by tlrs in other cell types. therefore, a feasible hypothesis is that tlr ligands may induce the production of such anti - inflammatory mediators in mscs resulting in an enhanced immunosuppressive phenotype. moreover, tlr signalling has been associated with the perpetuation of chronic inflammatory and autoimmune diseases such as crohn 's disease and rheumatoid arthritis [41, 42 ]. therefore, ad - mscs and bm - mscs employed in the treatment of such diseases will likely be exposed to tlr ligands, which may result in the modulation of mscs activity and therapeutic potency. therefore, it is very important to determine whether tlr signalling may modulate the immunosuppressive capacity of mscs. in recent years, several groups have reported inconsistent results regarding the role of tlr ligands on the modulation of mscs capacity to suppress immune responses. in this context, we tested the role of tlrs in the immunosuppressive capacity of human ad - mscs. to do so, we analyzed proliferation of activated cfse - labeled pbls, cd4 + t cells and cd8 + t cells in the absence or presence of increasing amounts of human ad - mscs precultured for up to 72 hours with medium alone or in the presence of lps, poly i : c or pgn. we found no significant effect of tlr activation on human ad - msc - mediated suppression, indicating that activation through tlr2, tlr3, and tlr4 do not significantly interfere with the capacity of human ad - mscs to modulate immune responses in vitro. supporting these results, ido, a key mediator of human ad - mscs immunosuppression was weakly induced by a high concentration of poly i : c and was not induce upon tlr2 or tlr4 triggering. similar results were reported by pevsner - fischer., showing that tlr2 activation by pam3cys does not affect immunosupression mediated by mouse bm - msc. however, other groups have reported that tlr activation may modulate the immunosuppressive properties of human bm - mscs, although in very different ways. thus, liotta. found that tlr3 and tlr4 activation reduce the inhibitory activity of human bm - mscs on t - cell proliferation without influencing ido activity or pge2 levels. by using inhibitors of the notch signalling pathway and anti - jagged-1 neutralizing antibodies they found that tlr activation leads to the downregulation of the notch ligand jagged-1 in bm - mscs. based on these data, the authors suggested that notch signalling pathway mediates the cell contact - mediated immunosuppression by mscs. in contrast to these results, opitz. have recently reported that tlr3 and tlr4 engagement enhances the immunosuppressive properties of human bm - mscs through the indirect induction of ido1. induction of ido1 involved an autocrine ifn- signalling loop, which was dependent on protein kinase r (pkr) and independent on ifn-. a common characteristic of all mscs is that they constitutively express il-6 and il-8. the significance of a constitutive expression of il-6 and il-8, which can both be considered proinflammatory cytokines, in context to the immunosuppressive activity of mscs is still unclear. interestingly, it has been reported that mscs inhibit the differentiation of dendritic cells, at least in part, through the release of il-6 hence, it is tempting to speculate that induction of il-6 secretion by tlr activation may enhance mscs - mediated impairement of dendritic cells differentiation and maturation. these inconsistent and partly contradictory results demonstrate the complexity of the immune system, even under in vitro conditions. it is likely that differences in the experimental settings between laboratories might be behind these contradictory results (see table 1). for instance, the use of pbmcs versus purified t cells or the method of activation of these cells may play a role. employed purified cd4 + t cells stimulated with allogeneic t cell - depleted pbmcs and anti - cd3 mab, opitz. used a mixed lymphocyte reaction (mlr) with total pbmc from two unrelated donors, one of which was irradiated, and we used either whole pbmc samples or purified cd4 + and cd8 + fractions stimulated with beads loaded with anti - cd3, anti - cd2 and anti - cd28 mabs. another important aspect could be the time of treatment with tlr ligands and the concentration of them (very variable among studies). while some left tlr ligands in the cocultures or pretreated the mscs with tlr ligands for several days before initiating the coculture experiments [23, 57 ], others pretreated mscs for 24 hours, washed and cocultured them with the mlr. it has also been suggested that a long - term exposure to tlr ligands may lead to downregulation of factors induced shortly after activation of tlr. as several studies have reported benefitial effects of mscs treatment in animal models of lps - induced sepsis or lung injury [32, 38, 60 ], an inhibition of a therapeutic capacity of msc by tlr ligands does not appear to be the case. thus, it is unclear whether in vivo potency of mscs can be potentiated by tlr ligands, however it does not appear to be impaired. it is necessary to better define the role of tlr activation on msc biology in the context of the development of new therapeutic strategies on inflammatory or autoimmune diseases, simply because mscs are likely be exposed to activation through tlr ligands in the sites of injury or inflammation. the discrepancies shown by different authors need further investigation as it would be relevant to determine whether or not tlr activation interferes or enhances migration, biodistribution or immunosuppressive capacity of mscs. differences in the source of mscs, type and concentration of the stimuli used, the experimental settings and method of detection or culture conditions may explain these discrepancies. on the other hand, the recognition of endogenous ligands by tlrs is now thought to have an important role in the regulation of inflammation, both in infectious and noninfectious diseases. a number of endogenous ligands have been identified, including heat shock protein (hsp) 60, hsp 70, heparan sulfate, hyaluronan, fibronectin extra domain a, uric acid, oxidized ldl, intracellular components of fragmented cells, myeloid - related proteins-8 and 14, eosinophil - derived neurotoxin, and human defensin-3 [6172 ]. as these ligands are accessible to tlrs in the setting of injury or non - infectious threat, they have been called danger signals. a very important aspect that has not been studied in details so far is the activation and modulation of msc activity by these danger signals. given the capacity of mscs to modulate immune responses it would be interesting to determine whether resident mscs may be activated by danger signals in the settings of injury and if this activation results in a contribution of mscs to the process of healing and cure in homeostasis. | mesenchymal stem cells (mscs) are of special interest as therapeutic agents in the settings of both chronic inflammatory and autoimmune diseases. toll - like receptors (tlr) ligands have been linked with the perpetuation of inflammation in a number of chronic inflammatory diseases due to the permanent exposure of the immune system to tlr - specific stimuli. therefore, mscs employed in therapy can be potentially exposed to tlr ligands, which may modulate msc therapeutic potential in vivo. recent results demonstrate that mscs are activated by tlr ligands leading to modulation of the differentiation, migration, proliferation, survival, and immunosuppression capacities. however inconsistent results among authors have been reported suggesting that the source of mscs, tlr stimuli employed or culture conditions play a role. notably, activation by tlr ligands has not been reported to modulate the immunoprivileged phenotype of mscs which is of special relevance regarding the use of allogeneic msc - based therapies. in this review, we discuss the available data on the modulation of mscs activity through tlr signalling. |
high blood pressure occurs in around 80% of patients with acute ischemic stroke and usually decreases over the following 7 days. whether this elevated blood pressure is a physiological and protective reaction or a side effect of a generalised stress reaction that is in fact harmful to the newly damaged brain and whether it should be lowered or not is yet to be answered. american (aha) and european (eso) guidelines recommend not to treat elevated blood pressure unless above 220/120 mmhg and describe an urgent need for large well - designed trials to address this issue [4, 5 ]. currently there are two large ongoing trials : the efficacy of nitric oxide in stroke trial (enos) compares nitric oxide versus placebo and continuing versus stopping previously taken antihypertensive agents in acute stroke. enchanted compares low dose versus normal dose of recombinant tissue - type plasminogen activator (r - tpa) and intensive blood pressure reduction versus standard reduction in thrombolysed patients. this article focuses on currently available data, the contraries of the results, and potential solutions. a post hoc analysis of the international stroke trial found a u - shaped relationship between initial blood pressure and outcome, indicating that systolic blood pressure of around 140180 mmhg is associated with best outcome. these findings were confirmed in another study showing that very high and very low initial blood pressure is associated with increased death rates. high blood pressure was also independently associated with recurrent ischemic stroke and cerebral edema [1, 9 ]. patients with very low initial blood pressure had a higher incidence of heart failure and coronary heart disease. high blood pressure after acute ischemic stroke causes brain edema, and brain edema decreases cerebral blood flow [11, 12 ], thus aggravating cerebral ischemia. another mechanism could be that endogenous tpa release is reduced when blood pressure is high due to the damage at the endothelium [1315 ]. one critique of those studies is that blood pressure was measured only on admission, usually several hours after stroke onset [1, 8, 16, 17 ]. however, variability and duration of high blood pressure in the first hours after stroke onset may be more relevant for prognosis. several observational studies looked into the prognosis of spontaneous change in blood pressure during the acute phase with divergent results. the barcelona downtown stroke registry found that early decrease of systolic blood pressure by 2030% was associated with full recovery (or 2.9, 95% ci 1.36.3). a decrease in systolic blood pressure within 12 hours spontaneous decrease of blood pressure within 24 hours was associated with poor outcome and early neurological deterioration. there might be a bias when looking into spontaneous decrease of blood pressure and correlating it to outcome. rapid spontaneous decrease might reflect less severe stroke, recanalization of the vessel, or might be due to cardiac insufficiency, all of which have an independent effect on outcome. so it might be a sign for a rapidly resolving or rapidly deteriorating process, explaining the divergent results. several randomised controlled trials addressed the topic of blood pressure management in acute stroke (see table 1). the trials are heterogeneous regarding time to treatment, degree and speed of effective blood pressure reduction, substances used, and the stroke subgroups included (i.e., ischemic, hemorrhagic strokes, or both). beta - blockers given within 48 hours after hemispheric stroke increased mortality (best). the treatment groups, however, were unbalanced with more severely disabled patients in the group receiving beta blockers. also patients did not have to have elevated blood pressure to be included in the trial. a metaanalysis of nine trials showed a significant benefit from oral nimodipine versus placebo when given within 12 hours of symptom onset. in contrast, intravenous nimodipine given as 1 mg / h and 2 mg / h increased the rate of poor neurological and functional outcome in ischemic stroke patients compared to placebo (inwest). a correlation was found between the amount of diastolic blood pressure reduction in the high - dose group and poor functional outcome. intravenous magnesium administered within 12 hours lowered systolic blood pressure by 4 mmhg and showed a trend towards improved outcome in all stroke patients (odds ratio : 0.95, 95% ci 0.801.13, p = 0.59). for the subgroup of patients with elevated blood pressure (mean arterial pressure > 108 mmhg), improvement was significant (odds ratio of 0.78, 95% ci 0.610.99). smaller trials with glyceryl trinitrate showed no significant difference in functional outcome [26, 27 ] but were also not powered to do so. the early administration of labetalol and lisinopril lead to a significant reduction in blood pressure as compared to the placebo group (mean systolic difference 10 mmhg, 95% ci 317, p = 0.004) (chhips). death and dependency at 3 months did not differ, but 3 months mortality was halved in the treatment group to 9,7% from 20,3% (hr 0.4, 95% ci 0.21.0, p = 0.05). the cossacs trial looked into the question whether previously taken antihypertensive agents should be stopped or continued in patients with acute stroke. the medication reduced blood pressure significantly (13/8 mmhg lower at two weeks). no difference was seen in the primary outcome death or dependency at two weeks, but there was a significant functional improvement in the subgroup of patients with confirmed acute ischemic stroke (relative risk 0.70, 95% ci 0.510.99, p = 0.045). another trial (scast) compared candesartan to placebo within 30 hours after onset looking at a combined vascular endpoint. blood pressure reduction was only moderate, and no significant differences in the endpoints were seen. a trend favouring placebo was noticed. a metaregression on actively lowered blood pressure in acute stroke including 9008 patients from randomized trials showed the lowest odds ratio (0.95, 95% ci 0.111.72) for death or dependency with a decrease of systolic blood pressure by 14 mmhg. two cochrane analyses concluded that there is insufficient evidence to evaluate the effect of blood pressure changes on outcome after acute stroke [32, 33 ]. the management of blood pressure in acute ischemic stroke remains unclear due to divergent results of the studies. the effect might be dependent on level of initial blood pressure, time to treatment, stroke severity, and history of hypertension, as well as intensity of blood pressure lowering, substances used, and administration form (e.g., i.v. or oral). it is evident that lowering blood pressure of normotensive patients in acute stroke may lead to hypotensive events, thus deteriorating outcome. the negative effect of the best trial might partly be explained by inclusion of normotensive patients with systolic blood pressure > 100 mmhg. in a subgroup of images only those patients with higher blood pressure levels had a benefit (mean arterial pressure > 108 mmhg). this matches with chhips and cossacs in which mean systolic level was high (181 mmhg and 150 mmhg), and decreased mortality and improved functional outcome in the subgroup with ischemic stroke were seen, respectively [28, 29 ]. observational data showed that low initial blood pressure increased poor outcome [1, 8 ]. however, there are also trials in which treating elevated blood pressure did harm (inwest) or showed a trend favoring placebo (scast) [23, 30 ]. significant benefit from oral nimodipine versus placebo was seen when given within 12 hours after onset, no benefit between 12 and 24 hours, and worse outcome when initiated after 24 hours. in images, magnesium was given early at a mean of about 7 hours after onset (max. 12 hours) and showed a benefit in those with elevated blood pressure. in scast, treatment was started after about 17 hours and did no benefit. in fact, the later treatment was started, the more did candesartan seem to harm the patient regarding the composite vascular endpoint. other trials showed the opposite. nimodipine was started early (11 hours after onset) and had a negative effect. chhips and cossacs started treatment late and were neutral, even with some positive secondary endpoints [28, 29 ]. although the start of treatment is known for most of the trials, the lag until achieving effective blood pressure reduction is not. it is agreed that a rapid and large decrease in blood pressure is dangerous for acute stroke patients. this opinion is largely based on the data from inwest and some observational data [19, 20, 23 ]. high - dose (but not low - dose) intravenous nimodipine was associated with death and dependency when diastolic blood pressure was lowered by > 20% within the first two days. a secondary analysis of scast showed that a large systolic decrease of > 28 mmhg was significantly associated with poor outcome. magnesium reduced blood pressure within 24 hours by 4/3 mmhg and showed a benefit for those with higher blood pressure. in chhips the systolic blood pressure difference was 10 mmhg within the first 24 hours, and mortality was halved after 3 months. a decrease of even 16 mmhg was achieved within 4 hours with labetalol compared to placebo, and this was found to be safe. so the rapid reduction does not seem to be dangerous, as long as reduction is moderate. some trials included various classes [28, 29 ], and others looked into the effect of a single one. the calcium channel blocker nimodipine had a positive effect when given orally within 12 hours, but a negative effect when given intravenously. the angiotensin - converting - enzyme inhibitor lisinopril had a positive effect on 3 months mortality, and the angiotensin receptor antagonist candesartan possibly has an injurious effect on the brain. nitric oxide donors found stable cerebral blood flow albeit reducing blood pressure effectively [27, 36 ], and the mixed alpha / beta - receptor antagonist labetalol showed reduction of 3-month mortality. several data showed that lowering blood pressure in acute ischemic stroke is safe in terms of early neurological deterioration and functional outcome [2629 ], even in the presence of carotid artery stenosis and that it does not decrease perfusion in the region of the infarct [27, 36, 3840 ]. trials looking at the effect of blood pressure reduction in the acute phase of stroke are heterogeneous in design, classes of antihypertensive agents, achieved blood pressure reduction, and influence on outcome. although the guideline recommendation is not to lower blood pressure in the range up to 220/120 mmhg, there might be some neuroprotective effect of very early (first 710 hours after stroke) and rather moderate (1020 mmhg) blood pressure lowering. substance classes may differ in that respect, but care should be taken with intravenous calcium channel blockers and angiotensin receptor antagonists. adequately powered, randomized trials, however, are urgently warranted to substantiate those speculations. | high blood pressure is common in acute stroke patients. very high as well as very low blood pressure is associated with poor outcome. spontaneous fall of blood pressure within the first few days after stroke was associated both with neurological improvement and impairment. several randomized trials investigated the pharmacological reduction of blood pressure versus control. most trials showed no significant difference in their primary outcome apart from the inwest trial which found an increase of poor outcome when giving intravenous nimodipine. nevertheless, useful information can be extracted from the published data to help guide the clinician 's decision. blood pressure should only be lowered when it is clearly elevated, and early after onset, reduction should be moderate but may be achieved rapidly. no clear recommendations can be given on the substances to use ; however, care should be taken with intravenous calcium channel blockers and angiotensin receptor antagonists. two ongoing randomized trials will help to solve the questions on blood pressure management in acute stroke. |
local canonical correlation analysis (cca) is a multivariate statistical method in fmri that uses the joint time course of a group of neighboring voxels, usually in a 3 3 in - plane voxel grid, to determine the significance of activation. the value of a suitable test statistic is used as a measure of activation. since the joint time course of the neighborhood is used, it is not immediately clear to which voxel the measure of activation should be assigned. for example, if a 3 3 voxel neighborhood is chosen and the measure of activation is significant, without further assumptions one can only conclude that activation occurred somewhere within the 3 3 voxel neighborhood. if the activation is assigned to all voxels of the neighborhood, loss of spatial specificity will occur. to increase spatial specificity, it has been proposed to assign the measure of activation to the center voxel of the 3 3 neighborhood [1, 2 ]. a center voxel assignment is usually justified by mathematical convenience but can also be reasoned on the fact that the fmri bold response leads to patches of activation patterns that are most likely of convex shape and simple connectivity (without any holes in the interior neighborhood). however, this center voxel assignment proved to be prone to yield artifacts as activations tend to bleed to the neighboring voxels of strongly active voxels. the smoothing artifact is not only common in conventional cca, but also in any analysis technique that involves spatial low - pass filter kernels, such as univariate (single voxel) analysis where the data have been preprocessed using gaussian spatial smoothing. in conventional data smoothing, the smoothing artifact has been intentionally induced to increase the signal - to - noise ratio at the cost of reduced specificity and occurrence of typical spatial low - pass artifacts such as blurring of edges of activation patterns. to compensate for the smoothing artifact in conventional cca for example, a minimum relative weight for the center voxel was used to restrict false activations. in another study using a more adaptive approach, the smoothing artifact was reduced by utilizing the spatial dependence among voxels as much as possible and assigning the significance of activation to the dominant voxel of local maxima. this method was shown to be effective in eliminating the smoothing artifact in motor activation data that is known to have large contrast - to - noise ratio (cnr), however, in data where the activation is more subtle (such as hippocampal activation using an episodic memory paradigm), the method has the disadvantage of being less sensitive, according to our studies. to reduce the smoothing artifact in cca, it is necessary to constrain the spatial weights properly and impose the condition that the center voxel always has the largest weight. use nonnegative spatial weights with maximum weight of the center voxel in order to ensure spatial low - pass filter properties of ccca. this has the additional benefit of constraining cca to eliminate spurious correlations occurring in conventional cca where spatial filters can have positive and negative coefficients. recently, we provided a mathematical framework for ccca and computed roc properties of ccca with different linear constraints and a nonlinear constraint for activation patterns of motor data and episodic memory data [7, 8 ]. in this paper we expand our previous research and investigate in detail the smoothing artifact that is associated with each spatial constraint in ccca. furthermore, we provide a novel approach of how to correct the measure of activation for the smoothing artifact. parts of this paper have been published in abstract form (one page) at a recent conference. in the following we briefly review cca and ccca, and explicitly consider the constraints introduced recently. mathematically, cca is a generalization of the general linear model (glm) by allowing the incorporation of spatial basis functions according to (1)(1f1()++sfs())y(,t) = 1x1(t)++rxr(t)+(t), where the data are given by y(, t), is the vector representing the spatial coordinates x, y, and z, and t is time. the functions fi(), i = 1,, s represent the spatial basis functions modeling the activation pattern in a neighborhood. the functions xj(t), j = 1,, r are the temporal basis functions modeling the signal observed (which is the result of a convolution of the hemodynamic response function and the stimulus function). the coefficients i and j are the spatial and temporal weights, respectively, that are being determined and optimized by the data for each individual neighborhood using an optimization routine. the symbol denotes spatial convolution and (t) is a gaussian - distributed random error term. if the number of spatial basis functions is reduced to a single function, (1) becomes (2)f1()y(,t)=1x1(t)++rxr(t)+(t). when f1() is a simple gaussian function, we obtain the conventional glm used frequently in fmri. we assume that the functions fi(), i = 1,, s are spatial dirac delta functions defining a local neighborhood within a 3 3 pixel neighborhood (s 9). let y be the matrix representing s voxel time courses with dimension t s and x the conventional design matrix of size t r for the r temporal regressors. furthermore, let and be two unknown vectors of size s 1 and r 1, respectively. in cca, we look for the linear combinations of voxel time courses y and temporal regressors x such that the correlation between both quantities is maximum. this leads to an eigenvalue problem with min(s, r) solutions from which the solution with the largest eigenvalue (i.e., maximum canonical correlation) is being chosen. without constraints on the i, the specificity of the activation pattern obtained by cca is low and could result in artifacts (see e.g.,). to put constraints on the spatial weights in order to restrict the space of unreasonable solutions for fmri, we consider the following four scenarios for the components i of, where 1 is the weight for the center voxel and the other i 's represent the weights for the s neighborhood voxels. one has (6)1max(i)>0, i0 i2.note that the neighborhood size s is not a fixed quantity, but is determined from the data by ccca and can differ for each center voxel. one has (4)1i=2si>0, i0 i2. constraint 3 (average constraint). one has (6)1max(i)>0, i0 i2. the smoothing artifact in cca is defined as the probability of incorrectly declaring the center voxel of a configuration of size s (s 9 for a 3 3 neighborhood) to be active. in the following, we outline how to compute the posterior probability to detect the smoothing artifact in real data using a bayesian framework. the posterior probability, p, that a center voxel is not active when it was in fact declared active, is given by (7)p = p(center voxel is not active >0,cnr, m, cnr, s), where > 0 indicates that the center voxel was declared active (statistic > threshold 0 with [0,)), cnr is the univariate contrast - to - noise ratio of the center voxel, m labels the method of data analysis, cnr is the contrast - to - noise ratio of the entire configuration defining the neighborhood within a 3 3 pixel region, and s is the size of the configuration (i.e., number of declared active voxels 9 within the neighborhood). for abbreviation, we define the set of parameters,, to be (8)={cnr, m, cnr, s}. then, according to bayes ' theorem for conditional probabilities, (7) can be written as (9)p = p(>0 center voxel is not active,)p(center voxel is not active)(p(>0))1, which is of the form (10)p = p1p3p2, where (11)p1=p(>0 center voxel is not active,),(12)p2=p(>0),(13)p3=p(center voxel is not active). the term p1 is called the bleeding artifact because it represents the probability that an inactive voxel is declared as active. we determine p1 as a function of the size, s, of the configuration only and not as a function of the geometrical shape of the configuration. note that the dependence on s is an approximation, because in reality there are 2 = 256 possible configurations that can contain 0 to 8 active voxels (corresponding to s { 1,, 9 } since s labels the neighborhood size within a 3 3 pixel grid, which always includes the center voxel, independent if the center voxel is active or not). each configuration of size s has, depending on its distance of all voxel members to the center voxel, a slightly different value for p1. for example, configurations with s = 7 leads to 3 different classes based on a distance measure, that is, class 1 = { center voxel, 4 corner voxels, and 2 midedge voxels }, class 2 = { center voxel, 3 corner voxels, and 3 midedge voxels }, class 3 = { center voxel, 2 corner voxels, and 4 midedge voxels}. according to our simulations, p1 is strongly dependent on s but not on a particular configuration of s. only a weak dependence based on different class memberships exist, which we neglect for the purpose of this research. to estimate p1, it is thus reasonable to group all configurations for a particular s together and compute an average value of p1 over all possible configurations with size s. the term p1 can be estimated from simulations using a mixture of resampled resting - state data and activation data at given using kernel density estimation. resampled resting - state data are considered null data with respect to any task fmri function since the temporal structure is destroyed by resampling using the wavelet transform. this resampling, however, does not destroy the autocorrelations inherent in resting - state data. furthermore, the resampling does not affect the spatial correlations within the data because the permutations of the wavelet coefficients are kept the same for each voxel time series in a particular simulation ; however, different simulations use different permutations [11, 12 ]. the simulated data are superpositions of time series from a 3 3 pixel neighborhood of null data and activation data. since the entire neighborhoods are used from resting - state data, realistic spatial correlations of the simulated data are obtained. in particular, for a configuration of s active voxels in the 3 3 neighborhood, the simulated voxel time courses, yi(t), are obtained by (14)yi(t)={yi(0)(t),for i=1,x(t)+yi(0)(t),for i {2,,s }, where i = 1 refers to the center voxel and all other i to the surrounding voxels of the configuration of size s within the 3 3 neighborhood. all yi(t) correspond to resampled resting - state time courses and represent spatially and temporally correlated null (noise) data. thus, p1 is a strong function of cnr of the configuration but not of the value cnr (which is the contrast - to - noise ratio of the inactive center voxel), and the dependence of p1 on cnr can be neglected. the activation is determined by the hemodynamic response function, x(t), of interest multiplied by factor so that the configuration has a given cnr. in order to compute the cnr we use the general definition (15)cnr=(ii)1/2, where i and i are the eigenvalues of the covariance matrix of the activation signal and noise, respectively. note that (15) can be used for a single voxel time series or an entire neighborhood of arbitrary size. to determine the activation signal and noise of a configuration using ccca, we convert the ccca problem into a multivariate multiple regression problem of the form (16)y=xb+e, where y are the data (size t s), is the optimum spatial weight vector (size s 1), x is the design matrix (size t r), b is the matrix of regression weights (size r s), and e is a residual error matrix (size t s). for a given contrast vector c, we reparameterize the design matrix x and obtain a transformed design matrix x~ such that (17)x~=[xeff x ], where (18)xeff = x(xx)1c(c(xx)1c)1 is the first regressor of the new design matrix x~ that is associated with a parameter estimate equivalent to the original contrast cb [8, 14 ]. the matrix x is perpendicular to xeff and plays no role in the estimation of c. then, the signal s(t) is obtained by (19)s = xeffb, and the noise n(t) is obtained by (20)n=(yxeffb). this term can be estimated directly from the real data. in this case, for each m and s > 1, p2 is a 2d function of cnr and cnr, but depends strongly only on cnr so that the dependence on cnr can be neglected. note that for s = 1, cnr = cnr, and in this case p2 is a 1d function of cnr only. it is possible to determine first the joint probability density p(, cnr | s, m) using 2d kernel density estimation with a 2d gaussian kernel, which then can be integrated numerically to obtain p2 according to (21)p2(0,cnr, s, m)=0p(,cnr s, m)d0p(,cnr s, m)d. note that p2(0, cnr, s, m) for fixed { 0, s, m } has a sigmoidal shape approaching the value 1 for cnr > 0.6. thus, voxels that are declared active at a family - wise error rate (fwe) 0,)p(>0 center voxel is not active,)p3(cnr)p(>0)p1(0,cnr, m, s)p3(cnr), since p(> highly active (i.e., fwe 0.5 and assign zero to the measure of activation if this statement is true. fmri was performed for 6 normal subjects with irb approval (according to institutional requirements) in a 3.0 t ge hdx mri scanner equipped with an 8-channel head coil and parallel imaging acquisition using epi with imaging parameters : asset = 2, ramp sampling, tr / te = 2 sec/30 ms, fa = 70 deg, fov = 22 cm 22 cm, thickness / gap = 4 mm/1 mm, 25 slices, and resolution 96 96. we briefly describe the paradigms and refer the reader for more detail to our previous article. the first data set was collected during resting - state where the subject tried to relax and refrain from executing any overt task with eyes closed. the second data set was collected while the subject was performing an episodic memory task with oblique coronal slices collected perpendicular to the long axis of the hippocampus. specifically, this task consisted of memorization of novel faces paired with occupations and contained 6 periods of encoding, distraction, and recognition tasks as well as short instructions where words on the screen reminded subjects of the task ahead. the third data set was obtained by performing an event - related motor task involving bilateral finger tapping while the subject was looking at a screen. all fmri data were realigned using statistical parametric mapping (spm5, http://www.fil.ion.ucl.ac.uk/spm/) and maximum motion components were found to be less than 0.6 mm in all directions. in a preprocessing step, all voxel time series were corrected for different slice timings and high - pass filtered by regression using a discrete cosine basis with cut - off frequency 1/120 hz. no temporal low - pass filtering was carried out. all voxels with intensity larger than 10% of the mean intensity this threshold effectively eliminated all nonbrain voxels leading to an average of about 4500 voxels per slice. all activation maps were thresholded using a fwe 0.6). these two ccca methods have very high sensitivity but can lead to false activations when the configuration size is large. the function p2(0, cnr, s, m) = 0p(, cnr | s, m)d/0p(, cnr | s, m)d was calculated in matlab (http://www.mathworks.com/) by 2d kernel density estimation of p(, cnr | s, m) using an optimum bandwidth estimator according to sheather and jones. in general, p(, cnr | s, m) has a bimodal distribution for configuration sizes s { 2,3, 4,5, 6,7}. for lower s, the larger mode of the density occurs at lower values of {, cnr }, whereas for larger values of s, the larger mode occurs at higher values of {, cnr}. for s { 8,9 }, the density becomes unimodal with mode located at large values of {, cnr}. also note, that is strongly correlated with cnr, which is expected. an example of p(, cnr | s, m) is given in figure 2 for s = 5 and ccca with the maximum constraint. the shape of p2(0, cnr, s, m) obtained by numerical integration of 0p(, cnr | s, m)d/0p(, cnr | s, m)d and density smoothing is shown in figure 3 for all s and 0 cnr 1. note the s - shaped form obtained for p2(0, cnr, s, m) for all integrations of bimodal distributions involving p(, cnr | s, m), whereas for s = 1 the function p2 is zero for cnr 1 and for s { 8,9 } p2 has the value 1 for 0 0.1 confirming that ccca with the sum constraint has largest specificity of the proposed ccca methods. the activation patterns that are corrected for the smoothing artifact show small changes compared to the uncorrected ones, however, these changes can provide important information of the activation profile. for example, in figures 8 and 9 we show a magnified region of the left motor cortex and the right hippocampus, respectively, for selected analysis methods (single voxel with and without gaussian smoothing, ccca with the maximum constraint). here we see that correction for the smoothing artifact leads to a separation of the right motor cortex (see green arrows in figure 8). this result is consistent with the activation pattern from single voxel analysis without gaussian smoothing. we believe that for the motor activation data, single - voxel analysis is already accurate due to the high cnr of the bold response for motor activation. regarding the hippocampal activation, we see that the correction for the smoothing artifact leads to a clear separation of hippocampal activation into three focal regions (see blue arrow in figure 9). it is conceivable that the corrected activation maps are more accurate representations of true hippocampal activations in this high - resolution study because it is known that the hippocampus is composed of the ca fields (ca1, ca2, ca3, and ca4), the dentate gyrus and subiculum, and each of these subregions has a specific function in memory. the obtained corrections of the activation pattern are more probable than a continuous elongated activation pattern obtained with ccca without correction for the smoothing artifact. this condition is still a conservative correction for activation maps. to obtain better specificity but at a cost of losing sensitivity tables 1 and 2 show the number of voxels affected by the smoothing artifact for thresholds 0.1 to 0.5. note that lowering the threshold for p to 0.2 leads to a dramatic increase in the number of voxels. thus, p > 0.2 should be avoided. the choice p > 0.3 is probably a good compromise of achieving better specificity and still maintaining high sensitivity for the examples shown here. however, the decision to use a lower threshold than 0.5 will primarily dependent on the particular application of the research. we preferred p > 0.5 which lead to a relatively small number of voxels that needed to be corrected. with this threshold the sensitivity of the methods is still very large and mostly voxel configurations of sizes 4 to 8 in motor data and 3 to 7 in memory data were affected by the smoothing artifact (figure 10). note that ccca with the max constraint leads to larger configuration sizes (mean value s = 5.6) that are affected by the smoothing artifact than ccca with the average constraint (mean value s = 4.3). this fact is expected due to the increased freedom of the spatial constraints in ccca with the maximum constraint leading, on average, to larger configuration sizes which are more probable to induce a smoothing artifact than the other constrained ccca methods. we summarize the ideas introduced in this study and results obtained as follows.we investigated the smoothing artifact in cca and proposed a new technique to reduce this artifact in fmri data analysis. using data from a motor activation paradigm and an episodic memory paradigm, we showed examples of activation maps obtained with constrained cca methods, the corresponding magnitude of the smoothing artifact, and activation maps corrected for the smoothing artifact.for all data studied, we found no appreciable smoothing artifact for ccca with the sum constraint. the best overall performance was obtained by ccca with the maximum constraint corrected for the smoothing artifact. we recommend this technique for fmri data analysis to obtain high sensitivity and good specificity. we investigated the smoothing artifact in cca and proposed a new technique to reduce this artifact in fmri data analysis. using data from a motor activation paradigm and an episodic memory paradigm, we showed examples of activation maps obtained with constrained cca methods, the corresponding magnitude of the smoothing artifact, and activation maps corrected for the smoothing artifact. for all data studied, we found no appreciable smoothing artifact for ccca with the sum constraint. the best overall performance was obtained by ccca with the maximum constraint corrected for the smoothing artifact. we recommend this technique for fmri data analysis to obtain high sensitivity and good specificity. | a wide range of studies show the capacity of multivariate statistical methods for fmri to improve mapping of brain activations in a noisy environment. an advanced method uses local canonical correlation analysis (cca) to encompass a group of neighboring voxels instead of looking at the single voxel time course. the value of a suitable test statistic is used as a measure of activation. it is customary to assign the value to the center voxel ; however, this is a choice of convenience and without constraints introduces artifacts, especially in regions of strong localized activation. to compensate for these deficiencies, different spatial constraints in cca have been introduced to enforce dominance of the center voxel. however, even if the dominance condition for the center voxel is satisfied, constrained cca can still lead to a smoothing artifact, often called the bleeding artifact of cca, in fmri activation patterns. in this paper a new method is introduced to measure and correct for the smoothing artifact for constrained cca methods. it is shown that constrained cca methods corrected for the smoothing artifact lead to more plausible activation patterns in fmri as shown using data from a motor task and a memory task. |
the use of visible and near - infrared (nir) light, penetrable to deep tissue, is an attractive method of spatiotemporally controlling drug release from various drug delivery forms, such as prodrugs, liposomes, polymers, and other nano- and macrodelivery systems. however, because of its lower energy, it is difficult to directly cleave a chemical bond using such light. thus, novel mechanisms using lower energy light to trigger the release of biologically active compounds have been a major topic of interest. the photodynamic process and the unique chemistry of singlet oxygen (so) were adopted to mediate lower energy light release of drugs. so is formed during the photodynamic process and reacts with electron - rich olefins to form unstable dioxetanes. we named the cleavage of aminoacrylate by so photo - unclick chemistry, and demonstrated the release of the anticancer drug combretastatin a-4 (ca4) from its prodrug using this method. we prepared cmp - l - ca4, a ca4 prodrug that can be activated by far - red light (690 nm), by combining a so - labile aminoacrylate linker, a core - modified porphyrin (cmp) photosensitizer, and ca4. the prodrug released ca4 and enhanced cytotoxicity upon illumination. the released ca4 damaged cancer cells, demonstrating our system s ability to generate bystander effects in vitro. we found that the antitumor effects of cmp - l - ca4 were superior to the effects of its pseudo - prodrug cmp - ncl - ca4 (ncl = noncleavable linker). the ability to optically image the light - activatable prodrug is useful for determining an illumination time when the prodrug is at its maximum concentration at the target site. if the prodrug accumulates in the target sites, we can use optical imaging to detect the target areas, as well as to treat the disease. we expected that we could optically image our photounclickable prodrug in vivo using a fluorescent photosensitizer. thus, we could track the prodrugs and then treat the tumor with the combined effects of photodynamic therapy (pdt) and local chemotherapy (figure 1a). the released drug could damage the cancer cells that survived the initial pdt damage through bystander effects (figure 1b). (a) multifunctional prodrug for optical imaging and combined treatment with pdt and local chemotherapy. (b) bystander effects from the released drugs can kill cancer cells that survive pdt damage. [the lifetime of so is short (submicrosecond scale). thus, direct cell damage by so occurs only during illumination. the light dose used for ] we generated pc-(l - ca4)2, an advanced multifunctioning ca4 prodrug, for both fluorescence optical imaging and combination therapy with pdt and released ca4. we chose phthalocyanine (pc) because pc is a fluorescent photosensitizer that can generate both fluorescence and singlet oxygen. although fluorescence emission and so generation are competing processes, pc has uniquely balanced yields of both functionality (i.e., si - pc : o2 = 0.22 and f = 0.4) with a high molar extinction coefficient (). its brightness (bt) is greater than that of cmp (e.g., = 150,000 m cm at 675 nm, bt = 6000 m cm for pc vs = 5000 at 690 nm, bt = 50 m cm for cmp). this pseudo - prodrug is similar to pc-(l - ca4)2 in structure, but can not release ca4 upon illumination. it will mimic the pdt effects of pc-(l - ca4)2, but can not induce damage from released ca4. we evaluated the cytotoxic effects of these two prodrugs with and without illumination, the inhibition of tubulin polymerization, the in vitro bystander effects, tumor localization using optical imaging, and the antitumor effects. we developed a synthetic scheme using high - yield reactions, such as esterification, nucleophilic substitution, and the yne - amine reaction, to make the process easily adaptable to other alcohol - containing drugs (scheme 1). a nucleophilic substitution reaction of silicon phthalocyanine dichloride (pc - cl2) yielded compound 3. pc-(l - ca4)2 was synthesized through a click (yne - amine) reaction of compounds 1 and 3. under the basic condition, pc-(ncl - ca4)2 overall, the synthesis was straightforward and all steps gave high yields (> 73% each step). reagents and conditions : (i) propynoic acid, dcc, dmap, ch2cl2, room temp, 24 h ; (ii) 1,3-dibromopropane, anhydrous k2co3, acetone, reflux, 12 h ; (iii) 2-(piperazin-1-yl)ethanol, pyridine, toluene, reflux, 4 h ; (iv) 1, anhydrous thf, 30 min ; (v) 2, anhydrous k2co3, acetone, reflux, 12 h. we formulated the prodrugs using peg pla [poly(ethylene glycol)-poly(d, l - lactide) ] copolymer micelles to take advantage of the enhanced permeability and retention (epr) effect to enhance the delivery to tumor. the nanosized peg pla polymer micelle was expected to provide three advantages : (1) passive targeting to tumors via the epr effect, (2) prolonged circulation in the plasma, and (3) solubilization of the nonpolar prodrug. the zeta potentials and mean diameters of the micelles of pc-(l - ca4)2 and pc-(ncl - ca4)2 were determined by dynamic light scattering (dls) (zeta potential = 11.64 1.38 mv, 16.81 1.67 mv and mean diameter = 71.96 1.34 nm, 75.07 1.45 nm, respectively). to visualize the formation of the polymeric micelles, we used transmission electron micrographs (tem). tem images of the micelles showed consistent particle sizes (6178 nm for pc-(l - ca4)2 and 6580 nm for pc-(ncl - ca4)2 micelles). the stability of the micelles was monitored by their particle sizes and zeta potentials at 4 c under dark conditions. these values remained within 95% of the initial values for up to 21 days. (a) particle size distribution and tem images (inset) of micelles of (a) pc-(l - ca4)2 and (b) pc-(nlc - ca4)2. ca4 is known to inhibit tubulin polymerization by binding to the colchicine binding pocket of tubulin. because the bulky groups (pc - l and pc - ncl) are attached to ca4, we expected lower inhibitory activity of tubulin polymerization. we determined the effects of these prodrugs using the tubulin polymerization assay, in which fluorescence emission increases as tubulins polymerize (figure 3a). consistent with our data on the previous ca4 prodrug cmp - l - ca4, both pc-(l - ca4)2 and pc-(ncl - ca4)2 had significantly (p 800 mm in 12 days (figure 7b). the pdt effects resulting from pc-(ncl - ca4)2 treatment (g3) had a significant impact on tumor size until day 3 (p g3 > g4 during day 1 to day 4 [p 95% by high - performance liquid chromatography (hplc) (figures s9 and s10). 1-(2-hydroxyethyl)-piperazine (1.91 g, 14.70 mmol) and pyridine (2.5 ml) were added to a solution of silicon(iv) phthalocyanine dichloride (pc - cl2, 1 g, 1.63 mmol) in 50 ml toluene. the reaction mixture was refluxed for 12 h. the solvent was then removed under reduced pressure. the solvent of the combined organic layers was removed by evaporation, and the crude was recrystallized with chcl3/n - hexane (1:4 v / v) to give a blue solid compound 3 (1.22 g, 94%). h nmr (300 mhz, cd2cl2) 1.19 (t, j = 5.8 hz, 4h), 0.82 (t, j = 5.8 hz, 1h), 0.28 (m, 8h), 1.75 (m, 8h), 8.308.40 (m, 8h, pc - h), 9.559.69 (m, 8h, pc - h) ; c nmr (300 mhz, cd2cl2) 123.4, 131.0, 135.9, 149.2 ; hrms esi (m / z) : [m + h ] calculated for c44h43n12o2si, 799.3401 ; found, 799.3395. compound 1 (46 mg, 0.13 mmol) and compound 3 (50 mg, 0.06 mmol) were dissolved in 20 ml dry thf, and the solution was stirred at room temperature for 15 min. the solvent was removed under reduced pressure to yield the crude product, which was then recrystallized from chcl3/n - hexane (1:5 v / v) to give pc-(l - ca4)2 (83 mg, 87%). h nmr (300 mhz, cd2cl2)) 1.96 (t, j = 5.6 hz, 4h), 0.55 (t, j = 5.0 hz, 4h), 0.29 (br s, 8h), 2.20 (br s, 8h), 3.67 (s, 12h), 3.72 (s, 6h), 3.77 (s, 6h), 4.34 (d, j = 12.8 hz, 2h), 6.45 (d, j = 4.9 hz, 4h), 6.62 (s, 4h), 6.84 (d, j = 8.1 hz, 2h), 6.96 (s, 2h), 7.00 (s, 2h), 7.09 (d, j = 12.8 hz, 2h), 8.388.43 (m, 8h, pc - h), 9.659.70 (m, 8h, pc - h) ; c nmr (300 mhz, cd2cl2) 55.8, 56.6, 60.4, 81.8, 105.9, 111.8, 123.5, 123.9, 126.6, 128.7, 129.1, 129.8, 131.2, 132.5, 135.8, 137.2, 140.3, 149.3, 151.1, 152.2, 153.0, 167.2 ; hrms esi (m / z) : [m + h ] calculated for c86h83n12o14si, 1535.5921 ; found, 1535.5914. anhydrous k2co3 (68 mg, 0.500 mmol) and compound 3 (200 mg, 0.25 mmol) were added to a solution of compound 2 (218 mg, 0.50 mmol) in 10 ml dry dmf. the reaction mixture was stirred at room temperature for 12 h. the k2co3 was removed by suction filtration and the solvent was removed under reduced pressure. the crude product was then recrystallized from chcl3/n - hexane (1:5 v / v) to give pc-(ncl - ca4)2 (302 mg, 80%). h nmr (300 mhz, cd2cl2) 1.96 (t, j = 5.6 hz, 4h), 0.78 (t, j = 5.8 hz, 4h), 0.41 (br s, 8h), 1.50 (m, 2h), 1.90 (m, 2h), 2.18 (m, 2h), 2.49 (m, 2h), 3.32 (s, 4h), 3.60 (br s, 8h), 3.65 (s, 12h), 3.75 (s, 6h), 3.80 (s, 6h), 6.46 (m, 4h), 6.51 (m, 4h), 6.74 (m, 2h), 6.81 (m, 2h), 6.92 (m, 2h), 8.38 (m, 8h, pc - h), 9.66 (m, 8h, pc - h) ; c nmr (300 mhz, cd2cl2) 55.8, 60.4, 105.1, 111.4, 113.6, 115.2, 117.5, 123.4, 126.7, 126.8, 126.9, 130.9, 132.8, 135.9, 149.1 ; hrms esi (m / z) : [m + h ] calculated for c86h91n12o12si, 1511.6649 ; found, 1511.6612. briefly, 3 mg of pc-(l - ca4)2 or pc-(ncl - ca4)2 was dissolved in 1.3 ml of thf. pla (cat # ak09, vendor : polyscitech) was dissolved in 1 ml of thf. 600 l of the 3 mg of pc-(l - ca4)2 or pc-(ncl - ca4)2 dissolved in 1.3 ml thf was added to the mpeg - pla - thf mixture. the volume of the resulting mixture was reduced to 300 l under reduced pressure. the 300 l of the mixture of pc-(l - ca4)2 or pc-(ncl - ca4)2 and mpeg pla was added to 3 ml of distilled water dropwise while stirring. the mixture was stirred for 3 h, after which the organic solvent was evaporated under reduced pressure at 40 c. the concentration of pc-(l - ca4)2 or pc-(ncl - ca4)2 micelles was determined by diluting the micelles in thf : the absorbance was measured by absorbance of pc group. the concentration was calculated from the molar extinction coefficient (ec) of pc-(l - ca4)2 or pc-(ncl - ca4)2 at 675 nm in thf (ec of pc-(l - ca4)2 = 205,000 ; pc-(ncl - ca4)2 = 206,110 m cm) using the beer lambert law. the concentrations of pc-(l - ca4)2 or pc-(ncl - ca4)2 micelles were determined to be 211 and 210 m, respectively. freshly prepared pc-(l - ca4)2 and pc-(ncl - ca4)2 micelles were used for all the experiments. 2.0 mm stock solutions of pc-(l - ca4)2 and pc-(ncl - ca4)2 prepared in thf were further diluted with 5% cremophor el in pbs to achieve appropriate concentrations. the pc-(l - ca4)2 and pc-(ncl - ca4)2 micelles in an aqueous solution were characterized by measuring their hydrodynamic diameter and zeta potential via dynamic light scattering (dls). the size measurement was carried out at a concentration of 1.0 mg / ml of pc-(l - ca4)2 or pc-(ncl - ca4)2. pc-(l - ca4)2 and pc-(ncl - ca4)2 micelles were also imaged by transmission electron microscopy (tem) at 20,000 operating at 80 kv. tem samples were prepared by depositing 20 l of diluted pc-(l - ca4)2 and pc-(ncl - ca4)2 micelle solution on a 300 mesh copper tem grid with a carbon film. a fluorescence - based tubulin polymerization assay was conducted using a kit supplied by cytoskeleton, inc. the basic principle is that an increase in fluorescence will occur as a fluorescence reporter is incorporated into microtubules during the course of polymerization. the assay was performed following the experimental procedure described in version 2.1 of the tubulin polymerization assay kit manual. briefly, a drug in dmso stock solution was added to a mixture of tubulin and gtp in a buffer solution, to give a final concentration of 3 m. fluorescence was monitored (excitation = 360 nm and emission = 450 nm) every 2 min for 1 h. pcx and ca4 were included in the assay as positive controls, as well as a vehicle - only negative control. the cytotoxicity of pc-(l - ca4)2 and pc-(ncl - ca4)2 was determined with and without illumination. mcf-7 cells were maintained in minimum essential medium (-mem) supplemented with 10% bovine growth serum, 2 mm l - glutamine, 50 units / ml penicillin g, 50 g / ml streptomycin, and 1.0 g / ml fungizone. mcf-7 cells (5000 cells / well) were seeded on 96-well plates in the medium and incubated for 24 h at 37 c in 5% co2. stock pc-(l - ca4)2 and pc-(ncl - ca4)2 micelle solutions (200 m) were prepared in distilled water. the stock solutions were further diluted with medium to obtain the necessary final concentrations. the diluted solution (10 l) the plates were incubated for 24 h and then removed from the incubator. for the phototoxicity study : the uncovered plate was illuminated for 30 min using a diode laser (690 nm, 5.6 mw / cm). to ensure uniformity of the light during the illumination, each plate was shaken gently on an orbital shaker (lab - line, barnstead international). for the dark toxicity study : plates were kept in the dark for 30 min and then returned to the incubator. briefly, a 10 l solution of mtt (10 mg in 1 ml pbs buffer) was added to each well and the plate was incubated for 4 h at room temperature or 37 c. then, the mtt solution was removed and the cells were dissolved in 200 l of dmso. the absorbance of each well was measured at 570 nm with background subtraction at 650 nm. cell viability was quantified by measuring the absorbance of the treated wells compared to that of the untreated control wells, and expressed as a percentage. colon-26 cells were seeded at 5000 cells / well on 24-well plates and incubated for 24 h. stock pc-(l - ca4)2 and pc-(ncl - ca4)2 micelle solutions (200 m) were prepared in distilled water. 200 m of the stock solutions were added to wells (1 ml) to obtain appropriate final concentrations of both pc-(l - ca4)2 and pc-(ncl - ca4)2 at 25 nm. after 24 h incubation, the plates were illuminated from the bottom with a 690 nm diode laser at 11 mw / cm for 15 min. during illumination, half of each well was blocked with black masking tape (cat # t743 - 1.0, vendor : thorlabs). the illuminated plates were incubated for an additional 48 h. then, a calcein am live cell staining assay (cat # 4892 - 010-k, vendor : molecular, probes tervigen) was performed. the cells were washed once with 1 ml calcein am wash buffer, then 250 l fresh wash buffer and 250 l working reagent were added to the wells. fluorescent images were obtained with an olympus ix51 inverted microscope with a green fluorescence channel to visualize live cells. the concentrations of pc-(l - ca4)2 and pc-(ncl - ca4)2 micelles in aqueous solution were determined by diluting 10 l of the formulation stock in 1 ml of thf and measuring the absorbance of phthalocyanine. the concentration was calculated from the ec of pc-(l - ca4)2 at 672 nm in thf (ec of pc-(l - ca4)2 = 205,000 ; pc-(ncl - ca4)2 = 206,110 m cm) using the beer lambert law. we used four- to six - week - old balb / c mice to investigate the biodistribution and tumor targeting ability of the polymeric micelles. the mice were shaved before the imaging experiments, and were imaged using the ivis imaging system. the mice were injected with pc-(l - ca4)2 and pc-(ncl - ca4)2 in the micelle formulation (2 mol / kg, i.v.). as a comparison, fluorescence images were taken 0, 3, 6, 12, 24, 48, and 72 h after retro - orbital injection. before taking the images, the mice were anesthetized in an acrylic chamber with a 2.5% isoflurane / air mixture. the following parameters were used to acquire images with living image software : fluorescence mode, exposure time : 2 s, binning : medium, f / stop : 2, excitation : 675 filter (660690 nm), and emission : 720 filter (710730 nm). during post processing, image counts were adjusted to 3 10 as minimum and 6.0 10 a.u. as maximum color scale. four- to six - week - old balb / c mice (1820 g) were used for the murine tumor model. the mice were implanted sc with 2 10 colon 26 cells in pbs (100 l) on the lower back of the neck. the longest axis of the tumor (1) and the axis perpendicular to l (w) were used to calculate tumor volume (lw/2). we used stock solutions of pc-(l - ca4)2 and pc-(ncl - ca4)2 micelles in aqueous solution and further dilutions to achieve final doses as follows : [ca4, (1 mol / kg each) ], [pc-(ncl = ca4)2, (1 mol / kg each) ], [pc-(l - ca4)2, (1 mol / kg each) ], and [pc-(l - ca4)2, (2 mol / kg each) ]. to each mouse, 200 l of sample was injected via iv once on day 1. twenty - four hours later mice were anesthetized by ip injection of 80 mg / kg ketamine and 6 mg / kg xylazine. tumors were illuminated with a 690 nm diode laser at 100 mw / cm or 200 mw / cm for 30 min 180 or 360 j / cm, respectively. tumor size was measured every day after the treatment. to evaluate antitumor effect, mice from various groups the specimens were fixed in 10% buffered formalin, embedded in paraffin, and 4 mm diameter tissue sections were stained with hematoxylin and eosin following a standard procedure at the tissue pathology core facility at ouhsc. the sections were viewed and photographed by bright - field microscopy at 4 magnification. | we recently developed photo - unclick chemistry, a novel chemical tool involving the cleavage of aminoacrylate by singlet oxygen, and demonstrated its application to visible light - activatable prodrugs. in this study, we prepared an advanced multifunctional prodrug, pc-(l - ca4)2, composed of the fluorescent photosensitizer phthalocyanine (pc), an so - labile aminoacrylate linker (l), and a cytotoxic drug combretastatin a-4 (ca4). pc-(l - ca4)2 had reduced dark toxicity compared with ca4. however, once illuminated, it showed improved toxicity similar to ca4 and displayed bystander effects in vitro. we monitored the time - dependent distribution of pc-(l - ca4)2 using optical imaging with live mice. we also effectively ablated tumors by the illumination with far - red light to the mice, presumably through the combined effects of photodynamic therapy (pdt) and released chemotherapy drug, without any sign of acute systemic toxicity. |
anterior cervical discectomy and fusion (acdf) is the most frequently performed surgical treatment for several cervical spinal diseases, including herniated disc, compressive myelopathy, trauma and degenerative disease5,8). after decompression of spinal cord or nerve roots, interbody fusion should be performed for spinal stabilization. there are several fusion materials which have been used for several decades with effectiveness and safety10). most classical fusion grafts are autologous iliac bone grafts and tricortical iliac crest bone block. autologous bone has achieved favorable fusion, but it results in an additional wound of the harvest site with risk of morbidity2). another fusion material is polyetheretherketone (peek) cage and has been used with or without anterior cervical plate augmentation. without the anterior plate, higher subsidence rate has been reported12). besides the peek cage, allograft or autograft acdf many studies regarding the peek cage and autograft have been reported but there have been fewer studies about allograft in cervical spine3,14). allografting has been used for decades and the safety and effectiveness of allografts in acdf was reported11,20). in addition, we also studied the anterior screw insertion angle with two types of screws. between november 2010 and june 2012, a total of 55 patients who were diagnosed with degenerative cervical disc disease underwent acdf using allograft bone substitute and combined reinforcement with rigid plate system were selected. exclusion criteria was less than 10 months follow up period, posterior instrumentation after acdf, and inability to measure radiologic parameters. the patients were divided into 2 groups according to used screws because the mean screw insertion angle was significantly different between the two screws - fixed and variable types. group a consisted of 13 patients who underwent anterior plate fixation with fixed type screws. insertion angle was limited at 12 degrees (atlantis) and 8 degrees (vectra) in each direction from cephalad to caudal. the mean age was 55.310.32 years (range, 44 to 63 years) in group a, and 52.610.32 years (range, 44 to 63 years) in group b. there were 9 males and 5 females in group a, and 9 males and 7 females in group b. the mean follow - up period was 14.576.09 months (range, 12 to 34 months). there were 3 one - level fusions and 8 two - level fusions and 4 three - level fusions. there were 2 segments of c3 - 4 fusion (zero in group b), 7 segments of c4 - 5 fusion (6 in group b), 11 segments of c5 - 6 fusion (15 in group b), 7 segments of c6 - 7 fusion (7 in group b) (table 1). a single surgeon performed all operations with a standard smith - robinson anteromedial approach using a surgical microscope5,16). after discectomy and decompression of the neural component, the graft bone was inserted into the disc space during gentle distraction of vertebral bodies. the allograft was commercially used freeze - dried bone from a cadaver donor. vertebral plating system was selected according to the fusion level, from single to maximally 3 levels. regular follow up was provided immediately after surgery, at 1 month, 6 months, and 1 year after surgery, and the last follow - up. flexion - extension lateral views were also obtained at 6 months and 1 year after surgery. two independent neurosurgeons measured the following parameters with pacs digital software system(piviewstar, infinitt co., ltd, seoul, korea). segmental height, segmental lordotic angle and segmental interscrew angle in each time and change of segmental interspinous distance between flexion and extension in the last follow up dynamic radiography. three dimensional computed tomography (3d ct) was obtained at 1 year follow - up and compared with plain radiographs of bony fusion. segmental height was measured on the radiographs, which was the mean value of anterior and posterior height of fusion segment (fig. the subsidence was defined as a 2 mm reduction in the segmental height at 1 level fusion, a 4 mm reduction in the segmental height at 2 level fusion and a 6 mm reduction in the segmental height at 3 level fusion between immediately at the last follow - up. segmental lordosis was measured using cobb 's method to assess the sagittal alignment. also, the angle between the most upper and lower screws was also measured serially. the segmental screw angle was defined as the measured value by cobb 's methods between the upper and lower screws. nonunion was defined as the appearance of segmental instability with 2 mm widening of the interspinous distance on the flexion - extension lateral views at the last follow - up. we used the bridwell 's fusion grading system in ct scan4) : grade i : complete fusion, grade ii : partial fusion, grade iii : unipolar pseudoarthrosis, grade iv : bipolar pseudoarthrosis. between november 2010 and june 2012, a total of 55 patients who were diagnosed with degenerative cervical disc disease underwent acdf using allograft bone substitute and combined reinforcement with rigid plate system were selected. exclusion criteria was less than 10 months follow up period, posterior instrumentation after acdf, and inability to measure radiologic parameters. the patients were divided into 2 groups according to used screws because the mean screw insertion angle was significantly different between the two screws - fixed and variable types. group a consisted of 13 patients who underwent anterior plate fixation with fixed type screws. insertion angle was limited at 12 degrees (atlantis) and 8 degrees (vectra) in each direction from cephalad to caudal. the mean age was 55.310.32 years (range, 44 to 63 years) in group a, and 52.610.32 years (range, 44 to 63 years) in group b. there were 9 males and 5 females in group a, and 9 males and 7 females in group b. the mean follow - up period was 14.576.09 months (range, 12 to 34 months). there were 3 one - level fusions and 8 two - level fusions and 4 three - level fusions. there were 2 segments of c3 - 4 fusion (zero in group b), 7 segments of c4 - 5 fusion (6 in group b), 11 segments of c5 - 6 fusion (15 in group b), 7 segments of c6 - 7 fusion (7 in group b) (table 1). a single surgeon performed all operations with a standard smith - robinson anteromedial approach using a surgical microscope5,16). after discectomy and decompression of the neural component, the graft bone was inserted into the disc space during gentle distraction of vertebral bodies. the allograft was commercially used freeze - dried bone from a cadaver donor. vertebral plating system was selected according to the fusion level, from single to maximally 3 levels. regular follow up was provided immediately after surgery, at 1 month, 6 months, and 1 year after surgery, and the last follow - up. flexion - extension lateral views were also obtained at 6 months and 1 year after surgery. two independent neurosurgeons measured the following parameters with pacs digital software system(piviewstar, infinitt co., ltd, seoul, korea). segmental height, segmental lordotic angle and segmental interscrew angle in each time and change of segmental interspinous distance between flexion and extension in the last follow up dynamic radiography. three dimensional computed tomography (3d ct) was obtained at 1 year follow - up and compared with plain radiographs of bony fusion. segmental height was measured on the radiographs, which was the mean value of anterior and posterior height of fusion segment (fig. the subsidence was defined as a 2 mm reduction in the segmental height at 1 level fusion, a 4 mm reduction in the segmental height at 2 level fusion and a 6 mm reduction in the segmental height at 3 level fusion between immediately at the last follow - up. segmental lordosis was measured using cobb 's method to assess the sagittal alignment. also, the angle between the most upper and lower screws was also measured serially. the segmental screw angle was defined as the measured value by cobb 's methods between the upper and lower screws. nonunion was defined as the appearance of segmental instability with 2 mm widening of the interspinous distance on the flexion - extension lateral views at the last follow - up. we used the bridwell 's fusion grading system in ct scan4) : grade i : complete fusion, grade ii : partial fusion, grade iii : unipolar pseudoarthrosis, grade iv : bipolar pseudoarthrosis. there was no statistically significant difference between groups a and b in demographic data (table 1). fusion rates were 100% in group a and 75% in group b in our previous suggested definition, the interspinous distance of fusion segment less than 2 mm per level (table 2). there were 41 fusion segments assessed by 3 dimensional ct scan (table 3). complete fusion was achieved in 5 segments (29.4%) of group a and 13 (54.1%) of group b. grade ii fusion was seen in 2 segments (11.8%) of group a and 4 segments (16.7%) of group b. grade iii fusion, 4 segments (23.5%) of group a and 3 segments (12.5%) of group b. grade iv fusion, 6 segments (35.3%) of group a and 4 segments (16.7%) of group b. grade i fusion was more frequent in group b, but fisher 's exact test did not show statistically significance. subsidence was found 2 cases (15.4%) of group a and 3 cases (18.8%) of group b and did not showed significant difference. significant difference between the two groups was seen in the pre - operative segmental angle and immediate postoperative interscrew angle. between the preoperative and each follow - up period, many parameters showed significant differences (table 5). in group a the segmental angle was increased after fusion surgery and slightly decreased at the 1 year follow up period, but was not significant in later statistical analysis. segmental height was not changed after surgery, but decreased at 1 year follow up. b, there was no difference in segmental angles, but the segmental height was increased after surgery then decreased at 1 year follow up (fig. mean value of interscrew angle was decreased more in group b, but there was no significant difference between two groups. dense radiolucent line was identified around the allograft block, however intra graft bony bridge formation was seen. also, the interspinous distance change did not exceed 2 mm during flexion and extension. no graft malposition, migration, or mechanical failure of instruments was observed, and there was no revision surgery. no graft malposition, migration, or mechanical failure of instruments was observed, and there was no revision surgery. acdf is the most favorable and familiar method for treatment of cervical degenerative diseases, and also for trauma. myelopathy or radiculopathy is treated with decompression of neural elemetns, and osseous fusion is established to stabilize the cervical spine. there have been many studies about several fusion materials and plate augmentation, but they are still controversial. achievement of fusion without associated instrument complication may be the most favorable result in radiologic assessment, and this may also be correlated with clinical outcome. non - union or mechanical failure of instrument causes pain or neurologic symptoms, and rarely dysfunction and injury of the esophagus or prevertebral tissues. sometimes these complications may be treated by revision surgery which also has its own surgical risks. to assess the result of allograft fusion, we studied the radiologic analysis and also insertion orientation of anterior screws which are mandatory to bone graft fusion. allograft insertion causes distraction of the vertebral body because using small and loose grafts for fusion has a high risk of graft migration or malposition. segmental height increased after surgery and also lordotic angulation increased because of lordotic curvature of the allograft and anterior fixation of plate and screw. however, in the more angled screw group, postoperative segmental lordotic angle was slightly decreased and it was caused by pulling of the vertebral body by the screw insertion. loosening of screws and retropulsing movement is not a common complication of cervical acdf with plate augmentation, but it may be associated with soft tissue injury and non - union. in our study, the angled screw group showed slightly decreased segmental angles after operation. this may be explained by compression of vertebral bodies toward the screw and plate. according to our results, the screw angle or type does not have large role in fusion or subsidence. to maximize pull out strength, screws may be inserted with 90 degree angle to the plate and the longest size selected. experimental ex - vivo animal or cadaver models and finite element analysis may be needed for evidence which support our results. plain radiography is a simple exam which is cheap and with less radiation exposure than ct scans. dynamic view of the cervical spine provides segmental stability and additional information about adjacent segment degeneration. the most exact assessment is the 3-dimensional ct scan, but this is not always checked during follow up. in our study, a halo like area was seen on lateral plain radiographs, but there was a bony bridge through the graft cavity, and our 86.2% fusion rate is not significantly different from other reports13). many studies reported cage subsidence after fusion surgery and the subsidence rate varied between 43.1 - 50.5% by oh, and 23.4% in the study of yamagata.12,19). the risk factors associated with subsidence have been already reported. obesity, bone mineral density, and smoking are nonsurgical risk factors, while anteroposterior diameter of cage, and intraoperative distraction are surgical risk factors21). however subsidence does not always result in poor prognosis or aggravation of symptoms, and mostly does not cause symptoms or morbidity15,18,20). fusion may be achieved with subsidence of cage and nonunion may occur without subsidence14). medical conditions such as osteoporosis and obesity also affect nonunion or subsidence. repeated neck motion after surgery also affects cage subsidence. during the initial one or two months after surgery, recently, recombinant human bone morphogenic protein-2 (rhbmp-2) has been used in acdf. it still remains controversial with regard to safety in spine surgery, but additional use of rhbmp-2 may promote bone fusion after surgery7,17). another weak point of the allograft is the absence of an anchoring structure, such as metallic spikes in the peek cage. increasing compressive force between fusion segments may be helpful for fusion and prevent migration of the allograft, but it also increases the risk of allograft breaks. the relatively short follow up period in this study restricts evaluation of adjacent segment degeneration development. the retrospective nature of this review is another limitation of this study. to evaluate further information about allografts, further study may be needed with prospective, larger series and longer follow up period. genetic study or medical status which affects bone fusion may be added in further study. acdf with allograft bone block and plate augmentation achieves favorable radiologic results, which is not inferior to other fusion materials. also, the type or angle of screw fixation does not affect the fusion rate but may be associated with subsidence or decrease of segmental height. | objectiveto evaluate radiologic result of anterior cervical discectomy and fusion with allobone graft and plate augmentation, and the change of radiologic outcome between screw type and insertion angle.methodsretrospective review of clinical and radiological data of 29 patients. segmental angle, height and screw angles were measured and followed. the fusion rate was assessed by plain radiography and ct scans. we divided the patients into two groups according to screw type and angles. group a : fixed screw, group b : variable screw. interscrew angle was measured between most upper and lower screws with cobb 's methods.resultsoverall fusion rate was 86.2% on plain radiography. fusion was also assessed by ct scan and bridwell 's grading system. there was no difference in fusion and subsidence rates between two groups. subsidence was found in 5 patients (17.2%). segmental lordotic angle was increased from preoperative status and maximized at the immediate postoperative period and then reduced at 1 year follow up. segmental height showed similar increase and decrease values.conclusionacdf with allograft and plate showed favorable fusion rates, and the screw type and angle did not affect results of surgery. |
they have more conservative cavity designs which basically rely on the effectiveness of current dentin bonding agents. however, composite resins still have some limitations in use due to their physical properties,[3 - 5 ] polymerization shrinkage, microleakage, low wear resistance and color stability,[7 - 8 ] and lower bond strength to dentin compared with enamel. these properties are related to each other somehow ; polymerization shrinkage can cause composite debonding, leading to gap formation, microleakage, secondary caries and so on. thus composite resin bond strength to dentin plays an important role in restoration success since the major surfaces of a preparation to be bonded are in dentin, in different depths, sometimes without any enamel margins. although enamel and dentin are both highly hydrophilic, bonding mechanism seems to be different in each substrate because of basic differences in their organic and inorganic contents. therefore, bonding to dentin presents a much greater challenge than to enamel, especially in deeper dentin due to a decrease in inter - tubular dentin and an increase in tubular diameter as well as tubular fluid.[13 - 14]efforts to improve clinical performance of methacrylate - based composite resins have led to the development of new monomers such as ring - opening silorane and new filler technology such as nanofillers. filtek p90 (p90) and filtek z350 (z350) are two dental composite resins on the market that have lower polymerization shrinkage and improved mechanical properties, respectively when compared with other composite resins. ring - opening polymerization technique in the silorane system, like p90, is a recent important development in dental composite resins. the term silorane comes from siloxane and oxirane, which combines their two advantages, i.e. high hydrophobicity from siloxane and ring - opening monomer from oxirane, to make silorane, with a self - etch and primer adhesive system. the ring - opening process seems to be insensitive to oxygen - inhibiting action unlike methacrylate - based composite resins, which hinders polymerization by converting free radicals to stable species. generally, the mechanical properties of composite resins are related to their filler load ; the more filler load in the composition the higher the mechanical properties such as wear resistance, modulus of elasticity and less polymerization shrinkage. z350 is a recently introduced nanofilled composite resin with 6575 wt% of silica and zirconia nanofillers, which has been claimed to have a low shrinkage due to its high filler content. one strategy is to condition the dentin with disinfectants like ozone gas, oxalate desensitizer, naocl or edta and chx. in total etch adhesive systems, the adhesive may not reach the entire depth of demineralized dentin, thus using a penetration enhancer may improve adhesive penetration into demineralized dentin. recently tjderhane. evaluated the effect of dentin pretreatment with dimethyl sulfoxide (dmso : (ch3)2so) before application of the adhesive. this compound is one of the best known penetration enhancers for medical purposes due to its dipolar amphiphilic nature and small size.the potential for tissue penetration and excellent solvent properties make dmso an attractive solvent candidate for the dental adhesives.the few studies on dmso available in dentistry are limited to its cryoprotection ability in preservation of pulpal and periodontal ligament undifferentiated cells. to the best of our knowledge no studies have been carried out on deep and surface dentin pretreatment with dimethyl sulfoxide for the analysis of the shear bond strength. the aim of this in vitro study was to evaluate the effect of surface and deep dentin pretreatment with dimethyl sulfoxide on shear bond strength (sbs) of a silorane - based and a methacrylate - based composite resin. in this experimental in vitro study, 80 recently extracted human maxillary first premolars, without any caries and cracks, stored in 0.2% sodium azide, were used. the teeth were cleaned and mounted 2 mm below the cementoenamel junction in self - polymerizing acrylic resin (acropars, iran) to simulate tooth position in the alveolar bone. the teeth were randomly divided into two groups (n=40), for sections of surface dentin (group a) or deep dentin (group b). the occlusal surfaces of the teeth were sectioned using diamond discs (d&z ; germany) under water cooling to remove the occlusal enamel and reach the central groove to expose the surface dentin just beneath the dentinoenamel junction in group a and 2 mm below the central groove to meet the deep dentin in group b as in previous studies. the exposed dentin surface of all the specimens was ground with 600-grit abrasive paper to create a standardized smear layer. then the specimens in each group were randomly assigned to 4 subgroups (n=10), according to the type of composite resins used (two types) and pre - treatment (with or without pretreatment) (figure 1). composite resin build - up was done by using a 33-mm cylindrical translucent plastic mold, placed on the center of each specimen. a light - curing unit (coltolux ii ; coltene, usa) having a light output of 600 mw / cm was used to cure the adhesive systems and composite resins. the light cure unit has been checked frequently after preparation of 5 teeth by using a radiometer (cm 300 - 1000 ; halogen lightmeter, china). materials, manufacturers and chemical compositions statistically significant difference (p < 0.05). the mean values followed by at least one similar letter were not significantly different. subgroup a1 : silorane adhesive system primer (3 m espe, usa) was applied with a microbrush on dentin after shaking the bottle well, gently massaged for 15 s, dried with a gentle stream of air and light - cured for 10 s. then silorane adhesive system bond (3 m espe, usa) was applied, followed by a gentle stream of air and light - cured for 10 s. filtek p90 composite resin (3 m espe, usa) was placed in two increments of 1.5-mm thickness and each was light - cured for 40 s. subgroup a2:acid phosphoric etchant gel (etchant 37 ; denfil vericom co. ltd, korea) was applied on dentin for 15 s, rinsed with water thoroughly for 10 s and dried for 35 s with a gentle stream of air, leaving the surface slightly moist. two consecutive coats of adper single bond (3 m espe, usa) were applied and light - cured for 10 s after gentle drying. filtek z350 (3 m es pe, usa) was placed in two increments measuring 1.5 mm in thickness and each was light - cured for 40 s. subgroup a3:dentin was pretreated with 0.004% dimethyl sulfoxide (merck, germany), which was obtained from diluting 3.55 ml dmso with distilled water to have 100 ml 0.004% dmso, for 30 s and gently dried, followed by the steps described for subgroup 1. subgroup a4:etchant gel was applied on dentin for 15 s, rinsed thoroughly for 10 s and dried with a gentle stream of air, leaving the surface slightly moist. dentin was pretreated with 0.004% dmso for 30 s and gently dried, followed by the bonding and restoration steps described for subgroup 2. the teeth were stored in distilled water for 24 h at 37c to allow for water sorption and postoperative polymerization. the teeth were then thermocycled (pc300 ; vafaei, iran) in distilled water at 52/552c for 1000 cycles with a dwell time of 30 s. there was a significant interaction effect between composite resin type and dentin depth (p= 0.001), composite resin type and dmso pretreatment (p= 0.005) but not between dentin depth and dmso pretreatment (p= 0.82). each specimen was individually subjected to a shear load applied by a wedge - shaped blade with a thickness of 1 mm in a universal testing machine (zo20 ; zwick/ roell, germany) at a crosshead speed of 1 mm / min until failure occurred (figure 2). shear bond strength test the force (newton) at failure was recorded and the shear bond strength values (mpa) were calculated from the peak load at failure divided by the bonded surface area in mm (a= r, 3.141.51.5=7.065 mm). three - way analysis of variance (anova) and tamhane post hoc test were conducted by spss 15. in this experimental in vitro study, 80 recently extracted human maxillary first premolars, without any caries and cracks, stored in 0.2% sodium azide, were used. the teeth were cleaned and mounted 2 mm below the cementoenamel junction in self - polymerizing acrylic resin (acropars, iran) to simulate tooth position in the alveolar bone. the teeth were randomly divided into two groups (n=40), for sections of surface dentin (group a) or deep dentin (group b). the occlusal surfaces of the teeth were sectioned using diamond discs (d&z ; germany) under water cooling to remove the occlusal enamel and reach the central groove to expose the surface dentin just beneath the dentinoenamel junction in group a and 2 mm below the central groove to meet the deep dentin in group b as in previous studies. the exposed dentin surface of all the specimens was ground with 600-grit abrasive paper to create a standardized smear layer. then the specimens in each group were randomly assigned to 4 subgroups (n=10), according to the type of composite resins used (two types) and pre - treatment (with or without pretreatment) (figure 1). composite resin build - up was done by using a 33-mm cylindrical translucent plastic mold, placed on the center of each specimen. a light - curing unit (coltolux ii ; coltene, usa) having a light output of 600 mw / cm was used to cure the adhesive systems and composite resins. the light cure unit has been checked frequently after preparation of 5 teeth by using a radiometer (cm 300 - 1000 ; halogen lightmeter, china). materials, manufacturers and chemical compositions statistically significant difference (p < 0.05). the mean values followed by at least one similar letter were not significantly different. subgroup a1 : silorane adhesive system primer (3 m espe, usa) was applied with a microbrush on dentin after shaking the bottle well, gently massaged for 15 s, dried with a gentle stream of air and light - cured for 10 s. then silorane adhesive system bond (3 m espe, usa) was applied, followed by a gentle stream of air and light - cured for 10 s. filtek p90 composite resin (3 m espe, usa) was placed in two increments of 1.5-mm thickness and each was light - cured for 40 s. subgroup a2:acid phosphoric etchant gel (etchant 37 ; denfil vericom co. ltd, korea) was applied on dentin for 15 s, rinsed with water thoroughly for 10 s and dried for 35 s with a gentle stream of air, leaving the surface slightly moist. two consecutive coats of adper single bond (3 m espe, usa) were applied and light - cured for 10 s after gentle drying. filtek z350 (3 m es pe, usa) was placed in two increments measuring 1.5 mm in thickness and each was light - cured for 40 s. subgroup a3:dentin was pretreated with 0.004% dimethyl sulfoxide (merck, germany), which was obtained from diluting 3.55 ml dmso with distilled water to have 100 ml 0.004% dmso, for 30 s and gently dried, followed by the steps described for subgroup 1. subgroup a4:etchant gel was applied on dentin for 15 s, rinsed thoroughly for 10 s and dried with a gentle stream of air, leaving the surface slightly moist. dentin was pretreated with 0.004% dmso for 30 s and gently dried, followed by the bonding and restoration steps described for subgroup 2. the teeth were stored in distilled water for 24 h at 37c to allow for water sorption and postoperative polymerization. the teeth were then thermocycled (pc300 ; vafaei, iran) in distilled water at 52/552c for 1000 cycles with a dwell time of 30 s. there was a significant interaction effect between composite resin type and dentin depth (p= 0.001), composite resin type and dmso pretreatment (p= 0.005) but not between dentin depth and dmso pretreatment (p= 0.82). each specimen was individually subjected to a shear load applied by a wedge - shaped blade with a thickness of 1 mm in a universal testing machine (zo20 ; zwick/ roell, germany) at a crosshead speed of 1 mm / min until failure occurred (figure 2). shear bond strength test the force (newton) at failure was recorded and the shear bond strength values (mpa) were calculated from the peak load at failure divided by the bonded surface area in mm (a= r, 3.141.51.5=7.065 mm). three - way analysis of variance (anova) and tamhane post hoc test were conducted by spss 15. the mean values and standard deviations of shear bond strengths are summarized in table 2. the mean values and standard deviations of sbs statistically significant difference (p < 0.05) pretreatment with dmso increased sbs in subgroups a3, a4 and b4 in comparison with subgroups a1, a2 and b2, respectively, and the increase was statistically significant in subgroups a3 (p= 0.003) and b4 (p < 0.001) but not significant in subgroup a4 (p= 0.963). dmso did not affect sbs of b3 comparing with b1 (p= 1.00). asb exhibited significantly higher sbs values in subgroups a2 (p < 0.001), a4 (p= 0.002) and b4 (p < 0.001) than in subgroups a1, a3 and b3, respectively. subgroup b2 had higher sbs than subgroup b1, but the difference was not significant (p= 0.09). sbs was higher in all the surface subgroups compared with deep subgroups ; it was significantly different in subgroup a2 compared with subgroup b2 (p < 0.001) and in subgroup a3 compared with subgroup b3 (p < 0.001) but not statistically significant in subgroup a1 compared with subgroup b1 (p= 0.99) and in subgroup a4 compared with subgroup b4 (p= 0.14) dentin pretreatment with dmso had a significant effect on sbs of asb to either surface or deep dentin. one is to condition dentin surface with chemicals such as polyacrylic acid, chlorhexidine digluconate and antibacterial agents such as edta and naocl. evaluated the effect of 0.004% dmso on nanoleakage and microtensile bond strength of filtek supreme xt to dentin. a methacrylate - based (z350) and a silorane - based (p90) composite resin were used in this study with their respective adhesive systems : a total - etch system, adper single bond (asb), and a self - etch system, p90 adhesive bond. we used 0.004% dmso to condition the surface and deep dentin and evaluate its effect on bond strength of sa and asb. in this study dmso increased sbs in the surface dmso - treated sa subgroup, a3, and deep dmso - treated asb subgroup, b4, compared with respective control subgroups a1 and b2, consistent with a previous study that reported dmso can improve the resindentin bond. in surface dmso - treated asb subgroup, a4, sbs was higher than its control subgroup b2, and lower in deep dmso - treated subgroup b3 than its control subgroup b1, but the differences were not statistically significant. dmso is a chemical penetration enhancing34 amphiphilic solvent, fully miscible in all the solvents used in adhesive dentistry. this agent is able to penetrate biological surfaces and compete with water molecules in inter - peptide hydrogen bonding of the collagen matrix. this may explain why conditioning dentin surface with dmso before adhesive application improved adhesive penetration into the exposed collagen matrix. the increase in sbs in the surface dmso - treated asb subgroup was not as much as it was expected and since this is the first time that the effect of dmso on surface and deep dentin is studied, we recommend more studies with more specimens and different concentrations of dmso and sem microscopy to understand its behavior on different depths of dentin. on the other hand, the adhesive system of silorane is a self - etch one, exclusively used with this composite resin. [36 - 38 ] the acidic hydrophilic primer with a ph of 2.7 is considered a relatively mild primer. in the sa subgroups dmso it seems that the dmso entrapped in large intra - tubular spaces of deep dentin (subgroup b3) has a tendency to dilute the primer and weaken its already mild demineralization capacity, leading to lower sbs in deep dmso - conditioned p90 specimens (subgroup b3) than non - conditioned one (subgroup b1). in contrast, comparison of subgroup b4 and b2 did not show this compromising effect since dmso was applied after acid - etching in asb subgroups, as described in a previous study and this can be the reason why sbs was higher in the asb subgroups compared to the sa subgroups. changes in the steps in which dmso is applied, may lead to different results. different bond strength results have been reported with the use of total - etch and self - etch adhesives, with higher bond strength for either total - etch or self - etch adhesives. in the present study, asb subgroups were restored using a total - etch adhesive system with 37% phosphoric acid, a strong acid (ph=0.030.05) and the application of an adhesive, asb. this technique leads to a complete removal of the smear layer, demineralization of the dentin surface and exposure of collagen fibers,31 creating a demineralized zone measuring 5 to 8m in thickness but sa system s acidic hydrophilic primer with a ph of 2.7 leads to dentin decalcification limited to a few hundredths of nanometers. after the primer is light - cured, a more viscous and hydrophobic adhesive resin, must be applied and light - activated independently.[43 - 45 ] the larger quantity and depth of tags obtained with total - etch systems may promote deeper micromechanical interlocking and greater sbs compared to self - etch systems like silorane.[44 - 46 ] all the deep dentin subgroups presented lower sbs than the surface dentin subgroups, consistent with previous studies. however, the difference was not significant in two pairs ; first in the surface and deep sa subgroups without dmso which might be because of lower etching potential in silorane primer that can not demineralize the highly mineralized components of the surface dentin in comparison with deep dentin ; and second in surface and deep dmso - treated asb subgroups due to high demineralizing potential of phosphoric acid utilized in asb subgroups. therefore, it seems that dmso pretreatment is not as much effective on surface dentin as that on deep dentin when using asb. self - etch adhesives have a mild acid primer that partially demineralizes the dentin, despite of a total etch system that demineralizes the dentin to a higher extent and this can be the reason why we had higher sbs values in b2 than in b1 although the difference was not significant. due to insufficient data base on the mechanism by which dmso affects different depths of dentin and dentin bond strength, further studies are required to evaluate its behavior in contact with acetone-, ethanol- or water - based dentin bonding agents and in different steps of a bonded restorative procedure. furthermore, sem and tem analysis are recommended for better investigation of the depth of resin penetration with or without dmso. within the limitations of this in vitro study, pretreatment of dentin with dmso significantly improved sbs of asb in comparison with sa. dmso appeared to enhance sbs of asb to either surface or deep dentin and the increase was much higher in deep dentin than in surface dentin. therefore, when using asb, dmso might improve sbs in deep cavities with no enamel margins if the results of this study are confirmed by in vivo studies. in addition, dmso could not affect sbs values in deep dentin restored with sa compared with surface dentin. | statement of the problem : composite bond to dentin is crucial in many clinical conditions particularly in deep cavities without enamel margins due to insufficient penetration of adhesive into demineralized dentin. purpose : the aim of this study was to assess the shear bond strength (sbs) of a methacrylate - based and a silorane - based composite resin to surface and deep dentin after pretreatment with dimethyl sulfoxide (dmso). materials and method : eighty extracted human premolars were randomly divided into two groups of flat occlusal dentin with different cuts as a : surface group (sections just below the dentinoenamel junction (dej) and b : deep group (2 mm below dej). each group was randomly assigned to 4 subgroups and their samples were restored with adper single bond (asb) and filtek z350 or silorane system adhesive (sa) and filtek p90 composite resins, using a 33 mm cylindrical plastic mold. following these steps, the subgroups were assigned as subgroupa1 : surface dentin+ silorane system primer (ssp)+ silorane system bonding (ssb)+ p90 ; subgroup a2 : surface dentin+ 37% etchant (e37%) + adper single bond (asb)+ z350 ; subgroup a3 : surface dentin+ dmso+ ssp+ ssb+ p90 ; subgroup a4 : surface dentin+ e37%+ dmso+ asb+ z350 ; subgroup b1 : deep dentin+ ssp+ ssb+ p90 ; subgroup b2 : deep dentin+ e37%+ asb+ z350 ; subgroup b3 : deep dentin+ dmso+ ssp+ ssb+ p90 ; subgroup b4:dentin + e37% + dmso + asb + z350. the specimens were thermocycled at 5 2/55 2c for 1000 cycles and then tested for sbs. results : using dmso as dentin conditioner increased sbs of asb to deep dentin (p < 0.001) and sbs of sa to surface dentin (p= 0.003) but had no effect on sbs of sa to deep dentin (p= 1.00). conclusion : the ability of dmso to increase sbs of asb to deep dentin provides a basis for improving bonding of this composite resin in deep cavities. |
over 5.7 million americans have been diagnosed with heart failure, and with the aging population, this number is expected to increase to 8 million by 2030.13 heart failure is the most common diagnosis among hospitalized patients 65 years of age and older and the leading cause of readmissions in the medicare population.46 accompanying the increasing prevalence of heart failure in older adults is the high burden of treatment, which grows in complexity as the disease progresses and exacerbations occur.7,8 older adults with heart failure also have numerous noncardiac comorbidities (eg, diabetes, chronic pulmonary disease, depression, anemia, chronic kidney disease), which further complicate clinical care and amplify treatment burden.911 previous data suggest that on average, patients with heart failure take 6.8 prescription medications per day, resulting in 10.1 doses per day, not including over - the - counter (otc) or complementary and alternative medications.12 as a result, polypharmacy (often defined as the use of five or more medications) is a pervasive problem in this population, particularly in older adults.1216 considering this high medication burden, it is not surprising that medication nonadherence ranges from 40% to 60% in heart failure patients.5,17 poor adherence to heart failure medications is associated with deleterious clinical consequences and is a major cause of hospital readmissions.15,1721 a better understanding of factors that may contribute to medication nonadherence, such as medication regimen complexity, is urgently needed, particularly in the elderly population. medication regimen complexity is a term used to describe multiple characteristics of a patient s drug regimen, beyond just the number of medications.22 it includes such factors as number of doses per day, number of units per dose, dosage forms, and additional instructions (eg, take with food).22 high medication regimen complexity has been associated with medication nonadherence, poor quality of life, and increased health - resource utilization (eg, hospital readmissions).2327 the medication regimen complexity index (mrci) was a tool developed and validated by george in patients with chronic obstructive pulmonary disease to measure prescription medications associated with that disease.22 the tool was subsequently expanded and validated by libby to include all medications in a patient s drug regimen (ie, disease state - specific, other prescription, and otc), which is often referred to as patient - level mrci (pmrci).28,29 the pmrci tool has been used to quantify medication regimen complexity in numerous patient populations, such as geriatric depression ; hospitalized elderly ; residents in long - term care facilities ; hospitalized patients with heart failure ; heart, kidney, and liver transplants ; hiv ; hypertension ; diabetes ; and dialysis, among others.24,2844 although heart failure is a leading discharge diagnosis in older adults and polypharmacy is common in patients with heart failure, to the best of our knowledge medication regimen complexity has not been evaluated in the ambulatory setting for this population. therefore, the purpose of our study was to quantify systematically medication regimen complexity in ambulatory older adults with heart failure using the pmrci tool. the primary objective was to compare medication regimen complexity in patients with heart failure stratified by age : young - old (6074 years of age) versus old - old (7589 years of age). we hypothesized that medication complexity would be higher in the old - old versus the young - old patients, due to progression of heart failure, increasingly impaired physiologic function, and the presence of multiple comorbidities. the secondary objective was to compare medication regimen complexity in ambulatory older adults based on heart failure etiology, ie, ischemic cardiomyopathy (iscm) versus nonischemic cardiomyopathy (niscm). this cross - sectional study consisted of a retrospective electronic medical record review of men and women 6089 years of age with any clinical diagnosis of heart failure, as reported in the health record. included patients were required to have had at least one visit at the university of colorado hospital advanced heart failure outpatient clinic between october 2014 and august 2015. this time frame was chosen to represent the most contemporary heart failure treatment strategies at the time of the study. patients were excluded if they were 59 years of age and younger, 90 years of age and older, or did not have a clinical diagnosis of heart failure. patients were also excluded if they had a history of solid organ transplant or hiv, as these could be potential confounders due to known high medication regimen complexity. the study protocol, including a full waiver of consent, was reviewed and approved by the colorado multiple institutional review board. standard - of - care clinical data were extracted from the electronic medical record and recorded in a deidentified fashion for this retrospective study. deidentified medication lists and patient - demographic data were extracted from university of colorado hospital electronic medical records. medications were grouped into three categories : 1) disease - specific (heart failure - related) prescription medications, 2) other prescription medications, and 3) otc medications. prescription medications identified as disease - specific consisted of ace inhibitors or angiotensin - receptor blockers, -blockers (eg, carvedilol, metoprolol succinate, bisoprolol), diuretics (eg, furosemide), aldosterone antagonists (eg, spironolactone, eplerenone), digoxin, vasodilators (eg, hydralazine, isosorbide mononitrate or dinitrate), intravenous inotropes (eg, dobutamine or milrinone), intravenous vasodilators (eg, nitroglycerin or nitroprusside), and intravenous vasopressors (eg, norepinephrine, dopamine, or epinephrine).7 examples of other prescription medications were statins, antiarrhythmics, potassium supplements, anticoagulants, antihypertensive agents not specifically indicated for heart failure (eg, atenolol), antidepressants, antianxiolytics, sedative hypnotics, antidiabetic agents, gout medications, thyroid supplements, opioid or nonopioid analgesics, and asthma or chronic obstructive pulmonary disease medications. examples of otc medications were multivitamins, laxatives, calcium supplements, aspirin, fish oils, and herbal products. medications were counted, and dosage formulations, frequencies, and additional directions were entered into an electronic pmrci tool (microsoft access database), which automatically calculated pmrci scores.28 the electronic pmrci tool is freely available at : http://www.ucdenver.edu/academics/colleges/pharmacy/research/researchareas/pages/mrctool.aspx. an overall pmrci score was calculated for each patient, along with subscores for each medication type, ie, heart failure prescription medication, other prescription medication, and otc medication. the electronic pmrci tool consisted of three sections : dosage forms, dosage frequencies, and additional directions. a weight of 1 was given to each dosage form of tablet / capsule and a frequency of once - daily dosing. higher weights were assigned relative to the increased level of difficulty of administration (eg, other dosage forms, other frequencies, and additional instructions). in many patients, medications were encountered that could be categorized as both prescription and otc agents (eg, omeprazole 20 mg). since most patients in this study were eligible for medicare part d prescription coverage and a majority of plans covered products deemed both prescription and otc, these products were consistently coded under the other prescription medication category. micromedex solutions (truven health analytics, ann arbor, mi, usa) and facts & comparisons (wolters kluwer, philadelphia, pa, usa) were used to confirm prescription and otc status. in the event that a medication on a patient s list did not contain a corresponding strength or had a missing frequency, the medication was not included in pmrci scoring. as a surrogate for concomitant disease states, each drug data were analyzed with spss software version 23 (ibm, new york, ny, usa). descriptive statistics were generated and data expressed as number (%), mean, standard deviation, and/or range. total and subsection pmrci scores were analyzed as continuous variables, while medication counts were analyzed as both continuous and categorical variables (ie, 010 medications, 1115 medications, and 16 medications). pearson correlations were used to assess the relationship between variables (eg, pmrci score and medication count ; pmrci score and sex). data were compared between age groups (young - old versus old - old) using general linear model analysis, with heart failure etiology (niscm versus iscm), new york heart association (nyha) functional class, and sex as covariates. data were also compared based on heart failure etiology (niscm versus iscm) using general linear model analysis, with nyha functional class and sex as covariates. this cross - sectional study consisted of a retrospective electronic medical record review of men and women 6089 years of age with any clinical diagnosis of heart failure, as reported in the health record. included patients were required to have had at least one visit at the university of colorado hospital advanced heart failure outpatient clinic between october 2014 and august 2015. this time frame was chosen to represent the most contemporary heart failure treatment strategies at the time of the study. patients were excluded if they were 59 years of age and younger, 90 years of age and older, or did not have a clinical diagnosis of heart failure. patients were also excluded if they had a history of solid organ transplant or hiv, as these could be potential confounders due to known high medication regimen complexity. the study protocol, including a full waiver of consent, was reviewed and approved by the colorado multiple institutional review board. standard - of - care clinical data were extracted from the electronic medical record and recorded in a deidentified fashion for this retrospective study. deidentified medication lists and patient - demographic data were extracted from university of colorado hospital electronic medical records. medications were grouped into three categories : 1) disease - specific (heart failure - related) prescription medications, 2) other prescription medications, and 3) otc medications. prescription medications identified as disease - specific consisted of ace inhibitors or angiotensin - receptor blockers, -blockers (eg, carvedilol, metoprolol succinate, bisoprolol), diuretics (eg, furosemide), aldosterone antagonists (eg, spironolactone, eplerenone), digoxin, vasodilators (eg, hydralazine, isosorbide mononitrate or dinitrate), intravenous inotropes (eg, dobutamine or milrinone), intravenous vasodilators (eg, nitroglycerin or nitroprusside), and intravenous vasopressors (eg, norepinephrine, dopamine, or epinephrine).7 examples of other prescription medications were statins, antiarrhythmics, potassium supplements, anticoagulants, antihypertensive agents not specifically indicated for heart failure (eg, atenolol), antidepressants, antianxiolytics, sedative hypnotics, antidiabetic agents, gout medications, thyroid supplements, opioid or nonopioid analgesics, and asthma or chronic obstructive pulmonary disease medications. examples of otc medications were multivitamins, laxatives, calcium supplements, aspirin, fish oils, and herbal products. medications were counted, and dosage formulations, frequencies, and additional directions were entered into an electronic pmrci tool (microsoft access database), which automatically calculated pmrci scores.28 the electronic pmrci tool is freely available at : http://www.ucdenver.edu/academics/colleges/pharmacy/research/researchareas/pages/mrctool.aspx. an overall pmrci score was calculated for each patient, along with subscores for each medication type, ie, heart failure prescription medication, other prescription medication, and otc medication. the electronic pmrci tool consisted of three sections : dosage forms, dosage frequencies, and additional directions. a weight of 1 was given to each dosage form of tablet / capsule and a frequency of once - daily dosing. higher weights were assigned relative to the increased level of difficulty of administration (eg, other dosage forms, other frequencies, and additional instructions). in many patients, medications were encountered that could be categorized as both prescription and otc agents (eg, omeprazole 20 mg). since most patients in this study were eligible for medicare part d prescription coverage and a majority of plans covered products deemed both prescription and otc, these products were consistently coded under the other prescription medication category. micromedex solutions (truven health analytics, ann arbor, mi, usa) and facts & comparisons (wolters kluwer, philadelphia, pa, usa) were used to confirm prescription and otc status. in the event that a medication on a patient s list did not contain a corresponding strength or had a missing frequency, the medication was not included in pmrci scoring. as a surrogate for concomitant disease states, each drug was also assigned a therapeutic drug class using micromedex solutions and facts & comparisons. data were analyzed with spss software version 23 (ibm, new york, ny, usa). descriptive statistics were generated and data expressed as number (%), mean, standard deviation, and/or range. total and subsection pmrci scores were analyzed as continuous variables, while medication counts were analyzed as both continuous and categorical variables (ie, 010 medications, 1115 medications, and 16 medications). pearson correlations were used to assess the relationship between variables (eg, pmrci score and medication count ; pmrci score and sex). data were compared between age groups (young - old versus old - old) using general linear model analysis, with heart failure etiology (niscm versus iscm), new york heart association (nyha) functional class, and sex as covariates. data were also compared based on heart failure etiology (niscm versus iscm) using general linear model analysis, with nyha functional class and sex as covariates. the study included 145 patients (64.1% men, 35.9% women, mean age 738 years, range 6189 years). age was categorized as young - old (6074 years, n=80 [55.2% ]) and old - old (7589 years, n=65 [44.8% ]). the racial distribution was 80.7% caucasian, 10.3% african - american, 2.1% asian / pacific islander, and 6.9% other ; 4.8% of patients classified their ethnicity as hispanic. there were 60 patients (41.4%) with niscm and 85 patients (58.6%) with iscm. the percentage of patients with nyha functional class i, ii, iii, and iv heart failure was 16.6%, 27.6%, 27.6%, and 28.3%, respectively. in the entire study cohort, the mean total medication count was 13.34.8 (range 230) and the mean total pmrci score was 32.114.4 (range 384). the percentage of patients taking 010, 1115, or 16 total medications was 28%, 43%, and 29%, respectively. of note, there was only one patient in the cohort taking fewer than five medications. total medication count was significantly correlated with total pmrci score (r=0.85, p<0.001), heart failure etiology (iscm vs niscm, r=0.19 ; p=0.02), and nyha functional class (r=0.17, p=0.04), but not age, sex, or race (data not shown). similar significant correlations were observed between total pmrci score and heart failure etiology (r=0.19, p=0.02) and nyha functional class (r=0.18, p=0.03). heart failure prescriptions, other prescriptions, and otc medications accounted for 24%, 50%, and 26% of the total medication count, respectively, and 22%, 56%, and 22% of the total pmrci score, respectively (table 1). of the pmrci subsections (ie, dosage form, dosing frequency, or additional directions), dosing frequency accounted for the majority (61.6%) of the total pmrci score. the most commonly prescribed heart failure medications were -blockers (76.6% of patients), loop diuretics (66.2% of patients), aldosterone antagonists (60.7% of patients), ace inhibitors / angiotensin - receptor blockers (57.2% of patients), and digoxin (32.4% of patients). percentages do not total 100%, as patients could be taking medications from multiple classes. the most common other prescriptions were statins (64.1% of patients), anticoagulants (51% of patients), proton pump inhibitors (38.6% of patients), potassium supplements (28.3% of patients), and thyroid supplements (27.6% of patients). the most common otc medications were aspirin (66.9% of patients), vitamin d (29% of patients), multivitamins (27.6% of patients), calcium (20.7% of patients), fish oil (20.7% of patients), and acetaminophen (20.7% of patients). to address the primary objective, medication regimen complexity was compared between young - old versus old - old patients, with heart failure etiology, nyha functional class, and sex as covariates (table 2). medication counts and pmrci scores did not differ significantly between the age groups, with the exception of otc pmrci score, which was significantly higher in old - old than young - old patients (7.85.8 vs 6.24, p=0.04). for the secondary objective, medication regimen complexity was also compared between patients with niscm and iscm, with nyha functional class and sex as covariates (table 3). there were more caucasians in the iscm group than in the niscm group ; however, race was not significantly associated with medication counts or pmrci scores, and was thus not included as a covariate in the analysis. total medication count was significantly higher in patients with iscm than niscm (14.14.9 vs 12.24.5, p=0.008), which was primarily driven by differences in the number of other prescriptions (7.33.4 vs 5.73.7, p=0.008). total pmrci score was also significantly higher in patients with iscm than niscm (34.515.2 vs 28.812.7, p=0.009). this was a result of higher other prescription and otc medication pmrci scores in the iscm group than the niscm group. the frequencies of the other major prescription - drug classes that were significantly higher in patients with iscm vs niscm were statins, calcium channel blockers, antiarrhythmics, nonaspirin antiplatelet agents, and topical corticosteroids (data not shown). the study included 145 patients (64.1% men, 35.9% women, mean age 738 years, range 6189 years). age was categorized as young - old (6074 years, n=80 [55.2% ]) and old - old (7589 years, n=65 [44.8% ]). the racial distribution was 80.7% caucasian, 10.3% african - american, 2.1% asian / pacific islander, and 6.9% other ; 4.8% of patients classified their ethnicity as hispanic. there were 60 patients (41.4%) with niscm and 85 patients (58.6%) with iscm. the percentage of patients with nyha functional class i, ii, iii, and iv heart failure was 16.6%, 27.6%, 27.6%, and 28.3%, respectively. in the entire study cohort, the mean total medication count was 13.34.8 (range 230) and the mean total pmrci score was 32.114.4 (range 384). the percentage of patients taking 010, 1115, or 16 total medications was 28%, 43%, and 29%, respectively. of note total medication count was significantly correlated with total pmrci score (r=0.85, p<0.001), heart failure etiology (iscm vs niscm, r=0.19 ; p=0.02), and nyha functional class (r=0.17, p=0.04), but not age, sex, or race (data not shown). similar significant correlations were observed between total pmrci score and heart failure etiology (r=0.19, p=0.02) and nyha functional class (r=0.18, p=0.03). heart failure prescriptions, other prescriptions, and otc medications accounted for 24%, 50%, and 26% of the total medication count, respectively, and 22%, 56%, and 22% of the total pmrci score, respectively (table 1). of the pmrci subsections (ie, dosage form, dosing frequency, or additional directions), dosing frequency accounted for the majority (61.6%) of the total pmrci score. the most commonly prescribed heart failure medications were -blockers (76.6% of patients), loop diuretics (66.2% of patients), aldosterone antagonists (60.7% of patients), ace inhibitors / angiotensin - receptor blockers (57.2% of patients), and digoxin (32.4% of patients). percentages do not total 100%, as patients could be taking medications from multiple classes. the most common other prescriptions were statins (64.1% of patients), anticoagulants (51% of patients), proton pump inhibitors (38.6% of patients), potassium supplements (28.3% of patients), and thyroid supplements (27.6% of patients). the most common otc medications were aspirin (66.9% of patients), vitamin d (29% of patients), multivitamins (27.6% of patients), calcium (20.7% of patients), fish oil (20.7% of patients), and acetaminophen (20.7% of patients). to address the primary objective, medication regimen complexity was compared between young - old versus old - old patients, with heart failure etiology, nyha functional class, and sex as covariates (table 2). medication counts and pmrci scores did not differ significantly between the age groups, with the exception of otc pmrci score, which was significantly higher in old - old than young - old patients (7.85.8 vs 6.24, p=0.04). for the secondary objective, medication regimen complexity was also compared between patients with niscm and iscm, with nyha functional class and sex as covariates (table 3). there were more caucasians in the iscm group than in the niscm group ; however, race was not significantly associated with medication counts or pmrci scores, and was thus not included as a covariate in the analysis. total medication count was significantly higher in patients with iscm than niscm (14.14.9 vs 12.24.5, p=0.008), which was primarily driven by differences in the number of other prescriptions (7.33.4 vs 5.73.7, p=0.008). total pmrci score was also significantly higher in patients with iscm than niscm (34.515.2 vs 28.812.7, p=0.009). this was a result of higher other prescription and otc medication pmrci scores in the iscm group than the niscm group. the frequencies of the other major prescription - drug classes that were significantly higher in patients with iscm vs niscm were statins, calcium channel blockers, antiarrhythmics, nonaspirin antiplatelet agents, and topical corticosteroids (data not shown). our retrospective study quantified medication regimen complexity in older adults with heart failure in the ambulatory setting using the pmrci tool. medication regimen complexity did not differ significantly in old - old versus young - old patients. however, total medication counts and pmrci scores were significantly higher in patients with iscm compared with niscm. this finding is not surprising, as patients with iscm have a greater burden of comorbidities, such as hypertension, angina, diabetes, peripheral vascular disease, renal dysfunction, and hyperlipidemia, compared to those with niscm.45 these comorbidities contribute additional drugs to the pharmacotherapeutic treatment regimen and increase the total pmrci score. together, our data highlight the substantial medication burden associated with heart failure, especially in older adults, and reveal multiple opportunities to address polypharmacy in this population. polypharmacy is a major risk factor for medication non - adherence, and 72% of the patients in our ambulatory cohort were taking eleven or more medications.5,24 from a public health perspective, this finding is of considerable importance, as the probability of an adverse drug reaction increases to 82% when seven or more medications are prescribed.46 to date, most assessments of medication regimen complexity in heart failure patients have been conducted in the hospital setting.36,38,39 for example, yam reported a mean pmrci score and medication count of 35.519 and 12.96.3, respectively, at the time of hospital admission in a predominantly male (97%) cohort of us veterans with heart failure. our findings are consistent with these data, and indicate that high treatment burden extends to a more heterogeneous population of heart failure patients (64% men) in the ambulatory setting. when we compare our findings to pmrci studies in other disease states, older adults with heart failure have higher medication regimen complexity and/or medication counts than patients with heart transplant, depression, hiv, diabetes, and hypertension.28,30,31,33,35 for example, in our previous work with heart transplant recipients, the mean pmrci score was 30.47 and mean medication count was 13.53.2 at 1 year posttransplant.31 therefore, older adults with heart failure are likely to be among the highest - risk patients, on par with heart transplant recipients, in terms of medication regimen complexity. it has been suggested that simpler methods to evaluate medication regimen complexity, eg, medication count, are needed in the clinical setting. we observed a strong correlation between total medication count and total pmrci score in older adults with heart failure. however, 28% of the variability in pmrci scores was not explained by medication count alone. as the evaluation of medication complexity continues to be documented in the literature and linked to clinical outcomes, the pmrci may be a useful tool to identify patients for more enhanced medication - therapy management interventions, such as those by a pharmacist.29,47 along these lines, a clinical science statement from the american heart association addressing medications that can exacerbate or cause heart failure suggested that while not currently associated with improved outcomes, the use of medication - complexity tools should be considered in potentially reducing polypharmacy (class iia, level of evidence c).48 there are several limitations of our study that deserve to be acknowledged. we retrospectively evaluated medication lists from the electronic medical record ; therefore, we could not assess medication adherence. the retrospective design and electronic data collection also precluded the ability to capture additional instructions that were given to the patient verbally or in writing at the time of the clinic visit. we did not compare pmrci scores between those with heart failure with reduced ejection fraction versus heart failure with preserved ejection fraction, as these classifications were not consistently documented in the clinical notes during the study time period (eg, recent echocardiograms were not always available in relation to the patient s most recent medication list). along the same lines, our retrospective study included only patients who had a heart failure diagnosis in the electronic medical record, possibly missing qualifying patients who were sick, yet did not have an established heart failure diagnosis. our study consisted of patients from one heart failure clinic, and may not accurately address prescribing patterns across different clinics or regions. the study sample size was also relatively small ; however, post hoc power analysis revealed that this sample size provided 80% power to detect a clinically meaningful difference of 2.3 medications between the young - old and old - old groups and a 6.8-point difference in pmrci score, a moderate - to - large effect. it is important to note that the pmrci tool does not take into account other factors that may contribute to medication regimen complexity in older adults, such as vision impairment, decreased manual dexterity, cognitive impairment, patient - education level, patient perceptions of treatment burden, and socioeconomic status (eg, insurance coverage).49 while nyha functional class was included as a covariate in statistical analysis, assessment of the severity of other comorbidities and the contribution to medication regimen complexity was not evaluated in this retrospective electronic medical record review. we also did not assess the relationship between medication regimen complexity and clinical outcomes (eg, hospitalizations for heart failure) in this cohort, and further research in this area is warranted. medication regimen complexity is high in older adults with heart failure, and differs based on disease etiology. opportunities exist for pharmacists and other health care professionals to address polypharmacy and medication regimen complexity in patients with heart failure, which may include extended counseling for patients and caregivers, frequent patient follow - up, simplification of medication regimens (eg, evaluation of the necessity of otc agents), and clinic - based adherence assessments. additional studies are needed to address the impact of these interventions on clinical outcomes in older adults with heart failure. | purposeheart failure prevalence is increasing in older adults, and polypharmacy is a major problem in this population. we compared medication regimen complexity using the validated patient - level medication regimen complexity index (pmrci) tool in young - old (6074 years) versus old - old (7589 years) patients with heart failure. we also compared pmrci between patients with ischemic cardiomyopathy (iscm) versus nonischemic cardiomyopathy (niscm).patients and methodsmedication lists were retrospectively abstracted from the electronic medical records of ambulatory patients aged 6089 years with heart failure. medications were categorized into three types heart failure prescription medications, other prescription medications, and over - the - counter (otc) medications and scored using the pmrci tool.resultsthe study evaluated 145 patients (n=80 young - old, n=65 old - old, n=85 iscm, n=60 niscm, mean age 737 years, 64% men, 81% caucasian). mean total pmrci scores (32.114.4, range 384) and total medication counts (13.34.8, range 230) were high for the entire cohort, of which 72% of patients were taking eleven or more total medications. total and subtype pmrci scores and medication counts did not differ significantly between the young - old and old - old groups, with the exception of otc medication pmrci score (6.24 young - old versus 7.85.8 old - old, p=0.04). with regard to heart failure etiology, total pmrci scores and medication counts were significantly higher in patients with iscm versus niscm (pmrci score 34.515.2 versus 28.812.7, p=0.009 ; medication count 14.14.9 versus 12.24.5, p=0.008), which was largely driven by other prescription medications.conclusionmedication regimen complexity is high in older adults with heart failure, and differs based on heart failure etiology. additional work is needed to address polypharmacy and to determine if medication regimen complexity influences adherence and clinical outcomes in this population. |
lung cancer has become a disease of great global impact and remains the leading cause of death from cancer in the world.1 as smoking and environmental pollution can not be controlled in the short term, the incidence of lung cancer continues to increase, especially in females.2,3 in brazil, the estimate for 2010 is approximately 18 new cases per 100 000 men and 10 per 100 000 women, corresponding to 17 800 and 9 930 new cases of lung cancer among men and women respectively.4 advances in lung cancer therapy have been improving survival rates, although the prognosis remains poor. the 5year survival rate is around 15% in developed countries1 and 10% in brazil.4 therefore, the impact of both disease and treatment on the health and psychosocial functioning of these patients should be considered.5 in this context, quality of life assessment and the analysis of the main symptoms that lead to functional capacity limitation have become issues of utmost importance in the evaluation of lung cancer patients. however, in brazil, there are few studies that evaluate this aspect of the disease, mainly because of the lack of specific tools adapted to and reproducible for the brazilian portuguese language. several instruments are currently used to evaluate the quality of life in patients with lung cancer. the functional assessment of cancer therapylung (factl) was generated by the functional assessment of chronic illness therapy (facit) group. it is a specific, multidimensional questionnaire widely used in clinical studies.6 - 9 moreover, the factlung symptom index (flsi), which is a brief measure involving five common symptoms in lung cancer patients, can be applied in combination with the factl or alone. although the factl is currently used in brazil, mainly in international multicenter trails, there is no study assessing the reliability of this questionnaire in the brazilian portuguese language. therefore, the purpose of this study was to evaluate the reproducibility of the factl and the flsi for brazilian lung cancer patients. a convenience sample comprising 30 patients with lung cancer was recruited from the outpatient lung cancer clinic of the so paulo hospital federal university of so paulo. this study was approved by the institutional review board of our center, and a term of informed consent was signed by all patients. the following inclusion criteria were used : histologically proven lung cancer ; 18 years of age or older ; a minimum score of 21 on the mini mental state examination (mmse);10,11 out of chemotherapy / radiotherapy treatment ; and clinical stability during the study and at least 10 days before the beginning of the evaluations. clinical stability was defined as the absence of change in cough, sputum, and dyspnea, assessed by a structured form filled out during outpatient followup, and no hospitalizations or modifications in the therapeutic regimen. the sample size was based on previous reliability studies of other quality of life questionnaires related to respiratory diseases in brazil.1215 clinical evaluation and physical examination were performed by a team of physicians, based on a structured form. the drug regimen used by the patients remained unchanged during the 15day interval between questionnaire applications. in the first visit, the following independent variables were collected : gender (male and female proportion), age (in years), history of tobacco use (yes or no) and consumption (pack years), histologic subtypes (adenocarcinoma, squamous cell, small cell lung cancer and others),16 staging according to the 1997 tnm classification for nonsmall cell lung cancer (nsclc) patients (stratified from ia to iiia and from iiib to iv),17 karnofsky performance status (kps),18 spirometry (forced expiratory volume in the first second (fev1) and forced vital capacity (fvc) percentage of predicted and fev1/fvc percentage),19 mmse, and factl and flsi scores.9 the portuguese version of the factl and flsi was released for use by the facit group (the developer of the questionnaire). the translation into brazilian portuguese, back translation and review by an expert committee to access the semantic, conceptual, idiomatic, cultural and metabolic equivalences were previously done by that group.20,21 the factl, version 4, is a combination of the 27item factgeneral (factg) and the 9item lung cancer subscale (lcs). a total factg score is calculated by summing the physical wellbeing (pwb), social / family wellbeing (swb), emotional wellbeing (ewb), and functional wellbeing (fwb) subscale scores, with a score ranging from 0 to 108. a total factl score is obtained by summing the factg score with the lcs (two of the nine items are not scored). the factl score ranges from 0 to 136.6 the factl trial outcome index (factl toi) is, a priori, an index that sums the pwb, fwb, and lcs into a 21item scale. its score ranges from 0 to 84.6 the flsi is a symptom index with six questions regarding the five most frequent symptoms reported by lung cancer patients, especially in the advanced stages : dyspnea, fatigue, pain, weight loss, and coughing. the score on each aspect of the factl and flsi is obtained according to the option chosen by the patient., a little bit, somewhat, quite a bit, or very much. the higher the score obtained by each patient in question, the greater the final score of the subscale and the better the quality of life. however, these scores have a nonlinear behavior. patients answered the questionnaire after being read each question, all by the same interviewer. the questionnaires were reviewed at the end of the interview to avoid any missed questions. the response time was timed in the two visits. all doubts expressed by patients during the questionnaire patients were also asked not only about what they felt in terms of question content, but also about the length of the questionnaire. the test the scores obtained on different scales and subscales of factg were compared with reference values established by the facit group.22 for this comparison, we used the minimal clinically significant difference, determined by the same group.6 variables were expressed as mean and standard deviation. the intraclass correlation coefficient (icc) and kappa reliability coefficient were calculated to assess the reliability of the questionnaire and questions respectively. to compare the two groups, we used the chisquare test for categorical variables, t test for parametric continuous variables, and mann whitney test for nonparametric continuous variables. for the correlations between spirometry and questionnaire scores, the spearman 's correlation coefficient was used. the intraclass correlation coefficient (icc) and kappa reliability coefficient were calculated to assess the reliability of the questionnaire and questions respectively. to compare the two groups, we used the chisquare test for categorical variables, t test for parametric continuous variables, and mann whitney test for nonparametric continuous variables. for the correlations between spirometry and questionnaire scores, the spearman 's correlation coefficient was used. the main characteristics of the 30 patients who completed the study are shown in table 1. there was no statistically significant difference between genders regarding age, kps, spirometry, staging, and histological type. among patients who never smoked, there was a prevalence of smoking habits in males (p = 0.04), with a higher tobacco smoke load for men than for women, consuming a mean of 53.2 pack years (sd = 31.6) and 29.6 pack years (sd = 25.5) respectively (p = 0.02). eight patients (26.7%) were diagnosed with chronic obstructive pulmonary disease (copd), according to the gold guideline.23 the mean values for each scale of the factl and flsi are illustrated in table 2. the intraclass correlation coefficient values for the different scales of the factl and flsi showed excellent correlations (table 2). the kappa coefficient was used to test question reliability, which was less than 0.4 on questions gp1, gp5 (pwb), gs3, gs4, gs5 (swb), gf1, gf2, gf3, gf5, gf6 (fwb), and lcl4 (lcs). as for the flsi, the kappa coefficient was moderate on question b1. in the other questions, the kappa coefficient was less than 0.4. the mean score of the factg scales was similar to the reference values for all scales that have these values established.22 table 3 shows the mean values for the scales of factg and factl described in several studies of reliability and cultural adaptation into other languages. the time spent by patients in answering the questionnaire the response time on the second application was significantly lower (p = 0.001). the focus of this study was to analyze the reliability of the factl and the flsi, a specific instrument for assessing the quality of life of lung cancer patients in the brazilian population. the factl is an instrument that has been widely used in phase i, ii, and iii clinical trials. it has been translated and adapted into several languages and cultures.2,3,7,22,2427 a feature of this instrument is the nonlinearity of the scores of its scales, a factor that complicates the interpretation of isolated study data. to facilitate the explanation of the results, the authors of the general questionnaire (factg) have developed normative values from two reference groups, one from normal adults and another from adults with cancer in general.22 however, these normative values are only for the general questionnaire (factg) and its scales. when comparing the results obtained in our study with the reference values, we observe that the score was similar in all scales and for the total value. a possible explanation for the fact that our patients have quality of life similar to patients with other cancer types is that, in the study for the establishment of benchmarks, besides including patients with various types of cancer, such as breast cancer, colon and rectum cancer, and cancer of the head and neck, they also included patients with lung cancer. furthermore, 76.7% of the sample comprised patients treated and in remission from the underlying disease and, therefore, with better quality of life. when our factl results are compared with those from other studies using the same questionnaire, we find that there is great variability between countries and different cultures,2,3,7,22,2427 which may be due to differing perceptions of the questions according to each culture and also differences in patient characteristics, such as staging, age, socioeconomic status, among others. the flsi is an index designed to identify the presence of the major symptoms related to lung cancer, including dyspnea, pain, fatigue, cough, and weight loss.28 these symptoms can negatively influence quality of life. in this study, in addition to the medical evaluation, flsi was also used to assess the patient 's clinical stability. the reliability for all scores of the factl showed icc values greater than 0.75, ranging from 0.78 (swb) to 0.96 (factl toi). these values show excellent reliability and are similar to those found in other studies. in the validation study of the korean version of the factl,25 the icc ranged from 0.52 to 0.84. the chinese version3 varied from 0.76 to 0.82. in the reliability study of the original version, the icc ranged from 0.56 (swb) to 0.89 (factl toi).7 the flsi also showed high reliability, which confirms the stability of the sample. most of the factl questions showed adequate reliability, analyzed by the kappa coefficient. in this study, the reliability analyses were conducted with a convenience sample similar to other studies evaluating the reliability and cultural adaptation of other questionnaires to the portuguese language.1215 in our sample, males predominated, in agreement with the worldwide prevalence of lung cancer.1 the mean age was 61.3 years, which is consistent with most studies that include patients with lung cancer.1,5 regarding histological type, adenocarcinoma was the most prevalent, consistent with several epidemiological studies in developed countries.1 unfortunately, regarding staging, there was a predominance of stage iii and iv, stages with less chance of survival after treatment.29 the mean fev1 (% predicted) and fev1/fvc ratio in our study were similar to the results reported by young., who observed a fev1 (% predicted) mean of 73% and fev1/fvc mean of 64% in smokers with lung cancer.30 in our study, there was no correlation between the questionnaire and the analyzed lung function parameters, consistent with other studies that found no significant correlation when investigating the effects of altered pulmonary function on the quality of life of cancer patients.31,32 the process of translating the factl questionnaire and flsi into the portuguese language (brazil) was performed by the facit group. only a few doubts were reported by the patients, which warrants its use in the current form. few patients had difficulty in interpreting the word muitssimo ; however, many of the patients interviewed understood the meaning of the term not because it is a familiar word, but because they realized that it was a scale with a progressive score in which the numerical value assigned to the word muitssimo alterations to this tool were proposed only for question q1 independentemente do seu nvel a(c)tual de a(c)tividade sexual, favor de responder pergunta a seguir. se preferir no responder, assinale o quadrculo e passe para a prxima seo. despite not raising doubts, the words atual and atividade do not require the letter (c), and there is no need to use the preposition we conclude that the brazilian version of the factl questionnaire and flsi is reproducible, fast, and of simple application, and that they are capable of measuring the quality of life in lung cancer patients in brazil. | objectives : the purpose of this study was to assess the reliability of the brazilian version of the functional assessment of cancer therapylung (factl) with the factlung symptom index (flsi) questionnaire.introduction:the assessment of quality of life in patients with lung cancer has become an important evaluative endpoint in current clinical trials. for lung cancer patients, one of the most common quality of life tools available is the factl. despite the amount of data available regarding this questionnaire, there are no data on its performance in brazilian lung cancer patients.methods:the factl with the flsi questionnaire was prospectively administered to 30 consecutive, stable, lung cancer outpatients at baseline and at 2 weeks.results:the intraclass correlation coefficient between test and retest for the factl ranged from 0.79 to 0.96 and for the flsi was 0.87. there was no correlation between these questionnaire dimensions and clinical or functional parameters.conclusions:the brazilian version of the factl with flsi questionnaire is reliable and is quick and simple to apply. this instrument can now be used to properly evaluate the quality of life of brazilian lung cancer patients. |
the concept of total mesorectal excision (tme), which was introduced by heald and ryall during the 1980s, has significantly improved the outcome for patients with rectal cancer, particularly with regard to local recurrence. the results of some studies showed that tme was a superior surgical modality for the treatment of rectal cancer because the local recurrence rates were reduced from between 30% and 40% to 5% after tme. therefore tme has been generally accepted as the gold standard for rectal cancer surgery. in the early days of minimally invasive surgery, there was concern about the oncologic safety of the laparoscopic approach for the treatment of colorectal cancer. however, recent reports derived from randomized comparative studies, most of them multicentric, have shown that laparoscopy for the surgical treatment of colon cancer is associated with morbidity, mortality, and long - term cancer - related survival rates similar to those seen with the open approach. the advantages of laparoscopic surgery have translated into smaller incisions and shorter recovery. however, the confines of the narrow bony pelvis and angling limits in current stapling technology, along with the standard practice of autonomic nerve sparing tme, have made laparoscopic surgery more challenging in the treatment of low rectal cancer. because of the absence of long - term (5-year) data on survival and recurrence, the role of laparoscopy in rectal cancer resection has been debated. macroscopic evaluation of the completeness of the mesorectum provides significant information about the quality of surgery for low rectal cancer. the aims of this study were to observe the short - term outcomes and to evaluate the macroscopic quality of specimens acquired after laparoscopic versus open tme in patients with low rectal cancer. from may 2010 to may 2012, a series of 88 patients with biopsy - proved rectal cancer received laparoscopic radical resection at the department of general surgery of our university hospital. conversion was defined as a laparoscopic procedure that had to be abandoned and replaced with median laparotomy. the pathologic specimens were prospectively examined and matched with the resection specimens from 90 patients who had undergone open tme. patients with rectal tumors located within the distal 10 cm of the anal verge were included in the study. the 2 groups were matched for sex and type of resection, which included low anterior resection (lar) and abdominoperineal resection (apr). the decision to perform either lar with stapled colorectal anastomosis or apr was made according to tumor localization above the anal verge, size, and histologic type. digital examination and fiberoptic colonoscopy were performed to assess the extent of tumor at the periphery of the rectum, the distance from the anal verge, and the degree of fixation. computed tomography was performed to evaluate the extent of tumor infiltration to the other organs. patients with tumors extending to the pelvic walls or organs were excluded from the study. the mean age of the patients was 62 years (range, 3982 years) in the laparoscopic group and 64 years (range, 4183 years) in the open group (table 1). the results of intraoperative and postoperative follow - up were compared between the 2 groups, including operative time, intraoperative and postoperative complications, time of bowel function rehabilitation, and postoperative hospital stay. patients and tumor characteristics : laparoscopic versus open total mesorectal excision special care was taken regarding the macroscopic judgment concerning the completeness of the mesorectum. pathologic examination of each specimen was performed in a standardized fashion by the technique based on the procedure originally described by parfitt and driman. after the mesorectal surface had been inked, the specimen was sliced at approximately 3- to 5-mm intervals. the depth of tumor spread in relation to the circumferential margin, as well as the presence of discontinuous tumor deposits and lymph nodes involved by tumor, was evaluated. macroscopic quality assessment of the resected specimen was performed in all patients by a single colorectal surgeon trained in tme and included evaluation of the following : the quality of the mesorectum was graded as described by quirke. both the specimen as a whole (fresh) and cross - sectional slices (fixed) were examined to make an adequate interpretation (table 2).if there was at least 1 cm of peritoneal edge at the cul - de - sac, the cut edge of the peritoneal reflection anterior to the rectum was considered adequate.the denonvilliers fascia was intact at the level of the middle rectum, especially for anterior cancers.in the case of apr of the rectum, the resected bowel wall below the mesorectum was graded as 1 of the 4 following groups as described by quirke. : substandard, a defect of the muscularis propria or perforation of the tumor ; standard, intact muscularis propria of the specimen ; enhanced, circumferentially covered by a slice of levator ani musculature ; or radical, circumferentially covered by a levator ani muscular cuff 1 cm thick. both the specimen as a whole (fresh) and cross - sectional slices (fixed) were examined to make an adequate interpretation (table 2). if there was at least 1 cm of peritoneal edge at the cul - de - sac, the cut edge of the peritoneal reflection anterior to the rectum was considered adequate. the denonvilliers fascia was intact at the level of the middle rectum, especially for anterior cancers. in the case of apr of the rectum, the resected bowel wall below the mesorectum was graded as 1 of the 4 following groups as described by quirke. : substandard, a defect of the muscularis propria or perforation of the tumor ; standard, intact muscularis propria of the specimen ; enhanced, circumferentially covered by a slice of levator ani musculature ; or radical, circumferentially covered by a levator ani muscular cuff 1 cm thick. grading of quality and completeness of mesorectum in total mesorectal excision specimen values are expressed as median and range. statistical analysis was performed with spss software for windows (version 13 ; ibm, armonk, ny, usa). comparisons between the 2 groups were made by applying either the student t or mann - whitney u test for unpaired values as appropriate. the test with yates correction for small samples was applied to compare differences in variables expressed as proportions. from may 2010 to may 2012, a series of 88 patients with biopsy - proved rectal cancer received laparoscopic radical resection at the department of general surgery of our university hospital. conversion was defined as a laparoscopic procedure that had to be abandoned and replaced with median laparotomy. the pathologic specimens were prospectively examined and matched with the resection specimens from 90 patients who had undergone open tme. patients with rectal tumors located within the distal 10 cm of the anal verge were included in the study. the 2 groups were matched for sex and type of resection, which included low anterior resection (lar) and abdominoperineal resection (apr). the decision to perform either lar with stapled colorectal anastomosis or apr was made according to tumor localization above the anal verge, size, and histologic type. digital examination and fiberoptic colonoscopy were performed to assess the extent of tumor at the periphery of the rectum, the distance from the anal verge, and the degree of fixation. computed tomography was performed to evaluate the extent of tumor infiltration to the other organs. patients with tumors extending to the pelvic walls or organs were excluded from the study. the mean age of the patients was 62 years (range, 3982 years) in the laparoscopic group and 64 years (range, 4183 years) in the open group (table 1). the results of intraoperative and postoperative follow - up were compared between the 2 groups, including operative time, intraoperative and postoperative complications, time of bowel function rehabilitation, and postoperative hospital stay. pathologic examination of each specimen was performed in a standardized fashion by the technique based on the procedure originally described by parfitt and driman. after the mesorectal surface had been inked, the specimen was sliced at approximately 3- to 5-mm intervals. the depth of tumor spread in relation to the circumferential margin, as well as the presence of discontinuous tumor deposits and lymph nodes involved by tumor, was evaluated. macroscopic quality assessment of the resected specimen was performed in all patients by a single colorectal surgeon trained in tme and included evaluation of the following : the quality of the mesorectum was graded as described by quirke. both the specimen as a whole (fresh) and cross - sectional slices (fixed) were examined to make an adequate interpretation (table 2).if there was at least 1 cm of peritoneal edge at the cul - de - sac, the cut edge of the peritoneal reflection anterior to the rectum was considered adequate.the denonvilliers fascia was intact at the level of the middle rectum, especially for anterior cancers.in the case of apr of the rectum, the resected bowel wall below the mesorectum was graded as 1 of the 4 following groups as described by quirke. : substandard, a defect of the muscularis propria or perforation of the tumor ; standard, intact muscularis propria of the specimen ; enhanced, circumferentially covered by a slice of levator ani musculature ; or radical, circumferentially covered by a levator ani muscular cuff 1 cm thick. both the specimen as a whole (fresh) and cross - sectional slices (fixed) were examined to make an adequate interpretation (table 2). if there was at least 1 cm of peritoneal edge at the cul - de - sac, the cut edge of the peritoneal reflection anterior to the rectum was considered adequate. the denonvilliers fascia was intact at the level of the middle rectum, especially for anterior cancers. in the case of apr of the rectum, the resected bowel wall below the mesorectum was graded as 1 of the 4 following groups as described by quirke. : substandard, a defect of the muscularis propria or perforation of the tumor ; standard, intact muscularis propria of the specimen ; enhanced, circumferentially covered by a slice of levator ani musculature ; or radical, circumferentially covered by a levator ani muscular cuff 1 cm thick. statistical analysis was performed with spss software for windows (version 13 ; ibm, armonk, ny, usa). comparisons between the 2 groups were made by applying either the student t or mann - whitney u test for unpaired values as appropriate. the test with yates correction for small samples was applied to compare differences in variables expressed as proportions. the 177 patients were divided into 2 groups : 87 patients (49 men [56% ] and 38 women [44% ]) underwent tme by the laparoscopic approach, whereas 90 patients (52 men [57% ] and 38 women [43% ]) underwent tme by the open approach. there was no difference between the 2 groups with regard to age, sex, tumor size, type of carcinoma, histologic type, or postoperative clinical staging. rectal tumors were located at a significantly lower level from the anal verge in the laparoscopic group than in the open group (6 cm for laparoscopy vs 8 cm for open, p 90 laparoscopic surgeries as the attending physicians, should have acquired the skills needed to perform laparoscopic resection for rectal cancer. the surgical time for rectal cancer is closely associated with the surgeon 's experience and the pathology. the mean surgical times for laparoscopic resection for rectal cancer, including lar and apr, commonly were reported to be between 180 and 260 minutes. in this study the mean surgical time of 160 minutes for laparoscopic apr was not significantly different from that for open resection. the similar surgical time was attributed to the fact that the laparoscopic group always underwent surgery by the same surgeons, who have performed 90 laparoscopic surgeries before this study. few comparative studies and randomized controlled trials have been conducted to show that laparoscopic techniques may be associated with less surgical blood loss and reduced perioperative transfusions. blood loss has ranged between 90 and 320 ml for laparoscopic resection in these studies. with regard to our study, the mean operative blood loss was only 28 ml in the laparoscopic group, which was closely associated with factors of the surgeons ' skill. among other iatrogenic injuries (ureter, bowel, vascular system), injuries to the pelvic autonomic nervous system have been much more debated. injuries to the pelvic autonomic nervous system were recorded in only 4 cases in the laparoscopic group compared with 12 cases in the open group. laparoscopy, provided with the characteristics of amplifying the local view, may help in eliminating the blind zone of naked eyes in an open procedure. thus the identification of the operating plane and the protection of the autonomic nerves could also be beneficial. in contrast to breukink., who reported an anastomotic leakage rate of 9% in the laparoscopic group versus 14% in the open group, the rate of anastomotic leakage for either approach ranged from 1% to 2% in our study., laparoscopy for tme in this study has shown consistent advantages over open tme, including a reduced hospital stay, a shorter disability period, less postoperative pain, and a lower rate of chest infection. however, the aforementioned advantages of laparoscopic tme are beneficial to patients only when the oncologic cure rate for this technique is at least similar to that for open tme. the adequacy of oncologic resection in laparoscopic tme has been shown in various studies. in most of these studies, only surgical margins, lymph node yield, and the length of bowel resected were evaluated. most laparoscopic tme series report lower median numbers of harvested nodes. however, our study comparing laparoscopic and conventional resections showed comparable node clearance between the 2 approaches. in this study a high tie of the inferior mesenteric vessels, which is the preferred technique during laparoscopic tme, has led to improved lymph node harvest, thus facilitating accurate tumor staging. it has been shown that a technically poor tme in patients with negative circumferential resection margin (crm) is associated with increased recurrence and worse survival compared with patients having negative margins in whom complete tme has been achieved. therefore the quality of the excised mesorectum is of additional prognostic value in patients who do not have crm involvement. in general, the laparoscopic approach was associated with more complete tme and more intact denonvilliers fascia in all aspects compared with the open approach. furthermore, moderate or incomplete tme at the lateral and posterior aspects was significantly more common after open surgery than after laparoscopic surgery. we conducted a study of 177 patients (87 laparoscopic procedures and 90 open procedures). in all specimens the cut edge of the peritoneal reflection at the anterior mid rectum, the denonvilliers fascia, the visceral fascia covering the posterior and lateral mesorectum, and the bowel wall below the mesorectum were assessed macroscopically. the denonvilliers fascia was violated in 16 patients after open surgery, and a more complete tme with intact visceral pelvic fascia was performed with laparoscopic versus open surgery. however, it is known that the outcome for rectal cancer treatment is improved with both specialized surgical training and a higher volume of rectal cancer procedures. in our study the operations for all the patients were performed by 2 experienced, specialized laparoscopic colorectal surgeons. furthermore, more complete tme after laparoscopy is attributed to the perfect deep pelvic view offered by the approach : dissection of the rectum and the mesorectum at all aspects down to the pelvic floor can be carried out under direct vision, thus preventing inadvertent entering of incorrect planes (figure 2). the better view and the ease with which lar of the rectum can be achieved may also explain the increased percentage of patients with lower colorectal anastomoses in the laparoscopic group compared with the open group. the advantage of a better view and more complete tme by use of the laparoscopic approach has also been emphasized by other authors. a macroscopically complete specimen after tme for rectal cancer is more likely to be acquired by laparoscopy because of the better pelvic view offered by the approach. nevertheless, a randomized controlled trial comparing laparoscopic tme with open tme is required to verify any possible additional benefits of laparoscopy. | it is suggested that laparoscopic total mesorectal excision of low - lying rectal cancer has advantages over an open procedure with less blood loss, reduced hospital stay, and a shorter disability period. complete macroscopic surgical resection appears to be aided by improved pelvic view offered by laparoscopy. |
a research report from the chinese center for disease control and prevention showed that the incidence of paraquat poisoning increased by about 47.35% a year on average from 2002 to 2010. although the survival rate of pq poisoning increased with the improvement of medical levels, there were still a considerable number of patients who died after large doses of oral pq. moderate and severe pq poisoning was mainly characterized by acute lung injury in the early stage and pulmonary fibrosis in the advanced stage. when the patients showed ali / ards or progressive pulmonary fibrosis, the conventional treatment was ineffective. used continuous positive airway pressure (cpap) to treat pq poisoning patients with respiratory failure and the patients died in 15 days. histopathology showed overexpansion of pulmonary parenchyma, suggesting that although cpap ventilation could increase lung volume, it might cause lung overexpansion and aggravate lung injury. lung protective strategies of ventilation (lpvs) has been proposed to treat ali / ards in recent years, which advocated small vt ventilation (6~8 ml / kg), permissive hypercapnia, and maintaining alveolus opening by using peep. at present, treating pq - induced acute lung injury by lpvs still lacks support from basic and clinical research. the present study was conducted to investigate the effects of different vt peep on pq - induced acute lung injury, blood gas analysis indexes, oxygenation index, and hemodynamics and aimed to provide theoretical guidance for the clinical application of mechanical ventilation. eighteen female piglets (6570 days, 25.02.1 kg) were obtained from the experimental animal center of henan province, zhengzhou, china. the piglets were provided with water ad libitum and fasted 12 hours before the operation. after intramuscular injection of atropine (0.05 mg / kg) (harvest pharmaceutical co. ltd, shanghai, china) and ketamine (15 mg / kg) (fujian gutian pharmaceutical co. ltd, gutian, china), the piglets were placed supine on a table with continuous ecg monitoring and ear - vein injection of ringer lactate solution (10 ml / kgh). the piglets received mechanical ventilation and the settings of volume controlled ventilation (vcv) were : vt=12 ml / kg, rr=30 breath / min, inspiratory / expiratory=1:2, fraction of inspired oxygen (fio2)=30%, peep=0 the central venous catheter was set by jugular vein and the picco catheter was set by femoral artery. each piglet received intraperitoneal injection of 20 ml 20% pq solution (sigma, st. louis, mo, usa) and the arterial blood gas analysis was measured every 30 minutes until pao2/fio2300 mmhg. the pao2/fio2300 mmhg in 30 minutes was considered to be successfully developed the ali / ards models. the piglets received intravenous injection of propofol (2.5 mgkgh) (pfizer, new york, ny, usa) and sufentanil (0.025 gkgh) (humanwell pharmaceutical co. ltd. meanwhile, the piglets were given cis atracurium (0.1 mgkgh) (glaxosmithkline, london, uk) intermittently to maintain muscle relaxation. the piglets were then randomly divided into three groups : small vt group (vt=6 ml / kg, n=6), middle vt group (vt=10 ml / kg, n=6), and large vt group (vt=15 ml / kg, n=6), with the peep set as 10 cmh2o. this study was performed in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health (bethesda, md, usa) eighth edition, 2010. the animal use protocol was reviewed and approved by the institutional animal care and use committee (iacuc) of the first affiliated hospital of zhengzhou university. the arterial blood was drawn at t2 (2 hours), t4 (4 hours), and t6 (6 hours) after mechanical ventilation for blood gas analysis. the picco temperature sensor was connected with the jugular vein catheter and the picco detector was connected with the femoral artery catheter. we rapidly injected 10 ml ice saline (04c) via the jugular vein catheter and recorded elwi, pvpi, heart rate (hr), and mean arterial pressure (map) each time at baseline (before ali / ards modeling), t0, t2, t4, and t6. tissues of the right lower lobe of the lung were fixed in 4% paraformaldehyde at room temperature, embedded in paraffin, sectioned serially at 10 m, and stained with hematoxylin and eosin (he). the data were analyzed by unpaired student s t test for comparison of same group at different time and test for comparison of different groups at same time. eighteen female piglets (6570 days, 25.02.1 kg) were obtained from the experimental animal center of henan province, zhengzhou, china. the piglets were provided with water ad libitum and fasted 12 hours before the operation. after intramuscular injection of atropine (0.05 mg / kg) (harvest pharmaceutical co. ltd, shanghai, china) and ketamine (15 mg / kg) (fujian gutian pharmaceutical co. ltd, gutian, china), the piglets were placed supine on a table with continuous ecg monitoring and ear - vein injection of ringer lactate solution (10 ml / kgh). the piglets received mechanical ventilation and the settings of volume controlled ventilation (vcv) were : vt=12 ml / kg, rr=30 breath / min, inspiratory / expiratory=1:2, fraction of inspired oxygen (fio2)=30%, peep=0 the central venous catheter was set by jugular vein and the picco catheter was set by femoral artery. each piglet received intraperitoneal injection of 20 ml 20% pq solution (sigma, st. louis, mo, usa) and the arterial blood gas analysis was measured every 30 minutes until pao2/fio2300 mmhg. the pao2/fio2300 mmhg in 30 minutes was considered to be successfully developed the ali / ards models. the piglets received intravenous injection of propofol (2.5 mgkgh) (pfizer, new york, ny, usa) and sufentanil (0.025 gkgh) (humanwell pharmaceutical co. ltd. meanwhile, the piglets were given cis atracurium (0.1 mgkgh) (glaxosmithkline, london, uk) intermittently to maintain muscle relaxation. the piglets were then randomly divided into three groups : small vt group (vt=6 ml / kg, n=6), middle vt group (vt=10 ml / kg, n=6), and large vt group (vt=15 ml / kg, n=6), with the peep set as 10 cmh2o. this study was performed in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health (bethesda, md, usa) eighth edition, 2010. the animal use protocol was reviewed and approved by the institutional animal care and use committee (iacuc) of the first affiliated hospital of zhengzhou university. the arterial blood was drawn at t2 (2 hours), t4 (4 hours), and t6 (6 hours) after mechanical ventilation for blood gas analysis. the picco temperature sensor was connected with the jugular vein catheter and the picco detector was connected with the femoral artery catheter. we rapidly injected 10 ml ice saline (04c) via the jugular vein catheter and recorded elwi, pvpi, heart rate (hr), and mean arterial pressure (map) each time at baseline (before ali / ards modeling), t0, t2, t4, and t6. tissues of the right lower lobe of the lung were fixed in 4% paraformaldehyde at room temperature, embedded in paraffin, sectioned serially at 10 m, and stained with hematoxylin and eosin (he). all data were analyzed using spss17.0 software (spss inc., chicago, il, usa). the data were analyzed by unpaired student s t test for comparison of same group at different time and test for comparison of different groups at same time. as shown in table 1, hr and map in the three groups increased after modeling success (t0) compared with baseline, with significant difference (p3 can distinguish between hydrostatic pressure and pulmonary edema [1618 ]. in this study, elwi increased significantly after modeling success compared with baseline, which reflected the degree of pulmonary edema to a certain extent. pvpi was much higher than the baseline after modeling success, which illustrated that pq - induced pulmonary edema was a permeability edema. lpvs has been proposed to treat ali / ards in recent years, which advocated small vt ventilation, permissive hypercapnia, and maintaining alveolus opening by using peep. since the currently recommended small vt ranged from 6 to 8 ml / kg, we used 6 ml / kg for the small vt in our study. the normal vt of pigs ranges from 10 to 12 ml / kg and we used 10 ml / kg for middle vt. the recommended large vt ranges from 1520 ml / kg and we used 15 ml / kg. peep is mainly set as 10 cmh2o in animal models, which had little effect on hemodynamics. lpvs combined with peep can increase alveolar pressure, promote the reabsorption of alveolar exudate, and reduce inflammation. application of appropriate peep could avoid alveolar collapse, improve hypoxemia, and increase the ventilation efficiency. as reported, the patients in the lpvs group had lower incidence of ventilator - associated lung injury (vali) and slower progression compared with the conventional mechanical ventilation (cmv) group. paco2 and pao2/fio2 in the lpvs group was much higher than in the cmv group, with a significant difference. in our study, pao2 and oxygenation index in the small vt group had a more obvious increase than in the other two groups. extravascular lung water in the small vt group was less than in the other two groups, which confirmed the treatment effects of small vt ventilation on pq - induced lung injury. small vt ventilation can cause co2 retention and paco2 rise, which can lead to acidosis. we found that paco2 rose and ph decreased after ventilation and the ph in the small vt group was the lowest of the three groups. it was considered that the permissive hypercapnia had protective effects on lungs, which might be related with enhanced antioxidant activity and attenuate lipid peroxidation of the lungs. our study showed that pip and pplat increased gradually after mechanical ventilation in the middle and large vt groups. lung histopathology showed obvious injuries in the middle and large vt groups, which might be associated with airway pressure increase and alveolar overinflation - induced lung injury. menendez. reported that overlarge vt ventilation could cause vasoconstriction, reduce endothelium dependent vascular relaxation, and promote hypoxic pulmonary vasoconstriction, which would aggravate lung injury and increase mortality by reducing the expression of -adrenalin. small tidal volume ventilation combined with appropriate peep can alleviate acute lung injury induced by paraquat, promote gas exchange, and improve oxygenation. lpvs may be a good choice to alleviate the degree of pulmonary edema and improve the oxygenation status after the pq poisoning patients showed acute lung injury and autonomous breathing was difficult to maintain. considering that our study was only a short - term experiment, further animal experiments and clinical practice | backgroundthe aim of this study was to explore the effects of different tidal volume (vt) ventilation on paraquat - induced acute lung injury or acute respiratory distress syndrome (ali / ards) in piglets.material/methodswe developed ali / ards models in piglets by intraperitoneal injection of paraquat (pq). the piglets were randomly divided into three groups : small vt group (vt=6 ml / kg, n=6), middle vt group (vt=10 ml / kg, n=6), and large vt group (vt=15 ml / kg, n=6), with the positive end - expiratory pressure (peep) set as 10 cmh2o. the hemodynamics were monitored by pulse - indicated continuous cardiac output (picco) and the airway pressure changes and blood gas analysis indexes were recorded at different time points. the pathological changes were observed by lung puncture.resultsthe piglets showed ali / ards in 4.50.8 hours after pq intraperitoneal injection. ph, pao2 and oxygenation indexes in the three groups all decreased after modeling success compared with baseline, and paco2 increased significantly. ph in the small vt group decreased most obviously after ventilation for 6 hours. pao2 and oxygenation indexes in the small vt group showed the most obvious increase after ventilation for 2 hours and were much higher than the other two groups after ventilation for 6 hours. paco2 increased gradually after mechanical ventilation and the small vt group showed most obvious increase. the elwi increased obviously after ventilation for 2 hours and then the small vt group clearly decreased. pip and plateau pressure (pplat) in the small vt group decreased gradually and in the middle and large vt group they increased after ventilation. the lung histopathology showed that the large vt group had the most severe damage and the small vt group had only minimal damage.conclusionssmall tidal volume ventilation combined with peep could alleviate the acute lung injury induced by paraquat and improve oxygenation. |
at present kidney diseases are a major problem across the globe. as per global and regional overview, 1783000 patients are suffering from end stage renal disease (esrd), of which dialysis has been given to 1371000 patients and kidney has been transplanted on 412000 patients suffering from renal failure whom live under particularly pro - oxidative conditions causing uremia (1, 2). treatment option for uremia includes kidney transplantation and dialysis, which are very expensive and not free from side effects. many scientists have tried to find out different phytomedicines to manage uremia (3). typically, urea builds up in the patient s blood as the result of inefficiently operating kidneys, which usually results from either acute or chronic kidney failure (4). oxidative stress is defined as an imbalance between formation of reactive oxygen species (ros) and anti - oxidative defense mechanisms. considering the profound biological effects of ros, in recent years numerous clinical and experimental studies have been focused on detection of signs of oxidative stress in renal patients (5). there is good evidence indicating that uremia in general is associated with enhanced oxidative stress (5). loss or deficiency of antioxidant activity (e.g. vitamin e deficiency) could also contribute to enhanced oxidative stress in uremia. interstitial inflammation and oxidative stress may participate jointly in the development and reduction of the number of nephron units, which thereby limits sodium filtration (6). it is known that low density lipoprotein (ldl) from uremic patients, presents an elevated susceptibility to oxidation (7). uremic oxidative stress is characterized from a biochemical point of view as a state of reactive aldehyde and oxidized thiol group accumulation, together with depletion of reduced thiol groups, which are particularly important as part of antioxidant defense (7). as a consequence of diminished renal catabolism and function, uremic oxidant mediators accumulate, favoring vascular cell dysfunction and progression to kidney failure and many other diseases (7). terminalia arjuna (t. arjuna) is also an important medicinal plant widely used in the preparation of ayurvedic formulations for over three centuries, primarily as a cardiac tonic in india (8). clinical evaluation of this plant indicates that it can be of benefit in the treatment of coronary artery diseases, heart failure and possibly hypercholesterolemia (9). however, most of the beneficial works on this plant have been carried out on hepatic or renal disorders (10). in this particular study, protective role of aqueous extract of t. arjuna bark was evaluated against dehydration induced oxidative stress and uremia in male rats. this work will also cover the basic physiological process for the management of uremia by loss of the antioxidant status in both blood and kidney and uremic profiles in blood. in contrast, this work has also a clinical dimension, because the results may be disseminated to the society, after proper investigation on the renal failure patients. animal selection and care this study was conducted on twenty four healthy adult male wister strain rats, with a body weight of 108 3 g. they were housed in cage (1 rats /cage) and acclimatized to laboratory conditions for 2 weeks prior to experimentation at constant temperature of 22 3 c, with 12 - 12 h light - dark cycle (8.00 - 20.00h light : 20.00 - 8.00 h dark) at a humidity of 5010%. they were supplied with an adequate dry food (pellet diet) and water ad libitum. the principle of laboratory animal care (nih 1985) was followed duration the experiments and our university ethics committee approved the experimental protocol (11). the bark of t. arjuna was collected from gopali, indian institute of technology, kharagpur, paschim medinipur district of west bengal, india. taxonomist of botany department, raja n. l. khan women s college, midnapore identified the material and voucher specimen (number - bvs-7) was deposited in the department of botany, raja n. l. khan women s college. preparation of aqueous extract of t. arjuna bark at first, t. arjuna bark was dried at 40 1 c in incubator and the dried parts were crushed using an electric grinder and the resulting powder was then separated. next, 25 g of the fine powder was dissolves in 250 ml of distilled water and kept in an airtight glass jar. this mixture was incubated at 37 1 c for 72 h in a soxhlet extraction apparatus. then this extract was dried in a vacuum desiccator to obtain a dry mass, stored in a refrigerator at 4oc and used for the next 7 days of our experiments. as per necessity, the extract was again prepared throughout the experimental period. when needed, the extract was suspended in de - ionized water and used in the study (12). experimental design twenty four healthy adult male wistar strain rats were divided into four groups on the basis of matching the body weights of the animals. the treatment schedule of each group was as follows : (i) group i or the control group animals were subjected to control groups feed dry food (pellet diet) and an adequate amount of water. rats of this group received de - ionized water for 15 days prior to experimentation, followed by the next 15 days of experimental period through forceful oral route at 8.00 a.m. through gavage. (ii) group ii or the control plus the extract (t. arjuna) treated group animals were subjected to forceful oral administration of the aqueous extract of this plant parts at a dose of 400 mg / kg body weight /day / rat in 0.5 ml deionized water for 15 days prior to the commencement of experiment followed by the next 15 days of experimentation without dehydration. (iii) group iii or the dehydration group initially, rats were supplied with a normal diet and adequate amount of water for the first 15 days of experimentation. these rats were then induced to dehydration (according to the dehydration protocol) for the next 15 days of experimentation and 0.5 ml of de - ionized water was provided forcefully through oral route at 8.00 a.m. through gavage. (iv) group iv or the pretreatment followed by dehydration and extract administration group rats were subjected to preconditioning by oral administration of the aqueous extract of this plant parts for 15 days, prior to the induction of dehydration at the same dose as group ii. from the 16th day, animals were subjected to dehydration for the next 15 days and all the animals in this group were subjected to oral administration of the aqueous extract of these plant parts at the same dose as group ii. animals sacrificed for plasma and organ collection the whole experimental design was continued for 30 days and animals were sacrificed and then their blood and kidney collected from aorta and peritoneal cavity, respectively. dehydration protocol group i and group ii animals were randomly placed as 1 rat / cage, with free access to dry food (pellet diet) and adequate water. groups iii and iv animals were randomly placed as 1 rat / cage, with free access to dry food (pellet diet). dehydration was achieved by withdrawing the drinking water bottle for 24 h and by providing 2 ml water to each rat after an interval of 24 h, throughout the 15 days dehydration period of experimentation (13). statistical analysis analysis of variance (anova) followed by a multiple two - tail t - test with bonferroni modification, was used for statistical analysis of the collected data. antioxidant enzymes (i) biochemical assay of catalase activity (cat) catalase activity was measured biochemically. for the evaluation of cat activity in plasma,, they were homogenized separately in 0.05 m tris hydrochloric acid (hcl) buffer solution (ph = 7.0) at a tissue concentration of 50 mg / ml. these homogenates were centrifuged separately at 10,000 g at 4 oc for 10 min. in a spectrophotometric cuvette, 0.5 ml of hydrogen peroxide (h2o2) and 2.5 ml of distilled water were mixed and absorbance was determined at 240 nm. forty l of tissue supernatant and plasma were separately added, and the subsequent six reading were noted at 30 sec intervals (14). (ii) biochemical assay of superoxide dismutase (sod) kidneys were homogenized in ice - cold 100 mm tris - cocodylate buffer to give a tissue concentration of 50 mg / ml and blood centrifuged at 10,000 g for 20min at 4 oc. the sod activity of these supernatants was estimated by measuring the percentage of inhibition of the pyragallol auto - oxidation by sod. the buffer was 50 mm tris (ph = 8.2) containing 50 mm cocodylic acid (ph = 8.2), 1 mm ethylene diamine tetra acetic acid (edta) and 10 mm hydrochloric acid (hcl). in a spectrophotometric cuvette, 2 ml of buffer, 100 l of 2 mm pyragallol and 10 l of supernatant were poured and the absorbance was noted at 420 nm for 3 min. one unit of sod was defined as the enzyme activity that inhibited the auto - oxidation of pyragallol by 50 % (15). estimation of lipid peroxidation from the levels of malondialdehyde (mda) and conjugated dienes (cd) the kidneys were homogenized separately at a tissue concentration of 50 mg / ml in 0.1 m of ice - cold phosphate buffer (ph = 7.4) and the homogenates and blood samples were separately centrifuged at 10,000 g at 4 c for 5 min. supernatant and plasma were used for the estimation of mda and cd. for the measurement of mda, 0.5 ml homogenate and plasma were mixed separately with 0.5 ml normal saline and 2 ml of tba - tca mixture (0.392 g of tba in 75 ml of 0.25 n hcl with 15 g of tca, with the final volume of the mixture being made up to 100 ml with ethanol) and, then boiled at 100 c for 10 min. the mixture was then cooled at room temperature and centrifuged at 4000 g for 10 min. the whole supernatant and plasma was transferred into a spectrophotometer cuvette and read at 535 nm. calibration was performed by using the acid hydrolysis of 1, 1, 3, 3 tetra - methoxy propane, as a standard. the mda present within the sample was calculated by using the extinction coefficient of 1.56 105 m / cm and expressed as the unit of nm / mg of tissue or nm / ml of plasma (16). the lipids were extracted with the chloroform - methanol (2:1) mixture, followed by centrifugation at 10,000 g for 5 min. the lipid residue was dissolved in 1.5 ml of cyclohexane and the absorbance was noted at 233 nm to measure the amount of hydrogen peroxide formed (16). blood uremia profile (i) biochemical estimation of blood urea the collected blood was centrifuged and plasma fraction was separated. urea level of the plasma measured by commercially available standard blood urea kit (merck, japan), using a semi - autoanalyser (merck, microlab-300, japan) as per the standard protocol for phtotometric determination of urea according to the urease gldh method (kinetic uv test). first, 10 l of urea standard (50 mg/ 100ml) was mixed with 1000 l of the monoreagent (composed of tris ph 7.8 120 mmol / l, 2-oxoglutarate- 7 mmol / l, adp 0.6 mmol / l, rease 6 1ku / l and nadh 0.25 mmol / l) and incubated for around 60 sec at 25 c, and absorbance was read at 37 oc for standardization. then, 10 l samples were used for the experimentation, as described before (17). (ii)biochemical estimation of blood creatinine the collected blood was centrifuged and plasma fraction was separated. creatinine level of plasma was measured using commercially available standard blood urea kit (merck, japan) and a semi - autoanalyser (merck, microlab-300, japan) as per standard protocol for phtotometric determination of creatinine, based on jaffe kinetic method without deproteinization. first, 100 l of creatinine standard (1 mg/ 100ml) was mixed with 1000 l of the monoreagent (buffer : naoh 313 mmol / l and picric acid 8.73 mmol / l) and incubated for around 5 min at 25 oc and then absorbance was read at 37 oc for standardization. toxicity study via biochemical estimation of glutamic oxaloacetic transaminase (got) and biochemical estimation of glutamic pyruvic transaminase(gpt) for the assessment of toxicity in blood and kidney, got and gpt were measured, based on the method of goel (19). water intake and dehydration procedure during the first 15 days of experimentation, all groups of rats were supplied with normal water. they were given a maximum of 16.4 0.6 ml water / day / individual as intake. then, during the next 15 days of experimentation, groups i and ii were supplied with an adequate (normal) amount of water. however, groups iii and iv were supplied with only 2 ml water / rat/24h (table 1). water intake (ml) /rat / day in the first 15 days, followed by the next 15 days of experimentation. dehydration procedure followed by providing a little amount of drinking water to male albino rats for the next 15 days of experimental period. the statistical test used was anova followed by multiple two - tail t - test. data with the same superscripts (a) for a specific data did not differ from each other significantly (p > 0.05). group ii : control + extract treated group group iii : dehydrating group group iv : pretreatment followed by dehydration and extract coadministration body weight and somatic indices of kidneys body weight increased at the end of experiment in groups i, ii and iv compared to their initial body weight (table 2). in group iii, the percentage of elevation in body growth was dramatically less than the other groups, due to the dehydration induced oxidative stress (table 2). renal somatic decreased significantly in group iii, in comparison to groups i, ii and iv. after administration of the t. arjuna extract in group iv, these indices were resettled towards the control level (table 2). protective effect of pretreatment followed by coadministration of aqueous extract of t. arjuna bark on body growth and renal organo - somatic indices in dehydration induced oxidative stress in rat. anova followed by multiple two - tail t - test and data with different superscripts (a, b) in a specific vertical column differed from each other significantly (p 0.05). group - ii : control + extract treated group group iii : dehydrating group group iv : pretreatment followed by dehydration and extract coadministration activities of catalase and sod in group ii, cat activities in blood and kidney were elevated, compared to group i, iii and iv. after dehydration (group iii), the activity of this enzyme in blood and kidney was decreased significantly, compared with group i. there was a significant protection in catalase activity after pretreatment with the extract, followed by its coadministration (group iv) compared with the animals in the dehydrating group (group iii) (table 4). protective effect of pretreatment followed by coadministration of aqueous extract of t. arjuna bark on plasma and kidney catalase and sod activities in dehydration induced oxidative stress in rat. data are expressed as mean se (n=6). the statistical test used was anova followed by multiple two - tail t - test and data with different superscripts (a, b, c) in a specific vertical column differed from each other significantly (p < 0.05). group ii : control + extract treated group group iii : dehydrating group group iv : pretreatment followed by dehydration and extract coadministration administration of the herbal mixture to non - dehydrating animals (group ii) resulted in a significant elevation in the activity of sod in blood and kidney, compared to the animals in groups i and iv. the activity of this enzyme was decreases significantly in blood and kidney in group iii, in comparison with group i (table 4). pretreatment followed by coadministration of the extract to the animals in group iv resulted in a significant restoration of the sod activity, compared with group iii, and the values were resettled to the control level. quantification of mda and cd quantities of mda and cd were increased in blood and kidney of group iii animals, compared to groups i, ii and iv (table 5). in the dehydrated rats (group iii), 15 days of dehydration resulted in a significant elevation in the values of both parameters in blood and kidney. however, in group iv administration of the extract significantly decreased mda and cd quantities. protective effect of pretreatment followed by coadministration of aqueous extract of t. arjuna bark on mda and cd of plasma and kidney in dehydration induced oxidative stress in rat. data are expressed as mean se (n=6). the statistical test used was anova followed by multiple two - tail t - test and data with different superscripts (a, b, c) in a specific vertical column differed from each other significantly (p < 0.05). group ii : control + extract treated group group iii : dehydrating group group iv : pretreatment followed by dehydration and extract coadministration mda : melondialdehyde cd : conjugated dienes levels of blood urea and creatinine urea and creatinine levels were significantly increased in group iii animals (the dehydration group), compared to groups i and ii. but in group iv (pretreatment, dehydration and coadministration of extract) significantly low levels of urea and creatinine were observed, compared to group iii, and the values resettled to the levels of control group. protective effect of pretreatment followed by coadministration of aqueous extract of t. arjuna bark on plasma urea and creatinine activities in dehydration induced oxidative stress and uremia condition in rat. the statistical test conducted was anova followed by multiple two - tail t - test and data with different superscript (a, b, c) in a specific vertical column differed from each other significantly (p < 0.05). group ii : control + extract treated group group iii : dehydrating group group iv : pretreatment followed by dehydration and extract coadministration dehydration is the risk factor at the point when urine production declines and finally results in no urine out put (anuria). here, dehydration is only used for causing oxidative stress, elevation of blood urea and creatinine levels (3). without urinary excretion of waste products, dangerous levels of urea decreased blood volume occurs with deficient fluid intake, which causes a reduced blood flow in kidney resulting in a decreased glomerular filtration rate (gfr). this can consequently lead to acute renal failure (arf) (4). hence prolonged increase in urea and creatinine levels due to a decreased gfr, causes chronic renal failure (crf) (4). when gfr decreases down to 90%, then end stage renal disease (esrd) may occur. dehydration induced oxidative stress in blood and kidney has been established in this study to cause low activities of sod and cat. the decrease in the activity of antioxidant enzymes, as a result of dehydration, might be due to their use against the free radicals destruction and their inhibition by free radicals species (22). it is well established that sod activity is inhibited by hydrogen peroxide, that reduces cu to cu in sod (23).the reduction of hydrogen peroxide is catalyzed by cat, that protects tissues from highly reactive hydroxyl radicals (24). increase in the levels of oxidative stress products like mda and cd in blood and kidney in the dehydrating group, again indicated the low level of antioxidant enzymes activities which causes progression of lipid peroxidation. other possibilities for such elevation in mda and cd could be the ischemia - reperfusion phenomenon (25, 26) or the high rate of catecholamine secretion that generates free radicals either through auto - oxidation or through metal ion or superoxide - catalyzed oxidation (12, 27). recent studies have shown that oxidative stress is highly present in patients with renal disease (7). uremic oxidative stress is characterized from a biochemical point of view as a state of reactive aldehyde and oxidized thiol group accumulation, together with depletion of reduced thiol groups, which are particularly important as part of antioxidant defense (28). as a consequence of diminished renal catabolism and function, uremic oxidant mediators accumulate urea and creatinine in blood (29). in this study, the t.arjuna extract was used due to its traditional of this plant extract caused a significant elevation in the levels of antioxidant status. moreover, this extract diminished the lipid peroxidation in blood cells and kidney of dehydrated animals. salvage pathway and transfer of urea from plasma to colon, where it is broken down to ammonia by bacterial urease. oxidative stress appears to increase as ckd progresses, and correlates significantly with the level of renal function (30). in the present study, the levels of urea and creatinine were elevated due to the increase in oxidative stress in only the dehydrated animals. however, administration of plant extract decreased the uremic parameters near the levels of control animals. the extract had no general and metabolic toxic effect, as reflected from the insignificant variation in body growth and activities of sgot and sgpt in blood and kidney tissues. hence, we could conclude that correction and protection of the oxidative stress and uremia in the experimental animals have been found by applying the aqueous bark extract of t. arjuna. further complex studies are needed to fully characterize the responsible active ingridents present in the plant and elucidate their possible mode of action and mechanism that is in progress. | the present study has been designed to find out the protective effect of aqueus extract of terminalia arjuna against dehydration induced oxidative stress and uremia, protection by plant extract in male wister strain albino rats, and therefore to find out the scientific basis of local use of terminalia arjuna bark extract by village ayurved doctors to protect the progressive kidney disorder (renal failure) relating to dehydration and other related problems. water withdrawing for 15 days in male wister strain albino rats resulted in a significant elevation in the level of blood nitrogenous products (i.e. urea and creatinine). on the other hand, it increased the levels of free radicals, melondialdehyde (mda) and conjugated dienes (cd) along with a significant diminution in the activities of superoxide dismutse (sod) and catalase in blood. all these water markers were significantly prevented after administration of aqueous extract of terminalia arjuna bark. these results suggest that dehydration induced oxidative stress and uremia in male rats may be protected by using the above mentioned medicinal plants extract. this herbal extract showed no toxic effect on blood and kidney, based on the measurements of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase activities (data not shown). |
behet s disease (bd) is the systemic vasculitis of small and large vessels, which affects both veins and arteries. the clinical features of bd indicate that there are distinct geographical differences ; while vascular involvement is detected in 40% of patients in the middle east, the percentage of vascular involvement is 5% - 10% in the far east (japan) (1, 2). thrombosis of the major veins such as the superior and inferior vena cava can be seen occasionally. budd - chiari syndrome (bcs) is a rare occurrence in the course of bd and carries a high mortality rate. bcs is caused by an outflow obstruction in any part of the hepatic venous system, from the small hepatic veins (hv) to the inferior vena cava (ivc) (3). while thrombosis is more prominent in the west, webs are prominent in the east and in japan. bd is a more prominent etiological factor in turkey than in other countries (4). although there are studies that demonstrate the prognostic significance of the portal vein (pv), hv, and ivc in bcs, there are no data in the literature explicating the prognosis and long - term outcomes for bcs that is secondary to bd (5). in this retrospective study, we aimed to present the long - term follow - up outcomes for patients with bcs due to bd through clinical findings, laboratory test results, and the determination of vascular involvement. the data for 402 patients diagnosed with bd (according to the diagnostic criteria for bd given by the international study group in 1990), who received regular follow ups at the rheumatology department at eskisehir osmangazi university medical faculty between 1998 and 2014, were evaluated retrospectively (6). of the 402 patients with bd, 96 (23.9%) doppler ultrasound (dusg) and computed tomography (ct) were used as the first - line imaging modalities for the diagnosis of bcs. dusg and magnetic resonance imaging (mr) or ct (for patients for whom mr was not suitable) were the preferred techniques for the long - term follow up of the patients. ultrasonography - guided (usg) liver mass biopsies were performed on the patients in whom mass lesions were detected during surveillance (cases 3 and 4). the full biochemistry results, complete blood counts, erythrocyte sedimentation rates (esr), and c - reactive protein (crp) results for the patients were determined at the initial presentation stage and throughout the follow up. the congenital (protein c [pc ], protein s [ps ], and antithrombin iii [atiii ] levels, and factor v leiden mutations) and acquired causes (anticardiolipin and lupus antibodies) of thrombophilia were evaluated in all patients initially, at the time of bcs diagnosis. if the levels of pc, ps, and at iii were below the normal range, the study was repeated twice - at both 6 months and 1 year after the initial diagnosis (cases 2, 3, 4, and 5). the activity of the bd at the time of bcs diagnosis was determined using the european bd activity index (7). for the detection of long - term complications, the patients were monitored via physical examinations, laboratory investigations, imaging modalities, and endoscopies. the patients were aged between 23 and 54, and all five were male. the duration of bd in the patients ranged from 1 to 24 years (median : 12.4 years). the interval between the onset of bd and the development of bcs ranged from 1 to 8 years (mean : 3.8 years). all the patients had genital ulcers, deep vein thrombosis (dvt), superficial thrombophlebitis (stp), and skin involvement at the time of diagnosis. the patients presented with hepatomegaly, abdominal pain, ascites, and mild or moderate hyperbilirubinemia, leading to a diagnosis of bcs. the diagnoses of bd and bcs were made simultaneously in two of the patients (cases 1 and 4). all the patients except one presented with severely increased serum aspartate aminotransferase (ast) and alanine aminotransferase (alt) levels. esr levels were increased in three of the patients only, but crp levels were elevated in all the patients. in terms of potential underlying thrombophilic conditions apart from bd, pc deficiency was detected in four of the patients. in addition to pc deficiency, factor v leiden mutation was determined in case 2. three types of venous outflow obstructions were recorded (cases 1 and 5 : involvement of 3 hvs and the vci ; cases 3 and 4 : involvement of 2 hvs and the vci ; case 2 : involvement of 1 hv and the vci). in case 1, in addition to the involvement of 3 hvs and the vci, intra - cardiac thrombus and pulmonary embolism were detected. portal venous thrombosis (pvt) was detected in only one of the patients (the detailed characteristics of the patients are given in table 1). abbreviations : bd, behcet s disease ; bcs, budd - chiari syndrome ; m, male ; dvt, deep vein thrombosis ; stp, superficial thrombophlebitis ; paa, pulmonary artery aneurysms ; ast, aspartate aminotransferases (normal range 7 - 39 u / l) ; alt, alanine aminotransferases (normal range 2 - 40 u / l) ; ggt, gamaglutamil transferases (normal range 8 - 50 348u / l) ; esr, erythrocyte sedimentation rate ; crp, c - reactive protein (normal range 0 - 0.5 mg / dl) ; fv, factor v ; pc, protein c ; pv, portal vein ; hep, hepatic encephalopathy ; hv, hepatic vein ; ivc, inferior vena cava ; lrns, large regenerative nodules ; nrh, nodular regenerative hyperplasia one of the patients with bcs died during the acute period due to acute liver failure (alf). the survival time for the other four patients after bcs diagnosis ranged from 4 to 16 years. hepatic masses were determined by radiological surveillance in case 3 (figure 1) and case 4 (11 and 4 years after the initial diagnosis of bcs, respectively). liver - mass biopsies revealed there to be large regeneration nodules (case 3 ; figure 2) and cirrhosis (case 4). during endoscopic surveillance for the evolution of esophago - gastric varices, esophageal varices were determined in case 4 only. with the exception of the patient in case 1, who died during the acute period, all of the patients are still alive. in case 4, in the acute stage, all the patients received pulse methyl prednisolone and cyclophosphamide, in addition to anticoagulants with heparin. this was followed by the prescription of warfarin and azathioprine, these being general treatments for the condition. the patients were evaluated for radiological or surgical interventions but, unfortunately, all patients with bcs caused by bd have icv thrombosis, which results in an unsuitable pressure gradient occurring between the portal vein and the inferior vena cava, and so radiological interventions and porto - caval shunt operations are technically impossible. the patients were aged between 23 and 54, and all five were male. the duration of bd in the patients ranged from 1 to 24 years (median : 12.4 years). the interval between the onset of bd and the development of bcs ranged from 1 to 8 years (mean : 3.8 years). all the patients had genital ulcers, deep vein thrombosis (dvt), superficial thrombophlebitis (stp), and skin involvement at the time of diagnosis. the patients presented with hepatomegaly, abdominal pain, ascites, and mild or moderate hyperbilirubinemia, leading to a diagnosis of bcs. the diagnoses of bd and bcs were made simultaneously in two of the patients (cases 1 and 4). all the patients except one presented with severely increased serum aspartate aminotransferase (ast) and alanine aminotransferase (alt) levels. esr levels were increased in three of the patients only, but crp levels were elevated in all the patients. in terms of potential underlying thrombophilic conditions apart from bd, pc deficiency was detected in four of the patients. in addition to pc deficiency, factor v leiden mutation was determined in case 2. three types of venous outflow obstructions were recorded (cases 1 and 5 : involvement of 3 hvs and the vci ; cases 3 and 4 : involvement of 2 hvs and the vci ; case 2 : involvement of 1 hv and the vci). in case 1, in addition to the involvement of 3 hvs and the vci, intra - cardiac thrombus and pulmonary embolism were detected. portal venous thrombosis (pvt) was detected in only one of the patients (the detailed characteristics of the patients are given in table 1). abbreviations : bd, behcet s disease ; bcs, budd - chiari syndrome ; m, male ; dvt, deep vein thrombosis ; stp, superficial thrombophlebitis ; paa, pulmonary artery aneurysms ; ast, aspartate aminotransferases (normal range 7 - 39 u / l) ; alt, alanine aminotransferases (normal range 2 - 40 u / l) ; ggt, gamaglutamil transferases (normal range 8 - 50 348u / l) ; esr, erythrocyte sedimentation rate ; crp, c - reactive protein (normal range 0 - 0.5 mg / dl) ; fv, factor v ; pc, protein c ; pv, portal vein ; hep, hepatic encephalopathy ; hv, hepatic vein ; ivc, inferior vena cava ; lrns, large regenerative nodules ; nrh, nodular regenerative hyperplasia one of the patients with bcs died during the acute period due to acute liver failure (alf). the survival time for the other four patients after bcs diagnosis ranged from 4 to 16 years. hepatic masses were determined by radiological surveillance in case 3 (figure 1) and case 4 (11 and 4 years after the initial diagnosis of bcs, respectively). liver - mass biopsies revealed there to be large regeneration nodules (case 3 ; figure 2) and cirrhosis (case 4). during endoscopic surveillance for the evolution of esophago - gastric varices, esophageal varices were determined in case 4 only. with the exception of the patient in case 1, who died during the acute period, all of the patients are still alive. in case 4, in the acute stage, all the patients received pulse methyl prednisolone and cyclophosphamide, in addition to anticoagulants with heparin. this was followed by the prescription of warfarin and azathioprine, these being general treatments for the condition. the patients were evaluated for radiological or surgical interventions but, unfortunately, all patients with bcs caused by bd have icv thrombosis, which results in an unsuitable pressure gradient occurring between the portal vein and the inferior vena cava, and so radiological interventions and porto - caval shunt operations are technically impossible. the worldwide geographical distribution of primary and secondary bcs is highly variable, and there is scarcity of data in developing countries due to its rarity. the third most common reason for the occurrence of bcs in turkey (where the incidence of bd is extremely high) is bd (4). the bcs occurrence rate during the course of bd was reported as being 0.35% in a study conducted in turkey, in which the diagnosis of bd was made at the same time as the diagnosis of bcs in 71.4% of the patients (8). in our series, the bcs occurrence rate during the course of bd was 1.2%, and the diagnosis of bd was made at the same time as the diagnosis of bcs in 40% of the patients. bcs results from occlusion of the three hepatic veins in about 2/3 of cases, isolated occlusion of the ivc in about 10% of cases, and combined occlusion in almost 1/3 of cases (9). uskudar. and harmanci. found there to be 30% combined venous occlusion in bd patients with bcs (4, 5). in our series, the combined venous occlusion rate was 100%. in a study by bayraktar., which followed 14 bd patients with bcs within a series of 493 bd patients (3%), the authors concluded that the extent of vascular thrombosis within the inferior vena cava, rather than the presence of the hepatic vein thrombosis per se, was the major determinant of survival (10). nowadays, in addition to the involvement of hvs and the vci, pvt has come into prominence as a prognostic indicator in bd patients with bcs (4, 5, 11, 12). found that none of the aforementioned prognostic parameters (i.e., vascular involvement sites) were correlated with mortality, except for the presence of pvt (which had an estimated instantaneous risk of 8.4). they explain that pvt may indirectly reflect the degree of hepatic distress produced by outflow production due to the hepatoportal venous reflex (5). darwish murad. undertook a large international study, investigating patients diagnosed with nonmalignant bcs between 1984 and 2001 and classifying them into isolated bcs cases (n = 204), bcs - pvt cases without spleno - mesenteric vein thrombosis (smvt ; n = 15), and cases of bcs - pvt with smvt (n = 18). the survival rate after five years was 59% (95% with a ci of 39% - 80%) in bcs - pvt, versus 85% (95% with a ci of 76% - 88%) in isolated bcs (p = 0.11) (12). the data obtained from the literature on bcs patients reflects that the results come from heterogeneous, general patient groups. further analysis reveals that pvt was present in just one of the eight bcs patients with bd in harmanci.s research (p = 0.667) and in none of the nine bcs patients with bd in the study by uskudar. although both studies conclude that pvt is a negative factor in the prognosis for bcs generally, it was impossible to determine the effect of pvt upon bcs patients with bd specifically due to limited patient numbers (4, 5). in light of the worsening prognosis for bcs patients with pv involvement, it might be argued that determining the factors that can contribute to the development of pvt would be benefical in terms of seeking methods of prevention and treatment. bagheri lankarani. found, however, that there were no significant risk factors for pvt patients with cirrhosis awaiting liver transplantation in shiraz, iran (13). in our study, pvt was determined in one patient only, who died during the acute period due to alf ; the other patients, all of whom survived, tested negative for pvt. a study including large numbers of bcs patients with bd and demonstrating pvt would be helpful in determining the long - term prognostic significance of pvt in bcs patients with bd. the survival time of patients after diagnosis of bcs can vary from between 3 months and 16 years, according to the literature (14). our data, therefore, with a survival time of 1 month to 16 years after diagnosis of bcs, was consistent with the literature. hepatic masses might be detected during long - term follow - ups for bcs patients. to date, several studies from different regions have reported that a proportion of cases of bcs, especially in patients with vci obstruction, are complicated by the development of hepatocellular carcinoma (hcc) in the long term (14, 15). as patients with hcc and concomitant hepatitis were excluded from these studies, the pooled prevalence of hcc in bcs was 15.4%. the risk factors for hcc in bcs included the hepatic venous pressure gradient and being female (in two asian studies), and factor v leiden mutation, being male, and inferior vena cava obstruction (in one european study) (14). as is evident the risk factors for hcc in bcs vary, depending on the geographical origin of the studies. as well as hcc, large regenerative nodules (lrns) caused by outflow obstructions in the hepatic veins or vena cava are the masses most commonly associated with bcs (16).. ames. found that nrh was often associated with organ transplantation, myeloproliferative disease, and autoimmune processes. in their study, ct and mri showed no enhancing liver masses in any of the patients with nrh. in contrast, lrns were often associated with bcs (17). in accordance with the literature, hepatic masses were detected in 2 patients in this study through radiological surveillance and liver biopsies (lnrs in case 3 and nrh with cirrhosis in case 4). currently, clinical judgment and local expertise play important roles in the management of bcs. medical therapy emerging technologies have offered new minimally invasive treatment modalities, such as percutaneous catheter - directed thrombolysis, angioplasty, stenting, and tips (9). although there is no controlled study in the existing literature regarding the benefits of immunosuppressive drug therapy for patients with bcs caused by bd, treatments used in the literature for bd with bcs include immunosuppressive and anticoagulation drugs, as well as the relief of hepatic venous outflow obstructions in order to preclude hepatocellular necrosis. unfortunately, most patients with bcs caused by bd have inferior vena cava thrombosis, resulting in an unsuitable pressure gradient existing between the portal vein and the inferior vena cava, thus rendering a porto - caval shunt operation technically impossible. another difficulty with managing these patients is that they usually have long - segment thrombosis, as well as thrombosis of other vascular structures (4). the most significant are the retrospective design and the small sample size, which make it impossible for us to generalize our results for clinical practice. another limitation is the absence of a disease control group. to conclude, as a result of the involvement of prominent vascular settlement sites in bd although there are no exact data in the literature about vascular involvement sites as prognostic indicators in bcs patients with bd, it has been demonstrated that portal venous thrombosis is prominent in general in bcs patient groups. despite limited patient numbers, in addition, the risk of cirrhosis and/or hcc development emerge during long - term follow - up investigations. thus, the occurrence of cirrhosis and hcc must be taken into consideration as measures of surveillance, in addition to the presence of portal venous thrombosis. furthermore, patients must be followed up at regular intervals via physical examinations, laboratory tests, and imaging procedures. | backgroundbudd - chiari syndrome, which is a rare complication of behcet s disease, carries a high mortality rate.objectivesthe aim of the study was to present our long - term follow up experience with patients suffering from budd - chiari syndrome due to behcet s disease.methodsthe records of 402 patients with behcet s disease were evaluated retrospectively. to facilitate detection of the long - term complications caused by budd - chiari syndrome, the patients were evaluated via physical examinations, laboratory tests, imaging modalities, and endoscopy results.resultsthe data for 402 patients diagnosed with behcet s disease, who were followed up at our hospital over 16 years, were analyzed retrospectively. five of these 402 patients (1.2%) were diagnosed as having budd - chiari syndrome. the patients with budd - chiari syndrome were aged between 23 and 54, and all five were male. the interval between the onset of behcet s disease and the development of budd - chiari syndrome ranged from 1 to 8 years. all the patients had combined venous occlusion (affecting the hepatic vein and inferior vena cava). portal venous thrombosis was detected in only one patient (case 1), who died 1 month after the diagnosis of budd - chiari syndrome. the survival time for the other four patients after the diagnosis of budd - chiari syndrome ranged from 4 to 16 years. during the long - term follow - up, hepatic masses were detected via radiological surveillance in case 3 (in the form of large regenerative nodules) and case 4 (nodular regenerative hyperplasia and cirrhosis).conclusionsin our study, portal venous thrombosis was detected in the patient who died during the acute period only. a study including large numbers of budd - chiari - syndrome patients with behcet s disease and portal venous thrombosis would be helpful to determine the prognostic significance of portal venous thrombosis in budd - chiari - syndrome patients with behcet s disease. in addition, patients should be monitored regularly for the development of hepatic masses via a long - term surveillance program. |
the issue of child sex selection and gender preferences is as old as human life. it seems that the historical tendency to determine the sex of the child is rooted in religious, economic, cultural - social and medical issues. until recently, sex selection seemed impossible, but modern medical technologies in the area of reproduction and genetics such as pre - implantation genetic diagnosis (pgd), introduce some assured methods for the couples regarding embryo sex selection before embryo transfer to uterus. currently, the reasons for using child sex selection technology can be categorized into three groups : a) preventing from the birth of babies with severe sex - linked diseases, b) choosing the gender of the baby to balance the child sex combination in the family and c) the freeness to choose the gender of the baby (1, 2). when sex selection is used for medical care, a small number of ethical issues are mentioned. problems usually arise when these methods are used in non - medical cases (3, 4). the sex selection technologies may cause gender discrimination or the gender imbalance in community (57). the most countries, including european ones, accepted the prohibition of sex selection except in medical cases. furthermore, they concluded that there is not enough acceptable reason to prohibit sex selection in case of social causes such as family balancing (811). the decline in fertility in modern societies makes sex determination technology essential to prevent the repeated pregnancies (12). if couples have one or two children, they may be more inclined towards sex selection in order to have children of the preferred sex, or both sexes. investigating the causes of the couples tendency to embryo sex selection before transfer to the uterus, especially in non - medical cases are very important factors for consideration. social and ethical reasons of the tendency toward this attitude need to be assessed ; moreover, the relation between this determination and the parents demographic and social - economic situation should be examined (3). many institutions in europe, including the uk department of health, was studied the various aspects of the ethical, legal and social reasons for non - medical sex selection of embryos (13, 14). sex selection is carried out through pgd technique in some leading clinics in iran. although sex selection through pgd is allowed by religious scholars in iran but its application is limited (15). like many asian countries, there are some gender preferences in iran, and women with daughter are more likely to have an abortion. in a small - scale study of sheykhi (2012), 11.1% of women underwent induced abortion after sex determination (12). as induced abortion is illegal in iran except in certain medical conditions (16), the sex selection demands more attention. the main objectives of the present study were evaluating the gender preferences and the attitudes of couples towards sex selection, and also determination of the factors affecting attitudes in using this method in iran. this cross - sectional study was conducted in tehran, iran in 2010 - 2011. participants were 100 women aged 18 - 45 years who were referred to avicenna fertility clinic for sex selection. single child families (families formed of parents and a child) were chosen because the parents desire to have a second child or a baby of the other gender was more likely. the questions were designed by the researchers at the experts panel with focus on available scientific documents. to determine the validity of the questionnaire, the viewpoints of professors specialized in these issues were obtained. then inappropriate questions were excluded and the questionnaires were answered by 30 women referred to avicenna fertility center in the pilot phase of the study. based on the results of the pilot phase, some modifications were made in the questionnaire. each likert - type question was scored from 1 to 5 (score 3 was a borderline score for each question). attitudes toward gender preferences, religious values, ownership of body components and body products were assessed quantitatively by calculating the total marks obtained by each variable. the gender preferences section based on the importance of the role of the son or daughter from the perspective of parents included 5 questions with borderline score of 15. the gender based preferences section based on the importance of the role of the son or daughter from the perspective of the community included 13 questions with borderline score of 39. the religious values section included 12 items with borderline score of 36, ownership of body components section included 8 questions with borderline score of 24 and body products section included 6 questions with borderline score of 18. in body ownership section, the first item was the rights and control of the body owner over the components and parts of his body and the second item was the rights and control of the body owner over the products of its body. for religious values, moreover, answers were developed from a range of favorable response to undesirable and from 5 to 1 point. in gender preference section, the scores below the grade boundary indicate the positive attitude of respondents to male gender. scores above the grade boundary for religious values, ownership of body components and body products, indicate the positive attitude of respondents to embryo sex selection. the results of the variables including parents satisfaction with sex composition of children and familiarity with various methods of embryo sex selection were examined based on the frequency of each answer. the mean age of women recruited in the present study was 35.5 years (sd = 4.6) and their marriage duration was 15 years (sd = 5). the demographic data including ethnicity, religion and level of education of participants are listed in table 1. most of participants had no prior experience of sex selection (82%) ; and agreed to have embryo sex selection experience (93%). the parental gender preferences and the gender preferences based on the importance of the role of the son or daughter from the perspective of the community, religious values, ownership and possession of body parts and body products are summarized in table 2. based on the mean scores reported in the method, the parental gender preferences and the gender preferences based on the importance of the role of the son or daughter from the perspective of the community were toward the male sex (55.5% male,15.5% female and 28.5% no tendency). participants had a more positive attitude toward sex selection in terms of religious values, ownership of body part and body products. demographic characteristics of women parental gender preferences, gender preferences, religious values, ownership of body parts, body products in relation to ethnicity and the education level of women and their spouses the values related to satisfaction with sex composition of children, awareness about sex selection methods and the importance of sex selection were not statistically significant based on women 's ethnicity (p = 0.623, p = 0.302, p = 0.560) and their spouses (p = 0.095, p = 0.110, p = 0.502). thevalues related to agreements with sex determination with medical or non - medical reasons in women (p = 0.186, p = 0.970) and spouses (p = 0.576, p = 0.180) were not statistically significant. values regarding satisfaction with sex composition of children, awareness about sex determination techniques, the importance of sex selection in general, in terms of women 's education level (p = 0.791, p = 0.126, p = 0.876) and their spouses (p = 0.745, p= 0.303, p = 0.092) were not statistically significant. values regarding agreement with embryo sex selection with medical and non - medical reasons in terms of women 's level of education (p = 0.05, p= 0.001) and with medical reasons in terms of spouses level of education were statistically significant (p = 0.001) but the difference was not significant due to non - medical reason and spouses level of education (p = 0.286). regression analysis showed that higher levels of education affect sex selection with medical reasons. with higher education level, the average score for sex selection with medical reasons increased. the score was 0.21 for women and 0.24 for men. also with higher education level, the average score for sex selection with non - medical reasons increased 0.24 for women. in other words, level of education has positive effect on agreements with sex selection. satisfaction with sex composition of children, familiarity with various methods of embryo sex determination and agreements with embryo sex determination according to medical and non - medical reasons are summarized in table 3. a significant percentage of the participants were satisfied with their existing children 's gender composition. majority of participants had moderate knowledge about different methods of sex selection and agreed with medical sex selection reasons and non - medical sex selection in order to balance the family. in this research, participants were referred for sex selection and consequently most of them (93%) agreed to have the experience of embryo sex determination. in examining the parental gender preferences and the general gender preferences in participants, the tendency was toward the male sex. son preference is an important issue that has a significant impact on the risk of female birth and can change the reproductive behavior of couples. performed a study in pakistan on gender preference issues and evaluated the demand of pregnant women for sex selection of embryos before transfer to the uterus. demand for boys was so high in some classes that may have caused gender imbalances. study of statham in england, 58% and 82% of respondents did not have any gender preferences, respectively (17, 18). although in the present study, results revealed the tendency to male sex, the tendency was not so strong. therefore, it seems unlikely that this selection is subject to conditions such as gender discrimination and will result in gender composition changes of the population in future. tendency for abortion has been more in women with daughter (26.51%) in the study of sheykhi (2012) in iran (12). as son preference is a significant predictor for induced abortions in women (19, 20) and induced abortion is illegal in iran except in certain medical conditions (16), then it seems that allocating enough resources for sex selection services can reduce the incidence of illegal induced abortion in iran. in the present study, participants consent to embryo sex determination to balance the sex composition of their children has won a high percentage. in other words, most people would employ sex selection to have a balanced two - child family and balancing has more influence on the desire to sex selection than the male preference (21, 22). furthermore, it can be inferred from the results that agreement of people to embryo sex determination with non - medical reasons in the present study was for balancing the sex composition of their children and not for sex discrimination which is especially common in some asian countries. similarly, in a study that was conducted on 809 couples during 18 months in england, it was revealed that the aim of majority of the clients for using sex determination techniques was to have a balanced family, and 95% of couples went to the center only for this reason, to have a balanced family (23). in a study conducted by jain (2005), it was shown that infertile women seriously wanted to choose the sex of their fetus, especially those who had no children or who had children of the same sex (3). in the study of himmel. (2008) in germany, a positive attitude towards sex selection was more likely in infertile people if the respondents had a preference for either a boy or a girl and/or had an unbalanced family (24). preference for having at least one child of each sex is a common pattern of parental gender preferences in many different social, economic and cultural contexts (25). although the participants in this study were referred to the center for sex selection, they were satisfied with the sex composition of their existing children. it means that family balancing is more important than having a child with special sex composition. the hypothesis that parents religious values and their idea about ownership of body components may influence their attitude about sex selection was evaluated and based on the findings of the present study, it could be claimed that respondents had a positive attitude toward sex selection with respect to religious values, ownership of body parts and body products. higher education levels resulted in more agreements to embryo sex selection with medical and non - medical reasons. similarly, in the study of kippen (2006) in australia, respondents with bachelor degree or above were more likely to agree with this technology (26). in the study of gleicher and barad in usa (2007), chinese, arab / muslim and asian - indian couples preferred male children more than female (27). however, sex selection in terms of different ethnicities was not significant in the present study. in examining parental gender preferences and gender preferences, tendency was toward the male sex but it was not so strong. most participants agreed to have the experience of sex selection and wanted to use this technique for gender balance in the family. only women 's level of education had positive effect on agreements to embryo sex selection with medical and non - medical reasons. the participants were satisfied with their existing child sex composition, although they were referred to the center for sex selection. in fact, providing sex selection services to iranian couples seeking family balancing is unlikely to result in some conditions such as gender discrimination or severe sex imbalance. it seems that allocating enough resources for sex selection services can reduce the incidence of illegal induced abortion in iran. | backgroundgender preference is prevalent in some communities and using medical techniques to choose the baby 's sex may cause the gender discrimination and gender imbalance in communities. therefore, evaluating the gender preferences and attitudes towards using sex selection technologies seems to be necessary.methodsthis cross - sectional study was conducted in avicenna fertility center. participants were 100 women with one child who were referred for sex selection. data were collected through self - developed questionnaires. the questions were designed by the researchers at the experts panel. to determine the validity of the questionnaire, the viewpoints of professors specialized in these issues were obtained. the statistical analysis of the data was performed using spss software (version 11.5), and p < 0.05 was considered significant.resultstendency toward the male was more than female sex (55.5% male, 15.5% female and 28.5% no tendency). majority of participants agreed with sex selection with medical reason and sex selection in order to balance the family. women 's level of education had positive effect on agreements to fetal sex selection with medical and non - medical reasons (p < 0.001).conclusionalthough gender preferences were toward the male sex but this preference was not very strong. most participants agreed with non - medical sex selection for balancing the sex composition of their children. it does n't seem that non - medical sex selection for family balancing causes severe sex imbalance in iran. |
normal pressure hydrocephalus (nph) is characterized by a clinical triad of symptoms including cognitive impairment, gait difficulty, and urinary incontinence along with ventricular enlargement in brain imaging. nph is considered as idiopathic (inph) when there are no known predisposing factors such as subarachnoid haemorrhage or brain trauma. nph can be treated by shunt but the response rate is highly sensitive for selection of patients [2, 3 ]. alzheimer 's disease (ad) is along with vascular dementia (vad) the most frequent differential diagnosis for inph. several supplementary diagnostic tests of cerebrospinal fluid (csf) dynamics are used in the selection of patients to shunt surgery. the csf tap test or external lumbar drainage test (eld) can predict the shunt response with high specificity and are widely used. infusion tests where usually ringer - solution is infused into csf space with simultaneous csf pressure monitoring to calculate outflow conductance or outflow resistance are also used. in addition, intracranial pressure monitoring alone or together with cortical brain biopsy to detect ad - related pathological findings has been suggested. csf biomarkers reflecting ongoing pathophysiological processes might further help in the evaluation of patients with suspected inph. previous reviews have pointed out several potential csf biomarkers associated with nph but all of them still requiring further verification. there are numerous experimental studies in both acute and chronic hydrocephalus that provide translational evidence for the role of metabolic changes and markers in parallel with hydrocephalus and disturbed csf dynamics, for example, as reported by kondziella.. tumour necrosis factor- (tnf-), a proinflammatory cytokine, seems to be one of the most promising since up to 45-fold increased csf concentrations compared with healthy controls have been observed in nph prior to treatment along with normalization after shunt. however, the patients with known aetiology that is secondary nph were mixed with idiopathic cases and no further studies on this marker have been published in nph. transforming growth factor- (tgf-) is associated with brain response to injury and inflammation, and increased csf tgf- concentrations are reported in ad patients. subarachnoid haemorrhage (sah) increased tgf- concentrations and correlated with the risk of shunt - dependent hydrocephalus but in inph the role of tgf- is somewhat controversial [14, 15 ]. neurofilament protein is a marker of neurodegeneration especially axonal injury, and clearly increased nf light (nfl) concentrations have been detected both in inph and snph patients [16, 17 ]. vascular endothelial growth factor (vegf) is associated with cerebral ischemia and has been correlated negatively with neurofilament heavy chain (nfh) protein in inph patients and increased during eld. increased total tau (t - tau) and phosphorylated - tau181 (p - tau181) in csf are associated with neurodegeneration and ad. in inph decreased amyloid-42 (a42) in csf is associated with ad and indicates risk of ad in mild cognitive impairment but may be normal in inph. in the present study, we correlated the concentrations of seven biomarkers, nfl, t - tau, p - tau181 a42, tnf-, tgf1, and vegf in the csf of 35 patients with suspected inph with the result of the lumbar drainage test. this study includes 35 patients referred to brigham and woman 's hospital (bwh) neurosurgery with suspected idiopathic normal pressure hydrocephalus (inph) according to clinical and radiological examination. continuous drainage was applied with targeted csf drainage rate of 5 to 10 ml / h. neurological (including folstein mini - mental status examination in patients with cognitive symptoms) and physical therapy (including timed up and go test the patient was timed while rising from a chair, walking 3 m, turning around, walking back to the chair, and sitting down) evaluations were completed prior to drainage and daily until the end of the test, which continued for a maximum of 5 days. if the patient demonstrated documented improvement before 5 days or if the patient experienced side effects of drainage refractory to conservative measures, the trial was considered complete and the drain was removed. clinical symptoms, history of any possible known cause of nph, and other neurological disorders were recorded as well as clinically evaluated response for treatment in shunted patients. shunt response was graded according to black scale (table 2) as no response, fair, good, or excellent response after 3-month followup. the cerebrospinal fluid (csf) samples (4 ml) were collected in the beginning of the eld immediately after the puncture, centrifuged, and stored in polypropylene tube at 80c until analysis to ensure the stability of the csf biomarker levels during the storage. measurements of csf were performed in bwh neurosurgery laboratory using commercially available solid phase sandwich enzyme - linked immunosorbent assays (elisa) according to the manufacturer 's protocol and blinded to the eld results (table 3). the study was approved by the partners human research committee. written informed consent was obtained from all patients. the csf concentrations of the analyzed markers were compared between the positive and negative eld groups by independent samples t - test. the biomarker concentrations in the csf of 26 patients with a positive eld result and nine patients with negative eld result are presented in table 1. nine patients had initially negative eld, and one of them was shunted with excellent response. the levels of all analyzed csf biomarkers were similar between the groups, and none of them could predict the eld result in these patients. nfl concentrations were increased similarly both in patients with positive (range 280>10000) and negative (range 4389306) eld (table 1). tnf- concentration was equally low around 1 pg / ml in both groups (table 1). in 10 cases the concentration was even under the mean detection limit (0.106 pg / ml). tgf1 concentrations were similar (mean 61 versus 71 pg / ml, range 20228, p =.52) in both groups (table 1). tgf1 concentration had positive correlation with t - tau (r = 0.413, p =.014, table 4). mean a42 concentrations did not differ significantly between the groups (250 versus 209 pg / ml) (table 1). mean t - tau concentration was 274 pg / ml (range 441860) in positive and 291 (range 89983) in negative eld groups without significant difference (p =.90, table 1) and increased with age (r = 0.38, p =.023, table 4). also p - tau181 concentrations were equal between positive and negative eld groups (55 versus 53 pg / ml, p =.88, table 1). vegf concentrations were under the detection limit (5 pg / ml) in all except two cases and therefore were not included in the correlation analysis. the most important finding of this study is the unexpectedly low csf tnf- concentrations observed in inph patients and the inability of csf biomarkers to predict the eld result. using a standard commercial elisa, the csf tnf- levels between 0 and 5.0 pg / ml close to the standard levels previously observed in serum (0.52.8 pg / ml)were detected. concentrations up to 700 pg / ml would have been expected according to one previous study. differences in sampling and analyzing processes (different elisa was used in the previous study) might have effect on the results although likely not crucial. the most probable explanation could be that in the previous study the idiopathic cases were not separated from the secondary cases. this indicates that the inflammatory reaction would be associated with secondary nph rather than idiopathic form. our study did not include healthy controls, and our negative result on tnf- should be reproduced in study were nph patients with possible known cause of the disease are separated from idiopathic cases and compared with healthy controls. it would also be very interesting to study the possible role of inflammation in the formation of secondary hydrocephalus for example after sah or trauma. experimental studies clearly indicate the increased production of tnf- as well as other proinflammatory cytokines due to accession of blood products to csf. notably increased tgf1 levels after sah indicated risk of persistent hydrocephalus. we detected rather low tgf1 levels (varied from 20 to 228 pg / ml) in inph patients contrary to a previous observation of increased concentrations but supporting other studies with low concentrations. interestingly tgf1 levels correlated with t - tau but not with the levels of other studied biomarkers. this is contrary to a previous study in ad patients and controls where tgf1 levels correlated with a42 but not with tau. this can be explained by different patient population and by rather small number of cases in both studies. the clear correlation between p - tau181 and total tau is expected since phosphorylated tau is an isoform included into total tau. the correlation analysis can be justified to indicate obvious associations between different biomarkers but has to be interpreted cautiously because of low statistical power due to rather small number of cases. equally increased csf vegf levels are detected both in ad and vad patients also correlating with tgf1 levels. decreased cerebral blood flow associated with chronic hydrocephalus potentially leads to hypoxia, which could induce increased vegf formation also in inph. in an experimental study the increased csf vegf levels were seen only in a short - term model of chronic hydrocephalus but not in the long - term model. therefore low vegf concentrations detected here are not very surprising. based on studies comparing ad patients and healthy subjects it could be expected that inph patients with low a42 and increased t - tau and/or p - tau might have concomitant early ad or could be in the risk of developing ad. csf tau and a42 protein levels might help in the detection of the patients who have ad instead of nph or may have significant risk for concomitant ad. the low a42 together with increased tau concentrations (t - tau / a42 ratio > 1.15) seems to indicate increased risk of ad. thus total tau / a42 ratio over 1.15 could be a potential cut - off limit for increased risk of ad and according to that one - third of the current inph patients diagnosed by ldt would be in increased risk of future ad. however, increased csf tau and decreased a42 could be detected also in other neurodegenerative disorders than ad. in any case, shunted inph patient with still progressing amnestic cognitive impairment and the profile of increased tau and decreased a42 should be noted and managed interdisciplinary with a thorough dementia workup. specific csf biomarkers of inph indicating shunt response and the possible point of no return in the course of the disease would be valuable. also indicators of the long - term prognosis especially those predicting risk of dementia despite of shunting would be useful. however, this would need a battery of biomarkers and long - term follow - up studies since causes of dementia in these patients are variable from ad to dementia with vascular origin and perhaps dementia due to inph itself. markers indicating possibility of ad are already available but their predictive value of concomitant ad in inph patients still needs to be shown in follow - up studies. currently there seems to be no csf biomarker available which could clearly predict the result of lumbar csf drainage test. it should also be kept in mind that the sensitivity of eld is not 100% and as also in this series there can be patients who benefit from shunt despite negative test result. therefore we can not exclude the possibility that some of the markers analyzed here could somehow predict response for shunt treatment in case of negative eld. in tau and nfl the current results are in line with the previous study where they did not correlate with outcome of shunt. further research is needed to evaluate the molecular biological basis of idiopathic nph and to obtain csf biomarkers for the clinical diagnosis of inph. the csf, blood, and even brain tissue samples which are possible to obtain without significant additional risk for the patient during diagnosis and treatment of nph may be useful in differential diagnosis and further research. also they can be useful for validation of less invasive methods to detect for example a and other possible markers of neurodegeneration and help in the discovery of new surrogate markers. neurosurgeons are encouraged to collect blood, csf, and tissue samples for future biobanks with detailed clinical characterisation of patients with careful history taking and use of standardized outcome scales. it is also obligatory to present the results of the analyses separately in different entities of nph since idiopathic and secondary forms of the disease have different molecular biological background. the secondary forms of nph should also be separated depending on the specific aetiology. in conclusion | the diagnosis of idiopathic normal pressure hydrocephalus (inph) is still challenging. alzheimer 's disease (ad), along with vascular dementia, the most important differential diagnosis for inph, has several potential cerebrospinal fluid (csf) biomarkers which might help in the selection of patients for shunt treatment. the aim of this study was to compare a battery of csf biomarkers including well - known ad - related proteins with csf from patients with suspected inph collected from the external lumbar drainage test (eld). a total of 35 patients with suspected inph patients were evaluated with eld. csf was collected in the beginning of the test, and the concentrations of total tau, ptau181, a42, nfl, tnf-, tgf1, and vegf were analysed by elisa. twenty - six patients had a positive eld result that is, their gait symptoms improved ; 9 patients had negative eld. the levels of all analyzed csf biomarkers were similar between the groups and none of them predicted the eld result in these patients. contrary to expectations lumbar csf tnf- concentration was low in inph patients. |
in 2004, denosumab was under development for treating osteoporosis, progressing from proof of concept to registration trials.1 questions existed that could not be practically addressed with clinical studies due to the protracted dosing interval (q6 m) and required trial duration, nor by traditional pkpd models. they included questions about possible effects of drug regimen changes, treatment discontinuation, prior treatments, and sampling schemes. these questions, together with general uncertainty in the scientific community regarding the physiologic links between clinical markers (e.g., serum calcium, parathyroid hormone [pth ], bone turnover markers), clinical endpoints (e.g., bone mineral density [bmd ]), and complex linkages between bone and calcium homeostasis, led the authors to embark on creating a model that incorporated much of the known bone physiology and was dependent on maintaining calcium 's narrow physiologic range. the representation of calcium homeostatic mechanisms was paramount to ensuring model extensibility to calcium and pth - related mechanisms. the initial scientific objective was to arrive at a qspm connecting bone turnover markers with bmd using known links between organs involved in calcium homeostasis, including bone and relevant cellular mechanisms (figure 1). thus, the necessary level of complexity and the nature of datasets mandatorily described simultaneously by the model were crucial questions. data used included measures of longitudinal system perturbations by drug therapies (e.g., teriparatide) and disease states (e.g., progressive renal failure). interestingly, while constructing the qspm, scientific knowledge gaps became vividly clear, and produced an unexpected value. the gaps forced assumptions to be made about a pathway 's importance, a diseases ' influence, or a drug 's potential, and subsequent sensitivity testing of those assumptions. in doing so, knowledge limitations became clearer and true understanding of the system became more transparent. schematic of calcium homeostasis and bone qspm (reprinted with permission from peterson and riggs, 2010). this arises from a qspm 's foundation in human physiology for understanding a drug 's effects (attempting to understand causality), rather than simply drawing a direct line between administration and endpoint observation (sometimes causality, often correlation). this aspiration of qspms aligns with the concept of precision medicine, and offers a tool by which myriad genomic level data may, in future, be used to understand disease and treatment. in our opinion, since the overall aim of pharmacology is to modify an underlying disease, understanding is increased by first generating a best estimate of the (patho)physiology and then adding the drug to that quantitative construct. it seems most relevant to consider investment in qspm development for drugs impacting physiology with substantial system feedback, forward branching or redundant pathways, or for biologics with long half - lives, to support confidence in target and steering research toward question - based development goals. additionally, as precision medicine generates further mechanistic understanding, a qspm can be extended, refined, and reapplied with relative rapidity. the bone and calcium homeostasis qspm provides examples of these extensions, refinements, and adaptable reapplication. the first bone and calcium homeostasis qspm publication described ca, pth, calcitriol, and bone marker changes associated with denosumab and teriparatide treatment in postmenopausal women, with theoretical changes expected during hypo- and hyperparathyroid disease states.2 subsequently, the model was expanded to include additional disease states, a range of therapeutic interventions, and middle - up extensions describing bmd changes associated with bone marker changes3 and fracture risk associated with bmd changes.4 qualification has included external validation of bone marker and bmd predictions.5 disease state expansions and applications have included : 1) secondary hyperparathyroidism associated with chronic kidney disease6 and 2) estrogen - related effects on bone and calcium during menopause transition.7 notably, this latter work was used to investigate bmd loss associated with treatments of endometriosis - related symptomatic pain and perform target mitigation. gnrh modulation therapies used for this purpose result in an estrogen depravation causing markedly decreased bmd and limiting the duration of approved therapy. results from this work7 were later stated to direct not just a particular compound in development but pfizer 's entire gnrh modulation research program : this work identified target levels for estrogen that would provide symptomatic pain relief with minimal impact on bmd. targeting the gnrh pathway to achieve the desired range of serum estrogen levels would be difficult to achieve ; therefore, the research program was halted before any compound entered the clinic.8 another example of extensibility was in assisting the understanding of clinical findings from orally administered calcium - sensing receptor antagonism (casra) programs. casra would conceptually result in a pth spike similar to that resulting from subcutaneously administered teriparatide without exogenous administration. casra programs, however, historically reported bmd increases less than those achieved from teriparatide, often with hypercalcemic events consistent with nontransiently elevated pth. using public - source clinical data and preclinical sponsor data, the model was used to describe a capacity - limiting effect in the pt gland that limited the maximum achievable pth cmax while describing prolonged pth elevations with continued dose escalations. these predictions were confirmed in a phase i single - dose study ; the results were used to support development criteria with expectations for maximal bmd changes achievable through casr antagonism.9 on september 12, 2014, the fda endocrine and metabolic drugs advisory committee met and discussed the biologics license application (bla) 125511, proposed trade name natpara (recombinant human parathyroid hormone (rdna) or (rhpth[1 - 84 ]), submitted for the proposed indication of replacement for endogenous parathyroid hormone (1 - 84) and long - term treatment of hypoparathyroidism.10 as part of the fda briefing document, a clinical pharmacology assessment of the adequacy of natpara dosage regimen in the treatment of hypoparathyroidism was rendered ; this review used an open - source version of the qspm2 (available in online appendix7) to evaluate alternate dosing regimens. the fda reviewers ' recommendation, based on their independent model qualification using phase i data and subsequent simulations representative of the registration trial, was that the dose regimen should be further optimized to address the safety concerns for hypercalciuria.10 the significance of this event will take some time to coalesce. this use of a qspm in a regulatory meeting, however, is an important first demonstration of a qspm extension beyond the research space, and lends a further element of credibility to the discipline. it is therefore reasonable to consider the event itself as marking a change in how we view the use - horizon for qspms and a watershed moment in the maturing qsp discipline. while today we have this first example of regulatory agency qspm use, there are numerous cases of qspm uses in early development decisions, trial design discussions, and program advancement decisions. it will be important to the success of qsp as a discipline to make sure these influential qspm uses are clearly visible to the pharmaceutical community and their relevance understood as we move beyond this landmark event. at the same time, due in part to the complexity of qspms, and the required fit - for - purpose status needed to address regulatory questions, we should not expect examples to amass rapidly. additionally, there are at least three further reasons why examples will likely emerge slowly. first, without question, clinical study and traditional modeling and simulation will continue to provide sufficient data and understanding to support the majority of regulatory discussions / decisions. second, unlike population pharmacokinetics / pharmacodynamics (pk / pd), there is no regulatory guidance speaking to the application of qspms. third, regulatory acceptance of a qspm will require comfort by users and consumers with the underlying physiology, mathematics, and mechanistic assumptions supporting the application. therefore, parameter sourcing and validation needs to be suitably scrutinized to ensure that the model behavior is appropriate. in this example, pth was an integral part of the qspm, and so questions asked were well within the model domain. the fda then used a phase i sponsor study to perform an independent model evaluation. this step undoubtedly promoted confidence in the subsequent simulations that were used to suggest alternative dosing regimens. while this represents a groundbreaking example of qspm use in a regulatory setting, future qspm applications will need to continue building case - based evidence to further the scientific community 's overall confidence in qspm application. in 2004, two questions motivated construction of the bone and calcium homeostasis qspm that would, a decade later, be used by the fda : 1) can the physiologic link between bone markers and bmd be captured within a model ? and 2) can a physiologically based model be used to explore development questions and guide clinical study ? moreover, the authors believed there was broader potential for a model covering multiple temporal scales, from cellular mechanisms to protracted clinical outcomes, and rooted in understood physiology. for instance, qspms can generate hypotheses, frame assumptions about mechanism and physiology, and provide a communication tool for answering questions about why and how a drug might or might not produce certain effects. the qspm extensions described above clearly demonstrate how a model based on physiology (rather than exclusively program - centered clinical observations) can have applications across therapeutic areas and diseases, addressing unanticipated questions at the time of conception. this sets up a condition of non - forecastable return on investment and underscores the disciplines needed to highlight and communicate the types of questions that can be addressed via qsp modeling. therefore, it can be useful to cast qspm uses in terms of addressable questions to help clarify the rationale(s) for qspm development / investment. a sample list of questions is proposed that a qsp modeler might seek to answer via a qspm (table 1). in many ways, our discipline 's goals should clearly articulate the value and utility of qspm investment as we move forward. published examples, such as the ones provided here, predicated on the triad of physiology - pharmacology - pathology, are crucial tools for such advocacy. therefore, we are encouraged by the fda 's use of a qspm to generate scientific deliberation at an advisory meeting and hope that this truly represents a watershed moment for qsp as a discipline. quantitative systems pharmacology model example use questions with most likely domain of interest within the pharmaceutical research and development (r&d) process | in the evolving discipline of quantitative systems pharmacology (qsp), qsp model (qspm) applications are expanding. recently, a qspm was used by us food and drug administration (fda) clinical pharmacologists to evaluate the appropriateness of a proposed dosing regimen for a new biologic. this application expands the use - horizon for qspms into the regulatory domain. here we retrace the evolution of the model and suggest a question - based approach to directing model scope, identifying applications, and understanding overall qspm value. |
the use of cardiopulmonary bypass (cpb), bleeding (during and after surgery), frequent blood analyses (before, during and after surgery), hemodilution, significant shift of intravascular volume, mechanical trauma of blood cells, therapeutic hypothermia, co - morbidities, the use of anticoagulant and antiplatelet drugs (before, during and after surgery), transfusion of blood products, cause significant changes in the three major cellular components of the hematopoietic system (1). experience and literature have shown that the values of peripheral blood parameters (number, size, function) undergo significant changes during the early phase of the surgery, gradually recovery during the postoperative period and reach the preoperative (baseline) values 2 - 6 months after surgery (2). removal of pro inflammatory stimuli after surgery, wound closure and the regenerative ability of the bone marrow will ensure a gradual recovery to preoperative values of the hematological parameters. the aim of this study was to assess the hematological changes after artery bypass graft surgery (cabg). between january 2012 and january 2013, 164 consecutive patients (138 men and 26 women) underwent elective cabg surgery at our division of cardiac surgery. patients undergoing emergent surgery or off - pump cabg were not included in this study. patients were reviewed for their preoperative demographic, clinical (coronary artery disease severity and co - morbidities) and laboratory variables and then followed to record their postoperative data and outcomes. hematological parameters were assessed before surgery (d-1) and after surgery : at the first day (d+1 ; the second day (d+2), the patient was transferred from the intensive care unit to the ward ; the third day (d+3) ; the fourth day (d+4) ; the sixth day (d+6), the patient left the hospital ; at the end of the second week (d+14), the third week (d+21), the fourth week (d+28) and the twelfth week (d+90). to achieve a complete blood count analysis, 4 ml of venous blood was taken by venipuncture technique, by vacutest ref 13030 k3 edta 7.2 mg, and evaluation done on the equipment sysmex xs-1000i automated hematology analyzer. the purpose of this study was to assess changes in hematological parameters of peripheral blood after cardiac surgery (the count of red blood cell, leukocytes, neutrophils, lymphocytes, platelets and the values of haemoglobin, hematocrit, mpv), analyzing changes in trend over time until 90 days after cardiac surgery and comparison with values before surgery. all patients included in the study were managed according to the hospital s current policies regarding preoperative preparation, intraoperative surgical and anesthetic management and postoperative care. all cardiac medications were continued until the day of surgery, except antiplatelet drugs, which were stopped 5 days prior to surgery in elective patients. patients were premedicated with 7.5 mg midazolam orally the night before surgery and 10 mg morphine intramuscularly 30 minutes before they were sent to the operating theatre. anesthesia was induced with 0.050.1 mg / kg midazolam, 510 g / kg fentanyl and 0.1 mg / kg pancuronium to facilitate endotracheal intubation and mechanical ventilation. after intubation, patients remained on mechanical ventilation with intermittent positive pressure with a tidal volume of 810 ml / kg, positive pressure at the end of expiration of 58 cm h2o and fraction of inspired oxygen of 0.61 to maintain arterial oxygen saturation > 95%. nitroglycerine and sodium nitroprusside were used as vasodilators, dobutamine and dopamine as inotropes, and noradrenaline and adrenaline as vasopressors. all surgical procedures, i.e., on - pump and off - pump, were performed via median sternotomy. packed red blood cells (rbc) were transfused according to the needs of each patient. blood transfusion was used to maintain hematocrit (hct) > 25% and hemoglobin (hb) > 8.5 g / dl during cardiac surgery. activated clotting time (act) all on - pump patients required 2 g tranexamic acid as an antifibrinolytic agent at the start of anesthesia. the anticoagulation was achieved with an initial dose of 300 u / kg heparin injected into the central venous system with act > 400 seconds (35 minutes after administration of heparin). at the end of the bypass procedure, the effect of heparin was reversed with protamine chloride at a ratio of 1:1. platelet count and homeostasis test we used autologue vein grafts (saphenous vein) or artery grafts (mammary, internal thoracic, and radial artery). on average, on - pump surgery lasted 2.54 hours and was easily accomplished with systemic hypothermia (3234c) ; off - pump surgery lasted 23 hours and was achieved with normothermia. patients were transferred to the icu, intubated, and mechanically ventilated until they were ready to be awoken. statistical methods : group statistics were expressed as mean sd. the paired - sampled t test bonferroni was performed for the statistical analysis between groups. the mean age of the patients was 61.8 9.0 years (range, 34 - 82 years). time trends of the average values of rbc(mm), hb(gr / dl), and hct(%), standard deviation(sd), percentage changes of these values versus d-1 (preoperative value taken as the basic value) are presented in figure 1. comparison with preoperative values of erythrocytes (d-1) standard deviation (sd) for pairs : d-1, d+1 ; d-1, d+2 ; d-1, d+3 ; d-1, d+4 ; d-1, d+6 ; d-1, d+14 ; d-1, d+21 ; d-1, d+28 ; d-1, d+90 ; are presented in the table 2. the same univariate analysis was employed for the hb level, and hct level (table 2) postoperatively versus preoperatively. average values of rbc, hb and hct declined to reach their lower values on day 3 after surgery (-33.6 %, -33.1 %, -32.6 % respectively from the preoperative value, p<0.001) and then gradually increased to reach normal values after one month and the preoperative values after three months. time trends of the average values of white blood cells (wbc) (mm), neutrophils (mm), lymphocytes (mm) and sd, percentage changes of these values versus d-1 (preoperative value taken as the basic value) are presented in figure 2. comparison with preoperative values of wbc (d-1)sd for pairs : d-1, d+1 ; d-1, d+2 ; d-1, d+3 ; d-1, d+4 ; d-1, d+6 ; d-1, d+14 ; d-1, d+21 ; d-1, d+28 ; d-1, d+90 ; are presented in the table 3. the same univariate analysis was employed for the neutrophils and lymphocytes postoperatively versus preoperatively (table 3). the average values of leukocytes and neutrophils increased rapidly (neutrophil leukocytosis) to achieve the highest value on day 2 after surgery, while the average value of lymphocyte decreased quickly to achieve lower value on day 1 after surgery (+ 74.7 %, + 127.1 %, -52.4 % respectively from the preoperative value, p<0.001). once these values were reached the average values of leukocytes and neutrophils were reduced gradually and the average value of lymphocytes increased gradually to reach the normal levels on day 21 and the preoperative values on day 28. comparison with preoperative values (d-1) standard deviation (sd), for red blood cells, hemoglobin and hematocrit. legend : rbc - red blood cells, hct - hematocrit, hg - hemoglobin, sd - standard deviation time trends of percentage values of white blood cells, neutrophils, lymphocytes. comparison with preoperative values (d-1) standard deviation (sd), for white blood cells, neutrophils, and lymphocytes. legend : wbc - white blood cells, sd- standard deviation time trends of the average values of platelets (mm) and mean platelet volume (mpv)(fl), standard deviation (sd), percentage changes of these values versus d-1 (preoperative value taken as the basic value) are presented in figure 3. the average platelet count decreased gradually to reach the lowest value on day 2 after surgery (-26.4 % from the preoperative value, p<0.001), after which gradually increased up to + 100.8 % of the preoperative value on day 14 (p<0.001) and then gradually decreased to reach normal values on day 21 and preoperative values after three months. the average values of mpv change in inverse way according to the average values of platelets (in regenerative bone marrow response). time trends of percentage values of platelets and mean platelet volume. comparison with preoperative values (d-1) standard deviation (sd), for platelets and mean platelet volume. legend : mpv - mean platelet volume, sd - standard deviation time trends of the average values of rbc (mm), wbc (mm), platelets (mm), percentage changes of these values versus d-1 (preoperative value taken as the basic value) are presented in figure 4. rbc get to their lower average value in d+3 (-33.6 % from the preoperative value) ; in d+2 leukocytes get to their higher average value (+ 74.7 % from the preoperative value), and the platelets to their lower average value (-26.4 % from the preoperative value). leukocytes regain their normal level in d+21, erythrocytes and platelets regain their normal level in d+28. all three elements recover totally in their preoperative levels within three months (d+90). time trends of percentage values of red blood cells, white blood cells, platelets. systematic assessment of peripheral blood hematologic parameters after cardiac surgery is important for the assessment of their changes and correction. anemia is a major concern in patients undergoing cabg and may be present in over 90 % of cases (3) and it can be explained by acute blood loss during and after surgery. about 75 - 90% of intra - operative and early post - operative bleeding is associated with technical factors and due to cpb which is associated with hemodilution (4). anemia is associated with a range of postoperative consequences (stroke, acute myocardial infarction), major side effects, re - hospitalization, duration of stay in the intensive care unit and hospital stay, mortality 30 days after the intervention (5). westenbrink (6) noted that every 1 mg / dl decrease in hb was associated with a 13% increase in cardiovascular events and 22 % increase in all - cause mortality. van straten (7) demonstrated that postoperative anemia is a risk factor for early and late mortality in patients undergoing cabg. the acute reduction in hct produced a reversible platelet dysfunction, manifested by an increase in bleeding time (bt) and a decrease in the shed blood thromboxane b2 level at the template bt site (8). blood loss is a common problem in cardiac surgery, which requires some re - interventions, while massive blood loss (the replacement by transfusion of more than 5 units prbc within 1 day of surgery) was associated with an 8.1-fold in the odds of death (9). clinical studies emphasize the paradox that both anaemia and transfusion are associated with organ injury and increased morbidity and mortality across a wide span of disease states and surgical interventions (10). there was mild anemia in 1.2 % of the cases ; moderate anemia in 66.5 % and severe anemia in 32.3 % of the cases. the lowest value of hct is associated with renal function damage, more cardiac damages, longer hospitalization and higher mortality. ranucci (12) noted that median values of the lowest hct on cpb below 25 % were associated with an increased major morbidity rate. the lower average value of hct in our study was 26.9 % in the third day after surgery. karkouti (13) demonstrated that a decrease of hb concentration by 50% according to the basal value was independently associated with increased risk for adverse outcomes. even in the absence of bleeding, intravascular fluid cause shifts in hemoglobin levels to drift postoperatively, possibly confounding the decision to transfuse. tschaikowksy (14) have shown that after cardiac surgery, by postoperative day 4, the circulating blood volume has reached its maximum and thus hb levels reach their nadir. in our study, the average hb level falls after surgery in 100 % of cases and reaches the lowest value (nadir) on day 3 (9.0 g / dl ; 33.1 % decreases from preoperative level). after nadir, the average hb level increases gradually and on day 6 patients exhibited hemoglobin recovery of 0.981.27 g / dl (p<0.001). santa ursula (15) noted that at the end of cardiac surgery a marked and sustained leukocytosis was found. in the differential count there were significant increase in neutrophils, associated with band and immature forms, corresponding with significant reductions in lymphocytes. leukocytosis is also very common after surgery and generally represents an acute response to the stress. the count of leukocytes and neutrophils increased significantly (neutrophilic leukocytosis) by the end of the surgery, until 24 hours thereafter. this rapid increase of the total count of leukocytes and neutrophils comes from the mobilization of the marginalized neutrophils and release of new neutrophils from bone marrow and the pulmonary circulation. the normal response to inflammation will be accompanied by an increase of leukocyte count, primarily to neutrophils and less mature forms (left shift). acute bleeding (4 - 5), physical and emotional stress, anesthesia and drugs also increase circulating leukocytes (16). as a rule, it will improve in further measurements and if growth persists or occurs after a period of decline it is thought to be an infection. the number of neutrophils markedly increases at the end of cardiac surgery and remains elevated for 48 hours (17). cpb induces a whole body inflammatory response that sometimes leads to postoperative organ dysfunction and neutrophil activation plays an important role in this reaction. neutrophil activation is implicated in postoperative complications in patients having cardiac surgery with cpb (18). leukocytosis was identified as an alarming sign for mortality among patients admitted to general hospital wards at early stages of admission (19). high wbc count before cabg surgery is an independent risk factor for ischemic events one year after the surgery (20). activated neutrophils stimulate platelet activation. close contact between platelets and neutrophils modulates their cellular interactions in thrombotic and inflammatory states, with stimulation of p - selectin expression on platelets by agonists such as thrombin and neutrophil - derived cathepsin g. neutrophils enhanced aggregation of human platelets and neutrophil cathepsin g is a physiologic modulator of platelet thrombus formation in vivo (21). despotis (22) have demonstrated that patients who exhibit the greatest increase in wbc count during the course of cpb are most likely to bleed during the first 24 postoperative hours. on - site measurements of wbc count as an index of the inflammatory response to cpb may be useful in identifying patients at increased risk for excessive bleeding. by using this information physicians may be able to intervene with anti - inflammatory medications and blood preservation techniques. in our study, the average values of leukocytes and neutrophils increased rapidly (neutrophil leukocytosis) to achieve the highest values on day 2 after surgery, while the average value of lymphocytes decreased quickly to achieve lower value on day 1 after surgery. 90.2% of the patients had neutrophilic leukocytosis after cardiac surgery ; 98% of these patients had mild leukocytosis and 2% had moderate leukocytosis. once these values were reached, the average values of leukocytes and neutrophils were reduced gradually and the average value of lymphocytes increased gradually to reach the normal levels on day 21 and the preoperative values on day 28. the relative decrease in platelet counts within the first 3 to 4 days after major surgery is informative about the magnitude of the trauma or blood loss, whereas the dynamic of the platelet count course thereafter shows whether or not the physiologic compensatory mechanisms are working. the platelet count nadir is typically reached by days 3 and 4 for major surgery and is nearly always related to postoperative consumption and dilution (23). in the vast majority of patients platelet counts will increase thereafter, reaching the presurgery level at about postoperative days 5 to 7. in another study, miyauchi (24) noted that platelet count was reduced markedly with the initiation of bypass and the low level was maintained until the 3 postoperative day. thrombocytosis following cabg has been described to occur frequently (2030%) and to be associated with thrombotic complications. postoperative thrombocytosis is a potentially dangerous complication, with an increased risk for postoperative myocardial infarctions, late symptomatic vein graft occlusion (25). khuri (26) in their study evaluated that the platelet count fell significantly during cpb, while the mpv decreases significantly after the institution of cpb and reaches its nadir approximately 2 hours later. there is a progressive and significant increase in mpv between 2 - 72 hours postoperatively, accompanied by a significant rise in platelet mass, suggesting that larger platelets are selectively removed during the cpb. slavka (27) in their study demonstrated that patients with an increased mpv11fl are at higher risk of death due to ischemic heart disease, with hazard ratios comparable to those reported for obesity or smoking. an increased mpv as an indicator of larger, more reactive platelets, resulting from an increased platelet turnover, may represent a risk factor for overall vascular mortality, including myocardial infarction. platelet volume, and therefore platelet activation, appears to play a causal role in late vein graft disease ; hence, mpv may be useful as a post - operative marker of graft success (28). in our study, the average platelet count decreased gradually to reach the lowest value on day 2 after surgery after which it gradually increased and then gradually decreased to reach normal values on day 21 and preoperative values after three months. the average values of mpv changed in inverse way according to the average values of platelets (in regenerative bone marrow response). interesting data could be the difference of this subgroup of patients versus patients undergoing off - pump surgery, which should be a matched and randomized study. in conclusion, we found that the average values of the three peripheral blood cells parameters, undergo mild to moderate changes after cabg and return gradually to normal and preoperative values after 1 - 3 months from surgery, when the compensatory function of the bone marrow is preserved and there are no post surgery complications associated with continuous consumption or loss of peripheral blood cellular elements. our study shows that in on pump cabg, a stable model of response of the erythrocytes, leukocytes and the platelets happens. analysis of change over time in the average values of the hematological parameters can be predicted according to the median curve. | objectives : removal of pro inflammatory stimuli after cabg, wound closure and the regenerative ability of the bone marrow will ensure a gradual recovery of hematological parameters. the aim of this study was to assess the hematological changes after cabg.materials and methods : a prospective cohort study included 164 consecutive patients undergoing on pump cabg surgery between january 2012 and january 2013. patients with primary hematologic disease, emergent or urgent cabg and off - pump cabg were not included. a time line protocol was employed.results:all patients survived surgery. average values of erythrocytes, hemoglobin and hematocrit declined, to reach lower values on day 3 after surgery (-33.6 %, -33.1 %, -32.6 % versus preoperative value, p<0.001) and then gradually increased to reach normal values after one month and the preoperative values after three months. the average values of leukocytes and neutrophils increased rapidly to achieve the highest value on day 2, while the average value of lymphocytes decreased quickly to achieve lower value on day 1 after surgery (+ 74.7 %, + 127.1 %, -52.4 % respectively from the preoperative value, p<0.001). the average platelet count decreased to the lowest value on day 2 after surgery (-26.4 % from the preoperative value, p<0.001), after which gradually increased up to + 100.8 % of preoperative value on day 14 (p<0.001) and then gradually decreased to reach normal values on day 21 and preoperative values after three months.conclusions:average values of the three peripheral blood cells parameters undergo important changes after cabg, but not life threatening, and regain normal and preoperative values after 1 - 3 months after surgery. |
the precision of the excimer laser introduced a new era for the correction of myopia. studies have demonstrated that photorefractive keratectomy (prk) and laser in situ keratomileusis (lasik) for myopia are safe and predictable. the application of excimer laser for the treatment of hyperopia started in the late 90s.13 in this process, a paracentral meniscus of the corneal stroma is removed to steepen the central cornea. initially, prk with the excimer laser was used to corrected hyperopia. however, postoperative corneal haze restricted prk for hyperopia, and eventually lasik became the most common method for the correction of hyperopia. lasik, however, has certain limitations and drawbacks such as flap related complications,4 dry eye,5 and keratectasia.6 in addition, patients at risk of trauma and/or those with thin corneas are not eligible for lasik. the prevention of corneal haze with mitomycin - c (mmc)7 has resulted in the resurgence of prk for keratorefractive surgery. the outcomes of hyperopic prk have been promising ; however, the predictability is lower, compared to myopic prk. published results indicate comparable efficacy for prk and lasik for the correction of up to 4.00 d of hyperopia.891011121314151617 however, few studies have assessed the outcomes of hyperopic prk with the application of mmc (prk - mmc). here, we present 1-year outcomes and complications with hyperopic prk - mmc for the treatment of mild to moderate hyperopia. from december 2009 to december 2010, consecutive cases of hyperopia undergoing prk - mmc were approached. all excimer ablations were done using either the allegretto wave concerto (alcon inc., fort worth, tx, usa) or the technolas 217-z (bausch and lomb inc. preoperatively, all patients were at least 20 years of age, and they had stable refraction in the past 18 months (< 0.50 diopters [d ] change). none of the patients had any history of ocular surgery, connective tissue disorders, or diabetes, and they were not using any medication that might interfere with wound healing. soft contact lens users stopped wearing lenses three days before examinations, patients wearing hard contact lenses were advised to stop using the lenses for at least 3 weeks before their appointment. preoperative examinations included assessment of visual acuity with and without correction, manifest and cycloplegic refraction, measurement of the central corneal thickness with an ultrasound pachymeter (nidek, us-1800 echoscan), slit lamp examination, corneal topography (eyesys vision, eyesys 3000), fundus examination, and measurement of the intraocular pressure. in cases with a considerable discrepancy between manifest and cycloplegic refraction, manifest refraction was repeated after the effect of cycloplegic drops had receded to allow greater correspondence between refraction. the maximum acceptable difference between manifest and cycloplegic refraction was 1.00 d. surgery was performed under topical anesthesia with tetracaine 0.05% eye drops. a hockey knife spatula was used to mechanically remove the epithelium in the central 8.0 mm of the cornea, and the laser ablation was delivered to the surface. for those over 40 years of age, the correction was based on full cycloplegic refraction, and in younger patients, we used the average of cycloplegic and subjective refractions. treatments with the concerto excimer laser were programmed for an optical zone of 6.5 mm and a transition zone of 1.25 mm. treatments with the technolas laser were programmed for an optical zone of 6.0 mm with 1.0 mm for the transitional zone. once the ablation was completed, 0.02% mmc solution was applied to the ablated area for 120 s,15 and then the entire corneal surface and conjunctiva was rinsed with 20 ml cold normal saline. subsequently, chloramphenicol drops were instilled, and surgery was concluded by applying a bandage contact lens (air optix ; novartis ag, basel, switzerland). postoperative medications included 0.5% chloramphenicol eye drops, four times daily, until complete re - epithelialization, and 0.1% betamethasone eye drops four times daily for the first two weeks which there replaced with 0.1% fluorometholone drops four times daily for the next 6 weeks (a total of 8 weeks). patients had follow - up visits every day until re - epithelialization was complete and were scheduled to be examined at 1, 3, 6, and 12 months, postoperatively. at each follow - up visit data were collected on the best corrected distance vision acuity (bcva) and uncorrected distance vision acuity (ucva), manifest refraction, and slit lamp examination, and any complication such as infection, ectasia, or haze formation. the protocol of this study was approved by the institutional review board of noor ophthalmology research center, and all patients signed informed consents before enrollment. quantitative variables are summarized as a mean and standard deviation. to compare results at preoperatively and postoperatively we used the repeated measures analysis of variance. the mean age of the patients was 44.8 11.3 years (range, 2055 years) and eight cases (19%) were under 40. preoperatively, mean manifest refractive spherical equivalent (mrse) was + 2.00 d 0.76 d (range, 0.54.0 d) and mean spherical error was + 2.57 d 0.87 d (range, 1.255.5 d). figure 1 presents the changes in corrected and uncorrected visual acuity, and figure 2 demonstrates changes in mean mrse. meansd (range) of spherical and cylinder error (diopters) at different visits during a 1-year follow - up logarithm of the minimum angle of resolution uncorrected distance visual acuity and best spectacle - corrected distance visual acuity preoperatively and at 1, 3, 6, and 12 months after surgery stability of hyperopic photorefractive keratectomy with mitomycin - c out to 12 months postoperatively table 1 presents changes in sphere and cylinder errors due to each laser. changes in spherical (0.073) and cylindrical (0.197) errors were not significantly different between devices [table 1 ]. one month after surgery, a mean spherical error was myopic but decreased toward emmetropia. there were significant changes in the spherical error preoperatively to 1-month postoperatively (p < 0.001), as well as from 1 to 3 months postoperatively (p = 0.024). postoperatively, both spherical error and mrse were significantly different from the preoperative values (p < 0.001) ; 69.0% were within 0.50 d, and 98.0% were within 1.00 d of emmetropia. at this time, 18 eyes (42.9%) had ucva of 20/20, and in 39 eyes (92.8%) ucva was 20/40 or better. the best spectacle - corrected distance visual acuity (bcva) increased by two lines in three eyes (7.1%) and one line in two eyes (4.7%), 31 eyes (73.8%) showed no change, three eyes (7.1%) lost one line, and three other eyes (7.1%) lost two lines of corrected vision [figure 3 ]. thirty - one eyes showed no change, two lost two lines, and two gained two lines no stromal haze was observed in the central cornea. during the first 6 months, two eyes (5.0%) had peripheral haze (hanna grade 1) which resolved by the 12-month follow - up visit. slight haze was detected in 15 eyes (36.0%) and resolved in all cases within a few months. there was no case of ectasia or need for retreatment during the 1-year follow - up. microbial keratitis occurred in one eye which was eliminated from the study and final analysis. in this study, we evaluated the safety, efficacy, stability, and predictability of hyperopic prk over 1-year. unlike myopia, one of the challenges in making hyperopic corrections is the difference between manifest and cycloplegic refractions, especially in prepresbyopic patients. this issue is less significant in hyperopia over 5.0 d, where the latent component is lower. in a young patient, corrections based on manifest refraction will result in a late hyperopic shift while treating the full cycloplegic refraction will result in myopia. esquenazi and mendoza12 suggested using manifest refraction when there is more than 0.50 d discrepancy between manifest and cycloplegic refractions, but this could result in residual latent hyperopia in young patients. jackson.18 recommended using only manifest refraction in a young patient and accept some regression and latent hyperopia. spadea.19 based the laser correction according to the cycloplegic refraction in patients under 40 years of age, allowing for up to 0.5 d difference between the cycloplegic and manifest refractions. in cases over 40 years, they19 based corrections on manifest refraction. we used the cycloplegic refraction as the correction target in patients over 40 and the average of cycloplegic and manifest refractions in younger cases, and we allowed for up to 1.00 d of difference between cycloplegic and manifest refractions. it seems that less difference between the two refractions should be allowed before correction. at 12 months postoperatively, 43% of our patients had 20/20 ucva, which is comparable to jackson. 's study18 (52% at 12 months) and spadea. s study19 (46% at 24 months). in this study, the safety index (postoperative bcva / preoperative bcva) was 1.00, and the efficacy index (postoperative ucva / preoperative bcva) was 0.88, which were comparable to 0.95 and 0.87 values reported by leccisotti.14 however, despite a safety index of 1.00, we had six eyes with one or two lines loss in bcva which could partly be due to loss of previous linear magnification of spectacles14 or irregular astigmatism caused by some apical nodular subepithelial haze.20 stability of refraction after hyperopic prk has been reported in most studies to occur sometime between 3 and 12 months after surgery. in our report, however, stevens and ficker,22 reported that it took about 12 months for the refraction to stabilize. these discrepancies can be due to different magnitudes of preoperative hyperopia in these studies because higher hyperopic corrections require longer stabilize. review of our study in comparison to other studies on the correction of hyperopic photorefractive keratectomy mrse within 1.00 d of the intended refraction has been reported in different studies to be between 67% and 100%. we found it was 98%, this can be attributed to better predictability with newer generations of excimer laser machines with larger ablation zones and improved ablation profiles.1223 in hyperopic prk, the central corneal stroma largely spared, and the main site of ablation is the periphery. thus, in the absence of serious complications, such as decentered ablation or infection, there should be no significant haze formation or loss bcva.16 however, haze has been reported and can cause decreased corrected vision and regression after prk. without mmc, grades 1 and 2 haze (hanna 's scale) has been reported.7 in our study, with the use of mmc, there were just two cases of grade 2 peripheral haze which resolved by 12 months postoperatively. high values of root mean square higher order aberrations in a 7 mm pupil size have been reported,16 which were largely due to coma and negative 4 order spherical aberration, especially with higher corrections. we found hyperopic prk is an effective technique with good stability to correct low to moderate hyperopia, with some degrees of overcorrection during the first few months with regression toward emmetropia by 1-year. safety, predictability, and stability are acceptable and comparable to that of hyperopic lasik after 1-year follow - up. | purpose : to evaluate refractive and visual outcomes of photorefractive keratectomy with mitomycin - c. (prk - mmc) for the treatment of mild to moderate hyperopia.materials and methods : this case series enrolled 21 patients with up to + 5.50 diopters (d) of hyperopia. all 42 eyes were treated with the concerto (wavelight) or the technolas 217-z (bausch and lomb) excimer laser. outcome measures included best corrected distance vision acuity (bcva) and uncorrected distance vision correction and refraction at 1, 3, 6, and 12 months postoperatively.results:mean patient age was 44.8 11.3 years. preoperatively, mean manifest refractive spherical equivalent (mrse) was + 2.00 d 0.76 d and mean spherical refractive error was + 2.57 d 0.87 d (range, + 1.25 d to + 5.50 d). at 12 months postoperatively, mean mrse was + 0.1 d 0.61 d. mrse was within 0.50 d of emmetropia in 29 eyes (69%), and 18 eyes (43%) had 20/20 uncorrected distant visual acuity. bcva increased by two lines or more in three eyes (7.1%) and one line in two eyes (4.7%) ; 31 eyes showed no change, three eyes (7.1%) lost one line, and three other eyes (7.1%) lost two lines of bcva. no eyes lost more than two lines of bcva. complications included grade 2 peripheral haze in two eyes which cleared by 12 months postoperatively.conclusion:prk-mmc was a safe and predictable method for the correction of mild to moderate hyperopia. |
the incidence of kidney stones has been increasing in korea with the rise in dietary and lifestyle changes influenced by western countries. nephrolithiasis is one of the common causes of morbidity and deterioration of quality of life in the united states, with a lifetime prevalence of 5% to 10%. in addition, urolithiasis is a recurrent disease, with lifetime recurrence risks reported to be as high as 50%. given this high incidence and recurrence, technological advances have been made to dramatically improve minimally invasive techniques for the management of kidney stones, such as percutaneous nephrolithotomy (pcnl), extracorporeal shock wave lithotripsy (eswl), and retrograde intrarenal surgery (rirs). although eswl and rirs are currently widely used as less - invasive treatment modalities for renal stones, pcnl still has a role depending on the size, position, shape, and composition of the stones. pcnl is recommended as the treatment of choice for large renal stones (> 20 mm) and for lower calyceal stones sized 10 to 20 mm with unfavorable factors for eswl according to updated european association of urology guidelines. since its initial introduction in 1976 by fernstrom and johansson, pcnl has been widely performed for the management of large and complex renal stones and it remains the gold standard approach for difficult - to - treat renal stones (staghorn calculi, hard stones, stones associated with abnormal renal anatomy, and stones that have failed treatment with other less - invasive methods). however, although pcnl is considered as a minimally invasive procedure for stone removal, it is still a challenging surgical technique and can be associated with critical complications such as bleeding and septicemia. in addition, as the indications for rirs have expanded, it has supplanted eswl and pcnl for the treatment of some renal stones. nevertheless, many efforts have been made to reduce morbidity and increase the efficiency and effectiveness of pcnl (table 1), because pcnl remains indicated as a first - line treatment for many cases. herein, recent advancements in pcnl in surgical technique, instrumentation, and perioperative care are reviewed from the current literature. traditionally, pcnl has been performed with the patient in a prone position, and retrograde ureteral catheter placement is usually performed in the dorsal lithotomy position before the main lithotripsy procedure. the prone position provides a larger surface area for the choice of puncture site, including upper pole puncture, a wider space for manipulating the nephroscope and lithotriptors, and a lower risk of other perirenal visceral injury. however, there are also disadvantages to performing pcnl in a traditional prone position, such as patient discomfort, a relatively longer overall operation time owing to repositioning, and anesthesiological risks including circulatory problems, ventilatory difficulties, suboptimal airway control, increased sympathetic activity, ocular and pharmacokinetic effects, and possible lesions of the cervical spine or peripheral nerves. moreover, repositioning of the anesthetized patient is inevitable for retrograde access if it is needed, and this can lead to wasted time and increased risk of iatrogenic articular and peripheral nerve inadvertent pressure injury. to overcome these limitations, various modifications of patient positioning have been proposed over the years. the prone split - leg position was introduced as a modification of standard prone pcnl to increase efficiency and reduce the operation time. this position obviously decreases the operation time and the need for operative staff for multiple patient transfers, but it still has disadvantages related to anesthesiological risks. the distance from the posterior iliac crest to the 12th rib is reported to be increased by 2.9 cm with the prone - flexed position compared with the standard prone position. this means that this position has an advantage for easier access to upper pole puncture. although standard and modified prone positions have been successfully performed, they still have drawbacks related to increased cardiopulmonary risks. in particular, obese patients and those with cardiopulmonary comorbidities may have increased risks of cardiopulmonary - related complications for long operations in the prone position. to overcome these drawbacks, first performed supine pcnl in 1987, several studies have reported favorable outcomes and the technical benefits of this technique. a recent systemic review and meta - analysis comparing supine and prone pcnl reported a shorter operation time in supine pcnl and similar complication and transfusion rates between supine and prone pcnl. theoretically and practically, the advantages of supine pcnl compared with standard prone pcnl are as follows : (1) optimal cardiovascular and airway control, (2) better in high - risk patients with heart failure or in obese patients, (3) shorter operation time due to no need for repositioning, (4) opportunity for a combined retrograde approach, (5) better stone fragment washout due to horizontal dorsal sheath angle, and (6) less radiation exposure to the surgeons ' hands. however, supine pcnl also has several limitations, as follows : (1) limited space for renal puncture and nephroscope mobility (fig. 1) ; (2) upper pole calyx puncture is more challenging ; (3) more complex tract dilation due to high kidney mobility ; (4) increased risk of spleen and liver injury during upper pole puncture ; (5) decreased filling of the collecting system, which is constantly collapsed ; and (6) spinal interposition in the fluoroscopic field during anteroposterior projections. in addition to supine pcnl, various modified positions have recently been proposed, such as supine oblique, semisupine, lateral decubitus, split - leg modified lateral, flank, and flank prone position. the advantage of these positions is the possibility for simultaneous antegrade and retrograde access to the kidney without repositioning. however, there are still limited studies available to validate the efficacy and safety of pcnl in these various positions, including supine pcnl. therefore, a large, prospective, multicenter, randomized controlled trial is necessary to verify the feasibility and safety of pcnl in modified positions. one of the most important steps for successful pcnl is well - placed access into the kidney. traditionally, intraoperative percutaneous access was most commonly performed by use of fluoroscopic guidance. however, this approach has some drawbacks, including increased radiation exposure for the surgeon, operating room staff, and patient, and possible iatrogenic visceral injury such as to the colon, liver, spleen, and pleura. moreover, it is hard to perform fluoroscopic - guided access in patients in whom retrograde ureteral catheter placement is difficult or impossible, such as in patients with urinary diversions or renal transplants. to overcome these limitations of fluoroscopy, the advantages of this approach are less radiation exposure and the ability to identify nearby organs. several studies have reported satisfactory outcomes and fewer complications with ultrasound - guided access compared with fluoroscopic - guided access. according to these studies, the success rate in the targeted calyces of ultrasound- and fluoroscopic - guided access ranges from 90% to 100% and from 96% to 100%, respectively. reported intraoperative bleeding rates of 10% (5 of 50) in an ultrasound - guided access group and 6% (3 of 50) in a fluoroscopic - guided access group (p=0.5). however, ultrasound - guided access is surgeon - dependent and has some limitations for delineating fine details of the renal anatomy, especially in patients with nondilated collecting systems or in obese patients. in this condition, my preference for percutaneous access is a combined approach using ultrasound and fluoroscopic guidance simultaneously. with fluoroscopic guidance, i can easily focus on the targeted calyx, and then percutaneous access can be performed by using ultrasound guidance, resulting in less radiation exposure and reducing the risk of nearby organ injury. moreover, large branches of the renal artery can be identified by using the doppler ultrasound setting, which may reduce the risk of bleeding by avoiding punctures to that area. if the patients have a collecting system that is difficult to access or a spinal deformity such as spina bifida or scoliosis, fluoroscopic - guided access can cause adjacent organ injury and may not guarantee successful pcnl. in these situations, matlaga. reported six cases of ct - guided access due to spinal deformity, retrorenal colon, or transplanted kidney and showed favorable outcomes. they reported that percutaneous access was achieved successfully without complications in all cases and the stone - free rate was 83% (5 of 6). because fluoroscopic - guided or ct - guided access can have concerns related to ionizing radiation exposure, and ultrasound - guided access may obscure fine details of the renal anatomy, magnetic resonance imaging (mri)-guided access has been introduced as an alternative means of nonionizing radiation.. performed percutaneous access using open - configuration, c - arm shaped mri and reported success in seven of eight patients. they demonstrated that a drawback of this approach is difficulty visualizing the guidewire with mri. besides, the need for specially designed equipment to perform this technique can also be an obstacle to widespread use of mri - guided access. in addition to these various imaging - guided access methods, the development of endoscopy such as flexible ureteroscopy and optical puncture systems has introduced modified techniques for safe and desirable access. used a retrograde approach that can be applied by directing a steerable catheter in a retrograde fashion into the desired calyx and then advancing a puncture wire out through the catheter to the skin. and kidd and conlin reported simultaneous retrograde ureteroscopic and fluoroscopic - guided percutaneous access. reported successful use of an optical puncture needle named an " allseeing needle " to confirm the optimal percutaneous access. after successful percutaneous access and tract dilatation, efficient stone fragmentation and evacuation depend on the effectiveness of the lithotriptors. ultrasonic lithotriptors can effectively fragment most stones except very hard stones, such as cystine or calcium oxalate monohydrate stones, and can simultaneously aspirate stone fragments. pneumatic lithotriptors can fragment hard stones more efficiently than ultrasonic lithotriptors but have no or limited suctioning function. to combine the characteristics of both ultrasonic and pneumatic lithotriptors, dual - modality devices, such as the swiss lithoclast ultra (boston scientific corp., natick, ma, usa) and the cyberwand dual ultrasonic lithotriptor (gyrus acmi, southborough, ma, usa) have been developed that provide efficient stone fragmentation with concomitant aspiration. pietrow. performed a randomized controlled study comparing the swiss lithoclast ultra with a standard ultrasonic lithotriptor and demonstrated a better stone clearance rate (40 mm / min vs. 17 mm / min, p=0.028) and shorter mean time to stone clearance (21 minutes vs. 44 minutes, p=0.036) with the swiss lithoclast ultra. however, another multicenter randomized clinical trial showed no advantage of the cyberwand dual ultrasonic lithotriptor compared with a single - probe ultrasonic lithotriptor with regard to time to stone clearance, stone - free rate, and complication rate. the vaporizing and bursting effect of the holmium : yag laser can fragment the stone quickly regardless of stone composition ; therefore, it may reduce the lithotripsy and operation time. recently, a cordless lithotripsy device, the lma stone breaker pneumatic lithotripter (cook medical, bloomington, in, usa), was developed. the advantage of this device is that it is portable and can generate high velocity at the probe tip, but no clinical trials have compared the efficiency and safety of this device with those of other lithotriptors., several reports have suggested that the risk of bleeding is associated with sheath size. thus, to decrease morbidity related to larger tracts, such as bleeding, postoperative pain, and potential renal damage, modifications to technique and the size of the instruments have been made. this technique of using a small caliber working sheath, known as mini - pcnl or miniperc, was originally developed for the management of large renal stones in pediatric patients. first reported miniaturized pcnl for 2-year - old pediatric patients with renal stones with the use of a 15-fr peel - away vascular sheath. then, jackman. accomplished mini - pcnl using an 11-fr access sheath. in their series, the mean decreases in operation time, estimated blood loss, and hematocrit were 176 minutes, 83 ml, and 6.6%, respectively. the stone - free rate was 89% (8 of 9) without any procedure - related complications. however, there is still no exact definition as to what should be called mini - pcnl or mini - perc. since the first trial of miniaturized pcnl, several authors have utilized various sizes of working sheaths ranging from 11 to 20 fr. thus, the term mini - pcnl is used for any pcnl that utilizes access tracts of 20 fr or smaller. in addition, desai. recently introduced the smallest sized access tract (4.8 fr), which was named " microperc. " definite indications for mini - pcnl have not been clearly defined. however, mini - pcnl can be the first - line treatment for renal stones with a diameter larger than 2 cm in children, and general indications for mini - pcnl in adults may include renal stones less than 2 or 2.5 cm in size with previous failure of eswl or rirs, cystine stones, and anatomical abnormalities inhibiting retrograde access or distal passage of stones. mini - pcnl can also be used in patients with a narrow or long infundibulum or as a secondary access for inaccessible or residual stones after standard pcnl. nevertheless, mini - pcnl has not only been used for the management of smaller renal stones or pediatric renal stones but for large impacted upper ureteral stones and staghorn calculi as well. the advantages of mini - pcnl, based on several prospective trials, are less bleeding, increased maneuverability, lower postoperative pain, and reduced hospital stay. however, the longer operation time due to the need to disintegrate stones into small fragments to evacuate through a small - sized sheath is a limitation of mini - pcnl. most studies reported similar stone - free rates and comparable overall complication rates between mini - pcnl and standard pcnl for the management of renal stones less than 2.5 cm in size. compared with eswl, mini - pcnl has been reported to have significantly higher stone - free rates, especially for renal stones greater than 1 cm, although it is more invasive. compared to rirs, mini - pcnl has shown better stone - free rates for the management of larger renal stones (2 - 3 cm) and large impacted upper ureteral stones, but similar effectiveness was reported when treating smaller renal stones between both procedures. in these studies, mini - pcnl was superior in terms of operation time for the management of comparably sized stones, but was inferior with respect to invasiveness. however, the combination of rirs and mini - pcnl showed better outcomes than did mini - pcnl alone for large renal stones (> 3 cm) with shorter operation times. microperc, which uses the smallest sized access tract, can be applicable for small or intermediate - sized lower calyceal stones, especially stones that are eswl - resistant or retrograde inaccessible. specifically, microperc can also be used for the management of renal stones in calyceal diverticula and in horseshoe and ectopic kidneys. however, further prospective randomized controlled trials will be needed to validate the safety, efficacy, and applicability of this technique. after completing the lithotripsy, the final step in pcnl is sealing the nephrostomy tract. traditionally, a large - caliber nephrostomy tube, such as a 24-fr council catheter, a reentry malecot catheter, or a nephroureteral stent, was placed to provide hemostasis of the tract and maintain adequate urine drainage. the nephrostomy tube was historically left indwelling for several days, which required a prolonged hospital stay, increased analgesic use owing to patient discomfort, and resulted in urinary extravasation around the tube. the first such trial involved the placement of a smaller sized nephrostomy tube instead of a large - bore one. maheshwari compared a patient group who underwent pcnl and had either a 28-fr nephrostomy tube or a 9-fr pigtail catheter postoperatively. in that study, the incidence of hematuria was comparable between the two groups, but the 9-fr pigtail catheter group showed a lower analgesic requirement, less urine leakage, and a shorter hospital stay. pietrow. also reported similar results from their randomized prospective trial comparing a 22-fr council catheter with a 10-fr pigtail catheter. f irst described no placement of a nephrostomy tube after pcnl in 1984, tubeless pcnl was not actively attempted for over 10 years because postoperative hemorrhage was reported. the first series of tubeless pcnl was reported in 1997 by bellman.. in that study, the tubeless pcnl group showed comparable stone - free and complication rates, a lower analgesic requirement (11.58 mg vs. 36.06 mg of morphine sulfate, p=0.0001), a reduced hospital stay (0.6 days vs. 4.6 days, p=0.0001), and an earlier return to normal activity (17.85 days vs. 26.6 days, p=0.0004) compared with the standard pcnl group with a 22-fr nephrostomy tube. desai. compared three different randomly divided groups (22-fr nephrostomy tube, 9-fr pigtail catheter, and 6-fr double - j stent without nephrostomy tube) and reported no significant differences in blood loss among the three groups. they also found that analgesics were most frequently required in the 22-fr nephrostomy tube group, and the duration of urine leakage and hospital stay was the shortest in the tubeless group. marcovich. also reported no significant difference in blood loss or the transfusion rate when comparing three different groups according to the size of the nephrostomy tube (24-fr reentry catheter, 8-fr pigtail catheter, and tubeless). however, analgesic use and hospital stay were also not significantly different in their series. meta - analysis also showed that tubeless pcnl was associated with lower analgesic requirements, shorter hospital stay, and comparable blood loss compared with standard pcnl. although the indications for tubeless pcnl have not been clearly defined, initially, tubeless pcnl was performed only in highly selected cases such as uncomplicated stones, smaller stones (less than 3 cm), normal renal function, single - tract procedure, short operation time, complete stone removal, no collecting system perforation, and no active bleeding from the tract at completion. since then, there have been several efforts to apply tubeless pcnl in more complicated cases, including multiple, complex, and bilateral stones ; patients with elevated serum creatinine ; children ; obese patients ; and elderly patients. on the basis of these current experiences and analyses, many investigators have concluded that tubeless pcnl should be avoided in case of multiple access, serious intraoperative bleeding, collecting system perforation, and need for early second - look surgery. although many studies have reported that tubeless pcnl can be safely performed in well - selected cases, there are still concerns associated with tract bleeding. limb and bellman reported a postoperative transfusion rate of 5.4% (6 of 112) and 2 cases (1.8%) of renal pseudoaneurysm, which were managed by selective angioembolization, in their first 112 tubeless renal surgery cases. to overcome these concerns, several techniques for tract sealing, such as electrocauterization of bleeding points, applying fibrin glue, and placement of hemostatic matrix into the tract, most of these studies reported favorable outcomes in terms of safety and efficacy for the tract control, but routine use of these tract - sealing techniques in tubeless pcnl to prevent tract bleeding or urine leakage is still controversial. moreover, the possibility of stone formation in the collecting system due to misplacement of these biological sealants or worsening of bleeding with further renal parenchymal injury due to inappropriate electrocauterization are possible side effects of these techniques. thus, meticulous care must be taken when the tract - sealing technique is considered for tubeless pcnl. initially, tubeless pcnl was performed with placement of an internal double - j stent for the purpose of preventing urinary extravasation. the internal stent can also cause patient discomfort owing to bladder irritation symptoms and urine reflux. in addition, the most bothersome fact is the need for postoperative stent removal by use of cystoscopy. to avoid this procedure, several attempts such as leaving the string attached to the stent and using externalized ureteral stents have been tried, but there is the risk that patients may pull out their stents prematurely by accident, and the risk of urinary tract infection may be increased. to overcome these drawbacks, " many investigators have suggested that the best available urinary drainage method from the kidney is the normal peristaltic ureter and the only indication for ureteral stent placement is ureteral obstruction due to edema, inflammation, presence of residual stones, or stricture. several studies have demonstrated that totally tubeless pcnl is safe, effective, and well tolerated in selected patients and is associated with a shorter hospital stay, less postoperative pain, and decreased analgesic requirements compared with standard pcnl. there are limited prospective randomized study data comparing totally tubeless and tubeless pcnl, but istanbulluoglu. found that totally tubeless pcnl had comparable blood loss, success rates, operation time, hospital stay, analgesic requirements, and complication rates with the advantage of no need for stent removal even compared with tubeless pcnl in their retrospective study. to validate these data, further prospective randomized trials will be needed to compare safety and efficacy between totally tubeless and tubeless pcnl. pcnl was developed to reduce the morbidity and mortality associated with open renal surgery, and it currently remains the first - line treatment for large renal stones. however, it represents the most morbid of the minimally invasive endoscopic surgeries for renal stones. over the years, with the development of endoscopic instruments and techniques, the role of eswl and rirs has increased. currently, many efforts and trials to reduce morbidity and increase the efficiency and effectiveness of pcnl have furthered this procedure technically and have broadened its indications. recent advancements that make pcnl a less - invasive technique promise higher success rates and less perioperative complications. further efforts will be needed to validate the many promising retrospective data by use of large - scale prospective studies and to develop this procedure more safely and effectively. | since its initial introduction in 1976, percutaneous nephrolithotomy (pcnl) has been widely performed for the management of large renal stones and currently is recommended for staghorn calculi, kidney stones larger than 2 cm, and shock wave lithotripsy - resistant lower pole stones greater than 1 cm. however, except for open and laparoscopic surgery, pcnl is the most invasive of the minimally invasive stone surgery techniques. over the years, technical and instrumental advances have been made in pcnl to reduce morbidity and improve effectiveness. a thorough review of the recent literature identified five major areas of progress for the advancement of pcnl : patient positioning, method of percutaneous access, development of lithotriptors, miniaturized access tracts, and postoperative nephrostomy tube management. this review provides an overview of recent advancements in pcnl and the outcomes of each area of progress and notes how much we achieve with less invasive pcnl. this information may allow us to consider the future role and future developments of pcnl. |
the growth of flexible ureteroscopy (urs) in the asian subcontinent has not been as fast as its potential appears to be. the factors responsible for this are perhaps the high initial cost and the maintenance cost involved in flexible urs. it is a well - known fact that the damage to the instrument is more so during the initial learning curve. the high maintenance cost is probably related to the wear and tear of the flexible endoscope. the learning curve reduces with the utilization of a skills laboratory for the human body. the higher the fidelity of a model, i.e. the ability to simulate a more life - like situation, the better it is supposed to be. high - fidelity simulators are those that are more lifelike, often with the ability to move beyond simple skill or task training and simulate partial or whole operations. commercially available high - fidelity simulators such as the uro - scopic trainer ; limbs and things and endo - urologie - modell ; karl storz offer the advantages of reusability, realistic anatomy and the ability to use real surgical instruments such as flexible endoscopes. a relatively new category of simulation, vr, has arisen as a result of significant improvements in computing and graphics capabilities. while expensive and requiring maintenance, vr simulators such as uro mentor offer the opportunity to practice basic skills or entire surgical procedures in virtually rendered environments. the cost of a high - fidelity model, especially the vr model, is high and prohibitive. this enables them to acquire the necessary psychomotor skills and confidence to start the procedure for a real human scenario. surgical skills laboratory relies on bench models and virtual reality (vr) simulators, which serve as surrogates. we determined the closeness to reality of each model and also whether any had advantages in terms of hastening skill acquisition. for this, we used two high - fidelity non - vr bench models and a vr model. the inclusion criteria were : a participant with a board - certified urology degree involved in a private practice without association to a teaching institute and willing to learn flexible urs. all the 21 subjects received an intensive teaching session involving a total of 3 h regarding the tips and tricks of performing flexible urs before the study day. an initial warm - up period of 2 h was provided to the participants to overcome operative and climatic inhibition. on the next day (study day), all the subjects watched a 20-min power point presentation reviewing the instruments, models and flexible urs video demonstrating the finer tricks involving the psychomotor movements on the two non - vr bench models and a vr model. high - fidelity non - vr - based bench models [figure 1 ] involved for the training included an uro - scopic trainer ; limbs and things and endo - urologie - modell ; karl storz while the vr model was a uro mentor(simbionix, israel). both uro - scopic trainer and endo - urologie - modell consist of a mannequin of the male genitourinary tract through which standard instruments may be passed. there is an obvious advantage as trainees practice with the real - time flexible ureteroscope, which they will be using subsequently in their operating rooms. the trainer allows the user to simulate several endourological techniques, including examination of the urinary tract, stent and guide wire insertion, lithotripsy and stone retrieval. uro mentor is a vr - based simulator specifically developed for training in percutaneous renal access and urs. it allows simulation of cytoscopic and ureteroscopic procedures performed using either flexible or semirigid endoscopes. the users interact with the haptic interface device containing flexible endoscopes and ancillary equipments, such as baskets and intracorporeal lithotripsy devices, linked to force feedback mechanisms. geometric models of urinary tract anatomy and devices used during urs provide tissue - tool interactions. (readers can view website for more details ; www.simbionix.com/uro_mentot.html,http://limbsandthings.com/404.shtml) study design - flow chart the study design is as depicted in figure 1. the participants were divided into three batches of seven participants each with rotation on each of the three models. once the training session of 30 min was over, they proceeded to the next station. in this way, all the participants received a total of 90 min of training. the rotations of each batch were different but the overall time utilized by each batch was 90 min (30 min at each station). station a had an uro - scopic trainer, station b an endo - urologie - modell and station c a uro mentor. a flex - x ; karl storz flexible ureteroscope was used at stations a and b while vr flexible urs (in - built uro mentor) was used at station c. batch 1 rotated from stations a to b to c, batch 2 rotated from stations b to c to a and batch 3 from stations c to a to b. an expert ureteroscopist first performed the simulation exercise at all the stations for a period totaling 3 h. he then devised a specific task for each station of similar complexity assumed to take a similar time. the purpose of doing this was to make the tasks comparable for evaluation. at station a, the task given to the trainee was introducing the flexible ureteroscope into the ureteric orifice without a guide wire across and reaching the mid ureter on both sides sequentially. the task at station b was introducing the flexible ureteroscope into the middle calyx of either kidney sequentially without a guide wire. at station c, a specific task scenario (task no. 9) of stone manipulation in the training module of the uro mentor software was selected. it consisted of passing the simulation - flexible ureteroscope to the middle calyx of the left kidney, retrieving the stone with dormia basket, placing the stone in the upper calyx and fragmenting the stone with holmium laser. at all the stations, the performance of the trainees was evaluated with a single experienced, non - blinded expert ureteroscopist using a global rating score (grs) and pass rating. the grs, adapted from matsumoto., was modified as per white. to exclude bladder and urethra and standardize the models for flexible urs [table 1 ]. the evaluator assigned a value of 1 - 5 for each of the seven aspects on a grs. the higher the score, the better was the participant performance. if the participant could perform the desired task, he was given a pass score. pass rating of the batch at a particular station was defined as percentage of participants passing with respect to total batch strength. a validated global rating score card (minimum 7, maximum 35) face validity is defined as the judgment of novices regarding realism and usefulness of the simulator. the participants rated a standardized face validity questionnaire based on likert 's scale 1 - 10 at each station to evaluate the realism and usefulness of each training model. domains of overall realism, flexible endoscope, training model, tissue feel, respiratory movements, urethra and bladder, negotiating ureteric orifice and negotiating endoscope in the ureter and pelvi - calyceal systems were evaluated to assess the realism of the training model. the objectiveness of the face validity results was compared using numerical data on the likert 's scale as provided by the participants. the results of face validity of the models and grss were calculated using student 's t - test. the pass rating on each station by the batch was analyzed by the chi square () significance test for comparing two proportions (with continuity correction). the inclusion criteria were : a participant with a board - certified urology degree involved in a private practice without association to a teaching institute and willing to learn flexible urs. all the 21 subjects received an intensive teaching session involving a total of 3 h regarding the tips and tricks of performing flexible urs before the study day. an initial warm - up period of 2 h was provided to the participants to overcome operative and climatic inhibition. on the next day (study day), all the subjects watched a 20-min power point presentation reviewing the instruments, models and flexible urs video demonstrating the finer tricks involving the psychomotor movements on the two non - vr bench models and a vr model. high - fidelity non - vr - based bench models [figure 1 ] involved for the training included an uro - scopic trainer ; limbs and things and endo - urologie - modell ; karl storz while the vr model was a uro mentor(simbionix, israel). both uro - scopic trainer and endo - urologie - modell consist of a mannequin of the male genitourinary tract through which standard instruments may be passed. there is an obvious advantage as trainees practice with the real - time flexible ureteroscope, which they will be using subsequently in their operating rooms. the trainer allows the user to simulate several endourological techniques, including examination of the urinary tract, stent and guide wire insertion, lithotripsy and stone retrieval. uro mentor is a vr - based simulator specifically developed for training in percutaneous renal access and urs. it allows simulation of cytoscopic and ureteroscopic procedures performed using either flexible or semirigid endoscopes. the users interact with the haptic interface device containing flexible endoscopes and ancillary equipments, such as baskets and intracorporeal lithotripsy devices, linked to force feedback mechanisms. geometric models of urinary tract anatomy and devices used during urs provide tissue - tool interactions. (readers can view website for more details ; www.simbionix.com/uro_mentot.html,http://limbsandthings.com/404.shtml) study design - flow chart the study design is as depicted in figure 1. the participants were divided into three batches of seven participants each with rotation on each of the three models. once the training session of 30 min was over, they proceeded to the next station. in this way, all the participants received a total of 90 min of training. the rotations of each batch were different but the overall time utilized by each batch was 90 min (30 min at each station). station a had an uro - scopic trainer, station b an endo - urologie - modell and station c a uro mentor. a flex - x ; karl storz flexible ureteroscope was used at stations a and b while vr flexible urs (in - built uro mentor) was used at station c. batch 1 rotated from stations a to b to c, batch 2 rotated from stations b to c to a and batch 3 from stations c to a to b. an expert ureteroscopist first performed the simulation exercise at all the stations for a period totaling 3 h. he then devised a specific task for each station of similar complexity assumed to take a similar time. the purpose of doing this was to make the tasks comparable for evaluation. at station a, the task given to the trainee was introducing the flexible ureteroscope into the ureteric orifice without a guide wire across and reaching the mid ureter on both sides sequentially. the task at station b was introducing the flexible ureteroscope into the middle calyx of either kidney sequentially without a guide wire. at station c, a specific task scenario (task no. 9) of stone manipulation in the training module of the uro mentor software was selected. it consisted of passing the simulation - flexible ureteroscope to the middle calyx of the left kidney, retrieving the stone with dormia basket, placing the stone in the upper calyx and fragmenting the stone with holmium laser. at all the stations, the performance of the trainees was evaluated with a single experienced, non - blinded expert ureteroscopist using a global rating score (grs) and pass rating. the grs, adapted from matsumoto., was modified as per white. to exclude bladder and urethra and standardize the models for flexible urs [table 1 ]. the evaluator assigned a value of 1 - 5 for each of the seven aspects on a grs. the higher the score, the better was the participant performance. if the participant could perform the desired task, he was given a pass score. pass rating of the batch at a particular station was defined as percentage of participants passing with respect to total batch strength. a validated global rating score card (minimum 7, maximum 35) face validity is defined as the judgment of novices regarding realism and usefulness of the simulator. the participants rated a standardized face validity questionnaire based on likert 's scale 1 - 10 at each station to evaluate the realism and usefulness of each training model. domains of overall realism, flexible endoscope, training model, tissue feel, respiratory movements, urethra and bladder, negotiating ureteric orifice and negotiating endoscope in the ureter and pelvi - calyceal systems were evaluated to assess the realism of the training model. the objectiveness of the face validity results was compared using numerical data on the likert 's scale as provided by the participants. the results of face validity of the models and grss were calculated using student 's t - test. the pass rating on each station by the batch was analyzed by the chi square () significance test for comparing two proportions (with continuity correction). the inclusion criteria were : a participant with a board - certified urology degree involved in a private practice without association to a teaching institute and willing to learn flexible urs. all the 21 subjects received an intensive teaching session involving a total of 3 h regarding the tips and tricks of performing flexible urs before the study day. an initial warm - up period of 2 h was provided to the participants to overcome operative and climatic inhibition. on the next day (study day), all the subjects watched a 20-min power point presentation reviewing the instruments, models and flexible urs video demonstrating the finer tricks involving the psychomotor movements on the two non - vr bench models and a vr model. high - fidelity non - vr - based bench models [figure 1 ] involved for the training included an uro - scopic trainer ; limbs and things and endo - urologie - modell ; karl storz while the vr model was a uro mentor(simbionix, israel). both uro - scopic trainer and endo - urologie - modell consist of a mannequin of the male genitourinary tract through which standard instruments may be passed. there is an obvious advantage as trainees practice with the real - time flexible ureteroscope, which they will be using subsequently in their operating rooms. the trainer allows the user to simulate several endourological techniques, including examination of the urinary tract, stent and guide wire insertion, lithotripsy and stone retrieval. uro mentor is a vr - based simulator specifically developed for training in percutaneous renal access and urs. it allows simulation of cytoscopic and ureteroscopic procedures performed using either flexible or semirigid endoscopes. the users interact with the haptic interface device containing flexible endoscopes and ancillary equipments, such as baskets and intracorporeal lithotripsy devices, linked to force feedback mechanisms. geometric models of urinary tract anatomy and devices used during urs provide tissue - tool interactions. (readers can view website for more details ; www.simbionix.com/uro_mentot.html,http://limbsandthings.com/404.shtml) study design - flow chart the participants were divided into three batches of seven participants each with rotation on each of the three models. once the training session of 30 min was over, they proceeded to the next station. in this way, all the participants received a total of 90 min of training. the rotations of each batch were different but the overall time utilized by each batch was 90 min (30 min at each station). station a had an uro - scopic trainer, station b an endo - urologie - modell and station c a uro mentor. a flex - x ; karl storz flexible ureteroscope was used at stations a and b while vr flexible urs (in - built uro mentor) was used at station c. batch 1 rotated from stations a to b to c, batch 2 rotated from stations b to c to a and batch 3 from stations c to a to b. an expert ureteroscopist first performed the simulation exercise at all the stations for a period totaling 3 h. he then devised a specific task for each station of similar complexity assumed to take a similar time. the purpose of doing this was to make the tasks comparable for evaluation. at station a, the task given to the trainee was introducing the flexible ureteroscope into the ureteric orifice without a guide wire across and reaching the mid ureter on both sides sequentially. the task at station b was introducing the flexible ureteroscope into the middle calyx of either kidney sequentially without a guide wire. at station c, a specific task scenario (task no. 9) of stone manipulation in the training module of the uro mentor software was selected. it consisted of passing the simulation - flexible ureteroscope to the middle calyx of the left kidney, retrieving the stone with dormia basket, placing the stone in the upper calyx and fragmenting the stone with holmium laser. at all the stations, the performance of the trainees was evaluated with a single experienced, non - blinded expert ureteroscopist using a global rating score (grs) and pass rating. the grs, adapted from matsumoto., was modified as per white. to exclude bladder and urethra and standardize the models for flexible urs [table 1 ]. the evaluator assigned a value of 1 - 5 for each of the seven aspects on a grs. the higher the score, the better was the participant performance. if the participant could perform the desired task, he was given a pass score. pass rating of the batch at a particular station was defined as percentage of participants passing with respect to total batch strength. each batch as a whole was evaluated rather than evaluating individuals. a validated global rating score card (minimum 7, maximum 35) face validity is defined as the judgment of novices regarding realism and usefulness of the simulator. the participants rated a standardized face validity questionnaire based on likert 's scale 1 - 10 at each station to evaluate the realism and usefulness of each training model. domains of overall realism, flexible endoscope, training model, tissue feel, respiratory movements, urethra and bladder, negotiating ureteric orifice and negotiating endoscope in the ureter and pelvi - calyceal systems were evaluated to assess the realism of the training model. the objectiveness of the face validity results was compared using numerical data on the likert 's scale as provided by the participants. the results of face validity of the models and grss were calculated using student 's t - test. the pass rating on each station by the batch was analyzed by the chi square () significance test for comparing two proportions (with continuity correction). most of the realistic domains were higher for the vr model as compared with the non - vr training models, although these were not statistically significant. the only significant change observed in the vr model was the realism of respiratory movements. one interesting observation made in the face validity results was the higher realism of the flexible endoscope domain in the non - vr - based models. face validity result of the models on likerts scale 1 - 10 figure 2 shows the task results of batches 1, 2 and 3, respectively. this was observed in all the batches, irrespective of whether it was a vr or non - vr model to start with. there was not much improvement in the grss when the second and third rotations were compared ; however, it was more marked when the third rotation was compared with the first rotation. the global rating scores improved statistically at evaluation performed after the second rotation (3.84) for all training models when the third and first rotations were compared. batch 3, i.e. the batch that was trained initially on the vr - based training model, had more improvement on evaluation performed at the end of the second rotation as compared with the batches that had initial training on non - vr training models, although this did not achieve statistical significance at the 0.05 confidence interval. pass ratings of the batches at successive stations overall, the results show that both the non - vr- and vr - based training models provide almost a comparable level of training skills. most of the realistic domains were higher for the vr model as compared with the non - vr training models, although these were not statistically significant. the only significant change observed in the vr model was the realism of respiratory movements. one interesting observation made in the face validity results was the higher realism of the flexible endoscope domain in the non - vr - based models. face validity result of the models on likerts scale 1 - 10 figure 2 shows the task results of batches 1, 2 and 3, respectively. this was observed in all the batches, irrespective of whether it was a vr or non - vr model to start with. there was not much improvement in the grss when the second and third rotations were compared ; however, it was more marked when the third rotation was compared with the first rotation. the global rating scores improved statistically at evaluation performed after the second rotation (3.84) for all training models when the third and first rotations were compared. batch 3, i.e. the batch that was trained initially on the vr - based training model, had more improvement on evaluation performed at the end of the second rotation as compared with the batches that had initial training on non - vr training models, although this did not achieve statistical significance at the 0.05 confidence interval. pass ratings of the batches at successive stations overall, the results show that both the non - vr- and vr - based training models provide almost a comparable level of training skills. most of the realistic domains were higher for the vr model as compared with the non - vr training models, although these were not statistically significant. the only significant change observed in the vr model was the realism of respiratory movements. one interesting observation made in the face validity results was the higher realism of the flexible endoscope domain in the non - vr - based models. figure 2 shows the task results of batches 1, 2 and 3, respectively. this was observed in all the batches, irrespective of whether it was a vr or non - vr model to start with. there was not much improvement in the grss when the second and third rotations were compared ; however, it was more marked when the third rotation was compared with the first rotation. the global rating scores improved statistically at evaluation performed after the second rotation (3.84) for all training models when the third and first rotations were compared. batch 3, i.e. the batch that was trained initially on the vr - based training model, had more improvement on evaluation performed at the end of the second rotation as compared with the batches that had initial training on non - vr training models, although this did not achieve statistical significance at the 0.05 confidence interval. pass ratings of the batches at successive stations overall, the results show that both the non - vr- and vr - based training models provide almost a comparable level of training skills. flexible urs is a relatively new technology, which often requires cognitive specific motor skills by the operator. it is often difficult for urologists in training and practice to acquire adequate experience because of limited opportunities in the operating room. the simulators offer an opportunity for the trainees to perfect their skills in an inanimate but dynamic model in which anatomical structures are accurately reproduced and the feel of the actual procedure is captured. a lot of models on ascending scale of cost are available to offer training modules in a skills lab. vr models have a higher number of individual procedure constructs and offer real life - like situations. they are limited by the exorbitant prices. in developing countries like india, there are only handful skill labs. even more rare is the availability of vr simulators in these labs. in any training module, if we do away with the high - cost vr model, do we compromise in the training ? if not, this may be more cost - effective. to address this issue simulators can be categorized as low- and high - fidelity simulators depending on the ability to reproduce life - like scenarios. the main disadvantages are the lack of realism and reduced number of constructs required for the operation. high - fidelity simulators are those that simulate life - like situations more realistically, often with the capability to move beyond the simple skill or task training and to simulate partial or whole operations. we hypothesized that performance of the necessary basic skills in the skills lab improved the cognitive motor skills and boosted the confidence level to start the flexible urs ; irrespective of the model status. to study whether it was the impact of training on a specific model or the number of hours of training, the batch as a whole was evaluated. we measured the operative performance by objective structured assessment of technical skills examination, relying on global rating scales for the evaluation of particular tasks and characteristics. time comparisons to assess the completeness of the task were checked with pass rating. as expected, the improvement was more marked and statistically significant during the first two bench rotations, irrespective of the bench model. the skills acquisition was slow during the rotation from the second to the third model, although the overall improvement was marked when the first and third rotations were compared. as the task is performed repeatedly, the results improve marginally ; we may not get statistically significant improvement when comparison of the last few procedures is carried out. beyond this level batches trained initially on vr model did not show any difference with respect to other models, although the pass rating at rotation 2 was more than that in the other models. this may have been due to a higher number of individual constructs of the real procedure incorporated in the model. the other reason could also be the fact that due to smaller width of the traversing road map on this model, it made subsequent interventions traversing in wider traversing passage in non - vr models easier. the face validity results in our study and the results were similar between all the models. this could have been due to eagerness on the part of novices to learn the procedure, irrespective of its fidelity status. the non - vr models have high realism of the endoscope scores as compared with the vr models. one of the possible explanations could be that the novices being impressed by their working with the real endoscope provided high scores. the limitation of the study includes the short number of participants, an overlap of test - retest reliability in improvement of successive scores, lack of validated instruments for assessing skill acquisition and the unblinded expert reviewer. but, the remarkable differences in the p - value could definitely account for improvement in skills occurring during the practice sessions. the validation instrument, the global rating scale, was altered to account for the lack of the bladder and the urethra. the experienced ureteroscopist was not blinded and, thus, bias could have been introduced. also, currently, there are no current publications and public acknowledgement on the cost implications of various models imparting training in flexible urs. even the non - vr models in this manuscript were funded and provided by the sponsors (see acknowledgement). therefore, the cost of setting up and running each model is largely unstudied. further studies looking at the cost - effectiveness should be an interesting area of research. the high - fidelity simulators are significantly more expensive than low - fidelity models with more advantages. matsumoto. found no significant difference between students who trained on the low - fidelity model and those who trained on the high - fidelity model. the low - fidelity model (20 $) was produced at less than 1% of the purchase cost of the high - fidelity model (3700 $). chou. examined the ability of two simulators, a high - fidelity bench model (uro - scopic trainer) and a vr simulator (uro mentor), to teach basic ureteroscopic skills to inexperienced medical students. the authors found no significant difference between the two groups in their ability to perform the steps of the procedure and concluded that either of these training modalities may improve the initial clinical performance of urological trainees. matsumoto. compared vr simulation with the uro mentor to a high - fidelity bench trainer (uro - scopic trainer) for the assessment of endourological skills. the authors concluded that performance on the vr simulator was comparable to performance on the high - fidelity bench model and that the uro mentor urs simulator is a useful tool for the assessment of resident performance. all the models used for training flexible urs in the current study were effective with respect to increasing the grss and pass ratings. it is spending time on the high - fidelity models that matters in acquiring the skills rather than the type of the model. the vr models have the advantage of assimilating a higher number of individual constructs required for the procedure, thereby increasing the confidence level much higher. also, once the training in the vr model was imparted, subsequent training in the non - vr model increased pass ratings of the batch more than vice versa, but the findings were not statistically significant. the advantage of using non - vr - based models is its higher realism of the real - time flexible endoscope, which makes training on these models attractive to the novices, but the disadvantages are use of fragile flexible endoscopes, maintenance of the endoscope and recurring costs. | objective : we performed a comparative study of high - fidelity training models for flexible ureteroscopy (urs). our objective was to determine whether high - fidelity non - virtual reality (vr) models are as effective as the vr model in teaching flexible urs skills.materials and methods : twenty - one trained urologists without clinical experience of flexible urs underwent dry lab simulation practice. after a warm - up period of 2 h, tasks were performed on a high - fidelity non - vr (uro - scopic trainer ; endo - urologie - modell) and a high - fidelity vr model (uro mentor). the participants were divided equally into three batches with rotation on each of the three stations for 30 min. performance of the trainees was evaluated by an expert ureteroscopist using pass rating and global rating score (grs). the participants rated a face validity questionnaire at the end of each session.results:the grs improved statistically at evaluation performed after second rotation (p<0.001 for batches 1, 2 and 3). pass ratings also improved significantly for all training models when the third and first rotations were compared (p<0.05). the batch that was trained on the vr - based model had more improvement on pass ratings on second rotation but could not achieve statistical significance. most of the realistic domains were higher for a vr model as compared with the non - vr model, except the realism of the flexible endoscope.conclusions:all the models used for training flexible urs were effective in increasing the grs and pass ratings irrespective of the vr status. |
in terms of enteral nutritional support, percutaneous endoscopic gastroscopy (peg) is widely accepted as a safe and effective approach. however, some general complications have been reported, such as skin infection of peri - gastrostomy site, ileus, leakage, tubal obstruction, stomach perforation, hemorrhage, peritonitis, pulmonary aspiration and buried bumper syndrome etc.1 - 6 gstrocolocutaneous fistula, defined as an epithelial linkage between the mucosa of the stomach, colon and skin, is rare but also is included as one of the complications. the typical symptoms of gastrocolocutaneous fistula are severe diarrhea, malnutritional status, gram negative pulmonary infection and feculent vomiting,7 and the fistula is diagnosed by complementary work - up of fistulography using barium or gastrografin injection through the peg tube, abdominal computed tomography (ct), gastroscopy and colonoscopy. this report will present the case of a patient who had a gastrocolocutaneous fistula after peg procedure and treated by endoscopic procedure. a peg tube insertion was performed to a 72-year - old female patient who was suffering from medullary infarction for the purpose of enteral nutrition (fig. three days after the peg, however, yellowish fecal materials were drained through the peg tube during gastric remnant volume examination before feeding. six days after the procedure, we performed the endoscopy, and the fecal materials were attached to gastric wall and the end of the peg tube was away from the location where it had been originally positioned, and was partially buried (fig.. the tube was immediately removed, and peripheral total parenteral nutrition without oral intake was started. at the follow - up the day after removing the peg tube, abdominal ct was conducted, and colonoscopy was also conducted 3 days after the abdomen ct since there was no spontaneous closure of gastrostomy site and fecal drainage was sustained. on the abdomen ct, the transverse colon was located in antero - superior site of the stomach (fig. 2) and the fistula of the middle of transverse colon was found during colonoscopy. however, the gastrocolocutaneous fistula was not shown during fistulography which was conducted after the injection of contrast media through nasogastric tube. the day after the colonoscopy, some of the end of hemo - clip located at the transverse colon was shown from gastric cavity side. this fistula at the gastric side was also sutured with hemo - clips, and then a detachable snare (maj-254 ; olympus, tokyo, japan) was applied for concrete suture (fig. the closure of the fistula was confirmed at the gastroscopy conducted 6 days after the endoscopic procedure (fig. peg was suggested in the early eighties as a new approach to substitute operative methods in the equipment of a gastrostomy.8 the majority of peg procedures are applied for neurological disorders such as stroke or traumatic irreversible brain damage, but it can be also applied in cases of anatomical problems such as a correction of cleft lip and palate, and in cases of head and neck injury caused by radiation therapy. peg requires minimal sedation, not general anesthesia, and can be performed within 15 to 30 minutes with 95% success rates,9 but approximately 17% out of total cases shows relevant complications according to a previous research.10 specifically, gastrocolocutaneous fistula caused by peg procedure was also reported in 2% to 3% of the total incidences.11 in order to prevent the gastrocolocutaneous fistula, croaker.12 suggested that excessive inflation of air into the stomach should be refrained during peg procedure since too much air in the stomach makes the greater curvature of the stomach rotate forward and makes the gastrocolic omentum and the transverse colon, originally located inferior to the stomach, move anterior to the stomach. in this condition, peg tube can be placed into the stomach, penetrating the transverse colon. according to patwardhan.,13 among 12 cases of gastrocolocutaneous fistula after peg insertion, all of the cases showed peg entrance site located in the posterior wall of the stomach. in our cases, however, peg entrance site was the at the greater curvature of the stomach. when the gastrocolocutaneous fistula occurs after peg procedure, general symptoms such as diarrhea however, symptoms like fecal drainage through the peg tube can be appeared after several days of asymptomatic period as described in our case study. there are insufficient data on the duration for the peg tube removal site to be collapsed and closed. also, clinically, it is not quite certain how long is needed for the closure of the removal site when removing the tube due to inappropriate peg function. it is assumed that the spontaneous closure of fistula occurs probably within a couple of days, but the spontaneous closure can be disturbed by delayed gastric emptying, slow recovery of the wounded site, and the leakage of gastric juice through fistula.14 also, there is not enough data in the literature to propose standard therapeutic approaches to peg malposition associated with gastrocolocutaneous fistula. the decisions on therapeutic timing and methods are based on the experience and discretion of the endoscopist. in our case, the fistula, which was not spontaneously closed 5 days after the peg tube removal, was first sutured with hemo - clips at the transverse colon side, then sutured the next day with hemo - clips at the gastric side and ligated with a detachable snare. with an increase of the elderly, the incidence rate of neurologic diseases such as stroke, brain hemorrhage, and cases of central nerve injury by trauma due to traffic accident is also increasing. accordingly, the need for enteral feeding by peg is on the rise and the cases of complications associated with peg are expected to increase. the progression to a fatal situation can be minimized by early detection of these complications. if fecal materials are drained in the evaluation of remnant stomach volume after a few days in patients undergoing peg, the possibility of gastrocolocutaneous fistula, though rare, should be kept in mind. it is meaningful that endoscopic treatment can be feasible if the complications were detected early by examinations such as the upper and lower gastrointestinal endoscopy, abdominal ct or fistulography. | as a rare complication of percutaneous endoscopic gastroscopy (peg), a gastrocolocutaneous fistula may occur after peg placement. this paper reports an interesting case which peg tube unintentionally penetrated transverse colon during peg. a 72-year - old female patient who suffered from medullary infarction underwent peg procedure for enteral nutrition, and fecal materials were observed 6 days after the procedure. transverse colon located in antero - superior site of stomach was observed through abdominal computed tomography, and also the wrong inserted tube was found through gastroscopy and colonoscopy. endoscopic treatment for the fistula was performed by the use of hemo - clip and detachable snare, closure of the fistula was finally confirmed 6 days after the endoscopic procedure. therefore, the gastrocolocutaneous fistula should be considered as one of the complications of peg when fecal material is observed through peg tube in a few days after peg procedure and endoscopic treatment can be feasible in this case. |
the closing article in the series celebrating the 30th anniversary of diabetes control and complications trial / epidemiology of diabetes interventions and complications (dcct / edic) study reflects on how past investigations have shaped the study s future directions. the preceding articles on retinopathy, nephropathy, neuropathy, and cardiovascular disease have provided detailed updates on the state of the complications of diabetes in the cohort over the past 30 years. collectively, the message is clear : chronic glycemia is the major modifiable factor driving the development and progression of the complications of type 1 diabetes (16). with intensive management of their diabetes, patients can achieve lower glycemia and reduce the risk of developing complications, including severe disease (7). in turn, patients free of complications report a good and sustained diabetes - related quality of life (8). the substantial effects of intensive therapy on primary prevention during dcct, compounded by metabolic memory established during edic, make it imperative to intervene with intensive therapy as early after diagnosis as possible to effectively slow the course of complications. as we move forward, the durability of our intervention will continue to be monitored : will the effects of intensive therapy metabolic memory decrease over time ? edic will continue to monitor the trajectory of the development and progression of microvascular and cardiovascular disease. dcct / edic core studies have been the foundation for many collaborations, each initiative advancing our understanding of the multisystem effects of diabetes and guiding future clinical and basic science investigations (fig., several new ancillary studies are planned to investigate clinical topics across diverse fields, such as residual -cell function, hearing dysfunction, and gastric disturbances. the dcct / edic core investigations have been the center of a diverse array of supplemental studies and collaborations over the past 30 years. the black circle denotes the major outcomes (i.e., retinopathy, nephropathy, neuropathy, and cardiovascular disease) studied during the dcct (19831993) and throughout edic (1994present). extending from these core investigations, a multitude of supplemental studies have been performed, starting from 19902005, the late dcct / early edic period ; continuing with 20062013, the first edic extension period ; and expanding to 2013forward, the current extension of edic. family genetics study, first - degree relative genetic studies (12) ; cvd - cac, coronary calcium ct scan (13) ; cvd - nmr lipid, lipid studies (14) ; uro - edic 1, study of urological dysfunction (15) ; cvd - imt, carotid ultrasounds (1618) ; ages, advanced glycation end products (19) ; cognitive function, neurocognition testing (2022) ; can, cardiac autonomic neuropathy testing (23) ; cardiac mri, enhanced cardiac mri (11) ; dermal ages, dermal advanced glycation end product study (24,25) ; retention, participant retention study (26) ; glycated albumin measurements (27,28) ; haptoglobin levels (29) ; cardiovascular biomarkers ; epigenetics ; mobility, joint mobility ; and uro - edic 2, repeat study of urological dysfunction. as reviewed previously, the dcct interventional study was conducted between 19821993, and since 1994 edic has continued as an observational follow - up of the consenting dcct participants, comprising 95% of the surviving members of the original dcct cohort (9,10). retention during edic has been consistent (9396%) whether considering original recruitment into primary prevention or secondary intervention groups or random assignment to intensive or conventional treatment. ancillary studies are offered to all subjects, and compliance has routinely been > 90% for studies involving minor procedures and/or questionnaires. even with procedure - intensive ancillary studies, such as the cardiac magnetic resonance imaging (mri), participation rates were 81% or better (11). for each dcct / edic participant, the current core protocol is performed on an annual basis and includes the major data collection form that reviews the participants health history by system and updates intercurrent events ; denotes critical diabetes - related health information including all treatments and hypoglycemia severity, awareness, and frequency during the prior 3 months ; and records the results of the standardized annual physical examination focusing on diabetes complications fasting lipid profiles every other year, urine albumin - to - creatinine ratio every other year, and ophthalmologist exam and fundus photographs once every 4 years. core data collection of hba1c, fasting lipids, and renal collections has exceeded 89%, 90%, and 91%, respectively, on an annual basis. in addition to the core data collection, the breadth of dcct / edic investigations has been increased through supplemental studies proposed by the dcct / edic study group and collaborations with experts in various fields of diabetes research (fig. data from the supplemental studies and collaborations have been incorporated into the longitudinal dataset and will be used in the planned analyses. this includes data from the following studies : first - degree relative genetic studies (12), coronary calcium computed tomography scan (13), lipid studies (14), urological dysfunction (uro - edic 1) (15), carotid ultrasounds (1618), advanced glycation end products (ages) (19), neurocognition testing (2022), peripheral nerve conduction testing (5,2022), cardiac autonomic neuropathy testing (23), enhanced cardiac mri (11), dermal age study (24,25), participant retention study (26), glycated albumin measurements (27,28), and haptoglobin levels (29). more recent supplemental studies with ongoing analyses include cardiovascular biomarkers, epigenetics, joint mobility, and repeat of urological dysfunction (uro - edic 2). as reviewed previously, the dcct interventional study was conducted between 19821993, and since 1994 edic has continued as an observational follow - up of the consenting dcct participants, comprising 95% of the surviving members of the original dcct cohort (9,10). retention during edic has been consistent (9396%) whether considering original recruitment into primary prevention or secondary intervention groups or random assignment to intensive or conventional treatment. ancillary studies are offered to all subjects, and compliance has routinely been > 90% for studies involving minor procedures and/or questionnaires. even with procedure - intensive ancillary studies, such as the cardiac magnetic resonance imaging (mri), participation rates were 81% or better (11). for each dcct / edic participant, the current core protocol is performed on an annual basis and includes the major data collection form that reviews the participants health history by system and updates intercurrent events ; denotes critical diabetes - related health information including all treatments and hypoglycemia severity, awareness, and frequency during the prior 3 months ; and records the results of the standardized annual physical examination focusing on diabetes complications fasting lipid profiles every other year, urine albumin - to - creatinine ratio every other year, and ophthalmologist exam and fundus photographs once every 4 years. core data collection of hba1c, fasting lipids, and renal collections has exceeded 89%, 90%, and 91%, respectively, on an annual basis. in addition to the core data collection, the breadth of dcct / edic investigations has been increased through supplemental studies proposed by the dcct / edic study group and collaborations with experts in various fields of diabetes research (fig. data from the supplemental studies and collaborations have been incorporated into the longitudinal dataset and will be used in the planned analyses. this includes data from the following studies : first - degree relative genetic studies (12), coronary calcium computed tomography scan (13), lipid studies (14), urological dysfunction (uro - edic 1) (15), carotid ultrasounds (1618), advanced glycation end products (ages) (19), neurocognition testing (2022), peripheral nerve conduction testing (5,2022), cardiac autonomic neuropathy testing (23), enhanced cardiac mri (11), dermal age study (24,25), participant retention study (26), glycated albumin measurements (27,28), and haptoglobin levels (29). more recent supplemental studies with ongoing analyses include cardiovascular biomarkers, epigenetics, joint mobility, and repeat of urological dysfunction (uro - edic 2). as presented in the earlier accompanying manuscripts, the data through edic year 18 suggest that the beneficial risk reductions afforded by intensive relative to conventional therapy, while persistent and significant, are decreasing over time (3033). declining risk reductions are evident across microvascular complications : risk reduction for further progression of retinopathy falling from 70% to 53% to 46% at edic year 4, year 10, and year 18, respectively ; risk reduction for confirmed clinical neuropathy decreased from 64% at dcct closeout to 30% at edic year 14 ; and risk reduction for albuminuria with intensive therapy dropping from a high of nearly 60% at edic year 8 to nearly 40% at edic year 18, which is identical to the risk reduction seen at dcct closeout. the fall in relative benefit of intensive therapy is almost certainly due to the adoption of intensive therapy by the original conventional therapy group and the waning of metabolic memory over time. the relative contribution of dcct hba1c, which is also declining in the case of retinopathy, to these risk reductions over time has important clinical as well as biological implications. the underlying mechanistic link(s) between hyperglycemia and the development of complications in various organ systems remains a critical unanswered question. the potential mechanisms for the lasting effects of metabolic memory may involve genetic factors, epigenetic changes, and/or glycation of proteins, all of which have been and continue to be explored in dcct / edic. the dcct / edic family genetic study has identified several loci that regulate risk of developing retinopathy (34), nephropathy (35,36), and erectile dysfunction (37). initial epigenetic investigations were presented during the 2013 american diabetes association scientific sessions and suggest intriguing links between glycemia - associated histone acetylation and activation of recognized pathogenic inflammatory cascades. in previous dcct / edic publications, ages have been shown to correlate with the presence of microvascular complications at dcct closeout and to predict the development and progression of complications in edic, all independent of the hba1c (19,38). the potential genetic factors predisposing to accelerated age formation are currently under investigation. the 30-year longitudinal data collected and the remarkable retention of our research participants provide an invaluable diabetes resource, and the dcct / edic and its collaborators hope to address several novel questions in diabetes care in the next few years. defining the relative time course of the development of retinopathy, nephropathy, and neuropathy (triopathy) to answer such clinical questions as : can the rate of progression of retinopathy predict individuals that will progress to more advanced renal disease ? and what is the frequency of triopathy and what are the dominant risk factors ? establishing the evidence - based frequency of screening of retinopathy and nephropathy refining our prior 7-point self - monitoring of blood glucose data to investigate the role of glycemic variability on outcomes as well as perform a current cross - sectional examination of glycemic variability using continuous glucose monitoring exploring the contributions of nonglycemic risk factors to risk reduction for macrovascular as well as microvascular outcomes monitoring for effects of glycemic control on neurocognition : will the rate of age - related cognitive decline in our cohort be different when compared with the general population ? continuing to monitor the bottom line that intensive diabetes management prevents costly complications and is economically sage building on these core initiatives, additional ancillary studies are planned. first, prior dcct data demonstrated that intensive therapy preserved insulin secretion, measured as c - peptide (39). those with preserved c - peptide secretion had lower hba1c with less frequent hypoglycemia and reduced development of retinopathy and nephropathy. now, in edic year 20 using modern, ultrasensitive assays, is there residual -cell function after an average diabetes duration of 30 years ? what factors influence residual -cell function ? what is the physiologic significance of low levels of c - peptide ? will there remain a beneficial reduction in rates of complications associated with preserved insulin secretion ? based on pilot data obtained with 4-h mixed - meal tolerance tests on 58 dcct / edic subjects, three different ultrasensitive c - peptide assays will be used to provide comparative analyses of detection limits. the c - peptide results, in combination with the extensive, historical database, will extend our understanding of the relationship of long - term c - peptide production with hba1c levels over time, insulin dose requirements, hypoglycemia rates, and complications. of importance, the study will also seek to identify the factors that mediate -cell preservation and provide hope for future treatment options for type 1 diabetes. a second initiative stems from the observation that hearing impairment is more common in type 2 diabetes than in the nondiabetic population (40,41). early detection of hearing loss allows for earlier intervention, which preserves quality of life. the current proposal includes standardized hearing tests at all edic centers with oversight by a centralized audiology unit. the dcct / edic participants nondiabetic partners / spouses will be used as control subjects. the prevalence of hearing impairment in type 1 diabetes, its potential correlation with other microvascular complications, and the relationship with glycemia and other risk factors will be investigated. third, abnormal gastric emptying is a manifestation of autonomic neuropathy that challenges many patients daily routine and their glycemic control. this initiative aims to advance our understanding of the prevalence of gastroparesis, symptomatic and asymptomatic as well as delayed and rapid types. in addition to symptom - related questionnaires, participants will complete the validated stable isotope c - spirulina platensis gastric emptying breath test (42). like the c - peptide study, a pilot investigation with 80 participants across seven edic centers is currently in progress. as presented in the earlier accompanying manuscripts, the data through edic year 18 suggest that the beneficial risk reductions afforded by intensive relative to conventional therapy, while persistent and significant, are decreasing over time (3033). declining risk reductions are evident across microvascular complications : risk reduction for further progression of retinopathy falling from 70% to 53% to 46% at edic year 4, year 10, and year 18, respectively ; risk reduction for confirmed clinical neuropathy decreased from 64% at dcct closeout to 30% at edic year 14 ; and risk reduction for albuminuria with intensive therapy dropping from a high of nearly 60% at edic year 8 to nearly 40% at edic year 18, which is identical to the risk reduction seen at dcct closeout. the fall in relative benefit of intensive therapy is almost certainly due to the adoption of intensive therapy by the original conventional therapy group and the waning of metabolic memory over time. the relative contribution of dcct hba1c, which is also declining in the case of retinopathy, to these risk reductions over time has important clinical as well as biological implications. the underlying mechanistic link(s) between hyperglycemia and the development of complications in various organ systems remains a critical unanswered question. the potential mechanisms for the lasting effects of metabolic memory may involve genetic factors, epigenetic changes, and/or glycation of proteins, all of which have been and continue to be explored in dcct / edic. the dcct / edic family genetic study has identified several loci that regulate risk of developing retinopathy (34), nephropathy (35,36), and erectile dysfunction (37). initial epigenetic investigations were presented during the 2013 american diabetes association scientific sessions and suggest intriguing links between glycemia - associated histone acetylation and activation of recognized pathogenic inflammatory cascades. in previous dcct / edic publications, ages have been shown to correlate with the presence of microvascular complications at dcct closeout and to predict the development and progression of complications in edic, all independent of the hba1c (19,38). the 30-year longitudinal data collected and the remarkable retention of our research participants provide an invaluable diabetes resource, and the dcct / edic and its collaborators hope to address several novel questions in diabetes care in the next few years. defining the relative time course of the development of retinopathy, nephropathy, and neuropathy (triopathy) to answer such clinical questions as : can the rate of progression of retinopathy predict individuals that will progress to more advanced renal disease ? and what is the frequency of triopathy and what are the dominant risk factors ? establishing the evidence - based frequency of screening of retinopathy and nephropathy refining our prior 7-point self - monitoring of blood glucose data to investigate the role of glycemic variability on outcomes as well as perform a current cross - sectional examination of glycemic variability using continuous glucose monitoring exploring the contributions of nonglycemic risk factors to risk reduction for macrovascular as well as microvascular outcomes monitoring for effects of glycemic control on neurocognition : will the rate of age - related cognitive decline in our cohort be different when compared with the general population ? continuing to monitor the bottom line that intensive diabetes management prevents costly complications and is economically sage first, prior dcct data demonstrated that intensive therapy preserved insulin secretion, measured as c - peptide (39). those with preserved c - peptide secretion had lower hba1c with less frequent hypoglycemia and reduced development of retinopathy and nephropathy. using modern, ultrasensitive assays, is there residual -cell function after an average diabetes duration of 30 years ? what factors influence residual -cell function ? what is the physiologic significance of low levels of c - peptide ? will there remain a beneficial reduction in rates of complications associated with preserved insulin secretion ? based on pilot data obtained with 4-h mixed - meal tolerance tests on 58 dcct / edic subjects, three different ultrasensitive c - peptide assays will be used to provide comparative analyses of detection limits. the c - peptide results, in combination with the extensive, historical database, will extend our understanding of the relationship of long - term c - peptide production with hba1c levels over time, insulin dose requirements, hypoglycemia rates, and complications. of importance, the study will also seek to identify the factors that mediate -cell preservation and provide hope for future treatment options for type 1 diabetes. a second initiative stems from the observation that hearing impairment is more common in type 2 diabetes than in the nondiabetic population (40,41). early detection of hearing loss allows for earlier intervention, which preserves quality of life. the current proposal includes standardized hearing tests at all edic centers with oversight by a centralized audiology unit. the dcct / edic participants nondiabetic partners / spouses will be used as control subjects. the prevalence of hearing impairment in type 1 diabetes, its potential correlation with other microvascular complications, and the relationship with glycemia and other risk factors will be investigated. third, abnormal gastric emptying is a manifestation of autonomic neuropathy that challenges many patients daily routine and their glycemic control. this initiative aims to advance our understanding of the prevalence of gastroparesis, symptomatic and asymptomatic as well as delayed and rapid types. in addition to symptom - related questionnaires, participants will complete the validated stable isotope c - spirulina platensis gastric emptying breath test (42). like the c - peptide study, a pilot investigation with 80 participants across seven edic centers is currently in progress. the dcct / edic set the standard of care for clinical management of type 1 diabetes, which has forever changed the course of its complications. intensive diabetes management remains the primary approach to prevent or slow the progression of complications. now entering the fourth decade of study, the dcct / edic core objectives continue to emphasize the rigorous assessment of microvascular and cardiovascular disease and traditional and novel risk factors. the comprehensive, longitudinal data collection and continued successful collaboration between the dcct / edic investigators and research partners should provide answers to questions that will guide the clinical care of type 1 diabetes. the improvement in the long - term prospects for people with type 1 diabetes brought about through dcct / edic should continue with these efforts. | objectivethe diabetes control and complications trial / epidemiology of diabetes interventions and complications (dcct / edic) study continues to address knowledge gaps in our understanding of type 1 diabetes and the effects of intensive therapy on its long - term complications.research design and methodsduring the dcct (19821993), a controlled clinical trial of 1,441 subjects with type 1 diabetes, and the edic (1994present), an observational study of the dcct cohort, core data collection has included medical history questionnaires, surveillance health exams, and frequent laboratory and other evaluations for microvascular and macrovascular disease. numerous collaborations have expanded the outcome data with more detailed investigations of cardiovascular disease, cognitive function, neuropathy, genetics, and potential biological pathways involved in the development of complications.resultsthe longitudinal follow - up of the dcct / edic cohort provides the opportunity to continue monitoring the durability of intensive treatment as well as to address lingering questions in type 1 diabetes research. future planned analyses will address the onset and progression of microvascular triopathy, evidence - based screening for retinopathy and nephropathy, effects of glycemic variability and nonglycemic risk factors on outcomes, long - term impact of intensive therapy on cognitive decline, and health economics. three new proposed investigations include an examination of residual c - peptide secretion and its impact, prevalence of hearing impairment, and evaluation of gastrointestinal dysfunction.conclusionswith the comprehensive data collection and the remarkable participant retention over 30 years, the dcct / edic continues as an irreplaceable resource for understanding type 1 diabetes and its long - term complications. |
the role of protein dynamics in the reaction catalyzed by dihydrofolate reductase from the hyperthermophile thermotoga maritima (tmdhfr) has been examined by enzyme isotope substitution (15n, 13c, 2h). in contrast to all other enzyme reactions investigated previously, including dhfr from escherichia coli (ecdhfr), for which isotopic substitution led to decreased reactivity, the rate constant for the hydride transfer step is not affected by isotopic substitution of tmdhfr. tmdhfr therefore appears to lack the coupling of protein motions to the reaction coordinate that have been identified for ecdhfr catalysis. clearly, dynamical coupling is not a universal phenomenon that affects the efficiency of enzyme catalysis. |
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the species richness of flowering plant lineages shows tremendous variation amongst clades, indicating that certain traits influence speciation and/or extinction rates. in plants, many traits have been associated with evolutionarily success, such as self - incompatibility and floral asymmetry. the reasons why certain traits are associated with increased diversification are often intuitive : some traits inherently encourage speciation via increased genetic diversity (self - incompatibility) or an association with increased opportunities for the evolution of specialization (floral asymmetry). while geographical distribution might be also influence diversification rates [3, 4 ] because speciation may accompany the establishment in new ecozones, the effects of geographical area are far from fully understood. on the one hand, increased geographical extent provides more opportunities for allopatric speciation (i.e., the geographical potential for speciation). if increased geographical extent is simply caused by high dispersal rates, however, gene flow is maintained and hinders speciation. the optimal conditions of dispersal for speciation appear to be met in island systems, where many classic examples of adaptive radiations are found. recent evidence indicates that island - like systems can also be found in mountainous areas, suggesting that mountain peaks also provide the right balance between dispersal and isolation to accelerate speciation, yet these patterns are not as well characterized. one reason why mountain peaks may differ from true island systems is that speciation may occur between lowland and high elevation bands as well as between isolated mountain peaks. in flowering plants, the gene flow between lowland and high elevation bands may decline because pollinators differ along elevation clines, potentially leading to floral isolation [11, 12 ]. pollinating fauna exhibit geographical heterogeneity and these differences in composition could play a central role in why angiosperms display such extreme diversity in floral colour and morphology. lineages of flowering plants can evolve floral colours and shapes that encourage visits from the most efficient pollinator, a process that leads to the observation of pollination syndromes. while the absolute nature of pollinator syndromes is debatable and there are numerous examples of exceptions, the bee functional group is attracted to flowers that are white, yellow, and purple, and birds are noted for their preference for red flowers. because beetles and flies also often visit white and yellow flowers and infringe on the bee portion of the spectrum, the red or reddish hues of the colour palette (including deep oranges, magenta) may be perceived as catering to a specialized clientele. the selection pressures that are involved in the evolution of specialization are not currently well characterized nor are the downstream consequences of evolutionary transitions between specialization and generalization. for instance, it has been suggested that hummingbirds are more common at high elevations and this may lead to a speciational pollinator shift accompanied by the transition towards reddish flowers at high elevations. while floral colour is a noted cue to the most common pollinator of a given species, there are other reasons why the evolution of floral colour is observed to be a labile trait. research on the genetic architecture of floral colour indicates that drastic colour shifts can occur with a number of single loss - of - function mutations. macroevolutionary analyses that examine trait conservatism versus node - based shifts in floral colour can shed light on (1) whether any particular floral colour is associated with increased diversification or (2) whether shifts in floral colour are involved in the speciation process itself. here, we examine these patterns within the genus mimulus, which comprises approximately 120 species, mostly restricted to north america. it has been previously shown that there have been multiple shifts from bee pollination to hummingbird pollination within the genus. with ample sequences and robust species - level phylogenies attainable for the genus, the time is ripe for macroevolutionary analysis of diversification patterns in mimulus within a biogeographical context. with a well - resolved phylogenetic tree, mimulus presents an ideal system in which to then test whether (1) transition rates to red / orange / fuschia (reddish) flowers are significantly different from the reverse pathway, (2) reddish flowers are associated with higher elevations, and (3) reddish flowers are associated with differences in speciation and/or extinction rates in mimulus. we searched the literature and e - floras (e.g., california native plant society (cnps,), calflora, encyclopedia of life (eol), and jepson herbarium) for qualitative assessments of floral colour of all species of mimulus. where these were not available, we scanned online pictures and made our own qualitative assessments of colour. in most cases, it was a very straightforward exercise to discern whether the floral colour was in what we considered reddish (ro : red, orange, or fuchsia) or not (no - ro), in other words, possessing both carotenoids and anthocyanins. quantitative assessments of elevation were obtained from e - floras as well, with the admittedly arbitrary cut - off of being restricted to high elevation similarly, e - floras were also used for assessments of distribution (e.g., calflora, encyclopedia of life (eol), florabase, united states department of agriculture (usda) - natural resources conservation service). we compiled the available dna sequence markers for mimulus species on the national center for biotechnology information (ncbi) genbank website (http://www.ncbi.nlm.nih.gov/genbank/). our review showed that there are high numbers of species sequenced for 3 markers : (1) trnl gene and trnl - trnf intergenic spacer (trnlf) ; (2) the internal transcribed spacer region its1, the 5.8s coding region and its2 (its) ; and (3) the external transcribed spacer (ets). the trnl - f marker is located in the chloroplast, and ets and its markers are located in the nucleus. these three markers were used for our phylogenetic analyses, which included 94 mimulus species (table 2). mohavea breviflora was used as an outgroup for all analyses, as in previous molecular work. dna sequences for ets, its, and trnl - f markers were aligned using mesquite version 2.75 with opal package using default settings. analyses were made with combined nuclear markers (ets and its) and for all markers (ets, its, and trnl - f). for partitioned analysis of the combined data, nexus files were manually edited for partition setting, in which each dna marker was set as an individual partition. the three dna markers were analyzed both individually and in combination for phylogenetic reconstruction analyses. rapid bootstrap analysis and search for a maximum likelihood (ml) tree were performed with randomized axcelerated maximum likelihood (raxml) software version 7.4.2 under the general time reversible (gtr) substitution model with gamma rate heterogeneity for all individual and combined data sets. mohavea breviflora was used as an outgroup, and the ingroup was assigned to be monophyletic prior to analysis. hasegawa, kishino, and yano (hky) model with gamma site heterogeneity was used as the substitution model due to its general reduction of assumptions. we used the node splitting mimulus from the outgroup mohavea as our calibration point for our phylogenetic tree. in order to take calibration uncertainty into account, we used normal distribution as prior to our calibration node with a mean of 76 ma and standard deviation of 1 the rest of the node ages were estimated using uncorrelated lognormal relaxed clock model with the mean distribution prior set to gamma. we selected the yule speciation model [35, 36 ] with uniform distribution as our tree prior. for the markov chain monte carlo (mcmc) analyses, the chain length was set to 10,000,000 and we logged parameters every 1,000 generations (i.e., at the end of the run, we had 10,000 samples). for runs with combined data sets, trees were generated using treeannotator version 1.7.4 (part of the beast package) with the first 50 steps discarded at the start of the run. we used the binary - state speciation and extinction (bisse) method implemented in the diversitree package of the r statistical software to examine differences in transition rates between character states as well as differences in speciation rates. we used this method to estimate the following parameters : speciation and extinction rates 0 and 0 of nonred lineages and 1 and 1 of red lineages and two transition rates representing the evolution of red flowers (q01) and the backwards transitions of evolving nonred flowers (q10), and then compute the likelihood of character states at the tips of our phylogenetic tree, given the maximum likelihood values of speciation, extinction and transition rates (the unconstrained model). we then constrain certain parameters to be equal to each other and test alternative models of evolution against the unconstrained model with likelihood ratio tests. we examined the fit of a model with (1) equal speciation rates (1 = 0), one with equal extinction rates (1 = 0), one with equal speciation and extinction rates (1 = 0 and 1 = 0), and, finally, one with equal transition / migration rates (q10 = q01). we further formalize ancestral reconstructions in mesquite version 2.75 to characterize the evolutionary history of mimulus. for the second analysis, we performed ml reconstruction with markov k - state 1 model for standard categorical data. mcc tree obtained from the bayesian analysis with combined dna sequence data is used for both ancestral reconstructions. red (or reddish) floral colour is denoted as 1, and all the other floral colours are denoted as 0 in the character matrix. finally, we established whether the origins of floral colour coincided with entry into specific biogeographical origins using reconstruct ancestral state in phylogenies (rasp) version 2.1 beta software. three alternative reconstruction methods were used : (1) statistical dispersal - vicariance analysis, (2) bayesian binary method, and (3) dispersal - extinction - cladogenesis model. four discrete states were used for present - day continental geographical distribution : north america, south america, asia, and oceania (table 2). analyses were conducted on the maximum clade credibility tree generated on beast using combined sequence data, and the results were summarized as tree graphs and pie charts. the geosse functions within diversitree can be used to test differential speciation and extinction within different geographical regions as well as the speciation that might accompany range shifts. we implemented geosse within diversitree to examine whether clades at high - elevation bands experienced greater speciation. the framework is very similar, but the parameters are speciation within low elevation regions (with range restricted to 1000 m, sb), between - region speciation (sab), extinction from low elevation regions (xa), extinction from high elevation regions (xb), dispersal from a to b (range expansion, da), and dispersal from b to a (db). we coded the species in three categories : a = restricted to low elevations, b = restricted to high elevations, and ab = ranges that span low and high elevation. by setting certain constraints we can test for whether speciation, extinction, or range expansion is different between regions. we also examine the variation in the estimates of these rates with markov models. a final analysis, musse, was run to test whether red flowers are correlated with high elevation zones. in the musse analysis, trait combinations use the following notation : 1 : low elevation, no - ro ; 2 : low elevation, ro ; 3 : high elevation, no - ro ; 4 : high elevation, ro. we test whether the evolution of the ro phenotype depends on the background of elevation (does 1 2 differ from 3 4 or 1 3 from 2 4, etc.) by constraining transition rates (q12 = q34, q13 = q24, q21 = q43, and q31 = q42) and comparing the likelihood of the tip character states on our phylogeny with log - likelihood tests. we provide the compiled traits of mimulus species in table 2. we were able to obtain high sampling of sequence data for mimulus, covering 94 of the ~120 species (~79% of the species as well, we had unbiased covering of the species in mimulus ; 10.6% of the species in our phylogeny are ro, identical to the approximately 10.6% in the genus as a whole that we estimate to be ro (see table 2). we had two nuclear markers : ets and its with sequence data for 90 and 91 taxa for these markers, respectively. after the alignment of all the taxa, the total length of the ets and its regions analyzed was 477 and 688 bp, respectively. for the chloroplast marker trnl - f, we had sequence data for 85 taxa. once all the taxa were aligned, the analyzed trnl - f region was 1122 bp long. when we align and combine all three markers (two nuclear and one chloroplast) together, the resulting matrix contained 2287 characters, comprising 94 taxa excluding the outgroup. ml and bayesian analyses of the individual dna markers resulted in largely congruent topologies for the genus mimulus (data not shown). when we compare individual marker data with the combined sequence data, combined molecular analysis provided higher resolution and more consistent tree topologies. we used partitioned and nonpartitioned combined data for ml analyses, and they both provided the same tree topology with slightly different likelihood values (data shown only for nonpartitioned analysis). overall, for the combined dna sequence data, mcc tree from the bayesian analysis is largely congruent with the ml analysis (figure 1), with the bayesian tree having higher resolution (see supplementary figure 1 in the supplementary material available online at http://dx.doi.org/10.1155/2014/382453, supplementary figure 2). overall, the phylogeny inferred for mimulus is very similar to the previously published phylogenies using alternative sampling and methods [1720, 44 ]. all three biogeographical origin reconstruction methods show strong support (93.83%) for the north american origin for the genus mimulus (see supplementary figure 3 for bayesian binary mcmc analysis). both ml and bayesian analyses highly supported the clade with oceanic distribution consisting m. prostratus, m. repens, m. uvedaliae, and m. gracilis, with bootstrap value (bs) of 100% and posterior probability (pp) of 1.0. there is high support for the oceania origin for m. prostratus, m. repens, and m. uvedaliae (pp : 98.53%), but pp values are not decisive with respect to the origin of the whole clade (45.58% : oceania origin, 30.35% : north america origin). highly supported clade consisting of m. tenellus, m. nepalensis, m. szechuanensis, and m. bodinieri is found in both ml and bayesian analyses (bs : 92%, pp : 1.0). bayesian binary mcmc analysis of biogeographic origin strongly suggests an asian origin for this clade (pp : 93.58%). another species with asian distribution, m. sessiflorus, is not clustered with the rest of the asian taxa in either ml or bayesian analysis, and the position of this species is poorly resolved in both trees. m. depressus, m. cupreus, and m. luteus form a strongly supported clade in both ml and bayesian analyses (bs : 97%, pp : 1.0). for this clade, a south american origin was suggested by bayesian mcmc analysis of biogeographic origin (pp : 82.67%). an alternative to a south american origin, the clade might also have originated in the americas (north and south america together), but the pp values are too weak to support this view (pp : 13.40%). both mp and ml analysis showed that the ancestor of the genus mimulus was unlikely to have reddish flowers. likelihood values for 1 (reddish) is 0.02 and for 0 (not reddish) is 0.98. out of 95 mimulus species, 14 of them there are 2 major clades with ro flowers : m. bifidus, m. flemingii, m. aurantiacus, m. longiflorus, m. puniceus (ro), and m. clevelandii (not ro) form one of the red clades, with a potential ro - flowered common ancestor (1 : 0.622, 0 : 0.378). the other clade with a potential ro - flowered ancestor includes the species m. rupestris, m. cardinalis, m. verbenaceus, m. eastwoodiae, and m. nelsonii (ro) and m. lewisii, and m. parishii (not ro) (1 : 0.628, 0 : 0.372). according to our ancestral reconstruction analysis, all the ro flowered mimulus taxa have appeared later than 20 ma, which is intriguingly consistent with the approximate time for the hummingbird origins in the americas. we find that specialization, measured narrowly as having the trait state of reddish flowers, is not associated with higher speciation rates (table 1 ; p = 0.196). (i.e., the ability to evolve red flowers) is different from the rate at which a lineage transitions away from reddish flowers (table 1 ; p = 0.02895) ; that is, losing red coloration happens far more frequently than gaining it. finally, we could not find any evidence suggesting that clades with reddish flowers experience differential extinction rates than those without reddish flowers (p = 0.7996)., we can reject the model of equal speciation and extinction at different elevational bands, concluding that high elevation regions experience increases in diversification (figure 2 ; p = 0.002). with this being an intriguing, yet somewhat surprising result, we reran the analyses with a small subset of bootstrap trees and found the result to be robust to phylogenetic uncertainty (p values ranging from 0.002 to < 0.0001, n = 10). including the between - region mode of speciation does not, however, we also find that high elevation lineages experience increased range expansion into lower elevations more so than the reverse (figure 2 ; p = 0.02). our musse analysis tested whether certain transitions were more common than others (e.g., we can test if ro flowers evolve more often in high elevation environments), yet we did not find any evidence that these transition rates were unequal (p = 0.21) in such a way that would result in a disproportionate number of ro - flowered species in high elevation locations (see figure 1). we hypothesized that red flowered lineages might be restricted to high elevation environments as others have posited and that this might contribute to lower speciation rates. surprisingly, we found no indication that red flowered species are more prevalent at high elevation, yet high elevation bands were actually regions of increased speciation. previous studies have also found that diversification rates increase in mountainous regions [10, 46 ] and our study in mimulus corroborates these findings. finally, the species that originate in high elevation environments show an increased propensity to experience range expansion into low elevation environments, supporting other studies that suggest that the increased diversification of alpine regions provides a species pump for global species richness [46, 47 ]. floral colour can be considered as a secondary sexual characteristic in flowering plants, somewhat analogous to exaggerated ornaments in animals. because floral colour provides an important cue for pollinators, differences in colour can translate to attracting an increased number of visits from efficient pollinators, which often results in increased pollen transfer and a reduction in the amount of inbreeding. reddish flowers are typically associated with pollination by birds, though this relationship is far from absolute. in mimulus, a primarily north american lineage, while hummingbird pollination is thought to be an extremely efficient means of dispersing pollen and evolving reddish flowers may be an adaptation for bird pollination, we did not find that the evolution or red / orange flowers was associated with increased speciation rates or decreased extinction rates, and the character state appears to be more ephemeral than a non - ro phenotype. also, ro lineages were not restricted to high elevation environments as has been supposed, yet we find that high elevation environments were hotspots for speciation, possibly due to the topographical heterogeneity presented by isolated mountain peaks. potential reasons for the increased transition rates away from the ro phenotype involve scenarios that incorporate the ease at which a mutant can arise and the probability that such a mutation can fix. one scenario that might be included is that the flowers themselves are costly (red - hued flowers are associated with hummingbird pollination and these flowers might also be large and nectar rich), and so the phenotype is only selected in environments where other pollinators (e.g., bees) are scarce. furthermore, there are many nonpollinator agents of selection as well as genetic arguments that may be responsible for this pattern and the ease of transitions away from an ro phenotype is not devoid of empirical evidence. yellow morphs have been observed to be easily established in more than one population of mimulus cardinalis. while transitions from blue to red have been observed to be more common than the reverse, there has been little work done on the transitions between red and yellow flowers (the most common alternate floral colour in mimulus). transitions from pigmented to nonpigmented flowers are thought to be more common than the reverse generally, and the agents of selection may or may not include pollinators. phylogenetic comparisons in ipomoea revealed that transition rates between the two character states were roughly equal but nonpigmented flowers had lower speciation rates. white flowers are potentially an adaptation for moth pollination, yet might also be selectively favoured if the resources for costly pigments can be coopted for other functions. because mimulus includes few nonpigmented (white) flowered species, direct comparisons with previous studies are difficult because yellow flowers still contain flavonol pigments. another potential explanation to why transitions to red flowers are rare in mimulus could be that the hummingbird family (a potential selective agent) originated about 20 mya, which is relatively recent when compared to the history of mimulus. in other words, the ecological opportunity for adapting to the hummingbird pollination syndrome was not a possible transition for much of the tree space we analyzed in mimulus. further studies on pollination syndromes that examine transition rates (and speciation rates) before and after the origins of hummingbirds with direct observations of pollinator visitation rates might be able to refine the role that the origin of a new mutualist has had on north american angiosperm genera. | the vast diversity of floral colours in many flowering plant families, paired with the observation of preferences among pollinators, suggests that floral colour may be involved in the process of speciation in flowering plants. while transitions in floral colour have been examined in numerous genera, we have very little information on the consequences of floral colour transitions to the evolutionary success of a clade. overlaid upon these patterns is the possibility that certain floral colours are more prevalent in certain environments, with the causes of differential diversification being more directly determined by geographical distribution. here we examine transition rates to anthocyanin + carotenoid rich (red / orange / fuschia) flowers and examine whether red / orange flowers are associated with differences in speciation and/or extinction rates in mimulus. because it has been suggested that reddish flowers are more prevalent at high elevation, we also examine the macroevolutionary evidence for this association and determine if there is evidence for differential diversification at high elevations. we find that, while red / orange clades have equivalent speciation rates, the trait state of reddish flowers reverts more rapidly to the nonreddish trait state. moreover, there is evidence for high speciation rates at high elevation and no evidence for transition rates in floral colour to differ depending on elevation. |
cofactors involved in respiratory tract carcinogenesis were studied in syrian golden hamsters or in rats using benzo(a)pyrene as the carcinogenic agent. these factors included severe tissue damage induced by electro - coagulation, glass fibers administered by intratracheal instillation, acetaldehyde as irritant vapor, food restriction, and nutrients such as vitamin a and saturated and unsaturated fats. in addition, the effects of a combined exposure to four different major gaseous cigarette smoke components -- methyl nitrate, isoprene, methyl chloride and acetaldehyde -- and to one solid cigarette smoke component -- norharman -- were examined in short- and long - term inhalation studies. an interesting finding was the carcinogenicity of acetaldehyde, of which the possible mechanism is briefly discussed. another conspicuous observation was the substantial increase in number and size of lipid droplets in alveolar fibroblasts of hamsters fed a high vitamin a diet.imagesfigure 1.figure 2.figure 3.figure 4.figure 5.figure 6.figure 7. |
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the maldi - tof ms spectrum of c. massiliense is available online (http://www.mediterranee-infection.com/article.php?laref=256&titre=urms-database). strain marseille - p3295 was deposited in the collection de souches de l'unit des rickettsies (csur, wdcm 875) under number p3295. | we report the main characteristics of a new genus, congobacterium, and a new species, congobacterium massiliense, strain marseille - p3295 (csur p3295), a new member in the order coribacteriacea, which was isolated from a stool sample of a healthy 50-year - old pygmy (baka) woman. |
jaundice presenting after cholecystectomy may be the initial manifestation of a serious surgical misadventure and requires rigorous diagnostic pursuit and therapeutic intervention. a 23-year - old female underwent laparoscopic cholecystectomy for symptomatic gallstones and three weeks postoperatively developed painless jaundice. radiographic and endoscopic studies revealed a subhepatic biloma causing extrinsic compression and obstruction of the common hepatic duct. percutaneous catheter drainage of the biloma combined with endoscopic sphincterotomy successfully relieved the extrahepatic biliary obstruction and resolved the intrahepatic ductal leak responsible for the biloma. although heretofore undescribed, post - cholecystectomy jaundice due to extrahepatic bile duct obstruction caused by biloma may occur and can be successfully treated by means of standard radiologic and endoscopic interventions. extrahepatic biliary obstruction following cholecystectomy is a worrisome occurrence that mandates immediate investigation to determine its cause. when choledocholithiasis is found not to be responsible, the etiology is usually a serious iatrogenic injury to the common bile duct, common hepatic duct, or both. less frequently, the obstruction is due to an obscure and self - limited process, such as inflammatory swelling of the duct. bilomas occurring after cholecystectomy are due to major or minor biliary leaks and virtually always imply an operative technical error. most patients with bilomas present with right upper abdominal pain and/or fever, and only rarely do they suffer gastrointestinal obstructive symptoms, such as nausea and emesis. although bilomas have frequently accompanied biliary ductal injuries, to our knowledge biloma has not been previously reported to cause biliary obstruction by extrinsic compression of the adjacent bile duct. a 23-year - old hispanic female underwent an apparently uneventful laparoscopic cholecystectomy for symptomatic cholelithiasis. no anatomic abnormalities of the common hepatic duct, common bile duct or cystic duct were noted at operation. she had no nausea or emesis, but her stools were light in color, and her urine was dark. liver function tests revealed marked elevations of the total and conjugated bilirubin and alkaline phosphatase suggestive of biliary obstruction. an abdominal computed tomography (ct) scan demonstrated a subhepatic fluid collection in the gallbladder fossa (figure 1). also noted was mild dilatation of the intrahepatic but not the extrahepatic biliary ductal system. a ct - guided needle aspiration of the subhepatic collection produced bilious fluid, and a percutaneous pigtail drainage catheter placement yielded 200 ml of bile. two days later, a diagnostic endoscopic retrograde cholangiopancreatography (ercp) study revealed a tapered narrowing of the common duct with posterior displacement consistent with residual extrinsic mass effect and associated inflammatory changes (figure 2, 3). multiple air bubbles were noted to be present within the bile duct that were felt by the endoscopist to have been introduced during the procedure. the metal clips placed at surgery were seen not to be in unusually close proximity to the common hepatic duct. an endoscopic sphincterotomy was performed, and no stones were retrieved from the common duct. nevertheless, the procedure was complicated by acute pancreatitis of moderate severity that subsided spontaneously over two weeks. the biliary drainage from the percutaneous catheter initially decreased and then remained steady at about 60 ml per day. a catheter fistulagram demonstrated a small right hepatic duct branch in the area of the gallbladder fossa (figure 4). after four weeks of drainage, there was complete resolution of the subhepatic biloma, and a repeat ercp demonstrated a normal common duct with absence of the narrowing seen on the initial study (figure 5). the bile drainage gradually decreased to nil over six weeks, and the percutaneous catheter was removed. ercp cholangiogram demonstrating a residual tapered stenosis of the common duct due to compression by the biloma and associated inflammation. lateral oblique view of ercp cholangiogram showing posterior displacement of the common duct consistent with an extrinsic mass effect exerted by the biloma. fistulagram through the percutaneous drainage catheter demonstrating a communication between the decompressed biloma cavity and a small branch of the right hepatic duct in the area of the gallbladder fossa. repeat ercp cholangiogram showing resolution of the stenosis of the common duct following closure of the biliary fistula and collapse of the biloma cavity. bilomas typically present as right upper quadrant abdominal pain and may be accompanied by nausea, emesis and fever. they most often are located in the subhepatic space but may occur intrahepatically or, rarely, in the retroperitoneal space. the frequency of biloma as a complication of cholecystectomy has been reported to range from 0.14% to 2.65%. the etiology is most often bile leakage from either an accessory bile duct, a duct of luschka in the gallbladder fossa of the liver, or an inadequately secured cystic duct stump. the diagnosis is supported by an abdominal ct, ultrasound (us) or magnetic resonance imaging (mri) scan showing a homogenous density fluid collection in the right upper abdomen and can be confirmed by a radionuclide (hida) scan demonstrating continuity of the collection with the biliary ductal system. ercp and transhepatic cholangiography are the most accurate techniques for delineating the exact anatomy of the biliary leak. initial management options include percutaneous catheter drainage, ercp with sphincterotomy and/or biliary stent placement, or a combination of these techniques. if initial percutaneous aspiration of the biloma yields a large volume or infected fluid, a drainage catheter should be placed. some authors recommend an algorithmic approach with percutaneous drainage followed by endoscopic stent placement only for persistent leakage. relaparoscopy has also been advocated based on its ability to cleanse the peritoneal cavity, replace cystic duct clips, electrocoagulate the gallbladder fossa and accurately place drainage catheters. surgical control of the biliary leak is reserved for cases of failed conservative management or major bile duct injury requiring biliary reconstruction. biliary obstruction manifesting after laparoscopic cholecystectomy most often is due to either a retained common duct stone or an unrecognized surgical misadventure, such as transection or clipping of the common hepatic or common bile duct. retained stones may cause postoperative cholangitis or pancreatitis but frequently manifest only as uncomplicated jaundice or elevated liver function tests. as iatrogenic injuries to the biliary tree have similar symptoms, signs and laboratory abnormalities, imaging of the ducts is essential. abdominal us, ct, mri and hida scans all have the ability to detect biliary obstruction, but differentiation between obstructing calculi and bile duct injury may be problematic. therefore, ercp and/or transhepatic cholangiography are usually required for accurate diagnosis of postoperative biliary obstruction. while common duct stones are typically successfully extracted by endoscopic or interventional radiologic means, biliary ductal injuries may or may not be amenable to nonsurgical techniques. early ductal strictures, partial transections and nonocclusive clips may sometimes be definitively treated by biliary dilatation and stenting, but complete transections or occlusions invariably require surgical reconstruction, usually by means of biliary - enteric anastomosis. on occasion, partial biliary obstruction appears to be temporary and self - limited, with spontaneous resolution over a period of days to weeks. this may be attributed to localized inflammation or even ductal spasm, although the etiology usually remains speculative. in the case presented, the specific cause for the partial biliary obstruction was the extrinsic pressure and inflammatory changes imparted by the adjacent biloma. this was manifested on ercp by the long, smoothly tapered stricture and displacement of the common duct. decompression of the biloma by percutaneous catheter drainage and treatment of the small intrahepatic ductal leak by sphincterotomy successfully relieved the extrinsic pressure and resulted in resolution of the biliary obstruction. although biloma has previously been reported to cause gastric outlet obstruction, this to our knowledge, is the first documented instance of extrahepatic biliary obstruction due to a biloma. ironically, the pressure exerted on the extrahepatic bile duct by the biloma served to increase the pressure within the more proximal biliary system, thus exacerbating the leakage of bile from the intrahepatic ductal branch and further contributing to the size of the biloma. the process thus became a self - perpetuating vicious cycle of ductal compression leading to higher biliary pressure leading to increased bile leakage and even greater ductal compression. decompression of the biloma was probably critical to relieving not only the biliary obstruction per se but also the pressure - head contributing to the perpetuation of the biloma itself. though obviously rare, extrahepatic biliary ductal obstruction caused by biloma may follow laparoscopic cholecystectomy and should be recognized as a possible etiology of postoperative jaundice. | background : jaundice presenting after cholecystectomy may be the initial manifestation of a serious surgical misadventure and requires rigorous diagnostic pursuit and therapeutic intervention. biloma is a well recognized postcholecystectomy complication that often accompanies biliary ductal injury.case report : a 23-year - old female underwent laparoscopic cholecystectomy for symptomatic gallstones and three weeks postoperatively developed painless jaundice. radiographic and endoscopic studies revealed a subhepatic biloma causing extrinsic compression and obstruction of the common hepatic duct.results:percutaneous catheter drainage of the biloma combined with endoscopic sphincterotomy successfully relieved the extrahepatic biliary obstruction and resolved the intrahepatic ductal leak responsible for the biloma.conclusion:although heretofore undescribed, post - cholecystectomy jaundice due to extrahepatic bile duct obstruction caused by biloma may occur and can be successfully treated by means of standard radiologic and endoscopic interventions. |
a 59-year - old womon presented with an abnormal mass on chest x - ray that was discovered during a routine check - up. we obtained a biopsy though endobronchial ultrasound and pathologically diagnosed a thymoma (type a). contrast - enhanced computed tomography showed a smooth, solitary homogeneously enhanced mass measuring 33.52 cm in the right paratracheal area, which had no calcification (fig. the resected specimen was a firm light - gray tumor with a smooth capsule that measured 4.532.5 cm. postoperative follow - up proceeded without any problems, and the patient was discharged after 5 days. microscopy revealed that the tumor was a thymoma (world health organization type a+b2, masaoka staging i) (fig. the majority of thymomas are found in the anterior mediastinum. only a few thymic masses arising out of the anterior mediastinum have been described in the literature, and these have been found in an ectopic thymus location such as the neck, pulmonary hilus, or posterior mediastinum. the thymus arises embryologically from the third pharyngeal pouch and branchail cleft on each side. the thymic masses from each side then move toward each other and migrate from the midline to the anterior mediastinum and their final position. the incidence of ectopic thymic tissue is 3% to 5% in the retroinnomiate vein (i.e., a paratracheal site). surgical resection has been used for both a firm diagnosis and treatment in previously reported cases of a middle mediastinum thymoma. sakurai. reported that 18f - fluorodeoxyglucose positron emission tomography (pet) scan and 11c - acetate pet are useful for diagnosing a thymoma from the middle mediastinum as ectopic thymic tissue. castleman disease is an atypical lymphoproliferative disorder, and masses are commonly located in the chest. most patients are asymptomatic and lesions are accidentally found on chest x - ray as rounded mediastinal masses, often mistaken for a thymoma. thymomas rarely arise in the middle mediastinum, and thymoma is not considered in the differential diagnosis of middle mediastinum masses. but thymomas appear to have malignant potential, and the overall rate of thymoma recurrence is approximately 20%. | thymoma is a common anterior mediastinal mass, although thymomas have occasionally been found in the neck, pulmonary hillus, or posterior mediastinum. but a thymoma within the middle mediastinum has rarely been reported. we report a thymoma arising in the middle mediastinum with a review of the literature. |
one of the major goals for scientists is to identify biomarkers for patients, thus ultimately providing them with personalized medicine. personalized medicine provides a patient - specific means by which to target one 's disposition to a disease or condition. recent developments in this area include molecular profiling technologies which may include metabolomic analysis, genomic expression analysis, and proteomic profiling. specifically, within proteomic profiling, there are several different techniques used to isolate and quantify the proteins within a subject 's proteome. the raw data, however, are abundant with technical and structural complexities, making statistical analysis a difficult task. preprocessing (including normalization, background correction, gel and/or spectral alignment, feature detection, and image registration) is therefore often required to account for the systematic biases present in the technology and to reduce the noise in the data. feature detection (i.e., the detection and quantification of data features, such as peaks in spectral data, or spots in two - dimensional images) is a particularly important component of low - level analysis, because it works to reduce data size and ease subsequent computations. feature detection falls under the general subject of mathematical morphology (mm), which began in the 1960s and encompasses methods from statistics, machine learning, topology, set theory, and computer science [14 ]. mm is the science of analyzing and processing geometric structures (e.g., local maxima) in digital images. examples of common mm functions include opening, closing, thinning, binning, thresholding, and watershed techniques. a key component in mm is the structuring element, that is, the shape used to interrogate the image. in digital images, the structuring element scans the image and alters the pixels in the window content using basic operators. the goal of processing images with mm methods can be to preserve the global features of the image, preserve large smooth objects in an image, denoise images, and detect objects within an image. situations where mm methods are employed for detection include pedestrian detection, tumor mass detection, and facial feature detection [7, 8 ]. this manuscript outlines feature detection methods used via data preprocessing, specifically to detect and quantify the data associated with peptides (or proteins) in various technologies, particularly stemming from gel electrophoresis or mass spectrometry. section 3 explains the general importance of low - level analysis procedures to be performed on the raw data. with the focus for this manuscript being on feature detection, section 4 discusses proposed approaches for time - of - flight mass spectrometry data, while section 5 discusses recent work with regard to two - dimensional (2d) gel data. proteomics is the study of the proteome, that is, the entire complement of proteins expressed by a genome or organism. from a developmental standpoint, high throughput analysis in the realm of science began with gene expression microarrays [9, 10 ]. following the advancements in microarrays, researchers began to develop high - throughput techniques to analyze the proteome. the overarching biological research goals are similar, namely, to detect statistically significant differential expression (with regard to genes for microarrays, and with regard to proteins in proteomic data) between samples in different treatment groups. further, there are analogous technological ideas and image processing techniques used to produce the image data. there exist, however, several significant differences that make preprocessing proteomic data and subsequent proteomic data analysis complex. biologically, a major difference between a genome and proteome is that the genome can be characterized by the sum of sequences of genomic bases, while the proteome requires knowledge of the structure of the proteins and the functional interaction between the proteins. the primary technical difference between these approaches is the means by which the data are provided. while spots from microarray images are arranged in a systematic matrix fashion, protein spots in a gel image or peaks in protein spectra can be more variable with regard to their location, given the procedure that is used to separate proteins. as well, there is a poor correlation between protein and mrna abundance and, while both methods address the question of differential expression, only proteomic data analysis can also address differential modification (i.e., where the protein is present in both treatment groups, yet its makeup is slightly altered via methylation or phosphorylation). a nice overview of differential proteomic approaches is provided in, with specific emphasis on mass spectrometry approaches and challenges discussed in. in this paper, we examine the most common approaches used to analyze protein abundance, namely, two - dimensional gel electrophoresis (2-de) and difference gel electrophoresis (dige), and time - of - flight mass spectrometry (tof - ms) ; tandem mass spectrometry (ms / ms) is also growing in prominence as a means for studying protein differentiation and modification. sections 2.1, 2.2, and 2.3, respectively, provide further details surrounding these techniques. meanwhile, nongel - based alternative methods exist for quantitative protein analysis and also make use of ms or ms / ms for feature detection and quantification. analysis of quantitative changes in a specific proteome (i.e., complement of proteins expressed in a particular tissue or cell at a given time) is commonly carried out using two - dimensional gel electrophoresis (2-de). o'farrell introduces two - dimensional polyacrylamide gel electrophoresis (2d - page), where protein samples are respectively dyed with a cyanine dye (e.g., cy2, cy3, or cy5) and separated in two directions : along the cartesian x - axis by their isoelectric point (pi) via isoelectric focusing, and along the cartesian y - axis by their molecular weight via sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds - page). the 2d - page technique can be very sensitive to experimental conditions such as laboratory humidity, voltage fluctuations, and gel matrix irregularities. suggest an alternative design (namely, the two - dimensional difference gel electrophoresis, or 2d - dige approach) to combat some of the inherent data variability that exists in the 2d - page method. here, after the respective samples are labeled with a particular dye, the samples are then mixed together to create one composite sample where the proteins are, in turn, separated in both directions as described above. in either case, the associated gel(s) is subsequently imaged via a charged - couple device (ccd) camera or a variable mode scanner to produce the raw image data, where the proteins appear as spots ; see, for example, figure 1. these images are then analyzed using an image analysis software tool (e.g., imagemaster, pdquest). 2-de methods such as 2d - page and 2d - dige are popular techniques for protein separation because they allow researchers to characterize quantitative protein changes on a large scale. thus, 2-de is frequently used as an initial screening procedure whereby results obtained generate new / subsequent hypotheses and determine the direction of ensuing studies. these technologies revolutionized the field of proteomics in their ability to detect protein differences via spot detection and quantification, either with respect to protein expression or modification. further, they are attractive because of their resolving power, sensitivity, and the low equipment cost. 2-de analyses, however, require personnel with significant wet laboratory expertise and can be time - consuming, thus potentially limiting the sample size for gels. furthermore, in some cases (e.g., aging studies, chronic drug treatment, screening for biomarker), replication of the study may be prohibitive. heterogeneities in different gels, the electric fields, ph gradients, thermal fluctuations, and so forth are all factors that make reproducible spot matching between gels a difficult task. as well, scientists are interested in better tools that allow for a completely automated approach to detect protein changes, particularly in low - abundance proteins. these factors not only make it critically important to correctly analyze the 2-de results, but also to maximize the information obtained from an experiment. mass spectrometry is an analytic tool used to identify proteins, where the associated instrument (a mass spectrometer) measures the masses of molecules converted into ions via the mass - to - charge (m / z) ratio. this technology can be used to profile protein markers from tissue or bodily fluids, such as serum or plasma in order to compare biological samples from different patients or different conditions. matrix assisted laser desorption and ionization time of flight (maldi - tof) is a popular tool used by scientists, where a metal plate with the matrix containing the sample is placed into a vacuum chamber that is excited by a laser, causing the protein molecules to travel (or fly) through the tube until they strike a detector that records the time - of - flight for the various proteins under study ; surface enhanced laser desorption and ionization time of flight (seldi - tof) is an analog of maldi - tof. the interested reader is referred to for discussion regarding the experimental design that creates the data, and elaboration on the maldi and seldi constructs. the resulting data are spectral functions containing the m / z ratio and associated intensity, where the peaks in the spectral plots correspond to proteins (or peptides) present in the sample ; see figure 2 for an example of a maldi spectrum. the appeal of mass spectrometry lies in its ability to produce high - resolution measurements with reasonable reproducibility. these procedures generate large amounts of spectral data and can detect protein differential expression and modification in different treatment groups. noisy data, however, can lead to a high rate of false positive peak identification. this is a significant issue when working to establish an unbiased, automated approach to detect protein changes, particularly in low - abundance proteins. tandem ms (ms / ms) is an extension of the ms procedure that allows for further fragmentation of protein mixtures. the setup for such a procedure can be physical where two mass spectrometers are assembled in tandem, or the machine may have the ability to store the ions of interest to run the subsequent separation. as a result, the second arrangement allows for continued subsequent operations to be performed. there are various experiments that warrant the use of ms / ms, including product - ion scans, precursor - ion scans, constant neutral - loss scans, and selected reaction monitoring. product - ion scans determine the product ions that result from decomposing the protein mixture. the precursor - ion scan can be thought of as solving the inverse equation, namely, determining the original mixture that could produce the specified product ions. selected reaction monitoring focuses on a preselected mass to identify the makeup of a protein mixture via the use of the associated product or precursor masses. just as there are various uses for the ms / ms technology, there exist a wide variety of tandem - mass spectrometers, including reverse - geometry ms, triple quadrupole ms, trapped - ion ms, and maldi - tof ms / ms ; see for details. ms / ms is valued by scientists for its ability to detect compounds in mixtures. in particular, ms / ms improves the detection limits for some compounds, and improves the signal - to - noise ratio relative to ms. on the other hand, however, the total ion current associated with an ms / ms spectrum is decreased compared to that from a ms spectrum. further, various ion activation methods affect the efficiency, reproducibility, and feature detection of the associated mass spectra. for a detailed description of the ms / ms procedure and associated technologies, non - gel based methods exist as an alternative for analyzing highly complex protein samples, for example, multidimensional protein identification technology (mudpit), isobaric tag for relative and absolute quantitation (itraq) and isotope coded affinity tags (icat). all of these technologies incorporate the use of ms or tandem ms (ms / ms) to analyze such mixtures. multidimensional protein identification technology (mudpit) analyzes proteomic data by first separating peptides via two - dimensional liquid chromatography, and then detecting protein information using a tandem mass spectrometer. strengths of this methodology include the orthogonality of the chromatographic phases in the separation process, and its robust representation of separated proteins from complex peptides. thus, mudpit is used for a wide range of proteomics experiments, from protein identification and protein cataloging, to quantitative analysis of protein expression. see for an overview of this technology. isotope - coded affinity tag (icat) is a gel - free, lc - based method for analyzing proteomic data that obtains accurate measurements of protein change, and can analyze sufficient amounts of low - abundance proteins. in the icat method, two samples are respectively labeled with either a heavy (i.e., with isotope) or light (without isotope) reagent. the samples are then mixed together and run through an ms or ms / ms machine. the interested reader is referred to [19, 20 ] for details regarding the icat procedure. while this technology provides an accurate measure of relative quantification, it has its share of drawbacks as well. proteins with little to no cysteine residue are not detected, information can be lost regarding posttranslational modification, and interpreting ms / ms spectra can be difficult because of the addition of the biotin group. isobaric tag for relative and absolute quantitation (itraq) is a nongel - based alternative to the icat method for identifying and quantifying proteins from different samples, having the ability to perform relative or absolute quantification in four or eight phenotypes. wu. found that itraq was more sensitive than dige and icat with regard to quantitation, but also more prone to errors when performing ion isolation. argue for the use of replicate and pooling samples in itraq studies. by decomposing the overall variation in itraq experiments as either technical or biological, they find that the biological variation outweighs the technical variation in the data, and propose including at least one biological replicate in any itraq experiment. various types of noise in the data make protein identification and quantification a difficult problem. several solutions have been proposed to resolve these issues, yet they remain open problems because of substantial limitations associated with these approaches. similar to the methods used to analyze gene expression microarrays, the general steps in preprocessing proteomic data include outlier detection, baseline or background subtraction, signal distribution normalization, protein (or peptide) alignment, feature (i.e., peak or spot) detection and quantification, and biomarker evaluation. concerns regarding these procedures are significant because all subsequent analyses relating to the proteomics data are contingent on these first steps being performed appropriately and optimally. the implications from different pre - processing pipelines are outlined in. thus, the goal in preprocessing proteomic data is to create an unbiased, reproducible, and automated approach toward identifying differentially expressed and modified proteins, via either spot or peak information differences. many of these low level analysis methods are directly integrated into the software that accompanies the mass spectrometer or gel imaging scanner. for example, the decyder 2d differential analysis software and imagemaster 2d softwares (all available through ge healthcare) are often purchased in combination with the 2d gel scanners. similarly, other commercial softwares available for gel image analysis include pdquest (bio - rad laboratories), progenesis samespots v3.0 (nonlinear dynamics), and dymension 3 (syngene). in [23, 24 ], these softwares are analyzed and compared on several levels including consistency, spot matching accuracy, and spot quantitation. other softwares and preprocessing methods such as z3 and melanie are analyzed and compared in [2527 ]. note, all softwares require user intervention to set parameters and filter settings in order to obtain the optimal preprocessing of the gel image data ; this limits the ability for an automated procedure using existing methods. meanwhile, there are several softwares for preprocessing ms data that are also generally combined with the associated technology. the preprocessing methods in these softwares are often specific to a particular ms structure with algorithms that differ greatly in complexity. recently, many of the ms preprocessing algorithms have become available to the statistics community through the bioconductor open source software of r libraries [28, 29 ]. for example, the bioconductor packages massspecwavelet, xcms, flagme, and targetsearch all offer various methods to compare and analyze ms - based datasets [3033 ]. while the nongel procedures differ in their protocol, the common denominator with all of these methods lies in their subsequent analysis via ms (or ms / ms). particularly for icat and itraq, they differ only in the number of labeling reagents used, and the distance between and within groups (i.e., peak pairs or groups, depending on the use of the icat or itraq method, resp.). low - lying peaks, however, still remain a problem in that (e.g., with icat data) it hinders identification of peak pairs. for both icat and itraq data acquired via lc - ms, this further emphasizes the need for accurate and precise peak / feature detection methods for data stemming from ms and ms / ms technologies. proposed procedures for feature (peak) detection in ms data range greatly in algorithm complexity. although, parsimonious methods should be favored, different variations of the ms technology (e.g., seldi - ms and maldi - ms) can require more complex methods to account for systematic biases. methods described in [35, 36 ] take the maximum value within the kth nearest neighbors to determine the location of a peak. apply this approach to preprocess the raw data into local peak / nonpeak binary data. in order to diminish the number of false positives that arose from their choice of k = 20, they further define a peak as having an intensity value larger than the average intensity level over a broad neighborhood as defined via the super - smoother method with five percent of all data points as the associated smoothing window. instead use k = 10 after considerable data preprocessing (including baseline correction, averaging the spectra, and spectral alignment via peak matching). their choice for a smaller k better aids in their ability to select peaks that are detected across spectra. approach the problem in this manner, because a peak detected in only one spectrum could arguably represent noise, while common peaks across patients may infer the existence of a true biomarker of interest. establish a simple peak finding (spf) algorithm for peak detection in one mass spectrum, where they use a change in slope (via first differences) to detect peaks in seldi - tof data. the median absolute value of the first differences is then used to determine the amount of noise in the data, and serves as a threshold for determining what peaks appear to be small enough that they actually represent noise as opposed to true signal. nearby peaks that fall within a nearness threshold are combined to represent one peak, and the associated peak locations are redefined as the nearest local minima surrounding the local maximum. finally, the upward and downward slopes are computed for all peaks to quantify peak size and remove small peaks that appear to represent noise. while the spf algorithm can identify peak location,. warn against solely using this algorithm for peak quantitation as the resulting peak intensity values are not baseline - corrected ; however it is sufficient for identifying peak locations. to address peak detection in multiple spectra, they also build from the spf algorithm to define a simultaneous peak detection and baseline correction (spdbc) algorithm. the spdbc algorithm, however does not necessarily identify the same peaks across all spectra. further, while both algorithms adjust for noise, it still potentially overestimates the number of real peaks in a spectrum depending on the user 's determination of certain parameter settings. advocate using an undecimated discrete wavelet transform (udwt) with hard thresholding to perform peak detection. perform the udwt with hard thresholding transform the data from the time domain to the wavelet domain, resulting wavelet coefficients equal to zero that are less than some predetermined threshold, and then transform back into the time domain. this preprocessed data is then baseline - corrected and run through the spf algorithm described in to locate all peaks in the spectrum. morris. also use a udwt to perform peak detection but via the mean spectrum. adapting the algorithm of, morris. compute the mean spectrum over all calibrated raw spectra, and apply the essence of the algorithm in to the mean spectrum to denoise, baseline correct, and find peaks. performing peak detection on the mean spectrum this algorithm also detects and quantifies peaks without the need for peak - matching algorithms to be applied across samples, because this is addressed inherently through the use of the mean spectrum. du. create a one - dimensional feature detection algorithm based on the one - dimensional continuous wavelet transform (1d - cwt) to detect peaks in mass spectrometry data. in, the 1d - cwt is applied to the raw spectral data thus moving from the time to wavelet domain (using the mexican hat wavelet as the mother wavelet), and cwt coefficients are obtained associated with corresponding scales, thus forming a coefficient matrix where, for each scale level, the associated cwt coefficients are maximized at the peak center. this matrix is then visualized via a false color image, matching the ridges in the image to the peaks in the mass spectrum to provide information on how the associated coefficients change across scales. the false - color image ridges correlate well with the spectral peaks, thus providing an alternative approach and visualization tool toward peak detection. the appeal of this approach is its ability to reduce the false positive rate with regard to peak detection and also its lack of dependence on any previous or subsequent preprocessing steps, thus improving the robustness of the results. assuming a slow - changing, locally monotonic baseline in the spectrum allows for the 1d - cwt to be applied directly to the raw spectrum, and statistical technologies have also been developed for use with ms / ms data or other non - gel - based methods described in section 2.4, for example, proicat, sequest, interact, or hardklr. see von haller., wu., and hoopmann. for details on these respective approaches a variety of low - level analysis algorithms exist to summarize information from 2-de data. below we focus on three recent algorithms that incorporate or focus on spot detection in gel images. have an elaborate, seven - step algorithm for feature detection, including region segmentation, region filtering, spot extraction, centroid estimation, spot merging or splitting, spot filtering, and centroid reestimation. using the watershed algorithm for initial segmentation, the regions are filtered to focus on regions of reasonable size or variability. the authors then apply k - means clustering to each region to differentiate foreground from background pixels, and apply morphological closing to remove noise. the authors then determine an initial spot center estimate for each region, and then reevaluate the initially determined spots via spot splitting and merging to address oversegmentation caused by the watershed procedure. finally, the algorithm performs another spot filtration procedure to remove features (e.g., dust) from future analysis, and spot centers are reestimated via a 2d gaussian function. the use of the 2d gaussian for spot center estimation can be disputed, given that it has long disputed that protein spots are not accurately modeled by a gaussian distribution. while the algorithm is apparently robust when applied to geometrically distorted simulated images, it has difficulties when applied to real gels, due to difficulty in handling images with differing illuminations, noise, and irregular spot structures ; see for details. langella and zivy have established an interesting algorithm that uses image topography to determine spot location and size. in this algorithm, one envisions beads placed at each pixel location within the image, and tracks each bead 's progression in the direction of maximal positive slope toward an associated spot 's maximum. the associated computer code, available at http://moulon.inra.fr/beads/beads.html, supplies the final image illustrating spot boundaries, along with intermediate grayscale images, including the paths of maximal slope for each respective bead associated with pixel locations in the original image (directions), the number of beads that arrive at each respective location (beads), the number of beads that travel through respective pixel locations (paths), and possible spot center locations (select). further, the probabilities image shows a bivariate normal distribution applied at every positive pixel in the select image to aid in final assessment of spot locations, and numbers contains number codes at pixel locations for spot identification and size quantification. this algorithm seems to perform well in simulated gels, but faces difficulties with regard to diffuse or saturated spots. further, this algorithm does not account for spot matching and, thus, can not be used for comparative analysis across gels ; see for details. apply a (hyper-)crossical - shaped structuring element (i.e., shaped like a multidimensional cross) of varying size to an image (creating a smooth image), and use the smoothing decomposition, data = smooth + rough to determine the associated residual (i.e., rough) image. this structuring element with arm - size c is combined with a median operation over the pixels within the cross shaped window. when the median operation is applied to the preprocessed image (as described in), the associated residual image contains crosses whose centers are the local maxima. focusing our attention on the rough image, isolates the positive intensities and applies mathematical morphology (erosion and dilation) to remove the noise and heighten the presence of the crosses. the nonlinear nature of the median operator allows this method to detect proteins of low intensity as well as nearby proteins within a gel. thus, with this method we have a means to identify spot centers and estimate spot sizes ; see [44, 46 ] for details. other spot detection procedures include modeling 2d - gaussians, applying diffusion equations, linear programming, and wavelet modeling as described in. the use of 2d - gaussians, however, is disputed due to the knowledge that spots can be oddly shaped, and thus can not be accurately represented via a 2d - gaussian model. ultimately, it is difficult to obtain the algorithm details for many of the proprietary softwares that are marketed in the industry. this severely limits the ability to understand exactly the feature detection methods employed to locate and quantify spots in a gel image. experiments utilizing the described proteomic platforms have the general goal of deriving knowledge of the biological system. through the experiments utilizing these platforms, common hypotheses to examine include differential expression, cluster analysis, association with a phenotype, and correlation with survival (or other censored variables). there are standard statistical methods designed to handle each of these situations ; for example, see for details. with all of these platforms in proteomic experiments, multiple testing can arise when (1) examining thousands of peptides for differential expression, (2) testing a peptide against several different contrasts, or (3) examining the significance of groups of peptides for association with a given phenotype. in these situations, scientists need to choose a type i error rate and a method to control it. the statistical aspects and the assumptions underlying the choice of error rate and control method are often critical to the success of proteomic experiments. before we can tackle such high - level analyses, we must first have sufficiently and satisfactorily determined the appropriate data summary information to address these problems. this matter, however, has not been addressed so that a uniform procedure is established and accepted. biological and medical communities have not uniformly accepted a low - level analysis procedure for preprocessing proteomics data and thus have many available methods from which to choose for performing low - level analysis of such data structures. proteomic technologies are not based on the hybridization of complementary dna strands, hence it is not possible to engineer quality control experiments for proteomic data as it is for microarrays. further, sample preparation, starting materials, and reagents and differences in ms machines and gel imagers have contributed to the wide variability in the data ; for example, the sensitivity of the techniques to specimen collection and handling is an issue. similar to the situation with preprocessing algorithms, inconsistent sample preparation and handling can lead to spurious results and conclusions. further confounding the problem is that many of the methods to analyze ms and gel data are proprietary, and thus not fully disclosed, while the field lacks a suitable gold standard to fully evaluate the available methods. these aspects of analyzing proteomic data are difficult to simulate and thus there is a need for a comprehensive set of experiments that can accurately assess each aspect of the data analysis pipeline in gel - based and non - gel - based experiments. none of these procedures are fully automated as they generally require additional user input to determine thresholding parameters or local window ranges for consideration. further, these input parameters can influence each stage in the sequence of preprocessing steps. algorithm results are generally inconsistent and unrecoverable, which causes great concern on its impact on the determination of scientifically significant proteins. as noted in regarding mass spectrometry data, different preprocessing algorithms could severely affect downstream analyses, and so the choice of procedure must be approached carefully ; the same is true for two - dimensional gel electrophoresis data, non - gel - based data or, more generally, any image or spectral data. another challenge for scientists that further complicates the field 's ability to establish a generally - accepted preprocessing approach is its ability to detect small proteins, that is, proteins that are present in low abundance but are differentially expressed or modified. the high - abundance proteins with large peaks are generally uninteresting as they are already extensively studied for their ability to serve as biomarkers. for example, in cancer biomarker research, studies commonly attempt to quantify proteins or metabolites that are shed into the blood stream by the tumor (see, e.g., [53, 54 ]). these proteins are present in relatively low abundance, and thus are represented in mass spectra by small or low - lying peaks, and analogously in gel data as low - lying or faint spots ; however, they represent a promising set of biomarkers in cancer diagnosis. many such peaks are usually undetected because of the signal - to - noise ratio, leading to larger false negatives. for example, in icat studies, noisy data hinder low - lying peak detection, and thus the identification of associated peak pairs. this is a significant problem for scientists since it limits their ability to detect and evaluate potential biomarkers and peptides. so, which approach is best for preprocessing proteomic data according to the respective methodology ? model accuracy, false discovery rates, the ability to detect low abundance protein peaks, and the user intervention required of the procedures are significant factors that play a role in addressing this question. these significant and substantial factors influencing preprocessing techniques for proteomic data therefore make this question difficult (if not impossible) to answer without a large, cohesive effort across the proteomics and statistics communities. only through such an endeavor can we truly make significant forward movement toward a generally accepted approach for data analysis. | numerous gel - based and nongel - based technologies are used to detect protein changes potentially associated with disease. the raw data, however, are abundant with technical and structural complexities, making statistical analysis a difficult task. low - level analysis issues (including normalization, background correction, gel and/or spectral alignment, feature detection, and image registration) are substantial problems that need to be addressed, because any large - level data analyses are contingent on appropriate and statistically sound low - level procedures. feature detection approaches are particularly interesting due to the increased computational speed associated with subsequent calculations. such summary data corresponding to image features provide a significant reduction in overall data size and structure while retaining key information. in this paper, we focus on recent advances in feature detection as a tool for preprocessing proteomic data. this work highlights existing and newly developed feature detection algorithms for proteomic datasets, particularly relating to time - of - flight mass spectrometry, and two - dimensional gel electrophoresis. note, however, that the associated data structures (i.e., spectral data, and images containing spots) used as input for these methods are obtained via all gel - based and nongel - based methods discussed in this manuscript, and thus the discussed methods are likewise applicable. |
drug addiction is a chronic relapsing brain disorder, manifested as an intense desire for the drug, with impairment of the ability to control the urges to take the drug, even at the expense of serious adverse consequences (cam and farr 2003). these behavioral abnormalities develop gradually with repeated exposure to a drug of abuse, and can persist for months or years after discontinuation of use, suggesting that addiction can be considered a form of drug - induced neural plasticity (nestler 2004). several compounds can lead to addictive behavior including opioids, psychostimulants, cannabinoids, alcohol and nicotine, and although their initial mechanism of action affects different neurochemical targets, the resulting neural dysregulation involves similar neurochemical and neuroanatomical pathways (hyman and malenka 2001). the limbic component of basal ganglia pathways, the endogenous opioid system and the brain - pituitary stress system are all essential for the addictive properties of most drugs of abuse, whose interaction with these circuits leads to a common dysregulation of brain motivational and reward pathways (maldonado 2006). the limbic component of the basal ganglia pathway is a common neuronal substrate for the reinforcing properties of drugs of abuse and drives the motivational, emotional and affective information on behavior (see for review koob 1992 ; di chiara 1999 ; koob 2004 ; pierce 2006). the mesocorticolimbic dopaminergic pathway (comprising dopaminergic neurons in the ventral tegmental area vta innervating the nucleus accumbens hippocampus, amygdala, medial prefrontal cortex and ventral pallidum), is a vital factor governing the fl ow of information through the limbic circuit comprising the interconnected nuclei. thus dopamine is considered one of the most important actors in the rewarding effects of drugs of abuse, as suggested by the finding that most of the drugs abused by humans raise dopamine levels in the nac, and blockade of dopamine transmission reduces the rewarding effect of psychostimulants (see for review pierce and kumaresan 2006). moreover, mesolimbic dopaminergic neurons communicate with cerebral areas involved in cognitive functions and dopamine release in the forebrain can be considered a learning signal. in the nac glutamatergic projections from the cerebral cortex, amygdala and hippocampus drive information about external situations and internal emotional and physiological states, thus contributing to addiction by consolidating reward - driven behavior (hyman and malenka 2001 ; kauer 2004). besides the importance of the mesocorticolimbic dopaminergic system in addiction, the shared mechanisms in the development of addictive behavior have not yet been fully identified so this review will focus on recent findings pointing towards a role of the endocannabinoid system in the circuitry underlying drug addiction. knowledge of the endocannabinoid system has been largely boosted since the cb1 receptor was cloned in 1990 and we now understand that the endocannabinoid system consists of cannabinoid receptors, endogenous ligands and several proteins responsible for their synthesis and degradation (see for review bisogno 2005). two cannabinoid receptors, cb1 and cb2 have been cloned and characterized, both belonging to the class of g protein - coupled receptors. cb1 has been located in the central nervous system and peripheral tissues and cb2 appeared mainly in the cells of the immune system (fride and mechoulam 2003) although it has now also been identified in brainstem, cortex and cerebellum neurons (van sickle 2005). the most fully characterized endocannabinoid substances isolated from brain tissue are anandamide (aea) and 2-arachidonylglycerol (2-ag) (fride and mechoulam 2003). endocannabinoids serve as neuromodulators in many physiological processes and once released from postsynaptic neurons upon depolarization, they activate presynaptic receptors, resulting in inhibition of the release of both excitatory and inhibitory transmitters (see for review fride 2005). in this capacity the endocannabinoid system may have important additional roles in the regulation of synaptic brain function. cb1 receptors are abundant in the brain reward circuitry, and the dopaminergic neurons of the mesocorticolimbic pathway are regulated by excitatory and inhibitory inputs influenced by activation of cannabinoid receptors (see for review gardner 2005). endocannabinoids released after depolarization in the nac and from dopaminergic neurons in the vta may possibly influence gabaergic and glutamatergic afferents by acting as retrograde messengers on cb1 receptors. wenger (2003) reported the presence of cannabinoid receptors in tyrosine hydroxylase - expressing neurons (most probably dopaminergic neurons) of the nac, vta, striatum and pyriform cortex, suggesting the endocannabinoid system might directly influence dopaminergic reward mechanisms. cb1 receptors are present in other areas related to reward and motivation (such as the basolateral amygdala and hippocampus) and endocannabinoids induce long - term depression (ltd) of the inhibitory synapses in the hippocampus, contributing to the synaptic plasticity involved in the learning processes related to addictive behavior (de vries and schoffelmeer 2005). the endocannabinoid system is certainly the primary site of action for cannabinoid addiction and in fact cannabinoids, like other drugs of abuse, induce tolerance and physical dependence and activate a rewarding system (see for review parolaro 2005 ; fattore 2005 ; gonzalez 2005). the exact sites and substrates of cannabinoid action in the core vta - medial forebrain bundle (mfb)-nac reward axis and on reward - related behaviors are still not clear but there is evidence that cannabinoids enhance brain reward substrates, acting on both dopamine - dependent substrates in the vta and dopamine - dependent / independent ones in the nac (lupica 2004). however, the endocannabinoid system certainly has an overall effect on the reward circuitry and participates in the rewarding and addictive properties of all prototypical drugs of abuse such as opioids, nicotine, alcohol and psychostimulants (cocaine and amphetamine). animal models of drug reward provide evidence of the endogenous cannabinoids role in the rewarding effects of several addictive drugs, and pharmacological manipulation of endocannabinoid tone with sr141716a (rimonabant, a specific cb1 receptor antagonist) in humans gave positive results (anthenelli and despres 2004). two complementary approaches have been used to demonstrate the endocannabinoid system s role in addictive behavior ; the first is a genetic approach evaluating changes to the addictive properties of several drugs of abuse in cb1 knockout mice, and the second is a pharmacological approach looking at the effect of sr141716a on drugs addictive properties. research in this field has also gained from the use of validated experimental models for the subjective effects of drugs (drug discrimination), their rewarding / reinforcing properties (intravenous self - administration, conditioned place preference cpp and intracranial self - stimulation), the influence of environmental factors on drug - seeking behavior (cpp, second - order schedules of self - administration, reinstatement of extinguished drug - seeking behavior and other relapse models), and the withdrawal states associated with abrupt termination of drug action (administration of a selective antagonist after chronic exposure). opioids and cannabinoids have several similar pharmacological effects, including analgesia and stimulation of brain circuitry, that are believed to underlie drug addiction and reward. there is ample evidence of a role for the endocannabinoid system in opioid dependence (table 1). cannabinoids attenuated morphine and methadone withdrawal signs (hine 1975 ; deikel and carder 1976 ; vela 1995 ; yamaguchi 2001 ; del arco 2002) and the cannabinoid antagonist sr141716a precipitated abstinence in morphine - dependent rats (navarro 1998 ; maldonado 2002). the severity of naloxone - precipitated morphine withdrawal was robustly attenuated in cb1 ko mice (ledent 1999 ; lichtman 2001) and long - term treatment with sr141716a reduced the intensity of naloxone - precipitated opioid withdrawal (rubino 2000 ; mas nieto 2001) ; however an acute dose of sr141716a just before naloxone did not affect the incidence of withdrawal signs (mas - nieto 2001). thus it appears that chronic blockade of cb1 receptors is needed to alleviate the main signs of morphine abstinence, suggesting that chronic treatment with sr141716a might be useful in the opiate withdrawal syndrome. in addition changes in cb1 receptor function (in terms of receptor binding and coupling with g protein) and/or in endocannabinoid levels were observed in specific brain areas of morphine - dependent animals, although the results ranged from no change to a decrease or even an increase (romero 1998 ; gonzalez 2003 ; vigan 2005). the opioid antagonist naloxone precipitated abstinence symptoms in rats tolerant to -tetrahydrocannabinol (-thc) (kaymakcalan 1977) and sr141716a - precipitated withdrawal was dose - dependently reduced by morphine (lichtman 2001). the somatic expression of cannabinoid withdrawal was attenuated in mice lacking pre - pro - enkephalin or mu opioid receptor genes (valverde 2000 ; lichtman 2001) whereas the deletion of mu, kappa and delta opioid receptors did not affect cannabinoid withdrawal (ghozland 2002). in contrast, in double ko mice deficient in mu and delta opioid receptors, cannabinoid withdrawal was significantly reduced (castane 2003), suggesting that a cooperative action of both receptors was required for the expression of -thc dependence. the traditional animal paradigm of drug dependence further confirmed the importance of the endocannabinoid system in opioid addiction. manzanedo (2004) reported that pre - exposure to the synthetic cannabinoid agonist win55212 - 2 increased the rewarding effect of morphine evaluated in a place preference paradigm, supporting the idea that using cannabis might make an individual more vulnerable to opiate addiction. more recently, goldberg s group (solinas 2005) reported that the reinforcing efficacy of heroin, measured in a progressive ratio schedule of i.v. heroin self - administration, was significantly enhanced by -thc and win55212 - 2 but not by compounds that raise the levels of endocannabinoids by blocking their uptake or metabolism ; this suggested that the interaction between cannabinoids and opioids might be mediated by their receptors and signaling pathways rather than by opioid - induced release of endogenous cannabinoids. in contrast, approaches involving the cb1 receptor antagonist sr141716a suggested the endocannabinoids had a facilitating effect on opioid reinforcement that was unmasked by cb1 receptor blockade. sr141716a reduced opioid self - administration and conditioned place preference in rodents (chaperon 1999 ; braida 2001 ; navarro 2001, 2004 ; mas nieto 2001 ; caille and parsons 2003 ; de vries 2003 ; fattore 2003 ; see for review fattore 2005 and maldonado 2006). sr141716a had more effect on heroin self - administration when more effort was required to obtain a heroin infusion ; for example it was efficacious under a progressive ratio schedule of reinforcement (high price of drug), weaker under a fixed ratio schedule 5 (low price of drug) and null under a fixed ratio schedule 1 (very low price) (solinas 2003). these results strengthen the idea that sr141716a attenuates the reinforcing effects of heroin and provide support for the potential efficacy of cannabinoid cb1 antagonists in the prevention and treatment of opioid reward. thus, morphine conditioned place preference (martin 2000) and self - administration (ledent 1999 ; cossu 2001) were abolished in cb ko mice although the results on morphine cpp in cb1 ko mice tended to vary. martin (2000) found morphine induced cpp in wild - type mice but there was no such response in ko mice, indicating that the drug had no rewarding effects in the absence of cb1 cannabinoid receptors. rice in 2002, reported that cb1 receptor ko mice developed a strong place preference to morphine, similar to that in wild - type swiss - webster mice ; this indicated that the brain cannabinoid system made no contribution to morphine reward. one explanation of these conflicting results might be found in the slightly more intensive conditioning paradigm and differences in the conditioning chambers used in the last study. in summary, the rewarding effects of -thc were suppressed in opioid - receptor ko mice (ghozland 2002 ; castane 2003) and attenuated by opioid antagonists (braida 2001 ; justinova 2004). it is interesting that sr141716a did not modify the dopamine releasing effect of heroin in the nac (tanda and di chiara 1997 ; caille and parsons 2003). caille and parsons (2006) have subsequently focused on an additional substrate in opiate reward, namely the ventromedial pallidum (vp), a cerebral area that receives dense gab - aergic input from the nac. opiates strongly reduced vp gaba efflux and the resulting disinhibition of the vp is thought to contribute to the positive reinforcing effects of opiates (caille and parsons 2004). sr141716a caused a dose - dependent blockade of the effect of morphine on vp gaba efflux without influencing morphine s dopamine - releasing effect. however, sr14716a did not alter cocaine self - administration, or cocaine - induced decrements in vp gaba efflux and increases in nac dopamine. this is consistent with evidence that selective inactivation of cb1 receptors reduces opiate-, but not psychostimulant - maintained self - administration. however, the cb1 receptor agonist win55212 - 2 reduced vp gaba efflux in a manner similar to morphine, and this effect was reversed by the opiate receptor antagonist naloxone. these results point to an interaction between cannabinoids and opioids in their effects on vp activity, and suggest that sr141716a attenuates opiate reward by reducing the opiates inhibitory influence on nac medium spiny neurons. finally, endocannabinoid tone seems to have a particularly interesting role in relapse to opiate abuse, especially in humans. detoxification from opiate addiction has been a medical problem for as long as opiates have been abused, as relapses occur even after long drug - free periods. the endocannabinoid system almost certainly plays a part in triggering or preventing reinstatement of drug - seeking behavior (see for review fattore 2006). the three synthetic cb1 receptor agonists win55212 - 2, cp55940 and hu210 promptly reinstate heroin - seeking after a long drug - free period (de vries 2003 ; fattore 2003). rimonabant, however, attenuates heroin - induced reinstatement of heroin - seeking behavior (de vries 2003 ; fattore 2003) suggesting that cb1 receptor blockade alters heroin s reinforcing consequences. further supporting the notion of a close reciprocal relationship between cannabinoid and opioid systems in relapse, blockade of opioid receptors by naloxone prevented relapse to cannabinoids (spano 2004) and a priming injection of heroin reinstated cannabinoid - seeking behavior after three weeks of extinction. interestingly, sr141716a per se did not reinstate responding but did prevent cannabinoid - seeking behavior triggered by heroin (spano 2004). there appears to be a functional interaction between the endogenous cannabinoid system and nicotine addiction (table 2). in the mouse cpp paradigm, co - administration of sub - threshold doses of -thc and nicotine induced rewarding effects (valjent 2002). acute -thc significantly lowered the incidence of several precipitated nicotine withdrawal signs and improved the aversive motivational consequences of nicotine withdrawal in mice (balerio 2004). these findings suggest that each drug enhances the rewarding effect of the other, and that cannabis might be used to reduce aversive reactions resulting from nicotine withdrawal. in contrast, valjent (2002) showed an enhancement in the somatic expression of withdrawal in animals co - treated with nicotine and -thc, suggesting an asymmetric relationship between the two compounds. nicotine had no rewarding effect in cb1 ko mice in a place preference paradigm (castane 2002). by contrast, cossu (2001) reported that the absence of cb1 receptors did not affect nicotine self - administration. methodological aspects such as the specific paradigm and the dose used may partly explain this discrepancy. the second approach used to demonstrate the endocannabinoid system s role in nicotine addiction involves sr141716a. pretreatment with this specific cb1 receptor antagonist reduced nicotine self - administration, nicotine - seeking behavior induced by conditioned cues in rats (le foll and goldberg 2004 ; cohen 2005), nicotine - induced dopamine release in the nac (cohen 2002) and the dopaminergic component of nicotine discrimination (cohen 2002). to conclude, since dopamine release in the nac is thought to play a major role in the positive reinforcement of nicotine, and although findings in cb1 receptor ko mice are inconsistent, sr141716a may well have some antismoking activity, by reducing the rewarding / reinforcing effect of nicotine. some recent papers have looked at the utility of sr141716a for reducing cues associated with nicotine - seeking behavior, one of the major causes of relapse in former smokers. forget (2005) showed that sr141716a impaired both the establishment and the short - term expression of cpp induced by nicotine, suggesting that endogenous cannabinoids are implicated in nicotine s motivational effects. interestingly, rimonabant did not affect the long - term expression of the incentive learning, suggesting other cannabinoid - independent mechanisms are involved (forget 2005). however, cohen (2005) found that rimonabant attenuated the long - term influence of environmental stimuliresponsible for relapse in nicotine - seeking behavior. after demonstrating the persistence of conditioned behavior in response to nicotine - related cues several weeks after nicotine withdrawal, cohen showed that rimonabant could reduce the responding maintained by these cues three months after stopping smoking. thus rimonabant may not only help stop people smoking but may also help them remain abstinent. biochemical investigations found altered levels of aea and 2-ag in several brain areas in animals chronically treated with nicotine. aea was elevated in the limbic area and brainstem, and 2-ag in the brainstem, but one or both were reduced in other regions such as hippocampus, striatum and cerebral cortex. cb1 receptor levels were not altered in any of these brain areas (gonzalez 2002 ; balerio 2004). to summarize, the pharmacological and cellular studies described indicate that the endogenous cannabinoid system has a specific role in nicotine responses related to its addictive behavior and open up new possibilities for the treatment of this major public health disorder. there are already some preliminary data from the stratus - us trial (smoking cessation in smokers motivated to quit) on the efficacy of rimonabant in humans (anthenelli and despres 2004). the study lasted ten weeks and the smokers were allowed to smoke during the first two weeks but were asked to abstain after this. the quitting rate in the 2-mg rimonabant group was double that with placebo and they showed markedly less weight gain during the ten - week study period (anthenelli and despres 2004). recent studies suggest that the endocannabinoid system has a major part among the neurotransmitter systems involved in regulating different alcohol - related phenomena, including tolerance, vulnerability, reinforcement, consumption and metabolism (table 2). thus acute administration of synthetic or endogenous cannabinoid agonists stimulated alcohol intake in sardinian alcohol - preferring rats (colombo 2002) and c57bl/6j mice (wang 2003), and dose - dependently increased break points (an indicator of motivation to drink alcohol) for beer in wistar rats (gallate 1999). all these effects were completely prevented by pre - treatment with the cannabinoid antagonist sr141716a. furthermore, genetic deletion of the faah enzyme (basavarajappa 2006) or an injection of urb597 (an irreversible faah inhibitor) into the prefrontal cortex enhanced motivation to drink alcohol (hansson 2006), pointing clearly to a role for the endocannabinoid system in alcohol addiction. it is in fact now established that sr141716a, given alone, has effects on alcohol - related behavior opposite to those of the cb1 receptor agonists. for example, sr141716a prevented the acquisition of alcohol drinking behavior in alcohol - nave sardinian alcohol - preferring rats with a free choice between alcohol (10%, v / v) and water (serra 2001). it also reduced ethanol consumption in c57bl/6 mice at doses only marginally affecting regular chow intake or water drinking ; this suggests that the endogenous cannabinoid system may affect the appetitive value of ethanol (arnone 1997). similar reductions in voluntary alcohol intake were obtained in sardinian alcohol - preferring rats (colombo 1998), and wistar rats (lallemand 2001) after chronic alcoholization (a model of the maintenance phase of human alcoholism). in the sardinian alcohol - preferring rats the cb1 receptor antagonist completely abolished the effect of alcohol deprivation (ie, the temporary increase in alcohol intake after a period of withdrawal, a model for relapse episodes in human alcoholics). this suggested that the cannabinoid cb1 receptor might be part of the neural substrate of the alcohol deprivation effect and that sr141716a may have anti - relapse properties (serra 2002). finally, sr141716a reduced oral self - administration and attenuated the appetitive properties of alcohol in unselected rats under operant procedures (gallate and mcgregor 1999 ; freedland 2001 ; colombo 2004). these data were confirmed by gessa (2005) using a second cb1 receptor antagonist, the newly synthesized sr147778, which suppressed acquisition and maintenance of alcohol drinking, relapse - like drinking and motivation to consume alcohol in sardinian alcohol - preferring rats.cippitelli (2005) provided clear evidence that blockade of cb1 receptors reduced both ethanol self - administration and conditioned reinstatement of alcohol - seeking behavior in marchigian - sardinian alcohol - preferring rats and wistar rats, the genetically selected rats showing higher sensitivity to rimonabant. these researchers also reported that at least in the strain of rats bred for its ethanol preference, the marchigian - sardinian alcohol - preferring rats, cb1 cannabinoid receptor mrna expression was increased in brain areas relevant for processing reward and reward - associated behavior, suggesting that alterations in the function of the cb1 receptor system may be linked to genetic vulnerability to alcohol misuse (cippitelli 2005). all these results further reinforce the concept that pharmacological blockade of the cb1 receptor may offer a novel approach to the treatment of alcoholism, not only for consumption but also for context - induced relapses to alcohol, one of the main problems in the treatment of this disorder. the cb1 receptor signaling system s participation in alcohol drinking and alcohol sensitivity was confirmed using cb1 receptor ko mice. / mice with cd1 background showed lower ethanol intake and less preference, effects associated with dramatic sensitivity to the hypothermic and hypolocomotor effects in response to low doses of ethanol (naassila 2004). these mice also had more severe withdrawal - induced convulsions. since previous studies in rodents have suggested that high levels of ethanol drinking are often associated with resistance to its intoxicant effects (schuckit 1994 ; kurtz 1996), the lower ethanol consumption in cb1 ko mice might be due to their greater sensitivity to ethanol s acute effects. chronic in vivo (gonzalez 2002) or in vitro (basavarajappa and hungund, 1999a ; basavarajappa 2003) ethanol exposure increased accumulation of aea. this was inhibited by pertussis toxin and the cb1 receptor antagonist sr141716a, and paralleled by the activation of ca - dependent and arachidonic acid - specific phospholipase a2, a key enzyme in the formation of endocannabinoids in neuronal cells and brain (basavarajappa 1997, 1998). the mechanism by which chronic ethanol exposure increases the endocannabinoid content remains to be established, though basavarajappa (2003) have reported that in cerebellar granular neurons chronic exposure to alcohol led to an increase in extracellular aea by inhibiting its uptake. this event was cb1 receptor - independent since it also occurred in cb1 ko mice and cannabinoid cb1 receptor antagonists did not alter the effects of chronic ethanol on aea transport. moreover, in rodents chronic exposure to alcohol impairs cb1 receptor function, lowering the levels of cb1 receptor binding, expression and cb1 receptor / g protein coupling (basavarajappa and hungund 1999b ; ortiz 2004). these studies suggest that during the development of alcohol tolerance there are changes in the steady state in the endogenous cannabinoid system, and this might explain the altered response to alcohol. these converging findings suggest that cannabinoid cb1 receptor blockade may be an effective therapeutic approach for alcohol dependence in humans but information on the clinical efficacy of rimonabant is still lacking. psychostimulants differ from the drugs of abuse since they affect mesolimbic dopaminergic terminals directly, raising dopamine levels in the nac by direct action on dopaminergic axon terminals ; the other drugs seem to induce rewarding effects by increasing dopaminergic neuron firing rates, acting in the vta, possibly through the release of endocannabinoids (lupica and riegel 2005). this may help us understand why endocannabinoids are not involved in cocaine s or amphetamine s primary reinforcing effects. in fact, there are several reports (table 3) that genetic deletion or pharmacological blockade of cb1 receptors does not alter cocaine or amphetamine self - administration (cossu 2001 ; de vries 2001 ; braida 2005 ; lesscher 2005) or the neurochemical correlate of this behavior, namely dopamine release in the nac (cossu, unpublished results, soria 2005). similarly, cocaine - induced cpp was not modified in cb1 ko mice (martin 2000 ; houchi 2005). these results clearly indicate that cb1 receptors are probably not involved in the primary reinforcing effects of psychostimulants. although acute reinforcing properties are essential for the establishment of drug addiction, other complex behavioral processes are involved in consolidating this chronic relapsing disorder (koob and lemoal 2001), so the acute reinforcing effects of the drug are only the first step in the acquisition of stable operant self - administration responding. besides the mesolimbic dopaminergic system, particularly the nac, it has been proposed that dopamine - independent neuronal circuits are involved in regulating reward - related processes (lupica 2004). cb1 cannabinoid receptors are highly expressed in other brain regions involved in the rewarding circuitry such as the basolateral amygdala, medial prefrontal cortex, and hippocampus, so mechanisms involving cb1 receptors in these structures might well participate in other aspects such as consolidation and relapse of cocaine - seeking behavior. in line with this hypothesis, soria (2005) found that only 25% of cb1 ko mice, compared with 75% of the wild - type, acquired reliable operant responding to self - administer the most effective dose of cocaine (1 mg / kg / infusion), and needed more sessions to attain this behavior. moreover, the maximal effort to obtain a cocaine infusion was significantly lower after genetic ablation of cb1 receptors, indicating decreased motivation for maintaining cocaine - seeking behavior. results were similar after pharmacological blockade of cb1 receptors with sr141716a in wild - type litter mates (soria 2005). the lack of motivation for cocaine in cb1 ko or sr141716a - pretreated mice might be due to impaired detection, association, and representation of the reward signal or to inadequate responding to the rewarding stimuli (soria 2005). in line with the idea of cb1 receptors involved in these other aspects of reward, fattore (1999) found that win 55212 - 2 reduced intravenous cocaine self - administration, suggesting that stimulation of cb1 receptors may potentiate cocaine s reinforcing effects. similar findings were reported with amphetamine (braida and sala 2002) : the combination of cp-55,940 with the maximal reinforcing unit dose of 3,4-methylendioxymethamphetamine (mdma) significantly lowered the mean number of drug - associated lever pressings in comparison with the drug alone, suggesting a synergistic action of cannabinoid agonists on mdma s reinforcing properties. finally, cb1 receptors play an important role in relapse to psychostimulants. de vries (2001) found that the cannabinoid agonist hu210 provoked relapse to drug - seeking in animals after prolonged withdrawal from cocaine self - administration, whereas blockade of the cb1 receptor by sr141716a attenuated the relapse induced by re - exposure to cocaine - associated cues or the drug itself. xi (2006) have now shown that the cb1 antagonist am251, administered systemically, selectively inhibited cocaine - induced reinstatement of reward - seeking behavior by a glutamate - dependent mechanism. cb1 receptor - mediated disinhibition of nac glutamate release could activate presynaptic mglur2/3 receptors, which then inhibited cocaine - enhanced nac glutamate release and cocaine - triggered reinstatement of drug - seeking behavior (xi 2006). along the same lines, sr141716a blocked the reinstatement of methamphetamine - seeking behavior in rats (anggadidiredja 2004). given the paucity of effective medications to treat psychostimulant addiction, and although the endocannabinoid system does not participate in the primary reinforcing effects of this class of drugs, cb1 receptor antagonists may offer hope for treating consolidated psychostimulant - seeking behavior and relapse. in the last 25 years the neurobiological and behavioral mechanisms that lead to drug dependence have been extensively investigated but clinical treatment is still unsatisfactory and ineffective in many subjects. experimental models are now providing evidence for the pharmacological management of endocannabinoid signaling not only to block the direct reinforcing effects of cannabis, opioids, nicotine and ethanol, but also to prevent relapse to these various substances of abuse, also including cocaine and metamphetamine. the endocannabinoid system can be manipulated by sr141716a and by all the new compounds that protect aea and 2-ag from deactivation and prolong the lifespan of these endocannabinoid substances in vivo. rimonabant reduces the motivational effect of drug - related stimuli and drug re - exposure, probably by altering synaptic plasticity, thus providing an effective means of preventing relapse and a new tool for the treatment of drug abuse. although further studies are needed to clarify the precise mechanism underlying the endocannabinoid system s role in addiction, some promising clinical findings have now been presented (eg, smoking cessation). a new question has now arisen from the discovery of cb2 receptors in the brain : are they involved in drug addiction ? | this review will discuss the latest knowledge of how the endocannabinoid system might be involved in treating addiction to the most common illicit drugs. experimental models are providing increasing evidence for the pharmacological management of endocannabinoid signaling not only to block the direct reinforcing effects of cannabis, opioids, nicotine and ethanol, but also for preventing relapse to the various drugs of abuse, including opioids, cocaine, nicotine, alcohol and metamphetamine. preclinical and clinical studies suggest that the endocannabinoid system can be manipulated by the cb1 receptor antagonist sr141716a, that might constitute a new generation of compounds for treating addiction across different classes of abused drugs. |
currently the influenza vaccine is recommended in the united states for all individuals over 6 months of age and by the world health organization for high - risk groups. these recommendations have, in part, the aim to reduce the worldwide estimated deaths 250,000500,000 each year caused by the seasonal form of the disease. pregnant women are included in these recommendations because vaccination with the inactivated virus has been shown to decrease the burden of suffering among neonates [46 ] and to reduce maternal morbidity of the infection. furthermore, vaccination in pregnancy has been documented as sufficiently safe to be recommended to be given regardless of trimester [4, 8 ]. despite these recommendations in pregnancy, coverage of pregnant women in the united states has been low in the years prior to the 2009 - 2010 influenza season (11% to 24%). reasons cited for this include not being offered the vaccine, not having the vaccine in the office, and maternal concerns for vaccine safety [9, 10 ]. in the spring of 2009, a new swine - origin form of the influenza a virus, causing substantial morbidity and mortality, was isolated : h1n1. as part of the pandemic strategy for this virus, a specific monovalent vaccine for h1n1 was developed and was ready for distribution in the fall of 2009. among the high - risk groups targeted to receive this vaccine, in addition to the seasonal vaccine, were pregnant women. the rationale for this included the high mortality rates associated with influenza among pregnant women in earlier pandemics and early reports of the severity of h1n1 illness in pregnancy, [1416 ] which proved to be true as the 2009 to 2010 influenza season progressed [17, 18 ]. recently, population - based data on vaccine coverage of pregnant women in the united states during the 2009 - 2010 pandemic were published by the centers for disease control (cdc). this report revealed less than full coverage during that season despite a robust public - health response, with self - reported coverage rates of 51% for the seasonal vaccine and 47% for the h1n1 vaccine. the purpose of this study is to compare these national rates to those in a public safety - net hospital system serving a large number of uninsured patients, using chart - verified documentation of vaccination. this was a retrospective cohort study of pregnant patients who delivered at maricopa medical center during the 2009 - 2010 influenza pandemic. in order to be eligible, subjects must have received prenatal care at the hospital or one of the nine associated prenatal clinics preceding their delivery. it serves as a public safety - net facility for maricopa county (population 3.8 million) which includes phoenix, arizona. efforts to promote influenza vaccine coverage for pregnant patients during the 2009 - 2010 influenza season included education of providers via grand rounds, small group in - service sessions, and emailed recommendations for clinicians providing prenatal and intrapartum care. standing orders for nursing staff to administer the vaccine without a physician signature were also written and distributed. indirect efforts to promote vaccination included activities to increase awareness, such as signage regarding influenza, distribution and implementation of influenza - like illness (ili) protocols, phone followup of ili patients not requiring admission, and backup call schedules for attending physicians, as well as infection control activities with screening of patients and visitors for ili in our health care facilities. we did not have an electronic medical record with a vaccine registry to identify prospectively those who had not been previously vaccinated. in order to be consistent with the cdc 's study we included births which occurred from when the vaccines were first available in our health system, september 15, 2009 for the seasonal vaccine and october 15, 2009 for the h1n1 vaccine. a random sample of subjects was selected for review from a billing database of 939 deliveries which occurred during the september 15, 2009 to april 30, 2010 time frame. charts were first reviewed for eligibility : receipt of prenatal care at mihs. at the beginning of the chart review, a sample of charts (the first 10 charts by each reviewer) was reviewed by a second reviewer to assure accurate capture of the study variables. in addition, in order to be certain that vaccine administration was not missed in the chart review, a second review of charts with no documented vaccination was undertaken by a different reviewer. predictor variables were also collected during the chart review and included age at delivery, gestational age at delivery, race / ethnicity, patient language, and gravidity and parity. the outcome was receipt of the seasonal vaccine and/or the h1n1 vaccine as determined by documentation on progress notes or vaccination forms. our targeted sample size was 200, which would allow us to be at least 95% certain that the observed vaccination rate falls within 6.9% of the true value. prevalence rates of vaccine coverage and 95% confidence intervals were calculated for the 2 vaccines for different time periods as noted in section 3. chi - square statistics were used to determine statistical significance with a p value of < 0.05 considered significant. this study was approved by the mihs institutional review board via expedited review on november 10, 2011. a random sample of 296 of the 939 deliveries which occurred in the study time frame was reviewed. of these the seasonal influenza sample comprised a total of 246 women who delivered between september 15, 2009 and april 30, 2010, and the h1n1 sample comprised. 217 who delivered between october 15, 2009 and april 30, 2010 table 1 provides a description of the seasonal influenza sample. the sample was representative of our population ; mainly hispanic, non - english speaking, without insurance, and of high parity. in the seasonal influenza sample, 132 of the 246 subjects (54%) received the seasonal vaccine, (95% confidence interval, 48% to 60%). in order to more directly compare the rates with national rates as presented by the cdc, the sample was restricted to births occurring until march 12, 2010. of these 216 subjects, 61% received the vaccine (95% confidence interval, 55% to 68%). in the h1n1 sample, 111 of 217 (51%) received the h1n1 vaccine, (95% confidence interval, 44% to 58%). in the sample restricted to births through march 12, 2010, 111 of 187 (59%) received the h1n1 vaccine (95% confidence interval, 52% to 66%). the corresponding rate for receipt of both vaccines from october 15, 2009 to april 30, 2010 was 33% (72 of 217) (95% confidence interval, 27% to 40%). when restricted to births through march 12, 2010, the corresponding rate was 39% (72 of 187 ; 95% confidence interval, 32% to 46%). our commitment to vaccinate against influenza required extra, nonroutine efforts in the form of education and mandatory separate written orders or standing orders, both of which involved separate paperwork. to understand the role of the additional documentation effort, we compared rates of influenza vaccination with rates of vaccination associated with the tetanus, diphtheria, and pertussis (tdap) vaccine that is written into all preprinted postpartum orders. a total of 201 of 246 (82%) received the tdap vaccine (95% confidence interval : 76% to 86%). none of the demographic and reproductive characteristics were significantly related to the receipt of either the seasonal or the h1n1 vaccine. this indicates that, in our health system, primary language, race / ethnicity, and even insurance status were not barriers to the receipt of the vaccine. the only significant predictor in table 2 was receipt of the other influenza vaccinse, that is, receipt of the seasonal vaccine was predictive of a higher likelihood of receiving the h1n1 vaccine and vice versa. in our health system during the 2009 - 2010 influenza season, we found rates of vaccination of pregnant women of 54% for the seasonal influenza vaccine and 51% for the h1n1 vaccine. when restricted in a manner similar to a 10-state sample (births through march 12, 2010) our rates were approximately 10% higher : seasonal vaccine coverage was 61% versus 51% and h1n1 coverage was 59% versus 47%. this is notable since our population serves pregnant women who have been shown in previous studies to be less likely to receive the vaccine, those who are disadvantaged, that is, uninsured, nonwhite, less educated, and of low income [2022 ]. other studies have reported on vaccination coverage of pregnant women in the 2009 - 2010 pandemic influenza season, many of which rely on self - report. in the united states, a nationally representative phone survey found coverage rates of 32% for the seasonal vaccine and 46% for the h1n1 vaccine. a study at a university hospital in denver, co, found higher coverage rates of 64% for the seasonal vaccine and 54% for the h1n1 vaccine. unlike our study, the denver study relied on self - report and demonstrated high rates of vaccination outside their health system, 15% for the seasonal vaccine and 29% for the h1n1 vaccine. a high rate of h1n1 vaccine coverage (76%) was achieved in a public hospital prenatal clinic in seattle, wa, using a system which included chart prompts and a vaccine registry. the highest rates were reported at the massachusetts general hospital with coverage rates of 88% and 86% for the seasonal and the h1n1 vaccines, respectively. their sample was restricted to 3 months of the influenza season (january 2010 to march 2010) which could have raised these estimates as compared to other studies, since both vaccines were more readily available at that time. a time difference might in part explain the lower coverage rates of the h1n1 vaccine (26%) reported in one health system, maimonides medical center in brooklyn, ny, as the sample began shortly after the h1n1 vaccine was available (time frame of november 12 to december 19, 2009). rates of seasonal coverage were reported to be 30% in alberta, while for h1n1 the rates have been reported to be between 63% and 76% [29, 30 ]. in france, rates of the h1n1 coverage of pregnant women were lower : 38% in a 3-hospital study in paris and 22% using a national insurance database. another insurance database from australia reported even lower estimated coverage rates of 10% for the h1n1 vaccine in pregnant women from october 2009 to january 2010. with one exception, all of the rates are lower than the 80% coverage rates targeted by the national healthy people 2020 objectives for pregnant women. however, most represent large increases over the prior year rates, which appear to be sustained with 49% pregnant women reporting being vaccinated during the 2010 - 2011 influenza season. in our health system we achieved rates higher than nationally reported rates ; we attribute this to a comprehensive approach to influenza. for example, we undertook many of the prevention strategies cited by goldfarb. in their successful program. our actions included standing orders and awareness activities, both of which have been demonstrated to increase vaccination rates. reasons for our rates being lower than the highest reported rates may have included not having chart prompts or not making use of a vaccine registry. furthermore, our system of prenatal care includes clinics spread over a large geographic area, making messaging and implementing programs challenging. however, streamlining the order process as we have been able to do with the tdap vaccine suggests that even higher rates can be achieved. it was based on chart review which indicates that we can be relatively certain of the lower limit of our vaccination rates. it is possible that actual coverage rates may be higher, especially in view of the high rates of vaccination outside of the health system reported by fisher.. however, not knowing the magnitude of the outside coverage in our population, we would not want to speculate on how much higher the real coverage rates would have been. another advantage of the chart review methodology is that many of the studies rely on self - report, which may have been an issue in the 2009 to 2010 season when 2 vaccines were available. our own experience with our population suggests this may have occurred, with a frequent response of, i already received the flu vaccine, when they had received only one of the recommended two vaccines. furthermore, many of the studies relying on self - report had substantial nonresponse rates of approximately 50% ; this included studies with high reported coverage rates or those that were nationally representative [23, 25, 27 ]. a limitation of our study is that it represents the cumulative efforts of one health system caring for the underserved, as such these results may not be generalizable to other populations or health systems. finally, we do not know which of our interventions impacted vaccination rates the most. our study has a number of implications. for other health systems it would appear that improved vaccination rates as compared to the national rates are possible, even for health systems that are safety - net systems that provide medical care to large numbers of underserved patients. our success with a population which usually experiences lower coverage rates minorities, uninsured, and with limited english - speaking ability could be a model for other systems. taken with goldfarb 's study, we also believe a comprehensive approach might be a rational strategy to achieve high coverage rates. given initial reports of continued high rates in pregnant patients over the subsequent influenza season to this study, it will be interesting to see if high vaccination rates continue in this population. finally, given that self - report might overestimate and chart review might underestimate coverage rates, it would be interesting to supplement chart review with self - report to further characterize this in our population. moreover, self - report in such a study would assist in determining the upper limit achievable by segregating those who represent missed opportunities versus those who refuse the vaccine. in summary, our public safety - net institution achieved influenza vaccine coverage rates, that were higher than those reported national samples by instituting a comprehensive strategy. this could be due to not capturing vaccinations done outside our health system, not including certain interventions in our vaccination strategy such as chart prompts, and not including an order for influenza vaccination on all postpartum orders. | the purpose of this study was to compare influenza vaccination rates of pregnant women in a public safety - net health system to national coverage rates during the 2009 - 2010 pandemic influenza season. a chart review of a random sample of deliveries was undertaken to determine rates of coverage and predictors of vaccine coverage of women who obtained prenatal care and delivered in our health system. rates were calculated from deliveries from when the vaccine was first available through april 30, 2010. coverage rates were 54% for the seasonal influenza vaccine and 51% for the h1n1 vaccine. race / ethnicity, insurance status and language spoken did not predict the receipt of either vaccine. when we included only births which occurred through march 12, 2010, as was done in a large population - based study, the rates were 61% and 59%, respectively. our rates are about 10% higher than the rates reported in that study. our comprehensive strategy for promoting vaccine coverage achieved higher vaccination rates in a safety - net health system, which serves groups historically less likely to be vaccinated, than those reported for the pregnant population at large. |
the recurrent laryngeal nerve (rln) injury during surgeries on thyroid and parathyroid remains the most significant commonly found complication of endocrine surgery in the neck, and it can result in significant morbidity including temporary or permanent paralytic dysphonia and dysphagia. rates of nerve injury published in the literature typically range from 1% to 2% and are significantly higher for re - operation. it has long been accepted that anatomic identification of both the rln and the external branch of the superior laryngeal nerve (ebsln) is the safest way to reduce nerve injury rates to a minimum and certainly injury rates are lowest when surgery is carried out by experienced endocrine surgeons or thyroid surgeons in specialized centers with high caseloads. nonetheless, identification and preservation of the rln an anatomically intact nerve may still show altered function postoperatively due to multiple factors, such as neural stretch during goiter retraction. the study group comprised of forty - seven patients who underwent thyroid or parathyroid surgery at tulane university medical center from october of 2007 to april of 2009. the protocol for assessment of the laryngeal nerves included routine preoperative and postoperative laryngoscopy at one to two weeks. stroboscopy and electromyography (emg) assessment were not used in this study. a standard mivat / p gasless approach to the thyroid gland was performed under general anesthesia. the surgeon stands at the lesion side, the camera operator on the other side, and an assistant at the patient 's head. all vessel ligation during the procedure was done using the harmonic scalpel (ethicon, nj). additionally, the harmonic scalpel was also used to divide the isthmus in lobectomies, to isolate the gland from the trachea. after routine attempted anatomical observation of the rln, stimulation of the nerve was done with a hand - held nerve stimulator with either the medtronic varistim iii nerve stimulator (medtronic inc., minneapolis, mi, usa) or the nervona system (nervona, ca, usa) at currents of 1 to 2 ma on completion of dissection [2, 5 ]. the rln was stimulated at the most proximal exposed site of the nerve (figure 1). data for continuous variables are expressed as median and range, unless specified otherwise. the outcome measures primarily the morbidity and mortality. a total of seventy - seven nerves in forty - seven patients were included in this study. there were twenty - three total thyroidectomies (48.9%), nineteen hemithyroidectomies (41.3%), 10.6% and ten parathyroidectomies (10.6%). out of seventy - seven rlns, there was one permanent unilateral rln injury (1.29%) in a patient with advanced papillary thyroid cancer managed by postoperative cord injection. no hemorrhage or cervical hematoma was observed, and none of our cases developed wound infection (figure 2). one of the most common operations throughout the world is thyroidectomy, and it has a low morbidity rate if performed by skilled surgical teams. conventional thyroidectomy requires a transverse cervical incision that leaves a visible scar on the anterior surface of the neck. the application of minimally invasive techniques for thyroid surgery was primarily motivated by the attempt to improve the cosmetic result of this operation. the aesthetic point of view is particularly important for young women, who constitute a large part of patients affected by thyroid diseases. most mivat / p allows for a prompt postoperative recovery and is performed as an outpatient procedure in some clinical settings. it has been recently demonstrated that endoscopic and video - assisted [911 ] procedures for thyroid and parathyroid surgery have some advantages over conventional methods of surgery in terms of not only cosmetic results but also analgesic requirements and postoperative recovery. on the other hand, it should be pointed out that these procedures are not easy or feasible in all clinical settings. they are technically demanding and require a surgical team skilled in both endocrine and endoscopic surgery. the endoscopic and video - assisted procedures require learning and a training period that can be time consuming, especially at the beginning of the experience. however, with training, learning, and experience, along with the continuous development in scientific medical technologies, mivat / p will be done in more centers and more candidates will be qualified for these kinds of surgeries. the recurrent branch of the laryngeal nerve (rln) innervates all of the laryngeal muscles except the cricothyroid muscle, which regulates the tension of the vocal cords and is innervated by the external branch of superior laryngeal nerve. if damage to the rln is unilateral, the patient may present with voice changes including hoarseness. bilateral nerve damage can result in breathing difficulties and aphonia, the inability to speak. the right recurrent laryngeal nerve is more susceptible to damage during thyroid surgery due to its relatively medial location. the nerve injury in this series was on the right side. such anomalies include nonrecurrence of the rln, rln displacement by thyroid nodularity or paratracheal lymphadenopathy, extralaryngeal branching of the rln, and variations of the nerve course in relation to the inferior thyroid artery and ligament of berry. in a recent analysis of rln anatomy in 491 thyroid surgery cases, 60.8% of rlns were found in the expected tracheoesophageal groove position, whereas 4.9% were lateral and 28.3% were posterior to the trachea. of greatest concern are those cases where the rln is found on the anterior surface of the thyroid gland, a particularly high - risk area for nerve injury. the surgical importance of this nerve relates to its proximity to the superior thyroid vessels. to preserve the rln, most surgeons tend to avoid rather than expose the nerve, suggesting selective ligation of the upper pedicle vessels. miccoli and colleagues introduced the minimally invasive video - assisted thyroidectomy procedure in 1998. during mivat /p, most surgeons do not use ionm to search for the rln. in our series consisting of forty - seven operations, we were able to visualize the rln in 100% of the cases. among these, the nerve ran entirely medial and separate to the superior thyroid artery in most of the patients. ionm of the rln during thyroid and parathyroid surgeries has been claimed in some studies to reduce the rate of nerve injury [15, 16 ] ; however, other studies have shown it to be of no benefit and no reduction in the risk of rln injury [1720 ] compared with anatomic identification. however, hermann. showed that ionm could be useful in identifying the rln especially when anatomically altered by prior surgery or large masses. numerous studies have shown that only routine exposure of the rln is associated with very low rates of injury in high - volume centers (less than 0.3%). during mivat / p, the surgical incision is really small in most cases to allow palpation of the nerve or the larynx as methods of identification. in our institution, we have been using ionm as a method of rln identification during mivat / p operations. now we are reporting our experience retrospectively. at the beginning of implementing both technologies together, we hypothesized that this method of monitoring the rln would decrease / prevent the risk of iatrogenic injury with lesser risk to the patient. our initial clinical results were very promising showing that the use of ionm is easy and feasible in mivat / p, and it was rather safe with no intraoperative complications or conversion to traditional open approach. though the operating space in these kinds of endoscopic surgeries is relatively narrow, there was no difficulty using the ionm simulator probe. the tip of the monopolar probe is flexible, which helped the surgeon to go beyond the view of his field. the rln may get injured while it still looks intact without the nerve being actually cut. this can be caused by different sorts of injuries, which include, but are not limited to, traction, pressure, ischemia, ligation, crush, electrical or suction injury [23, 24 ]. the role of ionm is not only during surgical dissection. its role extends after the operation as it can help detect how the rln is functioning at the end of the procedure. this provides the opportunity to stage contralateral surgery in case of suspected rln injury, thus avoiding the risk of rln injury bilaterally. we also would like to mention that in our experience, we never had device failure or any unexpected events with the use of ionm during mivat / p. our experience of ionm with mivat / p showed that ionm could be a very useful adjunctive method for rln identification besides endoscopic visualization and magnification. this possibly will decrease rln injury and improve the quality and outcomes of mivat / p operations. kern discussed an important point that nerve monitoring during surgeries can possibly decrease the medico - legal liability. however, the literature needs a study with a wider scale of patients to accurately prove our citations. to show a decrease in rln paralysis rates from 2% to 1% per nerves at risk, a study group of approximately 1,000 patients would be necessary. standardization of the technique and wider availability of these technologies in centers performing these kinds of operations would be of great benefit. | objective. our goal is to study the feasibility of using intraoperative neuromonitoring (ionm) in minimally invasive video - assisted thyroidectomy and parathyroidectomy (mivat / p) with emphasis given to the identification of recurrent laryngeal nerve (rln). methods. consecutive series of forty - seven patients with seventy - seven recurrent laryngeal nerves at risk undergoing both mivat / p and ionm were enrolled in this retrospective, nonrandomized analysis study. all operations were performed by the same surgeon within an academic institution setting. all patients underwent vocal cord evaluation postoperatively. demographics and intraoperative and postoperative complications following surgery were collected. results. out of seventy - seven rlns, there was one permanent unilateral rln injury (1.29%) in a patient with advanced papillary thyroid cancer, managed by cord injection. there was another transient rln paresis that resolved spontaneously (1.29%). there were no instances of equipment malfunction or interference. conclusions. to our knowledge, this is the first reported mivat / p series from the united states of america with a standardized ionm technique. the technical feasibility of ionm seems acceptable and may serve as a meaningful adjunct to the visual identification of nerves. neuromonitoring during mivat / p is effective in providing identification of laryngeal nerves and enables surgeons to feel more comfortable with mivat / p. comparative series are needed for further evaluation. |
it is a failure to report, respond to, or orient towards stimuli located on the contralesional side. it also occurs when converting not only a visual landscape but also body schema to a visual image2. spatial neglect is caused by a disturbance of both hemispheres after unilateral cortical lesions3. spatial neglect is more common after a right hemisphere lesion than after a left hemisphere lesion4. when the right hemisphere is damaged, spatial neglect leads to visual perception problems such as asymmetrical division of lines or failure to perceive the picture of the neglected side5. there are diverse methods that can be used to treat spatial neglect such as perception retraining, visual scanning treatment, and cognitive therapy but more effective and diverse interventions for the treatment of spatial neglect are necessary6. neglect patients show multiple eye movement impairments, including reduced saccade amplitude and difficulty in retaining spatial locations across saccades7. eye movements are increasingly being used as a tool for the elucidation of relatively complex neuropsychological processes relating to attention, spatial memory, motivation and decision - making8. oculo - motor exercise modulates many facets of neglect syndrome, and pursuit eye movement especially represents an effective and easily applicable technique for the treatment of neglect patients9. karnath reported that oculo - motor exercise was effective at improving body orientation in spatial neglect patients10. functional electrical stimulation (fes) has been developed as a method for artificially activating the sensory motor system after central nervous system injury11. it is useful for activating muscle paralyzed by upper motor neuron injury, and is used to strengthen weakened muscles, to decrease spasticity, and to enhance the range of motion of joints12. rushton noted that fes activates motor and sensory nerve fibers and promotes cortical reorganization through sensory stimulation of paralyzed muscle13. fes also activates a proprioceptive map within the right parietal lobe, and alleviates unilateral spatial neglect14. most stroke patients experience loss of proprioceptive sensation, which results in body sway increases, and failure to receive appropriate location information about the body reduces their efficiency of movement15. the proprioceptive neuromuscular facilitation (pnf) approach utilizes a typical diagonal pattern to stimulate proprioceptive sensation16. the pnf patterns may permit muscles to act in ways that are close to the actions and movements17. pnf can also have a positive effect on the active and passive ranges of motion18. silva and johnson reported that proprioceptive afferent input from the neck muscles plays an important role in postural control19. and kim and oh reported that the neck is an essential component in the regulation of head and body orientation in space, and is necessary for maintaining balance20. accordingly, this study applied fes and pnf to the neck area, to increase the proprioceptive input to the neck muscles and induce head movement. there have been many studies on balance and gait using oculo - motor exercise, fes, and pnf with stroke patients as subjects, but studies analyzing the visual perception of neglect patients are lacking. accordingly, this study examined the effects of fes and pnf with oculo - motor exercise on the visual perception of stroke patients with spatial neglect. this study was conducted at the rusk rehabilitation hospital and lohas hospital located in gyeonggi - do, and the subjects were 30 stroke patients with spatial neglect. in order to select neglect patients, patients scoring 11 or higher on the catherine bergego scale (cbs) were included21. the subjects did not have visual or hearing disorder, and their score in the korean version of the mini - mental state exam was 24 points or higher22. table 1table 1.general characteristics and cbs values of the subjectsomefomepomegendermale4 75female635age (years)60.27.856.77.761.18.1time after stroke (months)22.210.228.612.123.37.0stroke typeinfarction563hemorrhage547affected sideleft101010right000cbs (score)13.11.612.71.413.11.6ome : oculo - motor exercise group, fome : fes with oculo - motor exercise group, pome : pnf with oculo - motor exercise group, cbs : catherine bergego scale outlines the general characteristics and cbs values of the subjects. ome : oculo - motor exercise group, fome : fes with oculo - motor exercise group, pome : pnf with oculo - motor exercise group, cbs : catherine bergego scale this study complied with the ethical principles of the declaration of helsinki. the subjects agreed to participate in the study after receiving explanations regarding the purpose and procedures of the experiment, and signed an informed consent statement before participation. the protocol for this study was approved by the local ethics committee of yongin university (2 - 1040966-ab - n-01 - 201503-hsr-025 - 1). the subjects were equally and randomly divided into three groups : an oculo - motor exercise (ome) group, a fes with oculo - motor exercise (fome) group, and a pnf with oculo - motor exercise (pome) group. the intervention was conducted five times per week, for a total of six weeks. schematic diagram of the study the oculo - motor exercise was designed according to the method used by morimoto. a total of four different exercises were performed (saccadic eye movement, smooth pursuit exercise, and the adaptation 1 & 2 exercises). the saccadic eye movement exercise is a movement of the eyes horizontally between two stationary targets while keeping the head still. the smooth pursuit exercise involves moving the targets horizontally and tracking them with the eyes while keeping the head still. the adaptation 1 exercise involves moving the head horizontally while keeping the stationary target in focus. the adaptation 2 exercise requires movement of the head and a target in opposite horizontal directions while tracking the target with the eyes (fig. 2.oculo-motor exercise (1 : saccadic eye exercise, 2 : smooth pursuit exercise, 3 : adaptation 1 exercise, 4 : adaptation 2 exercise))23. those in the ome group each conducted two sets of each exercise, with each exercise performed 10 times per set. oculo - motor exercise (1 : saccadic eye exercise, 2 : smooth pursuit exercise, 3 : adaptation 1 exercise, 4 : adaptation 2 exercise) the fome group performed the oculo - motor exercise program and fes was additionally applied to the neck area of the paretic side, to induce movement of the head. the fes equipment used was a microstim (medel gmbh, germany) was used and two electrodes were attached to the origin and insertion of the paretic splenius capitis of the neck extensor. fes was applied for a total of 15 minutes and the electricity on - time and off - time were six and two seconds, respectively. the frequency was set at 30 hz, and the stimulation intensity was less than 15v, to avoid muscle contraction. the pome group performed the oculo - motor exercise program, and pnf additionally was applied to the neck area, to induce movement of the head. the pnf pattern used was a neck extensor pattern, and the technique used was a contract - relax technique. the starting posture was neck flexion, followed by right rotation, and left lateral flexion. the last posture was neck extension, followed by left rotation, and right lateral flexion. the subjects conducted exercises for a total of three sets, 10 exercises per set. the pnf training was conducted by a therapist who had completed pnf courses at levels iand ii. anova was used to evaluate the change in balance and head alignment. in all analyses, this study involved thirty subjects : 10 subjects in the ome group, 10 subjects in the fome group, and 10 subjects in the pome group. the line bisection test (lbt), motor free visual test (mvpt), and catherine bergego scale (cbs) were used as outcome measures. changes in visual perception within each group are shown in table 2table 2.comparison of the pre - test and post - test visual perception tests resultspre - testpost - testome grouplbt (mm)15.12.613.23.0mvpt (score)17.72.218.71.8cbs (score)13.11.611.31.4fome grouplbt (mm)16.11.315.71.5mvpt (score)16.51.316.81.2cbs (score)12.71.411.71.4pome grouplbt (mm)15.22.513.32.5mvpt (score)17.82.218.51.9cbs (score)13.11.611.81.7p0.05). however, a significant difference was found in the cbs results (p<0.05). lbt : line bisection test, mvpt : motor - free visual perception test, cbs : catherine bergego scale table 3table 3.comparison of the groups visual perception tests resultsmsdduncanlbt (mm)ome13.23.0b < a, cfome15.71.5pome13.32.5mvpt (score)ome18.71.8b < a, cfome16.81.2pome18.51.9cbs (score)ome11.31.4fome11.71.4pome11.81.7p<0.05. a : ome group, b : fome group, c : pome group presents a comparison of the results of the groups. in the ome and pome groups, the lbt and mvpt results were significantly different from those of the fome group (p<0.05). however, according to the cbs test, there were no significant differences among the three groups. spatial neglect is characterized by a lack of awareness of sensory events located towards the contralesional side of space. indeed, neglect patients often behave as if half of their world is no longer in existence24. this study conducted fes and pnf with oculo - motor exercises, and compared their results in order to verify their effects on the visual perception of spatial neglect patients. the ome group showed significant improvements according to the lbt, mvpt, and cbs results. noted that saccadic eye movement and pursuit eye movement are organized in the cerebral cortex and play an important role in spatial memory and concentration8. kerkhoff. reported that oculo - motor exercise activates multiple brain regions (temporo - parietal cortex, basal ganglia, brainstem, cerebellum) involved in auditory and visual space coding9. therefore, the physiological effects of oculo - motor exercise are considered to have positively affected the visual perception of the spatial neglect patients in this study. in the fome group, there were no significant improvements according to the lbt and mvpt results, but there was a significant difference according to the cbs result. reported that fes can enhance the recovery of upper extremity function in stroke patients25. fes has been developed to restore function to the upper extremity, lower extremity, bladder, bowel, and respiratory system26. however, in this study, fes with oculo - motor exercise did not have an effect on the visual perception of the spatial neglect patients. the pome group showed significant improvements according to the lbt, mvpt, and cbs results. karnath reported that visual input, together with vestibular and neck proprioceptive input has a positive effect on the body orientation of spatial neglect patients10. this supports the notion that pnf training applied to the neck area has a positive effect on visual perception. furthermore, hindle. reported that the contract - relax technique of pnf is effective at improving and maintaining range of motion18. it is considered that the contract - relax technique applied to the neck area in this study increased the range of motion of the neck, positively affecting visual perception. reported that head movement triggered coordination of the arms and legs, positively affecting postural balance27. therefore, head movement through the pnf technique is considered to have had a positive effect on postural adjustment as well as visual perception. in the comparison of the three groups however, the lbt and mvpt results showed there were significant differences between the ome and pome groups and the fome group. this indicates that the intensive application of oculo - motor exercises or pnf with oculo - motor exercise is more effective than fes with oculo - motor exercises in the improvement of the visual perception of spatial neglect patients. the number of subjects included in this study was insufficient for the generalization of the results to all spatial neglect patients. however, the results indicate that head movement through oculo - motor exercise and pnf is effective and more diverse interventions should be developed for spatial neglect patients. | [purpose ] the purpose of this study was to identify the effects of oculo - motor exercise, functional electrical stimulation (fes), and proprioceptive neuromuscular facilitation (pnf) on the visual perception of spatial neglect patients. [subjects and methods ] the subjects were randomly allocated to 3 groups : an oculo - motor exercise (ome) group, a fes with oculo - motor exercise (fome) group, and a pnf with oculo - motor exercise (pome) group. the line bisection test (lbt), motor free visual test (mvpt), and catherine bergego scale (cbs) were used to measure visual perception. these were performed 5 times per week for 6 weeks. [results ] the ome group and pome group showed significant improvements according to the lbt and mvpt results, but the fome group showed no significant improvement. according to the cbs, all 3 groups showed significant improvements. the ome and pome groups showed improvement over the fome group in the lbt and mvpt. however, there was no significant difference among the three groups according to the cbs. [conclusion ] these results indicate that oculo - motor exercise and pnf with oculo - motor exercise had more positive effects than fes with oculo - motor exercise on the visual perception of spatial neglect patients. |
today s world is a world of organizations, and their main operators are the people who invigorate them and prepare the grounds for the realization of their objectives. without people, the concepts of organization and management would become meaningless (1). therefore, professional satisfaction on the part of manpower is one of the most important factors in the efficacy of organizations. the issue of professional satisfaction has been studied repeatedly with respect to various organizations since the 1920s, and, according to many experts, it is one of the most challenging organizational concepts and the basis behind the many management policies adopted to improve organizational effectiveness (2). health care organizations and treatment centers are responsible for maintaining the health of all citizens in a society, and nurses have a key role. their success in fulfilling this role depends on the measures taken by competent managers who recognize the prominent factors involved in the activities of nurses and seek to create favorable work conditions in order to enhance their professional satisfaction (1). previous studies have shown that nurses should demonstrate a passion for their work and have the necessary proficiency and expertise to achieve the highest level of efficiency (2). it could be argued that any person would provide better services if he or she were motivated and enthusiastic about the work. despite the great significance of the professional satisfaction of nurses due to its considerable impact on them, their patients, their organizations, and the very profession of nursing (3), most previous studies have reported the satisfaction level to be low among nurses (4). the results from the study conducted by lou. indicated that only half of the nurses in the study were moderately or highly satisfied with their work (5). another study was conducted to compare the level of professional satisfaction among different groups of people in korea, and it was found that only 41.5% of nurses were satisfied with their work, whereas the percentages for other experts working at hospitals, social workers, and middle school teachers were 50.1, 58.2, and 89%, respectively. these statistics indicate that there is a lower level of satisfaction among nurses than other professionals (6). various studies have been conducted regarding the extent of professional satisfaction among the nurses, and, sadly, all have indicated low degrees of satisfaction (7). most nurses who work in internal medicine and surgical departments (54.4%) demonstrated low levels of professional satisfaction (8). overall, these studies suggested that the situation concerning nurses professional satisfaction is unacceptable. it should be noted that a lack of sufficient concern towards professional satisfaction will result in serious consequences in the long run. it could give rise to rebellion, diminished feelings of responsibility, and eventually resignation from the job (9). professional dissatisfaction among nurses could result in emotional detachment, depression, anger, evasion from work, and ineffectiveness (10). according to a qualitative study done in 2010 in india, the reasons nurses quit their jobs were determined to be professional exhaustion, stress at work, and lack of a friendly environment, all of which contributed to their diminished professional satisfaction. in addition, it was indicated that a strong relationship exists between professional dissatisfaction and quitting work (11). according to released statistics, the insufficient number of available nurses around the world is a tangible phenomenon. in australia, for instance furthermore, according to estimations, by 2010, canada will need an additional 113,000 nurses, and, by 2020, the united states would incur a 20% shortage in the number of nurses (approximately 581,500 nurses) (12, 13). in iran, one of the most problematic issues in nursing is the shortage of nursing staff. the shortage of nursing staff has different causes, including job dissatisfaction and organizational and sociocultural factors (14). in iran, many studies have reported that nurses are not happy because of the shortage of nursing staff, high numbers of patients, the lack of independence and authority, the lack of appreciation and acknowledgment, low income, heavy workloads, low level of involvement in management, disagreements with physicians, managerial issues, and a lack of appropriate work conditions in hospitals (15). iran regret having become nurses, whereas 43.9% of them feel disappointed and dissatisfied that they became nurses. the most challenging problem in nursing in iran is the shortage of nurses working in hospitals because of their dissatisfaction and lack of motivation, and, unfortunately, most nurses in iran would like to change their work (14). because satisfaction with nursing is related to improving the health system and patient satisfaction, this study was conducted to assess how the satisfaction of nurses with their work can be improved, their situation in hospitals can be improved, and also how patients satisfaction could be increased (15). the objective of this research was to determine iranian nurses experiences of the concept of professional satisfaction. today s world is a world of organizations, and their main operators are the people who invigorate them and prepare the grounds for the realization of their objectives. without people, the concepts of organization and management would become meaningless (1). therefore, professional satisfaction on the part of manpower is one of the most important factors in the efficacy of organizations. the issue of professional satisfaction has been studied repeatedly with respect to various organizations since the 1920s, and, according to many experts, it is one of the most challenging organizational concepts and the basis behind the many management policies adopted to improve organizational effectiveness (2). health care organizations and treatment centers are responsible for maintaining the health of all citizens in a society, and nurses have a key role. their success in fulfilling this role depends on the measures taken by competent managers who recognize the prominent factors involved in the activities of nurses and seek to create favorable work conditions in order to enhance their professional satisfaction (1). previous studies have shown that nurses should demonstrate a passion for their work and have the necessary proficiency and expertise to achieve the highest level of efficiency (2). it could be argued that any person would provide better services if he or she were motivated and enthusiastic about the work. despite the great significance of the professional satisfaction of nurses due to its considerable impact on them, their patients, their organizations, and the very profession of nursing (3), most previous studies have reported the satisfaction level to be low among nurses (4). the results from the study conducted by lou. indicated that only half of the nurses in the study were moderately or highly satisfied with their work (5). another study was conducted to compare the level of professional satisfaction among different groups of people in korea, and it was found that only 41.5% of nurses were satisfied with their work, whereas the percentages for other experts working at hospitals, social workers, and middle school teachers were 50.1, 58.2, and 89%, respectively. these statistics indicate that there is a lower level of satisfaction among nurses than other professionals (6). various studies have been conducted regarding the extent of professional satisfaction among the nurses, and, sadly, all have indicated low degrees of satisfaction (7). most nurses who work in internal medicine and surgical departments (54.4%) demonstrated low levels of professional satisfaction (8). overall, these studies suggested that the situation concerning nurses professional satisfaction is unacceptable. it should be noted that a lack of sufficient concern towards professional satisfaction will result in serious consequences in the long run. it could give rise to rebellion, diminished feelings of responsibility, and eventually resignation from the job (9). professional dissatisfaction among nurses could result in emotional detachment, depression, anger, evasion from work, and ineffectiveness (10). according to a qualitative study done in 2010 in india, the reasons nurses quit their jobs were determined to be professional exhaustion, stress at work, and lack of a friendly environment, all of which contributed to their diminished professional satisfaction. in addition, it was indicated that a strong relationship exists between professional dissatisfaction and quitting work (11). according to released statistics, the insufficient number of available nurses around the world is a tangible phenomenon. in australia, for instance furthermore, according to estimations, by 2010, canada will need an additional 113,000 nurses, and, by 2020, the united states would incur a 20% shortage in the number of nurses (approximately 581,500 nurses) (12, 13). in iran, one of the most problematic issues in nursing is the shortage of nursing staff. the shortage of nursing staff has different causes, including job dissatisfaction and organizational and sociocultural factors (14). in iran, many studies have reported that nurses are not happy because of the shortage of nursing staff, high numbers of patients, the lack of independence and authority, the lack of appreciation and acknowledgment, low income, heavy workloads, low level of involvement in management, disagreements with physicians, managerial issues, and a lack of appropriate work conditions in hospitals (15). approximately 45% of nurses in iran regret having become nurses, whereas 43.9% of them feel disappointed and dissatisfied that they became nurses. the most challenging problem in nursing in iran is the shortage of nurses working in hospitals because of their dissatisfaction and lack of motivation, and, unfortunately, most nurses in iran would like to change their work (14). because satisfaction with nursing is related to improving the health system and patient satisfaction, this study was conducted to assess how the satisfaction of nurses with their work can be improved, their situation in hospitals can be improved, and also how patients satisfaction could be increased (15). the objective of this research was to determine iranian nurses experiences of the concept of professional satisfaction. this was a qualitative study that was conducted with a targeted sampling of 10 nurses (4 men and 6 women), with a minimum of one year and a maximum of 25 years of work experience. all participants had earned a bachelor s degree and cared for patients directly as staff members in general or intensive care units at public or private hospitals in tehran. in this study, data were collected using semi - structured interviews. before conducting the interviews, the objective of the study was explained to the participants, and their consent to participate in the study was obtained in writing. all interviewees were ensured that their identities would not be disclosed in the reports and that they could have access to the results. the interviews were conducted in a place where the participants felt comfortable, such as in the break room in the hospital where they worked. in addition to covering the general questions of the study, attempts were made to personalize each interview depending on its flow. the qca is an analytical procedure that is utilized to provide a subjective interpretation of textual data contents (16). in this method, codes and categories are extracted from raw data directly and inductively through a systematic categorization process (17). content analysis is much more than the extraction of objective contents out of textual data, i.e., key concepts and hidden patterns may be revealed from the content of the participant - generated data through this method (16). in the qca, data collection and analysis are done simultaneously (17). in this method, the researcher collects and analyzes data back and forth in order to collect new data to be able to provide answers to the research questions (16). an analytical unit is a part of text that can be analyzed to help achieve the research objectives. initial codes were extracted from meaning units, which are important and reliable parts of the analytical units. initial codes could contain either the exact content of the participant s interview or the abstract of the content. based on their similarities and differences, the initial codes were reduced to sub - categories, which, in turn, were used to abstract categories and key concepts (17). in the first step of this study, the researchers listened to the interviews, and then they wrote the results of the interviews word for word. in the next step, then, based on similarities and differences, the initial codes were reduced to sub - categories, abstracts were prepared, and the key codes were identified. in order to achieve reliability in this research, the data were controlled by the participants and investigated by an investigation team. in addition to restoring the speech and experiences of the participants during the interviews by replaying them, the researcher gave them full typed texts of the first four interviews together with their initial codes to those with whom he or she had conducted the initial interviews for verified or revision. in addition, coding and initial categories with respect to content analysis were submitted to the researcher s supervisor, and verification was received on her / his part regarding the implementation, coding, and initial categories. in order to increase the reliability of the data, the researcher attempted to establish a deeper relationship with the participants and also allocated sufficient time for the interviews. the researcher also attempted to provide a friendly atmosphere in order to gain the participants trust, so that more accurate data and information could be elicited. the participants were willing to participate in the interviews, and they express their feelings, experiences, and thoughts with no fear of censorship or pressure. also, to increase the degree of reliability, the researcher attempted to avoid including his personal preferences in the study. from the qualitative analysis of the data collected from the interviewees, fair conduct was extracted as the turning point and basis upon which professional satisfaction of nurses could be achieved, which, in turn, consisted of three sub - categories, i.e., 1- expectation of fairness in social - professional settings, 2- expectation of fairness in receiving professional benefits, and 3- expectation of fairness in the area of professional interactions. many of the participants maintained that there was a gross distinction between what the society said and how people reacted, in that, even though nurses were academically educated, cared for patients, and dealt with their problems in a never - ending manner, they were not regarded by patients, their families, or even society as having an equal status to those engaged in other professions, including medicine. one of the nurses expressed her feelings by saying, nursing is a difficult job. you study for four years at university just like any other discipline, you get a bachelor degree, yet at the end, people regard nurses as a bunch of illiterates with no independence or literacy whatsoever, and who merely obey what doctors command them to do. society regards nurses as mere observers who tend their patients, but view doctors as even those engaged in other fields such as accounting, engineering, etc. with equivalent bachelor degrees enjoy a better salary and are held in greater esteem than the people employed in nursing. yet another participant stressed the significance of professional satisfaction in making nurses continue to do their jobs ; i become very upset when i see the respectful way by which people of other professions who have bachelor degrees just like we do, are referred to (e.g. the engineer man / lady), or when i compare the level of respect shown to doctors with that shown to nurses. people do not have high regards for the community of nurses ; they view them as ill - humored or bad people., nurses are shown wearing dressed with all kinds of colors running after doctors. yet, in those same movies, doctors are displayed as distinguished people helping patients. most of the participants stated that unfair practices at work with respect to financial matters had left them disappointed and lowered their professional satisfaction, in such a way that their attempts were directed towards quitting their job or finding a new one with better salary. concerning this matter, one of the nurses stated, in today s world my basic needs have to be fulfilled, and currently a nurse s salary is not in any sense proportionate to that earned by those working in other medical sectors. on lack of a financial fairness, another nurse maintained, it might be true that a doctor s education is higher than that of a nurse, but a nurse devotes more of his / her time to patients, in return. the thing that bothers the iranian nurses the most is their awareness of all professional benefits that nurses working in other countries enjoy. in advanced countries, nursing has a high status, while this is not the case for iran, and that is exactly the reason behind many dissatisfactions. a male nurse expressed his discontent of nursing as follows, with 35 years of age, i suffer from diabetes due to the stress at work, which i try to handle with strict regiments and such. i am trying to change my job, since no matter how hard i work i end up having nothing. i have to hold two or three jobs to be able to buy a house. yet, this is not the case for those engaging in other professions, with little or no difference than nursing from an academic point of view ? another male nurse expressed his discontent of nursing as follows, i know for a fact that, nurses in the great britain earn an equal amount of salary compared to surgeons, which is something to reflect upon. that how the monthly income of a nurse is equal to that of a surgeon is something that requires serious contemplation. furthermore, they merely work one shift and tend two patients all night long, unlike in iran, where two nurses tend 45 patients simultaneously. another nurse with two years experience said, with rising inflation, doctors salaries and fees increase day by day, whether officially or unofficially, and the authorities regard this as their inalienable right. however, nurses live off of a petty salary and then they complain about the few number of working nurses, and that why it is that they continually quit their job ! most of the participants viewed a number of factors as influencing the satisfaction they felt in doing their job, the most important of which was the viewpoint and attitude of hospital managers towards nurses. one of the participants in this study voiced her / his opinion on this matter as such, the treatment received by nurses from hospital management is unfair. they do not show them the level of respect they deserve, support doctors rights. one of the nurses pointed to decision making by doctors or nurses as one of the factors causing professional dissatisfaction, during all these years that i have worked as a nurse, i have seen with my own eyes that many decisions are made for us by those in the medical department. they are just not fair ! another participant stated, justice is not observed at iranian hospitals. one of the nurses working in the department of psychiatry pointed to the following as one of the reasons behind his / her professional satisfaction, since, as you know, working with mentally - challenged patients drives one to exhaustion. yet, despite all these hardships, our relationship with our managers is at a very high level. they act very reasonably, and this brings about satisfaction and makes us care for patients with greater motivation. the belief in establishing a kind of justice on the part of some of the participants was so strong that they viewed the existing difficulties in the nursing profession to be more bearable if some sense of justice was in place. the following statement could be brought to attention in this regard, despite the high level of physical and mental exhaustion that our work brings about, a fair treatment by managers and equality with respect to observing the rules would make the hardships of this job, including lack of sleep, intensity of work, low salary, etc. many of the participants maintained that there was a gross distinction between what the society said and how people reacted, in that, even though nurses were academically educated, cared for patients, and dealt with their problems in a never - ending manner, they were not regarded by patients, their families, or even society as having an equal status to those engaged in other professions, including medicine. one of the nurses expressed her feelings by saying, nursing is a difficult job. you study for four years at university just like any other discipline, you get a bachelor degree, yet at the end, people regard nurses as a bunch of illiterates with no independence or literacy whatsoever, and who merely obey what doctors command them to do. society regards nurses as mere observers who tend their patients, but view doctors as even those engaged in other fields such as accounting, engineering, etc. with equivalent bachelor degrees enjoy a better salary and are held in greater esteem than the people employed in nursing. yet another participant stressed the significance of professional satisfaction in making nurses continue to do their jobs ; i become very upset when i see the respectful way by which people of other professions who have bachelor degrees just like we do, are referred to (e.g. the engineer man / lady), or when i compare the level of respect shown to doctors with that shown to nurses. people do not have high regards for the community of nurses ; they view them as ill - humored or bad people., nurses are shown wearing dressed with all kinds of colors running after doctors. yet, in those same movies, doctors are displayed as distinguished people helping patients. most of the participants stated that unfair practices at work with respect to financial matters had left them disappointed and lowered their professional satisfaction, in such a way that their attempts were directed towards quitting their job or finding a new one with better salary. concerning this matter, one of the nurses stated, in today s world my basic needs have to be fulfilled, and currently a nurse s salary is not in any sense proportionate to that earned by those working in other medical sectors. on lack of a financial fairness, another nurse maintained, it might be true that a doctor s education is higher than that of a nurse, but a nurse devotes more of his / her time to patients, in return. the thing that bothers the iranian nurses the most is their awareness of all professional benefits that nurses working in other countries enjoy. in advanced countries, nursing has a high status, while this is not the case for iran, and that is exactly the reason behind many dissatisfactions. a male nurse expressed his discontent of nursing as follows, with 35 years of age, i suffer from diabetes due to the stress at work, which i try to handle with strict regiments and such. i am trying to change my job, since no matter how hard i work i end up having nothing. i have to hold two or three jobs to be able to buy a house. yet, this is not the case for those engaging in other professions, with little or no difference than nursing from an academic point of view ? another male nurse expressed his discontent of nursing as follows, i know for a fact that, nurses in the great britain earn an equal amount of salary compared to surgeons, which is something to reflect upon. that how the monthly income of a nurse is equal to that of a surgeon is something that requires serious contemplation. furthermore, they merely work one shift and tend two patients all night long, unlike in iran, where two nurses tend 45 patients simultaneously. another nurse with two years experience said, with rising inflation, doctors salaries and fees increase day by day, whether officially or unofficially, and the authorities regard this as their inalienable right. however, nurses live off of a petty salary and then they complain about the few number of working nurses, and that why it is that they continually quit their job ! most of the participants viewed a number of factors as influencing the satisfaction they felt in doing their job, the most important of which was the viewpoint and attitude of hospital managers towards nurses. one of the participants in this study voiced her / his opinion on this matter as such, the treatment received by nurses from hospital management is unfair. they do not show them the level of respect they deserve, support doctors rights. one of the nurses pointed to decision making by doctors or nurses as one of the factors causing professional dissatisfaction, during all these years that i have worked as a nurse, i have seen with my own eyes that many decisions are made for us by those in the medical department. they are just not fair ! another participant stated, justice is not observed at iranian hospitals. one of the nurses working in the department of psychiatry pointed to the following as one of the reasons behind his / her professional satisfaction, since, as you know, working with mentally - challenged patients drives one to exhaustion. yet, despite all these hardships, our relationship with our managers is at a very high level. they act very reasonably, and this brings about satisfaction and makes us care for patients with greater motivation. the belief in establishing a kind of justice on the part of some of the participants was so strong that they viewed the existing difficulties in the nursing profession to be more bearable if some sense of justice was in place. the following statement could be brought to attention in this regard, despite the high level of physical and mental exhaustion that our work brings about, a fair treatment by managers and equality with respect to observing the rules would make the hardships of this job, including lack of sleep, intensity of work, low salary, etc. more bearable. as was discussed, one of the main components of professional satisfaction is fair conduct, which consists of three sub - categories, i.e., expectation of fairness in social - professional settings, expectation of fairness in receiving professional benefits, and expectation of fairness in the area of professional interactions. the subject of fairness is one of the few fundamental topics relevant to various spheres of science and philosophy, and it can be applied to any situation in terms of people s needs and expectations. the concept of fairness is integrated with moral and social values and within the sphere of morality, in particular social morality, supporting fairness and equity is of the most fundamental moral values. researchers regard the establishment of justice as one of the most important factors affecting professional satisfaction (18). one of the long - term challenges of the nursing profession is its public image (19). there are various ups and downs to nursing due to the challenging nature of this profession from the initial education at university until retirement. dropping out of university, although low salaries and benefits and professional exhaustion are among the factors causing discontent among nurses, according to conducted studies, lack of consideration towards the status and public image of the nursing profession is responsible for 70% of professional dissatisfaction (20). generally, people form opinions or mental images of other individuals based on their look, way of clothing, reaction, communication, behavior and attitude (21). the image is the perception or assumption that the general public makes about an individual, profession, or organization. the people within those organizations use this image as a tool to communicate with people, and they, in turn, interact with those organizations based on the projected image, whether positive or negative (20). the social status of a profession and the society s perspective towards it are of effective motivational factors influencing the extent of professional satisfaction, which, in turn, leaves an impact on the attitude shown by individuals in fulfilling their professional functions. these statuses include placing a high value on a profession and its required proficiencies and the value and status of a profession in a particular society. whenever the status is damaged on a societal scale, the status of the profession is also diminished (22). in nursing, one of the most important concerns with regards to professional status is lack of a high value and social status within society. with regards to the importance of this issue, stevens stated that public opinion acts very strongly in determining the social structure and norms of a society (23). a positive public image will leave a considerable effect on gaining social and situational support. as was understood from the findings of this study, most nurses in this research regarded professional satisfaction as manifested through expectation of fairness in social - professional settings. regarding the second important finding of this study, i.e., the expectation of fairness in receiving professional benefits, the participants stated that unequal salaries among the personnel of the medical department and a gross distinction between the payments to the medical staff have been sources of disappointment and dissatisfaction. markovsky was among the first theoreticians to recognize the significance of the issue of salary satisfaction in observing fairness (24). therefore, the findings of this study, that is, feeling a sense of fairness in receiving professional benefits has an intermediary role in bringing professional satisfaction, is in line with markovsky s theory. furthermore, expectation of fairness in receiving professional benefits gives the indication that, if individuals deem a condition to be unfair, they will have an emotional response to it (25). yet another important finding of this study was the expectation of fairness in professional interactions. the participants of this study stated that the treatment they receive from managers is unfair and disrespectful compared to other health teams. defined interactional justice as the feeling experienced by employees when interacting with their supervisor and state ; cooperation with supervisors and other members of the work team and respectful behavior, and providing the required information would result in a high satisfaction level among employees (25). kim. maintained that interactional fairness, fair treatment by managers towards employees, and instituting behavioral justice significantly affect professional satisfaction (26). regarding the issue of fairness, the results of the present study were in line with those obtained by lou. (21), in which it was demonstrated that british nurses were more displeased compared to australian nurses. the british nurses felt that their professional statuses were lower, their relationships with hospital managers were weaker, and their work conditions were less favorable than those of australian nurses. the results of this study confirm the point that the existence of a relative social status to profession, and of justice within the work environment, and also respectful treatment towards nurses by management. generally speaking, fair conduct is an integral part of professional satisfaction in the nursing profession, which could encourage nurses to employ the related measures and interventions with a higher degree of motivation (18). furthermore, a review of the previous studies indicated that management and administrative experiences, which refer to organizational structures, such as (de)centralization and fair and transparent organizational policies and procedures are among the factors affecting professional satisfaction. researchers have found out that decentralization and all kinds of cooperative management models are more to nurses liking, and that they resent being looked at with indifference, and this leads to a lower professional satisfaction level in their eyes (12). the study conducted by farsi. indicated that the low public image of nursing in the iranian society has caused nurses to feel unappreciated and disrespected (16). as was demonstrated in other studies conducted in iran, the motivation level of iranian nurses in fulfilling their professional tasks, which to a large degree is dependent upon the public image, is not acceptable (20). in general, since professional satisfaction is a single concept that can not be deconstructed, it is possible to be defined by individuals in vague manners. therefore, professional satisfaction can be viewed as a general feeling, or as the attitude taken by an individual with respect to part of her or his job. overall, it is possible to improve the professional satisfaction of nurses through emphasizing the (in)visible aspects of professional satisfaction, such as fair conduct, removing tension - generating factors, improving the attitude of society towards the nursing profession, establishing justice among all hospital staff, improving communication, organizing the nursing profession, and enhancing the payment system and making it fair. | introductionthe professional satisfaction of staff is one of the most challenging organizational concepts that can enhance the efficiency level of organizations. in a similar vein, the professional satisfaction of nurses is of considerable importance, in that, professional dissatisfaction among nurses could result in emotional detachment, depression, anger, evasion from work, and inefficacy and would negatively impact the organization s work rate. the aim of this study was to understand iranian nurses experiences of the concept of professional satisfaction.methodsthis was a qualitative study conducted with a targeted sampling of 10 nurses (4 men and 6 women) in 2015. the data were collected through conducting in - depth interviews, and textual data were analyzed subsequently using the qualitative content analysis (qca) method.resultsthe findings of this study pointed to fair conduct, which was comprised of three sub - categories, i.e., expectation of fairness in social - professional settings, expectation of fairness in receiving professional benefits, and expectation of fairness in the area of professional interactions.conclusionsthere are various ups and downs in nursing due to the challenging nature of the profession, from the initial education at the university until retirement. according to the findings of this study, a lack of fairness in social - professional settings, a lack of fairness in receiving professional benefits, and a lack of fairness in the area of professional interactions were among the factors that have great impacts on the degree of professional dissatisfaction among nurses. |
crc cases were selected from patients who underwent surgical treatment at eulji university hospital from january 2000 to june 2005. all cases were histologically confirmed to be primary colorectal adenocarcinoma and hematoxylin and eosin slides were re - evaluated by two independent pathologists. the tumor grade of the adenocarcinoma was classified into low grade (50% of tumor glands) and high grade (< 50% of tumor glands). for signet ring cell carcinoma and mucinous adenocarcinoma, tumor budding was defined as a single or group of less than five detached tumor cells and classified into two grades. tumor recurrence was designated as tumor occurring at the anastomosing site, in the regional lymph nodes, and the pelvic cavity diagnosed by radiology, colonoscopy, exploratory surgical, and/or histological examination. in addition, metastasis was defined as the presence of tumor cells outside the area of resection, including the liver, pancreas, lung, and other organs. all cases of crc tissue with accompanying normal mucosal tissue were fixed in 10% buffered formalin for 24 hours and embedded in paraffin. tissue sections of 34 m thickness were cut and mounted on probeon slides (fisher scientific, pittsburgh, pa, usa). sections that contained both tumor and adjacent uninvolved colonic mucosa were selected for immunohistochemistry (ihc) in most cases. in a few cases, sections were trimmed in order to decrease the surface area for an even distribution of antibodies, so that only the tumor portion was included in the ihc evaluation. paraffin embedded tissue sections were deparaffinized and rehydrated through a series of xylene and alcohol. slides were then treated with 10 mm / l sodium citrate buffer (ph 6.1) for 15 minutes and autoclaved at 120c for antigen retrieval. all slide sections for ihc were incubated in 3% h2o2 for 10 minutes to inactivate endogenous peroxidase, washed with 10 mm / l phosphate buffered saline buffer (ph 7.4), and then incubated with normal bovine serum to reduce false - positive staining. saco, me, usa) and hif-1 (1:50, novus biologicals, littleton, co, usa) were used as primary antibodies. slide sections were incubated with primary antibodies overnight at 4c in a wet chamber and stained with diaminobenzidine as the substrate using an envision - hrp kit (dako, glostrup, denmark). sections were counterstained with mayer s hematoxylin solution and then mounted. to evaluate the expression of thymosin 4 and hif-1 in association with various clinico - pathological factors, the immunoreactivity of both thymosin 4 and hif-1 were analyzed in a semi - quantitative manner by two independent pathologists who were blinded to outcomes. immunoreactivity for thymosin 4 and hif-1 was observed primarily in the cytoplasm and nuclei of normal mucosal epithelium and tumor cells, respectively. the intensity of immunohistochemical staining was scored as 0 to 2 (0, weaker staining than the normal mucosal epithelium ; 1, staining similar to the normal mucosal epithelium ; and 2, stronger staining than the normal mucosal epithelium). the percentage of positive cells was scored as 1 (< 25% of tumor cells), 2 (25%49% of tumor cells), 3 (50%74% of tumor cells), and 4 (75% of tumor cells). to evaluate the statistical significance between thymosin 4 and hif-1 expression and clinico - pathological factors, the median value (25% of tumor cells showing a strong positive reaction than normal epithelium) of the series was used as the cutoff value to distinguish between tumor cells with low expression (< 25% tumor cells) and high expression (25% of tumor cells). the correlation between thymosin 4 and the various clinico - pathological parameters were analyzed with the pearson s chi - square test or fisher exact test. to evaluate statistical analysis, recurrence - free survival was defined as the duration from the date of surgery to the first date of recurrence or the date of last follow - up. similarly, overall survival was defined as the duration from the date of surgical therapy to the date of death or date of last follow - up. the mean follow - up duration for all patients was 53.3 months, ranging from 0.6 to 121.9 months. using the kaplan - meier method, the recurrence - free survival curve and the overall survival curve were formulated. to examine the statistical significance of the differences in survival distribution, log - rank test was utilized. multivariate analysis for overall survival and recurrence - free survival was performed using cox proportional hazard regression analysis. in all statistical analyses, p - values less than.05 were considered statistically significant. the institutional review board of eulji university hospital approved the study protocol and provided all necessary ethical permission. crc cases were selected from patients who underwent surgical treatment at eulji university hospital from january 2000 to june 2005. all cases were histologically confirmed to be primary colorectal adenocarcinoma and hematoxylin and eosin slides were re - evaluated by two independent pathologists. the tumor grade of the adenocarcinoma was classified into low grade (50% of tumor glands) and high grade (< 50% of tumor glands). for signet ring cell carcinoma and mucinous adenocarcinoma, tumor budding was defined as a single or group of less than five detached tumor cells and classified into two grades. tumor recurrence was designated as tumor occurring at the anastomosing site, in the regional lymph nodes, and the pelvic cavity diagnosed by radiology, colonoscopy, exploratory surgical, and/or histological examination. in addition, metastasis was defined as the presence of tumor cells outside the area of resection, including the liver, pancreas, lung, and other organs. all cases of crc tissue with accompanying normal mucosal tissue were fixed in 10% buffered formalin for 24 hours and embedded in paraffin. tissue sections of 34 m thickness were cut and mounted on probeon slides (fisher scientific, pittsburgh, pa, usa). sections that contained both tumor and adjacent uninvolved colonic mucosa were selected for immunohistochemistry (ihc) in most cases. in a few cases, sections were trimmed in order to decrease the surface area for an even distribution of antibodies, so that only the tumor portion was included in the ihc evaluation. paraffin embedded tissue sections were deparaffinized and rehydrated through a series of xylene and alcohol. slides were then treated with 10 mm / l sodium citrate buffer (ph 6.1) for 15 minutes and autoclaved at 120c for antigen retrieval. all slide sections for ihc were incubated in 3% h2o2 for 10 minutes to inactivate endogenous peroxidase, washed with 10 mm / l phosphate buffered saline buffer (ph 7.4), and then incubated with normal bovine serum to reduce false - positive staining. saco, me, usa) and hif-1 (1:50, novus biologicals, littleton, co, usa) were used as primary antibodies. slide sections were incubated with primary antibodies overnight at 4c in a wet chamber and stained with diaminobenzidine as the substrate using an envision - hrp kit (dako, glostrup, denmark). to evaluate the expression of thymosin 4 and hif-1 in association with various clinico - pathological factors, the immunoreactivity of both thymosin 4 and hif-1 were analyzed in a semi - quantitative manner by two independent pathologists who were blinded to outcomes. immunoreactivity for thymosin 4 and hif-1 was observed primarily in the cytoplasm and nuclei of normal mucosal epithelium and tumor cells, respectively. the intensity of immunohistochemical staining was scored as 0 to 2 (0, weaker staining than the normal mucosal epithelium ; 1, staining similar to the normal mucosal epithelium ; and 2, stronger staining than the normal mucosal epithelium). the percentage of positive cells was scored as 1 (< 25% of tumor cells), 2 (25%49% of tumor cells), 3 (50%74% of tumor cells), and 4 (75% of tumor cells). to evaluate the statistical significance between thymosin 4 and hif-1 expression and clinico - pathological factors, the median value (25% of tumor cells showing a strong positive reaction than normal epithelium) of the series was used as the cutoff value to distinguish between tumor cells with low expression (< 25% tumor cells) and high expression (25% of tumor cells). the correlation between thymosin 4 and the various clinico - pathological parameters were analyzed with the pearson s chi - square test or fisher exact test. to evaluate statistical analysis, recurrence - free survival was defined as the duration from the date of surgery to the first date of recurrence or the date of last follow - up. similarly, overall survival was defined as the duration from the date of surgical therapy to the date of death or date of last follow - up. the mean follow - up duration for all patients was 53.3 months, ranging from 0.6 to 121.9 months. using the kaplan - meier method, the recurrence - free survival curve and the overall survival curve were formulated. to examine the statistical significance of the differences in survival distribution, log - rank test was utilized. multivariate analysis for overall survival and recurrence - free survival was performed using cox proportional hazard regression analysis. in all statistical analyses, p - values less than.05 were considered statistically significant. the institutional review board of eulji university hospital approved the study protocol and provided all necessary ethical permission. the median age of the 143 crc patients (75 men and 68 women) at surgery was 62.2 years (range, 28 to 86 years) and the median tumor size was 5.2 cm (range, 0.8 to 12.0 cm) in maximum diameter. the majority of crcs were moderately differentiated adenocarcinoma and 111 cases (77.6%) were classified as low grade and 32 cases (22.4%) as high grade (poorly differentiated 19, signet ring cell carcinoma 3, and mucinous carcinoma 10). one hundred and four cases (72.7%) showed lymphovascular tumor invasion and 106 cases (74.1%) exhibited high - grade tumor budding. according to the seventh edition of the ajcc tnm system, 26 patients (18.2%) were diagnosed with early - stage tumor invasion (five cases of pt1 and 21 cases of pt2) and 117 patients (81.8%) were diagnosed with advanced - stage tumor invasion (105 cases of pt3 and 12 cases of pt4). seventy - six patients (53.1%) presented with regional lymph node metastasis and 22 patients (15.4%) with distant metastasis. twenty - one patients (14.7%) were at ptnm stage i, 45 patients (31.5%) were at stage ii, 55 patients (38.5%) were at stage iii, and 22 patients (15.4%) were at stage iv. twenty - seven patients (18.9%) were treated with chemoradiation after the first surgery (data not shown). clinico - pathological characteristics of the 143 crc patients are summarized in table 1. in the normal colonic epithelium, immunoreactivity for thymosin 4 and hif-1 while thymosin 4 expression was found primarily in the cytoplasm of cancer cells, hif-1 was stained predominantly in the nuclei of tumor cells (fig. a high level of thymosin 4 and hif-1 expression was observed in 66 of the 143 patients (46.2%) and in 67 of the 143 patients (46.9%), respectively. we found that predictive factors for prognosis, such as lymphovascular invasion, invasion depth (pt), regional lymph node metastasis (pn), distant metastasis, and tnm stage showed statistically significant correlations with thymosin 4 immunoreactivity (table 1). patients with high thymosin 4 expression levels showed a significantly greater presence of lymphovascular invasion, more frequent regional lymph node metastasis, deeper invasion depth, and more advanced tumor stage than in those with low thymosin 4 expression levels (p<.001). we found a statistically significant correlation between high hif-1 immunohistochemical expression and clinico - pathological factors such as lymphovascular invasion (p=.006), invasion depth (p=.002), regional lymph node metastasis (p=.007), distant metastasis (p=.029), and tnm stage (p=.002) (data not shown). we performed multivariate analysis to examine the correlation between thymosin 4 expression levels with recurrence - free survival and overall survival. forty patients (28.0%) presented with cancer recurrence during follow - up and 55 patients (38.5%) died of crc with or without metastasis. seven patients (4.9%) died of unknown causes, 17 patients (11.9%) were alive with local recurrence and/or distant metastasis, and 64 patients (44.8%) remained alive and recurrence - free. kaplan - meier analysis showed that high thymosin 4 expression was significantly correlated with decreased recurrence - free survival (p<.001) (fig. recurrence - free survival was shorter in patients with high expression levels of thymosin 4, with a mean duration of 37.3 months (95% confidence interval [ci ], 27.833 to 46.684), and was longer in patients with low levels of thymosin 4 expression, with a mean duration of 84.6 months (95% ci, 73.538 to 95.571). we also found that high thymosin 4 expression significantly correlated with worse overall survival (p=.001). thymosin 4 expression status also significantly split the cumulative overall survival curves (fig. 2b). while the overall survival of crc patients with high thymosin 4 expression was mean duration of 51.7 months (95% ci, 41.381 to 61.939), the overall survival of crc patients with low thymosin 4 expression was longer with mean duration of 96.8 months (95% ci, 86.952 to 106.732). multivariate analysis was also performed to assess the prognostic value of thymosin 4 expression for recurrence - free survival and overall survival using various clinico - pathological parameters. statistically significant clinico - pathological factors that were correlated with overall survival were high thymosin 4 expression (p=.005), high tumor grade (p=.013), high tumor budding (p=.047), and presence of distant metastasis (p=.001). the relative risk (rr) of death in patients with a high expression level of thymosin 4 was more than two times greater (rr, 2.457 ; 95% ci, 1.315 to 4.592) than those with low thymosin 4 expression levels. high thymosin 4 expression was also an independent and relevant factor of decreased recurrence - free survival (p=.001). the rr of recurrence for patients with high thymosin 4 expression level was 2.540 (95% ci, 1.479 to 4.362). a statistically significant correlation between high hif-1 expression and high thymosin 4 expression was also found (p<.001). specifically, of the 66 cases exhibiting high thymosin 4 expression, 49 cases (74.2%) also showed high nuclear immunoreactivity of hif-1. of the 77 cases expressing low thymosin 4 expression, 59 cases (76.6%) revealed corresponding low hif-1 expression (table 3). the median age of the 143 crc patients (75 men and 68 women) at surgery was 62.2 years (range, 28 to 86 years) and the median tumor size was 5.2 cm (range, 0.8 to 12.0 cm) in maximum diameter. the majority of crcs were moderately differentiated adenocarcinoma and 111 cases (77.6%) were classified as low grade and 32 cases (22.4%) as high grade (poorly differentiated 19, signet ring cell carcinoma 3, and mucinous carcinoma 10). one hundred and four cases (72.7%) showed lymphovascular tumor invasion and 106 cases (74.1%) exhibited high - grade tumor budding. according to the seventh edition of the ajcc tnm system, 26 patients (18.2%) were diagnosed with early - stage tumor invasion (five cases of pt1 and 21 cases of pt2) and 117 patients (81.8%) were diagnosed with advanced - stage tumor invasion (105 cases of pt3 and 12 cases of pt4). seventy - six patients (53.1%) presented with regional lymph node metastasis and 22 patients (15.4%) with distant metastasis. twenty - one patients (14.7%) were at ptnm stage i, 45 patients (31.5%) were at stage ii, 55 patients (38.5%) were at stage iii, and 22 patients (15.4%) were at stage iv. twenty - seven patients (18.9%) were treated with chemoradiation after the first surgery (data not shown). clinico - pathological characteristics of the 143 crc patients are summarized in table 1. in the normal colonic epithelium, immunoreactivity for thymosin 4 and hif-1 while thymosin 4 expression was found primarily in the cytoplasm of cancer cells, hif-1 was stained predominantly in the nuclei of tumor cells (fig. a high level of thymosin 4 and hif-1 expression was observed in 66 of the 143 patients (46.2%) and in 67 of the 143 patients (46.9%), respectively. we found that predictive factors for prognosis, such as lymphovascular invasion, invasion depth (pt), regional lymph node metastasis (pn), distant metastasis, and tnm stage showed statistically significant correlations with thymosin 4 immunoreactivity (table 1). patients with high thymosin 4 expression levels showed a significantly greater presence of lymphovascular invasion, more frequent regional lymph node metastasis, deeper invasion depth, and more advanced tumor stage than in those with low thymosin 4 expression levels (p<.001). we found a statistically significant correlation between high hif-1 immunohistochemical expression and clinico - pathological factors such as lymphovascular invasion (p=.006), invasion depth (p=.002), regional lymph node metastasis (p=.007), distant metastasis (p=.029), and tnm stage (p=.002) (data not shown). we performed multivariate analysis to examine the correlation between thymosin 4 expression levels with recurrence - free survival and overall survival. forty patients (28.0%) presented with cancer recurrence during follow - up and 55 patients (38.5%) died of crc with or without metastasis. seven patients (4.9%) died of unknown causes, 17 patients (11.9%) were alive with local recurrence and/or distant metastasis, and 64 patients (44.8%) remained alive and recurrence - free. kaplan - meier analysis showed that high thymosin 4 expression was significantly correlated with decreased recurrence - free survival (p<.001) (fig. recurrence - free survival was shorter in patients with high expression levels of thymosin 4, with a mean duration of 37.3 months (95% confidence interval [ci ], 27.833 to 46.684), and was longer in patients with low levels of thymosin 4 expression, with a mean duration of 84.6 months (95% ci, 73.538 to 95.571). we also found that high thymosin 4 expression significantly correlated with worse overall survival (p=.001). while the overall survival of crc patients with high thymosin 4 expression was mean duration of 51.7 months (95% ci, 41.381 to 61.939), the overall survival of crc patients with low thymosin 4 expression was longer with mean duration of 96.8 months (95% ci, 86.952 to 106.732). multivariate analysis was also performed to assess the prognostic value of thymosin 4 expression for recurrence - free survival and overall survival using various clinico - pathological parameters. statistically significant clinico - pathological factors that were correlated with overall survival were high thymosin 4 expression (p=.005), high tumor grade (p=.013), high tumor budding (p=.047), and presence of distant metastasis (p=.001). the relative risk (rr) of death in patients with a high expression level of thymosin 4 was more than two times greater (rr, 2.457 ; 95% ci, 1.315 to 4.592) than those with low thymosin 4 expression levels. high thymosin 4 expression was also an independent and relevant factor of decreased recurrence - free survival (p=.001). the rr of recurrence for patients with high thymosin 4 expression level was 2.540 (95% ci, 1.479 to 4.362). a statistically significant correlation between high hif-1 expression and high thymosin 4 expression was also found (p<.001). specifically, of the 66 cases exhibiting high thymosin 4 expression, 49 cases (74.2%) also showed high nuclear immunoreactivity of hif-1. of the 77 cases expressing low thymosin 4 expression, 59 cases (76.6%) revealed corresponding low hif-1 expression (table 3). growing tumors require oxygen and nutrient delivery through neovasculature. however, intratumoral hypoxia induced by imbalance between tumor growth and insufficient angiogenesis can lead to expression of hif-1, which is a transcriptional factor that activates tumor survival in an unstable hypoxic tumor microenvironment [29 - 32 ]. recent reports have shown that thymosin 4 stabilizes hif-1 in human cancer cells and that thymosin 4 also induces migration and metastasis of colon cancer cells via the ilk / iqgap1/rac1 signal transduction pathway. few studies have evaluated whether overexpression of thymosin 4 influences clinical prognosis and whether thymosin 4 is related to hif-1 in crcs. to better understand the relationship, we analyzed the clinical significance and expression status of thymosin 4 and hif-1 in crc patients. our study demonstrates that high thymosin 4 expression has a significant association with lymphovascular invasion, nodal status, distant metastasis, and tumor progression in crc patients (p<.001). this finding is consistent with our previous study showing hypoxia - induced high expression of thymosin 4, which also significantly correlated with regional lymph node metastasis in breast cancer. in a previous study, we found thymosin 4 to be up - regulated under hypoxic conditions (5% o2) using an in vitro hypoxia - induced model to generate transcription profiles in human crc. based on these findings, for this study we examined the association between thymosin 4 expression and hif-1 expression in crc specimens. we then discovered that the overexpression of thymosin 4 in crc is closely related to the restricted overexpression of hif-1 in the crc cells (p<.001). recently, there have been reports regarding the association between thymosin 4 expression with tumor development and epithelial mesenchymal transition (emt). in particular, nemolato. reported high expression of thymosin 4 at the invasive front in colon cancer and discussed its associated with emt as well as invasion and metastasis of tumor cells. however, from our study, since we were unable to find an association between thymosin 4 expression and tumor budding (p=.118) and tumor border (p=.560), we found the direct association of thymosin 4 expression with emt to be weak. the hif complex, which involves various hypoxia - regulated genes, is a group of critical gene products in the tumor microenvironment of hypoxic adaptation and in angiogenesis. the hif complex is also an essential mediator in coordinating transcription of various factors in the tumor cells to survive in the hypoxic environment and its overexpression has been associated with increased mortality in various cancer types [31,35 - 37 ]. among hif whether hif-2, hif-3, and hif-1 also play critical roles in the hif pathway and regulate hif target genes is not yet clearly known [38 - 41 ]. hif-1 is frequently upregulated in various cancer cells and the overexpression of hif-1 correlates with advanced cancer progression or aggressiveness. however, the clinical significance of hif-1 in crc has not been extensively studied. in this study, we observed a significant association between thymosin 4 expression and hif-1 expression (p<.001). this result coincides with previous studies that found overexpression of hif-1 to be associated with poor prognosis. thymosin 4 has various functional roles in normal cell biology and its mechanism of action has recently been studied in various tumors. in this study, we found that high cytoplasmic expression of thymosin 4 is clinically important and an independent prognostic factor for crc patients. as our results demonstrate that high thymosin 4 expression significantly correlates with tumor recurrence and worse overall survival, we suggest that high thymosin 4 expression may be a useful prognostic factor in crc. our results demonstrate that hif-1 is correlated with overexpression of thymosin 4 in human crc. although further studies are necessary to further validate our findings, we suggest that thymosin 4, has potential as a prognostic biomarker and has potential as a hif pathway target in human crc. | backgroundthymosin 4 is a multi - functional hormone - like polypeptide, being involved in cell migration, angiogenesis, and tumor metastasis. this study was undertaken to clarify the clinicopathologic implications of thymosin 4 expression in human colorectal cancers (crcs).methodswe investigated tissue sections from 143 patients with crc by immunohistochemistry. in addition, we evaluated the expression patterns and the clinico - pathological significance of thymosin 4 expression in association with hypoxia inducible factor-1 (hif-1) expression in the crc series.resultshigh expression of thymosin 4 was significantly correlated with lymphovascular invasion, invasion depth, regional lymph node metastasis, distant metastasis, and tnm stage. patients with high expression of thymosin 4 showed poor recurrence - free survival (p =.001) and poor overall survival (p =.005) on multivariate analysis. we also found that thymosin 4 and hif-1 were overexpressed and that thymosin 4 expression increased in parallel with hif-1 expression in crc.conclusionsa high expression level of thymosin 4 indicates poor clinical outcomes and may be a useful prognostic factor in crc. thymosin 4 is functionally related with hif-1 and may be a potentially valuable biomarker and possible therapeutic target for crc. |
the caudate lobe is located anterior to the inferior vena cava (ivc) and may envelop this structure circumferentially. it extends to the hilum of the liver just posterior to the bifurcation of the portal vein. cephalad, the caudate lobe lies posterior to the confluence of the left and middle veins as they enter the ivc on the left (figure 1). the caudate lobe is generally divided into three regions : the left spiegel lobe, the process portion, and the paracaval portion. the anatomy of the paracaval portion includes the liver parenchyma ventral to the hepatic ivc and the area between the spiegel lobe and the right lobe adjacent to the middle hepatic vein ventrally.1 this portion was classified by couinaud as segment ix (2). our aim in this study is to describe the standard technique for a caudate lobectomy procedure involving an anterior hepatic transection (aht) via a review of the literature. a literature search was conducted using ovid medline for the terms caudate lobectomy and aht covering 1992 to 2007. resection of the caudate lobe involves three major steps : controlling the inflow caudate blood supply from the left portal vein and the left hepatic artery at the base of the umbilical fissure, as well as controlling other inflow by lowering the hilar plate and dividing any vessels encountered ; dissection of the retrohepatic veins ; and in the case of an isolated caudate resection, it is necessary to divide the liver parenchyma between the base of segment iv and the left side of segment vii to allow removal of the lobe. when an aht of the caudate lobe is used, an anatomic resection of segment i or / and segment ix (an isolated or complete resection of the caudate lobe) may be performed. some authors have split the liver anteriorly through the glissonian plane, separating the right and left liver, and approaching the caudate lobe by dividing the liver along the right margin of segment iv. the combination of these techniques allows for the preservation of middle hepatic venous flow and good control of bleeding. after cholecystectomy, the right lobe is mobilized from the retroperitoneum, and the ivc is mobilized by ligating and dissecting the short hepatic veins caudocranially. the right hepatic vein (rhv) is secured after division of the vena cava ligament.2 on the left side, after dissecting and bending up the lateral segment and dividing the ligamentum venosum (arantius duct) at its junction with the left hepatic vein, the spiegel lobe is separated from the ivc, and the trunk of the left and middle hepatic veins is taped. the liver is completely separated from the ivc except for the major hepatic veins. prior to liver transaction, the outer confines of the caudate process and the paracaval portion against the posterior segment are identified using a counterstaining technique.3,4 under the pringle maneuver (a hepatic inflow occlusion time of 15 minutes and a reperfusion time of 5 minutes), transaction is performed along the left side of the middle hepatic vein (mhv), opening the interlobar plane and exposing the anterior surface of the paracaval portion and the hilar plate. the paracaval portion is detached from the hilar plate by ligating and dividing the ascending caudate portal branches. the transaction then proceeds in two directions : to the left side toward the sulcus of the ligamentum venosum (arantius ligament) and to the right side just behind the mhv. on the left side, after dividing the ligamentum venosum and the spiegel branches, the spiegel lobe is liberated from the left lobe. on the right side, the hilar plate is exposed up to the bifurcation of the anterior and posterior portal pedicles. with the right lobe bent medially, liver transaction is conducted from the border of the posterior segment and the caudate process, exposing the dorsal surface of the rhv. resection of the caudate lobe involves three major steps : controlling the inflow caudate blood supply from the left portal vein and the left hepatic artery at the base of the umbilical fissure, as well as controlling other inflow by lowering the hilar plate and dividing any vessels encountered ; dissection of the retrohepatic veins ; and in the case of an isolated caudate resection, it is necessary to divide the liver parenchyma between the base of segment iv and the left side of segment vii to allow removal of the lobe. when an aht of the caudate lobe is used, an anatomic resection of segment i or / and segment ix (an isolated or complete resection of the caudate lobe) may be performed. some authors have split the liver anteriorly through the glissonian plane, separating the right and left liver, and approaching the caudate lobe by dividing the liver along the right margin of segment iv. the combination of these techniques allows for the preservation of middle hepatic venous flow and good control of bleeding. after cholecystectomy, the right lobe is mobilized from the retroperitoneum, and the ivc is mobilized by ligating and dissecting the short hepatic veins caudocranially. the right hepatic vein (rhv) is secured after division of the vena cava ligament.2 on the left side, after dissecting and bending up the lateral segment and dividing the ligamentum venosum (arantius duct) at its junction with the left hepatic vein, the spiegel lobe is separated from the ivc, and the trunk of the left and middle hepatic veins is taped. the liver is completely separated from the ivc except for the major hepatic veins. prior to liver transaction, the outer confines of the caudate process and the paracaval portion against the posterior segment are identified using a counterstaining technique.3,4 under the pringle maneuver (a hepatic inflow occlusion time of 15 minutes and a reperfusion time of 5 minutes), transaction is performed along the left side of the middle hepatic vein (mhv), opening the interlobar plane and exposing the anterior surface of the paracaval portion and the hilar plate. the paracaval portion is detached from the hilar plate by ligating and dividing the ascending caudate portal branches. the transaction then proceeds in two directions : to the left side toward the sulcus of the ligamentum venosum (arantius ligament) and to the right side just behind the mhv. on the left side, after dividing the ligamentum venosum and the spiegel branches, the spiegel lobe is liberated from the left lobe. on the right side, the hilar plate is exposed up to the bifurcation of the anterior and posterior portal pedicles. with the right lobe bent medially, liver transaction is conducted from the border of the posterior segment and the caudate process, exposing the dorsal surface of the rhv. when the liver parenchyma dorsal to the rhv is transected, resection is complete. this study analyzed the records of 110 patients who were submitted to caudate lobectomy involving an anterior transhepatic approach between 1990 and 2007, as shown in table 1. isolated caudate lobectomy was performed on 28 (25.4%) patients, while complete caudate lobectomy was carried out on 82 (74.5%) patients. of the patients treated, 106 (96.3%) had hepatocellular carcinomas, 3 (2.7%) had metastatic caudate tumors, and 1 (0.9%) had hemangioma. aht was used in 108 (98.1%) caudate resections, while aht associated with a right - sided approach was performed in 2 (1.8%) cases. in the past, resection of the caudate lobe has been performed infrequently, partly due to the difficult dissection required and partly due to the perception of frequent early tumor involvement in the ivc or the portal vein. the caudate lobe can be the origin of primary liver tumors or the sole site of metastases to the liver.5,6 the paracaval portion of the caudate lobe is in front of the intrahepatic ivc just to the right of the spiegel lobe and is closely associated with the right and middle hepatic veins. as a result of this unique anatomical location, caudate lobe resection is technically challenging, especially in cases of isolated caudectomy, because it is easy to damage the hilum or the bile duct in dissecting the caudate lobe s anterior. additionally, an error in dissecting the posterior of the caudate lobe can cause uncontrolled bleeding from the inferior vena cava. the most difficult areas of the caudate lobe from which to remove tumors are the protruding portion and the portion near the inferior vena cava. the key issues in performing a safe resection of the caudate lobe include meticulous dissection and maintaining control of the retrohepatic veins in the posterior area and exposing the branches joining the middle hepatic vein in the anterior area. the ultrasound dissector is a valuable tool to enable the precise transaction of the hepatic parenchyma and the denudation of venous branches.7 the choice of procedure is essential to the success of a caudate lobectomy. there are different procedures for performing a caudate lobectomy, but only three are effective : the anterior procedure, in which the hepatic parenchyma is resected from the middle line and the right and left porta hepatitis are pulled aside to expose the caudate lobe,8 and right and left approaches to the caudate lobe.6 this review has shown that, when using aht most caudate lobectomies are complete caudate lobectomies (75%) due to hepatocellular carcinoma (96%). additionally, the anterior hepatic approach is a safe strategic alternative for isolated caudate lobectomies,9,10 but only 25% of the cases reviewed were submitted to this procedure. the combination of aht and a right - sided approach accounted for less than 2% of cases, which proves that, for tumors located in the paracaval portion of the caudate lobe (segment ix), the best approach is an anterior transection of the liver parenchyma. caudate lobectomy is sometimes associated with a left or right hepatectomy ; for instance, right hepatic trisectionectomy with caudate lobectomy and resection of couinaud s hepatic segments 1, 4, 5, 6, 7, and 8 is a surgical option usually indicated in advanced hilar cholangiocarcinoma, which mainly involves the right intrahepatic bile ducts in continuity with the left medial sectional bile duct. the relative volume resected in this hepatectomy averages 81% of the liver, which is an extensive liver resection.11 opening the hepatic parenchyma overlying the caudate lobe exposes the major hepatic veins and the hilar plate to direct view, facilitating control of venous bleeding and the interruption of the ascending paracaval portal branches along the hilar plate (figures 2 and 3). by separating the liver from the ivc, a safer transection can be carried out along the confines of the dorsal liver guided by the operator s left hand, which is placed behind the caudate lobe. there have been some reports of caudate lobe resections employing a combined right and left postero - lateral approach.12,13 in such procedures, liver transaction is performed caudocranially. as a result, it is important to divide the ascending paracaval portal branches that originate from the craniodorsal aspects of the hilar plate, as well as to maintain control of the bleeding from the hepatic venous branches, especially when the tumor is large or when it is difficult to bend the liver because of cirrhosis. using this method, liberation of the liver, securing the major hepatic veins, and complicated liver transaction all require a considerable amount of time, which is a significant shortcoming of these procedures.14 possible congestion of the medial segment caused by interruption of the branches of the mhv along its left margin did not significantly impair the remaining hepatic function. anterior hepatic transection is a rarely required but safe and potentially curative surgical option for isolated resection of the caudate lobe, especially when cirrhosis is apparent. additionally, in cases of metastatic liver carcinoma, it is possible to spare innocent hepatic parenchyma using this approach. anterior hepatic transection is recommended for tumors located in the paracaval portion of the caudate lobe (segment ix). | resection of the caudate lobe (segment i- dorsal sector, segment ix- right paracaval region, or both) is often technically difficult due to the lobe s location deep in the hepatic parenchyma and because it is adjacent to the major hepatic vessels (e.g., the left and middle hepatic veins).a literature search was conducted using ovid medline for the terms caudate lobectomy and anterior hepatic transection (aht) covering 1992 to 2007.aht was used in 110 caudate lobectomies that are discussed in this review. isolated caudate lobectomy was performed on 28 (25.4%) patients, with 11 case (11%) associated with hepatectomy, while 1 (0.9%) was associated with anterior segmentectomy. complete caudate lobectomy was performed on 82 (74.5%) patients. hepatocellular carcinoma was observed in 106 (96.3%) patients, while 1 (0.9%) had hemangioma and 3 (2.7%) had metastatic caudate tumors. aht was used in 108 (98.1%) caudate resections, while aht associated with a right - sided approach was performed in 2 (1.8%) cases. aht is recommended for tumors located in the paracaval portion of the caudate lobe (segment ix). aht is usually a safe and potentially curative surgical option. |
there are some commonly held beliefs that aging is associated with increased dependency, loss of self - control, incapacity, ailment, social isolation, and disengagement from life.1 misconceptions about older people and aging are called ageism, gerontophobia, or the elderly mystique.2 according to butler s definition of ageism, it has been defined as a process of systematic stereotyping and discrimination against people because they are old, just as racism and sexism accomplish this for color and gender.3 the ageism concept, which appears at the individual, institutional, and societal levels,4 is a composite of three components, including cognitive (stereotypes), affective (prejudice), and behavioral (discrimination) components.5 there are several cultural myths that tend to encourage ageism. the first myth is chronology, which considers people elderly by virtue of their age. the second myth is that of an inflexible personality, which means that as people age, they become more intolerant and conservative. third is the myth of misery, which suggests that elderly people are unhappy just because they are older. fourth, there is the myth of rejection and isolation, which holds that elderly people are rejected from society. fifth is the myth of dependence and unproductiveness, which declares that older adults are not productive members of society because they are not usually engaged in paid employment. sixth is the myth of physical ill health, which suggests that illness is part of the aging process and that old age is automatically linked to deterioration. last is the myth of mental deterioration, which suggests that all older people suffer from mental health problems.6 unfortunately, these myths are reinforced by mass media and gerontological studies, wherein elderly people are generally depicted in a negative light. for example, they are portrayed as immobile ; lacking in sex drive ; having poor eyesight and hearing ; being unable to take care of themselves ; being slow, confused, bent, and dowdy ; napping ; and lacking cognitive abilities.7,8 in addition to the mass media, several gerontological studies reinforce, rather than challenge, such ageist stereotypes.2 for example, the studies focusing on changes in physical and cognitive functioning of elderly people have extremely portrayed old age as a period of losses, loneliness, and declining physical and cognitive functioning.911 these negative and prevalent impressions toward aging have dramatically influenced health professional decisions and practices, which consequently have resulted in a failure to deal with treatable symptoms.1,12,13 for example, findings from a number of studies show that health professionals often prefer younger people to older adults, as they believe that young people are more productive and have a greater potential to live longer and healthier than older adults do. they may also see health - related problems of elderly people as normal signs of aging, rather than considering signs of illness that may be curable. unfortunately, these misconceptions have contributed to negative psychological, physical, and health outcomes14 and, consequently, to premature deaths of older adults.12 all these misconceptions state that elderly people have more problems than younger people. because this negative conception of older adults is becoming prevalent, resulting in the golden years of life becoming years of frustration and resentment, there is an urgent need for research to highlight the possible positive aspects of old age in society. therefore, it is imperative to do research that may dispel these myths about aging and give a better idea of old age. considering the above - mentioned misconceptions, this article compares emotional well - being as an important psychosocial marker of health and well - being15 between older adults and young people. emotional well - being has been defined as the possession of the flexibility to deal with the inevitable challenges of life conditions16 and having a positive feeling state comprising the affective aspect (happiness) and cognitive aspect (life satisfaction).1719 it also refers to the quantitative and qualitative experiences of joy, fascination, sadness, anxiety, anger, and affection that make life pleasant or unpleasant.20 the present study compares emotional well - being between older adults and adolescents. the alternative hypothesis of the study was that there would be a significant difference in emotional well - being between older adults and adolescents. the theoretical framework for this study was developed from carstensen socioemotional selectivity theory (sst) and wisdom theory. the sst comprises three central concepts : selectivity, emotion, and perception of time.21 the awareness of lifetime as a fundamental human characteristic plays an essential role in motivation. goals are often set within temporal contexts, and goal selection depends fundamentally on the perception of time. the first comprises expansive goals, such as acquiring knowledge or making new social networks. in contrast, the second encompasses goals related to feeling states, such as pursuing positive emotional experiences.22 according to the sst, people s goal selection is based on their perspectives regarding time. since most elderly people have already lost their parents and other ascending kin, their awareness of the fact that life is finite increases, and they become more selective about how to spend their remaining lifetime. instead of trying to spend their time in pursuit of varied experiences, they tend to use their time on activities that enhance their positive emotions and maximize their emotional well - being.21 in contrast, when people consciously or unconsciously perceive time as open - ended (in the case of young people), they are likely to be less selective with their time. they seek to gain resources to build their future, even though the experience is stressful and unpleasant.23 the sst maintains that perception of time horizons influences the selection of goals. when perceived as open - ended, time is associated with seeking new experiences and achievement - oriented stimuli ; for example, gathering new information and experiencing new knowledge. in contrast, when time is perceived as finite, it is linked to a stronger focus on emotionally relevant stimuli and interpersonal connectedness. for example, goals emphasize feeling states, mostly regulation of emotional states to optimize well - being.24 according to this theory, aging often involves a decrease in the experience of negative affect and is linked to a stabilization or even a slight increase in the experience of positive affect.25 as people get older and their perceived remaining lifetime is decreased, current emotional goals associated with well - being become more important. because people selectively attend to information that is consistent with their current goals, information processing in the elderly might be characterized by a positivity bias. research shows that older adults are more likely to forget negative and more likely to remember positive information compared with younger adults.26 compared with young people, the elderly were less likely to reserve negative information, but not positive information.25 older adults people have a more positive perspective than younger people.27 another theory that may explain possible generational differences in emotional well - being is wisdom theory. wisdom as a key factor in the creation of a good life and has been defined as expertise in the conduct and meaning of life that is associated with a high degree of personal and interpersonal competence, such as the ability to listen, evaluate, and give counsel.28 although wisdom has been widely defined, there are some agreements that it involves the use of certain kinds of pragmatic reasoning to navigate important life challenges.29 baltes and smith also define wisdom as having useful knowledge to deal with life problems, including an awareness of the varied contexts of life, acknowledgement that values and life goals vary among individuals and groups, and recognition of the uncertainties of life, along with ways to deal with them.30 this reflects that wisdom influences people s approach to life conflicts. because growing older may be related to the acquisition of wisdom - related concepts about life development,3134 it is believed that older persons are aware of ontogenetic changes, which may include the positivity effect. within this theory, a wise person is someone who can understand what is most important in life and knows the way to reach it. the wise person knows the things that make life worthwhile and meaningful.28 according to the earlier discussion, it could be speculated that older adults may have higher levels of emotional well - being compared with adolescents. data for this study were obtained from two surveys in malaysia : mental and spiritual well - being among adolescents (mswba)35 and patterns of social relationship and psychological well - being among older persons in peninsular malaysia (psrpwo).36 in both of these surveys, emotional well - being was measured using the world health organization (who)-five well - being index (who-5).35,36 the mswba was conducted from 2009 to 2010. the survey employed a self - group - administered questionnaire, which was distributed to 1,200 malay students selected using a multistage proportional stratified random sampling method. finally, the survey questionnaires were administered to all students in the selected schools, resulting in a sample of 1,200 respondents. the focus of the study was on a pattern of social relationship and psychological well - being in peninsular malaysia. the details of methodology have been published in full elsewhere.35,36 briefly, a multistage cluster sampling design was used to obtain a representative sample of community - dwelling elderly persons living in peninsular malaysia. the psrpwo employed a face - to - face interview technique to collect data in respondents homes with standardized questionnaires by trained interviewers. a subsample of 1,403 elderly malay aged 60 years and older was used for the present study. emotional well - being was measured using the who-5, a well - validated and widely used measure of positive well - being. the scale covers positive mood (good spirits, relaxation), vitality (being active and waking up fresh and rested), and general interests (being interested in things).37 because the who-5 well - being index was originally developed for adults, it is important to assess the readability of the measure. for this purpose, the fre rates text on a 100-point scale on the basis of the average number of syllables per word and words per sentence. the higher the fre score, the better the readability.38 the fre score obtained was 85.58, indicating that the who-5 could be read and understood by those with a ninth - grade reading level. the elderly group comprised 1,403 community - dwelling elderly persons aged 60 years or older, with approximately equal sex distribution (50.8% women, 49.2% men). the young group included 534 male and 656 female secondary school students with a median age of 16 years. elderly people scored significantly higher levels of emotional well - being (mean, 62.3 ; standard deviation, 22.55) than younger participants (mean, 57.9 ; standard deviation, 18.46 ; t=5.32 ; p0.001). using the commonly used criterion of a who-5 score lower than 52, suggesting poor emotional well - being,39,40 29.4% of older adults compared with 34.7% of young people reported poor emotional well - being (=8.46 ; p<0.01). a multivariate analysis of covariance (mancova) was used to compare the emotional well - being of older adults and young people (positive mood, emotional vitality, and general interests) after controlling for sex. before conducting the mancova, a series of pearson correlations were conducted between all of the dependent variables to examine the assumption that the dependent variables would be moderately correlated with each other.41 as shown in table 2, a meaningful pattern of correlations was observed among all of the dependent variables, suggesting the appropriateness of a mancova. in addition, box s test of equality of covariance was significant (box s m=74.94 ; p<0.001). this means that the assumption of equality of covariance matrices has been violated, which tests the null hypothesis that the covariance matrices between the groups are equal. therefore, because pillai s criterion is most robust for violations of test assumptions,42 it was used in interpreting the mancova results. as presented in table 3, the results of mancova revealed a statistically significant difference between older adults and young people on the combined dependent variables [f(3, 2587)=120.21 ; p0.001 ; =0.122 ]. the main effect of sex was also significant [f(3, 2587)=18.06 ; p0.001 ; =0.02 ]. however, the multivariate effect size for main effect of sex was small. finally, univariate analyses for each dependent variable as follow - up tests to the mancova, using the bonferroni method for controlling type i error rates, were conducted to examine individual mean difference across two groups and well - being subscales. as shown in tables 3 and 4, the results revealed a significant mean difference between older adults and young people in positive mood [f(1, 2591)=125.63 ; p0.001 ; =0.05 ], vitality [f(1, 2591)=25.09 ; p0.001 ; =0.01 ], and general interests [f(1, 2591)=57.70 ; p0.001 ; =0.02 ]. overall, mancova results indicate significant group differences in emotional well - being, wherein elderly people score higher on the emotional well - being scale than young respondents. the current study was designed to compare emotional well - being between older adults and young people. the findings from this study, as hypothesized, revealed that older adults had a higher level of emotional well - being compared with young people. our findings supported and extended existing research attempting to dispel misconceptions about aging. for example, lee and knight,43 in their study on a sample of 103 young people (mean age, 19.95 years ; standard deviation, 2.12) and 44 older adults (mean age, 71.75 ; standard deviation, 6.35) found that older persons have significantly lower anxiety scores than the young people.43 likewise, the findings from another study aiming to compare the psychological well - being of elderly people (mean age, 73 years) with that of young people (mean age, 20 years) showed that older adults significantly scored lower than young people on several indices of psychopathology including trait anxiety, neuroticism, schizotypal personality, rumination, and thought suppression.44 our results are also consistent with findings from a recent study conducted on a random large sample size among adult americans. it is generally believed that elderly people suffer from sleep problems more than younger people, but the findings from grandner s study reveal that sleep quality appears to improve over a lifetime.45 the results are also consistent with carstensen s study46 showing that older adults, compared with young people, have a higher ability to escape bad moods. similarly, our findings are also consistent with the recent study conducted by stone,47 who found that older persons were happier and less stressed than younger people. these findings can also support the hypothesis that emotional well - being may remain stable and intact in old age, and that despite having social losses, loneliness, declining physical and mental functioning, and cognitive impairments, elderly people have higher emotional well - being.48,49 the higher levels of emotional well - being of older adults compared with adolescents can be explained by carstensen sst, which maintains that as people age and perceive their time horizons become shorter, they focus on more immediate goals that elicit positive emotions. young people, in contrast, often need to explore or take risks to achieve longer - range goals, and they experience stress and frustration.50 the results also agree with baltes51 theory, in which wisdom and emotional intelligence increase with age. in other words, as people get older, their abilities to understand and develop strategies to avoid or at least lessen the possible emotional conflicts increase. a further explanation to justify why elderly people compared with younger adults have a higher level of emotional well - being has been provided by isaacowitz,52 who found that elderly people, when compared with young people, prefer positive outlook patterns and show the most positive outlook, which helps them regulate their mood and, consequently, leads to better emotional well - being.52 he also found that in certain situations, young people mostly pay more attention to emotionally negative information, and elderly people are more likely to pay attention to emotionally positive information. compared with young age, elderly people are more able to process positive stimuli while ignoring spontaneous reactions to negative stimuli.53 as people get older, they are more emotionally balanced and better able to solve emotional problems. there is also evidence that older adults are more positive and less negative than younger adults.54 there is also more evidence that as people grow older, negativity preference reduces compared with increased positivity preference. in other words, elderly people tend to see the good things in life more easily and are less likely to be affected by negative events. this evidence implies that aging is not characterized by an overoptimistic view of life but, instead, by an increasing ability to be less affected by negative events.55 the theory maintains that increased attention to emotional goals leads to greater complexity of emotional experience and, consequently, better regulation of emotions experienced in everyday life.22 another possibility that can explain higher levels of emotional well - being among older adults compared with young people is that they are more religious than young people and attach a high value to religious beliefs that strengthen them to enable effective coping with stress and losses.56,57 in malaysia, where culture and religion overlap, the elderly malays view the process of aging as god s will and believe that this world is temporary and there is a life after death, which enables them to view stressful experiences as less threatening and to cope more effectively with age - related changes and losses. therefore, their religious beliefs and practice positively influence their emotional well - being.58,59 although the results of this study show that elderly people had higher levels of emotional well - being compared with adolescents, it is worth mentioning that the pattern of greater well - being in old age may represent the well - being paradox, which refers to the notion that older adults report high psychological well - being despite increased age - related changes and life challenges.60 the review of the literature focusing on the well - being in old age reveals several reasons that many older adults paradoxically report high levels of well - being and life satisfaction. first, the high pattern of well - being in old age may not generalize to all dimensions of well - being. second, this pattern of the association between well - being and age may exist only in rich countries. another reason that older adults may, paradoxically, report higher levels of well - being is as a result of the inevitable influence of survivorship and the fact that people with lower well - being die earlier.61,62 contrary to negative ageist stereotypes of miserable elderly, the findings from the present study showed that elderly people compared with young people have better emotional well - being. the findings support sst. with regard to the well - known powerful influence of mass media on people s opinions, it is suggested that the findings from the present study be employed by the media to demonstrate more positive images of older people, which may promote positive attitudes toward aging. as a last word, it is noteworthy that although negative perceptions of older people are not totally without foundation,63 the problems of the elderly are greatly exaggerated. in other words, it can be argued that increasing problems with age are a nonlinear phenomenon that may be mediated and moderated by factors other than physiologic aging. the most important limitation of the present study that should be highlighted is its cross - sectional design, which makes it difficult to make a precise conclusion regarding emotional well - being trends over time. therefore, a further study using a longitudinal approach is needed to assess changes of emotional well - being across the life span. | backgroundthere are several negative stereotypes about older adults that have negatively influenced people s attitude about aging. the present study compared emotional well - being between older adults and adolescents.methodsdata for this study came from 1,403 community - dwelling elderly persons and 1,190 secondary school students and were obtained from two national cross - sectional surveys. emotional well - being was measured using the world health organization - five well - being index. data analysis was conducted using a multivariate analysis of covariance with spss software version 20 (ibm corporation, armonk, ny, usa).resultselderly people significantly scored higher levels of emotional well - being (mean, 62.3 ; standard deviation, 22.55) than younger people (mean, 57.9 ; standard deviation, 18.46 ; t, 5.32 ; p0.001). the findings from the multivariate analysis of covariance revealed a significant difference between older adults and younger people in emotional well - being [f(3, 2587)=120.21 ; p0.001 ; 2=0.122 ] after controlling for sex.conclusioncontrary to negative stereotypes about aging, our findings show a higher level of emotional well - being among older adults compared with younger people. |
we selected patients who were diagnosed with impending central retinal vein occlusion from january 2003 to march 2007 in wonju christian hospital, and we retrospectively studied their medical records. by gass ' definition, impending central retinal vein occlusion has mild or no loss of vision with venodilation and retinal dot hemorrhages under fundus examination. in all patients, slitlamp examination, intraocular pressure, gonioscopy, refraction, visual field, funduscopy, and fluorescein angiography were performed as part of a physical examination. to determine any underlying systemic disease, blood test, neurologic examination including brain computed tomography scan, carotid artery ultrasonography in ten patients with impending central retinal vein occlusion, eight were male (80%) and two were female (20%). the age distribution was between 18 and 48 years, with impending central retinal vein occlusion usually affecting a relatively young age group averaging 31.09.1 years of age. the ocular pressures in all subjects were found to be normal. on fundus examination, mild venous dilation and retinal hemorrhage were observed. the initial vision for the affected eyes was logmar 0.30, and the final vision was logmar 0.04. one case had progressed to central retinal vein occlusion, and after treatment with vitrectomy, radial optic neurotomy and intravitreal injection of triamcinolone, the patient 's vision improved. the other six patients had no specific systemic disease and no abnormalities on neurologic examination. an 18-year - old male patient with no specific history was admitted due to complaints of amaurosis fugax in his left eye. on fundus examination, venous tortuosity and dilation in the central retinal vein were found. the logmar vision after one month was improved to 0.0, and venous tortuosity and congestion in the central retinal vein disappeared completely (fig 1). a 31-year - old male patient with complaints of mild vision loss in his left eye visited the hospital. the initial vision was logmar 0.2, and on fundus examination, venous tortuosity, venous dilation, dot hemorrhages, and cotton - wool spots were observed. the patient was followed for two months without treatment, after which his vision improved to 0.0 and the previously observed central vein and retinal lesions improved (fig 2). a 39-year - old male patient without any specific history visited the hospital with the chief complaint of vision loss in his right eye. under fundus examination, venous tortuisity, dilation, and retinal h emorrhages were observed. after two weeks of admission, the retinal hemorrhage was aggravated, and the vision was down to logmar 1.0. the patient was treated with vitrectomy, radial optic neurotomy and intravitreal injection of triamcinolone. the retinal venous tortuisity and dilation in the central retinal vein improved, and the logmar vision also improved to 0.2. a 38-year - old male patient with a history of bronchiolitis visited the hospital due to vision loss in the right eye. on fundus examination, papilledema was discovered together with retinal venous tortuisity, dilation, and retinal dot hemorrhages in central retinal vein occlusion. two weeks after the visit, the vision was aggravated to 0.2, with macular edema and papilledema (fig 4). an 18-year - old male patient with no specific history was admitted due to complaints of amaurosis fugax in his left eye. on fundus examination, venous tortuosity and dilation in the central retinal vein were found. the logmar vision after one month was improved to 0.0, and venous tortuosity and congestion in the central retinal vein disappeared completely (fig 1). a 31-year - old male patient with complaints of mild vision loss in his left eye visited the hospital. the initial vision was logmar 0.2, and on fundus examination, venous tortuosity, venous dilation, dot hemorrhages, and cotton - wool spots were observed. the patient was followed for two months without treatment, after which his vision improved to 0.0 and the previously observed central vein and retinal lesions improved (fig 2). a 39-year - old male patient without any specific history visited the hospital with the chief complaint of vision loss in his right eye. under fundus examination, venous tortuisity, dilation, and retinal h emorrhages were observed. after two weeks of admission, the retinal hemorrhage was aggravated, and the vision was down to logmar 1.0. the patient was treated with vitrectomy, radial optic neurotomy and intravitreal injection of triamcinolone. the retinal venous tortuisity and dilation in the central retinal vein improved, and the logmar vision also improved to 0.2. a 38-year - old male patient with a history of bronchiolitis visited the hospital due to vision loss in the right eye. on fundus examination, papilledema was discovered together with retinal venous tortuisity, dilation, and retinal dot hemorrhages in central retinal vein occlusion. two weeks after the visit, the vision was aggravated to 0.2, with macular edema and papilledema (fig 4). central retinal vein occlusion has a wide range of manifestations, from mild and sporadic retinal hemorrhage to vitreous hemorrhage. since leber first described the clinical manifestation of central retinal vein occlusion as retinal apoplexy in 1854, scholars have suggested multiple classifications of the disease. classified central retinal vein obstruction into two categories, venous stasis retinopathy and hemorrhagic retinopathy. venous stasis retinopathy is a mild form without central retinal vein occlusion, and hemorrhagic retinopathy is a severe form with central retinal vein occlusion. magargal. added an intermediate category into the original classification of hayreh. and at the same time classified it into three types based on ischemic and hyperpermeable conditions. gass. reported impending or incipient central retinal vein occlusion. by gass ' definition, impending central retinal vein occlusion has no or mild loss of vision with venodilation and retinal dot hemorrhage under fundus examination. considered impending central retinal vein occlusion as a risk factor for the development of central retinal vein occlusion. however, no confirmed theory has been published regarding the natural progress of impending central retinal vein occlusion. mancall suggested that in the case of central retinal vein occlusion, the left eye is more frequently involved in males. however, raitta reported no discrepancies between left and right involvement or in male and female prevalence. in this study, we found that impending central retinal vein occlusion is more prevalent in males. factors known to cause central retinal vein occlusion include hypertension, diabetes mellitus, atherosclerosis, hyperlipidemia and primary open angle glaucoma. reported that hypertension, atherosclerosis and diabetes mellitus accompany central retinal vein occlusion in 57%, 34%, and 34% of cases, respectively. other studies have discovered that hematologic disease, paraproteinemia, vasculitis, and autoimmune disease accompanying changes in coagulation mechanism and viscosity of blood are often associated with central retinal vein occlusion [10 - 13 ]. in our study, four patients had systemic disease including hypertension, hypotenstion, bronchiolitis and pulmonary tuberculosis. hypertension, diabetes mellitus, hyperlipidemia, and other factors are relatively less associated with the occurrence of central retinal vein occlusion than they are with increased age. most of our patients were rather young, so they did not have general systemic risk factors of central retinal vein occlusion, except for hypertension. central retinal vein occlusion in young individuals usually has a good prognosis and a decreased risk for serious vision loss. in this study, we found that most patients recovered without any specific treatment, with only two patients developing aggravated disease. in the case of one patient, treatment with vitrectomy, radial optic neurotomy, and intravitreal injection of triamcinolone improved the condition. we conclude that most cases of impending central retinal vein occlusion have a good prognosis. on the other hand, we found that the disease can advance to the prodromal phase of central retinal vein occlusion with intensifying obstruction and hemorrhages. | purposeimpending central retinal vein occlusion is associated with mild or no loss of vision ; however, its progress and vision prognosis have not been clearly defined until now. therefore, we studied the progress and prognoses in patients with impending central retinal vein occlusion.methodsfor this study, we selected ten subjects who had been diagnosed with impending central retinal vein occlusion, and we retrospectively reviewed their progress and prognoses.resultsthe average age of the subjects was 31.0 years (18 to 48 years). eight patients were male and two were female. the average observational period was 5.5 months. six out of ten subjects were found to have no underlying systemic disease, four subjects had underlying disease. all ten patients were affected unilaterally. when initially tested, the affected eyes showed an average vision of logmar 0.30. the final vision test revealed an average of logmar 0.04, which indicates good progress and prognosis. in one patient, retinal hemorrhage and macular edema progressively worsened after the diagnosis, and the patient was treated with radial optic neurotomy.conclusionsthe cases of impending central retinal vein occlusion that we observed seemed to primarily affect young patients with generally good prognoses. however, in some cases, the degrees of obstruction and hemorrhage increased as time progressed. this suggests that impending central retinal vein occlusion could develop into the prodromal phase of an acute attack. |
radiofrequency (rf) catheter ablation has proven efficacy for treating many types of supraventricular arrhythmias and has become an increasingly important clinical tool for treating atrial fibrillation (af).1 electrical isolation of the pulmonary veins (pvs) from the left atrium is the cornerstone for most af ablation procedures and is considered the best interventional method to treat af in the paroxysmal af population.2 efforts to improve the efficacy and safety of rf ablation (rfa) led to the development of the open irrigation 6-hole catheter, which was the first catheter approved for ablation of af in 2009.3,4 the catheter design has now been improved with 56 irrigation holes to enhance cooling and reduce irrigation flow rate.5 the new porous tip provides uniform cooling over the entire tip rather than only localized cooling at the distal end, allowing more efficient heat dissipation and rf delivery. park. reported on better procedural efficiency to reach complete pulmonary vein isolation (pvi), as compared to the 6-hole catheter, with a significant reduction in irrigation volume administered (approximately 500 ml less).5 until now, safety data for the new catheter have largely been limited to unmonitored, single - center experiences.58 the primary objective of this paper is to report real world acute safety data from 2 prospective us observational registry studies using the porous tip catheter for the treatment of drug refractory, recurrent, symptomatic paroxysmal af. it is believed that these registry results represent a wider range of techniques, operator experience, and patient profiles than past studies, and as such will be generalizable to a broader range of clinical practice. a secondary objective of this paper is to report on any differences in acute safety between patients age 65 and older and younger patients, as af prevalence increases with age and adults age 65 years and older comprise an estimated 82% of all af patients in the united states.9 this analysis includes data from 2 prospective, multicenter observational registry studies (sfaf and iuaf) performed in the united studies and involving subjects who underwent rfa of drug - refractory recurrent symptomatic paroxysmal af. the sfaf study is currently ongoing and all available safety information at the time of the database lock is included in this analysis, which includes a minimum of 120 days follow - up on all patients. the iuaf study included only 7-day follow - up and is closed to enrollment, with all follow - up completed and all of the available safety information at the time of database lock included in this report. both studies were approved by institutional review boards at all clinical sites, and all patients provided informed consent before collection of any study related data. acute events that were predefined as potentially related to the device and/or procedure (primary events) were adjudicated by an independent safety committee. eligible study subjects were patients 18 years or older with drug - refractory, recurrent, symptomatic paroxysmal af. both studies excluded patients with af secondary to electrolyte imbalance or thyroid disease, reversible, or of noncardiac cause ; previous ablation for af ; af episodes that lasted 30 days or longer ; uncontrolled heart failure, or nyha class iii or iv heart failure ; documented atrial thrombus or other abnormality on preablation imaging ; contraindication to anticoagulation ; stroke, cardiac surgery, unstable angina, myocardial infarction (mi) or percutaneous coronary intervention within the past 3 months ; awaiting cardiac transplantation ; heart disease for which corrective surgery was anticipated within 6 months ; enrollment in other investigational drug or device study ; or who were pregnant. pvi was performed in both studies using 3-dimensional electroanatomical mapping (carto 3 system ; biosense webster, inc.) and a 56-hole porous tip ablation catheter (navistar thermocool sf catheter ; biosense webster, inc.) according to standard of care at each institution. the iuaf study also required utilization of the soundstar catheter for 3-d intracardiac echocardiography (ice). a minimum irrigation flow rate of 8 ml / minute was recommended for power levels 30w and 15 ml / minute for power levels > 30w. procedural details were recorded, including ablation targets, fluid delivered, maximum power, validation method used to verify complete pvi, and procedure time. following ablation, anticoagulation therapy and the use of other medications were at the discretion of the study physician. the safety evaluations in this analysis included the collection of all adverse events (aes) occurring over the course of the study, whether or not they were related to the device or procedure. exacerbations of preexisting conditions, if observed at follow - up and considered by the study physician to be clinically significant, were also included. the reporting period for aes began with access (opening of the femoral vein). any adverse changes to the subject 's health that occurred before access, but after the screening period, were considered to be a part of the patient 's medical history. all aes that were ongoing at the time of the 7-day follow - up contact were followed until the events were considered resolved or stable. electrocardiogram findings were also collected at baseline and discharge and were reviewed for safety as a part of both studies. the primary focus of this report is on serious adverse events (saes) related to the device and/or procedure. this includes any events occurring within 7 days postprocedure in the iuaf study or out to at least 120 days in the sfaf study. these primary events included any of the following saes occurring within 7 days postprocedure, except as otherwise specified : any event resulting in death, atrioesophageal fistula (discovered at any time postprocedure), atrial perforation, cardiac tamponade, mi, stroke or cerebrovascular accident (cva), thromboembolism, transient ischemic attack (tia), diaphragmatic paralysis, pneumothorax, heart block, pv stenosis (70% reduction in diameter, discovered at any time postprocedure), respiratory insufficiency, and pericardial effusion. the number and percentages of events were summarized for categorical variables (e.g., complications). means, standard deviations, medians, minimums and maximums were calculated for continuous variables (e.g., ejection fraction). pearson 's chi - squared tests were used to compare the counts of events across age cohorts. this analysis includes data from 2 prospective, multicenter observational registry studies (sfaf and iuaf) performed in the united studies and involving subjects who underwent rfa of drug - refractory recurrent symptomatic paroxysmal af. the sfaf study is currently ongoing and all available safety information at the time of the database lock is included in this analysis, which includes a minimum of 120 days follow - up on all patients. the iuaf study included only 7-day follow - up and is closed to enrollment, with all follow - up completed and all of the available safety information at the time of database lock included in this report. both studies were approved by institutional review boards at all clinical sites, and all patients provided informed consent before collection of any study related data. acute events that were predefined as potentially related to the device and/or procedure (primary events) were adjudicated by an independent safety committee. eligible study subjects were patients 18 years or older with drug - refractory, recurrent, symptomatic paroxysmal af. both studies excluded patients with af secondary to electrolyte imbalance or thyroid disease, reversible, or of noncardiac cause ; previous ablation for af ; af episodes that lasted 30 days or longer ; uncontrolled heart failure, or nyha class iii or iv heart failure ; documented atrial thrombus or other abnormality on preablation imaging ; contraindication to anticoagulation ; stroke, cardiac surgery, unstable angina, myocardial infarction (mi) or percutaneous coronary intervention within the past 3 months ; awaiting cardiac transplantation ; heart disease for which corrective surgery was anticipated within 6 months ; enrollment in other investigational drug or device study ; or who were pregnant. pvi was performed in both studies using 3-dimensional electroanatomical mapping (carto 3 system ; biosense webster, inc.) and a 56-hole porous tip ablation catheter (navistar thermocool sf catheter ; biosense webster, inc.) according to standard of care at each institution. the iuaf study also required utilization of the soundstar catheter for 3-d intracardiac echocardiography (ice).. a minimum irrigation flow rate of 8 ml / minute was recommended for power levels 30w and 15 ml / minute for power levels > 30w. procedural details were recorded, including ablation targets, fluid delivered, maximum power, validation method used to verify complete pvi, and procedure time. following ablation, anticoagulation therapy and the use of other medications were at the discretion of the study physician. the safety evaluations in this analysis included the collection of all adverse events (aes) occurring over the course of the study, whether or not they were related to the device or procedure. exacerbations of preexisting conditions, if observed at follow - up and considered by the study physician to be clinically significant, were also included. the reporting period for aes began with access (opening of the femoral vein). any adverse changes to the subject 's health that occurred before access, but after the screening period, were considered to be a part of the patient 's medical history. all aes that were ongoing at the time of the 7-day follow - up contact were followed until the events were considered resolved or stable. electrocardiogram findings were also collected at baseline and discharge and were reviewed for safety as a part of both studies. the primary focus of this report is on serious adverse events (saes) related to the device and/or procedure. this includes any events occurring within 7 days postprocedure in the iuaf study or out to at least 120 days in the sfaf study. these primary events included any of the following saes occurring within 7 days postprocedure, except as otherwise specified : any event resulting in death, atrioesophageal fistula (discovered at any time postprocedure), atrial perforation, cardiac tamponade, mi, stroke or cerebrovascular accident (cva), thromboembolism, transient ischemic attack (tia), diaphragmatic paralysis, pneumothorax, heart block, pv stenosis (70% reduction in diameter, discovered at any time postprocedure), respiratory insufficiency, and pericardial effusion. the number and percentages of events were summarized for categorical variables (e.g., complications). means, standard deviations, medians, minimums and maximums were calculated for continuous variables (e.g., ejection fraction). pearson 's chi - squared tests were used to compare the counts of events across age cohorts. the safety data analysis included 742 enrolled patients (sfaf = 508, iuaf = 234). approximately half of all patients were over 65 years old (sfaf = 48.4%, iuaf = 48.7%), and mean age was 62.1 years. the mean time since diagnosis of af was 1,594 days in sfaf and 1,619 in iuaf. a majority of the patients were on anticoagulation therapy before ablation (sfaf = 84.4%, iuaf = 81.2%) and had taken at least 1 antiarrhythmic drug (sfaf = 91.1%, iuaf = 86.8%). in general, cardiac history, incidence of comorbidities and antiarrhythmic drug medication history were similar among the patients enrolled in the 2 studies. baseline patient characteristics sfaf = registry identifier ; iuaf = registry identifier ; dx = diagnosis ; af = atrial fibrillation ; lvef = left ventricular ejection fraction ; la = left atrial ; nyha = new york heart association. of the combined 742 patients in the 2 studies, 85 (11.5%) experienced a total of 102 saes during the procedure or within the 7 days postprocedure, including 23 patients in the iuaf study (9.8%) who experienced 24 events and 62 patients (12.2%) in the sfaf study who experienced 78 events. only 30 (4.0%) of the 742 patients experienced an acute sae that was related to the device or the procedure. by study, there were 21 patients (4.1%) in the sfaf study and 9 patients (3.8%) in the iuaf study with acute device or procedure related saes. older patients (age 65 and over) experienced more saes within 7 days of ablation than younger (age 65 years old (range : 7177 years) with significant comorbidities and all deaths occurred greater than 7 days postprocedure. the results from these 2 large multicenter observational studies demonstrate the safety of the new catheter design in a real world setting with a large, heterogeneous patient population involving multiple clinician operators with various levels of experience. overall rates of device or procedure related saes were similar to rates published in other large studies of catheter ablation for af.1,10 in addition, there was no difference between older patients (age 65 and over) and younger (age < 65) patients with respect to overall rates of these events. there were no device or procedure related deaths in these registries, as reviewed and adjudicated by the independent safety monitoring committee. in 2009, a retrospective case series reported on the prevalence and cases of fatal outcome in catheter ablation of af. this large series reported that death is an uncommon complication associated with catheter ablation of af, with the incidence of death being 1 of every 1,000 patients.11 the absence of device and/or procedure related death in these studies further supports the past results. the safety data presented in this report are consistent with the published safety data on open irrigation catheter ablation for af. in a 2009 worldwide survey of 182 centers performing catheter ablation for af, major procedure - related complications occurred in 4.5% of patients.1 also, an analysis of 6 studies utilizing the 6-hole irrigation design reported a combined 4.9% (63/1275) procedural complication rate within 7 days postprocedure.10 the current studies, with a combined 30 patients of 742 (4.0%) who experienced procedure and/or device related events within 7 days, demonstrated a comparable level of acute safety for the new porous tip catheter. reported on initial experience with the new catheter in a cohort of 20 af patients at a single site, and observed major complications including 3 cardiac tamponade / perforations and 2 atrioesophageal fistulas.12 park. reported on a prospective multicenter study that compared the new porous tip catheter to the 6-hole thermocool catheter (biosense webster, inc.) using different ablation modes. the investigators observed no differences in ae rates between the 2 catheter technologies even when using different ablation modes.5 reported events included 1 patient of 78 in the thermocool sf catheter (biosense webster, inc.) group with a tia 2 days after pvi and 1 patient of 82 in the thermocool catheter group with a cardiac tamponade requiring thoracotomy. in the other studies reporting on use of the porous tip catheter, 1 tamponade and no deaths were reported in a total of 207 patients ablated with the thermocool sf catheter (biosense webster, inc.).57,1315 in the current studies, involving multiple sites and operators with various levels of experience, the combined rate of cardiac tamponade / perforation events recorded after index ablations (1.2%) is consistent with 2 large worldwide surveys that reported a 1.2% tamponade rate in 2005 and a 1.3% rate in 2009.11,16 although no patient in the current studies developed an atrioesophageal fistula, there was 1 patient with an esophageal injury. incidence of esophageal ulcer or thermal lesion has been reported to be as high as 1114% in patients who have undergone an af ablation.17,18 although the rate of thermal esophageal injury is unknown in the current studies because routine endoscopy was not performed, it is clear that esophageal injury is not an uncommon finding and does not lead to a fistula in the vast majority of cases. therefore, it is difficult to draw any conclusions from this isolated case. in these 2 studies, there was just 1 related sae for a congestive heart failure (chf) event in 742 patients (0.1%). a 2012 safety analysis including 1,275 patients from 6 fda clinical studies utilizing the 6-hole navistar thermocool catheter (biosense webster, inc.) reported heart failure or chf exacerbation events in 13 of 1,275 patients (1.0%).10 the average fluid volume delivered through the catheter in the current studies was 957.8 ml, as compared with 1,591.0 ml in the pivotal study for the 6-hole catheter design.19 it is likely that the reduction in the type of saes that typically result from fluid overload, such as chf exacerbation, is related to the reduction in fluid delivery. one of the limitations of these studies is that they are observational studies with no comparison arm. although a control arm is not typical of post - fda approval studies, the studies are closely monitored for any safety signals that would warrant additional evaluation. another limitation of the iuaf study is that it is limited to a 7-day follow - up and thus does not allow for full knowledge of late - occurring complications. this safety analysis demonstrated that catheter ablation utilizing a new open irrigation rfa catheter with 56-hole porous tip is safe for the treatment of drug - refractory recurrent symptomatic paroxysmal af, with no unanticipated device or procedure related aes. in addition to demonstrating overall safety in a heterogeneous population representative of what is seen in normal clinical practice, safety was consistent in the subpopulation of patients age 65 and older. | acute safety from 2 af ablation registriesintroductionthis report presents safety data on the use of a new open - irrigation radiofrequency ablation (rfa) catheter with a 56-hole porous tip in 742 patients enrolled in 2 us prospective, multicenter observational registry studies representing real - world use of the catheter.methodsthis analysis is comprised of patients who underwent rfa of drug - refractory recurrent symptomatic paroxysmal atrial fibrillation (af). acute adverse events (aes) were collected and categorized by seriousness, timing, and relatedness, with 7 days of follow - up data in one study and at least 120 days of data from a 1-year follow - up in the other. acute serious adverse events (saes) that were identified as potentially related to the device and/or procedure were adjudicated by an independent safety committee.resultsa total of 30 patients (4.0%) in the combined studies experienced an acute sae related to the device and/or procedure, which was similar in the subset of patients age 65 and over (4.2%). these saes included 1.2% cardiac tamponade / perforation, 0.7% pericarditis, 0.5% pulmonary events, and 0.8% vascular access complications. no myocardial infarction, stroke, transient ischemic attack, or atrioesophageal fistulas within 7 days postprocedure were reported. in the study with extended follow - up, 1 pulmonary vein stenosis and 1 esophageal injury were seen beyond 7 days postprocedure (0.2% each). there were no device or procedure related deaths.conclusionresults from 2 large observational studies demonstrated that a new porous tip rfa catheter was safe for the treatment of drug refractory, recurrent, symptomatic paroxysmal af, including treatment of older patients (65 years). |
solar energy is considered as one of the most important alternative energy sources and solar cell has been actively studied as promising solar energy conversion device. for its practical use, however, there are numbers of technical barriers to be overcome such as high cost and low - conversion efficiency. accordingly, numerous researches have been performed on organic solar cells for low - cost manufacturing and antireflection surface of the solar cells to improve the energy absorption efficiency [2 - 14 ]. the formation of antireflection surfaces reduces the reflection of incident light and increases its transmission into solar cells. a thin film layer on the surface can diminish the reflection of the incident light by the destructive interference between the reflected lights from the top and bottom surfaces of the coated layer when the film thickness is about a quarter wavelength of incident light. to induce this effect for a range of different wavelengths, multiple layers of thin films however, inevitable thermal mismatch between each thin film layer often causes adhesion and stability problems in the thin film type antireflection surfaces. to avoid these stability problems, antireflective nano structures with a period smaller than the wavelength of light reflection occurs when the light propagate through the interface of two materials of different refractive indices due to their discontinuous change. at the interface of the nano - structured material and the air, an effective refractive index at any cross - section orthogonal to the direction of incident light is determined by the areal fraction of the structural material and the air, and the tapered sas can make the continuous and monotonous change of the effective refractive index from air to solid surface. therefore, the array of tapered nano structures reduces the reflection of incoming light for a wide range of wavelengths [3 - 6 ]. previous works to make the etch mask relied on costly and complicated nano patterning techniques such as e - beam and nanoimprint lithography (nil). simpler methods to generate etch mask patterns in subwavelength scale on a large area were developed recently, including thermal dewetting of ni film on sio2 surfaces or on gan layers, ag deposition on heated substrates, and dispersion of nanospheres [12 - 14 ]. thermal dewetting of the ni film resulted in non - tapered and irregular - shaped sas array, where the refractive indices can not be changed monotonously giving relatively high reflectance. both approaches of ag deposition and consequently, they showed relatively inferior antireflectance compared with tapered sas fabricated by e - beam lithography. besides, the necessity of additional sio2 etch masks in the method using dewetted ni etch masks increases the number of fabrication steps, and therefore, reduces the cost - effectiveness. in this paper, pt / pd alloy thin films are thermally dewetted, and thus, hemispherical shape pt / pd nanodot arrays are formed. using these nanodot arrays as dry etch masks, capacitively coupled plasma - reactive ion etching (ccp - rie) using cl2 and n2 gases is then performed to form tapered sas arrays with narrower width at the top and wider at the bottom. our tapered sas fabricated by the simplest method reported so far using agglomerated pt / pd nanodots maintain as low reflectance as nil - based approaches achieved. schematic diagram of fabrication process and scanning electron microscope (sem) images for each step of tapered sas formation are shown in fig. pt / pd alloy thin film of 10 nm thickness is deposited on (100) si substrate by sputtering. the samples are then heated at 1,073 k for 90 s in rapid thermal annealing system to induce thermal dewetting of deposited pt / pd film. thermal dewetting occurs due to the increased surface energy of the metal film by heating. when the surface energy of the pt / pd thin film is bigger than the sum of the surface energy of si substrate and the interfacial energy between two layers, the film begins agglomerating to be minimum energy state with uniform contact angle. figure 2 shows the sem and atomic force microscope (afm) images of the thermally dewetted pt / pd film of 10 nm thickness. in the afm image, c, since the longer heating time enhances the surface energy of thin film. as a result, the pt / pd thin film is completely agglomerated into hemispherical nanodot array with subwavelength period fig. 2c, d. fabrication process flow for tapered subwavelength antireflection structures : apt / pd alloy thin film deposition with a thickness of 10 nm;bthermally dewetted pt / pd alloy nanodot etch mask formed at an elevated temperature ; andcformation of tapered subwavelength antireflection structures after rie thermal dewetting process of pt / pd alloy thin film for different heating time (a c) and afm image of dewetted nanodots (d) the array of hemispherical nanodots is then used as an etch mask for ccp - rie using cl2and n2gases at the flow rate of 50 sccm for each and the rf power of 300 w. during the rie process, the etch mask nanodots are also etched slowly, while the silicon is etched much faster. moreover, since the nanodots are in hemispherical shape, the edges of nanodots are consumed faster in the rie, exposing silicon under the nanodots. the size of nanodots becomes smaller as the rie is proceeded, and the rie time difference between the unmasked silicon and the silicon exposed later due to the nanodot etching makes the angled sidewall of sas as shown in fig. figure 3 shows sem images of thermally dewetted pt / pd nanodots generated from three different film thicknesses. 3a, c, and d, the pt / pd thin film is completely dewetted, but in fig. 3b, the pt / pd thin film is not completely dewetted due to the insufficient thermal energy. as the thickness of pt / pd alloy thin film is increased, more thermal energy is needed for complete agglomeration and bigger pt / pd nanodots are formed. thick pt / pd thin film leads to increased distance between nanodots and decreased number of nanodots in the same area. to achieve small reflectance, the period of antireflection structure should be less than the wavelength divided by the refractive index of substrate. considering that and from the repeated fabrication results, we decided to use 10 nm thickness pt / pd film for further etching process. thermal dewetting of pt / pd alloy thin film for different thickness figure 4a d are the sem images showing different height and shape of sas for different etching time. as the etching time of rie is increased from 60 to 110 s, the average height of tapered sas is also increased from 230 to 470 nm. these samples are used to find the optimum fabrication process that produces the lowest reflectance. the etch rate of si substrate in the experiment ranges from 4 to 5 nm / s. when a sample is etched for 110 s, the nanodot etch masks almost disappear and the average height of tapered sas reaches the maximum of 470 nm. 2d, the typical height of pt / pd nanodots after thermal dewetting step falls within the range of 80130 nm. therefore, the calculated etching selectivity of si substrate to pt / pd nanodots in our fabrication is about 45:1. in fig. 4d after the rie for 120 s, it is recognized that the height has been slightly reduced compared to the etching result for 110 s and the sidewall of tapered sas is roughened. the reason for the reduced heights is that the tips of the structures are etched faster than the bottom of the structure when the nanodot etch masks are completely removed. the measured average heights of sas are : a230 nm, b370 nm, c470 nm, andd450 nm for the repeated tests to find optimized recipe for thermal dewetting and rie process, the si wafer is broken into number of pieces and sas were processed on them as shown in fig. 5. the surface of the si substrate with fabricated sas array in fig. 5a seems black due to the low reflectance while the bare si wafer in fig. 5b reflects the image of the camera that took this picture due to the high reflectance. however, considering the fabrication process is composed of only metal thin film deposition, heating and rie without costly and time - consuming nano patterning steps such as electron beam lithography or nanoimprint lithography, the tapered sas fabrication could be easily extended to wafer scale larger area. in fig. 6, the sem image of the angled view of the tapered sas array fabricated in the large area is shown. si substrate (a) with fabricated sas array is compared to bare si substrate (b). due to the low reflectance of the sas array, substrate (a) seems completely black, while the highly reflective bare si substrate (b) reflects the image of the camera that took this picture an sem image (60 angle) of a high aspect ratio, large area subwavelength antireflection structures array since the tapered sidewall and height of sas decide antireflective property, not only the formation of nanodot arrays by thermal dewetting but also the control of etching process is critical. rie etching characteristics strongly depend on plasma density. as ion density and its energy differ between ccp - rie and icp (inductively coupled plasma)-rie, they result in different etching rate and selectivity. since rie with chlorinated plasma does not have large loading effect compared to cf4 plasmas, the chemical reaction during the silicon rie in cl2 plasma is not as much as in cf4 plasma. less chemical attack means the etching is relatively more physical, giving less chance of undercut. this is important for tapered sas formation, and therefore, cl2 plasma - based ccp - rie is adopted in our fabrication. as shown in figs. 4 and 6, the diameter of the tapered sas continuously increases from top to bottom and thus the refractive index also continuously increases. consequently, it is expected that the reflectance is very small for a wide range of wavelengths of light. to assure the aforementioned, the reflectance of the fabricated sas was measured by uv - vis - nir spectrophotometer (varian cary 500). figure 7shows the reflectance measured as a function of the wavelengths of the light irradiated on five different sets of tapered sas array with different heights generated by different etching time. the reflectance of bare silicon wafer is also presented for comparison. in the wavelength range from 350 to 1,800 nm, all of the differently processed samples show average reflectance under 5.5%. on the other hand, the measured average reflectance of bare si wafers in the same wavelength range is 35%. in infrared range (7001800 nm), the 420 nm height sas array shows the smallest average reflectance value of 3.17%. in visible wave range (400700 nm), the smallest reflectance is achieved on the sas array with the heights of 370 nm and the average reflectance is 1.12%. moreover, this sample shows extremely low reflectance value under 0.01% in a specific visible range of 570650 nm. all samples show the smaller reflectance in visible wave range than in infrared or ultra violet wave range. this result is meaningful especially for solar cell applications since 46% of solar energy is in the visible wave range. in theory, the reflectance of the structure is expected to be decreased as the height of the structures increases. however, the measured average reflectance in the whole wavelength range from 350 to 1,800 nm decreases only to the height of 420 nm where the average reflectance is 2.80% and higher reflectance is observed for 450 and 470 nm structures. the measured average reflectance in visible and infrared range also shows the similar result. as a possible reason for this, it is presumed that the measured reflectance value could be slightly different depending on the specific location on the substrate due to the nonuniformity of the fabrication process. as described earlier, the reflectance is decided not only by the height of the sas, but also by the period and taper angle. since our sas array is formed on a large area, it is possible that each fabrication step contains nonuniformity such as different pt / pd film thickness between center and edge of the substrate, which will lead to different sizes and periods of nanodot etch masks on the identical substrate. nonuniform rie may also result in the variation of the taper angle of sas array between different locations of the substrate. considering the further application of the sas array as nano mold that could be replicated to polymers for low - cost antireflective surface formation, our monotonously tapered sas is also advantageous, since previous works adopting thermal dewetting and etching produced mushroom - like shape of nanopillars with which demolding process is difficult. the reflectance measured as a function of the wavelengths of light irradiated on subwavelength antireflection structures with different height in this paper, we presented simple and large area fabrication methods for tapered sas without expensive and complicated nano patterning processes. by using the thermally dewetted pt / pd nanodots as etch mask and performing ccp - rie with cl2and n2gases, tapered the monotonously tapered shape of fabricated sas gives continuous and smooth increase of refractive index along the incident light path, resulting in very low reflectance < 5.5% for 3501,800 nm range of wavelength. especially for visible light range, the measured reflectance of 1.12% is as low as the sas fabricated by e - beam or nanoimprint lithography. the proposed method is expected to be applied not only to solar cell but also to optical and optoelectronic devices such as display screens and light sensors. this research was supported by nano r&d program through the korea science and engineering foundation funded by the ministry of science & technology (2008 - 02916), and partially by a grant - in - aid for new and renewable energy technology development programs from the korea ministry of knowledge economy (no. | we have demonstrated lithography - free, simple, and large area fabrication method for subwavelength antireflection structures (sas) to achieve low reflectance of silicon (si) surface. thin film of pt / pd alloy on a si substrate is melted and agglomerated into hemispheric nanodots by thermal dewetting process, and the array of the nanodots is used as etch mask for reactive ion etching (rie) to form sas on the si surface. two critical parameters, the temperature of thermal dewetting processes and the duration of rie, have been experimentally studied to achieve very low reflectance from sas. all the sas have well - tapered shapes that the refractive index may be changed continuously and monotonously in the direction of incident light. in the wavelength range from 350 to 1800 nm, the measured reflectance of the fabricated sas averages out to 5%. especially in the wavelength range from 550 to 650 nm, which falls within visible light, the measured reflectance is under 0.01%. |
brown tumors (bts), also called as osteoclastomas, are rare nonneoplastic lesions that arise in the setting of hyperparathyroidism319). hyperparathyroidism is overactivity of the parathyroid glands resulting in excess production of parathyroid hormone (pth). excessive pth secretion may be due to problems in the parathyroid glands such as adenomas, hyperplasia or carcinoma. however, it may also occur in response to low calcium levels in such conditions as vitamin d deficiency or chronic renal disease that is known as secondary hyperparathyroidism319). bts can arise as solitary or multiple lesions of any bone, more common in extremities, clavicle, ribs and pelvis., we report a rare thoracic bt case that occured in the setting of primary hyperthyroidism. 50-year - old man was admitted to our neurosurgery department with the chief complaint of difficulty in standing and walking due to leg weakness for nearly 2 days. thoracic magnetic resonance imaging (mri) and computerized tomography (ct) revealed expansile mass lesion that was compressing the spinal cord at t9 level. homogenously enhancing mass lesion was found to originate from the anterior portion of the spinous process and both laminas of t9 vertebra (fig. routine blood tests were uneventful except a calcium value of 14.3 mg / dl (8.4 - 10.2). as blood parathormone (pth) test also revealed a very high value of 547.45 pg / ml (15 - 68.3), endocrinology consultation was ordered to rule out primary hyperparathyroidism. however, any cystic or solid pathologic lesion compatible with adenoma were not seen on the parathyroid regions. parathyroid scintigraphy with tc-99 m mibi revealed focal activity retention on the inferior portion of the right thyroid lobe. endocrinology department insisted for the urgent parathyroidectomy in order to minimize hypercalcemia - related complications. total thyroidectomy was also performed since frozen examination of a suspected thyroid nodule was reported as micropapillary carcinoma. the day after parathyroid and thyroid surgery blood calcium and pth levels decreased to 10.2 mg / dl and 10.42 pg / ml, respectively. patient underwent spine surgery. wide posterior decompression (t8 partial / t9 total laminectomy+bilateral t9 transvers process and pedicle excision) was followed by t8 - 10 posterior instrumentation and fusion (fig. absolute pathology reports were consistent with the parathyroid adenoma and spinal brown tumor (fig. brown tumors occur due to increased osteoclastic activity in the setting of either primary or secondary hyperparathyroidism3819). parathyroid adenomas or hyperplasia constitute the major bt source in primary hyperparathyroidism while chronic renal failure is the leading cause in secondary hyperparathyroidism3). bts are localized form of osteitis fibrosa cystica (ofc) that is commonly associated with hyperparathyroidism. ofc is a process that is characterized by hyperstimulation of osteoclastic proliferation via longstanding excessive pth production causing bone resorption and bone marrow fibrosis371014). histopathologically, bts are composed of osteoclast - like giant cells and hemosiderin with a fibrovascular stroma. the name brown tumor comes from the accumulation of hemosiderin that gives the surrounding stroma a brown color38). bts look like similar histologically to other giant cell lesions such as giant cell tumor, aneursymal bone cyst and reparative giant cell granuloma8). only the clinical manifestation and endocrinologic status help to differentiate bts from other giant cell lesions. bts usually develop in the third or fourth decades of life8). in the beginning, bts that develop in primary hyperparathyroidism were seen much more common. however, they are seen more often recently as a result of secondary hyperparathyroidism that is closely related with increased life expectancy of chronic renal disease patients who require hemodialysis38). most of the patients with the diagnosis of primary hyperparathyroidism present with kidney stones or isolated hypercalcemia3). however, nearly one third of patients are asymptomatic and hypercalcemia is found incidentally. skeletal involvement such as bone resorption, bone cysts, bts and osteopenia is seen on the late phase of hyperparathyroidism311). the symptoms of spinal bts include axial pain, radiculopathy, myelopathy and myeloradiculopathy according to their locations13121314151617181920). it can mimic multiple myeloma, metastases, sarcomas and other giant cell lesions671019). bone cortex might be thinned and expanded but it is not penetrated by the tumor frequently. on ct scan, mri is the diagnosis modality of choice in evaluating the location, extent of spinal tumor and neural compression. on mri, bts are commonly seen hypointense on t1-weighted images, hypointense or hyperintense on t2-weighted images and homogenous enhancement is noted after contrast injection. plasmocytoma, lymphoma, giant cell tumors and metastates should be ruled out in the differential diagnosis of bts36819). when a solitary lesion that correspond to bt is found on the spine, differential diagnosis might include true giant cell tumor, aneurysmal bone cyst and giant cell reparative granuloma. also, hyperparathyroidism associated bone changes such as loss of lamina dura of the teeth roots and generalized demineralization of the medullary bone of the jaw could help the surgeon to differentiate bt from other giant cell lesions8). treatment of bts involve both the management of hyperparathyroidism and neural decompression. total or subtotal parathyroidectomy is the gold standard for the treatment of primary hyperparathyroidism371119). parathyroid surgery rapidly decreases the excessive amount of pth thus achieving complete regression of the lesions with remineralization346). neurological status is the main determinant in case of neural compression in the treatment of bts37). unless the decompression procedure does lead to instability, patients can be mobilized in braces without spinal fixation. however, spinal instrumentation and fusion is needed if the tumor is too large, affects multiple levels or pathological spinal fractures exist7101219). our search in english literature demonstrated 20 spinal bt patients with primary hyperparathyroidism (table 1). the patients ' age ranged from 16 to 69 with a mean of 45.3 years. thoracic spine was the most affected part of the spine (55%) followed by lumbar, sacral and cervical regions. 15 patients (75%) underwent double surgeries for both removing the mass and to treat the primary hyperparathyroidism. 6 patients (35%) underwent instrumentation and fusion surgery whilst 11 patients (65%) underwent only decompression surgery. bts should be kept in mind in the differential diagnosis of lytic, expansile spinal tumors, especially with the histopathological diagnosis of giant cell tumors. treatment consists of both treating the underlying primary pathology and decompression of the neural structures, if necessary. | brown tumors also called as osteoclastomas, are rare nonneoplastic lesions that arise in the setting of primary or secondary hyperparathyroidism. parathyroid adenomas or hyperplasia constitute the major brown tumor source in primary hyperparathyroidism while chronic renal failure is the leading cause in secondary hyperparathyroidism. most of the patients with the diagnosis of primary hyperparathyroidism present with kidney stones or isolated hypercalcemia. however, nearly one third of patients are asymptomatic and hypercalcemia is found incidentally. skeletal involvement such as generalized osteopenia, bone resorption, bone cysts and brown tumors are seen on the late phase of hyperparathyroidism. the symptoms include axial pain, radiculopathy, myelopathy and myeloradiculopathy according to their locations. plasmocytoma, lymphoma, giant cell tumors and metastates should be ruled out in the differential diagnosis of brown tumors. treatment of brown tumors involve both the management of hyperparathyroidism and neural decompression. the authors report a very rare spinal brown tumor case, arisen as the initial manifestation of primary hyperparathyroidism that leads to acute paraparesis. |
interestingly, the trajectory of a proton in the lumen of the so - called m2 channel of the influenza a virus is an example from biology. the m2 integral membrane protein is a tetramer assembled from four identical subunits, each with 97 residues. as a prerequisite for release of genetic material to the cytoplasm, the interior of a viral particle must become acidified as a means to detect its own entrance in the endosome. the m2 protein channel fulfills this acidification function by conducting protons through its membrane pore. the gating of this essential proton transport step in the infection cycle is controlled by ph. among the pore - lining residues, his37 and trp41 were identified as the most important residues in the proton transfer through the m2 channel. the proton selective conductance in m2 is achieved by the presence of the his37 cluster : substitution of his37 with residues such as gly or ala induces a nonselective channel, indicating a mechanistic role of his37 in proton relay. a transfer cycle was proposed where a tautomerization of the his37 side chain occurs between the acceptance of a proton from the viral exterior and the release of a proton to the viral interior, to reestablish in full the original configuration for the next cycle. on replacing the imidazole ring of the his37 side chain with a 4-thiazolyl group, which has the n1 as the only proton acceptor with pka 2.5, this mutant shows inward proton flux resembling the wild type, thus demonstrating that tautomerization is not necessarily required for proton transport. however, rotation of the imidazole ring during proton translocation can not be ruled out. besides the functional role in proton selectivity, his37 is associated with channel activation : binding of a third proton to the his37 cluster under acidic conditions triggers a detectable proton flux, with a peak conductance rate of 10 protons per second. substitution of trp41 with smaller residues, like gly, ala and phe, induces higher proton conductivity compared to the wild - type channel. despite the relatively small size of the m2 protein, and several high - resolution structures being available, the mechanism of proton conduction through the channel is not completely understood (figure 1a). previously, we studied the addition of a proton to the his37 cluster from the extraviral side, and compared the energetics and dynamics of two configurations of the doubly protonated state at neutral ph a 4-fold symmetric histidine - box and a 2-fold symmetric dimer - of - dimersand proposed a multiconfiguration model where these configurations are in moderately fast equilibrium. here, we focus our analysis on the configuration of the his37trp41 cluster in the triply protonated state, and its subsequent deprotonation. m2 channel and its two configurations of the his37trp41 quartet at low ph. (a) shown is the tm domain of m2 protein imbedded in a fully hydrated lipid bilayer. his37 and trp41 residues and lipids are colored in green, pink, and ice blue, respectively, as space filling spheres. waters are shown in gray surface. the d - model (b) and the h - model (c) of the quartet. system with the absence of cl ion are shown in stick mode with the same color code as in (a), and the system with cl ion are shown in white stick. (d) the principal components of the quartet,, in different systems as a function of time ; data for the 2 + state are shown for comparison. herein, quantum mechanics / molecular mechanics (qm / mm) molecular dynamics (md) simulations reveal insights into the function and mechanism of this protein. specifically, molecular simulations complemented by crystallographic and nmr (nuclear magnetic resonance) determined structural data, as well as physiological measurements provide mechanistic clues in understanding this rate - limiting step of proton conduction in m2, especially the proton trajectory during the translocation reaction. the present study elaborates our previous computational works, and explores the free energy profile with the explicit presence of the complete transmembrane domain of the protein as well as a fully hydrated membrane environment. moreover, to explore the effect of the proton filter and the channel gate, the entire his37trp41 cluster and the nearby water clusters are treated within a quantum mechanical methodology. in this manuscript, we document multiple calculations : first, we studied the structural integrity of the m2 protein with two models at low ph with qm / mm md simulations. we find that the structures of the his37 cluster starting from both models converge at low ph, and are stable within the simulation time scale : both models appear ready for the subsequent proton release toward the intraviral side. then, we calculated potentials of mean force (pmfs) of the deprotonation by constrained qm / mm md simulations : the pmfs of both models have barriers 6.5 kcal / mol. compared to previous simulation studies, the local electrostatic environment of the hydrated membrane significantly lowers the energy cost. finally, we explored the possible role of a cl anion in the pore at low ph, which was found to stabilize the highly charged 3 + histidine cluster, but also to impede proton release by stabilizing the hydronium species within the channel pore. configuration at the 2 + state (with 2 of 4 his37 side chains charged) at neutral ph as the d2-model, and the subsequent 3 + state obtained by adding a third proton as the d3-model (figure 1b). correspondingly, the histidine - box at 2 + state and the subsequent 3 + state structures are designated as the h2- and h3-models (figure 1c), respectively. the 1.65 resolution x - ray structure (pdb entry : 3lbw, residues two and three of the four his37 residues were initialized to be charged in the 2 + and 3 + states, respectively. the protein was then embedded in a previously equilibrated 80 80 lipid bilayer containing 171 1-palmytoyl-2-oleoyl - sn - glycero-3-phosphatidylcholine (popc) molecules, hydrated by 10,031 water molecules. potassium and chloride ions were added to neutralize the system, and to provide a solution buffer with ionic concentration of 150 mm. periodic boundary conditions were applied, and long - range electrostatic interactions were treated with the particle mesh ewald method. a time step of 2 fs was used, and all of the bonds involving hydrogen atoms were constrained with the shake method. after an equilibration period with gradually released harmonic restraints, production simulations were performed at 310 k and 1 atm using langevin temperature and langevin piston pressure coupling schemes. the tip3p force field was used for water molecules, and charmm force field was used for the protein and lipid atoms. simulations were performed with namd, and continued for 200 ns for the 2 + state, and 300 ns for the 3 + state. to prepare the his37trp41 quartet structures for the subsequent h3- and d3-models at the acidic condition with qm / mm md simulations, we followed the protocol used in our previous work to optimize the his37trp41 quartet structure at the 2 + state with the oniom method implemented in gaussian 03. on the basis of the optimized his37trp41 quartet structure in the h2- and d2-models, a third proton was added to a neutral his37 side chain, and oniom was used to optimize the system after this addition. then, the h3 and d3-models were constructed with the original quartet in the corresponding h2(d2)-model replaced with the newly optimized quartet at the 3 + state. therefore, the initial backbone of the h3(d3)-model is similar to the preceding h2(d2)-model. with the quartet structure held fixed, we then relaxed the remaining atoms of the h3(d3)-model with classical md simulations for 20 ns. we took the last snapshot from the md simulations, and minimized the system with the his37trp41 quartet fixed. the minimized structures were used for the following qm / mm md simulations. in the qm / mm scheme, as in our previous study, we included in the qm region the his37trp41 quartet and thirteen water molecules nearby (corresponding to the three water clusters identified in the 3lbw.pdb structure : six waters just above the his37 tetrad, two in the his37trp41 cavity, and five below the trp41 gate). these atoms were treated at the density functional theory (dft) level using a mixed gaussian and plane - waves treatment, while the rest of the system, including the protein, the lipids, and waters, was treated with the mm method (charmm force field and tip3p). the generalized gradient approximation (gga) functional blyp with the d2 level dispersion - correction (blyp - d2), the triple- basis set with two polarization functions, and the goedecker, teter, and hutter type pseudopotentials were used. a wavelet - based poisson solver was used to remove the spurious interactions of the qm region atoms with the periodic images. a box size of 32 32 32 was used for the qm region so that the buffer between qm atoms and the box edge is 8. we saturated the broken bonds with hydrogen atoms, and combined the qm and mm subsystems with the imomm methodology, where the scaling factors of 1.355, 1.384, and 1.416 were used to relate the qm c h bond distance to the mm cc, cn, and cc distances, respectively. for the qm / mm md simulations, after 2 ps equilibration for each model, we accumulated 9 ps trajectories for the d3-model with and without cl, and 4 and 7 ps for h3-model with or without cl, respectively. to characterize the free energy profile for proton release from the his37 cluster in m2 protein, we followed the well - tested protocol used in the previous work from our group to run constrained qm / mm md simulations. the starting configuration was adopted from the preceding 3 + state qm / mm md simulations. the reaction coordinate was defined as the distance between the n2 on one his37 side chain and its neighboring h2, in the range of 1.0 to 1.6. with the reaction coordinate fixed in each window, constrained qm / mm md simulations were carried out for times varying between 5 and 8 ps to obtain a converged pmf. forces after the initial 1 ps of each constrained simulation were collected to calculate the mean constraint force. free energy profiles for the proton release were estimated by thermodynamic integration of the mean force along the reaction coordinate. all of the qm / mm md simulations were carried out with the cp2k program with a time step of 0.4 fs. nbo was used to calculate the partial charge at the mp2/6 - 311++g(d, p) level, performed with gaussian 03. in those calculations, only the side chains of the his37 and trp41 residues the conformation of the his37 cluster in both the d3-model and the h3-model was quantified by a single parameter,, defined as the projection of the atomic coordinates of his37 along the displacement vector connecting the two models, where = 0 represents the dimer - of - dimers (d2-model) and = 1 the histidine - box (h2-model). upon binding the third proton, as shown in figure 1d, the -value in the h3-model remains at 1, indicating that the protonation does not change the conformation of the his37 cluster significantly. in the d3-model, his37 shifts from = 0 to = 0.4, showing that the d2-model and the corresponding d3-model are connected by a ph - dependent transformation. furthermore, this transformation appears to form an intermediate state of the one leading from the dimer - of - dimers (= 0) to the histidine - box this conformational change has been previously proposed for the dimer - of - dimers at acidic conditions, where one dimer is broken upon taking a third proton, to avoid electrostatic repulsion. the structure of the his37 cluster after this conformational change is closer to the histidine - box conformation. in both models, the presence of a cl ion in the his37trp41 cavity does not have a major impact on the protein s conformation (figure 1b d), but has more profound effects on proton conduction, which is further described below. we monitored the specific structural motifs of either model by exploring the rotamer structures of his37 and trp41. as disclosed by experimental determined structures at different conditions, these two key residues at both high and low phs adopt rotameric angle 1 = 180, whereas the angle 2 is relatively flexible. in particular, the side chain dynamics of the two residues are distinct between the d- and the h - models. in the qm / mm md simulations at the 2 + state (figure 2a), the his37 cluster in the d2-model adopts a 2-fold symmetry, where the two dihedral angles [1, 2 ] of the charged his37 side chains are [180, 360 ] and those of the neutral ones are [180, 260 ]. the [180, 360 ] configuration of his37 was rarely sampled in the classical md simulations (figure 2b and 2b), largely because of the inability of the classical force field to reproduce the strongly hydrogen bonded dimer - of - dimers of the d2-model. in the d3-model (figure 2a), one of the charged his37 side chain in the broken dimer remains at the [180, 60 ] configuration with the other one swings at [180, 150 220 ], and the intact histidine - pair resembles those in the d2-model. interestingly, this [180, 60 ] configuration is the characteristic of the low ph structure, which was occasionally sampled in the classical md simulations at the 2 + state, but never reached in the corresponding qm / mm md simulations. in both the h2- and h3-models, all of the four his37 side chains have [1, 2 ] values of [180, 180 ]. dihedral angle distributions of the his37 and trp41 side chains from the d- and h - models are plotted at neutral and acidic ph conditions. the top panel shows the plots at neutral conditions (2 + state), which are labeled as a f ; the corresponding plots at acidic conditions (3 + state) are labeled as af, shown in the bottom panel. panels a and b show the correlation between 1 and 2 on his37 side chain from the qm / mm and classical md simulations, respectively. panels c and d show the correlation between 1 and 2 on trp41 side chains. panels e and f show the correlation between 2 on his37 and 2 on trp41. the trp41 side chains feature a flip variant in the h - models (the n1-h1 bond of the indole group points toward the central pore) and a flop variant in the d - models (the same bond points away from the central pore). these variants correspond to 2 angles located at 80 (tp rotamer, the flip variant) and at 280 (tm rotamer, the flop variant), which are retained at both high and low ph conditions in each model (figure 2c and 2c). however, the 2 angle of trp41 at the low ph conditions in the d3-model is more diffuse than the congener in the d2-model, indicating a more dynamic trp41 gate in the d - model under acidic conditions. in contrast, this gate seems to be more rigid in the h - models, as evidenced by the similar distributions of 2 angle at neutral or acidic ph conditions. due to the inward orientation of trp41 in the h3-model, the excess proton is less easy to be released toward the intraviral end of the channel, as further described below. however, this orientation enables the trp41 side chains to form water - mediated hydrogen bonding interactions with asp44 (figure 3), which was proposed to stabilize the trp41 gate, a critical factor for the asymmetric proton flux. the outward orientation of the trp41 in the d3-model, on the other hand, points the amide group toward the hydrophobic wall, which seems to be a less favorable factor for its gating. shown is the top view of the channel, in which trp41, asp44 and water molecules are the sticks, and the backbone of the protein is the spiral. it should be noted that, in the classical and qm / mm md simulations of both d - models, only the tp rotamer of trp41 was observed (figure 2d and 2d), as the histidine - box x - ray structure was used as the initial structure and no indole ring flipping happened in the submicrosecond time scale simulations. the average h2h2 distance of neighboring trp41 residues in the d2- and d3-models are 4.7 and 7.2, respectively, which are consistent with the data derived from f nmr measurement on f(hnm) m2tm sample (3.2 at ph 8.0 and 8.0 at ph 5.3). in contrast, the average h3h3 distance is 11 in both the h2- and h3-models. this is in line with a nmr experiment under different conditions, in which the f(h3) m2tm sample was found to have the closest distance of 11 at both high and low ph conditions. as the m2 protein s structure is sensitive to the change in the solubilizing environment, the diversity of the his37trp41 contact depends on the balance between different rotamers. the nature of the interaction between his37 and trp41 differs between the d- and h - models, as the former is more sensitive to the protonation state (figure 2e, e,f, f). this difference in the his37trp41 packing and its response to the environment can be mainly attributed to the higher flexibility of the his37 side chain compared to trp41. in the h - model, the orientations of the two residues and their coupling are less dependent on ph condition, showing only small thermal motions for both the imidazole ring of his37 and indole ring of trp41. due to the different rotameric conformations, distances between his37 and trp41 are also distinct in the d- and h - models, which can be determined by different techniques. as shown in table 1, the distances measured in our md simulations are quantitatively consistent with experimental structures or nmr measurements. taking the c(his37)h3(trp41) distance as an example, at neutral ph condition, the average distances (figure 4a) in the d2- and h2-models are 6.8 and 4.7, respectively, corresponding to 6.5 in the 2l0j.pdb and 5.0 in the 3lbw.pdb. the dashed line connecting h1 on his37 and c on trp41 indicates the reaction coordinate used to scan the change of partial charge, as shown in panel b. nbo analysis was used to calculate the partial charge at the mp2/6 - 311++g(d, p) level. ensemble average of the eight snapshots of the nmr structure. among the eight snapshots of the nmr structures, data at ph = 8.5 and ph = 4.5 (in parentheses) are shown. ssnmr : solid - state nmr. to explore the free energy cost for the his37 cluster to release a proton at low ph conditions, we carried out constrained qm / mm md simulations on the d3- and h3-models imbedded in the fully hydrated lipid bilayers, in which the geometrical parameter of the distance between n2 on one his37 side chain and its neighboring h2 was selected as the reaction coordinate. among the three charged his37 residues, the one with the [1, 2 ] of [180, 60 ] was selected to release a proton, as this his37 has an orientation similar to those found in the low ph condition x - ray structure. the free energy profiles for deprotonation and the representative snapshots along the reaction pathways are shown in figure 5. the calculated pmfs was obtained by thermodynamic integration of the average force along the deprotonation pathway, where all free energy values are shown relative to the value of the lowest pmf calculated in each system (figure 5a). the deprotonation curves are quite similar between the d3- and h3-models, but the presence of cl imposes significant perturbation on his37 deprotonation (describe later). in the d3-model, the minimum of the free energy profile is located at = 1.024, corresponding to the triply charged his37 cluster state (figure 5a, b). along with the increase of the n2h2 bond until = 1.30, the pmf increases, showing that the proton moving from his37 to the neighboring water is energetically unfavorable (figure 5a, b). at = 1.30, the proton is shared by his37 and the neighboring water, with the average n2(his37)o(water) distance of 2.52, where the proton is closer to the water (the averaged h2o(water) distance is 1.24). the newly formed hydronium is stabilized by the surrounding hydrogen - bond network, which extends to the waters at the c - terminal side (figure 5b). at high ph conditions, these interactions are interrupted as the trp41 gate is closed. meanwhile, one proton of the hydronium points to the neighboring water, and is ready to be released. the additional proton on the hydronium is expected to release to the c - terminal side of the pore. at = 1.5, the proton transfer is complete (the averaged h2o(water) distance is 1.06), and the hydronium group is separated from his37 by three water molecules. c) is very similar to that in the d3-model, with the free energy barrier height of 6.63 kcal / mol, located at = 1.36 : at this point, a hydronium is formed next to the deprotonated his37 (with the n2(his37)o(water) and h2o(water) distances of 2.53 and 1.22, respectively). (a) shown are the free energy profiles for proton release under different starting configurations. both the d3- and h3-models have similar deprotonation pathways, while the presence of cl in the h3-model inhibits proton release. panels b d show the representative snapshots during the proton release in the d3-model (b b), the h3-model (c c), and the h3-model with cl (d). their locations are labeled in panel a. taking the d3-model as an example, along its deprotonation pathway, one proton originally attached to the n on a charged his37 residue gradually moved to the neighboring water cluster at the c - terminal side, with the nh distance increased from 1.024 (b) to 1.20 (b) and to 1.30 (b). at 1.30, further increase of the nh distance enables the fully release of the proton from his37, as shown on the red dashed line in (a). previously, we demonstrated that two alternative configurations (the d2- and h2-models) are capable of stabilizing the charges in the pore at neutral ph condition. here we find that, under acidic conditions, the mechanism of proton release from the his37 cluster is much less dependent on its configuration (the d3- or h3-models). in other words, for a charged his37 side chain, which is ready to lose a proton, it is largely unaffected by the configuration of the remaining three his37 residues. therefore, the interplay between his37 residues, the ph sensor of the channel, is ph dependent during the proton relay. the trp41 gate and the membrane environment, on the other hand, are likely to play a role in modulating the rate of deprotonation and thus the conductance. different computational approaches have previously been used to explore the deprotonation from the charged his37 cluster in the m2 protein. in one recent paper from our group, the deprotonation from a single histidine and from the histrp motif in water was investigated with dft - based md simulations. for a single histidine, the free energy barriers were calculated to be 10.010.1 kcal / mol at the n position, and 10.510.6 kcal / mol at the n position. a recent study from voth and co - workers combined an empirical valence bond and a dft - based qm / mm approach to study the proton release at different protonation states, reporting barrier heights of 13 and 10 kcal / mol for proton release from the triply and quadruply charged his37 cluster, respectively. when a single trp residue is included in the calculation with a position resembling that in the protein, the barrier for the n was found to be 9.1 kcal / mol, 1 kcal / mol lower than that in the isolated histidine in water box, suggesting a role of the trp41 gate in regulating the proton relay through his37, in addition to its known ability to generate an asymmetric flow. specifically, charge transfer from his37 to the bound trp41 through the cation interactions was witnessed, as assessed by the partial charge on each group (figure 4b), consistently with the calculated electrostatic potential. presumably, this charge delocalization decreases the energy cost to lose a proton. in the present work, the energy cost for deprotonation in the membrane environment is 6.5 kcal / mol, significantly lower than the one in the histrp motif or the isolated his. apparently, the present work provides a more realistic model in studying proton transfer, where the backbone of the transmembrane domain of the protein, the trp41 gate, and the explicit membrane environment have significant effects on modulating the dissociation of protons from his37. for the latter one, to be specific, the confined environment of the pore and the low dielectric constant of the membrane destabilize the charged cluster, and thus facilitate the deprotonation of the charged his37. by using the following equations and assuming g = 0 for simplicity between his37 and bulk water, the upper limit of the pka of his37 in the membrane environment is estimated to be 4.7 in the d3-model and 5.0 in the h3-model, respectively, based on the major barrier heights for deprotonation in both models. experimentally, the third pka of the his37 cluster when three of four residues are protonated was determined as 6.3 and 4.9 0.3, respectively, by using different protein constructs and lipid membrane compositions. the backbone conformations in both d3- and h3-models are very similar to those of the relatively compact protein structure at neutral ph. without significant expansion of the backbone, we demonstrate that both models are able to release a proton with a moderate energy cost. the presence of the c - terminal amphipathic helix anchoring at the lipid interfacial region is likely to limit the dynamic of the c - terminal segment, compared to what seen for the isolated the transmembrane helices from electron paramagnetic resonance (epr) experiments and the x - ray structure determined at low ph conditions. finally, we investigated the possibility of a role for cl or other halide anions in the proton relay mechanism through the pore. in electrophysiology studies, the proton conduction rate was found to be unaffected by the concentration of cl in both oocytes and liposomes. this finding is in apparent contrast with the attractive force that a triply charged his37 tetrad can exert on a cl ion : assuming for simplicity that the rate of proton transfer is only controlled by electrostatics, cl ions should increase the proton conduction rate. in a recent study of the calcium - release activated calcium channel (crac), a cation - conducing channel with a 6 - wide pore and a high density of basic side chains, we observed that cl anions in the pore significantly increase the rate of permeation of na cations. therefore, we extended the proton transfer calculations to a model including a cl ion bound to the his37 tetrad, to explore its impact on the proton conduction rate. as shown in the pmf calculations, the presence of cl makes the free energy cost of deprotonation much higher. following the increasing n h distance, the pmf keeps rising : at = 1.36, where the major barrier is located in the previous pmfs, the free energy value is 10.5 kcal / mol, 3.9 kcal / mol higher than that in the h3-model (figure 5a). this demonstrates that the presence of cl creates an unfavorable pathway for a proton to be released. during the simulations, the cl ion is located in the highly charged his37 cluster, and has stable interactions with the water in the cavity below, which is formed by the his37 and trp41 clusters (figure 5d). the arrangement of the hydrogen bonding network of water molecules in the cavity could compensate in principle the energy cost for bond breaking of the proton release. nevertheless, the electrostatic attraction between the anion and the proton attenuates the proton diffusion out of the cavity to the c - terminal even though the trp41 gate is occasionally open. therefore, the presence of anions in the his37trp41 cavity is an unfavorable factor for proton release, which the m2 channel can circumvent by maintaining its pore free of cl ions through its asp44 residues, two helical turns away from his37. this is confirmed by mutations of asp44, which significantly impact the proton conduction profile in a salt solution with a [cl]/[so4 ] ratio approximately 2:1. in this study, we explored the different configurations of the his37trp41 cluster in the m2 protein at low ph conditions, and the subsequent deprotonation via qm / mm md simulations. with regard to the conformation of the key residues, two alternative models (d3 and h3) converge to a single deprotonation mechanism, and feature similar free energy profiles to release a proton, with a barrier height of 6.5 kcal / mol. therefore, the multiconfiguration model proposed in our previous work, includes deprotonation as a mechanism not only for proton conduction, but also for conformational exchange connecting the multiple configurations of this protein. the confined region between his37 and trp41 also appears to significantly increase the proton affinity of a water molecule compared to the bulk solution. calculations with a bound cl ion in the pore show that halide anions may contribute to stabilizing the triply charged histidine cluster, but at the same time impede the release of protons. we suggest that only those viral strains capable of maintaining halide anions outside the pore can maintain a stable proton conduction rate irrespective of the anions bulk concentration. | the activity of the m2 proton channel of the influenza a virus is controlled by ph. the tautomeric state and conformation of his37, a key residue in the m2 transmembrane four - helix bundle, controls the gating of the channel. previously, we compared the energetics and dynamics of two alternative conformations of the doubly protonated state at neutral ph, namely, a 4-fold symmetric histidine - box and a 2-fold symmetric dimer - of - dimers, and proposed a multiconfiguration model for this charge state. here, we elaborate this model by further studying configurations of the his37 tetrad in the triply protonated state and its subsequent deprotonation via quantum mechanics / molecular mechanics (qm / mm) molecular dynamics (md) simulations, starting with the aforementioned configurations, to gain information about proton release in a viral membrane environment. interestingly, the two configurations converge under acidic ph conditions. protons can be transferred from one charged his37 to a neighboring water cluster at the c - terminal side of the channel when the trp41 gate is open transiently. with limited backbone expansion, the free energy barrier for proton release to the viral interior at low ph is 6.5 kcal / mol in both models, which is much lower than at either neutral ph or for an isolated his37 cluster without a membrane environment. our calculations also suggest that the m2 protein would seem to exclude the entrance of anions into the central channel through a special mechanism, due to the latter s potential inhibitory effect on proton conduction. |
patent foramen ovale, which is necessary in fetal circulation, is a potential route for emboli arising from the venous system to enter the systemic arterial circulation, resulting in paradoxical air embolism syndrome. we report a case of paradoxical air embolism as a complication of percutaneous approach for treatment of calyceal diverticular stone. a 37-yr - old man in good health was referred to us for right calyceal diverticulum with milk of calcium. after induction of general anesthesia, the patient was placed in the lithotomy position. a 6-fr ureteral catheter with ureteropelvic junction occlusion balloon was passed through the right ureter, and the patient was then turned to prone position. after confirming the position of the ureteral catheter under fluoroscopy, the pyelogram was performed by injecting contrast and a small volume of air into the right pelvocalyceal system to locate the diverticulum (fig. 1). direct puncture of the calyceal diverticulum was attempted several times without success. the total volume of air used during the procedure was about 25 ml. the patient remained cardiovascularly stable throughout the procedure. six hours after the surgical procedure, the patient complained of weakness of his right leg. eight hours after this episode, he had sudden tonic seizure and became unconscious ; he was transferred to the intensive care unit and treated, accordingly. the patient underwent magnetic resonance imaging of the brain including diffusion - weighted imaging, which was negative. twenty - four hours after the procedure, he recovered fully without any neurological deficit and regained consciousness. neurologic evaluation showed that cryptogenic arterial air embolism was the mostly likely cause of the seizure. cardiac workup with transthoracic echocardiogram failed to reveal a cardiac source of emboli. however, transesophageal echocardiography with provocation testing showed air bubbles in the left atrium revealing that the patient had right - to - left shunt due to patent foramen ovale (fig. 2). in this case, the clinical features were strongly suggestive of paradoxical air embolism during air pyelogram. air embolism, the entry of gas into the vascular structure, is mainly a iatrogenic problem that can result in serious morbidity and even death. venous embolism occurs when air is introduced to the central venous intravascular space and embolizes to the right heart or pulmonary arterial system. arterial embolism results from the entry of gas into the left heart chambers, as with paradoxical air embolism across an intracardiac shunt or during cardiac surgery, or directly into the arteries of the systemic circulation such as during decompression barotraumas or penetrating trauma involving an artery (1, 2). this phenomenon was first described by lopez, who noted the passage of fluid from the calyces into the renal veins (6). air embolism as a complication of retrograde pyelography, and air entering the hepatic veins after nephrolithotomy have also been reported (7). the factors that influence morbidity and mortality of air embolism include the volume of air entrained, rate of entrainment, position of patient during the event, and the cardiac status of the patient. patent foramen ovale is necessary in fetal circulation to sustain intrauterine life. the closure of the foramen ovale is normally completed by the age of 2 yr. in case of incomplete closure, the foramen ovale is kept sealed by the left atrial to right atrial pressure gradient but may be opened if the right atrial pressure increases and exceeds that of the left atrium (8). a persistent patent foramen ovale is thus a potential route for emboli arising from the venous system to enter the systemic arterial circulation. a patent foramen ovale and paradoxical air embolism syndrome has been increasingly implicated in cryptogenic embolic stroke. the most widely supposed mechanism for a patent foramen ovale related stroke is paradoxical air embolism of venous thrombotic material across the atrial right - to - left shunt (9, 10). the normal collecting system is known to have a capacity of approximately 5 - 10 cc. in this case, retrograde injection of 25 ml of air appeared to be the cause of physiologically significant air embolism, most likely via pyelovenous backflow phenomenon. in addition, clinical features of this case strongly suggested paradoxical air embolism associated with patent foramen ovale. a recent study reported that endoscopic operation under carbon dioxide pneumovesicum is effective and safe (11). although gas embolism is usually air embolism, the medical use of other gases such carbon dioxide, nitrous oxide, nitrogen, and helium can also result in embolism. thus, endoscopic operation using carbon dioxide, which is highly water - soluble and relatively safe, carries potential risk of embolism. to our knowledge, this is the first report of paradoxical air embolism associated with patent foramen ovale during percutaneous nephrolithotomy. at present, there are no consensual guidelines on the optimal management of patent foramen ovale in patients who have suffered from cryptogenic embolic stroke (12). urologists who prefer to use air during pecutaneous nephrolithotomy under general anesthesia should be aware of the potential risk of air embolism. injection of a smaller amount of air for pneumopyelography is strongly advisable, if necessary. | air embolism is a rare complication of percutaneous nephrolithotomy. patent foramen ovale, which is necessary in fetal circulation, is a potential route for emboli arising from the venous system to enter the systemic arterial circulation, resulting in paradoxical air embolism syndrome. a case of paradoxical air embolism during percutaneous nephrolithotomy is presented. to our knowledge, this is the first report of paradoxical air embolism associated with patent foramen ovale during percutaneous nephrolithotomy. |
results of the investigation on artificial radionuclides in the environmental objects provide for the assessment of possible affects of radiation exposure on human health. at the same time investigation of radionuclide distribution in the environment hence it is necessary to consider the basic sources of radioactive pollution, in particular in the baltic sea waters, as well as further behaviour of radionuclides under the influence of the external factors. artificial radionuclides have penetrated into the baltic sea for the first time from the global fallouts as a result of the nuclear and thermonuclear weapon tests in the atmosphere. during a long period of time this sea was contaminated by the north sea waters where radioactive waste was discharged from various nuclear objects. after the accident in the chernobyl power plant (chpp) in 1986, an average value of va of cs has increased by an order of magnitude in the baltic sea compared to the value which was formed by global fallout (12 bq / m). however, an increase of va of cs was observed in the coastal waters during some years after chpp accident. it is natural that radioactive fallout on the surface waters of the baltic sea was inhomogeneous. the biggest amount of cs occurred in the northern part of the baltic sea and in the gulf of bothnia. therefore, the levelling process of va of cs in the surface waters took place. it means that this radionuclide was transferred from more polluted to less polluted areas, and increased there va of cs. in particular, the increased values of cs va in the coastal waters of the baltic sea in lithuania were observed up to 1989 and 1990, when its average values were 113 and 117 bq / m accordingly. since 1991 a gradual decrease in average values of va of cs was observed. however, frequent variations in va values took place because of the secondary effects and also after the changes in the hydro meteorological situation. the variations of va values of artificial radionuclides after the change of external factors is presented in article. it is known that sr practically did not participate in the air mass movement from chernobyl. therefore its va values have changed slightly in the baltic sea and a primary source of pollution of this sea by sr turned out to be the global fallout. it is determined that va of cs and sr in the surface and near bottom waters is almost the same [4, 7 ]. since the behaviour of these radionuclides in the sea waters is diverse, their self - purification processes are different also, as well as the variation of va under the effect of external factors. individual observation results of sr and cs va demonstrate only an approximate estimation of their behaviour in the sea waters. in order to define their possible variation caused by the natural factors or to find the additional amount of radionuclide which could penetrate into the sea waters from accidental unidentified sources, it is necessary to execute large - scale observations. these facts were registered during the individual experiments and also in the average draft results, which were obtained before and after chpp accident [8, 9 ]. in particular since 1991 the va of the above mentioned radionuclides decreased in the coastal waters of the baltic sea near lithuania. however, obvious variations in the defined va measurements were observed. thus, in 2004 the extreme values of cs va were 94 and 49 bq / m and sr va were 17 and 7 bq / m. they indicate major changes in the absolute values of the radionuclide va. according to the average va of cs in the surface waters of the baltic sea at a present time and the predicted value, almost the same result has place (about 40 bq / m). the aim of the present study is to define possible values of variation of sr and cs va in coastal waters of the baltic sea which can be formed under the influence of natural hydro physical factors. water samples (40 l) for the determination of cs and sr volumetric activity (va) in the near - shore waters of the baltic sea (juodkrante) were taken in 20052009 (fig. 1). in 2008, water samples were taken near pervalka and nida. the temperature, specific electric conductivity, water current direction and wind direction were registered during the time of sampling.fig. 1observation stations the cs and sr were concentrated from the same sea water samples after adding the stable carriers by ferrocyanide - carbonate precipitation [11, 13 ]. 2.fig. 2scheme of radiochemical analysis of cs and sr scheme of radiochemical analysis of cs and sr after the radiochemical cleaning the yield of caesium was determined gravimetrically in the form of cs3sb2i9. the activity of cs samples was registered by gamma spectrometer (canberra) with hpge detector (resolution 2 kev, efficiency 15 %). a stable strontium yield was determined by the atomic absorption spectrometer and y - gravimetrically in y2o3 form. the yield values varied within a range of 6080 %. the determination error for sr va amounted to 15 %. it is known that contamination of the hydrosphere began in 1954, when sr was found in the waters of the atlantic ocean for the first time. the increase of the global fallout was observed up to 1963, when nuclear and thermonuclear weapon testing countries signed the treaty banning nuclear weapon tests in the atmosphere, hydrosphere and outer space. since then a gradual decrease of radioactive environmental pollution was observed. after the accident the radioactive pollution spread far from chernobyl and in particular toward the baltic sea. by that time va of cs has increased in the surface waters by a more than an order of magnitude and nowadays its average va has decreased 34 times. it means that the baltic sea is not completely purified from this radionuclide. during the above period sr va this fact can be explained by its negligible transfer to the baltic sea after the chpp accident. because of the process of levelling of va of these radionuclides in the surface water of the baltic sea, their absolute values varied in the coastal waters from time to time. 1) in 2005 are presented in table 1.table 1va of cs and sr, and data of some hydro meteorological parameters in coastal waters of the baltic sea (juodkrante) in 2005no.dateconductivity, ms / mprecipitations, mmdirection of windvelocity of wind, m / s sr, bq / m cs, bq / m 1.22.049700.0nw610622.23.049650.0n56693.25.049900.0e412744.27.049900.0e39665.29.0410500.0nw513756.01.0510500.0s412757.03.0510252.4s610488.05.0510500.0n614509.07.0510250.0w5155610.08.0510250.0nw3164611.28.069951.7nw6114512.01.079000.0w3145413.03.079000.00135514.05.079000.0e4115415.07.079600.0e3135416.09.079600.0e1135417.23.089900.0e4124918.25.089300.0s2115019.27.089603.9sw6115420.29.089550.0w5125121.31.089650.0nw2105722.02.099800.0sw2114823.04.099800.0e3134724.14.108800.0w6114625.15.109200.0n8154126.16.109400.0n4124627.18.109600.0nw3115328.20.109200.0s674129.22.109601.1s684530.23.1095011.7w7114431.25.109608.0sw7154832.27.109200.0n7133833.30.109200.0s7103034.31.109200.0sw5122735.01.119200.0se61130 va of cs and sr, and data of some hydro meteorological parameters in coastal waters of the baltic sea (juodkrante) in 2005 according to the obtained results (table 1), the marked variations of va of radionuclides have been observed from april to november, 2005. for sr the extreme values for this period of time were 6 and 16 bq / m and for cs27 and 75 bq / m accordingly ; the ratio of the above values was almost the same, i.e. about 2.5. however, there is a possibility for the additional penetration of radionuclides from the accidental sources. the average values were 12 bq / m (sr) and 51 bq / m (cs). according to average monthly values, the va of cs has a seasonal course with the highest values in spring and the lowest in autumn (fig. these results are approximate since various numbers of measures were carried out every month. however, such conformity to natural law concerning va of sr was nt observed.fig. 3average values of cs and sr va in coastal waters of the baltic sea (juodkrante) in 2005 average values of cs and sr va in coastal waters of the baltic sea (juodkrante) in 2005 similar measurements were carried out in april may, 2006 (table 2). then the extreme values of sr va were 9 and 14 bq / m and for cs37 and 52 bq / m. however, the difference between these values is smaller than for the same data in 2005 (table 1).table 2va of cs and sr, and data of some hydro meteorological parameters in the coastal waters of the baltic sea (juodkrante) in 2006no.dateconductivity, ms / mprecipitations, mmdirection of windvelocity of wind, m / s sr, bq / m cs, bq / m 1.19.049800.6w612372.20.049803.8s512373.21.049900.0nw213374.22.049900.0n614385.25.0410204.2nw311396.26.049900.0nw412417.27.0410800.0se79428.28.0411100.0n211439.29.0411000.0e7104410.02.0511000.8s2114411.03.0511000.0sw2134512.04.0510800.0n5134513.05.0511000.0n4144514.06.0510500.0e61152 va of cs and sr, and data of some hydro meteorological parameters in the coastal waters of the baltic sea (juodkrante) in 2006 a small number of measurements of va of sr and cs were carried out in the baltic sea near juodkrante in 2007. here there is small difference in va for sr and cs (since observations were carried out during a short span of time, i.e. from 21st to 27th august, 2007. it is necessary to note, that an average va of sr (12 bq / m) coincides with the results obtained in 2005 and 2006 (tables 1 and 2).table 3va of cs and sr and data of some hydro meteorological parameters in the coastal waters of the baltic sea (juodkrante) in 2007no.dateconductivity, ms / mprecipitations, mm sr, bq / m cs, bq / m 1.21.088301.410452.23.0892011.214473.25.089000.012404.27.089204.51247 va of cs and sr and data of some hydro meteorological parameters in the coastal waters of the baltic sea (juodkrante) in 2007 simultaneous observations in va of radionuclides near juodkrante, pervalka and nida (fig. 1) were carried out in july, 2008. in some cases a marked difference in va of radionuclides was observed. 4va of cs (bq / m) in the coastal waters of the baltic sea in july, 2008 in observation stations near juodkrante, pervalka, nida (fig. 5va of sr (bq / m) in the coastal waters of the baltic sea in july, 2008 in observation stations near juodkrante, pervalka, nida (fig. 1) va of cs (bq / m) in the coastal waters of the baltic sea in july, 2008 in observation stations near juodkrante, pervalka, nida (fig. 1) va of sr (bq / m) in the coastal waters of the baltic sea in july, 2008 in observation stations near juodkrante, pervalka, nida (fig. 1) the best coincidence of the obtained results near the above observation stations for cs was on 10th july and for sr the same results were on 4th and 8th july. these results show that the scale of va variations in these radionuclides does not coincide in time and can be diverse under identical changes of the external factors. during water sampling minor changes of water conductivity were identified, including the observation of insignificant variations in wind direction and velocity. the obtained results of radionuclides va during the second half of august 2009 are presented in table 4.table 4va of cs and sr and data of some hydro meteorological parameters in the coastal waters of the baltic sea (juodkrante) in 2009no.dateprecipitations, mmdirection of windvelocity of wind, m / s sr, bq / m cs, bq / m 1.18.089.6w913402.19.080.0nw913433.20.080.0e213384.21.080.0sw412375.22.081.1sw41441 va of cs and sr and data of some hydro meteorological parameters in the coastal waters of the baltic sea (juodkrante) in 2009 sr va practically did not change (table 4) and its variations did not exceed an experimental error despite the high velocity and changing wind direction. hence, the change of hydro meteorological situations determined different variations of va of sr and cs. the average values of va of these radionuclides in 20052009 are illustrated in fig. a minor increase in the average data of sr va from 11 to 13 bq / m was observed from 2005 to 2009. at the same time the average values of cs va decreased from 49 to 40 bq / m. however, this comparison should be considered as an approximate, since the number of measurements was different from year to year. it is also necessary to focus on the extreme values of the obtained results defining the limits of their possible deviations from average values. this was to be expected that the greatest difference of extreme values was appeared in 2005 when more measurements were carried out. here the difference in the extreme values of sr va was 10 bq / m and the same for cs48 bq / m. it means that external factors can change va of the radionuclides to a marked degree.fig. 6average values va (bq / m) of sr and cs, obtained in 20052009 in the coastal waters of the baltic sea (juodkrante) average values va (bq / m) of sr and cs, obtained in 20052009 in the coastal waters of the baltic sea (juodkrante) in the surface waters of the baltic sea an average va of sr was 24 bq / m and cs was 12 bq / m before the chpp accident. after this accident va of sr increased slightly and va of cs increased by more than an order of magnitude. hence, the process of self - purification of the baltic sea waters from these radionuclides has various features. according to theoretical results an average va of cs in the baltic sea has to be about 40 bq / m. nowadays, however, va of sr was almost the same during the last decade, i.e. 12 bq / m. sr va decreases because of radioactive decay, but also can increase due to global fallout and other factors. during 20052009, major variations of radionuclides va were observed and sometimes their absolute values differed from each other more than two times. however, in order to define the additional amount of radionuclides from other sources of radioactive pollution, it is necessary to know possible variations of va of radionuclides caused by the natural hydro meteorological situation. variations of va of artificial radionuclides in the coastal waters of the baltic sea are possible from inflowing bays and rivers. this causes water mass movement along the coast which depends on wind velocity and direction. besides, short - term change of radionuclides va is possible in spring time as a result of the melting snow and surface runoff, i.e. the water flowing over the land surface. during a storm it is necessary to take into consideration the intensity and duration of precipitation which can affect the content of radionuclides in the surface sea waters. after 3rd may, 2005, an obvious decrease of va of both radionuclides after precipitation (table 1) was observed. the decrease of radionuclides va in coastal waters was registered during the last days of october, 2005 when precipitation lasted long enough (table 1). however, such dependence is not always observed since during precipitation water mass transfer with higher radionuclides va is also possible. a short - term increase of radionuclides va can take place on the sea surface because of the air mass transfer after the forest fires and burning peat lands. it is known, that plants accumulated greater amount of artificial radionuclides after the chpp accident and global fallouts. therefore va of sr and cs in the surface waters of the baltic sea depends on the season of the year since sea water stratification differs in summer and winter time, and also on the transition period when change of water mass takes place. radionuclides from water are especially poorly accepted by the ice cover, whereas in summer time zooplankton, phytoplankton and sea vegetation accumulate radionuclides. thus, a change in artificial radionuclides va is possible because of the above processes. it is necessary to note that external effects on the baltic sea waters not always cause the identical tendency of sr and cs va change since these radionuclides behave differently in the sea waters. however, extreme variations of radionuclides va caused by the natural factors have their limits. average values of sr and cs va decrease in time because of their natural radioactive decay and the decrease of the environmental radioactive pollution. nowadays an average va of sr has to be considered 12 bq / m and cs40 bq / m in coastal waters of the baltic sea near lithuania. compared to these values the variations of radionuclides va exceeded them by 50 % in 2005 when long - term observations took place. marked variations of radionuclides va during short - term observations in 20062009 were not identified. therefore, the additional measurement results are necessary to define the limited values of va of sr and cs. more measurements must be executed in order to determine the effect of new radioactive sources. | the article presents the measurement results of the volumetric activity (va) of artificial radionuclides 90sr and 137cs in the coastal waters of the baltic sea near the curonian peninsula in 20052009. the annual average values for this period of time were 12 bq / m3 (90sr) and 40 bq / m3 (137cs). considerable variations in the va of the radionuclide in individual measurements compared to the average results were observed. the extreme values were 6 and 16 bq / m3 for 90sr and for 137cs27 and 75 bq / m3. it is proposed to allow such variations under the influence of a variety of external factors such as hydro meteorological situations, inflowing rivers and bays, storm activity and etc. besides, a possibility of penetration of radionuclides into the sea waters from the additional radioactive sources is not excluded. |
the vascular lesions of the skin are divided into congenital and acquired lesions. acquired and congenital vascular lesions are described as follows : the most significant acquired vascular lesions of infancy are hemangiomas. the lesions are composed of proliferating blood vessels and, although benign, have a potentially destructive character. after infancy, acquired vascular lesions are associated with aging (senile angiomas), trauma (arteriovenous fistulas), systemic conditions (spider angioma), and malignancy (kaposi 's sarcoma). vascular malformations can be classified based on their type of blood flow into slow - flow lesions (capillary, venous, and lymphatic), high - flow lesions (arterial), and lesions with a combined slow and fast blood flow. the lesions are composed of proliferating blood vessels and, although benign, have a potentially destructive character. after infancy, acquired vascular lesions are associated with aging (senile angiomas), trauma (arteriovenous fistulas), systemic conditions (spider angioma), and malignancy (kaposi 's sarcoma). vascular malformations can be classified based on their type of blood flow into slow - flow lesions (capillary, venous, and lymphatic), high - flow lesions (arterial), and lesions with a combined slow and fast blood flow. a 42-year - old female patient came to the department of oral and maxillofacial surgery as an outpatient with a chief complaint of swelling in the left side of the lower lip. the patient gives a history of small negligible swelling present since 5 years and gradually increased in size due to repeated trauma during mastication with a short span of bleeding when exposed to trauma and subsequent arrest of bleeding by itself. her blood report was within normal limits. local examination revealed a single, oval, well - defined, nonpulsating, soft and compressible swelling roughly measuring 1.6 1.5 0.8 cm [figure 1 ] involving buccal mucosa in the left side of the lower lip. on careful inspection, a fine capillary network of blood vessels seen in the mucosa mimicking a vascular lesion. the diascopy test was positive with escape of coloration upon compressibility resulting in blanching of the lesion when placing a microscope glass slide under pressure which gave us the impression of vascular lesion. preoperative appearance of the lesion for confirmatory diagnosis, the lesion was subjected to a duplex ultrasound color doppler study followed by magnetic resonance imaging (mri) and magnetic resonance venography (mrv) scans. an ultrasound color doppler scan reported with heterogeneously hypo echoic with multiple tiny cystic areas and mild internal vascularity within the lesion [figure 2 ], while the mri scan reported a homogenously hyperintense in t2w and stir sequences and hypointense in t1w sequences [figure 3 ]. on a postcontrast study, mrv, homogenous enhancement of the lesion was seen with no evidence of prominent vessel around the lesion and was diagnosed as slow - flow vascular malformation of the buccal mucosa involving the lower lip [figure 4 ]. ultrasound color doppler study showing hypo echoic with multiple tiny cystic areas and mild internal vascularity within the lesion mri scan showing a homogenously hyperintense in t2w and stir sequences and hypointense in t1w sequences mrv demonstrating a homogenous enhancement of the lesion with no evidence of prominent vessels around the lesion the patient was planned for conservative treatment by sclerotherapy with 3% sodium tetra decyl sulfate (sts). the area to be injected was cleansed and local infiltration (2% lignocaine with 1:2,00,000 adrenaline) was administered at the base and periphery of the lesion. sts was injected directly into the lesion through the mucosa at multiple sites without any radiological guidelines. in total, 1.5 ml of sts was injected. after withdrawal of the needle, manual compression for 1015 min was done over the lesion with a sterile gauze. the patient was advised to take anti - inflammatory and analgesics and recalled after 15 days. in the second visit, the patient was reviewed with complete resolution of the lesion [figure 5 ]. in our case, the patient experienced transient severe pain postoperatively with minor complications such as local inflammatory response including cutaneous blistering which gradually reduced. the patient was reviewed after 15 days and bimonthly interval, found the complete absence of the lesion and no evidence of recurrence. the diagnosis of vascular malformations is based on the patient 's medical history and a physical examination. vascular low flow lesions may present a progressive increase with age, trauma, and after partial resection. ectatic blood vessels and the reddish - blue surface are characteristically found in this lesion. change on pressure is a common finding with return to original color on withdrawal of pressure. we believe that our case corresponds to vascular low flow malformation due to their reddish - purple aspect, consistence, response to diascopy, and the absence of vascular pulsation. the appropriate combination of noninvasive to minimally invasive tests should follow in order to confirm or exclude the clinical impression. duplex ultrasound scanning is the first choice for noninvasive evaluation of patients with vascular malformations, doppler mode to assess flow characteristics. minimally invasive tests such as mri and mrv are useful to confirm the extent and type of venous malformations, delineates feeding and draining vessels, distinguishes between different soft tissues (muscle, fat) and the vascular structures. sclerotherapy, the mainstay of treatment is the injection of an agent to induce inflammation and obliteration of affected veins. for small cutaneous or mucosal lesions, local injection may be effective. intralesional sclerotherapy using liquid sclerosing agents, which is a palliative treatment in most types of vascular anomalies, produces good outcomes in smaller lesions. disrupt vein cellular membrane (protein theft denaturation) sodium tetradecyl sulfatepolidocanolsodium morrhuateethanolamine oleate sodium tetradecyl sulfate osmotic agents damage the cell by shifting the water balance through cellular gradient (osmotic) dehydration and cell membrane denaturation hypertonic sodium chloride solutionsodium chloride solution with dextrose hypertonic sodium chloride solution sodium chloride solution with dextrose chemical irritants damage the cell wall by direct caustic destruction of endothelium chromated glycerinpolyiodinated iodine sts at low concentrations are effective in stripping endothelium over a considerable distance, and is also able to induce a hypercoagulable state, possibly by selective inhibition of protein c, and can also promote platelet aggregation. we selected 3% sts as a sclerosing agent because of its high effectiveness and minor complications like the presence of small skin ulcers and superficial skin breakdown which responded well to the application of silver sulfadiazine. intralesional sclerotherapy can be performed without serious complications if the sclerosing agent is selected and injected cautiously. | vascular anomalies constitute some of the most difficult diagnostic and therapeutic enigmas in the head and neck region. it is of paramount importance that a modern classification system is established to differentiate vascular lesions from vascular malformations. vascular malformations are usually congenital and venous anomalies usually expand because of hormonal changes such as puberty, pregnancy, or secondary to trauma. here, we report a case which was diagnosed as low flow vascular malformation of buccal mucosa involving the lower lip and subsequently treated with 3% sodium tetra decyl sulfate. this paper provides overall understanding regarding the presentation and management of small vascular lesions in the orofacial region. percutaneous sodium tetradecyl sulfate when used either alone or as adjunct to surgery is a safe, effective, and inexpensive agent in the treatment of venous malformations. however, proper case selection, evaluation, and careful planning are necessary to reduce the unwarranted risks and complications. |
trichomycosis is asymptomatic, superficial infection, which is generally caused by a coryneform - actinomycetes bacteria which, from the very first reports, has been called corynebacterium tenuis. while on rare occasions the pubic, scrotal and intergluteal hairs may also be affected, this infection predominantly affects the hairs of the axilla, and thus the condition is referred to as trichomycosis axillaris. it is characterized by the appearance of concretions (hair - nodules), which grow around the hair shaft. trichomycosis is a rare condition and few cases are reported in the literature, its name is wrong because it is not a fungal infection but is a superficial bacterial infection, so it should be called trichobacteriosis. due to the fact that the condition is asymptomatic and causes practically no discomfort, patients generally do not seek medical attention. however, when a careful, deliberate search is performed in the clinical setting, it tends to appear more frequently. our principal objective is to present a series of cases, their clinical and microbiological aspects and emphasize that this clinical entity in axillar and genital hairs maybe is underestimated. all patients included had diagnosis of clinical trichomycosis and were confirmed microbiologically. a clinical history, an examination under wood 's light and microbiological tests was performed on each patient. in all cases, there took hair of the affected region, by means of court with scissors and the sample was divided in two parts for its study, for examination and culture. the samples were examined microscopically with koh 20% and cotton blue preparations, and cultured in brain - heart infusion agar, and chocolate - blood agar. positive cultures were gram - stained to identify coryneform - bacteria forms, and isolated cultures were subsequently identified by biochemical testing using the microscan - system. the main demographic characteristics and microbiological findings of the study are shown in table 1. it is important to point out that 32 out of the 56 patients (57.1%) sought medical attention because they felt a change in the texture of the affected hair, and the rest of the patients were diagnosed during careful, deliberate search by visual inspection and examination under wood 's light [figures 13 ]. eighteen of the fifty - six patients (32.2%) related their condition to athletic activities and to the use of clothing made of lycra. of the 56 cultures that were obtained, only 18 were identified by the microscan walkaway 9651 system, which identified corynebacterium centers for disease control (cdc) group g / ld ; according to bergey 's manual, this is indicative of corynebacterium flavescens [figures 4a & b and 5 ]. flava) yellow - fluorescence of trichomycosis pubic hairs (wood light) direct examination of trichomycosis hair (a) koh 10%, 10 ; (b) koh 10%, 40 culture of corynebacterium sp., in chocolate - blood agar plate there were two patients who, in addition to axillary trichomycosis, also presented with two related conditions affecting the inguinal zone (erythrasma) and the soles of the feet (pitted keratolysis) table 2. trichomycosis, a more correct term would be trichobacteriosis or bacterial trichonodosis is a superficial infection, primarily of the axillary hair, which can exhibit three different clinical presentations : the most common clinical variant is trichomycosis flava (yellow), while rubra (red) and nigra (black) variants occur much less frequently. from the earliest reported cases of trichomycosis, the causative agent was classified as c. tenuis. in light of the new taxonomic position of the genus corynebacterium, however, that particular species is no longer considered, and thus, the majority of the reports are left as corynebacterium sp. however, some studies have shown that the causative agent belongs to the so - called group 2 (ld2) (also referred to as the cdc - g / ld group), that it corresponds to the c. flavescens species, and that it is related to the flava variant. in the present study, no specific causative agent could be identified in the remaining 38 cases, which were simply classified as corynebacterium sp., according to their microscopic characteristics because presented gram - positive rod - shaped forms. there are indications that the rubra and nigra variants of trichomycosis are actually caused by other microorganisms, specifically micrococcus castelani and micrococus nigricans, although this has yet to be proven definitively. the causative agent of trichomycosis has not yet been isolated in nature, only from infected human hairs. the disease is quite cosmopolitan, however, it is reported more frequently in humid and tropical climates ; it is an affliction of adolescents and young adults. there are no differences in the rates of infection with respect to race or gender, although, in our culture it is seen with greater frequency in male patients since it is customary for women to shave their axillary hair. there have been reports of man - to - man transmission of trichomycosis, particularly in groups that live in very close quarters, such as soldiers and athletes. trichomycosis infection begins when the causative agent comes in contact with the hair shaft, and the bacteria adhere to the surface, or the cuticle, of the hair, using a cement - like substance, the chemical composition of which is not yet known, that is insoluble in water as well as in the other principal solvents (i.e., acetone, ethanol). electron microscopy studies have clearly shown that the microorganism does not penetrate to the medulla 's cortex of the hair ; instead, it only adheres strongly to the surface of the hair and develops slowly until it forms concretions around the hair shaft. levit has suggested that the adhesive substance is synthesized by both the apocrine glands of the human host and by the microorganism, which would explain why the disease develops in the areas of the body where it does (i.e., axillary, pubic and inter - gluteal hairs). as it pertains to our study, it is noteworthy that the majority of cases involve adult males, due to the fact that, in our culture, women generally tend to shave their axillary hair. there were cases involving both extremes of age, but by the mean age of all the patients, it can be deduced that trichomycosis appears more often in young adults. the specific results concerning the clinical location of the lesions are shown in table 1. out of all the cases studied, 97.4% presented with an infection of the axillary hairs, a figure, which is consistent with the majority of reports found in the literature. there were two particularly noteworthy cases in which trichomycosis affected three different areas of the body at the same time (axillary, pubic and inter - gluteal regions). according to some authors, a more thorough examination of trichomycosis patients is particularly important, since they feel that the figures reported of the frequency of genital involvement underestimate the actual number of cases. another remarkable case in this study was the one in which the eyebrows were also affected. this is truly an exceptional finding and we were not able to find a similar case reported in the literature, most likely resulted dissemination from the axillary region by means of auto - inoculation. de - almeida. have recently reported a case of trichomycosis capitis, in an 8-year - old boy, this report also indicates that there may be cases distant areas and apocrine glands that can be clinically similar to cases of white piedra. in general, trichomycosis is characterized by the formation of concretions around the hair shaft ; these are invisible at first and there is only a slight thickening, which can be felt on palpation. initially, the bacterial masses remain isolated or independent, and it is at this stage when it could be mistaken for pediculosis (nit - lice). as the infection becomes chronic, the concretions extend along the entire length of the hair until they form a sheath, causing the hair to thicken, turn a yellowish, red or black color, and become creamy, opaque and soft. it should also be mentioned that only 32/56 of patients (57.1%) sought medical attention as a result of having felt a change in the texture of the hair, odor and in most cases accompanied by increased sweating (hyperhidrosis), while the rest of the patients were diagnosed during a careful, deliberate search. practically, all of the patients (98%) presented with the flava variant, and only one patient, who sought medical attention after developing reddish sweat, presented with the rubra variant, and was and was proven by the isolation of cultures, which was due to a mixture of microorganisms : corynebacterium sp. (white - yellow growth) and serratia mascescens (red growth), and probably the red form due to the latter bacteria produce carotenoid pigments. there is a series of case reports in the literature involving patients with mixed infections caused by different corynebacteria, the so - called corynebacterial triad consisting of trichomycosis, erythrasma and pitted keratolysis ; in our study, there were two patients who also presented with erythrasma and pitted keratolysis, another example is the report of rho and kim, whom found a high incidence of trichomycosis in korean soldiers, co - existing with erythrasma (20.4% of cases) and with pitted keratolysis (13% of cases). as far as the microbiology is concerned, it is important to note that all of the cases in this study were confirmed by means of direct examination, in which the infection of the hair, in the form of concretions or bacterial masses consisting of coccoid and diphtheroid shapes measuring between 0.5 and 1 m, could be seen adhered to the hair, which rules out the majority of mycoses that affect the hair (tinea and piedra). wood 's light is extremely useful for diagnostic purposes, and above all, for delineating the extent of the infection, since the bacterial concretions emit fluorescence under low - intensity ultraviolet (uv) light, making it much easier to find the affected hairs. in terms of causative agents, was identified and isolated in all cases (single agent in 98.2% and mixture in 1.8%). microscopic examination revealed the presence of various gram - positive, coccoid and diphtheroid shapes or rod - shapes, measuring, on average, from 1.2 m to 1.8 m in length, and 0.4 - 0.6 m in width, and resembling little drum sticks. it is important to note that 18 strains were identified as c. flavescens, which is consistent with the results obtained by garca - martos. this organism is not generally considered virulent, although, it has been reported to cause endocarditis in some intravenous drug users. an interesting finding in our study involved the case of the rubra variant, where two different pathogens were isolated, corynebacterium sp. and serratia marcescens, the latter being an extremely opportunistic type of bacteria, with low virulence, and which produces a carotenoid pigment. in general therapy of trichomycosis, many authors consider that the most effective treatment consist in shaving of the affected area for a period of 2 - 3 weeks, but the use of a concomitant treatment, such as sulfur soaps, is also recommended. those patients who shave the affected area only once will generally experience a recurrence of the infection, since, the bacteria begin to develop the concretions once again as the hair grows back. topical treatments containing any of the following : 3% sulfur, 2% formalin, 1% mercury (ii) chloride (or mercuric chloride) or 2% sodium hypochlorite, as well as topical antibiotics with fusidic acid, erythromycin and clindamycin, may also be used. | background : trichomycosis is asymptomatic bacterial infection of the axillary hairs caused by corynebacterium sp.objective:to bring a series of cases of trichomycosis, its clinical and microbiological experience.materials and methods : this report consists in a linear and observational retrospective study of 15 years of cases of trichomycosis confirmed clinically and microbiologically.results:fifty six confirmed cases of trichomycosis were included in this report. the majority were men 53/56 (94.6%), mean age was 32.5 years. the most commonly affected area was the axilla (92%), trichomycosis flava was the principal variant 55/56 (98.2%) and signs and symptoms associated were hyperhidrosis (87.5%), hairs texture change (57.1%) and odor (35.7%). bacterial concretions were observed in all cases, and the predominant causative agent in 89.3% of all cases was corynebacterium sp. thirty patients were included in therapeutic portion of the study, and 28 (93.3%) of them experienced a clinical and microbiological cure.conclusion:trichomycosis is asymptomatic, superficial infection, which primarily affects axillary hairs. |
anthocyanins, as ubiquitous constituents of berries and coloured vegetables, are widespread in the plant dominion. much attention has been paid in recent years to their radicals scavenging activity in vitro and in vivo and possible health benefits. the main anthocyanins being analysed in elderberries are various glucosylated cyanidins (figure 1). besides glucose, sambubiose is the typical sugar conjugate in elderberries [1, 2 ]. rate and extent of absorption, metabolisation, and excretion of elderberry anthocyanins are to date not fully understood. first of all, controversially discussed hitherto is whether also conjugated glucuronides of cyanidin are formed and excreted in vivo. time - course plots (mean sd) of total anthocyanins (glucosides + glucuronides) and anthocyanin glucuronides in human urine following ingestion of 150 ml of elderberry juice. individual amounts excreted of total anthocyanins (glucosides + glucuronides) and anthocyanin glucuronides within 5 hours following ingestion of 150 ml of elderberry juice. urinary excretion of anthocyanins as unchanged glucosides and glucuronides after elderberry juice consumption (means sd). calculated as the ratio of amounts excreted (within 5 h) to amounts / doses ingested. the study protocol was approved by the ethics committee of the giessen university, germany. after an overnight fasting, seven healthy nonsmoking volunteers (6 female and 1 male with a mean bmi of 21.5) consumed a bolus quantity of 150 ml of concentrated elderberry juice (containing 3.57 g of total anthocyanins) together with rolls and cheese. the elderberry juice concentrate was obtained from wild (heidelberg, germany). urine samples were collected initially and in hourly intervals over a period of 5 hours. hplc - dad analyses of urinary samples (05 hours after ingestion) were performed before and after hydrolysis of glucuronide conjugates with -glucuronidase [3, 4, 5, 6 ]. the anthocyanins were extracted by using a solid phase extraction cartridge (sep - pakvac 12, waters, milford, mass). the cartridge was first washed with 5 ml methanol and equilibrated with the same volume of 5% aqueous formic acid. seven ml urine samples diluted with 2 ml formic acid were applied to the equilibrated cartridge. after washing with 5 ml of 5% formic acid, the anthocyanins were eluted with 5% formic acid in 5 ml methanol. the eluate was evaporated to dryness in a vacuum rotary evaporator at 30c and the extract redissolved with 200 l mobile phase before hplc analysis. the chromatographic conditions adopted from netzel comprised separation on a prontosil eurobond rp-18 (5 m, 250 4 mm i d, bischoff, leonberg, germany), protected by a guard column (lichrospher 100 rp-18, 4 4 mm, merck, germany), and isocratic elution with water / formic acid / acetonitril (v / v / v) = 81/10/9 (flow rate 0.5 ml / min) by using a high - precision pump (model l-6200, merck - hitachi, darmstadt, germany). the cyanidin glucosides were detected at 520 nm with the aid of a uv - vis detector (l-4200, merck - hitachi). cyanidin glucosides excreted were identified by spiking blank urine samples with authentic compounds and comparing the retention time in the hplc analysis and uv - visible spectra. standard curves were prepared for quantification prior to the preparation procedure. the detection limit (s / n 10) was between 0.13 ng and 0.60 ng / injection volume (100 l). aliquots of each urine sample were acidified with formic acid (2 ml/0.2 ml) and stored frozen at 80c until analysis. the urinary excretion of total anthocyanins (unchanged cyanidin glucosides and their glucuronide conjugates) within 5 hours was 0.053 0.030% of the administered dose. only 6.2 2.2% of the excreted amount consisted of glucuronides. based on this recovery, the percentage of glucuronides in urinary excretion was 0.003 0.001% (calculated as the ratio of anthocyanin glucuronides excreted to anthocyanin glucosides ingested). the excretion pattern of total anthocyanins in figure 2 demonstrates that a maximal excretion rate is reached 1 hour after intake, followed by a rapid drop to initial values about 5 hours after intake, resembling a first - order excretion kinetics. the metabolic conversion of anthocyanins within the organism remains to be elucidated. in principle, glucuronidation or conjugation with sulphuric acid is a common final metabolic step to facilitate urinary excretion. tsuda found no cyanidin glucuronides in livers and kidneys of rats after oral administration of cyanidin 3-glycoside, but cyanidin was converted to peonidin and protocatechuic acid. a different situation seems to exist to date in man. in the urinary excretion of elderly women, minor amounts of glucuronides of peonidin and cyanidin 3-glucoside could be deteced in half of the volunteers besides unchanged glucosides after elderberry consumption, which does correspond to our results [2, 5 ]. only glucosides of elderberry anthocyanins, however, could be found in plasma and urine of volunteers by other authors [8, 9 ]. to entirely estimate the extent of glucuronidation of anthocyanins as metabolic fate, it has to be considered that besides urinary excretion, biliary secretion may also serve as a possible way of elimination, particularly known for glucuronides. newer studies, however, revealed that after intake of elderberry extract, the identical pattern of glucosylated cyanidins could be detected in plasma as in urine. thus, the results of the quantification of excretion products in the present study demonstrate that, at least in the given dose range, glucuronidation of cyanidin obviously represents a negligible conversion step in the metabolism of cyanidin ingested from elderberries. the proportion of glucuronide conjugates seems to represent a rather constant but very small proportion despite interindividual differences of total anthocyanin excretion (figure 3). this may be in contrast to other fruits such as strawberry anthocyanins, being predominantly excreted in urine as glucuronides besides small amounts of sulfoconjugates. it remains to show the extent to which the administered dose level does determine the site of metabolism. there exists some body of evidence that large doses of polyphenols are primarily metabolised in the liver, whereas small doses may be metabolised by the intestinal mucosa, with the liver playing a secondary role to further modify the polyphenol conjugates. | in a pilot study with 6 females and 1 male, the metabolism of various cyanidin glucosides after oral administration of elderberry juice was investigated. the anthocyanin metabolites were detected in urinary excretion. after ingestion of a bolus quantity of 3.57 g total anthocyanins in a 150 ml elderberry juice concentrate, 0.053% of the administered dose was excreted in urine as glucosidically bound cyanidins within the first 5 hours. only 0.003% of the ingested anthocyanin glucosides was excreted as cyanidin glucuronide, suggesting that this conversion step might be of minor importance in urinary excretion. |
the trunk midline is the reference for orientation of the body in space and is the point of comparison for the synthesis of all tactile, kinesthetic, and pressure - related information from the two sides of the body1. thus, the trunk midline can be considered the axis of symmetry around which the body organizes motor behavior2. the corpus callosum is the principal interhemispheric commissure, and its functions include the exchange of information between the two central hemispheres and integration of the inputs that reach one or both of them3. all somatosensory callosal connections appear to provide a common anatomical substrate : this phenomenon is known as midline fusion. the body regions that are represented in the callosally connected zones of the first somatosensory area (si) were identified as the midline of the somatosensory space4. the subjective vertical midline of the body is the result of multisensory integration of vestibular, proprioceptive, visual, and tactile afferences5, 6. meilinger and colleagues hypothesized that every alteration of these afferences produces an abnormal representation of the body system, which receives incoherent information, and answers in a reflex and primitive manner7. the integration of spatial information that is perceived8, 9 from different viewpoints is a common yet largely unexamined cognitive ability10, 11. locomotion is supported by additional content, which is closely related to behaviors, such as modulation of the gait cycle, from vision control12,13,14. as logan and colleagues have pointed out, frequency response functions between visual scene motion (input) and trunk kinematics (output) change very little or not at all with gains in trunk orientation in the standing posture and under walking conditions15, 16. there is evidence of a correlation between heading and the rate at which strategic modifications in trunk yaw decrease, because adaptive recalibration of locomotor trajectory using optic flow stimuli depends on the rate at which the kinematic variability that is associated with strategic control is reduced15. however, logan and colleagues consider that the increased gain reflects a decline in stability, due to a change in the control problem from standing to locomotion. keeping the body upright with the use of vision during walking when vision is deprived, subjects operate not according to a pre - established pattern of action but in relation to its internal representation of the previously seen target. visual feedback allows a subject to have the same gait patterns in various surroundings, whereas visual deprivation has environment - specific effects on gait dynamic stability16. the significance of visual feedback underscores its importance in pathological conditions, such as patients with post stroke hemiplegia who might be unable to adapt to changing visual or surface conditions17. iosa and colleagues reported that the surrounding environment influences the performance of subjects who are asked to walk toward a memorized target. when fewer external cues were available, participants relied more on information on body mechanics and body feedback to complete the task accurately. conversely, in a small indoor environment that was rich in environmental cues, subjects perceived the target as a finish line that was not to be overshot18. mohapatra and colleagues indicated that under blindfolded conditions, anticipatory postural adjustments are not generated. they proposed that the increased emg activity in leg and trunk muscles after a perturbation, indicating lower postural stability, reflects a lack of anticipatory postural adjustments (and relative compensatory postural adjustments) when vision is unavailable19. other research suggests that the priority of whole - trunk control in the mediolateral direction is higher than in other directions and is linked to attention, whereas whole - trunk control in the vertical rotation and anteroposterior directions is passively regulated and requires minimal attentional control20. as recently demonstrated in chronic pain conditions, alterations in somatosensory perception can change the body s sense of posture21. the possibility of training a subject to understand his position with respect to the gravitational axis through a specific cutaneous training program opens new avenues in body posture perception and action22. also, the maintenance of chronic low back pain is linked to a disorder of altered perception of the trunk23. perceptual surfaces (pss) reduce static and dynamic balance impairments, even in patients with parkinson disease (pd)24. the function of the trunk is crucial in pd : other studies have shown how vibrotactile biofeedback of the trunk improves postural stability in pd25. thus, multiple sensory stimuli contribute to conscious awareness of the body, but the manner in which the central nervous system constructs and updates the body schema after injury or on visual deprivation is unknown. for example, pereira and colleagues demonstrated that local muscle vibration does not improve the sit - to - walk performance of healthy young adults26. the purpose of this study was to determine the effects of tactile and proprioceptive sensory stimulation of the back on perception of the body midline and on the capacity to walk toward a memorized distance without vision or trunk stability in healthy subjects. we hypothesized that training individuals perceptive capacity (pressure and somesthetic sensation) by providing dedicated instruction that is based on cognitive exercises that are performed using a specific rehabilitative tool would modify their estimation of walking distance and upright gait stability. ten healthy subjects (mean age 31.90 2.47 years, mean weight 64 11.4 kg, and mean height 170.4 7.56 cm) were recruited and matched with 10 healthy control subjects (mean age 30.5 2.8 years, mean weight 65 10.20 kg, and mean height 169.5 8.40 cm). the subjects gave their informed consent to participation in the study, and approval was obtained from the local ethics committee of the s. lucia foundation (n ce - prog 266 - 09). the subjects were asked to perform an experimental indoor test wearing comfortable shoes that they usually wore, not specialty shoes, such as boots, ballet shoes, high heels, and flip - flops. we examined the subjects abilities to perceive and estimate the length that they were required to walk over a given distance in an indoor environment. the subjects had to walk toward a memorized target 3 m, 6 m, or 10 m from a reference position (0 m) in a 20 by 5 m laboratory, without obstacles on a linear trajectory in the middle of the laboratory, with their eyes closed and prompted by an acoustic signal to begin walking. the environment was quiet, with good natural lighting, and there were no other items or furniture that could serve as external references for the subject. similar to the experiment that was conducted by iosa and colleagues27, before starting the test, the investigator positioned the subject at 0 m and showed him the distances that were set at 3 m, 6 m, and 10 m to facilitate memorization of the trajectories that the subject had to cover blindfolded. the target was a person who stood on 1 of 3 strips that were placed on the ground 3 m, 6 m, and 10 m from the starting line. the subjects were asked to memorize the position of the target, fixing it for several seconds ; blindfold themselves ; and, after an acoustic signal, walk to the target. participants were asked to stop walking when they believed that they had reached the target. the experimenter promptly warned a subject if he was going to hit a wall. the sequence of the 3 tasks (1 for each distance) was randomized among the subjects. at the end of each test when a subject stopped at one of the targets, a tape measure was used to determine the distance that was traveled and the distance that remained to complete the task. after each sequence, the subject, still blindfolded, was returned to the starting line by the investigator, preventing him from knowing whether an error had been committed to avoid learning opportunities. no side effects were recorded during the test, and the investigator never had to stop a subject. the entire study group was tested before starting treatment with pss (= t0) and after 10 treatment sessions with pss (= tend). five randomly chosen subjects repeated the test immediately after the first treatment session of sensory - motor evaluation with pss (= t1) to exclude the possible influence of learning of the task. to complete the test, an accelerometer was fixed at the level of the l2/l3 vertebrae with an elastic band (freesense, sensorize, sampling frequency 100 hz, weight 94 g). accelerometry is a technique that generates data on the dynamic stability of gait with regard to movements of the trunk during walking. the accelerometer provided acceleration data of the trunk along the 3 body axes (anteroposterior, laterolateral, and craniocaudal) to assess upright gait stability. upright gait stability has been defined as the capacity to minimize upper body accelerations18. upper body accelerations were analyzed after subtracting their mean values and low - pass filtering at 20 hz, and summarized in terms of acceleration root mean square (arms), which is a measure of acceleration dispersion (coinciding with the standard deviation due to subtraction of the mean signal). we computed the arms for each body axis, to obtain information on upright gait instability18. the arms was computed for each of the 3 acceleration measures along the 3 body axes and averaged over the 3 values of 3 consecutive steps in the central section of the walking pathway28. the number of steps that were performed was computed as the number of ap - acceleration negative peaks29, and the average step length was calculated as the ratio between the distance that was walked and the number of steps (step length refers to the distance between 2 successive placements of the 2 feet, whereas stride length refers to the distance between 2 successive placements of the same foot, formed by 2 steps). the perceptual surfaces protocol (ps) stimulation of awareness of the trunk midline is effected using a specific tool, called super (perceptual surfaces, ps), patented in 1997 by ennio spadini (reference 01291920, rome, italy). super is a therapeutic system that is based on the interaction between a subject s back and a support surface comprised of small latex cones of various dimensions (height : 38 cm ; base diameter : 24 cm) and rigidity (20 to 60%). these cones are applied with their inferior bases on a rigid wood surface through elastic strips, and typically, over 100 cones are used for each session. subjects were asked to lay supine on the surface that was formed by the smoothed apex of these cones, creating reaction forces to the patient s weight, generated by interaction with the cones. the base conformation of the pss comprises blue cones that represent the anteroposterior trunk midline (60% rigidity), yellow cones that represent the paravertebral, and the remaining areas that are symmetrically adjacent on either side of the midline (40% rigidity) (fig.1fig. 1.surfaces for perceptive rehabilitation in the basic configuration and below an examples of cones with varying dimensions.). we chose this conformation on the basis of the following principles : to provide greater stimulation to the skin of the posterior midline of the trunk (blue cones with greater rigidity than the yellow ones), and to provide symmetrical stimulation to other areas of the body. surfaces for perceptive rehabilitation in the basic configuration and below an examples of cones with varying dimensions. subjects were asked to lie supine on these cones, with their knees and hips flexed, so that their weight was supported by many reaction force vectors, 1 for each cone. these forces generate high pressure in the small areas of contact, resulting in intensive perceptive stimuli. the base conformation allowed us to determine spatial alterations in the back (subjects with antalgic postures and scoliosis), based on the distribution of pressures on the subjects backs and their perception of back pressures (i.e., sensory - motor evaluation in the first session). subjects were asked to relax and find the most comfortable position, breathing normally. subjects had to recognize the areas of support, indicating the surface of the body that was in contact with a particular area, describing and counting the number of cones, checking the distribution of the load on the bed and correcting it, and paying attention to posture. the subjects reported how they perceived and felt the cones, particularly if the load was distributed uniformly and symmetrically with respect to the trunk midline. after the evaluation session, each subject underwent 10 sessions in 2 weeks (5 days per week). subjects performed 45 minutes of a cognitive - perceptive task (divided into perceptual - motor and active phases) to improve their perception of the trunk and, in particular, their midline. the perceptual phase helps a subject become aware of the position of his body segments with respect to the various cones. the perceptual - motor phase is characterized by growing awareness of the trunk midline. diaphragmatic breathing, associated with retroversion of the pelvis in the expiratory phase, allows the curves of the spine to flatten and the muscles to stretch to increase the support surface. the actual exercise comprises a perceptual task that increases in difficulty, asking the patient to perceive the elasticities and heights of the surfaces. the active phase involves movement of the arms and legs, displacement, and weight control of the trunk and pelvis. the base conformation can be modified during the session to improve contact between the trunk and the surfaces of the cones. at the end of each session, the experimenter examined the interaction between the skin on the back and the surfaces that relieved the hyperemic area on the patient s back. in subsequent sessions, cones with varying elasticities were positioned by the therapist to improve the symmetry of contact between the surface and the patient s back, considering the hyperemia in the previous session. the experimenter measured perception through the symmetry, quality, and uniformity of the supports. empirically, in the static posture after sessions with the pss, the perceptive capacity of the trunk was assessed using quality representation and symmetry, with respect to the line of the spinous processes, of the hyperemic areas that were created by the support with the pss. quality was defined as the magnitude of the hyperemic area that is left by the cones on the skin and signs of pressure from the cones that remain on the skin of the trunk. the aim was to improve the subjects abilities to recognize, perceive, and discriminate the trunk midline and enhance their sensory experience. in the control group, subjects were asked to lay supine with the knees and hip flexed. subjects were asked to relax and find the most comfortable position, breathing normally. lumbar exercises were performed : lumbar bridging, lumbar pelvic tilt and hamstrings and piriformis supine stretching. all measures were continuous and distributed normally (lilliefors test) ; thus, parametric statistical methods were employed. to compare the results between pre- and post - treatment performances, repeated - measures analysis of variance was performed using time (pre- vs post - treatment or first vs second assessment) and distance (3 m, 6 m, or 10 m) as the main factors, and axis (ap, ll, or cc) as an added factor when acceleration was analyzed. before treatment, the subjects displayed the greatest errors in walking 10 m, followed by walking 6 m and 3 m. this error was always negative : subjects tended to walk less distance than required. by analysis of variance, there was a significant effect of time (pre- vs post- treatment, f=5.968, p=0.037, observed power 59%) and distance (f=8.055, p=0.012, observed power 91%) but not of their interaction (f=2.977, p=0.108, observed power 36%). the reduced error was related to a longer distance being walked, which was founded on longer steps (in mean + 5%) and more steps performed (in mean one more). although step length and number of steps did not improve significantly over time (analysis of variance, p>0.05 for both), their combination significantly improved the subjects performances. there were no significant changes in the error in walking distance estimates among the subjects who repeated the test immediately after the first treatment session (effect of trial : f=0.374, p=0.574) ; the absence of visual and verbal feedback did not allow the subjects to correct their mistakes. non - significant differences in these errors were observed at tend in those who undertook the test for the second time at t1 or at tend. despite the general increase in speed and acceleration after treatment, their changes were not statistically significant (table 1table 1.mean and standard deviation of the gait parametersparameterstime3 m6 m10 merrors (m)t00.150.110.700.291.570.45tend0.050.110.130.180.330.29ws (m / s)t00.840.200.820.130.680.18tend0.890.200.880.150.720.15step length (m)t00.430.090.510.090.520.09tend0.460.100.530.090.550.05number of stepst071102162tend71112171arms - ap (m / s)t01.160.351.230.321.180.27tend1.200.331.350.421.330.29arms - ll (m / s)t01.150.431.140.301.090.22tend1.170.391.320.501.260.31arms - cc (m / s)t01.400.561.600.511.470.37tend1.400.401.730.521.750.43). time did not affect self - selected walking velocities pre- and post - treatment (f=2.924, p=0.121). subjects walked slower over the 10-m distance than over the other 2 distances (factor distance : f=15.734, p<0.001), without any significant interaction with time (f=0.067, p=0.935) (table 1). similarly, by analysis of variance of arms values along the 3 body axes (table 1), there were no significant effects of time, pre- versus post - treatment (f=2.091, p=0.182), or distance (f=2.454, p=0.114) ; only axis had a significant effect (f=11.689, p=0.001), with greater acceleration along the cc axis. the interactions of time and distance (f=1.963, p=0.169) and between time and axis (f=0.141, p=0.869) were not statistically significant. the purpose of this study was to determine whether a training program that is based on tactile and proprioceptive sensory stimulation of the trunk, performed using perceptive surfaces, modified the locomotor body schema and improved estimation of walking distance and upright gait stability. after treatment with perceptive surfaces, the distances that were traversed were closer to the target than those before treatment, because the subjects increased their awareness regarding perception and estimation of the walking body. there was a non - significant increase in trunk acceleration, which were likely related to the increase in gait speed30, after training31. the concept of the trunk having a fundamental dynamic function during walking is a recent model. until the 1990s, the trunk was considered to be a static passenger unit of a locomotor apparatus that was located primarily at the lower limb level34. our study shows that tactile and proprioceptive stimulus of the trunk, with a new rehabilitation tool, can influence the behavioral pattern of locomotion. in particular, the most important result of our study was that stimulation of the trunk improved the ability of the locomotor body schema to walk toward a memorized target without visual support. this result is in conflict with the older model, in which the lower limbs are a locomotor unit and the upper part of the body is merely a passive passenger5. our findings are consistent with a recent suggestion that the upper body has an active function during walking32. thus, our results suggest that the locomotor body schema includes the upper body not just the lower limbs. in this model, the cns integrates many sensory inputs visual, vestibular, cutaneous, gravito - inertial, and proprioceptive inputs to compute spatial and body coordinates during a walking task, assigning a different weight to each input, depending on the environment and the constraints in which the movement is performed33,34,35. shenton suggested that proprioceptive inflow is an important sensory input in the online representation of the body in space36, and that the processing of proprioceptive information is context - dependent37. the presence of a body schema has been well described, as has the function of the location of tactile stimuli on body surfaces in promoting a body schema during the spatial representation of the body (position) and walking (locomotor body schema)38. the perception of tactile stimuli and proprioception is critical for conveying information about the relative positions of body parts when assuming a posture or during walking. the decrease in perception, for example, during pain conditions could drastically change the body schema39, 40. two studies of patients with chronic and non - specific low back pain reported that training with perceptual surfaces (pss), targeting back midline perception, reduces pain41 and improves postural control42. how the body schema is generated in the cns is unclear, but we know that the body schema and sensory information interact ; thus, their conflict might generate pain, as shown in the experiment conducted by mccabe and colleagues using healthy volunteers43. furthermore, the position of the body (sitting or standing) might alter the accuracy of the imagination of movements (i.e., mental simulation of an action without its actual execution) such as walking44. trunk biomechanics and thus trunk movement perception and action are fundamental for establishing self - body representation (position), and during body movement, efficiency of the locomotor body schema37. our proposed exercise, the use of the trunk midline, and tactile and proprioceptive stimulation of the trunk enhance the locomotor body schema by generating more accurate information regarding trunk perception, the trunk - body midline, and gravity perception, and the interaction between trunk - body midline perception and space during a specific task. this enhancement in the locomotor body schema is a likely reason why the subjects displayed fewer errors in estimating walking distances after the intervention. that the subjects showed no improvement in estimating errors in a second evaluation (after the first session) indicates that the learning effect was negligible during repetition of the task thus, the improvements are likely attributable to the increase in movement perception during walking (locomotor body schema) while blindfolded. the blindfolded walking test is a complex task in which perception of the distance that has been walked should match the actual distance that is covered without visual correction. as reported by schmidt, better trunk proprioception improves the efficiency of walking while blindfolded. schmidt and colleagues applied vibratory stimuli if the trunk muscle spindle noted changes in the trajectory direction during walking, even in the presence of vision45,46,47,48. overstimulation of perceptual and proprioceptive trunk sensory information, as occurs during trunk muscle vibration, might disrupt the steering of locomotion49. this was likely due to enhanced trunk proprioception, which was the target of our training program. further studies should confirm these preliminary results and our hypothesis with more rigorous experiments and a larger sample. our study highlights the importance of trunk perception in computing egocentric space information during body locomotion. an example of the relative dependence of postural control on visual, vestibular, and somatosensory inputs was discussed by horak and nashner, who measured changes in postural sway in the standing position. they proposed that the apparent displacement of visual information and postural vertical input was the result of a recalculation of the central orientation of the gravity vector, based on a model in which the central nervous system extracts and interprets afferent information to construct a picture of reality (motor imagery)50. before treatment, subjects made errors that were proportional to the distance that was to be traveled. the mistakes were always negative : subjects tended to choose a shorter distance than the distance required. this pattern might be due to fear of hitting a wall or the ground, resulting from decreased body perception during walking (the locomotor body schema). this hypothesis is supported by the finding that before treatment, subjects walked with shorter steps, which is typical of people who are fearful of falling51, and of those with reduced somatosensory information, as might occur during indoor blindfolded walking29. our results reinforce the hypothesis that working on the representation of the midline and motor imagery of the trunk creates a bridge between perception and movement, which can give rise to new functional strategies52. blindfolded subjects evaluate movements better, because the motor patterns that are learned for ambulation, compared with specific tasks, can be affected by and depend on visual representations of the same image and are related to motor imagery, which is linked to somesthetic perception. our results also show that after training, upright gait stability was unchanged, likely because upright gait stability is modified in pathological walking pattern conditions to a greater extent than in healthy subjects30. but, this hypothesis should be confirmed with an ad hoc sample with pathological conditions and age - matched healthy subjects. this study had several limitations, such as the absence of a control group ; however, the repetition of the test immediately after the first treatment session should eliminate any suspicion of a bias linked to the learning effect. furthermore, our small sample size did not allow us to examine the differences between genders29, 53, only within - subject comparisons were made pre- and post - treatment. we did not plan to measure lateral errors, but we noted lateral deviations during the tests. also, we did not measure joint kinematics during walking, losing important information, especially with regard to the function of the hips. finally, a potential bias of our test was the learning effect. although we did not find statistically significant differences in the performance of subjects who were tested and immediately retested, the absence of significant differences could be due to the reduced sample size (5 subjects) with which this sub - analysis was performed. nevertheless, the values of their performance during the retest were similar to those that were recorded in the first test, suggesting the absence of any effect. the potential bias of learning should be taken into account, and the interpretation of our results should be made with caution. in light of our results, future studies in this field should be performed with larger sample sizes to determine whether perceptive rehabilitation, integrated into traditional rehabilitation programs, improves locomotor body awareness and enhances stability and walking performance in pathological conditions (e.g., stroke, ataxia, cerebral palsy, juvenile idiopathic scoliosis, and low back pain). in conclusion, this pilot study demonstrated that perceptive stimulation of the trunk midline improves the estimation of walking distance, implicating the upper body in the locomotor body schema. | [purpose ] recently, there has been growing interest in the somatosensory system, but little data exist on the interaction between dynamic postural control and the somatosensory system. the purpose of this study was to determine whether a training program, based on tactile and proprioceptive sensory stimulation of the trunk with the use of perceptual surfaces, improved the estimation of walking distance by healthy subjects, the ability to walk toward a memorized distance without vision, and whether it increases upright gait stability. [subjects and methods ] ten healthy subjects with a mean age of 31.9 2.5 years were enrolled and participated in 10 daily sessions of perceptive training using perceptual surfaces, for 45 minutes each session. an experimental indoor test measured the subjects ability to perceive walking distances to a memorized target in an indoor environment. [results ] after treatment, the distances that were traversed were closer to the target than before treatment. trunk acceleration did not differ significantly between pre- and post - training and did not increase significantly after training. [conclusion ] treatment with perceptual surfaces stimulating the trunk midline improves the estimation of walking distance and modifies proprioceptive gait patterns, allowing various corrective strategies to be implemented during ambulation. |
the study was carried out between 2005 and 2007 at the all india institute of medical sciences (aiims), new delhi. amphotericin b deoxycholate (fungizone) was purchased from ambalal sarabhai enterprises ltd, vadodara, india. new formulation of the liposomal amphotericin b, kalsome10, was a kind gift from lifecare innovations pvt., the commercially available amb deoxycholate was reconstituted in sterile, chilled, triple - distilled water to obtain a stock solution of 5 mg / ml. all other reagents used were of analytical grade (sigma - aldrich, st louis, usa). experimental animals and strain of leishmania donovani : experiments were conducted on balb / c male mice (5 - 6 wk old, 2530 g) purchased from laboratory animal facility of national institute of nutrition, hyderabad, india. the animals were maintained by giving a standard pellet diet and water ad libitum. mice were checked daily for their mortality and waning prior to commencement of the study and only healthy mice were included. hm / in / ke16/1998 strain of l. donovani isolate from an untreated child from muzaffarpur district of bihar (eastern india) was used for infection establishment12. the parasite culture is being maintained in medium 199 with 25 mm hepes, ph 7.4 (hi - media, mumbai) supplemented with 10 per cent heat inactivated foetal bovine serum (sigma - aldrich, usa) at 22 c in a biological oxygen demand (bod) incubator. electron microscopic analysis of kalsome10 : to increase the plasma half - life of the liposomal drug kalsoma10 (10 mg / ml of amphotericin b) for facilitating better bio - distribution in the body bath sonication was carried out for 45 min on ice. sonicated and non - sonicated preparations of kalsome10 were observed under the scanning electron microscopy. analysis was carried out on leo scanning electron microscope available at electron microscopy facility of aiims. in vivo single dose drug toxicity study : groups of six mice each were injected with various concentrations of conventional fungizone (ranging from 1 - 3 mg / kg of body weight) or new liposomal amphotericin b formulation : kalsome10 (range, 15 - 20 mg / kg) and empty liposomes i.e. liposomes without amb (range, 20 - 100 l) as control, intravenously. the toxicity of the drug was evaluated in vivo by determining the 50 per cent lethal dose (ld50). for this, the mortality rate and activity levels of animals were monitored for 24 h. chronic toxicity study : the chronic toxicity was performed according to the organization for economic co - operation and development (oecd) test guidelines with minor modifications, as the purpose was not regulatory clearance but to provide an evidence of its efficacy13. healthy mice were randomly assigned into four groups : a, b, c and d (6 per group). daily administration of the ld50 as estimated above for fungizone and kalsome10 through the known route was given for 1 wk to group a and b, respectively. in group c normal saline was given while in group d empty kalsome (liposomes without amb) was given using the same route. biochemistry analysis of blood urea nitrogen (bun), creatinine and glucose level, was done using the commercially available kits in a semi - automated chemistry analyzer (techno-168) (i.s.e sr.t. establishment of infection and therapeutic protocols : to determine, the duration required to establish the l. donovani infection, group of six balb / c mice, were infected with l. donovani (mhom / in / ke16/1998) strain12. for infection, the log - phase promastigotes of the parasite were washed twice using pbs buffer (ph 7.4) at 2300 g at 4 c. finally, the pellets were re - suspended in pbs buffer for intravenous infusion and approximately 1107 promastigotes / ml were injected into the tail vein using 26 gauge needle. these included loss of hair, other signs of being unwell such as fever, loss of weight and appetite and enlargement of spleen and liver were observed for 3 wk post - infection. after visible splenic enlargement, and other symptoms, two mice were sacrificed on every alternate day. blood was collected and tested for the presence of antibodies against visceralising species of leishmania using the commercially available lc - rk39 (recombinant antigen prepared from l. chagasi) dipstick test kits (insure, in - bios int, usa) and ld - rke16 (recombinant antigen prepared from l. donovani) spot test (signal ka, span diagnostic ltd., liver and spleen samples were taken out under sterile conditions, homogenized in mortar and pestle and were subjected to pcr analysis for confirmation of infection. for dna isolation 300 l of homogenized sample was taken and re - suspended in two volumes of lysis buffer [50 mm nacl, 50 mm tris - hcl, 10 mm edta (ph 7.4), 1 per cent triton x-100, 200 g / ml of proteinase k ]. thereafter the genomic dna of leishmania was extracted using phenol - chloroform extraction and finally suspended in tris - edta (te) buffer or sterile water. a 1020 bp region of genomic dna was amplified, using the following primer set14. forward, itsf5ctggatcattttccgatg-3, and reverse, itsr5acactcaggtctgtaaac-3. since amplicon size of our product was 1000 bp (ideal 100 - 500 bp real time pcr), the rt - pcr was standardized with low salt concentration and adjusted reaction time. pcr was performed in 50 l reaction volumes containing 100 ng of genomic dna, 10 pmol each of the gene - specific forward and reverse primers, 10 m of each dntp, 2 mm mgcl2 and 5 units of pfu taq dna polymerase (bangalore genei, india). the conditions for pcr were as follows : 95c for 2 min, then 34 cycles at 95c for 20 sec, 53c for 30 sec and 72c for 1 min. leishmania infected mice were randomly assigned to three groups ; groups a, b and c (6 mice per groups) and treatment was started one week after establishment of infection. groups a and b mice were treated on each alternate day with fungizone (2 mg / kg of body weight) and 0.75x dose of kalsome (7.5 mg / kg of body weight, half of the ld50), respectively. three mice were sacrificed by cervical dislocation at day 7, and the remaining three at the termination of the study i.e. day 28 post - treatment. flowchart summarizing the experimental details of the study determination of parasite burden : detection of parasitic load in liver was carried out by blinded microscopic enumeration with giemsa - stained liver impression smear in three groups. leishman - donovan units (ldu) were calculated per organ for the liver by using the formula [ldu= number of amastigotes per 1,000 nucleated cells organ weight (in g) 2105, according to stauber 's formula15. (ii) quantitative real - time pcr for quantitative analysis of parasite load : both liver and spleen are well accepted system for assessment of l. donovani infection, however, due to larger volume of the tissue liver was used for quantitation of parasite load. a real - time hot - start pcr was performed with the smart cycler dna master sybr green kit (cepheid, usa) in a smart cycler (cepheid, usa) to determine the parasitic load in liver tissues of control group as well as in fungizone and kalsome -treated mice. the 25 l reaction mixture contained 12.5 l dna master sybr green, 0.5 m each primer and 1 l of template. for fluorescence signal acquisition, channel f1 was used and standard threshold of 30 fluorescence was set. the fractional cycle number reflecting a positive pcr result is called the cycle threshold (ct). statistical analysis : to determine the variability of the assays, intra- and inter - assay (repeatability) precision was measured. three replicates of eight different concentrations of l. donovani dna were tested simultaneously in the same run. variability is shown as the mean standard deviation (sd) and reported as the coefficient of variation (cv). statistical procedures were performed using microsoft excel and stata statistics package 9 (stata corp. statistical analyses were performed by using an unpaired t - test (two - tailed) followed by mann - whitney test using, graphpad prism5 version (graphpad software inc., la jolla, ca). determination of parasite burden : detection of parasitic load in liver was carried out by blinded microscopic enumeration with giemsa - stained liver impression smear in three groups. leishman - donovan units (ldu) were calculated per organ for the liver by using the formula [ldu= number of amastigotes per 1,000 nucleated cells organ weight (in g) 2105, according to stauber 's formula15. (ii) quantitative real - time pcr for quantitative analysis of parasite load : both liver and spleen are well accepted system for assessment of l. donovani infection, however, due to larger volume of the tissue liver was used for quantitation of parasite load. a real - time hot - start pcr was performed with the smart cycler dna master sybr green kit (cepheid, usa) in a smart cycler (cepheid, usa) to determine the parasitic load in liver tissues of control group as well as in fungizone and kalsome -treated mice. the 25 l reaction mixture contained 12.5 l dna master sybr green, 0.5 m each primer and 1 l of template. time and temperatures were same as used in normal pcr reaction. for fluorescence signal acquisition, channel f1 was used and standard threshold of 30 fluorescence was set. the fractional cycle number reflecting a positive pcr result is called the cycle threshold (ct). statistical analysis : to determine the variability of the assays, intra- and inter - assay (repeatability) precision was measured. three replicates of eight different concentrations of l. donovani dna were tested simultaneously in the same run. variability is shown as the mean standard deviation (sd) and reported as the coefficient of variation (cv). statistical procedures were performed using microsoft excel and stata statistics package 9 (stata corp. statistical analyses were performed by using an unpaired t - test (two - tailed) followed by mann - whitney test using, graphpad prism5 version (graphpad software inc., determination of parasite burden : detection of parasitic load in liver was carried out by blinded microscopic enumeration with giemsa - stained liver impression smear in three groups. leishman - donovan units (ldu) were calculated per organ for the liver by using the formula [ldu= number of amastigotes per 1,000 nucleated cells organ weight (in g) 2105, according to stauber 's formula15. (ii) quantitative real - time pcr for quantitative analysis of parasite load : both liver and spleen are well accepted system for assessment of l. donovani infection, however, due to larger volume of the tissue liver was used for quantitation of parasite load. a real - time hot - start pcr was performed with the smart cycler dna master sybr green kit (cepheid, usa) in a smart cycler (cepheid, usa) to determine the parasitic load in liver tissues of control group as well as in fungizone and kalsome -treated mice. the 25 l reaction mixture contained 12.5 l dna master sybr green, 0.5 m each primer and 1 l of template. time and temperatures were same as used in normal pcr reaction. for fluorescence signal acquisition, channel f1 was used and standard threshold of 30 fluorescence was set. the fractional cycle number reflecting a positive pcr result is called the cycle threshold (ct). statistical analysis : to determine the variability of the assays, intra- and inter - assay (repeatability) precision was measured. three replicates of eight different concentrations of l. donovani dna were tested simultaneously in the same run. variability is shown as the mean standard deviation (sd) and reported as the coefficient of variation (cv). statistical procedures were performed using microsoft excel and stata statistics package 9 (stata corp. statistical analyses were performed by using an unpaired t - test (two - tailed) followed by mann - whitney test using, graphpad prism5 version (graphpad software inc., electron microscopic features of the new formulation : the sonicated and non - sonicated drug samples revealed typical micro sphere structure of approximately 1.0 m in diameter. these micro spheres tend to aggregate in non - sonicated samples, while after sonication these got separated (fig. ld50 evaluation : the ld50 for standard drug fungizone was estimated to be 2 mg / kg of body weight of the mice. the ld50 for kalsome10 (10 mg / ml) was 15 mg / kg of body weight of the mice. hence, kalsome10 (10 mg / ml) was markedly less toxic to mice. study on empty kalsome revealed that the carrier molecule used was non - lethal to the animals (fig. toxicity in animal models : treatment with new liposomal formulation was found to be associated with less nephrotoxicity compared with treatment with fungizone. the level of serum creatinine and blood urea nitrogen increased significantly in mice treated with fungizone. creatinine value increased > 2-fold over base line values (0.70.04 and 0.280.02 mg / dl ; p>0.001) and blood urea nitrogen level increased by > 2.5 fold (64.8 1.04 and 22.4 1.74 p 35 was observed at day 30 for both the treated groups (table iii). in qrt - pcr evaluation the mice treated with kalsome10 reduced parasite burden by about 2.34 log units within one week and finally resulted in complete clearance of parasites. in all of the assays, the ct values of negative controls were always > 35. efficacy of new amphiphilic formulation of amphotericin b (kalsome10) against established l. donovani infection in balb / c mice results of real - time and conventional pcr assays for leishmania dna in mouse tissue samples efficient and cost effective short course treatment of leishmaniasis remains a much desired research question17. the major limitations of the existing treatment options for vl are the emergence of unresponsive strains1819 and cost of the treatment2021. amphotericin b is the current second line drug of choice with 97 per cent cure rate and no reported resistance2223. the limitation is infusion related toxicities, especially the renal toxicity2425 and other adverse effects restrict its use. however, the systemic side effects are reported to be considerably reduced by the particulate forms of liposomes which are highly effective as well as require a shorter course of 3 - 5 days therapy and facilitate targeted delivery of liposomal amphotericin b but, the stability and cost of these formulations seriously limit their widespread use. choice of sterols is a critical factor in designing of liposomal formulation of a drug specifically to prevent binding of liposomal amphotericin b to mammalian host cells. the therapeutic antimicrobial action as well as toxicity of amphotericin b to the host cells result from its binding with sterols of the cell membranes, resulting in pore formation and finally leading to cytolytic action of the drug. ergosterol in combination with certain lipids in liposomes appears to be a better choice since the drug has stronger affinity for the ergosterol or their precursor in leishmania26 than to cholesterol, present in most mammalian cell membranes. also, cholesterol is an essential requirement for the endurance of leishmania, and cholesterol containing liposomes may serve as a source of cholesterol for leishmania. in the present study the toxicity as well as efficacy of the new liposomal formulation of amphotericin b the study demonstrated a major reduction in the toxicity of amphotericin b upon incorporation in liposomes compared with amphotericin b deoxycholate. the evaluation of the toxicity of liposomal amphotericin b (kalsome10) in mouse model involved the study of ld50 profile of new drug. further, the safety study of lipid - based carrier i.e. empty kalsome (liposome without amphotericin b) hd in two volumes of lysowed it to be safe for application. in vivo single dose acute toxicity study showed that amphotericin b upon encapsulation in liposomes induced less toxicity in comparison with amphotericin b deoxycholate which would allow accommodation of much higher doses of the drug for in vivo use. mice treated with new liposomal formulations of amphotericin b showed a maximum tolerated dose that was 7-fold greater than amphotericin b deoxycholate which would be advantageous to less susceptible or drug - resistant isolates. in vivo renal toxicity was assessed by estimating serum creatinine, blood urea nitrogen and glucose level in mice. though the levels of serum creatinine and blood urea nitrogen were elevated in mice treated with amphotericin b deoxycholate, no renal or hepatic alterations occurred, as evidenced by normal levels of creatinine and blood urea nitrogen levels in liposomal amphotericin b treated mice. the present study showed in vivo efficacy of this new liposomal formulation of amphotericin b using the l. donovani infected balb / c mouse model by real time pcr. the conventional pcr and gel based visualization of amplified products has several limitations such as time, low sensitivity, short dynamic range < 2 logs, low resolution, non - automated, only size - based discrimination and non - quantitative results27. to overcome these limitations, highly sensitive and specific quantitative real time pcr method was used for anti - leishmanial efficacy. though, 100 - 500 bp is optimal size for amplicons of real time pcr, but up to 1000 bp was possible with low salt concentration and adjustment in reaction time. this method was used not only to detect the l. donovani infection in the experimentally infected animals but also to quantify the parasite load accurately up to a minimum detection level of 1 parasite per reaction. treatment of infected mice with kalsome10 revealed that it reduced parasite burdens more efficiently than amphotericin b deoxycholate. though, potential limitation of this study included the fact that daily dose- sub acute toxicity tests and subchronic toxicological evaluations could not be done as per oecd guidelines. hence, these results can not be used for regulatory purposes, as the purpose of this study was only collecting the evidence of efficacy and diagnostic modalities to assess the efficacy. single dose acute toxicity experiments and chronic toxicity experiments were performed according to the oecd test guidelines13 and the results showed that the new formulation was efficacious and displayed no major toxic effects in the animals. | background & objectives : current therapy for leishmaniasis is limited and unsatisfactory. amphotericin b, a second - line treatment is gradually replacing antimonials, the first - line treatment and is used as the preferred treatments in some regions. though, presently it is the only drug with highest cure rate, its use is severely restricted by its acute toxicity. in the present study novel lipid - amphotericin b formulations with lower toxicity than the parent drug were evaluated for the treatment of visceral leishmaniasis (vl) in a mouse model.methods:the toxicity and therapeutic efficacy of a new amphiphilic formulation of amphotericin b (kalsome10) was compared to that of amphotericin b deoxycholate (fungizone) in a mouse model of vl using quantitative real - time pcr (qrt - pcr).results : the toxicity of amphotericin b was significantly less with liposomal formulation as compared to the deoxycholate form, evidenced by reduced nephrotoxicity and higher tolerated dose in balb / c mice. the therapeutic efficacy was evaluated by quantitative real time (rt) pcr using primers highly specific for the its region of leishmania donovani. there was reduction in parasite load by 2 log unit after 7 days of treatment and finally resulting in complete clearance of parasite from infected mice after 30 days of treatment with kalsome10.interpretation & conclusions : this new formulation showed a favourable safety profile and better efficacy when compared to conventional amphotericin b. if production cost is kept low, it may prove to be a feasible alternative to conventional amphotericin b. |
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