text
stringlengths 0
868k
|
|---|
Atrial fibrillation (af) is commonly associated with congestive heart failure and strokes secondary to atrial thromboemboli.13 lone atrial fibrillation is a subtype that usually occurs in the younger population and represents af without pre - existing cardiovascular diseases . Episodes are often preceded by exercise or emotional stress.1 hyperthyroidism and pheochromocytoma are other rare causes of af.13 pathophysiology of af is not well understood but it is known that ectopic atrial beats stem from areas in the left atrium around the inlet of pulmonary veins.4 an overactive parasympathetic (vagal) nervous system may also cause af, such as in athletes . People with digestive problems could also develop af and potentially have weekly episodes which last from a few minutes to several hours . Af in general occurs at night, often ending in the morning following rest, digestive periods (after dinner), and alcohol consumption . Frequent urination (every 20 minutes or so) often occurs during the early phase of an episode and is due to the release of atrial natriuretic peptide from the fibrillating atria . The patients with af may first be treated with medications to slow the heart rate and to control heart rhythm . Anticoagulants are often given to prevent stroke, depending of the risk of embolic event . In addition, procedures such as synchronized electrical cardioversion may also be used to eliminate af and return the heart beat to a normal rhythm . Radiofrequency ablation may also be used to prevent recurrence of af in certain individuals . In this report, we described how a simple digital rectal examination led to the termination of typical af in a 29-year old male . The case report is valuable for practice in cardiology clinics and emergency medicine for diagnosis and treatment of af patients . A 29 year - old male with no significant past medical history complained of sudden onset of palpitations while walking home from the store on the night of admission . Onset of palpitations was 3 hours later, just prior to presentation in the emergency department . Patient denies shortness of breath, diaphoresis, chest pain, dizziness, loss of consciousness, fever, chills, malaise, recent illnesses, alcohol or recreational drug use . He smoked cigarettes (under a pack a day) for 5 years but quit five years ago . Patient s vitals were as follows, blood pressure: 144/99, pulse: 113 bpm, temperature: 97.0 f, saturation: 99% on room air . A pertinent physical examination revealed an athletic male with cardiovascular ausculatory findings of an irregularly irregular rhythm with no murmurs or rales . Twelve - lead ekg records showed a typical af with rapid and irregular rhythm, and coarse fibrillating p waves (fig . The patient was found to be in rapid atrial fibrillation in the emergency department . Predicted onset of atrial fibrillation, based on the patient s symptoms, is less than 48 hours . He was admitted and monitored by telemetry . A heparin drip was planned for the patient in anticipation of his undergoing cardioversion since anticoagulation is necessary prior to cardioversion to prevent development or further development of atrial thrombus and thereby preventing embolic events . However, prior to starting anticoagulation, a digital rectal exam (testing the stool for occult blood) is required to rule out a gi bleed . Before administering the rectal exam,, his rhythm converted to normal sinus and the rate decreased to 80 beats per minute . The patient no longer complained of discomfort and was discharged from the hospital . In addition, the patient remained asymptomatic after a 3-month follow - up . A 29 year - old male with no significant past medical history complained of sudden onset of palpitations while walking home from the store on the night of admission . Onset of palpitations was 3 hours later, just prior to presentation in the emergency department . Patient denies shortness of breath, diaphoresis, chest pain, dizziness, loss of consciousness, fever, chills, malaise, recent illnesses, alcohol or recreational drug use . He smoked cigarettes (under a pack a day) for 5 years but quit five years ago . Patient s vitals were as follows, blood pressure: 144/99, pulse: 113 bpm, temperature: 97.0 f, saturation: 99% on room air . A pertinent physical examination revealed an athletic male with cardiovascular ausculatory findings of an irregularly irregular rhythm with no murmurs or rales . Twelve - lead ekg records showed a typical af with rapid and irregular rhythm, and coarse fibrillating p waves (fig . The patient was found to be in rapid atrial fibrillation in the emergency department . Predicted onset of atrial fibrillation, based on the patient s symptoms, is less than 48 hours . He was admitted and monitored by telemetry . A heparin drip was planned for the patient in anticipation of his undergoing cardioversion since anticoagulation is necessary prior to cardioversion to prevent development or further development of atrial thrombus and thereby preventing embolic events . However, prior to starting anticoagulation, a digital rectal exam (testing the stool for occult blood) is required to rule out a gi bleed . Before administering the rectal exam,, his rhythm converted to normal sinus and the rate decreased to 80 beats per minute . The patient no longer complained of discomfort and was discharged from the hospital . In addition, the patient remained asymptomatic after a 3-month follow - up . The patient is a typical af subject as confirmed by the ekg with 140 beats per minute and the loss of the p wave (fig ., the second ekg revealed a normal sinus rhythm, which indicated that the exam eliminated the af . The termination of af is unlikely a result from medications, because the effect of the medication on the af occurred immediately during and after the rectal exam . This was very different than the medication - mediated effect which gradually returns the af to normal within minutes to hours . The mechanism for the effects of the rectal exam on the af is likely due to the increase in parasympathetic activity through cranial nerve x (vagus nerve), which also dampens the sympathetic stimulation to the heart . First, increased parasympathetic activity slows the rate of impulse formation of the sinus atrial (sa) node . Second, increased parasympathetic activity slows conduction and increases refractory through the atrioventriuclar (av) node . For example, in an earlier report, a digital rectal exam stimulated receptors in the rectum causing the increase in parasympathetic activity, after which the procedure was able to eliminate supraventricular tachycardia.5,6 the patient was also told to strain during the rectal guiac exam, causing increased vagal tone through the valsalva maneuver.7 one study compared 27 people with af against a control group of 27 people with no history of heart disease in their ekg response to valsalva maneuver . They monitored the p - wave dispersion of the two groups and found that the maneuver normalizes the p - wave dispersion and duration . They concluded that decreased sympathetic tone is beneficial in patients with af.8 another study measured more ekg parameters in patients with paroxysmal af in response to the valsalva maneuver.9 other similar mechanisms of increasing parasympathetic stimuli or decreasing sympathetic stimuli include the carotid massage . This method has been documented to be a diagnostic and therapeutic tool for patients with atrial flutter and atrial fibrillation.10,11 we could not completely exclude the possibility that the conversion to sinus rhythm was occurred by pre - administrated beta - blocker . However, in consideration of the limited half life of the beta - blocker, the possibility by the beta - blocker were less . In general enhanced vagal response may increase af episodes . But, in this case there was not further af episode during the three months follow - up . Thus, the reported digital procedure could be an additional (not replaced) method to treat af patient . In conclusion, the case report described how a digital rectal exam and/or valsalva maneuver was able to terminate typical af in a 29-year old male instantly . The rectal exam is a relatively simple procedure that presents little risks for af patients . Thus, this case report provides an implication to use the rectal exam as an additional therapeutic modality for selective af patients.
|
In early november 2001, a 57-year - old female laboratory worker (laboratory worker 1) began experiencing nonspecific symptoms of malaise, vomiting, headache, lower leg cramping, anorexia, and fever . One week after onset of symptoms, she was evaluated for severe headaches at a local emergency room, where cerebrospinal fluid (csf) and blood cultures were collected . Small, gram - positive bacilli were isolated and characterized as coryneform bacilli, which are usually interpreted as contaminants of unknown clinical importance . Despite multiple hospital admissions, approximately 5 weeks after symptom onset, colleagues from the hospital microbiology laboratory where she was employed (laboratory 1) drew her blood for culture again . After 5 days of incubation, gram - variable coccobacilli, the brucella serum agglutination test (sat) was reactive (1:640) at the nysdoh laboratory, wadsworth center . Laboratory worker 1 was initially treated with doxycycline and gentamicin, followed by doxycycline and rifampin, for 6 weeks of outpatient therapy . The isolate was later identified as b. melitensis by the wadsworth center and confirmed by the centers for disease control and prevention . The patient has not relapsed 18 months after completing treatment . In a second incident in mid - january 2002, a 48-year - old woman had nocturnal temperature spikes to 40c, chills, drenching sweats, and weight loss . Symptoms persisted, and uveitis developed . In early march 2002, a diffuse, erythematous rash appeared on the anterior aspect of both legs . A blood culture and serologic tests for lyme disease, ehrlichiosis, and rocky mountain spotted fever (rmsf) were performed . From the blood culture, gram - positive cocci were isolated and identified as micrococcus spp . By a commercial laboratory . Rmsf titers were immunoglobulin (ig) m - negative with a reactive igg of 1:256 . Subsequently, she was referred to an infectious disease specialist, who found repeat rmsf titers unchanged, which made acute rmsf unlikely . When interviewed by nysdoh staff, the patient reported that she was a laboratory worker (laboratory worker 2) at laboratory 2 . Her initial blood culture specimen, which had originally been identified as micrococcus, was reassessed by the commercial laboratory . The commercial laboratory referred the original isolate to the wadsworth center, where the isolate was identified as b. melitensis . . They denied traveling outside of the united states, consuming imported or domestic unpasteurized dairy products, knowing ill family or friends who may have traveled, attending events with potentially contaminated foods, or handling farm or laboratory animals . Both laboratory workers denied any accidental contamination or spills in the laboratory during the 6 months before their respective illnesses . Site visits to laboratories 1 and 2 were conducted by nysdoh . On the open bench at laboratory 1 a syringe was used to directly plate contents of the blood culture media bottle onto agar . Subsequent biochemical tests, including a catalase test, which may generate aerosols by introducing hydrogen peroxide to the specimen, were performed on the open laboratory bench . At laboratory 2, subculturing occurred in a similar manner, but it took place in a class ii biosafety cabinet . However, biochemical tests were performed on the open laboratory bench when gram stain results indicated that spores were not present . Laboratory workers 1 and 2 wore gloves when processing specimens, and both denied having dermatitis or skin lesions on their hands . After the diagnosis of brucellosis in these two laboratory workers, serum samples from their co - workers from laboratories 1 and 2 were tested by brucella sat; samples from seven of eight co - workers were nonreactive (<1:20). A co - worker of laboratory worker 1 had an initial agglutination titer of 1:40 (indeterminate) and 1 month later had a repeat titer of <1:20; she denied having symptoms . To determine the source of these laboratory workers' infections, each laboratory reviewed gram stains of blood culture specimens processed within 3 to 6 months of symptom onset . Laboratory worker 2 reviewed prior gram stain slides from laboratory 2 and identified one slide containing a questionable coryneform bacillus . This blood culture had been drawn from laboratory worker 1 during her visit to the emergency room of laboratory worker 2's hospital, early in november 2001 . Approximately 2 months before her illness, laboratory worker 2 had personally processed laboratory worker 1's blood culture but had characterized the isolate as coryneform bacilli . Wadsworth center staff reviewed the original gram stain from laboratory worker 1's blood culture specimen and noted gram - variable organisms with similar size and shape to brucella organisms . However, laboratory worker 2 did not identify any other gram stains that resembled brucella organisms during the period before her illness . The likely source of laboratory worker 1's infection was found after reassessing gram stains performed in laboratory 1 in august, september, and october 2001 . A 76-year - old woman visited the emergency room served by laboratory 1 in early september 2001 . The patient had had fever (temperature 38.3c) for 1 day and headache for 3 weeks . She received one dose of ciprofloxacin for a possible urinary tract infection and was discharged . After 4 days of incubation, a gram stain of this patient's blood culture specimen showed tiny, gram - negative coccobacilli, reported as coryneform bacilli . As part of the investigation for the source of infection for laboratory worker 1, this patient's prepared gram stain slide was referred to the wadsworth center, where small, gram - variable cocci were identified . More than 1 year after being seen in the emergency room, this patient was offered brucella sat and a repeat blood culture . She refused brucella sat, and her physician did not identify symptoms consistent with brucellosis . Six months after this identification, further experimental analysis by the wadsworth center with polymerase chain reaction tests performed on gram stain material detected b. melitensis dna (wadsworth center, nysdoh, unpub . The patient was again contacted, and brucella sat (> 1.5 years after her initial blood culture) showed a titer of 1:80 (indeterminate)., the patient denied any visits outside the united states since 1989, when she emigrated from peru . Although brucellosis is a rare disease in the united states, its potential use as a bioweapon highlights the need for accurate and rapid identification (15). In this investigation, brucellosis was diagnosed weeks to years after initial positive blood cultures were misidentified, and laboratory personnel were unknowingly exposed to the organism . This investigation suggests that transmission occurred from the 76-year - old index patient to laboratory worker 1, on the basis of b. melitensis dna found in the gram stain material . Processing the index patient's blood culture specimen on an open laboratory bench was most likely the reason for laboratory worker 1's illness approximately 5 weeks later . The same mechanism of transmission probably occurred when laboratory worker 2 handled laboratory worker 1's blood culture specimen on the open bench for biochemical testing . In these instances, blood culture bottle media were transferred to slides and agar without much risk for aerosolization, since contents were manipulated with a syringe . Biochemical tests, however, included the catalase test, which creates bubbles as a result of exposure to 3% hydrogen peroxide in positive specimens . Neither laboratory worker could identify any other possible sources of infection, and because brucellosis is a rare disease in new york, the connection between these three patients is plausible . With the initial interpretations of these gram stains as gram - variable, which resulted in misidentification of the organism by three different laboratories, nysdoh initiated an effort to educate clinical laboratories in new york state about the potential difficulties in characterizing the organism and the importance of primary gram stain interpretation . Additional investigation into the staining properties of brucella spp . Under various conditions is now in progress and may help differentiate brucella spp . From other organisms . Because of immigration and foreign travel, brucellosis remains an occupational hazard for laboratory personnel, even in industrialized countries where animal control efforts have virtually eliminated the disease . Because of the nonspecific symptoms and the rarity of the disease in the united states, healthcare providers may not consider brucellosis in a differential diagnosis . . Moreover, improved communication among healthcare providers and laboratory personnel should facilitate prompt and accurate identification and appropriate handling of the organism . Its potential use as a bioweapon necessitates that healthcare providers, as well as microbiologists in hospitals and commercial laboratories, be knowledgeable about the diagnosis, identification, and handling of brucella spp . Reporting brucellosis cases to public health officials is another component in protecting others from this disease . Public health officials should notify laboratory personnel who may have handled cultures taken from patients ultimately diagnosed with brucellosis . Early notification of exposed personnel could lead to their timely diagnosis and treatment, should symptoms occur, and could prevent further laboratory exposures.
|
A number of studies have examined the role of psychological stress in the development of masticatory muscle disorders (mmd). War - related psychological stress, the performance of mental arithmetic, and the solving of five - letter anagrams can increase masticatory muscle activity and are believed to be associated with mmd [13]. Moreover, psychological tension can induce muscle fatigue and spasm, resulting in mmd [46]. A relationship between stress and mmd has been reported in children, adolescents, and adults [79]. Additionally, most patients with mmd experience moderate to severe depression or anxiety [10, 11]. On the other hand, cognitive - behavioral, habit reversal, and biobehavioral treatment strategies are all known to be beneficial in the management of mmd, thus reflecting another line of evidence demonstrating a relationship between psychological stress and mmd . All of the studies conducted to date illustrate the importance of psychological factors in mmd [15, 16]. However, it is not yet possible to determine how the mental / psychological factors affect the muscle and even to facilitate the mmd . Clinical studies using traditional electromyography (emg) to assess the role of psychological stress in mmd have shown significantly increased emg activity of masticatory muscles under stress conditions [3, 1719]. Similar results have been observed in animals [20, 21]. Under the emotional stress, changes in the masticatory behaviour and hormones such as glucocorticoids were accompanied by a modification of particular myhc isoforms of the muscle, which showed evident faster phenotype due to a higher percentage of either pure myhc-2b containing fibres or hybrid myhc-2x / d and -2b expressing fibres . Furthermore, a decrease in oxygen saturation of muscle blood around the onset of mental stress proved the potential role for psychological stress in hemodynamic alterations, which might occur via their regulation by the sympathetic nervous system (sns) [23, 24]. An elevation of histidine decarboxylase activity in skeletal muscles during stress was induced, partly or wholly by muscle activity and/or muscle tension . However, muscular mechanism underlying emotional stress is not well understood and appears to be equally complex . The hypothesis of this paper was that psychological stress could alter the ultrastructure and energy metabolism of masticatory muscles . Using an emotional stress paradigm, which employed the use of intraspecies emotional communication within a communication box, we introduced experimental correlates of psychological stress, such as anxiety and depression, in rats and then tested our hypothesis on their masseter muscles . The masseter muscle is useful in this type of research because there are large amounts of spindles in these muscles, which are critical in determining muscle contraction and mandibular movement in response to stimulation . This study sought to assess alterations in the ultrastructure and energy metabolism of masticatory muscles of rats under psychological stress . Forty - eight male wistar albino rats weighing between 160 and 180 g (approx . 35 days old) were housed in 80 cm 45 cm 40 cm cages in a temperature - controlled room at 24c under a 12-hour light / dark cycle and were given free access to food and water . Before experiments, they were housed in a communication box one hour a day for five days to allow them to acclimatize to the box . The rats were then randomly divided into control (con), foot - shocked (fs), and psychological stress (ps) groups, with each group having 16 rats . Fs and ps rats were housed in one communication box during the procedure as described below, with the fs rats receiving electric foot shock and the ps rats being subjected to psychological stress . The rats in the control group were housed in another communication box under the same conditions but were not subjected to electric foot shock or psychological stress . The same study was performed twice, and the durations of psychological stimulation were three weeks and five weeks . Thus, 24 animals were used per study, with eight animals in each of the three groups . After three or five weeks of stimulation, the behaviors of the eight rats from the control or ps group were immediately evaluated by an elevated plus - maze apparatus . Blood was drawn from the ophthalmic artery, and serum samples were prepared for the measurement of serum stress indices . The rats were then sacrificed by intraperitoneal injection of an overdose of thiamylal sodium, and their bilateral deep masticatory muscles were removed . All muscle samples were obtained according to guidelines established by the university internal review board for use of mice subjects . The experimental procedures were reviewed and approved by the ethics committee of the fourth military medical university . The middle bellies of each muscle sample were dissected for ultrastructure observation by electron microscopy . The communication box (16) it consisted of 16 compartments that were 16 16 cm each and were separated by transparent plastic boards with several small holes . The boards prevented the animals from making physical contact with each other but allowed them to receive cues, such as visual, auditory, and olfactory sensations, from the neighboring animals . Each compartment was equipped with a grid floor of stainless steel rods, which were 5 mm in diameter and placed at intervals of 0.3 cm . A 48-v electric generator, which was made by the department of biomedical engineering of the fourth military medical university, was connected to the grid floor to produce an electric current and generate an electric foot shock every two seconds . The grid floors of eight of the compartments were covered by plastic plates to prevent electric foot shock and served as non - foot - shock compartments for the ps rats (figure 1). Prior to the day of stress stimulation, the ps rats, together with the fs rats, were individually confined in each compartment of the communication box for one hour per day without any electric foot shock for one week to allow them to acclimatize to the surroundings . The electric foot shock was then introduced to the fs rats (stress senders) for one hour a day at a fixed time in the morning . The ps rats (stress responders) confined in the non - foot - shock compartments were simultaneously exposed to emotional cues from the neighboring fs rats, such as shrieks, smells of urine or feces, and jumping responses . Consequently, the ps rats were assumed to be in a state of fear or anxiety . The purpose of this study was to explore the effects of the pure psychological stress upon the masseter muscles . But the foot - shocked group rats experienced a great part of physical stress, so we did not include this group in the experimental evaluations . The foot - shocked group was only used to induce psychological stress in the neighboring ps rats, and they were not included in the following investigations . The elevated plus - maze (epm) apparatus (rd1208, shanghai mobiledatum corporation, shanghai, china) consisted of two open arms (50 10 cm) and two enclosed arms (50 10 cm, with 50-cm high walls) extending from a central square platform (10 10 cm). The apparatus was elevated 50 cm above the floor in a quiet room with temperature controlled at 20c, with the light just bright enough to clearly observe the rats movement within a 1.5-meter range . Two of the opposing arms (50 cm 10 cm) were enclosed by 40-cm high side and end walls (enclosed arms), the other two arms were not installed with walls (open arms). In the beginning, animals were placed in the central area (10 10 cm) of the maze, facing an enclosed arm . Entry into one arm was recorded when an animal placed all four paws past the line dividing the central square from the open arms . The test arena was wiped with a damp cloth after each trial . The number of entries into the open / closed arms and the time spent in open arms / closed arms were measured by an observer blind to the treatment conditions of the animals . Then, the percent of open arm entries (100 open / open + enclosed entries, oe%) and time spent in the open arms (100 open / open + enclosed arm time, ot%) of the epm were calculated for each rat . We used oe% and ot% to analyze the anxiety level of each animal [2932]. The serum concentrations of cortisol and adrenocorticotropic hormone (acth), stress indices, were measured by radioimmunological analysis kits (northern bioengineering institute, beijing, china) according to the protocols provided by the manufacturer . The middle bellies of the bilateral deep masseter muscles were dissected and cut into 1-mm pieces and then fixed with 4% glutaraldehyde and 1% osmic acid . The samples were embedded in epon812, sectioned by lkb - v ultramicrotome (lkb, bromma, sweden), and stained with uranyl acetate and lead citrate . The ultrastructures of the muscles were then observed with transmission electron microscopy (jem-100sx, jeol company, japan). A sample of masticatory muscle (0.2 g) was minced and homogenized with 1.8 ml distilled water . Then, 0.1 ml homogenates were diluted 10-fold with water . After incubation for 15 min at room temperature, the mixtures were then centrifuged for 10 min at 3000 r / min . The activities of na - katpase, ca - atpase and lactate dehydrogenase (ldh) and the contents of lactic acid (ld) were measured by commercial kits according to the protocols provided by the manufacturer (a070 - 5, a019 - 2, a020, nanjing jiancheng bioengineering institute, nanjing, china) using a uv-754 spectrophotometer (shanghai third analytical instrument factory, shanghai, china). The protein concentrations were measured via the bradford method, with bsa as the standard . The substrate for atpases is atp, which is split by the atpase into adp and inorganic phosphorus (pi). Units of atpase activity were shown as the content of pi generated per hour (h) by decomposition of protein per gram (g), and the total protein content was determined by lowry method . Therefore, the amount of inorganic phosphorus was used to detect the activity of na - katpase and ca - atpase at the wavelength of 636 nm . With regard to ldh, it catalyzes the reaction that generates pyruvic acid from lactic acid; pyruvic acid can then react with 2,4-dinitrophenylhydrazine to produce pyruvate dinitrophenylhydrazone, which is brownish red in alkaline solutions . Enzyme activity can be calculated through colorimetry; the detecting wavelength is 440 nm . Further, by catalytic dehydrogenation of ldh, lactic acid changes into pyruvate and nad+ changes into nadh . By dehydrogenation of pms, nbt deoxygenizes purple - colored objects; the relationship between this change and ld is linear and occurs at an absorbance of 530 nm . Therefore, the wavelength for na - k, ca - atpase detection ldh detection, and ld detection was 636 nm, 440 nm, and 530 nm, respectively . The enzyme activities were expressed as the incorporation of total phosphate into the substrate per mg protein per hour . In this part, we stored the supernatants for no more than 6 hours at 4c and finished the detection of all samples in one day . Experimental data were analyzed by one - way analysis of variance (anova) across the control group, the three - week ps group, and the five - week ps group using spss, version 11.0 (spss co., chicago, illinois). The snk - q test was further used to calculate any differences between the two groups . A p value to confirm that the experimental rats were in a state of stress, we first analyzed the serum levels of cortisol and acth in the rats . As shown in figure 2(a), the concentrations of cortisol were 12.46 2.67 ng / ml and 11.78 2.35 ng / ml in the ps group after three or five weeks of psychological stimulation, respectively . These values were markedly increased compared with those of the control group (6.19 1.47 ng / ml and 5.93 1.54 ng / ml, resp . Similar changes were observed in acth levels . As shown in figure 2(b), in the ps group, the serum acth concentrations were 25.46 5.12 ng / ml and 23.49 4.95 ng / ml in rats after three and five weeks of stimulation . By contrast, during the same periods, the serum concentrations of acth in the control group were only 12.22 2.56 ng / ml and 12.54 2.79 ng / ml, which were significantly lower than those in the ps group (p <.05), indicating the rats in ps group were anxious . We then compared the behaviors of rats after three or five weeks of psychological stimulation with those of the control group by epm tests . As shown in figure 3(a), percentages (%) of entry into open arms (open arm entry, oe) in the ps rats were 34.02 2.89% and 24.66 1.99% after three or five weeks of stimulation, respectively, while those of control group were 48.94 2.94% and 48.65 3.81%, respectively, which were significantly higher than the ps groups . Similarly, as shown in figure 3(b), percentages of time spent in open arms (ot%) significantly decreased from 70.39 3.03% in the control group to 46.14 4.05% in the ps group after three weeks of psychological stimulation and from 72.25 1.35% in the control group to 25.05 3.07% in ps group after five weeks (p <.05), indicating that the rats in ps group were mentally under tension . Next, we examined the ultrastructures of the masticatory muscles from both the control and ps groups after three and five weeks of psychological stimulation . As shown in figure 4, the ultrastructures of the masticatory muscle cells of rats in the control and ps groups showed evenly distributed muscular nuclei under the sarcolemma around muscle fibers and no signs of hyperplasia, swelling, or pyknosis (panels 1, 4, and 7). Myofibril and myotome of the ps rats were as normal as the rats in the control group . The light i bands and dark a bands of myotomes in the ps rats were intact and in the right position (panels 4 and 7). Swollen mitochondria with cristae loss and reduction of matrix density were found in the 62.5% (5 of eight samples) and 87.5% (7 of eight samples) of the masticatory muscles samples of 3-w and 5-w ps rats, respectively, (as marked by black arrows in panel 5 and 8). These ultrastructural observations are consistent with the possibility that the mitochondria from muscles of stressed mice may have altered permeability characteristics . Moreover, dramatic vacuolar changes in mitochondria appeared in ps rats after five weeks of stimulation (as marked by white arrows in panel 8), which approximately were found in all samples (87.5%, 7 of eight samples). In contrast, there were nearly no swollen / abnormal mitochondria or vacuolar changes being observed in unstressed animal, which also gave the proof that the mitochondria changes observed in the ps rats has no relationship with the tem sample processing . The cytoplasm contained ribosomes, small strands of rough endoplasmic reticulum, and occasional small mitochondria . The outer membrane of each capillary was generally smooth, whereas their inner surfaces were often more convoluted with small cytoplasmic processes projecting into the lumen (panel 3). The outer capillary wall was enclosed within a basal lamina approximately 20 nm in thickness . The morphology of most capillaries in each specimen of 3-w ps rat was essentially normal, and the endothelial cell thickness appeared to be similar to that of the control specimens . But 2 of eight samples showed a sign of electron density decrease in the capillary cells (as marked by black arrow in panel 6). As to the capillaries ultrastructure of the 5-w ps rats, cytoplasm of some capillary cells appeared to be more electron - lucent than those in control specimens (as marked by black arrows in panel 9), with an increase in vesicle number and membrane ruffling . Some places of the capillary basal laminae seemed accidented or even discontinuous (as marked by white arrows in panel 9), which appeared in about 75% of all samples (6 of eight samples) and meant local hypoxia and increased vascular permeability to some extent . These results implied that the ultrastructures of masticatory muscle cells were distinctly affected in ps rats after three and five weeks of psychological stimulation . With regard to energy metabolism in the masticatory muscles, na - katpase activity in the masticatory muscles decreased significantly from 7.60 0.50 mol pi / mg protein / hour in the control group to 4.10 0.53 mol pi / mg protein / hour in the ps rats after three weeks of stimulation and even decreased from 7.15 0.35 mol pi / mg protein / hour in the control group to 2.50 0.32 mol pi / mg protein/ hour in the ps group after five weeks of stimulation (p <.05) (figure 5(a)). Similarly, ca - atpase activity of the masticatory muscles decreased from 8.38 0.23 and 8.47 0.37 pi / mg protein / hour in the control group to 3.82 0.58 mol pi / mg protein / hour (p <.05) and to 2.14 0.43 mol pi / mg protein / hour (p <.05) in the ps rats after three and five weeks of stimulation, respectively, (figure 5(b)). By contrast, the activities of ldh and the contents of ld, respectively, significantly increased from 32.41 2.35 (u / g protein) and 0.67 0.10 mmol / g protein in the control group to 85.13 9.63 (u / g protein) and 1.25 0.19 mmol / g protein in the ps group after three weeks of stimulation (p <.05) and even increased to 128.69 10.19 (u / g protein) and 1.97 0.33 mmol / g protein in the ps group after five weeks of stimulation (p <.05) (figures 5(c) and 5(d)). There were also significant differences in the activities of na - k atpase, ca - atpase, and ldh and in the contents of ld between the three - week and five - week ps groups (p <.05). To our knowledge, this is the first study to report significant changes in the ultrastructures and energy metabolism of the masticatory muscles of rats exposed to psychological stress . The communication box is a well - established method for introducing psychological stress to animals . In this paradigm, the animals that do not undergo physical stress are able to perceive the responses of their neighboring animals that are exposed to physical stress, which is delivered through an electric foot shock . The intraspecies emotional communication then signals the nonstimulated rats to become anxious and can cause increased plasma level of stress hormones, such as corticosterone, in the experimental subjects . In this paper, we successfully simulated a psychologically stressful environment using the communication box as indicated by the increased levels of cortisol and acth (figure 2) and the decreased ot% and oe% in the epm test (figure 3) after three and five weeks of psychological stimulations . It has been demonstrated that psychological stress is accompanied by regional modification of muscle morphology, possibly due to differential gene expressions or accessibility to hormones in certain regions of the masticatory muscle . The present study observed subtle but significant changes in the ultrastructure of the rat masticatory muscles under psychological stress . Transmission electron microscopy demonstrated swollen mitochondria with cristae loss and reduction of matrix density in rats under psychological stress for three weeks (figure 4, panels 2, 5, and 8) and severe vacuolar changes in the mitochondria of the rats under a longer period of psychological stress (figure 4, panels 3, 7, and 9), suggesting that there were likely some important changes in mitochondrial function secondary to psychological stress . Therefore, we further investigated energy metabolism in the muscle, which is primarily associated with mitochondrial function . Na - katpase and ca - atpase are key factors that help to maintain and modulate mitochondrial function . Increased atpase activity accelerates the rate of metabolism of muscle cells and improves muscle exercise capacity . Decreased atpase activity is one of the signs of cell damage, which is often associated with hypoxia, acidic metabolites, or free radical formation . Increases in lactate, concomitant with glycogen breakdown, are the result of an increased need by the muscle for an anaerobic energy supply . Ldh activity level plays an important role in the removal of lactic acid and maintaining normal ph in the tissues, and it is an important enzyme in anaerobic oxidation . We found that psychological stress results in decreased na - katpase and ca - atpase activity, increased ldh activity, and elevated ld content in masticatory muscles, indicating a decrease in aerobic glycolysis and hypoxia or ischemia of the muscles . With increased exposure to psychological stress, the aerobic metabolism of the masticatory muscle decreased (figures 5(a) and 5(b)), while anaerobic metabolism increased (figures 5(c) and 5(d)), implying correlations between mitochondrial ultrastructure and energy metabolism in rat masticatory muscles . Similar results have been observed in the atrial cardiomyocytes of rats exposed to audiogenic stress for 6 h . Psychological stress, such as anxiety and tension, has been reported to increase myoelectricity [3, 18] and excessive activities of the masticatory muscle, including bruxism . It has been suggested that the hemodynamics of jaw muscles are susceptible to mental stress via their regulation by the sympathetic nervous system (sns) [23, 24], which is generally beneficial in the acute state of stress as it helps the organism to cope with changing environmental conditions and to reobtain homeostasis . Observed a decrease in oxygen saturation of muscle blood around the onset of mental stress . This is consistent with our observations by energy metabolism analysis showing decrease in aerobic glycolysis and hypoxia or ischemia of the muscles by mental stress . Widegren et al . Reported that the vascular response of skeletal muscle in individuals under mental stress is mainly a vasodilation, rather than a vasoconstriction, which is consistent with the widened space around the capillaries and increased vascular permeability observed in this paper by tem . Emotional stressors induce masseter muscle contractions [3941] that may be based on a pathway and may, in part, be mediated as follows: sensory inputs occur via the thalamus (activation of the amygdale, especially basolateral complex). Efferents to various cns systems, included muscular contraction, via ventral amygdalofugal pathway to the brainstem, specifically the trigeminal motor neurons . The effect on cranial nerve nuclei and components induced by emotional stress (cranial nerve v, masseters; cranial nerve vii, muscles of facial expression) also lead to mouth opening and jaw movements that are supposedly facial expressions of fear . There also has been theorized that compression of the blood vessels caused by muscle contraction could result in the release of local myogenic and metabolic vasodilatation factors . Therefore, adequate blood flow is usually guaranteed in the working muscles, as the sympathetic vasoconstriction is antagonized and overridden by the powerful local vasodilator action of metabolites released by the contracting muscles . The imbalance between these actions, the excessive muscle contraction / metabolites, and an insufficient vasodilation induced by the sns may create ischemia, which might be responsible for the decreased aerobic metabolism and increased vascular permeability of masseter capillaries . Clinically, these results emphasize the importance of environmental influences on the emotional state, and these influences could in turn affect the ultrastructure and energy metabolism of masticatory muscles . Whether these alterations are reversible or reducible needs to be further investigated with countermeasures, such as drugs, physiotherapy, and psychological stress - free environments . Psychological stress can cause mitochondrial injuries and hyperemia of the masticatory muscle capillaries in rats . Further, such stress can result in dramatic alterations in the energy metabolism of the masticatory muscles.
|
Asthma is a chronic inflammatory disease characterized by variable airflow obstruction and bronchial hyperreactivity associated with airway remodelling . Although this narrowing is multifactorial in origin, abnormalities of airway smooth muscle (asm) structure and function have been identified as one of the main causes . Increased asm mass has long been recognized as a major component of airway remodelling [3, 4]. More recently, asthmatic asm was also found to be abnormal in its functional properties with increasing evidence showing intrinsic heightened contractility independent of other structural cells and independent of the asthma inflammatory milieu . In this paper we will examine the evidence of asm hypercontractility in asthmatics, explore the potential mechanisms driving it, discuss its relevance, and briefly suggest its role in future asthma therapy . Abnormalities of asthmatic asm structure and morphology have been described by huber and koesser more than 90 years ago when they reported increased asm mass in a small group of patients who died of status asthmaticus compared to asm form patients who died from nonpulmonary conditions . This structural association has since been extensively described although whether asthmatic asm is also abnormal in function and if so whether this abnormality is an inherent property or only a result of the asthma inflammatory milieu has long been an unresolved question . A few in vitro studies from the 198090s have tried to address this issue but the results have largely been conflicting . Compared to nonasthmatic controls, some studies suggested increased force generation in asthmatics asm preparations; others showed no difference and even some seem to suggest decreased force generation in asthmatics [613]. Most of these studies had major methodological and statistical limitations such as small sample size, failure to measure force per cross - sectional area (stress), failure to measure asm shortening, and failure to identify ideal lengths for maximal contraction . Furthermore, none of these studies examined asm contractility at a cellular level, thus the mechanical effect of the extracellular nonmuscular connective tissue, and the biological effect of inflammatory cells and cytokines present in asm preparations, on the final results could not be determined . The first robust evidence of asm hypercontractility in asthmatics was reported by ma et al . This was the first study attempting to assess contractility characteristics of asthmatic asm at cell level . Maximum capacity and velocity of shortening of zero loaded single asm cells in response to electrical field stimulation were measured under inverted phase - contrast microscopy . Asthmatics asm cells showed significantly increased maximum capacity and velocity of shortening compared to controls . Although in this study the maximum shortening capacity in asthmatic asm cells was increased by almost a third compared to controls, it should be considered that this shortening was measured at zero load and asm cells in vivo would very likely shorten by a much lesser degree . This observation is pivotal but needs to be interpreted with caution due to the small sample size of this study . Gel percentage contraction to histamine was measured using floating gels containing asm from 8 subjects with asthma and 9 nonasthmatic controls . These asm containing gels were incubated overnight using 2 methods: attached or unattached to casting plates . The study found, using both methods, that histamine - exposed gels containing asthmatic asm contracted more significantly . More recently, sutcliffe et al . Also used gel contraction assay to assess asm contractility in a much larger sample of 19 asthmatics and 8 healthy controls . Results again showed significantly increased agonist - induced contraction in the asthma group (figure 1). Importantly, phenotypic plasticity of structural cells in culture cannot be completely excluded from studies done on primary asm cultures, but we think this, if present, was minimal . The above evidence, in our opinion, confirms that asthmatic asm is fundamentally different and hypercontractile and that this hypercontractility is a basic property and is independent from other asthma structural cells and airway inflammation, although in vivo these may play an important role in modulating the hypercontractile response . As in all muscle cells, contraction in asm is initiated by increased cytosolic calcium ions (ca) level, though, unlike most other muscle cells, the source of this ca surge in asm is mainly from intracellular sarcoplasmic reticulum (sr) stores rather than from the usual extracellular ca influx through voltage - dependent calcium channels during depolarization seen in cardiac, skeletal, and vascular muscle cells . The sequence of events leading to the contraction of an asm cell starts with the interaction of a contractile agonist with its g - protein - coupled receptors (figure 2). This results in the activation of phospholipase c (plc), which in turn leads to the formation of the inositol triphosphate (ip3) through the hydrolyzation of phosphatidylinositol bisphosphate (pip2). Ip3 then binds to its receptor on sr membrane releasing ca stores which then, through forming a complex with calmodulin, activate myosin light chain kinase (mlck) which phosphorylates regulatory myosin light chains (rmlc) forming p - mlc . Finally, this leads to the activation of actin and myosin crossbridges resulting in shortening and contraction . After initiation of contraction, cytosolic ca levels return to normal through different mechanisms including pumping out of the cell by the plasma membrane ca - atpase (pmca) and the sodium calcium exchanger (ncx), binding to cytosolic proteins, uptake by mitochondria, and also reuptake to the sr through the action of the sarco / endoplasmic reticulum ca atpase (serca). Another mechanism for this is mediated by membrane cd38 and nucleotide metabolite cyclic adp - ribose (cadpr). The ryr channels are also activated through localized elevation of ca levels (ca induced ca release). The phosphorylation of rmlc is also regulated by myosin light chain phosphatase (mlcp) which converts p - mlc back to inactive rmlc . Mlcp activity is modulated through two agonist - induced mechanisms in a process called calcium sensitization . First, it is controlled by the inhibitory action of diacylglycerol (dag), another second messenger, which also results from the hydrolyzation of pip2 . Dag activates protein kinase c (pkc) which in turn inhibits mlcp through phosphorylation . Second, mlcp is also negatively controlled by rhoa and its target rho kinase, which deactivates mlcp similarly through phosphorylation . Exploring the possible mechanisms of asthmatic asm hypercontractility is a difficult task as the evidence is less well established with relatively few human studies . Hypercontraction of asthmatic asm could be due to abnormalities in one or more of these components or steps of asm contraction model . The complexity of investigating differences in signalling or contractile proteins is that the abnormality could be in a number of levels . This could be at gene, gene expression (epigenetics), or, more commonly, at protein phosphorylation level . The most characterized potentially abnormal component of the contraction apparatus in asthmatic asm is mlck, a key regulator of asm contraction . Increased mlck levels have been reported in sensitized animal and human airways [22, 23]. As part of the same contractility study described earlier, ma et al . Measuring mlck protein was not possible due to the small cell sample (1020 cells per subject). Mlck mrna was significantly increased in asthmatics compared to a group of both allergic and nonallergic nonasthmatic controls . Examined contractile protein expression in biopsies in asthmatics with different asthma severity compared to nonasthmatic controls and also compared to patients with copd . Although -actin and myosin heavy chain isoforms (sm1, sm2) expression was similar in all groups, mlck expression was increased in all patients with asthma and copd compared to controls . Furthermore, mlck expression was significantly more in patients with severe asthma compared to all other groups . Interestingly, although p - mlc, the active product from the action of mlck, was detected only in the asthmatic groups, this was not statistically significant . The authors did admit that this negative result could be due to possible degradation of mlck during the harvesting stage of asm cells . Moreover, woodruff et al . Showed no increased gene expression of any of the contractile proteins mlck, mch, sm22, or -actin in a sample of 11 asthmatics compared to 8 controls, although this could be due to the fact that the asthmatics in this study had only mild disease . In vivo, the degree of mast cell infiltration of asm, a histopathological feature of asthma [26, 27], has been shown to be positively associated with increased -actin expression . Moreover, in vitro coculture of human asm with human lung mast cell (hlmc), or -tryptase, a serine protease released by mast cells following activation, resulted in increased -actin expression and increased asm contraction . This has been shown to be mediated through autocrine upregulation of transforming growth factor 1 (tgf-1) in asm . Histamine release from mast cells in a piece - meal fashion as demonstrated by mast cells within the asm bundle in vivo by electron microscopy and in vitro following fibroblastoid differentiation [29, 30] might exert a direct spasmogen effect upon the asm in asthma . Mast cells localized in the asmbundle express il-13, particularly in severe disease which can prime asm to a more hyper - contractile state [3133]. Abnormalities of -actin expression in asthmatic asm have only been described in this context and previous studies examining -actin expression in asthmatics were mostly negative [24, 34]. There is an increasing pool of evidence showing abnormal calcium homeostasis in asthmatic asm, thus, suggesting that abnormal calcium handling, signaling, or storage as possible underlying mechanisms for asthmatic asm hypercontractility is a plausible argument . In general, factors leading to increased cytosolic ca levels result in increased asm contraction . The expression of serca2 isoform mrna, the main isoform expressed in human asm, was reduced in patients with moderately severe asthma . Reduction of serca2 expression in healthy control asm culture by sirna resulted in phenotypic shifting to an asthmatic asm type with increased motility, secretion, and slow ca recovery . Another signalling pathway that has the potential of altering calcium homeostasis and increasing contractility is the cd38/cadpr / ryr pathway . Cd38 deficiency in animals has been shown to inhibit airway hyperresponsiveness (ahr). In human asm, tnf-, il-1, il-13, and ifn- were all found to increase cd38 expression, cadpr activity, and ca response to various natural contractile agonists [37, 38]. Moreover, in another study, highlighting the possibility that cd38 abnormalities could be a fundamental characteristic in asthma, tnf- was shown to significantly increase cd38 expression in asthmatic asm than in controls . Altered calcium homeostasis in asthmatic asm is also due to altered extracellular calcium influx through non - voltage - dependent channels . Although this has been directly implicated in altered mitochondrial biogenesis and increased asm proliferation in asthmatics, its relevance to hypercontractility remains to be investigated . Upregulation of the calcium independent rhoa / rho kinase signalling pathway leading to inhibition of mlcp would result in increased levels of p - mlc and subsequently increased asm contraction force at the same ca concentration . Abnormalities of this signalling pathway have been described in animal models of various smooth muscle disorders including hypertension, coronary artery spasm, and preterm labour . Increased levels of rhoa protein and rhoa mrna were found in airway hyperresponsive rat models although this is probably medicated through inflammatory cytokines [4143]. We emphasize that most of the abnormalities of calcium homeostasis and calcium sensitization explored in this paper were only described in single studies which have not been replicated; thus their importance remains to be fully determined . Although reactive oxygen species (ros) play an important physiological role in different cellular functions, excessive production results in the tissue damage seen in a range of chronic and acute diseases . Oxidative stress burden is increased in bronchial asthma with recent evidence identifying an increase in the generation of ros in asthmatic asm in vivo and in primary asm cultures . More importantly, nicotinamide adenine dinucleotide phosphate oxidase type 4 (nox4) expression, an important source of ros, was increased in asthmatics asm . Moreover, increased asthmatics asm contractility seen in gel contraction essay was abolished by adding nox4 inhibiters or nox4 small interfering rna . Genome - wide association studies (gwass) have identified several associations between multiple single - nucleotide polymorphisms (snps) on a number of locations and asthma . Smad3 gene, located on chromosome 15, encodes for a similarly named protein, smad3 protein . This protein is a signal transduction and transcription modulator and is activated by tgf- which has a complex role in cell growth and proliferation and is involved in airway inflammation and remodelling in asthma . As mentioned earlier, upregulation of tgf-1 observed on coculture of asm and hlmc was associated with increased -actin expression and increased contractility of asm . Another association identified by gwass was on the ormdl3 gene [44, 45]. This gene encodes for the sr membrane protein ormdl3 which is thought to have an important role in calcium homeostasis possibly through its action on serca . Overexpression of ormdl3 was found to be associated with reduced serca activity as evidenced by higher basal cytosolic ca levels, lower sr ca levels, and slower ca reuptake into the sr . Thus, based on the above evidence, polymorphism of smad3 or ormdl3 could be implicated in the hypercontractility seen in asthmatic asm through their effect on -actin expression and calcium homeostasis, respectively . Although we know that ahr is predominantly a function of asm, how much of it is driven by asm hypercontractility is a difficult and controversial question to answer . We do recognize that some of asm hypercontractility is contributed to by airway inflammation and inflammatory cytokines, but there is also good evidence to suggest that there is more to ahr than inflammation . Although corticosteroids reduce ahr, studies using agents that target specific parts of inflammation, in the form of antibodies against il-5 and ige, significantly improved inflammation but did not affect ahr [4850]. Therapies that reduce asm contraction have been shown to improve asthma symptoms, lung function, and maybe even ahr . Bt, where radiofrequency energy is used on airways to reduce asm mass, was shown to improve asthma control, quality of life, and, in one study, ahr . Further studies are required to examine whether the efficacy of bt in asthmatics is related to changes in asm mass and how this relates to changes in airway structure, physiology, and clinical expression of disease . Asthma incidence is increasing with more than half of the patients failing to achieve adequate control . Furthermore, 510% of patients have persistent symptoms despite maximal treatment with conventional anti - inflammatory and bronchodilator therapy [1, 52]. Detailed discussion of the future of asthma treatment is beyond the scope of this paper . Targeted drugs that would act on specific aspects of inflammation and contractility are the way forward . Over the last few years, a huge research effort has been on trying to identify asthma phenotypes based on inflammation, but much less was dedicated to addressing contractility . Several chemicals have been found to reduce asm gel contraction in vitro including inhibitors of phospholipase c, myosin light chain kinase, rho kinase, and nox4 and thus this has identified these enzymes as potential targets for future novel asthma treatments [16, 17]. In conclusion, we believe, based on the evidence reviewed, that asm in asthmatics is hypercontractile . Indeed evidence presented here suggests that asm hypercontractility is an intrinsic abnormality in asthma that persists in primary culture in the absence of the asthmatic environment . Improved understanding of the mechanisms driving this hypercontractility will pave the way for future treatments that will address contractility, achieve better relief of airway obstruction, and impact on asthma control and exacerbations.
|
Renal cell carcinoma (rcc) is the most common primary malignant neoplasm of the kidney and accounts for about 3% of all adult neoplasms . The incidence of rcc has been increasing during the past two decades in all age groups . The greatest increase has been in patients with localized tumors as a result of incidental detection, which is thought to be the result of more widespread use of abdominal ultrasound and computed tomography . However, about 30% of patients with rcc already have metastatic disease at the time of diagnosis, and 10% to 14% of patients experience local recurrence or distant metastasis after nephrectomy despite a finding of pathologically confined disease at the time of nephrectomy [3 - 5]. Well - known pathological and clinical variables have been identified and sometimes integrated into nomograms to better predict disease recurrence, but with only partial success . With a better understanding of the biology of rcc, laboratory values and molecular markers are increasingly being investigated as potential predictive factors for rcc [6 - 8]. Disease progression appears to depend on a complex interaction between the tumor and host inflammatory responses . It is estimated that underlying infection and inflammatory responses are linked to 15% to 20% of all deaths from cancer worldwide . The serum level of c - reactive protein (crp), a nonspecific inflammatory acute - phase protein, is frequently increased in patients with metastatic rcc and is a predictor of prognosis . However, secondary thrombocytosis has been reported with many malignant diseases; the survival of such patients has been reported to be significantly shorter . The prognostic value of the platelet count has been studied in patients with localized and metastatic rcc; patients with thrombocytosis had a poorer survival than did patients with a normal platelet count . The purpose of this study was to investigate the association of preoperative crp elevation and thrombocytosis with the prognosis of patients with non - metastatic rcc . The medical records of 177 patients with non - metastatic rcc who underwent a radical nephrectomy between march 2000 and may 2008 and for whom preoperative crp and platelet levels were available for analysis were retrospectively reviewed . There were 130 men and 43 women, with a mean age of 53.5 years (range, 28 - 83 years). Preoperatively, all patients were evaluated with a physical examination, routine hematology and biochemical analysis, and radiology studies, including abdominal computed tomography and chest x - ray . The tumors were staged by using the 2002 tnm classification of the american joint committee on cancer (ajcc) and were graded according to fuhrman's nuclear grading system . All patients were evaluated postoperatively every 3 months for the first 2 years, every 6 months for the next 2 years, and yearly thereafter . Crp elevation was defined as a crp level 0.8 mg / dl, and thrombocytosis was defined as a platelet count 380,000/l in men and 370,000/l in women according to the normal reference range in our hospital . Disease recurrence was defined as local failure in the tumor bed or regional lymph nodes or distant metastasis . The chi - square test or fisher's exact test was used to analyze the correlation between preoperative crp elevation or thrombocytosis and the clinical and pathological variables, including age, gender, anemia, histology subtype, tumor size, t stage, nuclear grade, and metastasis during follow - up . Univariate and multivariate analyses were performed by using the log - rank test or the cox proportional hazards regression model . For all tests, the clinical and pathological characteristics of the 177 patients with non - metastatic rcc are summarized in table 1 . The mean tumor size was 5.12 cm (median, 4.50; range, 1 - 22 cm). Regional lymph node dissection was performed in 24 patients who showed suspicious lymph node metastasis on computed tomography . However, none had a pathological diagnosis of lymph node metastasis . There were 38 patients (21.5%) with crp elevations and 11 patients (6.2%) with thrombocytosis . Among patients with crp elevation, the mean follow - up period was 48.3 months (median, 48.0; range, 13 - 111 months). Twenty - three patients (13.0%) developed metastases and six patients died during the follow - up period . Preoperative crp elevation was significantly correlated with anemia (p=0.001), t stage (p=0.004), grade (p=0.025), and metastasis (p<0.001), but not with age, gender, tumor histology subtype, or tumor size (table 2). Preoperative thrombocytosis was significantly correlated with anemia (p=0.003), t stage (p=0.002), and metastasis (p=0.001), but not with age, gender, tumor histology subtype, tumor size, or grade (table 3). No significant correlation was found between preoperative crp levels and platelet counts when analyzed by using pearson's correlation coefficient (r=0.132, p=0.079). Kaplan - meier recurrence - free survival curves according to crp level showed that the survival rate of patients with crp elevation (crp0.8 mg / dl) was significantly lower than that of patients with normal crp levels (p<0.001) (fig . The univariate analysis identified anemia, crp elevation, thrombocytosis, tumor histology subtype, tumor size, t stage, and grade as significant prognostic factors for recurrence - free survival, whereas the multivariate analysis showed that crp elevation (p=0.033) and tumor size (p=0.007) were independent prognostic factors (table 4). Crp is an acute phase reactant that is produced exclusively by hepatocytes in response to cytokines such as interleukin (il)-6, which are known mediators of ongoing inflammatory processes . An elevation of the serum concentration of this protein indicates the presence of acute inflammation, which is observed 8 to 12 hours after the onset of infection . The relationship between cancer and crp elevation hepatic production of crp is induced by cytokines such as il-1, tumor necrosis factor (tnf), and primarily il-6, which is frequently overproduced by the tumor cells themselves . Experimental studies on rcc cell lines and expression studies of renal surgical specimens have shown that at least some renal tumors produce il-6, which functions as an autocrine growth factor of the rcc . Therefore, the presence of a systemic inflammatory response might be associated with aggressive rcc behavior . The prognostic value of crp levels has been reported in many recent studies of cancers, including those of the esophagus, ovaries, and colon [21 - 23]. However, the mechanism by which a systemic inflammatory response affects cancer - specific survival in patients with rcc is unclear . Hwang et al studied the relationship between crp and survival in patients with rcc who underwent a radical nephrectomy . They reported a statistically insignificant difference in the survival rates of patients with and without crp elevation and concluded that crp was not a significant prognostic factor for rcc when compared with tumor stage, grade, tumor size, and cell type . However, komai et al demonstrated that the 5-year and 10-year disease - specific survival rates (75% and 30%, respectively) in patients with high crp levels were significantly worse than the rates in patients with normal crp levels (both 93%, p<0.001) and suggested that the preoperative crp level is associated with poor survival in patients with localized rcc . In addition, they reported that the crp level is a useful preoperative screening tool in patients with localized rcc . Blay et al reported that pretreatment il-6 and crp were significantly associated with the effects of cytokine therapy and high il-6 and that the crp level indicated a poor response to cytokine therapy . In the present study, preoperative crp elevation was associated with a poorer prognosis and a higher recurrence rate in patients with non - metastatic rcc . This result confirms the importance of the preoperative crp level as an independent prognostic factor for tumor recurrence and is consistent with the findings of earlier reports showing that a high crp level is associated with poor survival in localized rcc . Secondary or reactive thrombocytosis has been associated with tumor metastasis and poor survival in cancer patients . The pathophysiologic mechanism of reactive thrombocytosis may be tumor - associated elevation of circulating platelets by tumor - derived humoral factors such as il-6 and macrophage colony - stimulating factor (m - csf) that may be responsible for the development of cancer - related thrombocytosis . Il-6 is a potent stimulator of megakaryocytopoiesis, and tumor cells have been shown to release il-6 both in vitro and in vivo . These observations suggest that il-6 plays a role in the development of tumor - associated thrombocytosis . Karakiewicz et al analyzed 1828 patients with rcc by univariable, multivariable, and predictive accuracy analyses with regard to rcc - specific mortality . In that study, they showed that the addition of thrombocytosis to the base model (age, tumor size, tnm stage, ecog - performance status, fuhrman grade, and histology subtype) increased the predictive accuracy by only 0.3% (from 85.3% to 85.6%); these changes in predictive accuracy were not statistically significant . Consequently, they concluded that patients who presented with thrombocytosis did not have poorer prognosis than did their counterparts who did not exhibit these apparently unfavorable characteristics, as long as the effects of the tnm stage, histology, tumor grade, and ecog - performance status were considered . However, many investigators have reported that thrombocytosis is related to a poor prognosis in patients with rcc ., they showed that patients with thrombocytosis had a mean survival of 45.2 months compared with 76.6 months in patients without thrombocytosis (p=0.0002) and concluded that preoperative thrombocytosis is associated with a poorer survival than is a normal platelet count . In the present study, we showed that preoperative thrombocytosis was significantly correlated with anemia, t stage, and metastasis . In addition, thrombocytosis was a significant prognostic factor associated with recurrence - free survival in the univariate analysis . Therefore, thrombocytosis was associated with a poorer prognosis and a higher recurrence rate in patients with non - metastatic rcc; these findings are consistent with those of previous studies . Ito et al showed that preoperative crp levels and platelet counts had a significant correlation and suggested that this was because the reactive thrombocytosis and crp elevation in rcc were both caused by the production of inflammatory cytokines such as il-6 . As in the study by ito et al, we analyzed the relationship between preoperative crp and platelet count . However, we did not find a statistically significant association between preoperative crp and platelet count (r=0.132, p=0.079). Therefore, the exact relationship between preoperative crp and platelet count remains to be determined . A limitation of this study is that several variables could not be considered in the current analysis . Nonetheless, all available variables were included, and our findings indicate that several known prognostic factors were valuable as prognostic factors the reason for this result was, to some extent, too many patients in stage t1 rather than other t stages and a small number of patients with grade 4 compared with other grades according to the early detection of rcc nowadays . Preoperative crp elevation and thrombocytosis were associated with a poorer prognosis and a higher recurrence rate in patients with non - metastatic rcc . Moreover, however, preoperative thrombocytosis was not an independent prognostic factor for tumor recurrence and prognosis.
|
Ainsi, les inhalothrapeutes (it) devraient tre des consommateurs, des utilisateurs et des producteurs efficaces de recherche scientifique portant sur la technologie en inhalothrapie et en physiologie respiratoire . Cependant, on ne sait pas grand - chose de lavis et des attitudes des it envers la recherche . De plus le prsent article contient les rsultats dun sondage auprs des it au sujet des attitudes envers la recherche, y compris lintrt, les comptences autoperues et les obstacles . Les chercheurs ont prpar un sondage en consultation avec les it et les chercheurs en enseignement . Les sondages ont t remplis et remis de manire anonyme . Les chercheurs ont examin les statistiques descriptives et les associations . Au total, 112 it ont rempli le sondage (taux de rponse de 26,9%). La majorit des rpondants (environ 80%) ont convenu que la recherche en inhalothrapie est importante, quelle peut faire progresser la profession et que les it possdent les comptences pour faire de la recherche en inhalothrapie . Plus de 70% souhaitaient faire de la recherche, pourvu que les obstacles soient limins . Parmi huit obstacles potentiels, le manque de temps tait class comme le principal dans 59% des cas . Le niveau dducation des it sassociait de manire positive leur volont de faire de la recherche et leur conviction de possder les comptences ncessaires pour en faire . Ils auraient besoin dun appui considrable, y compris une plus grande exposition la recherche pendant leur formation en inhalothrapie et plus de temps et de soutien de la part de chercheurs forms . The st michael s hospital (toronto, ontario) research ethics board approved the study . An extensive literature search for rt - appropriate measurement surveys and opinion questionnaires was conducted . The pubmed, medline, cinahl and proquest nursing and allied health databases were searched using the terms respiratory therapist, views, attitudes, opinions, interest, experience, research and survey . The search did not find an instrument suitable for rts; therefore, a paper - based survey was developed . The survey was pilot - tested for clarity and content with a small rt subgroup from one of the participating sites . The survey underwent minor revisions based on the feedback received and results from the pilot group were excluded from the final results . False to very true. One section included a forced - rank - order question in which respondents ranked a set of eight barriers . Another section asked respondents to select the best response from a list of five statements characterizing their belief about research when training to become an rt and the type of involvement they expected to have postlicensure . The demographic section did not request specification of practice areas and, thus, the results represent rts in various roles within the academic centres . The survey was distributed to all 416 rts practicing in six university of toronto - affiliated teaching hospitals (toronto, ontario). Distribution occurred via departmental mailboxes, handouts by departmental leaders or managers, or by leaving the survey in a common area used frequently by rts . An initial contact e - mail message accompanied the survey distribution as a formal invitation and explanation of the project . The survey broadly operationalized facets of rt research capacity and motivation using several question and response formats . The analysis was exploratory - descriptive, did not have classical test theory measurement objectives, and did not analyze validity and reliability of constructs . Responses were analyzed using spss (ibm corporation, usa) in consultation with a statistician . Descriptive statistics and frequency tables were produced and associations were examined using cross - tabulations and the statistic . The kruskal - wallis anova was used to test equality of group locations . Subgroup analyses were performed on educational attainment and years of work experience as an rt using the least significant difference post hoc test . The four education categories were: diploma; diploma plus advanced training (diploma+); diploma plus bachelor s degree; and master s degree . The five work experience categories were 0 to 5, 6 to 10, 11 to 15, 16 to 20 and> 20 years . All (100%) respondents either strongly agreed (84.8%) or somewhat agreed (15.2%) with the statement research is important (figure 1). The majority of the respondents either strongly agreed (78.6%) or somewhat agreed (20.5%) that respiratory therapy research is important. Research plays an important role in advancing respiratory therapy as a profession was answered strongly agree by 56.8%, somewhat agree by 39.6%, and neither agree nor disagree by 3.6% . For the statement, research plays an important role in my day - to - day practice as an rt, the responses were 33.9% strongly agree, 45.5% somewhat agree, 17.0% neither agree nor disagree, and 3.6% somewhat disagree . Three statements were used to identify rts opinions as to who they believe is best suited to conduct respiratory therapy research (figure 2). The statement rts are best suited to research respiratory therapy - related topics was answered as strongly agree by 50.9% and somewhat agree by 39.3% . For rns are best suited to investigate respiratory therapy - related topics, 52.7% strongly disagreed, 31.3% somewhat disagreed, and 12.5% neither agreed nor disagreed . The third statement was, md and other scientists are best suited to investigate respiratory therapy - related topics. These responses had some variability: 4.5% strongly agreed, 20.5% somewhat agreed, 25.0% neither agreed nor disagreed, 44.6% somewhat disagreed and 5.4% strongly disagreed . To differentiate between who should perform respiratory therapy research and how rts feel about their day - to - day practice, the statement, i trust in the staff mds to keep respiratory care practices in the icu up - to - date and current was used . Strongly agree was selected by 9.8%, somewhat agree by 42.0%, neither agree nor disagree by 17.9%, somewhat disagree by 21.4% and strongly disagree by 8.9% (table 1). The survey asked how rts view their colleagues relationship to research (table 1). The five - point response scale ranged from false to very true . The statement, there is a general lack of interest by the department s rts to do research, was believed to be false by 1.8%, somewhat false by 18.8%, somewhat true by 46.4% and very true by 7.1%; approximately 25.9% were unsure . For the item, respiratory therapy research would not be valued by anyone else other than rts themselves, 18.9% of respondents believed this statement was false and 36.0% somewhat false . Only 15.3% believed it was somewhat true and 0.9% very true; approximately 28.8% did not know . Lack of time was ranked as the most significant barrier by the largest proportion (59.0%). Each barrier s mean rank was calculated and barriers were sorted in ascending numerical order according to mean (ie, decreasing order of barrier relevance). The top three most significant barriers were lack of time (mean [m]=1.9), lack of incentive (m=4.0) and lack of skill (m = 4.1). Lack of interest was the least significant barrier (ie, it had the largest mean [m=5.8]). A large majority (71.8%) said that if barriers were eliminated they would be interested in pursuing a respiratory therapy research project and 17.3% said maybe (table 2). Cross - tabulations and the statistic were used to identify demographic characteristics associated with rt willingness and self - perceived ability to conduct research . Willingness was assessed by the statement if i had dedicated time away from clinical responsibilities, i would be willing to work on a research project. The five - point response scale ranged from strongly disagree = 1 to strongly agree = 5 . Educational attainment and willingness to work on a research project rts with more education were more willing to work on a research project (p=0.003) (figure 3). Rts with a bachelor s degree had a higher mean (m=4.3) than diploma respondents (m=3.5) and the difference was statistically significant (p=0.001). Rts with a master s degree were more willing to conduct research (m=5.0) than those with a diploma (m=3.5; p=0.001). The belief in having research skills (i have skills required to do research) was also associated with educational attainment (p=0.006). Rts with master s and bachelor s degrees were more likely to believe they had research skills than those with a diploma (p=0.006) (figure 4). Willingness to learn skills required to do research (i want to learn the skills required to do research) was associated with years of experience as an rt (pearson; p=0.048; kruskal - wallis; p=0.01). Respondents with 11 to 15 years were more willing to learn than others (m=4.4; 0 to five m=4.0; six to 10 m=3.9; 16 to 20 m=4.2;> 20 m=3.3). I want to be left to do my job, fulfill my clinical responsibilities and nothing else, those with six to 10 (m=1.8) and 11 to 15 (m=1.8) years of experience expressed more disagreement (pearson; p<0.001; kruskal - wallis; p=0.03) than those with 0 to five (m=2.0), 16 to 20 (m=2.4) and> 20 (m=2.9) years of experience . Rts were asked to share their expectations pertaining to research when they were training to become an rt by selecting one of five qualitative response options (figure 5). Research could be a part of the job if you were interested was selected by 47.6% of respondents . Research was not part of the job and not discussed was selected by 27.6% . Research was discussed as something people in other professions did was selected by 18.1% . Research would be encouraged was selected by 3.8%, and research would be an expected part of our job and duty was selected by 2.9% . The associations between these expectations and educational attainment (p=0.93) and years of experience (p=0.13) were not statistically significant . What was once a job that involved hauling tanks and titrating oxygen is now a skilled career in which professionals manage life support devices for critically ill patients . Rts now create recommendations for respiratory care plans and are valued members of the multidisciplinary team (46). They are bedside specialists who combine a technological understanding of machinery with an advanced knowledge of respiratory physiology and particular training to assess this interaction (46). It appears consistent that the group best suited to critically question and analyze respiratory therapy practice is the very group of people responsible for respiratory interventions . Without continuous critical assessment through research however, few rts have time to perform the research that establishes ebp (17). Although many rts are competent clinicians in academic hospitals, research barriers are numerous . By participating in the present study, rts in several academic hospital roles the most relevant result is that rts believe that research is generally important and that respiratory therapy research is particularly important . Rts also believe that research is necessary in their day - to - day practice and for advancing the profession . However, the level of agreement decreased the more specific the survey statements became about research in relation to respiratory therapy . Research is important, while 78.6% strongly agreed that respiratory therapy research is important. It is unclear why the strength of agreement decreased when respiratory therapy research was specified, or why rts believe that respiratory therapy - related research is slightly less important . The level of agreement continues to decrease with each successive statement and suggests that there is a sense of uncertainty within the profession regarding the association between respiratory therapy and research . The reason for the progressive change in response is speculative without further investigation . Despite this observation had the opposite been true, a starting approach to respiratory therapy research would require a shift in motivation . Consequently, academic hospital leaders should be aware that rts values with respect to research are similar to the organization s values . Academic hospitals should support rts who want to pursue research that is in accord with the institution s mission . Rts often facilitate other investigators research . However, to develop independent research capacity, rts must display professional responsibility toward research within their area of practice (25,12,14,16,19,25). Rts expressed a sense of ownership over respiratory therapy research; they believed that rts are best suited to conduct respiratory therapy research . Rts do not believe that registered nurses (rns) are best equipped to investigate respiratory therapy topics . They disagreed with the statement about rns with the same frequency that they agreed with rts suitability . In addition to the small percentage of respondents that disagreed with the statement that rts are best suited to investigate respiratory therapy topics, there was a small percentage that believed that rns are best suited to perform respiratory therapy research . Few rts have master s degrees and some respondents may perceive rts to be academically unprepared for research . The role rts believe rns should or should not have in respiratory therapy research requires further investigation to fully comprehend the ensuing dynamic it may also relate to how comparatively new respiratory therapy is as a discipline that has not secured as many academic avenues and research supports as nursing . Although many rts believed that physicians are best suited for respiratory therapy research, a near majority disagreed . This result further exemplifies rts sense of professional responsibility and it provides a small glimpse of how health care is changing . With the evolution of specialties, such as respiratory therapy, these respondents believe that medical doctors (mds) or scientists, despite having the skill to conduct research, are not necessarily best suited to research respiratory therapy topics . They work with the intricacies of the processes and procedures of patient care within their daily practice . They have clearly identified themselves as respiratory specialists and the candidates best suited to generate questions and add to the information base in this area of expertise . Although rts believe that they themselves should be asking questions and contributing to research, their views do not suggest a strong desire to perform it all themselves . Moreover, it does not imply that mds and other scientists should not research respiratory therapy topics . When asked whether they trust the staff mds to keep respiratory care practices in the intensive care unit [icu] up to date and current the majority of rts agreed with the statement . Clearly, these respondents believe that mds still play an important role in the process of establishing and implementing practice guidelines . At first glance, the responses to the statements about mds conducting research on respiratory therapy topics (mds are not best suited for it) and mds updating intensive respiratory care practices (they are trusted to do so) appeared to be contradictory . However, this indecisiveness as to what role rts believe the mds should have in respiratory practice is more likely a reflection of the professional responsibility that rts carry . As illustrated by the responses, there is a demarcation on what rts believe the role of the rt should be as it pertains to research . They have distinguished themselves as having a role in generating knowledge more so than implementing practices . We found that rts do have a sense of self - worth in relation to research . However, they also expressed apathy . One - half of the respondents perceived a general lack of interest among their department colleagues to conduct research . Although this is a rather negative opinion about their colleagues motivation for research, the perception may not match reality because, for example, lack of interest in research was self - ranked as the least significant barrier to research . Furthermore, nearly three - quarters said that they would be interested in conducting a research project related to respiratory therapy if barriers were eliminated . Approximately one - quarter of rts did not know whether their colleagues lacked interest in research and did not know whether respiratory therapy research would be valued by non - rts . These figures are reasons for concern because they suggest that some rts are substantially unaware of local norms in relation to research . We are concerned by the results regarding rts beliefs about research while training to become rts . Very few respondents reported impressions that research was encouraged or that it was an expected part of the job . For example, new rts and those with 20 years of experience shared the same belief about research when training to become an rt . Similarly, regardless of educational attainment, research exposure, encouragement and support during training were uncommon . Three main barriers to conducting research that rts encounter in standard practice were identified: lack of time, lack of incentives and lack of skill . Rts are busy clinicians and the foundation of the profession remains at the bedside (17). For this reason, rts need to have time available for research whether it be dedicated hours away from bedside, higher rt - patient ratios or a supported research program . Other incentives, such as flexibility in hours and scheduling, could be alternatives to monetary offerings . Lack of skill is a factor that employers, professional organizations and academic hospitals could jointly remediate by making resources available to rts who do not have formal training necessary to conduct their own research . The study identified two associations with educational attainment: first, a willingness to work on a research project; and, second, a belief in having the skills necessary for research . Respondents with more education were more willing to conduct research and assert that they have the necessary skills . Willingness to learn the skills required to conduct research was greatest in the 11 to 15 years experience range . Groups with moderate experience (eg, six to 15 years) were more willing to pursue activities in addition to clinical practice . It appears that these rts have become proficient in their bedside responsibilities and wish to have a greater impact on the profession in which they have become so adept . Less experienced rts were not as willing to conduct disciplinary research and are perhaps focused on developing their clinical bedside skills . The most experienced rts (> 16 years) were the least willing to learn research skills . Most clinicians in this group also do not want to take on additional duties outside of their clinical responsibilities . It is possible that this group lacks confidence to work in novel areas . For departments that want an active respiratory therapy research program, it may be helpful to recruit rts with higher educational attainment and moderate levels of work experience . The obtained sample size was low and there may have been a response bias favouring research . Most respondents held a bachelor s degree, and the survey should be fielded at community hospitals to assess the generalizability of our results . The items in some scale sections should be analyzed with statistical methods to judge the data s validity and reliability with a larger sample size and when formal measurement constructs are developed . The broadest challenges to respiratory therapy research are the lack of systematic exposure to research during respiratory therapy training, as well as within colleague networks and the work environments . If rts intend to conduct research, respiratory therapy curricula should be modified to convey research as a valuable activity . This will help foster a culture of inquiry through which new graduates, regardless of whether from an academic centre, can enter the profession with a view on research as an expectation rather than simply an afterthought . Administrative leaders of academic centres should acknowledge that their bedside clinicians share similar organizational values . With this knowledge, resources can be allocated to eliminate barriers and support the willingness and urgency of rts to conduct research . Rts could then perform at a level that is expected of them and which they desire.
|
When our hominid species evolved from several millions of years ago, ancient man was a hunter - gatherer, and survival required covering long distances . As well as stamina, homosapiens had to have sufficient strength to kill large animals for food . Ancient man would have sustained muscle injuries during hunting and tribal confrontations, and, from a darwinian viewpoint, natural selection would have resulted in generations of offspring with strong and adaptable musculature; this includes rapid and effective tissue repair as this was also a requisite for survival and the continuation of the species . However, over most of this time the average life expectancy for most homosapiens was only about 25 years, that is to say a little beyond the age of reproduction . For example, in ancient egypt the average life span was 24 years but now with developments in science and medicine this has increased by over 3-fold which presents problems for human society . In the more affluence society of today there are other factors such as overconsumption of food and alcohol and the failure to maintain an active, healthy life style . In scandinavian countries family doctors prescribe exercise to improve the general fitness which enables individuals to maintain an active life style and to live longer . Longevity and the increasing percentage of elderly in the populations in many developed countries including the usa, europe, and japan present its own major socioeconomic as well as medical care problems . Therefore maintaining independence has now to be very much focused on the aging processes of the musculoskeletal system . Mechanical tissues are designed to respond to mechanical forces, and it is important to determine why there is a decreasing sensitivity of the transduction of mechanical signals that maintain muscles and to what extent this is due to inactivity or intrinsic tissue changes as we get older . These are not simple questions to answer as such factors as neurological input, blood flow, and fatigue resistance that include tissues other than muscle may become limiting factors . From the prospective of the author the information in this paper concentrates on that acquired over the last decade on changes at the cellular and molecular levels in aging muscle tissue as present day molecular genetics and proteomics methods have provided us with tools for studying the age - related muscle growth, adaptation, and repair . Sarcopenia is the term that is often used to describe the syndrome of age - related muscle loss which is somewhat unfortunate as this implies that it is a disease rather than an attenuation of processes that develop and maintain muscle in young healthy people . Postnatal growth of muscle is very much influenced by hormones which include growth factors and androgens, the circulating levels of which decrease with age . This decrease in hormone levels in the elderly has sometimes been referred to as the somatopause as this occurs in males and females . Supplementing the levels of these hormones has been found to be beneficial, for example, oestrogen and progesterone replacement therapy in women and administration of testosterone in elderly men to improve muscle strength . The insulin - like growth factor (igf - i) system is beginning to receive considerable attention as it is involved in tissue growth, maintenance, and repair . Interestingly, an igf gene is present in invertebrate animals . This and its receptor gene have been studied in the nematode worm c. elegans as it is involved in determining the life span of the worm by suppressing cell death (apoptosis). Experiments have shown that the igf gene and its receptor gene represent a primitive system involved in maintaining terminally differentiated cells . In this way these determine lifespan in the nematode worm and have become a model for studying aging at the very basic level . The lifespan of vertebrates including man is of course much longer than the nematode worm . In higher animals the igf - i system is similar but more sophisticated in that the family of genes and the alternate splicing of genes in vertebrates result in a number of gene products . In vertebrates during aging, muscles decline in strength and adaptability . These are controlled by growth hormone (gh) produced by the pituitary gland and referred to as the gh / igf - i axis which controls body mass particularly muscle mass . However, space does not permit a discussion of the combined effects of growth hormone and androgens on muscle during aging although this topic is still receiving much attention and not reviewed here except where they might be involved in gh / igf - i axis . However, this paper will concentrate mainly on the adaptation mechanism(s) that are linked to the repair processes via which myofibers adapt and/or are repaired after being subjected to mechanical strain . As skeletal muscle postmitotic tissue and cell replacement is not a means of repairing damage as it is in most tissues and there has to be a somewhat different but effective mechanism, otherwise the cellular units would undergo cell death and not be replaced . This does happen to some extent in advanced old age when the maintenance and reinnervation of the muscle fibers begins to fail . In normal muscle events associated with local damage and repair result in adaptation whereby the muscles increase in strength but this decreases with age . In diseases such as the muscular dystrophies muscle repair is not initiated in the normal way as certain cytoskeletal assemblies are defective or missing as in duchenne muscular dystrophy that is the most severe type . In the mdx dystrophic mouse that is an animal model for human duchenne muscular dystrophy, the muscles are unable to respond to mechanical stimuli by initiating the repair signalling including splicing the igf - i gene towards the igf - iec . Because of the original confusion of what was called igf - ieb which has almost the same sequence of igf - iec in the human and because its expression increased in response to mechanical signals, the latter was called mechano growth factor (mgf). In experiments in which the dystrophin complex has been restored by cell transfer, the appropriate signalling including the production of mgf is restored . During normal ageing this mechanotransduction system apparently becomes increasingly less sensitive as the fibers become less compliance due to increased amount and stiffness of the fibrous connective tissue in the muscles . In addition to muscle stiffness, another factor is the drop in circulating levels of some hormones which influence the expression of the genes that result in the local signals that are produced in response to physical activity . Studies of cellular changes associated with age - related muscle atrophy indicated that there is a considerable loss of muscle fibers, motoneurons, and motor units . In a more recent project studied in older men aged just over 70, with a follow - up study on the cohort when they approached 80 years of age, the muscle cross - sectional area and specific strength of the knee extensors had significant decreased . Interestingly, there was some increase in the size of the type iia fibers indicating that the recruitment pattern of the myofiber types changed, and this tended to compensate for the loss of fatigue resistance and strength . This paper will, however, concentrate on molecular biology and proteomic studies . By using these approaches we can begin to understand the atrophy processes associated with aging and the possibility of intervening in these processes of loss of muscle mass and strength, as we age . One of the changes during aging is that the circulating levels of gh drop markedly with age and one approach to counteract the effects of aging might have been to administer human gh as a recombinant peptide . In experiments with the author's group in collaboration with professor kjaer's group in copenhagen, it was found that strength improved with the administration of growth hormone which apparently increases the level of the primary igf - i transcript and there was a correlation between increased mgf levels and muscle mass measured by mri scanning [9, 10]. More recently our group has been involved in detecting the misuse of gh by athletes but it should be appreciated that young healthy individuals are not gh deficient . Therefore, the response is likely to be different to that in older muscle tissue, particularly in those individuals in whom the circulating levels are well below that of their age group . A study by yamaguchi et al . Showed that in hypophysectomized rat muscle the expression of igf - i splice variants was more influenced by mechanical factors than endocrine status . This reaffirmed a earlier study carried out a good number of years ago by goldberg who used hypophysectomized rats and showed when muscles are under mechanical strain they still underwent hypertrophy indicating that regulation of muscle mass and strength are regulated at the muscle cellular level . Systemic administration has associated problems as gh is produced episodically, and there is a negative feedback so that administration of a large bolus of recombinant human growth hormone (rhgh) shuts down its endogenous secretion by the pituitary which produces the opposite result . Also elevated gh levels are associated with an increased in igf - i and increased cancer risk as igf - i is carcinogenic at abnormally high levels although it has been reported as repressing protein breakdown . A higher risk of cancer is seen in acromegalic patients whose pituitary gland is overactive . These people are invariably tall and well muscled but have a high risk of cancer resulting from the higher circulating levels igf-1 . It is now becoming apparent that this may be a problem in young athletes and bodybuilders and the antidoping agencies are becoming concerned about this form of abuse . Hence there are considerable risks associated with elevating the serum gh levels beyond that which is normal for a particular age group artificial elevation of gh or igf . The levels of several hormones are known to drop markedly with age including testosterone as well as gh that are involved in the maintenance of muscle mass . It is not likely that these would be used as a general treatment for age - related muscle loss although it may be indicated for some elderly individuals but this has to be combined with exercise to obtain an increase in strength . As the endocrine system is complex and the chances of inducing unwanted side effects are reasons why the pharmaceutical industry is attempting to develop sarms, these compounds stimulate specific androgen receptors, in particular the testosterone receptor, and already they have found their way into doping in certain sporting events . The size of tissues in the body is determined mainly by a balance between the rate at which proteins are synthesised and the rate they are broken down . This is a way cells adapt to change in local environmental conditions and for removing nonfunctional, damaged proteins . Every few days approximately half the enzyme molecules within our body will be broken down and replaced . The turnover of the contractile proteins of muscle such as actin and myosin takes a little longer with about half of being replaced every two weeks or so . Using nonradioactive isotope methods it was found that as we get older our muscle proteins are degraded and rebuilt at less frequent intervals but still the process is very dynamic . Protein degradation requires energy as does the synthesis of muscle proteins, and it appears that the balance is more towards decreased protein synthesis except in some disease states, for example, endotoxin poisoning, in which muscle protein breakdown can be very rapid . Any proposal for increasing muscle mass during aging by slowing the degradation process during healthy aging would seem to be physiologically undesirable as continual replacement of proteins is particularly important in a mechanical tissue as this is the way of ensuring there is no build - up of nonfunctional proteins . Also in tissues such as muscle there would probably be a reduction in specific strength as increased mass does not necessarily mean an increased ability to generate muscle force, and there is no rationale for increasing body mass without commensurate increases in strength . There has been a lot of interest in a negative muscle regulatory factor that has been named myostatin . As the name implies this factor reduces muscle growth, and knocking this out therapeutically offered prospects of increasing muscle mass and strength . Double muscling in certain breeds of cattle, such as the belgian blue, which exhibit very considerable muscle hypertrophy and also in a few human subjects in which the gene is mutated . However, mcmahon et al . Reported that in the myostatin knockout mouse, the lack of myostatin did not ameliorate disuse atrophy . As discussed below skeletal muscle is a postmitotic tissue and in order to increase in size or to repair the myofibers have to obtain extra nuclei . Knock out with an increased number of satellite cells, and an increase in muscle mass reverses the quiescence state of these muscle progenitor cells . However, it was shown in these mice that the increased muscle mass resulted in decreased specific contractile force as there was apparently an accumulation of nonfunctional protein . The suggested therapy for muscle loss that entails partially knocking out / knocking down myostatin is also probably not a good strategy as this is associated with a loss of oxidative capacity of the muscles as well as impaired respiratory and cardiovascular function . In one young boy who has a myostatin double allele knockout, he has increased muscle mass and strength and this would obviously be an advantage in certain sports, but to be muscle bound is not necessarily an advantage in many activities that require power rather than maximum force nor would it be desirable for older people . Interestingly, a study on aging weight lifters did show that myostatin levels decrease when they resume exercise so this subject needs further investigation . Clearly if the use of an antimyostatin strategy results in an increase of mass without a commensurate increase in strength this would handicap rather than help elderly individuals . Even in individuals who exercise regularly, the ability to maintain muscle mass and strength diminishes with age, and this is associated with the marked drop in the circulating serum levels of igf - i . Muscle hypertrophy and wasting have been studied at the gene level based on the realisation that there must be local as well as systemic regulators of muscle growth . Approximately 15 years ago the author's group cloned a factor that proved to be involved in local regulation of muscle mass and which is derived from the splicing of the igf - i gene . To do this we used an animal model previous work had shown that the tibialis anterior in the mature rabbit whilst held in the stretched position by a plaster cast and when electrically stimulated using an implanted microcircuit increased in mass by 35% in just over 7 days . It was known that muscles adapt to an increased functional length by adding sarcomeres in series at the ends of the existing myofibrils, and if they are also subjected to electrical stimulation they also increased in girth and also added more sarcomeres in parallel as well as in series . Rna was extracted from these muscles that were undergoing rapid growth and using differential display, and we detected a rna transcript that is expressed in stretched / exercised but not in resting muscles . This mrna was converted to cdna, sequenced, and referred to the genome data base which showed it to be derived from the insulin - like growth factor (igf - i) gene . However, its 3 sequence was different to the liver or systemic type of igf - i (igf - iea). Later work showed that in human muscle 3 main types of igf - i can be spliced from the igf - i gene (figure 1), a systemic (liver) type of igf - i (igf - iea), igf - i eb, that is not the same as igf - ieb in rodents and the newly discovered splice variant igf - iec . As the terminology of the igf - is is a problem when attempting to apply it to nonhepatic tissues and different species we called this newly discovered splice variant mechano growth factor (mgf) as it is expressed in response to mechanical stimuli and muscle damage . As well as mgf expression was mechano sensitive its 3 sequence was unique and encoded a different e domain at the carboxy end (figure 1) that presumably has several distinct actions . The reason the c terminal end of mgf is different to the other igf - i splice variants is because during the splicing of the igf - i gene at exon 5 has 52 bases in the human (49 bases in rabbit and rat) and this introduces a reading frame shift at the 3 end . Following the initial splicing to mgf (human igf - iec) which has been found to initiate muscle growth and repair, the igf - i gene is later switched to igf - iea which is a main anabolic agent and some studies suggest that it initiates the fusion of satellite cells with the myofibers and the expression of myogenic genes [2224]. Mgf, however, appears to have a special role in muscle repair that involves its unique e domain sequence that arises from this reading frame shift . As well as activating and replenishing the muscle stem / precursor cell pool (see figure 1) this unique e domain peptide has also been found to have several roles in limiting tissue damage but space does not permit these to be reviewed here . There is good evidence that the age - related changes in muscle mass and strength result from a decreasing ability to produce mgf [2628], and this seems likely to be the case in other tissues . From a physiological point of view this is interesting as the age - related decline in the ability to respond to physical signals by producing mgf is presumably related to a dulling of the mechanotransduction system which is still not understood . As shown in the rat (figure 2) and human subjects a major intrinsic effect is increased load, but this response becomes dulled during aging . Also in studies on elderly human subjects this was found to be related to decreased mgf and igf-1ea which could be improved by administration of gh which increases the expression of the igf - i gene . We understand more about the endocrine aspects than the physical signalling and signalling molecules involved in muscle adaptation involved in maintaining and repairing our muscles as we age . The detection of mechanical strain is thought to involve focal adhesion kinases (faks) which are activated by mechanotransducers systems that link the very long titin molecules that run through the myofibrils to the tendons . These also connect in 3 dimensions as the myofibrillar system is linked to the surrounding basal lamina and response elements, and when these are missing, for example, dystrophin or defective is responsible for the muscle wasting conditions . The connective tissue of the tendons and ligaments also transmits the forces generated by sarcomeres of the muscles to tendons, bones, and ligaments . These mechanical forces generated by skeletal musculature are thus also involved in maintaining the whole of the musculoskeletal system . It seems that as we grow older this mechanotransduction system becomes less sensitive because of the decreased compliance of the connective tissue due to cross - linking of the collagen which seems to occur in all tissues . In some experiments on mice subjected to repeated exercise the author's group found that regular exercise improved muscle compliance during aging but it was still not as good as in young mouse muscles . These experiments preceded the discovery of mgf which now offers good prospects for its use as a therapeutic compound for treating age - related muscle loss as well as muscle cachexia in a range of diseases although there seems to be more interest in its use as a doping agent as it is available over the internet and it is now being produced using recombinant e. coli methods, and therefore it will become relatively inexpensive . Unfortunately, during aging the muscles become less compliant and become less able to produce mgf [2628, 31]. As with other tissues muscles become less compliant which apparently occurs due to cross - linking in the connective tissues and possibly other factors, and in some long running mouse experiments we showed that regular exercise improved muscle compliance during aging but it was still not as good as in young mouse muscles . Also in diseases such as the muscular dystrophies, the impairment is often the absence or incorrect conformation of the certain linking molecules such as dystrophin that apparently results from an inability to produce mgf . From a physiological point of view this is of considerable interest as the initiation of the activation of the igf - i gene and the switch in splicing to produce mgf must involve a mechanotransduction system . The detection of mechanical strain is thought to involve focal adhesion kinases (faks) which change conformation when stretched which phosphorylate other signal molecules . The impairment of mechanosignalling appears to be defective in diseases such as the muscular dystrophies in which splicing of the igf - i gene is deficient but transfer of normal mesenchymal stem cells into the dystrophic mouse restored its ability to produce mgf . Therefore, there are probably good prospects for use of therapeutic compounds such as mgf for treating age - related muscle loss as well as muscle cachexia in a range of diseases . Unfortunately, at the present there seems to be more interest in its use as a doping agent as it is available over the internet and it is now being produced using recombinant e. coli methods, and therefore it can become relatively inexpensive . Skeletal muscle and most neurological tissues are postmitotic tissues, and age - related deficits require a somewhat different understanding to that of other tissues . The term postmitotic tissue is used as after embryonic tissue is complete, the myofibers have residual myoblasts in the space between the plasma membrane and the basal lamina . These upon the appropriate signal undergo proliferation and one of the progeny fuses with the muscle fibres, and the other mononucleated muscle stem cell enters quiescence . It has been realized for some time that these cells provide the extra nuclei for postnatal growth and regeneration and in response to mechanical strain and local injury [3238]. Growing up mononucleated myoblasts for injection into muscles of patients has been the goal for several groups but as most of these die after injection, this does not seem preferable to introducing mgf as a stabilized peptide or gene construct as one injection provides many additional myofiber nuclei . The initial splicing of the igf - i gene to produce mgf is within the first few days after the mechanical challenge and coincides with the activation of the muscle stem cell pool [3638]. Indeed, the initiation of muscle stem cells resulting from exercise has been shown in human muscle after a single bout . Bamman's group found that after resistance exercise there was a correlation between cyclin d1 activity with mgf expression demonstrating cell replication in this postmitotic tissue . In another study it was shown that a decrease in myostatin levels results in an increase in specific strength so it is assumed that muscle satellite (stem) cell activation is positively regulated by mgf and not by removal of myostatin, the negative regulator . Following the expression of the igf - i gene the satellite cell pool undergoes periods of replenishment lasting just a few days, and the initial splicing to mgf expression following a mechanical challenge fits with the timing of the expansion of the muscle stem (satellite) numbers [30, 39]. Both mgf and igf - iea are apparently involved during muscle hypertrophy and repair as this involves replenishing the muscle satellite (stem) cell pool which kick starts the growth, and repair processes the anabolic response to igf - i ea . Stem cell therapy has been proposed for extending the life and the quality of life but in the case of muscle loss and frailty and in muscle cachexia in a range of diseases . However, transplanting muscle cells would not seem to be necessary if the existing stem cells can be induced to multiply by merely administering mgf as a stabilised peptide or by gene therapy . It has been shown that age - related sarcopenia is related to igf - i signaling [34, 35] and in particular reduced ability of muscle to express mgf, a splice variant of the igf - i gene and also igf - iea which is the main anabolic agent or increased muscle protein content [34, 36]. This has been associated with muscle aging in animals [22, 26] and humans [27, 28], and because of this, the need to activate the muscle stem / progenitor cells to increase muscle mass has been highlighted in a number of publications [3541]. For this purpose the unique e peptide sequence of mgf (mgf-24aa - e) was used to study the activation of the human satellite cells (mononucleated myoblasts) taken from patients with muscle wasting diseases including two muscular dystrophies and als . If igf - i was added together with the mgf peptide then it became apparent that the mononucleated cells went through the next stage of activation and fused with the muscle myofibers in the cultures . Therefore, it seems that both the unique mgf e domain peptide and igf - i are required for the repair process . Gillian butler - browne's group shed more light on the repair process in relation to aging . With this group we investigated the actions of the mgf e-24aa peptide using differentiated cultures and analyzed to estimate (1) the fusion index, (2) the percentage of unfused (desmin positive) reserve cells, and (3) the mean number of nuclei per myotube . A fusion index was determined by counting the number of nuclei in the differentiated myotubes with more than two myonuclei as a percentage of the total number of nuclei (mononucleated and multinucleated). The percentage of unfused positive cells was calculated, by counting the number of unfused, desmin - positive - single - nucleus cells as a percentage of the total number of nuclei (1000 nuclei per dish in triplicate). To measure the extent of cellular proliferation within a culture, the incorporation of 5-bromo-2-deoxyuridine (brdu) was used . During the replicative life span, proliferating cells were incubated for 72 h in the presence of 10 g / ml of brdu . To identify the cells that had incorporated brdu, using a monoclonal antibody directed against brdu, and the nuclei were counterstained with dapi . It was found that this mgf peptide significantly increases the proliferative life span of satellite cells isolated from neonatal and young adult but not from old adult muscle . However, it was noted that the mean number of nuclei per myotube and the fusion index were higher in cultures from older subjects . This was associated with a significant decrease in the percentage of reserve cells correlated with an increase in the number of nuclei in the myotubes that occurred when mgf-24aa - e peptide was added to cultures . It is concluded that only 24aa of the mgf isoform of igf-1 has a marked ability to initiate and enhance satellite (progenitor) cell replication and fusion for muscle repair and maintenance . Aging muscle has a declining ability to produce mgf but it was found that administration of the mgf peptide prevents satellite cells entering senescent and by activating these reserve progenitor cells to fuse, and this provides the extra nuclei required for continued tissue maintenance . Unfortunately as mentioned above as we become older our ability to produce mgf declines along with gh and igf - i . Although the older myoblasts cultures showed a dramatic decrease in the number of cycling cells following the addition of mgf-24aa - e, their ability to induce hypertrophy was assessed following a single dose of mgf-24aa - e which was given immediately or just after 3 days of establishing which resulted in the cultures developing myofibers and producing additional myosin heavy chain protein . Also it appears that mgf-24aa - e significantly delays the entry of cells into senescence and increases moderately the proliferative life span of satellite cells isolated from neonatal and young but not from old adult skeletal muscle . These experiments allow the distinction between two specific roles of mgf e-24aa peptide and its effects on proliferation and differentiation that are required for the maintenance and then repair of the musculature throughout life (see figures 4 and 5). It has been known for some time that during aging there is a marked decline in muscle mass and strength which can be only partly ameliorated by continuing with resistance type of training . Exercise is to be encouraged as it has health benefits and a feeling of well - being during the aging process . However, in old age many individuals will become partially or only partially immobile . In the aging population day- to - day care will increase as a percentage of the elderly, and this will result in an ever - increasing financial commitment particularly in the advanced nations . For this and other reasons the large pharmaceutical companies have recently taken a serious interest in possible ways to increase muscle mass and strength in the elderly and in the many disease states that are accompanied by muscle loss . Much of their recent research is kept confidential for obvious reasons but can be appreciated somewhat from filed patents some of which had been filed by academic institutions before the present financial crisis . However, it seems that in the reasonably near future, when we understand better the signaling and the role of igf - i growth factors, it should be possible to strengthen the musculature in the infirm so they can continue to carry out routine physical tasks . It has been known for some time that gh levels markedly decrease during aging and gh supplementation increases muscle mass in the elderly . Serum igf - i levels also decrease during aging but administration of igf - i is problematic because of its insulin - like effect on blood sugar levels, and the main consensus in a point: counterpoint discussion was that igf is not a major regulator of muscle mass . Over the past decade or so, studies have demonstrated alternative splicing events occur in the igf-1 gene in skeletal muscle in response to mechanical strain . This splicing results in the production of different 3 mrna and a different terminal amino acid sequence in main splice variants and isoforms . Initially there was confusion as the splice variants previously named igf - ieb in the rat but igf - iec in the human are essentially the same although shift in the reading frame shift occurs at a slightly different place and the c terminal peptides (e domain) have just a few amino acids difference . In animals and humans because of this confusion the igf - i splice variants were named mechano growth factor (mgf) as the splicing of the igf - i gene responses to mechanical signals [2128]. Experiments using a synthetic peptide of just 24 amino acids increased murine myoblast c2c12 cell cultures, and using myoblasts transfected with mgf cdna showed proliferation in these mouse muscle cell cultures . Some groups found the experiments with the e peptide difficult to repeat as unfortunately they used commercially available murine c2c12 cells which had been grown up so many times and very few, if any of these transformed and mutated cells behaved as muscle progenitor cells . Cloned the few progenitor cells by separating them from the transformed cells that continually multiplied whether or not they were exposed to mgf or igf - i . There was a lesson to be learnt from this, and the author's group concentrated on using human muscle mononucleated myoblasts obtained by biopsies from patients with muscle wasting diseases . It was that a 24 h treatment with this peptide increased the number of progenitor cells observed in primary cultures from healthy, als, and dystrophic human cells in vitro . As far as aging is concerned our studies have shown that older muscles are less able to produce mgf, and this seemingly results in an age - related loss in both muscle mass and strength . Recent studies by gillian butler - browne's group in paris and our group in london who used primary cultures found that human mononucleated / desmin - expressing myoblasts from young muscle biopsies reacted somewhat differently to those from elderly muscle when cultured . Muscle progenitor cells like other diploid somatic cells have a finite potential to divide before they become senescent but it was found that the myoblasts from older individuals still responded to the unique 24aa mgf peptide but in a somewhat different way . Treatment with the mgf-24aa - e peptide increased the proliferative capacity of the human myoblasts isolated from neonatal and young adult muscle . Those cultured from old adult muscle showed a marked increase in nuclei per myotube (see figure 5). In the old muscle there are considerable numbers of quiescent reserve cells, and their activation by the unique e domain peptide increased in the number of nuclei in the myotubes . The primary action of the mgf e-24aa peptide on elderly muscle was to push reserve myoblasts through the next step of fusion to start the hypertrophy process . It seems that as we grow older the system becomes less sensitive for detecting mechanical strain because of the decreased compliance of the tissue due to cross - linking in the connective tissues . There is some evidence that the compliance of the muscle connective tissue is increased more than in sedentary individuals and this helps to maintain power output as well as fatigability . Recent research has shown that there are mechanosensor molecules such as focal adhesion kinase systems . As their name denotes these faks are believed to involve activation of phosphatases which are possible part of the upstream activation of the splicing of the igf - i gene to produce mgf . It has been shown that the signalling associated with mgf is different to other splice variants of the igf - i gene from the human exercise studies carried out at mcmaster university in canada . The temporal response of mgf to acute damaging exercise results in myf5 and myod expression and the later response to igf - iea and igf - i eb to mrf-4 to the acute damaging repair phase which occurs after the increase in the number of extra nuclei triggered by mgf (human igf-1ec). Confusion has arisen in this field of study because of the temptation to use murine myoblast cell lines such as c2c12 cells . These transformed, mutated cells that readily multiply are no longer suitable for this kind of study, and this message has not yet been appreciated by pharmaceutical companies or academic research groups . The question remains, why does not exercise produce as much mgf and the same muscle gain in the elderly as in young individuals? We do need to understand the age - related amelioration in the upstream signalling at the cellular and molecular levels as this apparently holds the clues as to why loss of muscle mass and strength and to put this in the context of the musculoskeletal system as a whole . Eccentric exercise has been found to be associated with akt / mtor / p70 signalling but we still do not know why there is no apparent relationship in young individuals and rate of stretch in eccentric contractions, and at present this is presumed to be the case in older individuals . Second messengers relating to mgf expression have also been studied by the team in the bach institute of the russian academy of sciences, and protein kinases a and c and camp appear to be involved in mgf upregulation and the specific type of exercise that should be more beneficial for maintaining musculature in the elderly . Mgf is expressed in other tissues following mechanical strain and tissue damage and enhances myogenic precursor cell transplantation success . It is expressed in tendons following mechanical stress and increased osteoblasts in bone after damage . Mgf has been found to be very neuroprotective, and this includes protection against oxygen - free radical damage, and on this basis other aspects continuing to exercise as we get older can be recommended . Therefore, it seems that the splicing and expression of the igf - i gene and its isoforms is central to our understanding of the progressive inability of tissue repair, and there are broader aspects then just decline in skeletal muscle function [54, 56]. Although the stabilized mgf e 244aa peptide can be purchased over the internet it is expensive, and apart from research it is apparently being used by body builders and doping for improved athletic performance . It is predicted that its use will be more widespread as it can be produced using recombinant methods in the same way as human insulin . A group of russian scientists [57, 58] are producing a mgf peptide in this way and using it for bona fide reasons that include alcoholic myopathy and muscle atrophy resulting from space travel . There is also much interest in china where recombinant mgf is also being produced but less is known about its uses in these countries but it is clear that they appreciate its potential for treating in tissue atrophy and repair.
|
The principal risk factors are tobacco smoking and alcohol consumption, and the association of both factors is highly synergistic . Genetic factors, dietary habits, human papillomavirus (hpv) infection, consumption of hot mate (traditional south american infused drink), and poor oral health are also risk factors [35]. As lifestyles and human behaviour are directly affected by these factors, social inequalities are related to increases in risk . The most recent oropharyngeal cancer incidence data of spain reveals that the country is situated amongst those that present elevated incidence in men, with rates of 3.6 cases per 100,000 inhabitants / year (ci 95% = 3.43.8), along with france, slovenia, switzerland, and germany . For women, spain is amongst the countries with lowest incidences, with a rate of 0.2 cases per 100,000 inhabitants / year (ci 95% = 0.2 - 0.3). Oropharyngeal cancer is rarely described separately from others head and neck tumours . The anatomical proximity between the oral cavity and the oropharynx, along with the fact that the risk factors are the same, besides, some publications analyse these locations in conjunction with other types of cancers that present different behavior and risk factors, such as nasopharynx and hypopharynx cancers, and denominate the entire group as oral cavity and pharynx cancer . Other publications separate between oral cavity and oropharynx but include base of tongue cancers in the oral cavity classification, when the base of tongue should be analysed as a sublocation of the oropharynx . All these problems in the classification of oral cavity and oropharynx cancers hinder a more detailed analysis of the changes in epidemiological profiles, of the effect of treatment, and consequently, of the survival rates of both types of cancers . Recently, studies have proved that some risk factors are more related to specific locations, such as hpv infection and oropharynx locations, and tobacco and alcohol with oral cavity . The objective of this study is to determine oropharyngeal cancer survival from the population - based cancer registry (pbcr) of zaragoza, spain . Cancer survival analysis from population - based data constitutes a valuable tool for the evaluation of welfare services offered and allows for the orientation towards diagnose and treatment strategies . The population studied was diagnosed with oropharyngeal cancer in the province of zaragoza, spain, between january 1, 1978 and december 31, 2002 . Data was actively collected, analysed, and monitored by the pbcr of zaragoza until december 31, 2007 . The following locations were included: base of tongue, lingual tonsils, soft palate, uvula, tonsils, and oropharynx (icd codes: c01.9, c02.4, c05.1, c05.2, c09.0, c09.1, c09.8, c09.9, c10.0c10.4, c10.8 y c10.9). The difficulty in establishing survival rates at a population level lies firstly in obtaining reliable data on cancer incidence for the population, and secondly, in carrying out the monitoring with accuracy and integrity . Monitoring of cases was carried out by the cancer registry itself through the mortality registry of the aragon government, which is administered by the statistical institute of aragon . All new cases registered as major salivary gland cancer were included in the analysis, except for the cases diagnosed through death certificates . The pbcr data of zaragoza was the first spanish data to be published in cancer incidence in five continents, appearing in this publication since volume iii . A set of quality indicators evaluates the data prior to publication in ci5; the registry data of zaragoza fulfils the established quality standards and presents excellent quality [13, 14]. Calculation of the survival rate was carried out by the kaplan - meier method, and relative survival was calculated through the webpage of the catalan institute of oncology (cio). The relative survival rate is defined as the relationship between the observed survival and the expected survival in a group of healthy people of similar age and gender . In practice, the survival of people without cancer is difficult to predict, and for this reason the general mortality rate of the population is used . The cio webpage uses the hakulinen method to calculate the relative survival of the database sent by the user and is based on the mortality tables of the mortality registries of the autonomous communities and provinces of spain [15, 16]. When the number of risk patients was lower than 15, these results were not considered in the analysis, as the final estimations were unstable . Survival was studied by gender, age groups (4064 and over 65 years of age) and location (base of tongue, tonsils, soft palate, and oropharynx). In order to study the dynamics of survival, the data was stratified in three study periods (19781986, 19871994, and 19952002), and the survival indicators were compared . The effect of each prognostic factor (sex, age group, location, and time period) on the survival rates was evaluated by the log - rank test . The log - rank test is a statistical hypothesis contrast test, used to compare two or more survival curves, and the null hypothesis is that the survival of the groups under comparison is the same . The distribution of cases, number of deaths, and percentage of censored cases according to sex, age, and period are presented in table 1 . The number of incidence cases included in the study was 380, after exclusion of the cases registered through death certificates . It was observed that 87.6% of cases occurred in men; the censored percentage was 26.6% (24.3% men and 42.6% women). The observed survival after one year of diagnosis of oropharyngeal cancer was 61.3% (ci 95%: 56.466.2). One - year relative survival was 61.9% (ci 95%: 56.867.5) in men and 64.3% (ci 95%: 51.970.8) in women; five - year relative survival was 34.2% (ci 95%: 29.240.1) in men and 53.3% (ci 95%: 40.370.5) in women (table 2). Comparison of survival rates by sex revealed statistically significant differences (p value = 0.017) with better survival in women . There were no differences when comparing the three age groups (p value = 0.61), the locations (p value = 0.25), and the three studied time periods (p value = 0.17). The continuous increase in the number of cancer survivors along with population ageing in spain, with a consequent increase in the number of cases, translate into new challenges that have to be overcome by the health care system . Nowadays in spain, more than 50% of cancer patients are alive after five years of diagnosis and the trend is to increase this percentage . As a consequence of improvements in diagnose tests and treatments, an increase in cancer survivors is expected, and this situation creates new demands in welfare services, which must consider such complexity for patient monitoring . It is estimated that in 2015 in spain, 136,002 new cancer cases are diagnosed in men and 85,818 in women . Actually, knowledge on the health situation of long - term survivors is still limited . It is known that the adoption of healthy lifestyles, selfcare attitude, and sociocultural and psychological aspects play an important role in the duration and life quality of patients [20, 21]. The eurocare studies are the most important survival data used for comparison purposes against this study . Eurocare is a multicentric project that gathers population - based cancer registries for european countries . Survival data from this study shows that the highest survival rates are in western europe, independently from the period studied . For the 19901994 period, average survival for europe was 28.7% (ci 95% = 26.031.5) in men and 43.5% in women the highest survival rates occurred in sweden (46.6%, ci 95% = 40.753.4 in men and 56.2%, ci 95% = 47.566.4 in women), and the lowest rates were obtained in the czech republic, estonia, and slovakia . Spanish data is within the european average, with a 29% five - year survival rate for men (ci 95% = 24.035.0) and 44.1% (ci spanish data originates from grenada, majorca, murcia, navarre, and tarragona . In zaragoza, the data encountered for the 19871994 period corroborates these findings, with a survival percentage of 30.4% (ci, the average for all european countries reveals that 37.6% of men and 49.6% of women survived after five years of diagnosis . Sweden and denmark presented the highest survival rates, with five - year survival rates of 45.3% (ci 95% = 26.042.0), and the lowest survival rates were found in portugal and northern ireland . The economic situation of countries and the amount of resources destined to welfare assistance are the main reasons for such a variation in survival rates and explain the lowest survival rates found in eastern europe . Nevertheless, low survival rates in countries such as northern ireland or other regions of the united kingdom could also be associated with other aspects, such as the structure of the health care system, comorbidity, and risk factor patterns [24, 25]. Regarding gender, the results for zaragoza indicate that women present higher survival rates and most part of the studies found results in the same direction . The reasons for these higher survival rates in women could be associated with a biological superiority of women in response to illness and treatment, or a higher awareness in women concerning their bodies, and consequently, a higher percentage of early - state diagnosis [24, 26]. Higher survival rates in women were also found by another study, which investigated 89 population - based cancer registries in several european countries for the head and neck regions . Oropharyngeal cancer results demonstrate highest relative survival rates in women (47.67%) than in men (37.67%), in younger ages and in northern europe . The fact that along with an increase in age there is a significant decrease in survival has been published in all eurocare studies . The reasons are comorbidity and the therapeutics used in elder patients, where many times surgical treatments are not indicated [22, 23]. The results for the population of zaragoza did not show significant differences when comparing survival rates in the age groups of 4064 and over 65 years of age . The low number of diagnosed cases in patients with 39 years of age or less prevented the survival rate analysis for this group . Regarding location, it was observed that in zaragoza the highest survival rate was found for cancers located in the tonsils and the lowest, for those located in the tongue . A recent study carried out with data from the surveillance, epidemiology end results (seer), a program of the united states national cancer institute, compared cancers located in the oropharynx with those in the oral cavity . The results revealed a higher survival rate for cancers located in the oral portion of the tongue than for cancers located in the tonsils and base of tongue . This division was adopted to differentiate those locations that supposedly present a higher risk for the development of cancers associated with hpv, such as oropharyngeal cancer, from those situated in the oral cavity . When comparing the three studied period, no statistically significant differences were found in the survival rates for oropharyngeal cancer in zaragoza . Spanish results published in international studies do not demonstrate significant improvement in survival rates [22, 23]. An investigation using canadian cancer registry data has studies changes in survival rates for oropharyngeal and head and neck cancers for patients diagnosed between 1992 and 2001, totaling 10,860 cases in men and 4002 cases in women . The results revealed significant improvements only in men, with an increase of 13.5% in the survival rates after five years . The data presented herein suggest that zaragoza presents survival rates similar to other spanish registries already published . Nevertheless, no statistically significant changes have been identified when dividing and comparing the total study period of 25 years in three study periods . These results must be interpreted with caution because it is difficult to followup cases in such a long span study . Although there are limitations, the authors consider that that survival studies using data from population - based cancer registries must be carried out and published, as they allow for the evaluation of the results obtained when treating the illness in the studies population . Investigations such as the one presented herein can be the first step for the development of more effective treatment, prevention and control programs for cancer, improvement in the follow - up process of patients and future research . Advances in the treatment of cancer and the increase in the number of survivors call for a progressively wider monitoring of this part of population studies on cancer survival need to increase follow - up time and broaden perspectives, with the objective of knowing the physical, psychological, and social aspects associated with this illness [18, 30].
|
Palatal rugae are irregular, asymmetric ridges of mucous membrane extending laterally from the incisive papilla and the anterior part of the median palatal raphe . These structures have been used as internal cast reference points for quantification of tooth migration . A few investigators have shown the medial rugae region to be stable or show predictable changes post orthodontic therapy . The rugae patterns are completely formed by the 12 to 14 week of prenatal life and remain stable thereafter . These are unique to each person and show distinctiveness based on ethnic groups and hence are useful in forensic identification . Because they are stable landmarks the palatine rugae play a significant role in clinical dentistry as well . The objective of this study was to analyze and characterize the rugae patterns, compare the rugae dimensions in various age groups and to ascertain any relationship between dimensional analysis and palatal depth . Cross - sectional casts of 52 females and 48 males were selected from the archival section of the department of orthodontics, m. m. college of dental sciences and research, mullana (ambala) and studied employing following parameters: assessment of age (according to erupted teeth).gender differentiation (based upon records).division of medial palatal region into: a: distance between incisive papilla length and anterior limit of the anteriormost rugae.b: distance between incisive papilla and posteriormost rugae limits.lateral rugae dimensions . Palatal depth (measured from healthy gingival margin underneath the mesiolingual cusp to the deepest concavity of the palatal arch using a brass wire between two opposing points and measuring the vertical distance at the center).rugae patterns . Division of medial palatal region into: a: distance between incisive papilla length and anterior limit of the anteriormost rugae.b: distance between incisive papilla and posteriormost rugae limits.lateral rugae dimensions . A: distance between incisive papilla length and anterior limit of the anteriormost rugae . Palatal depth (measured from healthy gingival margin underneath the mesiolingual cusp to the deepest concavity of the palatal arch using a brass wire between two opposing points and measuring the vertical distance at the center). No patient details were disclosed and ethical guidelines as per the declaration of helsinki were followed . Correlations among a, b, lateral rugae patterns and palatal depth were calculated by mean s.d . Correlations among a, b, lateral rugae patterns and palatal depth were calculated by mean s.d . Followed by evaluation of p values . The rugae patterns identified were: common origin; separate origin; lateral branching; secondary rugae and fragmentary patterns . Females were found to have slightly higher predilection towards having the common, fragmentary and lateral branching rugae patterns . There was a stronger female predisposition for rugae with separate origin, whereas, the secondary rugae were equally existent in either gender . Significant correlation was found between the a (p=0.03) and b values (p=0.02) on comparing the mean s.d . Values of age groups between 12 - 13 years and> 14 years, respectively . Hence, there is a corresponding anteroposterior increase in palatal dimensions in these age groups [table 1]. This study showed no changes in the lateral rugae dimension and palatal depth with increasing age . The majority of the study cohort (57%) had a palatal depth in the range of 1.6 - 2.0 cm [table 2]. Palatal rugae have been used as reference points for many purposes such as evaluating tooth movement pre- and post - orthodontic treatment, population studies and forensic identification . Stability of medial palatal region has been a subject due to differences among various investigators . Christou and kiliardis evaluated the vertical changes in the medial aspects of the rugae and concluded that these changes over time are due to the alterations in the vertical positioning of maxillary incisors and increase in lower face height . Growth periods (12 - 13 years and> 14 years) studied in this paper, showed a downward and forward movement of the maxilla in relation to the cranial base and also, changes in the size and shape of maxilla by structural remodeling . These phenomena can be explained by the deposition of new bone on the oral surface of the palate and at the alveolar crest . Therefore, the changes in rugae dimensions can be the result of this differential growth in the palate and alveolar crest. [24] rugae patterns showed a strong female predilection for rugae with separate origins [figure 1] whereas slightly higher incidences were noted for rugae with common origin, fragmentary and lateral branching patterns . There was an equal percentage of gender with secondary rugae pattern in the ethnic segment of north indian population studied [table 1]. Photograph depicting rugae patterns with secondary origin and fragmentary nature the purpose of this paper was to evaluate a cross - sectional patient database to analyze the anteroposterior stability of the medial rugae region . Analysis of 100 study casts showed a significant difference in the medial rugae region (p= 0.03, 0.02) in a and b values . No significant changes were noted in the lateral rugae dimensions . The palate growth in this period was found to be non - significant . Hence, it can be surmised from the analysis of the results that there is a differential growth spurt in the anterior and posterior palate during adolescence as is marked by the significant differences in the measurement values obtained through this study . Palatal rugae can be studied as a strong indicator of ethnicity, gender differentiation and study of growth changes in the anterior maxilla . Thus, rugae are important tools in clinical investigations involving forensic anthropology and developmental biology.
|
The trial was conducted according to the declaration of helsinki, and was approved by the research & ethics council of the faculty of medicine, isfahan university of medical sciences . After full explanation of the study protocol, the whole program was offered free of charge . We have previously reported the results of this trial on vascular reactivity,5 and here we report the effects on biochemical markers of inflammation and endothelial dysfunction . This non - pharmacologic randomized controlled clinical trial was conducted among 30 adolescents, aged 12 - 15 years, with mets as defined by the international diabetes federation for the pediatric age group . Accordingly, mets was diagnosed in the case of central adiposity 90 percentile waist circumference (wc) or adult threshold if lower than the 90 percentile plus at least two of the following criteria: 1) triglycerides (tg) 150 mg / dl, 2) hdl - cholesterol (hdl - c) <40 mg / dl, 3) blood pressure (bp) 130 mmhg systolic or 85 mmhg diastolic, and 4) fasting plasma glucose (fpg) 100 mg / dl or previously diagnosed type 2 diabetes.6 participants were randomly selected from those who met the diagnostic criteria, and were referred from health care centers, schools, public and private clinics to the farhanguian medical clinic, isfahan, iranclinic . Those children with signs or symptoms of secondary obesity, endocrine disorders, presence of any physical disability, and history of chronic medication use, smoking, or chronic infection during the two weeks before the study were not included to the trial . The allocation was conducted from computer generated random numbers using the children's record numbers in the clinic . All measurements were made by a trained team of general physicians and nurses under the supervision of the same pediatrician, and used calibrated instruments and standard protocols . Height, weight, and wc were measured, and body mass index (bmi) was calculated as weight (kg) divided by height in meters squared (m). The readings at the first and the fifth korotkoff phase were considered as systolic and diastolic bp, respectively . The average of the last two bp measurements was recorded and included in the analysis.7 participants were instructed to fast for 12 hours before screening . While one of the parents accompanied his / her child, blood samples were taken from the left antecubital vein between 8:00 and 9:30 am for measurement of fpg, lipids and adhesion molecules . The blood samples were centrifuged for 10 minutes at 3000 rpm within 30 minutes of venipuncture . Fpg, total cholesterol (tc), hdl - c, ldl - c, tg and high - sensitive c - reactive protein (hs - crp) were measured by autoanalyzer . Serum adhesion molecules (sicam-1, svcam-1 and se - selectin) and interleukin-6 (il-6) were measured by enzyme - linked immunosorbent assay (elisa) method using standard kits (bender med systems, gmbh, vienna, austria). The same cardiologist conducted all studies for measurement of brachial arterial reactivity in the abovementioned clinic . Using the method previously described,25 the diameter of the brachial artery was measured from highresolution b - mode ultrasound images (aloka 5000 system, 7.5 megahertz transducer) at rest as basal brachial dimension, 90 seconds after cuff deflation to assess reactive hyperemia (endothelium - dependent dilation or flowmediated dilation), again at rest, and 3 to 4 minutes after administration of 400 micrograms sublingual nitroglycerin which led to endothelium independent dilation . The percent change of flow - mediated dilation was calculated as the ratio of the brachial artery diameter after reactive hyperemia to the baseline diameter; a similar calculation was done for nitroglycerin - mediated vasodilatation.5 after baseline measurements, adolescents were assigned into two groups of equal number using computer - based randomization . For the next month, participants of one group were asked to drink 18 ml / kg / day of natural grape juice;8 the second group was asked to drink 240 ml / day of natural pomegranate juice.9 we emphasized that children and their parents must only use home - made juice without adding any sweetener, not concentrated juices that usually have higher - calorie content . Compliance with regular drinking of the recommended type and amount of juice was determined by weekly phone calls to participants, and visiting participants at 2-week intervals . The baseline survey measurements including physical examination, measurement of flow - mediated dilation (fmd) of the brachial artery, and biochemical analyses were repeated 4 hours after initial juice consumption and 1 month later . Statistical analyses were performed using the spss for windows software (version 15; spss, chicago, il). We verified the normality of the distribution of variables with a kolmogorov - smirnov test . Statistical analyses of bmi, wc, tg and hs - crp were performed using log - transformed values because the distribution was skewed . To compare continuous at different stages of the trial between the two groups under study, age- and sex - adjusted analysis of variance (anova) with post hoc bonferoni test, as well as age and gender - adjusted linear regression analysis was used to assess the association between changes in biochemical parameters and changes in fmd90 after 4 hours and 1 month . This non - pharmacologic randomized controlled clinical trial was conducted among 30 adolescents, aged 12 - 15 years, with mets as defined by the international diabetes federation for the pediatric age group . Accordingly, mets was diagnosed in the case of central adiposity 90 percentile waist circumference (wc) or adult threshold if lower than the 90 percentile plus at least two of the following criteria: 1) triglycerides (tg) 150 mg / dl, 2) hdl - cholesterol (hdl - c) <40 mg / dl, 3) blood pressure (bp) 130 mmhg systolic or 85 mmhg diastolic, and 4) fasting plasma glucose (fpg) 100 mg / dl or previously diagnosed type 2 diabetes.6 participants were randomly selected from those who met the diagnostic criteria, and were referred from health care centers, schools, public and private clinics to the farhanguian medical clinic, isfahan, iranclinic . Those children with signs or symptoms of secondary obesity, endocrine disorders, presence of any physical disability, and history of chronic medication use, smoking, or chronic infection during the two weeks before the study were not included to the trial . The allocation was conducted from computer generated random numbers using the children's record numbers in the clinic . All measurements were made by a trained team of general physicians and nurses under the supervision of the same pediatrician, and used calibrated instruments and standard protocols . Height, weight, and wc were measured, and body mass index (bmi) was calculated as weight (kg) divided by height in meters squared (m). The readings at the first and the fifth korotkoff phase were considered as systolic and diastolic bp, respectively . The average of the last two bp measurements was recorded and included in the analysis.7 participants were instructed to fast for 12 hours before screening . While one of the parents accompanied his / her child, blood samples were taken from the left antecubital vein between 8:00 and 9:30 am for measurement of fpg, lipids and adhesion molecules . The blood samples were centrifuged for 10 minutes at 3000 rpm within 30 minutes of venipuncture . Fpg, total cholesterol (tc), hdl - c, ldl - c, tg and high - sensitive c - reactive protein (hs - crp) were measured by autoanalyzer . Serum adhesion molecules (sicam-1, svcam-1 and se - selectin) and interleukin-6 (il-6) were measured by enzyme - linked immunosorbent assay (elisa) method using standard kits (bender med systems, gmbh, vienna, austria). The same cardiologist conducted all studies for measurement of brachial arterial reactivity in the abovementioned clinic . Using the method previously described,25 the diameter of the brachial artery was measured from highresolution b - mode ultrasound images (aloka 5000 system, 7.5 megahertz transducer) at rest as basal brachial dimension, 90 seconds after cuff deflation to assess reactive hyperemia (endothelium - dependent dilation or flowmediated dilation), again at rest, and 3 to 4 minutes after administration of 400 micrograms sublingual nitroglycerin which led to endothelium independent dilation . The percent change of flow - mediated dilation was calculated as the ratio of the brachial artery diameter after reactive hyperemia to the baseline diameter; a similar calculation was done for nitroglycerin - mediated vasodilatation.5 after baseline measurements, adolescents were assigned into two groups of equal number using computer - based randomization . For the next month, participants of one group were asked to drink 18 ml / kg / day of natural grape juice;8 the second group was asked to drink 240 ml / day of natural pomegranate juice.9 we emphasized that children and their parents must only use home - made juice without adding any sweetener, not concentrated juices that usually have higher - calorie content . Compliance with regular drinking of the recommended type and amount of juice was determined by weekly phone calls to participants, and visiting participants at 2-week intervals . The baseline survey measurements including physical examination, measurement of flow - mediated dilation (fmd) of the brachial artery, and biochemical analyses were repeated 4 hours after initial juice consumption and 1 month later . Statistical analyses were performed using the spss for windows software (version 15; spss, chicago, il). We verified the normality of the distribution of variables with a kolmogorov - smirnov test . Statistical analyses of bmi, wc, tg and hs - crp were performed using log - transformed values because the distribution was skewed . To compare continuous at different stages of the trial between the two groups under study, age- and sex - adjusted analysis of variance (anova) with post hoc bonferoni test, as well as age and gender - adjusted linear regression analysis was used to assess the association between changes in biochemical parameters and changes in fmd90 after 4 hours and 1 month . This non - pharmacologic randomized controlled clinical trial was conducted among 30 adolescents, aged 12 - 15 years, with mets as defined by the international diabetes federation for the pediatric age group . Accordingly, mets was diagnosed in the case of central adiposity 90 percentile waist circumference (wc) or adult threshold if lower than the 90 percentile plus at least two of the following criteria: 1) triglycerides (tg) 150 mg / dl, 2) hdl - cholesterol (hdl - c) <40 mg / dl, 3) blood pressure (bp) 130 mmhg systolic or 85 mmhg diastolic, and 4) fasting plasma glucose (fpg) 100 mg / dl or previously diagnosed type 2 diabetes.6 participants were randomly selected from those who met the diagnostic criteria, and were referred from health care centers, schools, public and private clinics to the farhanguian medical clinic, isfahan, iranclinic . Those children with signs or symptoms of secondary obesity, endocrine disorders, presence of any physical disability, and history of chronic medication use, smoking, or chronic infection during the two weeks before the study were not included to the trial . The allocation was conducted from computer generated random numbers using the children's record numbers in the clinic . All measurements were made by a trained team of general physicians and nurses under the supervision of the same pediatrician, and used calibrated instruments and standard protocols . Height, weight, and wc were measured, and body mass index (bmi) was calculated as weight (kg) divided by height in meters squared (m). The readings at the first and the fifth korotkoff phase were considered as systolic and diastolic bp, respectively . While one of the parents accompanied his / her child, blood samples were taken from the left antecubital vein between 8:00 and 9:30 am for measurement of fpg, lipids and adhesion molecules . The blood samples were centrifuged for 10 minutes at 3000 rpm within 30 minutes of venipuncture . Fpg, total cholesterol (tc), hdl - c, ldl - c, tg and high - sensitive c - reactive protein (hs - crp) were measured by autoanalyzer . Serum adhesion molecules (sicam-1, svcam-1 and se - selectin) and interleukin-6 (il-6) were measured by enzyme - linked immunosorbent assay (elisa) method using standard kits (bender med systems, gmbh, vienna, austria). The same cardiologist conducted all studies for measurement of brachial arterial reactivity in the abovementioned clinic . Using the method previously described,25 the diameter of the brachial artery was measured from highresolution b - mode ultrasound images (aloka 5000 system, 7.5 megahertz transducer) at rest as basal brachial dimension, 90 seconds after cuff deflation to assess reactive hyperemia (endothelium - dependent dilation or flowmediated dilation), again at rest, and 3 to 4 minutes after administration of 400 micrograms sublingual nitroglycerin which led to endothelium independent dilation . The percent change of flow - mediated dilation was calculated as the ratio of the brachial artery diameter after reactive hyperemia to the baseline diameter; a similar calculation was done for nitroglycerin - mediated vasodilatation.5 after baseline measurements, adolescents were assigned into two groups of equal number using computer - based randomization . For the next month, participants of one group were asked to drink 18 ml / kg / day of natural grape juice;8 the second group was asked to drink 240 ml / day of natural pomegranate juice.9 we emphasized that children and their parents must only use home - made juice without adding any sweetener, not concentrated juices that usually have higher - calorie content . Compliance with regular drinking of the recommended type and amount of juice was determined by weekly phone calls to participants, and visiting participants at 2-week intervals . The baseline survey measurements including physical examination, measurement of flow - mediated dilation (fmd) of the brachial artery, and biochemical analyses were repeated 4 hours after initial juice consumption and 1 month later . Statistical analyses were performed using the spss for windows software (version 15; spss, chicago, il). We verified the normality of the distribution of variables with a kolmogorov - smirnov test . Statistical analyses of bmi, wc, tg and hs - crp were performed using log - transformed values because the distribution was skewed . To compare continuous at different stages of the trial between the two groups under study, age- and sex - adjusted analysis of variance (anova) with post hoc bonferoni test, as well as chi square tests for categorical variables were used . Age and gender - adjusted linear regression analysis was used to assess the association between changes in biochemical parameters and changes in fmd90 after 4 hours and 1 month . This trial was comprised of 30 adolescents (46.7% girls) with a mean (sd) age of 13.4 (1.1) years . They had a mean (sd) body mass index of 27.1 (1.1) kg / m (corresponding to more than the 95 percentile), and mean (sd) wc of 93.5 (9.8) cm (corresponding to the 95 percentile), without significant difference between the two groups studied and no significant changes after the trial (p> 0.05). The mean (sd) systolic and diastolic blood pressures were 115.14 (2.11) and 64.1 (1.4) mmhg, respectively; and did not change significantly during the trial (p> 0.05). Table 1 represents lipids and inflammatory markers at the baseline and the changes at 4-hours and at 1-month after drinking grape and pomegranate juices . Fasting lipids and glucose were also similar throughout the study . When both groups combined, a significant decline in se - selectin, sicam-1, and il-6 at one month follow up was seen . Moreover, in the group drinking pomegranate juice, significant declines in these three inflammatory markers were observed from baseline to the 4-hour - measurements and after one month follow up . Moreover, se - selectin and sicam-1 showed a significant decrease in grape juice group after one month . Mean (sd) of investigated factors at baseline, 4 hours and 1month after daily drinking juices in adolescents with metabolic syndrome other variables did not change significantly . In separate group analyses, the effect was confined to the pomegranate juice group only for sicam-1 but was seen in both groups for the other variables . For pomegranate juice, a beneficial effect was apparent at 4 hours and even increased over one month follow up . No significant differences was seen for crp or svcam . Considering baseline diameter, the percent changes of fmd of the brachial artery at 90 seconds after ischemia and after receiving nitroglycerin were significant at short - term (at 4 hours) in both groups (table 2). The improvement in fmd persisted at long - term (at 1 month), but the percent change at 1 month versus 4 hours was significant only in the grape juice group . The percent changes were not significantly different between the two groups receiving grape juice and pomegranate juice . Percent change of brachial artery diameter from baseline to 4 hours and 1month after daily drinking juices in adolescents with metabolic syndrome at baseline there were significant correlations between components of the mets and inflammatory markers . Hs - crp had significant correlation with bmi (r = 0.5, p = 0.03), wc (r = 0.7, p = 0.01), and tg (r = 0.7, p = 0.005). Se - selectin was also significantly correlated with bmi (r = 0.5, p = 0.02), wc (r = 0.6, p = 0.01), tc (r = 0.5, p = 0.04), and tg (r = 0.7, p = 0.01). Also, sicam-1 had significant correlation with fpg (r = 0.6, p = 0.01), tg (r = 0.5, p = 0.04), and hdl - c (r = -0.7, p = 0.01). Il-6 was significantly correlated with tc (r = 0.5, p = 0.04). Age- and gender - adjusted regression analysis of changes in fmd90 at 4 hours and 1 month with changes in biochemical parameters showed significant negative association of sicam-1 and se - selectin with fmd at both follow up times . Correlations between changes in mean flow mediated dilation (fmd) of the brachial artery with changes in biochemical parameters after 4 hours and 1 month drinking grape and/or pomegranate juices in adolescents with metabolic syndrome this study has shown that consumption of natural grape and pomegranate juice have short and one month benefits on endothelial function, soluble intercellular adhesion molecules and some markers of inflammation among obese adolescents with metabolic syndrome . We found weak but significant inverse associations between changes in sicam-1, se - selectin and il-6 and changes in fmd90 after drinking both types of juices . These sustained effects on markers of endothelium function are consistent with the findings of a recent trial among hypertensive adults, which documented a dose - response relationship between increasing fruit and vegetable consumption and improved fmd.10 mets may result from interactions of vascular abnormalities, oxidative stress, visceral fat, inflammation, adipocytokines, and cortisol, as part of the larger environment of obesity and insulin resistance, and under the influence of genetic and ethnic predispositions.11 in adults it has been documented that mets is associated with endothelial dysfunction as assessed by fmd of brachial artery.1213 recent studies confirmed this association in the pediatric age group.11415 up - regulation of endothelial adhesion molecules, including endothelial - leukocyte adhesion molecule (se - selectin), intercellular cell adhesion molecule-1 (sicam-1), and vascular cell adhesion molecule-1 (svcam-1), play a crucial role in the earliest phases of atherosclerosis.1617 inflammation markers and soluble adhesion molecules concentrations have been found to be higher in the obese than in the lean children.1819 higher levels of markers of inflammation and oxidative stress in children with mets and obesity2021 suggest early stages of endothelial dysfunction in obese children.2224 in this study, juices with anti - oxidant properties improved these markers and they might be beneficial for prevention and control of atherosclerotic diseases . Daily consumption of grape juice8 and pomegranate juice9 improve endothelial function and myocardial perfusion in patients with ischemic coronary heart disease . It is suggested that certain natural antioxidants or flavonoids are responsible for these effects on endothelial function, and ingesting moderate amounts of grape juice each day might supply these nutrients.25 in animal and human studies, grape products have been shown to produce hypotensive, hypolipidemic and anti - atherosclerotic effects, and also to improve an - tioxidant status as measured in terms of plasma antioxidant capacity, oxidation biomarkers, antioxidant compounds or antioxidant en - zymes.1026 the anti - atherosclerotic effects of grape juice are suggested to be mediated by its antioxidant content and influence on intracellular production of reactive oxygen species27 through possible indirect mechanisms such as changes in hdl paraoxonase 1 and 2 activity.28 a few adult trials have assessed the effect of juices on plasma intracellular cell adhesion levels and revealed both positive and negative findings . One type of antioxidant - rich juice (sea buckthorn) had no effect on plasma sicam-1 level.29 grape juice could improve fmd and reduce sicam-1 of hypercholes - terolemic individuals but had no effect on svcam-1.30 in a 2-week trial in patients undergoing hemodialysis, grape juice was not effective in reducing the concentration of markers of inflammation and adhesion molecules.10 in the current trial, both types of juices had beneficial effects on vascular reactivity, some adhesion molecules and markers of inflammation . Some of these beneficial effects on biochemical parameters were significantly greater in the group consuming pomegranate juice than in the group consuming grape juice . The pomegranate (punica granatum) is a fruit native of iran,31 and now it is cultivated in many countries . The antioxidant capacity of pomegranate juice is reported to be three times higher than that of red wine and green tea32 and higher than other juices including grape juice.3334 several studies confirmed its antioxidant and anti - inflammatory properties.3536 pomegranate juice may increase serum antioxidant capacity, decrease plasma lipids and lipid peroxidation, diminish oxidized - ldl uptake by macrophages, reduce intima media thickness, decrease atherosclerotic lesion areas, enhance biological actions of nitric oxide, lessen inflammation, decrease angiotensin converting enzyme activity, and lower systolic blood pressure.3537 in these adult trials of antioxidant juices, the process of aging and the presence of underlying chronic disease may have masked the effects of juices on early atherosclerosis . The findings of the current trial supplement the existing knowledge about antiatherogenic properties of pomegranate and grape juices . Given that dietary intake of fruits and vegetables is found to improve microvascular function in hypertensive subjects in a dose - dependent manner,38 trials with longer duration than the current one might show better results over time . In children, studies have shown that many factors including acute infections, inflammation, trauma, active and passive smoking, postprandial lipemia, and mental stress affect endothelial function.27 changes in dietary and physical activity habits239 and zinc supplementation40 have shown beneficial improvements in components of mets, markers of inflammation and endothelial dysfunction . Interestingly, as in this study was shown, these improvements are often with minimal or no change in body mass index . This suggests that at least for the short term, modest lifestyle changes alone may confer beneficial health effects . In the current trial, natural home - made juices without any added sweetener excessive consumption of sugar supplemented beverages, including fruit juices have been implicated in the obesity epidemic . This trial emphasizes the importance of considering the overall nutrition quality of the diet with attention paid to the food quality and overall food intake in relation to energy expenditure.41 daily consumption of diets rich in natural antioxidants may improve endothelial function in adolescents with metabolic syndrome . The beneficial effects of natural juices, particularly pomegranate juice should be considered in additional clinical research on lifestyle interventions in the pediatric age group to prevent atherosclerosis - related heart disease . We should acknowledge that one of the limitations of this trial was lack of control group . Rk participated in the design and conducting the study as well as drafting and editing the manuscript; ssg participated in the design and helped to edit the manuscript; mh participated in the design and conducting the study; mh participated in the design and conducting the study;az participated in the design and conducting the study; pp helped to draft and edit the manuscript.
|
Almost 30 years after its first description, hiv still remains a global pandemic, particularly affecting the countries of sub - saharan africa, southeast asia, and latin america . Hiv is an rna retrovirus which compromises the immune system, and renders the infected person susceptible to opportunistic infections and malignancy . The incidence of hiv infection in 2009 was 2.6 million, whilst the respective prevalence ranged between 31.435.3 million people . The prevalence of hiv had risen by 27% compared to the previous decade, although the annual rate of new cases had been steadily declining since the late 1990s . In addition, the estimated number of children living with hiv increased to approximately 2.5 million in 2009 . The increased incidence of hiv has resulted in a greater number of hiv - infected patients presenting to ent doctors . Indeed, up to 80% of hiv - infected patients eventually develop ent manifestations . Among the latter, oral disease seems to be the most common, occurring in approximately 4050% of hiv positive patients . Predisposing factors for hiv - related ent conditions include cd4 + cell count of less than 200/l, plasma hiv - rna levels greater than 3000 copies / ml, xerostomia, poor oral hygiene, and smoking . Although ent manifestations may not be diagnostic of hiv infection, they may be heavily suggestive of such an infection . In addition, the occurrence of certain oral manifestations in patients with known hiv disease who are not receiving treatment may be related to the progression of the disease . Finally, the presence of ent disease in patients on antiretroviral therapy could be the result of an increase in the plasma hiv - rna and suggest treatment failure . In this context, the provision of appropriate care to hiv patients may require a multi - disciplinary approach . The aim of the present paper is to review the current knowledge on ent manifestations of hiv infection, and present the available diagnostic and treatment options . The implications of the early identification of hiv - associated ent disease from a public health perspective are also discussed, along with clinical markers of immune compromise . Oral candidiasis, commonly known as thrush, is by far the most common oral manifestation of hiv infection . Candidal infection can occur in the oropharynx, hypopharynx, and larynx, and usually results in severe odynophagia and swallowing difficulties . The prevalence of candidiasis varies from 3090% among hiv positive adult patients whereas the respective percentage in children ranges between 22.5 and 83.3% . Oral candidiasis can present in three forms: pseudomembranous candidiasis, erythematous candidiasis, and angular cheilitis (figures 13). It has been associated with more frequent progression of hiv to aids, and also used as a clinical marker to define the severity of hiv infection . It appears as creamy, white, curd - like plaques on the buccal mucosa, tongue, and other oral mucosal surfaces . The most common organism involved is candida albicans; however involvement of non - albicans species, such as candida glabrata and candida dubliniensis, has also been described . Reproduced with permission from ias - usa . Top hiv med 2005;13:143 - 8 . Erythematous candidiasis, on the other hand, presents as a red, flat, subtle lesion on the dorsal surface of the tongue, or on the hard or soft palate . The lesion often involves two opposing surfaces, i.e. If a lesion is present on the tongue, the palate should be examined for a matching lesion, etc . Patients usually complain of a burning sensation, especially while eating spicy or salty food . When the hypopharynx, larynx, or esophagus are affected, symptoms may progress to severe odynophagia and swallowing difficulties . This may be especially true in children, in which candidal esophagitis may require hospital admission, and intravenous administration of amphotericin b. diagnosis is based on the clinical appearance of the lesions taking into consideration the history of hiv infection . However, candidiasis can be confirmed in challenging cases from the identification of fungal hyphae or blastospores in potassium hydroxide (koh) preparation . Treatment of mild to moderate cases of both erythematous and pseudomembranous candidiasis includes clotrimazole troches, nystatin oral suspension, and nystatin pastilles, whereas systemic administration of fluconazole, intraconazole and voriconazole is warranted in moderate to severe cases (table 1). Voriconazole should be reserved for cases of fluconazole resistance, due to more serious iteractions with other drugs . Antifungal therapy should last for two weeks to reduce the colony forming units to the lowest level possible and prevent recurrence . Table 1treatment regimens for hiv - associated ear, nose and throat (ent) manifestations in adults and children.hiv manifestationtreatment in adultstreatment in childrenoral candidiasistopical agentstopical agents clotrimazole troches 10 mg: dispense 70, nystatin 200,000 - 800,000 u: used qds, or 5 times a day dissolve 1 troche in mouth 5 times a day for 14 days miconazole 200,000 - 800,000 u: qds to 5 times a day . Nystatin oral suspension 500,000 u: swish 5 ml in mouth oral nystatin 200,000 u: in tablets dissolve as long as possible then swallow (optional), qds for 14 days 1 in the mouth 5 times a day nystatin pastilles 100,000 u: dispense 56, systemic agents dissolve 1 in mouth qds for 14 days fluconazole or ketoconazole 6mg / kg ofsystemic agents body weight for 5 - 7 days fluconazole 100 mg: dispense 15 tablets, take 2 tablets on day 1, clotrimazole 10 mg bd followed by 1 tablet od for the remainder of the 14-day treatment period itraconazole oral suspension 10 mg/10 ml: dispense 140 ml, swish and swallow 10 ml per day for 7 - 14 days . Voriconazole 200 mg: dispense 14 tablets, take 1 tablet bd for 2 weeks or at least 7 days following resolution of symptomsangular cheilitismiconazole cream apply qds for 14 daysmiconazole cream apply qds for 14 daysketoconazole cream apply qds for 14 daysketoconazole cream apply qds a day for 14 daysnecrotizing periodontitismetronidazole 500 mg: 1 tablet bd for 7 - 10 days.metronidazole 15 - 30 mg / kg / day orally in 3 dividedamoxicillin 500 mg: tds for 7 - 10 days.doses tds for 7 - 10 daysclindamycin 150 to 300 mg: qds for 7 - 10 days.amoxicillin 40 mg / kg / day in divided doses tdsfor 7 - 10 days.into 3 or 4 equal doses for 7 - 10 days.clindamycin 8 - 16 mg / kg / day (4 - 8 mg / lb / day) dividedoral hsvacyclovir 800 mg, 5 times a day for 7 - 10 daysacyclovir 10 mg / kg qds or 5 times per day.famciclovir 500 mg tds for 7 dayshsv prophylaxis: acyclovir10 mg / kg bd, or tdssinusitiscd4>200 cells / mmamoxicillin 40 mg / kg / day tds in divided doses amoxicillin 1.5 - 4 g / dayamoxicillin / clavulanate 125/31 tds between 1 - 6 years, amoxicillin / clavulanate: 1.75 - 4/250 g / day250/62 between 6 - 12 years cefuroxime axetil 500 mg bdcefuroxime axetil 20 - 30 mg / kg daily given in 2 divided trimethoprim / sulfamethoxazole:160 mg-800 mg orally bddoses in children> 4 weeks of age telithromycintrimethoprim / sulfamethoxazole erythromycin, clarithromycin, azithromycin6 - 10 mg / kg / day orally in children> 2 months of agecd4<200 cells / mm or failure of above therapyerythromycin, clarithromycin, azithromycin fluoroquinolone + clindamycin or metronidazoleod, once daily, bd, twice daily; tds, every 8 h; qds, every 6 hbd, twice daily; tds, every 8 h.*severe cases: acyclovir 10 mg / kg iv tds od, once daily, bd, twice daily; tds, every 8 h; qds, every 6 hbd, twice daily; tds, every 8 h. severe cases: acyclovir 10 mg / kg iv tds angular cheilitis presents as erythema, and/or fissuring in the corners of the mouth . It may co - exist with erythematous or pseudomembranous candidiasis, and persist for an extensive period of time if left untreated . Treatment involves the use of a topical antifungal cream directly applied to the affected areas four times a day for two weeks . It most commonly presents as plaques similar to the ones found in non - hiv populations . Linear gingival erythema (figure 4) presents as a red band along the gingival margin, accompanied by occasional bleeding and discomfort . It most frequently appears at the anterior teeth, but can also extend to the posterior teeth . It can also present on attached and non - attached gingiva as petechia - like patches . Treatment includes debridement by the dentist, mouth rinses with a 0.12% chlorhexidine gluconate suspension twice daily for two weeks, and home oral hygiene . In contrast, necrotizing gingivitis and necrotizing periodontitis (figure 5) can result in the rapid destruction of soft tissue in the former and hard tissue in the latter condition . It is characterized by severe pain (often described by patients as deep jaw pain), loosening of the teeth, bleeding, fetid odor, ulcerated gingival papillae, and rapid loss of bone and soft tissue . Intervention is usually intensive curettage and debridement of all involved tissues, and use of topical antiseptic agents, such as 0.12% chlorhexidine gluconate or 10% povidoneiodine lavage . More severe cases should be supplemented by a short course of systemic antimicrobial therapy, usually metronidazole . Oral infections with herpes simplex virus (hsv) (figure 6) occur in up to 9% of adults and 1.324% of children with hiv . Oral hsv presents as a small crop of vesicles which produce small, painful ulcerations extending onto the adjacent skin, and may coalesce to form giant herpetic lesions . Although their clinical features are similar to non - hiv infected patients, the lesions are often bigger in hiv patients, recur more frequently, and tend to be more persistent . Lesions most commonly appear on the lips, in the mouth, hard palate, and gums . Although they are typically self - limiting, the use of antiviral agents, such as acyclovir, is sometimes required . Hsv ulcers may become chronic in children with severe hiv, and convert into true membranes which may require hospital admission and intravenous administration of acyclovir (table 1). Figure 6oral herpes simplex virus . Oral hairy leukoplakia (figure 7) is a largely asymptomatic condition of the tongue caused by the epstein - barr virus . The prevalence of oral hairy leukoplakia varies from 0.4238% in hiv - infected adults to around 2% in children . The terminology of this condition arises from the appearance of elongated filiform papillae which can be accompanied by white plaquelike changes . In such cases, good results have been reported with topical application of trichloracetic or glycolic acid, podophyllum resin solution 25% and oral acyclovir . Oral hairy leukoplakia . Oral human papilloma - virus infection (hpv) (figure 8) has increased in the era of highly active antiretroviral therapy (haart therapeutic regimens). This suggests that a drug or combination of drugs used to treat hiv may be a risk factor for oral hpv infection . The most common hpv subtypes found in the oral cavity are subtypes 16 and 18, which may be related to oral sexual behavior . The warts may be cauliflower - like, spiked, or raised with a flat surface . Treatment involves surgery (laser, or cryotherapy) which may need to be repeated due to the frequent recurrence of the lesions . . Top hiv med 2005;13:143 - 8 . Oral hpv lesion . Reproduced with permission from ias - usa . Kaposi's sarcoma (figure 9) is still the most common oral malignancy seen among patients with hiv . The prevalence of oral kaposi's sarcoma of the mouth varies from 012% in africa and 038% in usa and europe . The oral cavity is commonly affected and is the first clinical site of kaposi's sarcoma in 20% of cases, while it occurs concomitantly with skin and visceral involvement in up to 70% of patients . Within the oral cavity, the hard palate is the most frequently involved, followed by the gingival and buccal mucosa, as well as the dorsum of the tongue . Kaposi's sarcoma - associated herpes virus was proven to be a co - factor in the presentation of kaposi's sarcoma in patients with hiv . Early lesions tend to be red, flat and asymptomatic, with the color becoming darker as the lesion ages . Following the diagnosis of kaposi's sarcoma, oral hygiene is necessary, and topical injections of chemotherapeutic agents, such as vinblastine sulfate, or even surgical removal or radiation therapy can be considered for treatment . Systemic chemotherapy should be reserved for patients with both oral and extra - oral kaposi's sarcoma . Non - hodgkin's lymphoma (nhl) is the second most common malignant condition associated with hiv infection . Lymphomas present as a focal, ulcerated soft tissue mass on the palate or gingival tissues, which may be red and inflamed . Many studies have shown that the chop regimen (cyclophosphamide, doxorubicin, vincristine and prednisone) can be considered the standard approach for patients with aggressive nhl in the context of hiv infection . Surgical debulking may be required for pain relief and improvement of chewing, swallowing, and speech in large exophytic or pedunculated lesions, whereas radiotherapy may be considered for large lesions which cannot be easily accessed . In addition to reactive lymphadenitis, cervical lymphadenopathy may result from tuberculosis, lymphoma, or kaposi's sarcoma in hiv patients . The term hiv lymphadenopathy describes the presence of diffuse lymphadenopathy in two or more sites of the neck for longer than three months . This can occur in up to 70% of hiv patients within the first few months after seroconversion, even before any other symptoms of hiv infection appear . The lymph nodes are soft and symmetrical, ranging from 1 to 5 cm in diameter . Fine needle aspiration is indicated in cases of asymmetry, rapidly enlarged lymph nodes, or any other suspicious features . Biopsy under local or general anesthetic may be necessary in cases of high suspicion for lymphoma . It usually involves the parotid glands, tends to be bilateral, sometimes cystic, and can be accompanied by generalized lymphadenopathy . Typically, the patient presents with a history of progressive parotid swelling with minimal tenderness over several months . Salivary gland enlargement occurs in approximately 3 to 30% of adult patients infected with hiv, and in up to 30% of infected children . Clinical examination should include assessment of the characteristics of the mass (i.e. Fixation) and the function of the facial nerve . The three common causes of parotid enlargement in hiv - infected patients are reactive hyperplasia of an intraparotid lymph node, benign lymphoepithelial lesions with ductal metaplasia, and benign lymphoepithelial cysts . Fine - needle aspiration is an effective method of distinguishing benign from malignant parotid lumps . Aspiration of the cystic lesions can be of some temporary benefit, and injections of tetracycline and doxycycline have been shown to be successful, although with limitations due to the presence of multiple cysts . Alternatively, external irradiation can be considered (24 gy in 1.5 gy daily fractions), with overall acceptable cosmetic and long - term functional results . It involves all three parts of the ear (external, middle, inner), with a cumulative frequency of 2080% in both adults and pediatric patients . Indeed, seborrheic dermatitis has been reported in up to 83% of patients, and usually involves the periauricular area . Otitis externa, on the other hand, is usually caused by pseudomonas aeruginosa, whilst candida albicans is often the cause of otomycosis . Otalgia is a very frequent symptom in hiv patients, which can be attributed to the disproportionately severe inflammatory changes in the mastoid air - cells even in otherwise asymptomatic carriers . Otitis media with effusion secondary to nasopharyngeal lymphoid hyperplasia or other nasopharyngeal masses is also not uncommon in hiv - positive patients . Acute otitis media may also occur, but is usually seen in patients with end - stage hiv disease . Finally, an increased prevalence of pneumocystis carinii - infected aural polyps has been reported in hiv patients with chronic otitis media . Treatment in cases of middle ear infection usually includes broad - spectrum antibiotics, whereas mastoid exploration may be necessary in cases unresponsive to conservative treatment (figures 1012). Please note the difference in the signal on the right (circle) compared to the left side . Please note the difference in the signal on the right (circle) compared to the left side . Sensorineural hearing loss, either unilateral or bilateral, occurs in 2149% of hiv - infected patients . Most patients show down - slopping hearing loss, usually moderate in the high frequencies, whereas speech discrimination is not significantly affected . This type of hearing loss cannot be easily explained, as a histological study of the organ of corti in hiv patients did not reveal any abnormality, except from some cystic changes in the spiral ligament and stria vascularis . Possible explanations include the involvement of either retrocohlear pathways, or the cochlear nerve itself . Indeed, hiv was shown to induce neuropathological changes and damage to the central nervous system, particularly subcortical demyelination, in a large percentage of infected individuals, even in the absence of gross neurological manifestations . Other causes of sensorineural hearing loss such as neoplasms, and ototoxic agents should also be excluded . If the hearing loss affects the patient's everyday listening activities and quality of life, the provision of digital hearing aids should be considered . Hiv patients also tend to experience significant disequilibrium, which is also often clinically attributed to central nervous system pathology . However, inner ear abnormalities have also been reported (i.e. Sub - epithelial elevation of the neurosensory epithelium of the saccule and utricle, inflammatory endolymphatic precipitations) and may also be important in the pathogenesis of vertigo in these patients . Finally, unilateral and bilateral facial nerve palsy is a condition that occurs with a 100-fold greater frequency in the hiv infected population (4.1% vs 0.04%) (table 2). It may precede the appearance of hiv antibodies, and seems to occur more frequently in hiv carriers than patients with fullblown aids . Peripheral facial nerve neuropathy is usually self - limiting, and may either be idiopathic, or due to herpes virus infection (ramsey hunt syndrome). Treatment includes acyclovir 800 mg five times daily for seven days, and administration of prednisolone 30 mg once daily for five days, with tapering of the starting dose in three - day intervals . Facial nerve palsy can also be seen in end - stage patients either as an isolated entity, or as part of multiple cranial nerve involvement . However, it is usually secondary to opportunistic infections or intracranial tumors . Table 2prevalence of hiv - associated ear, nose and throat (ent) manifestation in adults and children.common hiv - associated ent manifestationsprevalence in adultsprevalence in childrenoral manifestations oral candidiasis30 - 90%22.5 - 83.3% periodontal and gingival disease4%20% herpes simplex virus infection9%1.3 - 24% oral hairy leukoplakia0.42 - 38%2%neck manifestations cervical lymphadenopathy70%70% parotid gland enlargement3 - 30%30%nasal manifestations allergic rhinitis70%n.r . Sinusitis30 - 68%24%otological manifestations otitis externa5%4% otitis media13%46% snhl21 - 49%n.r . Nasal manifestations are not uncommon among hiv patients . Indeed, rhinosinusitis seems to occur with a prevalence of 1170% in different studies . Although cellular immunity is compromised in aids, studies have shown excessive production of ige, which can be suggestive of allergic rhinitis (in the absence of active parasitic infections). Reported a 2-fold increase in the incidence of allergic symptoms in hiv - infected men, which may reach 87% after the infection . Treatment is challenging, due to the risk of iatrogenic cushing's syndrome from the intranasally - administered steroids, in patients receiving ritonavir - containing antiretroviral regimens (table 3). Budesonide is preferred over fluticasone, due to the significantly longer half - life of the latter, whereas montelukast can be successfully used . Table 3interactions between commonly administered ear, nose and throat medications and antiretroviral drugs.ent medicationinteraction with antiretroviral drug / potential clinical effectsmanagementitraconazoleinhibition of cyp450 3a4 increased darunavir and itraconazole effects when itraconazole is combined with darunavir (darunavir also inhibits cyp450 3a4)decreased itraconazole effects when combined with didanosine, (decreased gastric acidity from the antacid buffer contained within didanosine tablets / suspension resulting in decreased itraconazole absorption)decreased itraconazole effects when combined with efavirenz (efavirenz induces cyp450 3a4)increased indinavir effects when combined with indinavir (due to cyp450 3a4 inhibition)increased lopinavir / ritonavir and itraconazole effects when combined with lopinavir / ritonavir (lopinavir / ritonavir also inhibits cyp450 3a4)increased ritonavir effects when combined with ritonavir (due to cyp450 3a4 inhibition)increased saquinavir and itraconazole effects when combined with saquinavir (saquinavir also inhibits cyp450 3a4)if co - administration with darunavir is required, the dose of itraconazole should not exceed 200 mg dailyadminister itraconazole capsules at least 2 h after didanosine tablets / suspensiondo not co - administer with efavirenzdecrease indinavir to 600 mg tdsdo not exceed itraconazole 200 mg bdmanufacturer recommends against using high doses of itraconazole (including drops) with lopinavir / ritonavir (200 mg daily)dose adjustment when combined with ritanovir is not establishedconsider reducing itraconazole to 100 mg bd when combined with saquinavirvoriconazoleinhibition of cyp450 3a4decreased yoriconazole effects when combined with atazanavir/ darunavir (due to possible induction of cyp450)possibly increased etravirine effects when combined with etravirine: (due to cyp450 3a4 inhibition)decreased voriconazole levels when combined with lopinavir / ritonavir (due to possible induction of cyp450 by ritonavir)decreased voriconazole effects when combined with ritonavir (due to induction of cyp450 3a4)decreased voriconazole effects and increased efavirenz effects when combined with efavirenz (efavirenz induces cyp450 3a4)do not co - administer with atazanavir/ darunavirno dose adjustment is necessary when combined with etravirinedo not co - administer with lopinavir / ritonaviravoid co - administration with ritonavirdo not co - administer with efavirenz at standard doses; increase voriconazole to 400 mg bd, and decrease efavirenz to 300 mg qhsfluconazoleinhibition of cyp450 3a4increased etravirine, nevirapine, saquinavir, tipranavir, zidovudine effectsno dose adjustment necessaryketoconazoleinhibition of cyp450 3a4increased darunavir and ketoconazole effects when ketoconazole is combined with darunavir (darunavir also inhibits cyp450 3a4)increased delavirdine effects when combined with delavirdine (due to cyp450 3a4 inhibition)when combined with didanosine, possibly decreased didanosine effects (decreased gastric acidity from the antacid buffer contained within didanosine tablets / suspension resulting in decreased ketoconazole absorption)decreased ketoconazole effects when combined with efavirenz (efavirenz induces cyp450 3a4)increased indinavir effects when combined with indinavir (due to cyp450 3a4 inhibition)when combined with lopinavir / ritonavir, increased ketoconazole effects, and decreased lopinavir / ritonavir effectsincreased maraviroc effects when combined with maraviroc (due to cyp450 3a4 inhibition)increased nelfinavir effects when combined with nelfinavir (due to cyp450 3a4 inhibition)decreased ketoconazole effects when combined with nevirapine (nevirapine induces cyp450 3a4)decreased ketoconazole effects when combined with rilpivirineincreased ritonavir/ saquinavir effects when combined with ritonavir/ saquinavir (due to cyp450 3a4 inhibition)dose adjustment when combined with darunavir is not establishedif co - administration is required, the dose of ketoconazole should not exceed 200 mg dailyno dose adjustment necessary when combined with delavirdineconsider didanosine ecadminister ketoconazole at least 2 h prior to didanosine tablets / suspensiondo not co - administer with efavirenzconsider decreasing indinavir to 600 mg tdsmanufacturer recommends against using high doses of ketoconazole (including drops) with lopinavir / ritonavir (200 mg daily)reduce maraviroc dose to 150 mg bd when used with ketoconazolno dose adjustment necessary when combined with nelfinavirdo not co - administer with nevirapineno dose adjustment necessary when combined with rilpivirine, but monitoring for potential failure of antifungal therapy is requireddose adjustment when combined with ritonavir/ saquinavir is not establishedcyp450, cytochrome p450 enzyme complex - family 3-subfamily a - polypeptide 4; bd, twice daily; tds, every 8 h; qhs, at bedtime . Cyp450, cytochrome p450 enzyme complex - family 3-subfamily a - polypeptide 4; bd, twice daily; tds, every 8 h; qhs, at bedtime . In addition to compromised immunity, impaired mucocilliary clearance has also been reported, and can be held accountable for the high prevalence of rhinosinusitis in hiv patients . There is no difference in bacteriology compared with the general population, including streptococcus pneumoniae, haemophilus influenzae, moraxella catarrhalis, and streptococcus viridans in acute episodes, whereas staphylococcus aureus, staphylococcus epidermidis, and anaerobes are seen in chronic cases . However, atypical bacteria can also play an important role and need to be considered in the treatment of these patients, especially in cases of decreased cd4 count (i.e. Alternaria alternata, aspergillus, pseudallescheria boydii, cryptococcus, candida albicans, acanthamoeba castellani, microsporidian, and legionella pneumophila). Standard outpatient medical therapy with oral antibiotics for three weeks and nasal decongestants are often sufficient . In chronic cases, oral antibiotics in cases of acute infection include amoxicillin (standard or high doses), co - amoxiclav (standard or high doses), or cefuroxime . Trimethoprim / sulfamethoxazole or macrolides can be used in -lactam - allergic patients, but failure rates are higher . If the response to antibiotic therapy is partial, or when the cd4 count is less than 200 cells per mm, and in cases of chronic infection, the coverage should be broadened to include pseudomonas, staphylococci, and anaerobic species . Appropriate oral treatment in these cases includes the combination of fluoroquinolone and clindamycin or metronidazole . The combination of two antimicrobial agents with activity against pseudomonas aeruginosa was shown to improve mortality in patients with hiv with pseudomonas infection, compared with monotherapy . Patients with persisting symptoms require nasal endoscopy with culture of the obtained swabs, and a ct scan of the sinuses, with the view of performing endoscopic drainage; intravenous antibiotics can also be used . Patients with no improvement after maximal medical treatment can be considered candidates for functional endoscopic sinus surgery, especially when anatomic variations of the nasal and paranasal cavities which predispose to sinus disease are present . Furthermore, surgical management is required in cases of invasive fungal sinusitis, with debridement of infected bone and tissue, correction of the conditions that predispose to infection, and antifungal medication . The use of liposomal amphotericin b which shows increased cure rates and decreased drug toxicity, compared with conventional amphotericin b therapy, should be preferred . Washings with solutions containing amphotericine - b and water for injection may also prove useful . Hiv is a global pandemic that affects millions of adults and children in the developed and developing countries . The prevalence of hiv has risen by 27% compared to the previous decade, although the annual rate of new cases had been steadily declining since the late 1990s . Up to 80% of hiv - infected patients eventually develop ent manifestations . Among ent manifestations, oral disease seems to be the most common, occurring in approximately 4050% of hiv positive patients . Clotrimazole troches, nystatin oral suspension, and nystatin pastilles can be used in the treatment of mild to moderate cases, whereas systemic administration of fluconazole, intraconazole and voriconazole is warranted in moderate to severe cases . Oral hpv infection has also increased in the era of haart therapeutic regimens, suggesting that a drug or combination of drugs used to treat hiv may be a risk factor for the former . Kaposi's sarcoma is still the most common oral malignancy seen among patients with hiv . This requires oral hygiene and topical injections of chemotherapeutic agents; surgical removal or radiation therapy can also be considered for treatment . Hiv lymphadenopathy may be the result of reactive lymphadenitis, tuberculosis, lymphoma, or kaposi's sarcoma, and is the most common manifestation of hiv infection in the neck . Salivary gland disease is also not uncommon, but its treatment still remains controversial in the presence of hiv disease . Superficial parotidectomy has been proposed, and external irradiation seems to provide overall acceptable cosmetic and long - term functional results . Acute otitis media is usually seen in patients with end - stage hiv disease and usually requires broad - spectrum antibiotics . Hiv patients also tend to experience a 100-fold greater frequency of unilateral and bilateral facial nerve palsy, which usually requires treatment with acyclovir and prednisolone (in a tapered dosologic regimen). Although there is no difference in the bacteriology of rhinosinusitis compared with the general population, atypical bacteria can also play an important role and need to be considered in patient treatment, especially in cases of decreased cd4 count . Oral antibiotics in cases of acute infection include amoxicillin, coamoxiclav, or cefuroxime, whereas trimethoprim / sulfamethoxazole or macrolides can be used in cases of allergy . In cases of chronic infection or low cd4 count patients with no improvement after maximal medical treatment, and cases of invasive fungal sinusitis are considered candidates for functional endoscopic sinus surgery . Although ent symptoms are not diagnostic of the disease, they might be suggestive of hiv infection, or related to its progression and the respective treatment failure . In the era of haart ent manifestations of hiv it is important that ent doctors are aware of the ent conditions associated with hiv disease, and the respective diagnosis and treatment . A multi - disciplinary approach may be required to provide the appropriate level of care to hiv patients.
|
Median arcuate ligament syndrome (mals, also called celiac axis compression syndrome or dunbar syndrome) is known to be caused by compression of celiac artery (ca) by a fibrous arch that originates from the diaphragmatic crura on either side of the aortic hiatus and passes superior to the origin of the celiac axis . Though it is still debated whether the compression of the ca can cause chronic mesenteric ischemia or not, there have been anecdotal case reports of surgical decompression of ca by dividing the medial arcuate ligament (mal). The common clinical features of mals are chronic postprandial abdominal pain characteristically augmented by full expiration, nausea or vomiting, weight loss and audible epigastric bruit . The diagnosis of mals usually depends on the clinical features and radiologic finding of the focal narrowing at the proximal celiac axis on a lateral view of conventional aortography or computed tomography (ct). While open surgical release of mal has been used for the treatment of this syndrome, laparoscopic release has been reported recently . However, it was usually ineffective owing to the refractory extrinsic compression of the ca by the tight ligament structure . We would like to report a case of surgical treatment of mals describing its clinical features, characteristic findings of diagnostic imaging study, and details of our surgical procedure . A 37-year - old female patient presented with chronic epigastric pain lasting 6 months, food phobia and weight loss of 10 kg during the previous 4 months . The pain was cramping aggravated after meals, and persisted for 20 - 30 minutes after meals . There were no other gastrointestinal symptoms such as diarrhea, constipation, gastrointestinal bleeding and nausea or vomiting . As past history, she underwent coil embolization of the bilateral ovarian veins at another hospital 2 months before visiting us under the impression of pelvic congestion syndrome, which did not improve her abdominal symptoms . Physical examination showed mild abdominal tenderness at both lower quadrants without muscle rigidity . Laboratory test showed normal range including serum amylase, liver enzyme and complete blood cell count . Gastro - duodenoscopy showed chronic atrophic gastritis and 18-fluorodeoxyglucose positron emission tomography - ct showed no spe cific lesion with abnormal hot uptake . 1) showed downward angulation of ca and superior mesenteric artery (sma) close to their origins by the compression . To decompress ca, we approached through the upper mid line incision and lesser sac . Postoperatively, epigastric postprandial pain was resolved and the patient could return to a normal diet . On a follow - up mal was first described by lipshutz as an anatomic structure that caused ca compression in 1917 . Thereafter, harjola and dunbar et al . Described it as a clinical syndrome causing nausea, vomiting and postprandial pain in 1963 and 1965 . The origin of ca and mal varies in its location from t11 to l1, and occasionally, their locations are in conflict with each other when mal extends inferiorly or ca originates su periorly . During deep inspiration in the erect position during expiration, the condition is opposite, and compression causes the symptom and is thus called mals . At this point, it is proposed that mals is related with neurogenic pain from the compression and intermittent ischemia of splanchnic nerve plexus . This pain can be caused either by nerve stimulation leading to vasoconstriction or by direct sympathetic fiber irritation . Splanchnic nerve plexus is an autonomic nerve plexus supplying upper abdominal organs (stomach, liver, gallbladder, pancreas). This is located in front of diaphragmatic crura around the origin of ca and sma . To make a diagnosis of mals, other common causes of abdominal pain routine laboratory blood tests including amylase, lipase and tumor markers for hidden malignancy, esophago - gastro - duodenoscopy, liver, pancreas and kidney ultrasonography are usually used . A typical feature is focal narrowing of ca with poststenotic dilatation aggravated during deep inspiration . Nowadays standard treatment of mals is an open surgical division of mal followed by a dramatic symptom relief . Recently, a lapa roscopic approach can be attempted but carries the risk of arterial injury and massive hemorrhage . Three series of case reports showed mean rates of open conversion due to bleeding at about 20%, but despite that, laparoscopic treatment showed no other morbidity or mortality and shortened hospital stay . This is probably due to the extraluminal compression by mal, which should be solved outside of the ca . In cases of recurrent symptom after surgical decompression of ca, angioplasty is beneficial.
|
Cyclodextrins (cds) are well - known objects of supramolecular chemistry and glycoscience (dodziuk 2006; philp and stoddart 1996; szejti and osa 1996; wenz 1994). They are products of amylopectin enzymatic destruction by the action of extracellular enzymes, cyclodextrin glycosyltransferases (cgtase, e.c . The more abundant and available of the cds are cyclic oligosaccharides, consisting of 6, 7, 8 d - glucose residues, connected with (1 4) links and named -, - and -cd, respectively . The shape of these molecules resemble a hollow truncated cone with a central cavity, containing c3h and c5h carbons atoms and ester - like o-4 and o-5 oxygen atoms . The cd structure provides an external hydrophilic region and a rather hydrophobic inner cavity (bender and komiyama 1978). The number of sugar rings defines the size of the cavity and the flexibility of the cds . Cds and their derivatives are well - known as molecular hosts capable of including, in their cavities, different guest molecules of appropriate size, shape, and polarity via non - covalent interactions (connors 1997; dodziuk 2006; harada 1997; saenger 1980; szejti 2004). The apolar nature of their cavities (harada 1997) allows cds to act as hosts preferentially for nonpolar guests, which include small molecules and surfactants (harada 1997; gonzlez - prez et al . The interactions of cds with natural and synthetic polymers bearing attached hydrophobic tags have also been intensively studied (beheshti et al . In addition, cd derivatives are being used as a core in the synthesis of star - like polymer systems (hoogenboom et al . Cyclodextrins ability to form inclusion complexes with appropriate sized hydrophobic guest molecules is the most frequently applied property of cds (hedges 1998; szejti 2004) and has been studied by spectroscopic, kinetic, and crystallographic methods (dodziuk 2006; saenger et al . Cds and their derivatives are considered to be potential carriers for hydrophobic pharmaceutical compounds (loftsson and duchene 2007; uekama et al . 1998). However, information about the properties of cyclodextrins molecules in diluted solution is still rather scarce (dodziuk 2006; nakata et al . 2003; longsworth 1953; szejti 1998). In this study, we present investigations on -, - and -cyclodextrin by macromolecular hydrodynamics . The cds studied, -, - and -cyclodextrin, were obtained commercially from sigma (purity 98%) (table 1). Velocity sedimentation, the translational diffusion, and viscosity as well as the increment of density were measured in three different solvents: water, dimethylformamide, and dimethylsulfoxide.table 1images of modeling structures, calculated molecular mass (mcalc), inner (dii) and outer (d0i) diameters and the values of molecular mass obtained with maldi - tof ms of cyclodextrin moleculescdimages of modeling structuresmcalc (g / mol)diia (10 cm)d0ia (10 cm)m (maldi) (g / mol)-cd (c6h10o5)6972.95.04.714.6972.4-cd (c6h10o5)71,135.06.56.015.41,134.5-cd (c6h10o5)81,297.18.37.517.51,296.6as measured on space - filling or cpk models (saenger 1980; corey and pauling 1953) images of modeling structures, calculated molecular mass (mcalc), inner (dii) and outer (d0i) diameters and the values of molecular mass obtained with maldi - tof ms of cyclodextrin molecules as measured on space - filling or cpk models (saenger 1980; corey and pauling 1953) sedimentation velocity experiments were performed on a beckman xli analytical ultracentrifuge at a rotor speed of 55,000 rpm and at 20c in al - double - sector cells of optical path 12 mm using interference optics . The evaluation program sedfit for continuous particle size distributions (schuck 2000) was used for data analysis . The regularization method used was the tikhonov - philips 2nd derivative, and the confidence level (f ratio) chosen was 0.80.9 . By fitting for (f / fsph) in a nonlinear regression, an estimate of the weight - average frictional ratio of all macromolecules in solution is obtained, where f is the frictional ratio of the solute macromolecule and fsph is the frictional ratio of the rigid sphere with the same anhydrous volume (free of solvent) as the macromolecule . The final result is the differential distribution (dc(s)/ds) of the sample, which is named c(s). It is scaled such that the area under the c(s) curve between the smallest s value, s1, and the largest one, s2, in the distribution will give the loading concentration of macromolecules between these sedimentation coefficients (expressed in number of fringes, j, in the case of interference optics). J, which is proportional to the polymer concentration in solution, was used to calculate the refractive index increment: (n/c) = j/kcl (pavlov et al . 2003), where is the wavelength (675 nm), k the magnifying coefficient and l the optical path . With k = 1 and l = 12 mm we obtain: n/c = 5.625 10(j / c) and c in g / cm . Translational diffusion was studied by the classical method of forming a boundary between the solution and the solvent on tsvetkov polarizing diffusiometer (tsvetkov 1989). The diffusion boundary was formed in glass cell of length h = 30 mm along the beam path . The optical system used for recording the solution - solvent boundary in diffusion analysis was a lebedev s polarizing interferometer (lebedev 1930). Translational diffusion coefficients were calculated from the equation: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\sigma ^2 = \sigma _ 0 ^ 2 + 2dt,$$\end{document}where is the dispersion of the diffusion boundary calculated from the maximum ordinate and the area under the diffusion curve, 02 is the zero dispersion characterizing the quality of boundary formation, and t is the diffusion time . Experiments were carried out at 25c, and the intrinsic diffusion coefficient, which depends only on the macromolecule properties, is calculated as: [d] = d00/t . The respective flow times, 0 and t, were measured at 25c for the solvent and polymer solutions, with relative viscosities r = t/0 . The extrapolation to zero concentration was made by using both the huggins and kraemer equations (cantor and schimmel 1980; tsvetkov 1989) and the average values were considered as the value of intrinsic viscosity . The density measurements were carried out in the density meter dma 5000 (anton paar, graz, austria) according to the procedure of kratky et al . The cyclodextrins were investigated also by matrix - assisted laser desorption / ionisation time - of - flight mass spectrometry (maldi - tof ms). Maldi - tof ms measurements were performed with an ultraflex iii tof / tof (bruker daltonics, bremen, germany) equipped with a nd: yag laser and a collision cell . For the ms / ms mode, argon was used as collision gas at a pressure of 2 10 mbar . The instrument was calibrated prior to each measurement with an external pmma standard from pss polymer standards services gmbh (mainz, germany) in the required measurement range . Ms and ms / ms data were processed using polytools 1.0 and an isotope pattern calculator . The velocity sedimentation and isothermal translational diffusion studies were made separately in three different solvents: water, dimethylformamide (dmf) and dimethylsulfoxide (dmso). The solubility of the cds studied in these solvents increases in the following order: h2o <dmf <dmso . The velocity sedimentation experiments were run overnight (1214 h), at a solute concentration c 4 mg / ml . Figure 1 represents the sedimentation interference profiles of -cyclodextrin in dmf and in dmso as well as the calculated distribution of the sedimentation coefficients, c(s), as obtained by the use of the sedfit program . Figure 2 shows the comparison of the normalized differential distributions for -cd, obtained in the different solvents . The density increment (/c), which is also required for the quantitative interpretation of the sedimentation data, allows the determination of the partial specific volume \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left ({\bar{\upsilon}} \right). $$\end{document} the value remained the same for different cds in the same solvent and was found to be 0.667, 0.632, and 0.649 cm / g in h2o, dmf, and dmso, respectively . The refractive index increment (n/c) also remained virtually the same for different cds in the same solvent: 0.148, 0.104, and 0.07 cm / g in h2o, dmf, and dmso, respectively . The translational diffusion was studied at an average solute concentration c of 12 mg / ml (fig . . 1velocity sedimentation of cds: experimental data and evaluations obtained with the sedfit program . A -cd with c = 4.2 10 g / cm in dmf, b the same solute with c = 4.8 10 g / cm in dmso . Panels at the top show the superposition of some interference profiles on the whole range of sedimentation time (12 h), those at the middle the corresponding residual plots . The panels at the bottom represent the distribution of sedimentation coefficients, c(s), obtained with a regularization procedure with a confidence level of 0.70fig . Cn(s), of -cd in h2o (1), dmf (2), and dmso (3), as obtained with the sedfit program . For clearness each distribution is normalized on the maximal value of cmax(s) such as cn(s) c(s)/cmax(s) to eliminate the influence of different increments of refractive indexesfig . 3time dependence of dispersion of the diffusion boundary 2 versus time t of diffusion in h2o (a) and dmf (b), for -cd (1), -cd (2), and -cd (3) velocity sedimentation of cds: experimental data and evaluations obtained with the sedfit program . A -cd with c = 4.2 10 g / cm in dmf, b the same solute with c = 4.8 10 g / cm in dmso . Panels at the top show the superposition of some interference profiles on the whole range of sedimentation time (12 h), those at the middle the corresponding residual plots . The panels at the bottom represent the distribution of sedimentation coefficients, c(s), obtained with a regularization procedure with a confidence level of 0.70 comparison of the normalized differential distributions of sedimentation coefficients, cn(s), of -cd in h2o (1), dmf (2), and dmso (3), as obtained with the sedfit program . For clearness each distribution is normalized on the maximal value of cmax(s) such as cn(s) c(s)/cmax(s) to eliminate the influence of different increments of refractive indexes time dependence of dispersion of the diffusion boundary 2 versus time t of diffusion in h2o (a) and dmf (b), for -cd (1), -cd (2), and -cd (3) the values of both the sedimentation coefficient s and the diffusion coefficient d obtained at the concentrations given above were assumed to be equal to the values extrapolated to zero concentration . The solute concentrations practically correspond to the limiting dilution as the values of the debye parameter (c[]), describing dilution of a solution, is within the limits of 0.01 c[] 0.03 . The plots of sp / c and ln r / c as a function of c allowed us to determine the intrinsic viscosity, [], of the cyclodextrins (fig . Table 2 represents the values of s, (f / fsph), d and [] obtained.fig . 4determination of the intrinsic viscosity values [] (which are the intercepts, at c = 0, of the plots of sp / c (solid lines, huggins plot) and ln r / c [dashed lines, kramer plot] vs. c) for -cd (1), -cd (2), and -cd (3) in dmso . The huggins parameter kh are 0.39, 0.22, and 0, and the kramer parameter kk are 0.11, 0.24, and 0.40 for -, -, and -cd, respectivelytable 2translational diffusion coefficient d, velocity sedimentation coefficient s, frictional ratio f / fsph, and intrinsic viscosity [], of cd in h2o, dmf, and dmsoh2odmfdmso10d (cm / s)s (s)f / fsph10d (cm / s)s (s)f / fsph[] (cm / g)10d (cm / s)s (s)f / fsph[] (cm / g)36.50.481.00340.491.335.59.70.151.385.429.10.511.00290.531.335.28.20.111.436.0270.531.0625.50.581.375.98.450.181.497.5 determination of the intrinsic viscosity values [] (which are the intercepts, at c = 0, of the plots of sp / c (solid lines, huggins plot) and ln r / c [dashed lines, kramer plot] vs. c) for -cd (1), -cd (2), and -cd (3) in dmso . The huggins parameter kh are 0.39, 0.22, and 0, and the kramer parameter kk are 0.11, 0.24, and 0.40 for -, -, and -cd, respectively translational diffusion coefficient d, velocity sedimentation coefficient s, frictional ratio f / fsph, and intrinsic viscosity [], of cd in h2o, dmf, and dmso the different experimental hydrodynamics measurements can be expressed as intrinsic values, [], [s], [d], [f], which are independent of the solvent properties (cantor and schimmel 1980; tsvetkov 1989). Each of them is related to common macromolecular characteristics such as molar mass m and mean - square radius of gyration <r>: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left [\eta \right] = {{\upphi^{\prime} <r^{2}> ^{3/2}} \mathord{\left/ {\vphantom {{\upphi^{\prime} <r^{2}> ^{3/2}} {\text{m}}}} \right . \kern-\nulldelimiterspace} {\text{m}}} $$\end{document}\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left [s \right] \equiv {{s_{0} \eta_{0}} \mathord{\left/ {\vphantom {{s_{0} \eta_{0}} {\left ({1 - \rho_{0} \upsilon} \right)}}} \right . \kern-\nulldelimiterspace} {\left ({1 - \rho_{0} \upsilon} \right)}} = m / n_{\text{a}} p^{\prime} <r^{2}> ^{1/2} $$\end{document}\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left [f \right] = k / p^{\prime} <r^{2}> ^{1/2} $$\end{document}\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left [f \right] \equiv f_{0} /\eta_{0} = p^{\prime} <r^{2}> ^{1/2} $$\end{document}with []: intrinsic viscosity; [s], [d], and [f]: intrinsic coefficients of velocity sedimentation, translational diffusion, and translational friction, correspondingly; na is the avogadro number, and and p are the flory hydrodynamic parameters . These relationships have a general meaning and are valid for any type of molecules / macromolecules . The values of the dimensionless parameters and p depend, however, on the shape and asymmetry of the solute molecules and, in addition, on the hydrodynamic interactions between the different parts of the same molecule which exercise the friction in the liquid medium . The theoretical values of these parameters are obtained by solving the hydrodynamic problem and depend on the models and mathematical approximations . A frequently used concept in biochemistry and polymer science is that of the hydrodynamic equivalent sphere . In this concept, the real molecule is modeled by a rigid sphere which has the same translational frictional coefficient . The radius of the hydrodynamic equivalent sphere is calculated from the stokes relation (5). In the case of the intrinsic viscosity the einstein relation (2) is applied.\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left [f \right]_{\text{sph}} = 6\pi r_{\text{sph}} $$\end{document}\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left [\eta \right]_{\text{sph}} = \left ({10/3} \right)\pi n_{\text{a}} \left ({r_{\text{sph}}^{3} /m} \right) $$\end{document} obviously the radius of a hydrodynamic equivalent sphere can differ considerably from the size of the real molecule . Nevertheless, this approach is useful, in particular when sizes in a series of polymerhomologues are to be compared, e.g., for proteins, dendrimers, or highly branched macromolecules . 3 and 4 leads to the svedberg equation (cantor and schimmel 1980; maechtle and boerger 2006; tsvetkov 1989) used for molar mass determination from the hydrodynamic data:\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$m_{\text{sd}} = \left ({rt/\left ({1 - \upsilon \rho_{0}} \right)} \right)\left ({s_{0} /d_{0}} \right) = \left ({n_{\text{a}} /\left ({1 - \upsilon \rho_{0}} \right)} \right)s_{0} f_{0} $$\end{document} the translational friction coefficient f0 of molecules may be expressed in our case by the following way:\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$f_{0} = \left ({f / f_{\text{sph}}} \right)_{0} f_{\text{sph}} = \left ({f / f_{\text{sph}}} \right)_{0} 6\pi \eta_{0} \left ({3{\text{m}}\upsilon_{\text{bar}} /4\pi n_{\text{a}}} \right)^{1/3} $$\end{document} from the eqs . 4 and 7 it is possible to calculate the translational diffusion coefficients and, correspondingly, the intrinsic translational diffusion coefficient:\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left [d \right]_{\text{sf}} = k \times (9\pi 2^{1/2}) ^ {- 1} \left ({\left ({f / f_{\text{sph}}} \right)_{0}} \right)^ {- 3/2} (\left [s \right]\upsilon)) ^ {- 1/2} $$\end{document} linking s0 and [d]sf in the equation obtained from eqs . 6 and 8 allows us to determine the molar mass of moving molecules using:\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$m_{\text{sf}} = 9\pi 2^{1/2} n_{\text{a}} \left ({\left [s \right]\left ({f / f_{\text{sph}}} \right)_{0}} \right)^{3/2} \upsilon^{1/2}. $$\end{document} this equation is the transformation of original svedberg equation 6 which, in turn, when f / fsph = 1 is transformed into the relationship describing the solid sphere model . Table 3 shows the molar masses and hydrodynamic radii of the cds studied, as determined from the experimental data by use of the equations given above.table 3molar mass (g / mol) and hydrodynamic radii rh 10 cm (9%) of cd in different solventscdh2odmfdmsomavmrhmrhmrh1,1006.79708.21,300101,100 1001,3007.71,1509.11,100111,180 801,5008.51,40010.11,90012.41,600 200 molar mass (g / mol) and hydrodynamic radii rh 10 cm (9%) of cd in different solvents comparison with the m values calculated from the chemical structure of the cds shows that, in all solvents, the experimental data are relatively close to the theoretical ones . The measurements in dmf yield by far the closest agreement, the deviations being below 10% for all samples . But even for the worst result, obtained with -cd in dmso, the deviation is below 50% . These results are highly surprising, since the s values on which they are based are around 0.5 s or even, in the solvent dmso, around 0.15 s. as far as we know, the possibility of correctly or nearly correctly determining such small velocity sedimentation coefficients on the basis of the numerical solution of the lamm equation has not been demonstrated before . It should be noted that, in the experiments described, the evolution of the sedimentation boundary occurs without appreciable boundary displacement . Elementary estimation shows that when the sedimentation coefficient is only 0.1 s the shift of the sedimentation boundary during 12 h of experiment at 55,000 rpm amounts to only 1 mm . To fix this shift by usual methods against a background of significant diffusion spreading obviously is not possible . In order to further strengthen the conclusions described, we have checked the validity of the theoretical m values by maldi - tof taking into account the mass of sodium ion the good correlation is observed between calculated and experimental values of molar masses (table 1).fig . 5maldi - tof - ms spectrum for the cyclodextrins (1 -cd, 2 -cd, 3 -cd) obtained on dihydroxybenzoic acid as matrix and natfa as a source of sodium ions . The figures at the peaks are the molar masses of cyclodextrine isotopes including the mass of one sodium ion maldi - tof - ms spectrum for the cyclodextrins (1 -cd, 2 -cd, 3 -cd) obtained on dihydroxybenzoic acid as matrix and natfa as a source of sodium ions . The figures at the peaks are the molar masses of cyclodextrine isotopes including the mass of one sodium ion the average values for the radii of the hydrodynamic equivalent sphere, calculated from the stokes (5) and einstein (2) relationships are also presented in table 3 . These values characterize certain external sizes of cds molecules . When comparing these data with those measured on space - filling or cpk models (table 1, r0cpk = d0/2) (saenger 1980; corey and pauling 1953), it is interesting to note that (1) both kinds of radii virtually agree in water, and (2) the ratios of the hydrodynamic to the radii r0cpk remain virtually constant in the series of studied cds but vary in different solvents: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$r_{{{\text{h}} _ {2} {\text{o}}}} $$\end{document}/r0cpk = 0.96 0.03 1.0, rdmf / r0cpk = 1.15 0.02, and rdmso / r0cpk = 1.41 0.02 . Since hydrodynamic interactions inside of cds molecules will contribute to the friction of the molecules, different ratios of radii in different solvents may reflect not only different sizes but also different hydrodynamic interactions . A more adequate model for the description of the hydrodynamic behavior of cds molecules can be toroidal particles / molecules . In recent years approaches which aimed at modeling the shape of biopolymers by bead - shell models were extended to the calculation and predictions of biopolymer hydrodynamic properties (garcia de la torre and bloomfield 1977, 1981; allison 1999; garcia de la torre 2001). Applying such methods to toroids, the dependency of the translational diffusion coefficient and of the intrinsic viscosity on the characteristic ratio of toroids, x = ri / r0, was derived (ri, r0: inner and outer radius, respectively) (garcia de la torre 2001). The following interpolating polynomials were obtained for dimensionless values [d]r0/k and 0.01[]m/(nar03):\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left [d \right]r_{0} /k = 0.0620 - 0.00143x + 0.0278x^{2} $$\end{document}\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$0.01[\eta] m/\left ({n_{\text{a}} r_{0}^{3}} \right) = 0.0701 - 0.0365x - 0.00629x^{2} $$\end{document} a comparison of the experimental and computational results is shown in fig . The space - filling models or cpk models data were used to characterize the outer and inner sizes of the cd molecules (saenger 1980; szejti 1998). The details of the deviation of the experimental points from the theoretical curves correlate to the above - obtained estimations concerning the hydrodynamic radii of the cd molecules in different solvents . In the plots shown, r0 is the more crucial parameter since the ordinate directly depends on it whereas the dependence on ri becomes apparent only as a ratio of ri / r0 in the abscissa . Both plots indicate that, in order to superimpose the experimental data to the computational results, it is necessary to increase the outer sizes of cd molecules . In practice, these sizes could be increased in solution by the absorption of a few solvent molecules to the external surface of the cd molecules, forming an absorbed layer . Thus, in solution the outer size of cd molecules may be characterized by an effective radius r0eff = r0 + r0, where r0 is the average thickness of solvent layer . The latter figure probably correlates to the size and the number of the solvent molecules . The size of the solvent molecules can be estimated by the relationship d = (6 m/0na). 3.86, 6.26, and 6.08 10 cm for h2o, dmf, and dmso molecules were obtained, respectively . The calculated results could be fitted to the experimental ones by assuming that the thickness of the solvent layer varies depending on the solvent, amounting for r0 0.5dsolv in water and dmf but r0 dsolv in dmso . Qualitatively the number of dmso molecules must be higher in comparison with both other solvents.fig . 6comparison of experimental hydrodynamic values (13) with the calculated hydrodynamic values for toroidal molecules (4): a characteristic translational diffusion coefficient (1, 1: in h2o, 2, 2: in dmf, 3, 3: in dmso) b intrinsic viscosity (1, 1: in dmf, 2, 2: in dmso) (13): the hydrodynamic values are plotted in function of the space - filling (or cpk) models radii ricpk (average inner) and r0cpk (outer) of the cd molecules (table 1). (13): the hydrodynamic values are plotted in function of effective outer radii r0eff (see text) comparison of experimental hydrodynamic values (13) with the calculated hydrodynamic values for toroidal molecules (4): a characteristic translational diffusion coefficient (1, 1: in h2o, 2, 2: in dmf, 3, 3: in dmso) b intrinsic viscosity (1, 1: in dmf, 2, 2: in dmso) (13): the hydrodynamic values are plotted in function of the space - filling (or cpk) models radii ricpk (average inner) and r0cpk (outer) of the cd molecules (table 1). (13): the hydrodynamic values are plotted in function of effective outer radii r0eff (see text) although, in reality, the cd structure in solution are flexible, whereby cd instant conformations probably differ from each other because of a variation of the valence angles of the glycosidic links and, possibly, a twist - conformation of the sugar rings (french and johnson 2007; saenger et al . Nevertheless, the cd time - average characteristics, as monitored by hydrodynamic methods, can be fully interpreted by a rigid toroid model . The velocity sedimentation and translational diffusion coefficients of cd molecules were measured in water, dmf, and dmso . In dmf and dmso also the intrinsic viscosities could be measured . It was possible to determine from the hydrodynamic data, by use of the sedfit program, relatively accurate values for cd molar mass and size, despite the fact that the sedimentation coefficients were as low as 0.10.5 s. the correspondence of the cd hydrodynamic values with the results calculated for toroids by use of a bead - shell model is demonstrated . This comparison also shows that the outer cd dimensions are solvent - dependent and larger than those obtained from the crystallographic data.
|
From 2000 through 2005, the french national reference centre for salmonella at the institut pasteur in paris reported 829 newport isolates among 69,759 salmonella clinical isolates . During this period and depending on the year, serotype newport ranked between 6th and 10th in prevalence among human serotyped isolates . From 2000 through 2005, the agence franaise de scurit sanitaire des aliments reported 2,160 newport isolates among 101,791 salmonella isolates collected from animals and food products . Antimicrobial drug susceptibility testing was performed on 585 human newport isolates and 342 nonhuman newport isolates by disk diffusion with 32 antimicrobial drugs (additional information available from fxweill@pasteur.fr). Of 585 isolates tested, 46 (7.9%) were resistant to third - generation cepalosporins . The geographic origin of the isolates was mainly the paris metropolitan area and northern france (appendix table). Resistant isolates during 2000 (15%) and 2003 (17.5% caused by a small outbreak). No third - generation cephalosporin resistance was detected in any of the nonhuman newport isolates tested . * studied; n, no . Of isolates received at the french national reference centre for salmonella (1 per patient). All but 1 of the newport isolates were resistant to cefoxitin (appendix table). These isolates showed 4 resistance phenotypes; most (41, 89.1%) were resistant to streptomycin, sulfonamides, chloramphenicol, and tetracycline . Pcr and sequencing showed that the 45 isolates resistant to cefoxitin were positive for the blacmy-2 gene, and cefoxitin - susceptible isolates contained the extended - spectrum -lactamase gene blactx - m-1 . Mg / l to> 256 mg / l, and ceftazidime mics ranged from 64 three isolates with additional resistance to aminoglycosides contained a class 1 integron with the 1-kb gene cassette aada24 (known to encode resistance to streptomycin and spectinomycin) (11). The chloramphenicol / florfenicol resistance gene flor was detected in all but 1 cmy-2producing newport isolate . Clonal relatedness of newport isolates was assessed by multilocus sequence typing (mlst) and pulsenet standard method pulsed - field gel electrophoresis (pfge) (figure 1). All 16 newport mdr - ampc isolates tested had a common sequence type (st), st45 . Xbai - pfge identified 10 distinct profiles (similarity 76.7%) among all 45 newport mdr - ampc isolates . Single enzyme matches were found for 3 of the profiles (15 isolates) in the us pulsenet national database (www.cdc.gov/pulsenet; appendix table; figure 2). Two pfge types (new6 and new8) were divided into 24 subtypes because of additional band(s) <100 kb . Isolates from the 2003 outbreak showed 4 similar but distinct pfge profiles that differed by 12 bands, migrated between 60 and 100 kb, and were attributed to plasmid(s) (additional information available from fxweill@pasteur.fr). If only cases with indistinguishable pfge profiles had been tested, potentially related cases would not have been linked to this outbreak . Therefore, during an outbreak investigation of newport mdr - ampc, analysis of plasmid content (either by alkaline lysis or s1 nuclease, depending on size of additional bands) might complete xbai - pfge profiles for isolates whose profiles differ by 1 or 2 additional bands of low molecular mass . Representative xbai pulsed - field gel electrophoresis (pfge) profiles of third - generation cephalosporin a dendrogram was generated with bionumerics software (applied maths, sint - martens - latem, belgium). The pfge profile (and if there were indistinguishable isolates in the pulsenet usa database [www.cdc.gov/pulsenet], the corresponding centers for disease control and prevention pulsenet profile), the number of isolates, and the -lactamase genes are indicated . Representative psti restriction profiles (a) and blacmy-2 southern hybridization (b) of plasmids from escherichia coli dh10b transformants of cmy-2producing salmonella spp . Clinical isolates . Lane 1, dh10b/00 - 7490; lane 2, dh10b/03 - 3349; lane 3, dh10b/03 - 3367; lane 4, dh10b/00 - 3525; lane 5, dh10b/00 - 4165; lane 6, dh10b/03 - 9969; lane 7, dh10b/03 - 9243; lane 8, dh10b/02 - 2049 . The gel is focused on the resolution of high molecular mass bands; smaller bands (in particular, the 0.8-kb band) are not well visualized . Alkaline lysis extraction showed that all but 1 of the newport mdr - ampc isolates harbored a plasmid> 125 kb that hybridized with a blacmy-2 probe; the remaining isolate harbored a plasmid of 100 kb (appendix table). Analysis with s1 nuclease showed that these plasmids were 100 kb370 kb . Up to 3 additional plasmids (3.5 kb100 kb) that did not have blacmy-2 were detected in most isolates (appendix table). Cephalosporin resistance was transferred by electroporation of plasmid dna to eschericha coli dh10b for all 38 cmy-2positive isolates tested . When present in the donor strain, resistance to sulfonamides, chloramphenicol, and tetracycline was also transferred . Restriction analysis of plasmids isolated from transformants showed 6 similar restriction profiles for newport isolates (r1r6) (figure 2, appendix table). Newport plasmids r1r6 and agona plasmid r8 were shown by pcr to contain variant a / c2 replicons (13), whereas typhimurium plasmid r7 contained the i1 replicon . Psti - digested plasmids analyzed by southern hybridization with a blacmy-2 probe (figure 2) showed 4 hybridization profiles among newport isolates . Profiles h2, h3, and h4 differed from h1 by 1 additional band (> 10 kb for h2, 3.2 kb for h3, and> 18 kb for h4), which indicated that the blacmy-2 gene was partially or totally duplicated . Newport mdr - ampc isolates have been the source of sporadic cases and small outbreaks in humans in france during 20002005 . All isolates had the same mlst type, st45, and highly similar xbai - pfge profiles . Their plasmids carrying blacmy-2 were homogeneous (same incompatibility group a / c2, a main restriction type r1, and a main hybridization type h1). These results support clonal expansion of 1 newport strain (or a limited number of genetically related newport strains) able to acquire and maintain a large inca / c2 mdr plasmid . However, this strain was not found in french food animals or domestically produced food products (additional information available from fxweill@pasteur.fr). Further investigation identified the source as a wholesaler who imported meat from belgium, the united kingdom, hungary, canada, brazil, argentina, uruguay, and australia (12). In contrast to europe, newport mdr - ampc has been frequently seen in the united states during the past decade . Furthermore, several characteristics were shared between us and french newport mdr - ampc isolates: st45 (15), pfge profiles new5, new6a, and new6b (displayed by 15 isolates among the 45 studied), and blacmy-2 plasmid hybridization type h1 (14). We can reasonably hypothesize that during 20002005 some isolates likely entered france from north america through imported food . Alternatively, they could have come to france and north america from some other country.
|
We conducted a retrospective cohort study of individuals with diabetes who were treated in general medical practices that participate with the health information network (thin) data system . Thin was created in 2002 and includes data from 300 physician practices in england and wales . Population and are similar in age, sex, and geographic characteristics (13). Thin includes records on 4.78 million patients, of whom 2.26 million are currently active participants . The patient population in thin is stable with only 3% of patients being lost per year attributable to leaving a practice or death . The database for thin, contains information on medical diagnoses (acute and chronic), as well as free text on these conditions . Thin also includes laboratory values, which are electronically captured, and some aspects of the physical examination, as well as hospitalizations, consultations, and prescription medications, which are electronically transferred to thin . The ability to ascertain the diagnosis of diabetes has been previously investigated by national health service investigators and is excellent (14). All subjects enrolled in this cohort had at least two database records for diabetes between january 2002 and january 2005 and were required to be at least 35 years of age by january 2002 . The egfr was estimated using the modification of diet in renal disease equation (15). Our estimate did not include a mathematical expression for ethnicity / race, which is a common practice in the u.k . We also explored via sensitivity analyses whether the inability to adjust for race / ethnicity would have had an affect on our results (see below). Ckd was defined on the basis of egfr and categorized into three levels corresponding to the u.s . National kidney foundation staging scheme for ckd . These three categories were defined by the national kidney foundation cut points between stage ii and stage iii ckd as well as between stage iii and stage iv (60, 30 to <60, and <30 ml / min per 1.73 m, respectively) (15). Outcomes were determined separately in each study subject for incident dfu and incident lea on the basis of the computerized medical records . Therefore, dfus in this study were chronic or severe enough to require a patient to seek medical care and may not represent all wounds on the feet of those with diabetes . For a dfu to be considered incident, the subject must have had no report of a dfu for at least 6 months before the database record of the egfr . For a lea to be considered incident if the subject had a prior lea, a new one must have occurred on the contralateral leg or a previous lea must have been converted from a minor to major (transtibial) amputation . Because of the potential for recording error, it was not possible to identify a new minor amputation on a foot with a previous minor amputation (e.g., the amputation of the fifth digit after a previous amputation of the first). If an observed dfu or lea did not meet these criteria it was categorized as history of . In 2002, a series of performance programs were established and became mandatory by 2003 . These required documentation and assessments by the general practitioner at least every 15 months of lower - limb pulses, neuropathy (usually recorded as a failure to perceive 10 g of monofilament pressure), a1c, cigarette use, and serum creatinine in those patients with diabetes ., a confounder must be associated with the risk factor of interest (i.e., ckd) and the outcome (dfu or lea). It should not be on the causal pathway between the risk factor and the outcome . Our confounders also included subject age at the time of entering our cohort, duration of diabetes as noted in the medical record, practice site, diagnosis of history of myocardial infarction (myocardial infarction or unstable angina), sex, and history of hypertension . A subject was assumed to have pad if he or she had an absence of pulses of both lower extremities (screening test frequently used in u.k . Clinical practice and epidemiological studies) or had a medical diagnosis consistent with lower - extremity atherosclerotic disease (16). It is possible that this diagnosis of pad lacks validity and therefore could result in an underestimate of the true association between ckd and lea / dfu . Person - time was estimated from the database date for the first egfr after 2002 until either an outcome occurred (lea and dfu separately), the study subject died, the study subject left the practice, or the last transaction date in the database . The unadjusted hazard ratio (hr) of the association between our ckd categories and dfu or lea with 95% ci was determined using proportional hazards regression analysis . All estimates were calculated for the full sample and separately for both those with and those without clinical history of pad (i.e., interaction). The final multivariable models (adjusted models) were developed by using variables deemed clinically important for lea and dfu and any variable that changed the effect estimate by> 15% . The fit of the models was assessed visually using cox - snell residuals and by graphic display . Sensitivity analyses were conducted by reestimating the egfr using the mathematical expression for african descent by assuming that all subjects were of african descent, by assuming that only those in the most severe ckd class (i.e., those with egfr <30 ml / min per 1.73 m) were of african descent, and by repetitive random sampling of 1% of the population and reclassifying them as if they were of african descent (1% is the u.k . We also conducted analyses excluding anyone with a past history of dfu or lea just in case those with a past history truly did not have a new dfu or lea after our measured egfr . All statistical analyses were performed using stata 9.2 (stata corporation, college station, tx). All subjects enrolled in this cohort had at least two database records for diabetes between january 2002 and january 2005 and were required to be at least 35 years of age by january 2002 . The egfr was estimated using the modification of diet in renal disease equation (15). Our estimate did not include a mathematical expression for ethnicity / race, which is a common practice in the u.k . We also explored via sensitivity analyses whether the inability to adjust for race / ethnicity would have had an affect on our results (see below). Ckd was defined on the basis of egfr and categorized into three levels corresponding to the u.s . National kidney foundation staging scheme for ckd . These three categories were defined by the national kidney foundation cut points between stage ii and stage iii ckd as well as between stage iii and stage iv (60, 30 to <60, and <30 ml / min per 1.73 m, respectively) (15). Outcomes were determined separately in each study subject for incident dfu and incident lea on the basis of the computerized medical records . Therefore, dfus in this study were chronic or severe enough to require a patient to seek medical care and may not represent all wounds on the feet of those with diabetes . For a dfu to be considered incident, the subject must have had no report of a dfu for at least 6 months before the database record of the egfr . For a lea to be considered incident if the subject had a prior lea, a new one must have occurred on the contralateral leg or a previous lea must have been converted from a minor to major (transtibial) amputation . Because of the potential for recording error, it was not possible to identify a new minor amputation on a foot with a previous minor amputation (e.g., the amputation of the fifth digit after a previous amputation of the first). If an observed dfu or lea did not meet these criteria it was categorized as history of . In 2002, a series of performance programs were established and became mandatory by 2003 . These required documentation and assessments by the general practitioner at least every 15 months of lower - limb pulses, neuropathy (usually recorded as a failure to perceive 10 g of monofilament pressure), a1c, cigarette use, and serum creatinine in those patients with diabetes ., a confounder must be associated with the risk factor of interest (i.e., ckd) and the outcome (dfu or lea). It should not be on the causal pathway between the risk factor and the outcome . Our confounders also included subject age at the time of entering our cohort, duration of diabetes as noted in the medical record, practice site, diagnosis of history of myocardial infarction (myocardial infarction or unstable angina), sex, and history of hypertension . A subject was assumed to have pad if he or she had an absence of pulses of both lower extremities (screening test frequently used in u.k . Clinical practice and epidemiological studies) or had a medical diagnosis consistent with lower - extremity atherosclerotic disease (16). It is possible that this diagnosis of pad lacks validity and therefore could result in an underestimate of the true association between ckd and lea / dfu . Person - time was estimated from the database date for the first egfr after 2002 until either an outcome occurred (lea and dfu separately), the study subject died, the study subject left the practice, or the last transaction date in the database . The unadjusted hazard ratio (hr) of the association between our ckd categories and dfu or lea with 95% ci was determined using proportional hazards regression analysis . All estimates were calculated for the full sample and separately for both those with and those without clinical history of pad (i.e., interaction). The final multivariable models (adjusted models) were developed by using variables deemed clinically important for lea and dfu and any variable that changed the effect estimate by> 15% . The fit of the models was assessed visually using cox - snell residuals and by graphic display . Sensitivity analyses were conducted by reestimating the egfr using the mathematical expression for african descent by assuming that all subjects were of african descent, by assuming that only those in the most severe ckd class (i.e., those with egfr <30 ml / min per 1.73 m) were of african descent, and by repetitive random sampling of 1% of the population and reclassifying them as if they were of african descent (1% is the u.k . We also conducted analyses excluding anyone with a past history of dfu or lea just in case those with a past history truly did not have a new dfu or lea after our measured egfr . All statistical analyses were performed using stata 9.2 (stata corporation, college station, tx). Person - time was estimated from the database date for the first egfr after 2002 until either an outcome occurred (lea and dfu separately), the study subject died, the study subject left the practice, or the last transaction date in the database . The unadjusted hazard ratio (hr) of the association between our ckd categories and dfu or lea with 95% ci was determined using proportional hazards regression analysis . All estimates were calculated for the full sample and separately for both those with and those without clinical history of pad (i.e., interaction). The final multivariable models (adjusted models) were developed by using variables deemed clinically important for lea and dfu and any variable that changed the effect estimate by> 15% . The fit of the models was assessed visually using cox - snell residuals and by graphic display . Sensitivity analyses were conducted by reestimating the egfr using the mathematical expression for african descent by assuming that all subjects were of african descent, by assuming that only those in the most severe ckd class (i.e., those with egfr <30 ml / min per 1.73 m) were of african descent, and by repetitive random sampling of 1% of the population and reclassifying them as if they were of african descent (1% is the u.k . We also conducted analyses excluding anyone with a past history of dfu or lea just in case those with a past history truly did not have a new dfu or lea after our measured egfr . All statistical analyses were performed using stata 9.2 (stata corporation, college station, tx). There were 125,933 individuals identified with diabetes between january 2002 and january 2005 in thin who were at least 35 years of age and who had at least two visits . At the start of our period of observation, the average age was 62.9 years (95% ci 62.863.0) and the median age was 64.6 years (52.682.6). Our subjects, after a documented egfr, were followed for about 200,675 person - years of time between january 2002 and july 2005 . The average time of follow - up was 2.2 years, and the median time was 2.4 years . During this time, 378 individuals had an lea and 2,619 individuals had a dfu . Before entry into our cohort 3,491 subjects had a diagnosis of dfu and 768 had an lea . Of those in our cohort who needed an amputation, 70 (18%) had a previous minor or contralateral lea and 126 (33%) had a previous dfu> 6 months before their entry into our cohort . Of those in our cohort who developed a dfu, 569 (22%) had a previous dfu before 6 months prior to entry into our cohort and 138 (5%) had a previous lea . Ckd (egfr <60 ml / min per 1.73 m) was noted in 23,350 (26%) of our cohort . In addition, several other factors were associated with the development of dfu or lea, such as hyperglycemia, peripheral neuropathy, hypertension, and history of myocardial infarction (table 1). Development of a dfu was associated with progressive ckd and many of our other variables (tables 1 and 2). Compared with our reference group (group 1 [egfr 60 ml / min per 1.73 m]) the hr for group 2 (egfr 30 and <60 ml / min per 1.73 m) was 1.85 (95% ci 1.712.01) and for group 3 (egfr <30 ml / min per 1.73 m) was 3.92 (3.234.75) (all p values <0.001). The fully adjusted associations between dfu and ckd were 1.51 (1.381.66) for group 2 and 3.22 (2.604.00) for group 3, both versus group 1 (all p <0.001). An interaction due to pad was also present (pinteraction = 0.08 for group 2; pinteraction = 0.003 for group 3), and, as a consequence, our ckd effect estimates are reported for those with and without pad (table 2). The need for an amputation was associated with progressive ckd and many of our other variables (tables 1 and 2). Compared with the reference group (group 1), the hr for group 2 was 2.08 (95% ci 1.682.58) and for group 3 was 7.71 (5.2911.26) (p <0.001). Our fully adjusted association between lea and ckd was 2.18 (1.702.78) for group 2 and 7.09 (4.5711.00) for group 3, respectively (all p <0.001). These estimates were influenced primarily by an interaction due to pad (pinteraction = 0.07 for group 2; pinteraction = 0.04 for group 3) (table 2). All ckd effect estimates are therefore reported for those with and without pad (table 2). As noted above, 35,316 (28%) of those in thin did not have the full data necessary for a calculation of egfr . There were many attributes that were similar between those in our cohort and those who did not have data allowing an estimate of egfr . For example, for our main confounders, the mean age was 63.9 years and 51% were women . When their full medical record and not just data obtained after 2002 was evaluated, 5% had evidence of a foot ulcer at some time in their medical record versus 4% for our cohort and 1% had a record of an amputation versus 1% for our cohort, 26% used insulin versus 23% of our cohort, and 0.9% had a history of esrd versus 0.8% of our cohort . The modification of diet in renal disease estimation includes a term for african ethnicity (15). This variable is frequently not recommended for use in the calculation of egfr in the u.k . Population is african . We were not able to measure ethnicity, but we did create sensitivity analysis to evaluate whether our inability to measure african ethnicity might have affected our results . Next we assumed that all of the individuals in the worst ckd category were african . For example, this resulted in point estimates of 2.08 (95% ci 1.682.56) and 7.18 (4.3711.78) for ckd and lea, respectively . Finally, we conducted an unmeasured confounders sensitivity analysis and were not able to eliminate the association of ckd with dfu or lea using previously demonstrated effect estimates for race / ethnicity . Several authors have noted a relationship between esrd and lea . Within one of the largest studied cohorts of diabetic subjects who have egfr measurements, we have shown a strong association, not just with esrd, but between the severity of ckd and the onset of both dfu and lea among those with diabetes . Whereas this association is greatest for those with the most severe ckd, even those with less severe ckd were approximately two times more likely to develop a foot ulcer or undergo an lea than those with minimal to no impairment (table 2). This association was present among those without clinically apparent pad . To confirm the appropriateness of our database and analysis, we were also able to show, as expected, an association between dfu or lea and hyperglycemia, pad, peripheral neuropathy, hypertension, history of myocardial infarction, age, previous history of dfu, previous history of lea, and esrd . Traditionally, the complications of diabetes have been divided into those due to microvascular disease and those due to macrovascular disease . We were able to explore dfu and lea in individuals with or without clinically apparent pad . Although there may be many potential mechanisms used to explain the onset of ckd, dfu, and lea in those with diabetes, it is fascinating to note that structurally in many ways the onset of ckd many be similar to the onset of dfu and/or lea . As a consequence of hyperglycemia different cell types, such as mesangial cells, which have some phenotypic properties similar to those of fibroblasts, and podocytes, these cells are damaged from hyperglycemia, but the damage is also physically attributable to the trauma induced by hypertension (18). Unlike renal cells, the cells of the dermis may be replenished from neighboring cells, transient amplifying cells local to the wound, and bone marrow derived cells . One might hypothesize that the onset and progression of ckd might serve as an early marker (not actually a risk factor) because severe damage occurs first in the kidney (cells most sensitive to hyperglycemia that cannot replenish) and then later in the skin, which can replenish, resulting in the onset of dfu and ultimately lea . In the setting of a dfu, local trauma (e.g., walking, poor - fitting shoes, plantar contact with hard objects, and others) interestingly, in a recent secondary analysis of a randomized clinical trial of footwear, which looked only at one form of trauma that was due to daily weight bearing, the authors noted that by itself this form of trauma was not directly associated with dfus (19,20). Our preceding hypothesis might help to explain this finding and to explain why trauma does not cause wounds in everyone with diabetes; i.e., before trauma results in a clinically significant wound, an individual's ability to repair must first be altered . This explanation does not diminish the importance of trauma as the likely cause of the initial insult that results in a dfu or lea, but it may be that ckd, neuropathy, pad, and many other complications of diabetes are on the same causal pathway related to the progressive inability to repair . Statistically, if this is true, then in our study we underestimated the magnitude of the associations that we report by adjusting our models for pad, neuropathy, and so on . As an alternative explanation, circulating factors that directly affect wound repair and ultimately are responsible for lea and dfu, may exist as a consequence of progressive ckd . With respect to selection bias we did not have egfr measurements for all subjects in thin, so perhaps the general practitioners preferentially measured creatinine in those with more severe renal disease who were most likely to develop a dfu or undergo an lea . In addition, the completion of the required examinations is a performance criterion of the general practitioners, which is linked to their pay (www.nhsemployers.org/pay-conditions/index.cfm) (21). Our study began in 2002, before mandatory compliance and egfr measurements were available for 72% of subjects . If this were true then our results would have been biased to the null unless the general practitioners a priori decided that an association between egfr, a value requiring calculation, and our outcomes existed . There could also be concerns that data are not always collected in the record for thin . It is important to realize that per agreement with the company that organizes thin, the record for thin is the general practitioner's only medical record . We could have mis - specified the degree of ckd because we did not know the subjects ethnicity / race . Ethnicity / race is an unmeasured confounder in our study . We were able to conduct several sensitivity analyses and found that our inability to determine race / ethnicity was not likely to have influenced our measurement of egfr 22) did note various rates of foot complications and pad and dfu in ethnic groups . However, these authors concluded that variable rates of pad and neuropathy at least partially explained the different rates of lea and dfu in these ethnic groups . It is possible that important assessments such as pad and neuropathy were measured with error . However, as in other studies they were measured by general practitioners in their practices, thereby making these assessments generalizable to other general practice settings and even to other studies such as that of abbott et al . Because we know of no reason that the accuracy of their measurement would have been influenced by the general practitioner's knowledge of egfr (a calculated blood test), this error is probably nondifferential, meaning that any bias due to this error should have resulted in an underestimate in the association between ckd and lea or dfu . In summary, we have demonstrated a strong association between ckd and dfu or lea among a population - based sample of individuals with diabetes who are cared for by general practitioners in the u.k . It is important to note that demonstrating an association is not the same as showing causation, which often requires an experimental design such as a randomized clinical trial and the demonstration of a common mechanism that causes ckd and failure of the skin to heal . On the basis of our study, it is likely that ckd and dfu or lea among those with diabetes are associated more tightly then was recognized previously . Clinically, our findings are important in that we have shown an association between even individuals with moderate ckd (egfr <60 ml / min per 1.73 m) and an increased risk for the onset of dfu and ultimately amputation.
|
Hepatocellular carcinoma (hcc) is the most common primary malignant tumor of the liver and the second leading cause of cancer - related death in the world . Almost 80% of hcc cases are associated with cirrhosis due to chronic hepatitis b or c virus infection . However, hccs that are very large in size and mass mostly develop in non - cirrhotic livers . Surgical resection is the only potentially curative treatment for patients with adequate liver functional reserve . We present the case of a patient with an extensive hcc, who clinically presented with thoracic pain due to an extrahepatic metastasis . A 33-year - old male presented to an ambulant orthopedic surgeon with left thoracic pain for six months . The patient s history offered no trauma, no alcoholism or smoking as well as no drug abuse or intake of steroid hormones . Clinical examination revealed selective pressure pain over the fifth rib near the left mammilla . A gadoxetic acid - enhanced mri was performed and revealed a giant inhomogeneous tumor originating from the left liver lobe with compression of surrounding organs (fig . Liver biopsy revealed a hepatocellular adenoma (hca) with conspicuous cytoplasmic yellow - brown granular pigments (fig . Tumor cells showed nuclear -catenin expression and overexpression of glutamine synthetase, leading to the diagnosis of hca with -catenin activation . Although in biopsy tissue transition to hcc was not evident, progression to hcc was suspected due to the big tumor size . Furthermore, a single - photon emission computed tomography (spect) showed suspect tumor in the left fifth rib (fig . 1b), and a ct - guided puncture revealed hcc metastasis, verifying the suspicion of malignant progression . A moderate increase of transaminases with alanine aminotransferase (alt) of 0.92 katal / l (normal: 0.220.77) and aspartate aminotransferase (ast) of 1.0 katal / l (normal: <0.59) was assessed by blood sample . The tumor markers alpha - fetoprotein (afp), cancer embryonic antigen (cea) and carbohydrate antigen 19 - 9 (ca19 - 9) were all within normal limits, whereas carbohydrate antigen 125 (ca125) was moderately increased with 24.8 u / ml (normal: <20). Preoperative ct volumetric measurement revealed a right liver lobe volume of 1367 cubic centimeter by using the summation - of - area method.fig . 1(a) gadoxetic acid - enhanced mri axial t2-weighted showed large inhomogeneous abdominal mass and continuation with the parenchyma of left hepatic lobe (arrows). (b) single - photon emission computed tomography (spect) showed metastasis of fifth left rib (arrow).fig . 1 (a) gadoxetic acid - enhanced mri axial t2-weighted showed large inhomogeneous abdominal mass and continuation with the parenchyma of left hepatic lobe (arrows). (b) single - photon emission computed tomography (spect) showed metastasis of fifth left rib (arrow). The huge tumor did not invade surrounding organs and hepatic cirrhosis or peritoneal metastasis was not observed (fig . After cholecystectomy and complete mobilization of the left liver, the left portal vein and left hepatic artery were ligated and divided . Liver transection of left liver including segment i and partial segment iv was performed by using endo gia 60-mm vascular with tri - staple technology (covidien, mansfield, massachusetts, usa), whereas larger vessels and biliary tracts were overstitched (fig . The resection surface was sealed with fibrin sealant patch (tachosil, takeda, osaka, japan) to avoid bleeding . The intraoperative course was uneventful, the tumor capsule was not disrupted and there was no need of blood transfusion . The giant tumor mass was found to be 34 24 24 cm in size and 7.2 kg in weight . Macroscopic examination showed a multinodular and multicoloured tumor with yellow - green as well as dark pigmented areas (fig . Histological evaluation revealed a well - differentiated hcc with trabecular growth pattern, invasion of portal vein branches and tumor - free resection margins . Tumor cells of the macroscopically dark coloured areas in histology contained brown cytoplasmic pigments (data not shown), just as observed in the cells of the hca . As a two - stage surgical procedure, partial en bloc resection of the left fifth rib was performed 4 weeks after hepatectomy . The defect of the thoracic wall was repaired by using peri - guard repair patch (synovis life technologies, saint paul, minnesota, usa), a biological tissue prepared from bovine pericardium . In summary, this case was classified as a pt3a pn0 pm1 stage iv, according to the 7th edition of the uicc tnm staging system for hcc.fig . (b) image of intraoperative situs after left hemihepatectomy showed prominent feeding vessel originating from the left gastric artery (arrow).fig . 3(a, b) multinodular and multicoloured appearance of the tumor with yellow - green areas, caused by bile production, and dark pigmented areas . (c) yellow - green appearance of the bone metastasis due to bile deposition.fig . 4upper line: (a) hepatocellular adenoma with cytoplasmic granular pigments, (b) nuclear -catenin expression, (c) conserved reticulin fiber meshwork and (d) very low proliferative activity . Lower line: (e) hepatocellular carcinoma with trabecular growth pattern, low nuclear atypia and bile plugs (arrows), (f) showing regular membraneous -catenin expression, (g) fiber - destructive growth and (h) medium proliferative activity . A, e: hematoxylin & eosin; b, f: -catenin immunohistochemistry; c, g: gomori silver staining; d, h: ki67 immunohistochemistry.fig . (b) image of intraoperative situs after left hemihepatectomy showed prominent feeding vessel originating from the left gastric artery (arrow). (a, b) multinodular and multicoloured appearance of the tumor with yellow - green areas, caused by bile production, and dark pigmented areas . (c) yellow - green appearance of the bone metastasis due to bile deposition . Upper line: (a) hepatocellular adenoma with cytoplasmic granular pigments, (b) nuclear -catenin expression, (c) conserved reticulin fiber meshwork and (d) very low proliferative activity . Lower line: (e) hepatocellular carcinoma with trabecular growth pattern, low nuclear atypia and bile plugs (arrows), (f) showing regular membraneous -catenin expression, (g) fiber - destructive growth and (h) medium proliferative activity . A, e: hematoxylin & eosin; b, f: -catenin immunohistochemistry; c, g: gomori silver staining; d, h: ki67 immunohistochemistry . The post - operative period was uneventful and the patient was discharged home one week after the second operation . A follow - up surveillance according to the recent guidelines of the european association for the study of the liver (easl) was initiated . A 33-year - old male presented to an ambulant orthopedic surgeon with left thoracic pain for six months . The patient s history offered no trauma, no alcoholism or smoking as well as no drug abuse or intake of steroid hormones . Clinical examination revealed selective pressure pain over the fifth rib near the left mammilla . A gadoxetic acid - enhanced mri was performed and revealed a giant inhomogeneous tumor originating from the left liver lobe with compression of surrounding organs (fig . Liver biopsy revealed a hepatocellular adenoma (hca) with conspicuous cytoplasmic yellow - brown granular pigments (fig . Tumor cells showed nuclear -catenin expression and overexpression of glutamine synthetase, leading to the diagnosis of hca with -catenin activation . Although in biopsy tissue transition to hcc was not evident, progression to hcc was suspected due to the big tumor size . Furthermore, a single - photon emission computed tomography (spect) showed suspect tumor in the left fifth rib (fig . 1b), and a ct - guided puncture revealed hcc metastasis, verifying the suspicion of malignant progression . A moderate increase of transaminases with alanine aminotransferase (alt) of 0.92 katal / l (normal: 0.220.77) and aspartate aminotransferase (ast) of 1.0 katal / l (normal: <0.59) was assessed by blood sample . The tumor markers alpha - fetoprotein (afp), cancer embryonic antigen (cea) and carbohydrate antigen 19 - 9 (ca19 - 9) were all within normal limits, whereas carbohydrate antigen 125 (ca125) was moderately increased with 24.8 u / ml (normal: <20). Preoperative ct volumetric measurement revealed a right liver lobe volume of 1367 cubic centimeter by using the summation - of - area method.fig . 1(a) gadoxetic acid - enhanced mri axial t2-weighted showed large inhomogeneous abdominal mass and continuation with the parenchyma of left hepatic lobe (arrows). (b) single - photon emission computed tomography (spect) showed metastasis of fifth left rib (arrow).fig . 1 (a) gadoxetic acid - enhanced mri axial t2-weighted showed large inhomogeneous abdominal mass and continuation with the parenchyma of left hepatic lobe (arrows). (b) single - photon emission computed tomography (spect) showed metastasis of fifth left rib (arrow). The huge tumor did not invade surrounding organs and hepatic cirrhosis or peritoneal metastasis was not observed (fig . After cholecystectomy and complete mobilization of the left liver, the left portal vein and left hepatic artery were ligated and divided . Liver transection of left liver including segment i and partial segment iv was performed by using endo gia 60-mm vascular with tri - staple technology (covidien, mansfield, massachusetts, usa), whereas larger vessels and biliary tracts were overstitched (fig . The resection surface was sealed with fibrin sealant patch (tachosil, takeda, osaka, japan) to avoid bleeding . The intraoperative course was uneventful, the tumor capsule was not disrupted and there was no need of blood transfusion . The giant tumor mass was found to be 34 24 24 cm in size and 7.2 kg in weight . Macroscopic examination showed a multinodular and multicoloured tumor with yellow - green as well as dark pigmented areas (fig . Histological evaluation revealed a well - differentiated hcc with trabecular growth pattern, invasion of portal vein branches and tumor - free resection margins . Tumor cells of the macroscopically dark coloured areas in histology contained brown cytoplasmic pigments (data not shown), just as observed in the cells of the hca . As a two - stage surgical procedure, partial en bloc resection of the left fifth rib was performed 4 weeks after hepatectomy . The defect of the thoracic wall was repaired by using peri - guard repair patch (synovis life technologies, saint paul, minnesota, usa), a biological tissue prepared from bovine pericardium . In summary, this case was classified as a pt3a pn0 pm1 stage iv, according to the 7th edition of the uicc tnm staging system for hcc.fig . (b) image of intraoperative situs after left hemihepatectomy showed prominent feeding vessel originating from the left gastric artery (arrow).fig . 3(a, b) multinodular and multicoloured appearance of the tumor with yellow - green areas, caused by bile production, and dark pigmented areas . (c) yellow - green appearance of the bone metastasis due to bile deposition.fig . 4upper line: (a) hepatocellular adenoma with cytoplasmic granular pigments, (b) nuclear -catenin expression, (c) conserved reticulin fiber meshwork and (d) very low proliferative activity . Lower line: (e) hepatocellular carcinoma with trabecular growth pattern, low nuclear atypia and bile plugs (arrows), (f) showing regular membraneous -catenin expression, (g) fiber - destructive growth and (h) medium proliferative activity . A, e: hematoxylin & eosin; b, f: -catenin immunohistochemistry; c, g: gomori silver staining; d, h: ki67 immunohistochemistry.fig . 4 (a) in situ appearance of the hepatocellular carcinoma mass . (b) image of intraoperative situs after left hemihepatectomy showed prominent feeding vessel originating from the left gastric artery (arrow). (a, b) multinodular and multicoloured appearance of the tumor with yellow - green areas, caused by bile production, and dark pigmented areas . (c) yellow - green appearance of the bone metastasis due to bile deposition . Upper line: (a) hepatocellular adenoma with cytoplasmic granular pigments, (b) nuclear -catenin expression, (c) conserved reticulin fiber meshwork and (d) very low proliferative activity . Lower line: (e) hepatocellular carcinoma with trabecular growth pattern, low nuclear atypia and bile plugs (arrows), (f) showing regular membraneous -catenin expression, (g) fiber - destructive growth and (h) medium proliferative activity . A, e: hematoxylin & eosin; b, f: -catenin immunohistochemistry; c, g: gomori silver staining; d, h: ki67 immunohistochemistry . The post - operative period was uneventful and the patient was discharged home one week after the second operation . A follow - up surveillance according to the recent guidelines of the european association for the study of the liver (easl) was initiated . Giant hepatocellular carcinomas have a diameter over 10 cm, whereas a diameter over 20 cm is extremely rare . Interestingly, very large hccs mostly develop in non - cirrhotic livers . At time of diagnosis an extrahepatic spread occurs in only about 515% of patients and mainly in advanced - stage primary tumors over 5 cm . The most common sites of hcc metastases are lung (49%), abdominal organs (24%) and bone (1316%). Skeletal metastases appear as expansive soft tissue masses with bone destruction and mainly involved sites are the vertebrae, pelvis, ribs, skull, humerus and sternum . In the presented case the progression from a hca with -catenin activation to hcc was documented . Molecular characterization of hcas has been practiced for few years now, resulting in an improved risk stratification of the heterogenous entity of hcas . These -catenin activated hcas are often associated with male sex and administration of androgenic hormones and have an increased risk of malignant transformation . In this case it is suggested that these pigments constitute lipofuscin deposits and several reports indicate an increased risk of malignancy in pigmented hcas, . The pigments were also found in the hcc cells, but, unexpectedly, the nuclear -catenin activation of the hca was not preserved in the hcc cells . However, pigmentation of hcas besides molecular characterization could serve as additional hint in evaluation of malignant potential of hcas . Currently, surgical resection is the only curative treatment with the best long - term survival . Preoperative liver function as a key parameter and the skill and experience of the surgeon significantly affect the success of resection in complicated hcc . Recurrence is the major challenge, as it occurs in approximately 70% of patients within 5 years after hepatic resection . Predictive factors of recurrence are disruption of tumor capsule, daughter nodules, positive surgical resection margins and blood transfusion . According to the literature, we recommend a therapy specific to the patient on a case - by - case basis . To our knowledge, this is the first case report of a two - staged surgical resection of a giant hcc with skeletal metastasis in a young adult . Bone metastasis of a hepatocellular carcinoma (hcc) is a very rare cause of thoracic pain . Hepatocelluar adenomas (hca) with an activating mutation of -catenin, which occurs in up to 15% of cases, have a high risk of malignant transformation . Molecular characterization and morphological feature of pigmentation are useful tools for risk assessment of hcas and hepatobiliary surgery with metastasis resection is the only curative treatment and can be safe and effective even in patients with giant hccs, as our case report shows . Written informed consent was obtained from the patient for publication of this case report and accompanying images . A copy of the written consent is available for review by the editor - in - chief of this journal on request . Sebastian lnse: study concept, data collection, literature research and writing the manuscript.
|
Developmental hip dysplasia (ddh) presents considerable technical challenges to the primary arthroplasty surgeon . Harris first described the technique of bulk autogenous grafting to achieve superolateral bone coverage in 1977, and while early - to - midterm results were promising, longer term outcomes have been mixed . In harris's series, 21% of patients had radiographic evidence of loosening at seven years, and outcomes at mean sixteen years showed a high rate of acetabular failure due to component loosening and graft collapse . Recently, however, more favourable long - term outcomes have been reported in ddh patients utilizing bulk autografts with both cemented [46] and uncemented implants . Achieving union and stability of the autogenous graft bulk autogenous grafts are known to incorporate slowly and often incompletely, limiting their ability to respond to stresses under cyclic loading . The main factors affecting incorporation of the graft are stability of the construct and host - graft bone contact [1, 8]. We describe a novel technique combining the use of bulk autograft with an iliac osteotomy which provides primary stability and optimises direct cancellous - cancellous bone contact and report mid - term results on 21 patients . During the period 19982001, 21 hips in 21 patients were managed with autologous bone graft in combination with an iliac osteotomy at our institution . There were 3 men and 18 women in the study group, and the average age at the time of surgery was 50.1 years (range 2677). The diagnosis in all patients was ddh, with a defective acetabular roof which would lead to uncovering of the acetabular component when placed in the desired position without augmentation . Preoperative radiographic planning with use of transparent overlays and socket templates is first performed to evaluate the position of the socket and autograft and potential coverage . A posterior approach was used, with the short external rotators divided at their insertion and reflected to protect the sciatic nerve . A full capsulectomy was performed, and a pin inserted above the acetabulum and the distance to a fixed point on the greater trochanter measured to give an estimate of leg lengthening once trial components were placed . The acetabulum was sequentially reamed with the aim of positioning the socket at the level of the true acetabulum . At the base of the deficient superolateral portion of the true acetabulum, a three - sided chamfered osteotomy is performed in the ileum and a wedge of bone is removed . The resected femoral head is then used as a graft with the base of the neck cut into a wedge matching the chamfer on the iliac osteotomy (figures 1 and 2). A tight fit is achieved by fine adjustments to the graft with a saw, and this is used as a stem to place the graft into the site of the iliac osteotomy . The graft is impacted into place before being finally secured with partially threaded screws with or without washers . This construct then is reamed in the usual way, and reamings are packed into any minor defects that remain . After a saline wash, all the polyethylene cup (lubinus, waldemar link, hamburg, germany) was then cemented in the desired position, aiming to achieve an inclination angle of 45 and 1030 degrees of anteversion . The femoral canal was then broached and sequentially reamed before a collared monoblock implant (lubinus sp - ii, waldemar link, hamburg, germany) was cemented in situ . Radiographs taken preoperatively and postoperatively at 1, 3, 5, 7, and 9 years were assessed by two observers (swy and fc). Postoperatively, the inclination angle of the socket was measured in relation to kohler's line, and the amount of coverage of the socket was expressed as a percentage of the horizontal distance between the most medial point and the most lateral edge of the socket [3, 6]. Graft union was assessed by observing disappearance of the graft - host bone interface and the appearance of bridging trabeculae . The horizontal (distance from the inferior point of the teardrop) and vertical (distance from the interteardrop line) locations of the cup were measured as described by russotti et al . . Resorption of the graft was assessed at each follow - up interval, and the cup - bone interface was assessed based on the zones of delee and charnley . Loosening the cup was assessed based on the criteria of mulroy and harris, with vertical or horizontal migration> 2 mm or cement fracture, inclination change> 4 degrees, or a continuous radiolucent line> 1 mm or noncontinuous radiolucency> 2 mm at the acetabular cement - bone interface considered evidence of loosening . The preoperative radiographic classification was crowe type i in 12 hips (57%), type ii in 4 hips, and type iii in 5 hips, and the mean sharp angle was 49.6 (range 4260). Immediate postoperative radiographs showed a mean acetabular inclination angle of 43 (range 2862), and the average coverage of the component by graft was 40.2% (range 2460%). The mean horizontal location of the hip centre was 35 mm (range 2242 mm) lateral to the teardrop . The mean height of the hip centre was 31 mm (range 2056 mm) vertically from the interteardrop line . Russotti and harris defined proximal placement of the socket as 35 mm of vertical displacement, and according to these criteria six patients had proximal placement of the socket ., all hips were functioning well with no clinical signs of loosening and no revisions had been performed (figures 3 and 4). There was no discernible graft resorption or collapse and no patient had acetabular loosening according to our criteria . No patient had vertical or horizontal migration> 2 mm or a change in the inclination angle> 3. two patients had noncontinuous radiolucent lines of <1 mm visible at seven - year followup which were not apparent at 3 or five year followup . Postoperative complications included transient neuropraxias in 3 patients, an episode of dislocation in one patient which was treated with closed reduction . Two patients showed evidence of heterotopic ossification, one brooker stage i and one brooker stage ii . There were no infective complications . A combination of a high local incidence of ddh in bolivia, possibly related to both genetic and environmental factors including swaddling of infants, together with limited primary care facilities in childhood leads to a high proportion of patients presenting in the 3rd or 4th decade of life with degenerative hip disease . Developmental dysplasia of the hip presents considerable difficulties in restoration of the acetabular anatomy and achieving coverage of the acetabular component, particularly in the superolateral region . Strategies to address this problem include proximal positioning of a smaller cup [13, 16] penetration of the medial wall (protrusio technique) [17, 18], an iliac sliding graft, and lateral bulk grafting [1, 46] with either autogenous or allogenic bone . While all of these techniques have reported satisfactory medium term results, bulk autogenous grafting has been favoured by many authors as it allows more anatomic cup placement and provides early structural support to the acetabular component, and there is a ready availability of autogenous graft in the form of the resected femoral head . A further advantage is the augmentation of pelvic bone stock in case of subsequent revision, an important consideration given the often early age of onset of secondary degenerative change in this population group . Published results of bulk autograft in ddh have been variable, probably because of differences in patient selection, the severity of dysplasia, bone quality, and the technique of bone grafting and components used . Reported good early results in 27 hips in 22 patients but subsequently reported a combined clinical and radiographic rate of 60% at 16 years with failures due to graft resorption and collapse . In contrast, kobayashi et al . Reported on 37 hips at a mean of 19 years after cemented arthroplasty with 100% survival of the socket, and other authors have also reported favourable outcomes [4, 5, 7, 2022]. Previous studies have identified the two most important factors in graft incorporation as the host - graft bone contact and the stability of the graft [1, 8, 9, 19]. Harris originally described scoring of the lateral cortex of the ileum before directly bolting the curvature of the femoral head against the ileum . Later authors have described using a cut surface of the femoral head [7, 22] as a graft to ensure cancellous bone is contact with the ileum . In addition, kobayashi described preparation of the ileum with multiple drill holes to obtain a bleeding bone bed to encourage graft incorporation . In this study, we describe a technique of graft and iliac preparation which maximises the area of cancellous - cancellous bone contact between graft and host bone . In addition, the bevelled nature of the osteotomy allows impaction of the graft and gives primary stability, meaning that the screws provide supplementary fixation only . This optimises the biological situation for revascularisation and incorporation of the graft, which hopefully will lead to an increase in bone stock and possibly reduced resorption and failure rates in the longer term . Ikeuchi et al . Described the use of a sliding iliac graft for acetabular reconstruction in ddh patients which utilised a similar osteotomy to that in this study . They reported excellent short term outcomes in 19 patients and noted rapid incorporation of the graft secondary to intimate host - graft contact and stability and a subsequent low rate of resorption . However, the maximum thickness of the iliac graft was 14 mm, limiting the technique in more severe cases of dysplasia . Our method retains the advantages of host - graft contact and stability without limitation in available graft size . Firstly, we used cemented implants only, due to availability and cost in the country where the study was performed . While early series on bulk autograft also used cemented implants, in recent years secondly we lack pre- and postoperative clinical outcome scores, partly due to the lack of cultural and language appropriate validated scoring instruments . In conclusion, our study has shown that this technique variation combining an iliac osteotomy with bulk autograft in cases of developmental hip dysplasia provides early stability and reliable graft incorporation, together with satisfactory clinical and radiological outcomes in the medium term.
|
Environmental stress due to salinity is one of the most serious factors limiting the productivity of agricultural crops, most of which are sensitive to the presence of high concentrations of salts in the soil . There are two main components to salinity stress in plants; an initial osmotic stress and a subsequent accumulation of toxic ions which negatively affects cellular metabolism . In addition, it can lead to secondary stresses such as nutritional imbalance and oxidative stress . The na cation is chaotropic and predominantly associated with the deleterious effect of salinity, and therefore, most research has focused on this mineral . Indeed, for species such as soybean, citrus, and grapevine where na is predominantly retained in the roots and stems, cl is considered more toxic since this ion is accumulated to high levels in shoot tissues, negatively impacting on essential processes such as photosynthesis . The osmotic component of salinity is caused by excess inorganic ions such as na and cl in the environment that decrease the osmotic potential of the soil solution and hence water uptake by the plant root . Uptake of abundantly available na and cl therefore, offers a comparatively cheap way to lower the tissue - osmotic potential . To avoid the risk of ion toxicity associated with this strategy, na and cl are generally compartmentalized in the vacuole and/or in less sensitive tissues . In parallel, adjustment of the cytoplasmic compartment is achieved via production of compatible osmolytes such as, proline, mannitol, sorbitol, and glycine betaine . The latter also acts as antioxidant and thus detoxifies reactive oxygen species (ros). However, when plants are growing in high salt concentrations, an adequate sequestration of ions in the vacuole can become a limiting factor, especially in the case of glycophytes . In this scenario, plants can accumulate excessive amount of na in the cytosol which negatively affects many aspects of cellular physiology . The most abundant inorganic cation in the cytosol is k, in plant as in animal cells . This might be due to the fact that this cation is less chaotropic than na, that is, more compatible with protein structure even at high concentrations . The physicochemical similarities between na and k lead to a competition at transport and catalytic sites that normally bind the essential cation k and maintaining a high cytosolic k / na ratio is believed to improve salt tolerance [4, 5]. Oxidative stress is another aspect of salinity stress which is in fact a consequence of salinity - induced osmotic and/or ionic stress . The salt - induced production of ros such as superoxide radicals (o), hydrogen peroxide (h2o2), and hydroxyl radicals (oh) has then a severe effect on cellular structure and metabolism negatively . Although considerable progress was made to increase and secure crop yield through conventional breeding, the goal of improving the resistance of crops to abiotic stresses has seen limited success because of the complex, multigenic nature of the traits, and the narrow genetic variation in the gene pools of major crops . Numerous genes and proteins have been shown to affect the tolerance to environmental stress in an array of plant species, which together compose a complex puzzle with a myriad of individual elements and crisscrossing signal transduction pathways . A common theme of tolerance is the adequate control of salt uptake at the root level, regulation of influx into cells, control over long distance transport, and the compartmentation at both cellular and tissue levels [8, 9]. These processes are mediated by membrane transporters and manipulating the activity of this class of proteins has therefore enormous potential to affect plant performance in saline conditions . Different approaches have been used to identify membrane transporters with putative functions in salt tolerance . Yeast complementation screens led to the isolation of plant transporters such as the vacuolar na / h antiporter atnhx1 and the plasma membrane k / na symporter tahkt1 . Loss of function mutants in the model system arabidopsis thaliana helped characterize many membrane transporters including athkt1:1 involved in long distance na transport, the plasma membrane na / h antiporter sos1, and the vacuolar pyrophosphatase atvp1 . It is well established that uptake, efflux, translocation, and compartmentation of toxic ions (mainly na and cl) provide important bases for salinity tolerance in plants, and hence, a potential avenue to improve crops . However, a lack of understanding regarding the molecular entities and complex interactions of the responsible membrane transport proteins has hindered progress in this respect . The present paper focuses on the main ionic constituents of salinity, na and cl and also analyses which specific membrane transporters have been shown, or are believed, to be involved in uptake, extrusion, long - distance transport, and compartmentalization of salt at the cellular and tissue level . Subsequently, the paper critically evaluates the reported data to assess which proteins may be particularly suitable as engineering targets to improve crop salt tolerance . The excess of salts in the soil solution poses a challenge to the plant . Na and other ions taken up by roots elevated concentrations of salts are built up in the apoplast and eventually inside the cell, as water evaporates . Whether plants have specific transport systems for low - affinity na uptake from soil remains an open question and the exact mechanisms responsible for root na and cl uptake are only partially clear and likely include transporters from several gene families and transport classes . In the last few years, evidence has been presented supporting the existence of weakly voltage - dependent nonselective cation channels (nscc) that are the main pathway for na entry into the roots, at high soil nacl concentrations [17, 18]. Although there are many candidate genes in the databases that could encode these nscc channels, their identity remains elusive . Two families of nonselective cation channels, cngcs (cyclic nucleotide - gated channels), and glrs (glutamate - activated channels) have been suggested to be candidate nscc channels (figure 1). The inhibition of na influx and nscc currents upon addition of membrane permeable cyclic nucleotide analogues provided correlative evidence for the operation of cngcs in plants, a family of plant channels that in arabidopsis comprise 20 members . To date, five atcngcs have been characterized (atcngc1, 2, 3, 4, and 10) [19, 2325]. Electrophysiological studies have suggested that atcngc1 and atcngc4 are equally permeable to k and na and when expressed in xenopus oocytes, they displayed activation by cyclic nucleotides [19, 23] atcngc10 rescued k transport defective mutants of e. coli, yeast, and arabidopsis akt1 - 1, suggesting that atcngc10 mediates the transport of k into the roots . Atcngc3 was recently characterized by functional complementation of yeast and by characterization of arabidopsis t - dna knockout mutants . Growth of the mutant seedlings in toxic nacl (and kcl) concentrations was improved, suggesting a restricted ion influx in the mutant plants . Ionotropic glutamate receptors (glrs) are proteins that interact with glutamate and form cation channels with a wide range of permeability's . In arabidopsis, glutamate - activated na and ca voltage - independent currents were characterized in arabidopsis roots . Noted that although the effects of environmental factors on apoplastic glutamate remain unclear, the concentrations of glutamate required for half activation of these channels correlated well with the range of apoplastic glutamate concentrations reported (0.20.5 mm), suggesting a role of these channels in na uptake . Sodium has a strong inhibitory effect on k uptake by cells, presumably by interfering with transporters in the root plasma membrane such as the shaker type k channels (kat1 and akt1 form the predominant inward k conductance observed in plant plasma membranes). Such channels generally have a high k / na selectivity and were generally regarded not to play a significant role in na [26, 27]. However, a more recent work suggests that the picture is more complex and there may be ecophysiological variations in this respect . Wang et al . Used apharmacological approach to characterize na uptake in the halophyte suaeda maritima and concluded that the low - affinity na uptake pathway in this species resembles an akt1 channel . Similarly, kader and lindbergh provide evidence that k channels mediate substantial na influx in a salt - sensitive rice cultivar but not in a tolerant one . In both cases the conclusions were derived from applying channel blockers and inhibitors which can be notoriously nonspecific, but these findings do suggest that k channels are potential pathways for root na influx . In addition, the study by wang et al . Suggests that basic processes such as na uptake may be considerably different in halophytes and such diversity could be an important contributor to salt tolerance . However, the scarcity in data from halophytes in this respect forms a large hindrance in testing this hypothesis . Hkts (high affinity potassium transporters) are carrier - type proteins that mediate na and k transport (figure 1). Members of the hkt gene family are na - specific transporters (although they were initially described as high - affinity k transporters and hence their name) that mediate either preferential na transport or na - k symport, partly depending on whether the specific transporter has a highly conserved serine (subfamily 1) or glycine (subfamily 2) residue in the first pore loop of the protein and on the extracellular na - k ratio [31, 32]. Generally, hkt members of subfamily 1 have a relatively higher na - to - k selectivity than subfamily 2 hkt transporters . In arabidopsis, loss of function of the only hkt1;1 gene encoding a na - selective transporter caused the accumulation of na in leaves but reduced na concentrations in roots, with little effect on the net uptake of na by the plant [13, 33, 34]. Athkt1;1 is preferentially expressed in the vasculature, where it is thought to regulate the na distribution between roots and shoots [13, 3135]. The phloem recirculation model posits that na is loaded into shoot phloem cells by athkt1;1 and then transferred to roots via the downward stream of phloem, preventing na over accumulation in shoots (table 1). However, there seems to be little (10% or less) retranslocation of na from leaves via the phloem relative to the amount imported in the transpiration stream via the xylem [17, 38, 39]. On the other hand, athkt1;1 is generally accepted to mediate the retrieval of na from the xylem sap, thereby restricting the amount of na reaching the photosynthetic tissues [34, 3739]. These two na transport processes could be functionally linked to achieve basi - petal translocation of na because ions that were unloaded by xylem parenchyma cells might be transported into the phloem via symplastic diffusion . Engineered expression of athkt1;1 in the root pericycle of arabidopsis enhanced inward na - transport in the targeted cells, reduced root - to - shoot transfer of na, and improved salt tolerance . However, it remains unclear whether the reduced activity of athkt1;1 was the sole basis for enhanced tolerance or if there were other processes that could also contribute to salt tolerance linked to enhanced na accumulation such as improved capacity for na sequestration in vacuoles . Similar studies in cereals have shown that natural variation in the activity or expression of hkt transporters may be a genetic resource for enhanced nacl tolerance . In rice, two of the nine members, oshkt2;1 and oshkt2;2, are expressed in roots amongst other tissues . Oshkt2;1 has been shown to catalyse high - affinity na uptake into roots under k - starvation conditions, and it appears that na can partially replace the function of k under such conditions . Oshkt2;1 has been shown to catalyse high - affinity na uptake into roots under k - starvation conditions, and it appears that na can partially replace the function of k under such conditions . Expression of oshkt2;1 is localized to the root epidermis, cortical cells, and vascular tissues of both roots and leaves [4345], and expression patterns in roots were found to be different in salt - tolerant and sensitive varieties in response to nacl stress . Loss of function mutants in oshkt2;1 shows reduced growth in low k conditions and accumulated less na . Thus, it appears that oshkt2;1 augments monovalent cation uptake by providing high - affinity na uptake in k deficient conditions . However, oshkt2;1 relevance in na uptake during salinity stress may be limited since it has a micromolar affinity for na, and its activity is rapidly downregulated at higher ambient concentrations of na . However, when heterologously expressed in yeast, hvhkt2;1 was shown to mediate na (or k) uniport, na - k symport, or a combination of both, depending on the construct from which the transporter was expressed [30, 46]. These characteristics suggest that the hkt transporters are potentially of importance in the regulation of na influx into roots . Several qtls responsible for variation of k and na content were mapped to hkt family genes . Qtls analyses showed that greater shoot k content of the relatively salt - tolerant rice cultivar nona bokra cosegregated with the presence of an allelic variant of skc1 (shoot k content) with greater activity relative to that of the salt - sensitive koshihikari variety . Skc1 (renamed oshkt1;5) is a plasma membrane k - independent, na - selective transporter that is preferentially expressed in the parenchyma cells surrounding xylem vessels . The greater na concentration in the xylem sap and leaves of the salt - sensitive variety would be a consequence of a weaker skc1 allele and reduced na reabsorption from the xylem . Quantitative genetic analyses in wheat have also led to the identification of two loci, nax1 and nax2, which reduced na accumulation in the leaf blade by excluding na from the xylem by two different mechanisms . The process controlled by nax2 was confined to the roots and had the effect of reducing the transport of na from root to shoot, presumably by improved discrimination between na and k at the point of xylem loading . The nax1 locus enhanced the retention of na in the leaf sheath, thus restricting further passage to the leaf blade . High - resolution mapping and sequencing analyses of known na transporter genes have suggested that the nax1 and nax2 loci are attributable to polymorphisms in wheat hkt genes encoding proteins of the subfamily 1 with preferred na transport [48, 49]. These results strongly indicate that na exclusion from the transpiration stream may be an important mechanism in the salt tolerance of cereals, similar to many other plant species . It should be pointed out, however, that most studies concerning qtl analysis for salt tolerance are based in na and/or k content in tissues or organs and not directly in salt tolerance . Often, higher na / k ratios are regarded as determinants of salt tolerance itself without considering any other agronomical or physiological traits . In fact, the skc qtl of rice did not show a significant correlation coefficient with survival to salt stress . A clear difference should be made between qtls responsible of ionic balance and qtls for salt tolerance . Some members of the high - affinity k uptake transporter family hak / kup / kt may transport na with low affinity in the presence of high na: k ratios . Furthermore, yeast expression studies revealed that the normal function of hak / kup / kts, high - affinity k uptake, is competitively inhibited by na, pointing to a shared transport pathway of the two monovalent cations [52, 53]. Several studies have shown substantial transcriptional regulation of hak / kup / kt isoforms by salt stress [5456]. For example, su et al . Observed that the expression of haks in mesembryanthemum crystallinum was upregulated during salt stress and k - starved conditions . However, whether this result and those for other haks relate to a potential role in na uptake or augmentation of k uptake during salinity stress remains to be established . The low - affinity cation transporter lct1 from wheat functions as nonselective cation carrier conducting k, rb, na, and ca transport in yeast [58, 59]. Expression of lct1 in yeast leads to an increase in cation influx and hypersensitivity to na . Addition of external ca was found to reduce na influx and sensitivity, but the cation profile of the influx caused by lct1 resembled that of endogenous ion transport in yeast, suggesting that lct1 might be stimulating the ion transporters already present . Chloride (cl) is a major osmotically active solute in the vacuole involved in both turgor and osmoregulation processes, with implications for the proper development of plants . Despite its importance in plant biology, cl is one of the less studied essential nutrients at the physiological and molecular levels . In contrast to na, cl uptake in most conditions must be energized, but although there is a substantial amount of information regarding k and na transport in plants, very little is clear about the molecular mechanisms behind the substantial cl influx that results from salinization . The attention has been mainly focused on the voltage - dependent cl channel clc family [6365]. Phylogenetic and functional analyses have shown that plant clc genes encode anion channels and active cl / h antiporters localized in endosomal compartments, which are involved in no compartmentalization and ph regulation in the trans - golgi system . Although the transcript abundance of several clcs is affected by salinity, they are unlikely to contribute to root cl uptake: firstly, plant clcs have only been detected at endomembranes which appear to exclude a role in cl uptake and secondly the thermodynamics of cl uptake role out passive - channel - type mechanisms . A second class of potential cl transporters is formed by the cation chloride cotransporters (cccs) encoding one gene in arabidopsis and two genes in rice . The arabidopsis thaliana cation - cl cotransporters (atcccs), expressed in root and shoot tissues, mediate the coordinated symport of k, na, and cl and have been postulated to participate in the long - distance transport of cl . Loss of function of atccc in arabidopsis led to a changed root: shoot cl ratio but also to a large increase in net cl uptake arguing against a role of atccc in the uptake of this ion . More recently, the arabidopsis slow anion channel associated 1 (atslac1) gene was shown to encode for the guard cell plasma membrane s - type anion channel involved in stomatal closure [69, 70]. Another member of this family, atslah1, is expressed in the root vasculature suggesting a potential involvement in the long - distant transport of anions . It is essential that plants possess adequate efflux systems to remove potentially dangerous ions such as na from the cytosol . Inevitably, the mechanisms to extrude na into the apoplast or vacuole have to be energized which typically occurs via h - coupled antiport [5, 71], whereas those for cl may be (partially) passive . Early studies on tonoplast antiporters showed significant upregulation of their pumping capacity after plant exposure to salt [72, 73]. In the plasma membrane too, evidence for h: na antiporters was obtained underlining the relevance of such systems to plant salt tolerance . Data dealing with cl efflux are scarce: using compartmental flux analysis, britto and kronzucker showed large cl efflux when plants were exposed to 100 mm nacl . Just as is the case for na, the majority (up to 90%) of cl that entered the symplast although some of the cl efflux could theoretically be mediated by anion channels, no data are available regarding the mechanistic details or regarding the identity of the proteins involved . Comparisons of unidirectional na fluxes and rates of net accumulation of na in roots indicate that 7095% of the na fluxed into the root symplast is extruded back to the apoplast, and that small differences in na exclusion capacity lead to major changes in the net accumulation of na . In arabidopsis, the plasma membrane na / h exchanger sos1 (salt overlay sensitive) facilitates na homeostasis by extruding the ion from root epidermal cells at the root - soil interface (table 1, figure 1) [76, 77]. Sos1 is preferentially expressed in xylem parenchyma cells, and analyses of the na root / shoot partitioning in roots of sos1 plants under different salt regimes indicate that sos1 participates in the redistribution of na between the root and shoot, likely working in concert with athkt1;1 at the plasma membrane of xylem parenchyma cells [34, 36, 76, 78]. Additional evidence of the involvement of sos1 in long - distance na transport has been produced recently in the halophytic arabidopsis relative thellungiella salsuginea (a.k.a . Lower net na flux was observed in the xylem sap of tomato plants with suppressed sos1 activity . Downregulation of thsos1 in thellungiella increased na accumulation in the root tip and in the stele . Maximal na accumulation, concomitant with a decrease in the k content, was found in the root xylem parenchyma . These cells presented a na - k ratio more than 12 times higher than equivalent cells in wild - type plants . Reduced or abolished activity of sos1 interferes with k nutrition and long - distance transport . Mutations in rice and arabidopsis hkt na transporters also reduce k accumulation in shoots during salt exposure [34, 47]. The activity of the sos1 exchanger is regulated through protein phosphorylation by the sos2-sos3 kinase complex in arabidopsis (table 1) [77, 81]. Sos3/cbl4 is a myristoylated membrane bound ca sensor belonging to the recovering - like family of scabps / cbls . Upon ca binding, sos3 binds to and enhances the protein kinase activity of sos2 . Besides activating sos2, sos3 was shown to recruit sos2 to the plasma membrane to facilitate interaction with sos1 . Sos2 also interacts with scabp8/cbl10 to form an alternative protein kinase complex that regulates sos1 at the plasma membrane . Sos2 has recently been shown to phosphorylate scabp8/cbl10 at its c - terminus, thus adding a new layer of regulation to cbl proteins besides ca binding and fatty acyl modifications . This phosphorylation was induced by salt stress, occurred at the membrane, stabilized the scabp8-sos2 interaction, and enhanced plasma membrane na / h exchange activity . Surprisingly, interaction of sos2/cipk24 with scabp8/cbl10 may also result in localization of the kinase complex at the vacuolar membrane where it mediates salt tolerance by regulating the accumulation of na in shoot tissues by an as yet undefined mechanism that may involve regulation of the na / h exchange at the tonoplast [86, 87]. Regulation of the tonoplast v - atpase by sos2 in the absence of cbl proteins has also been reported . Presumably, the posttranslational modifications of scabp8/cbl10 or the interaction of combinatorial protein kinase complexes with specific targets in different cellular membranes may ultimately define the localization of the protein kinase in vivo . The compartmentation of na ions into vacuoles provides an efficient mechanism to avert the toxic effects of na in the cytosol . The transport of na into the vacuoles is mediated by cation / h antiporters that are driven by the electrochemical gradient of protons generated by the vacuolar h - translocating enzymes, the h atpases and the h pyrophosphatase (h - ppase) (table 1, figure 1). Although the activity of these cation / h antiporters was demonstrated more than 20 years ago, their molecular characterization was only possible after the arabidopsis genome - sequencing project . Na compartmentation in the vacuole occurs in all tissues and is an important mechanism for osmotic adjustment and na detoxification in the cytosol . There are eight nhx gene family members in arabidopsis, and the functions of atnhx1, 4, 7 and 8 have been studied . Atnhx7 is also known as atsos1 and atnhx8 has been shown to be a li / h antiporter, although the biological relevance of li transport remains obscure . Atnhx4 is localized to the vacuole and might function in concert with atnhx1 (table 1). Several reports indicate that constitutive overexpression of the vacuolar transporters increases the salt tolerance of a variety or species . Constitutive overexpression of the arabidopsis vacuolar na / h antiporter, atnhx1, appears to increase salinity tolerance significantly in yeast, arabidopsis, tomato, brassica napus, and cotton . Similarly, constitutive overexpression of various cereal homologs has been reported to improve the salinity tolerance of arabidopsis, rice [97, 98], wheat, and barley . The overexpression of nhx1 appears to increase the capacity of the plant to regulate cytoplasmic and vacuolar ph [101, 102]. The overexpression of nhx1 in arabidopsis led to a small increase in shoot na accumulation, possibly allowing the cells to maintain a favourable osmotic balance, yet maintaining low cytoplasmic na levels due to sequestration of the na within the vacuole . The nhx1 mutant had significantly lower na / h and k / h exchange capabilities in isolated vacuoles, fewer large epidermal cells, and less overall leaf area, indicating that nhx1 also plays a developmental role . Overexpression and knockout of the nhx1 gene in arabidopsis have been shown to significantly and differentially alter the expression of a large number of genes involved in the response to salt stress, indicating that arabidopsis can respond to a change in one na transporter by regulating other genes [105, 106]. Yokoi et al . Reported that atnhx2 and atnhx5 could be important salt - tolerant determinants and observed that atnhx2 has a major function in vacuolar na sequestration . H pumps in the plasma membrane and tonoplast energize solute transport necessary to compartmentalize cytotoxic ions away from the cytoplasm and to facilitate the function of ions as signal determinants [107109]. That is these pumps provide the driving force (h electrochemical potential) for secondary active transport and function to establish membrane potential gradients that facilitate electrophoretic ion flux . The plasma membrane localized h pump is a p - type atpase and is primarily responsible for the large ph and membrane potential gradient across this membrane . A vacuolar type h - atpase and h - ppase generate the ph and membrane potential across the tonoplast [108, 110]. The activity of these h pumps is increased by salt treatment, and induced gene expression may account for some of the upregulation [108, 111]. Two cdna clones (ovp1 and ovp2) encoding vacuolar h - ppases isolated from rice were reported . Indeed, there are two genes in arabidopsis annotated as inorganic pyrophosphatase h - ppase (avp1, avp3) and a third loci encoding a pyrophosphatase like (avp2 = avpl1), more than five isoforms in rice, and at least three isoforms in barley [107, 113]. It has been previously demonstrated that overexpression of the h - ppase avp1 increases salinity tolerance and na accumulation in arabidopsis . The vacuolar na levels of the transformants were found to be higher than those of wild - type plants, indicating that overexpression of avp1 led to increased activity of the na / h antiporter . Functional characterization of wheat na / h antiporter tnhx1 and vacuolar pyrophosphatase tvp1 was reported by brini et al . . Transgenic arabidopsis lines overexpressing tnhx1 or tvp1 are much more resistant to high concentrations of nacl and to water deprivation than the wild - type plants . These transgenic plants grow well in the presence of 200 mm nacl and also under a water - deprivation regime, while wild - type plants exhibit chlorosis and growth inhibition . In barley, expression of the h - ppase hvp1, and the vacuolar na / h antiporter nhx1, was similarly upregulated by salt stress and is regulated by aba, auxin, and gibberellin . The simultaneous expression of nhx and avp genes in rice was found to increase salinity tolerance to a greater extent than expression of the genes individually . The overexpression of avp1 also appears to increase growth rates of plants due to an interaction with the auxin transporter pin1, which increases auxin transport resulting in more robust plants which are better able to survive stress conditions [24, 116]. In addition to na, cl compartmentation is also important for salt tolerance, as elevated levels of cl in the cytosol may be harmful, particularly in the case of citrus crops . Since the vacuole is moderately positive with reference to the cytoplasm, part of the vacuolar cl sequestration could proceed through ion channels, and several voltage - gated anion channels of the clc family have been detected in the tonoplast of various species . In arabidopsis, clca was recently shown to function primarily as a h - coupled antiporter to drive vacuolar nitrate accumulation, whereas clcc may also be involved in no homeostasis rather than vacuolar cl sequestration . However, clc transcription has been found to respond to salinity: in rice, osclca was significantly upregulated in salt - sensitive cultivars in response to salinity stress and osclcc, which is expressed in both leaves and roots, showed transcript reduction in the chloride accumulating salt - sensitive ir29 while transient induction occurred in the chloride excluding variety pokkali . Didhiou and golldack showed a coordinated regulation of anion and cation homeostasis in salt - treated rice and suggested a function for osclcc in osmotic adjustment at high salinity . Nakamura et al . Showed that the same clc channels partially complimented the yeast gef1 mutant which lacks the yeast clc channel . In conjunction, these findings suggest that clc - type anion channels are important in mediating cl sequestration in vacuole . An important issue in salt stress physiology is na translocation from the root to the shoot [120, 121]. Physiological evidence suggests that halophytes and salt - resistant glycophytes actively transport na from the root to the shoot, whereas salt - sensitive glycophytes appear to limit na entry into the transpirational stream to prevent na accumulation in the shoot [120, 121]. The transporter(s) responsible for na transport to and from the xylem vessels are not known, although plasma membrane na / h antiporters have been proposed to fulfil this role [111, 122]. It also is not known which cell layer(s) might be critical for controlling the extent of na entry or exit from the xylem . The plasma membrane antiporter sos1 is expressed in root parenchyma and in arabidopsis impacts on na loading into the xylem sap during moderate salt stress . However, its exact function may depend on the severity of the salinity stress and includes removal of na from the xylem stream when salt stress is excessive . In arabidopsis, loss - of - function mutations in the hkt1 gene lead to overaccumulation of na in shoots and rendered the plant na hypersensitive [37, 123]. Rna in situ hybridizations showed that hkt1 is expressed mainly in leaf phloem tissues and mediates na loading into the phloem vessels . In addition, hkt1 may be involved in na unloading from the phloem sap in roots thus providing a mechanism for na retranslocation from shoot to root . In rice, oshkt1;5 is a plasma membrane na transporter expressed in xylem parenchyma cells that retrieves na from the xylem sap . Heterologous expression revealed that oshkt1;5 is a na transporter, and whole plant analysis indicated that it functions in the root xylem parenchyma to retrieve na from the xylem stream, thereby reducing na accumulation in the shoot . Flux analysis of a salt - tolerant durum wheat landrace, line 149, revealed that na exclusion in this line is underpinned by the individual traits of decreased na transfer to the shoot and increased na retrieval to the leaf sheath tissue . Nax1 and nax2, two previously mapped qtls that have been linked to salinity tolerance in line149 were found to control the two transport traits . The nax2 locus coincided with a na transporter related to oshkt1;5 in rice, and this gene was shown to be responsible for removal of na from the xylem in the roots . Members of the h: monovalent cation exchanger family (chx) are also likely to contribute to na translocation . Atchx21 is mainly expressed in the root endodermis, and loss of function in this gene reduced levels of na in the xylem sap without affecting phloem na concentrations . In rice, salt - induced expression of oschx11 in roots was cultivar dependant and higher in a tolerant cultivar . The differential expression correlated with a higher k / na ratio in the tolerant cultivar suggesting that chx11 may be involved in long - distance transport of na and/or k. currently, a large number of potential gene targets are available to manipulate salt tolerance . This number has drastically increased through the many large scale transcriptomic studies over the past decade, but in many cases the validity of the reported findings has yet to be established . For the various processes that contribute to salt tolerance, regulating uptake of na and cl from the soil is of primary importance, particularly in glycophytes which appear to have unidirectional na and cl influx that greatly exceeds net uptake . Although several studies convincingly show that nonselective cation channels are involved, their molecular nature is largely unknown . Out of the substantial gene families that encode nonselective cation channels (cngcs and glurs), only cngc3 and cngc10 were shown to have a moderate impact on salt tolerance [25, 128]. The data available suggest that single cngcs do not play important roles in na uptake . However, creating multiple loss of function mutants, for example, for all cngcs or glurs expressed at the root periphery, may be required to provide more conclusive evidence in this respect . This might be possible by overexpressing na / h antiporters or na - atpases specifically in the outer root cells to improve na extrusion . The bryophyte physcomitrella patens is tolerant to a range of stresses and is consequently attracting significant attention as a potential source of genes to improve stress tolerance in higher plants [129132]. As in many algae and fungi, and unlike higher plants that do not have primary atp hydrolysing na pumps, p. patens has two na - atpases: ppena1 and ppena2 . Expression of ppena1 is significantly upregulated by na stress, and the wild - type physcomitrella maintained a higher k / na ratio and larger size than the ena1 knockout line at moderate naconcentrations (100 mm). The generation and characterization of plants with increased na efflux from the root epidermal and cortical cells are eagerly awaited . The potential for storage of na in vacuoles in the root should be maximized, as has been achieved by overexpression of nhx and vacuolar pyrophosphatase genes [11, 15]. Loading of na into the xylem by the inner stelar cells of the root should be minimized and retrieval of na from the xylem increased, as has been achieved through amplification of athkt1;1 activity in the root stele in arabidopsis . Once na has been transported to the shoot, strategies for tolerance to na become important . These include increased storage of na in the vacuole either through increased uptake or decreased efflux across the tonoplast . Several studies using overexpression of nhx and vacuolar pyrophosphatase have shown this strategy to be effective (table 1) [11, 15, 9396, 98100]. Several abiotic stresses cause changes in morphological, physiological, biochemical, and molecular processes in plants . The increasing prevalence of soil salinity is one of the most dangerous obstacles to improving crop productivity and quality . The adverse effects of saline soil include ion toxicity, nutrient constraints, osmotic stress, and oxidative stress . Many gene targets involved in salt tolerance have been identified through various approaches, particularly through transcriptomic studies . It is likely that such approaches generate many false positives, and this is born out by a lack of supporting evidence for an actual function in plant salinity tolerance . The accumulative data show importance of two particular classes of transporters: hkts which function in both na uptake and long - distance translocation and nhxs in their capacity as na: h antiport or by maintaining k homeostasis . The significance of these systems is often isoform dependent and may be further complicated by allelic variation between cultivars . Manipulation of several of the genes discussed above has been shown to alter uptake, efflux, translocation, and compartmentation of na . Knowledge gained through use of heterologous expression systems and model plant systems provides an extremely useful starting point for the development of salinity - tolerant crop plants . However, further work involving the transgenic expression of na transporters in cereal and broad leaved crop plants needs to be undertaken . Before any claims of salinity tolerance can be substantiated, robust data on yield measurements is required; preferably from field - based trials . Also, it is argued that phenotyping of arabidopsis should be carried out under the most relevant conditions possible, notably, under transpiring conditions rather than in sealed - agar - plate - based assays, as transpiration is a crucial factor in the transport of ions such as na through the plant.
|
, skilled observers cannot consistently detect hypoxemia until theoxygen (o2) saturation is below 80% . Thedifficulty that physicians have in detecting hypoxemia was recently exemplifiedin a study of over 14000 patients being evaluated at the ucla emergencydepartment . Patients were monitored by oximetry butrecordings were given to physicians only after they completed their initialassessment . Changes in diagnostic testing and treatment were most likely at ano2 saturation of 89%, and changes were actually less common at lowersaturations, probably because the physicians were able to detect evidence ofhypoxemia without requiring a pulse oximeter . With the proliferation of pulse oximeters in different locations ofthe hospital throughout the 1980s, several investigators demonstrated thatepisodic hypoxemia is much more common than previously suspected with anincidence ranging from 20 - 82% (fig . 1). Thesignificance of episodic desaturation on patient outcome is largely unknown . In patients admitted to a general medical service, bowton et al . Found that o2saturation <90% of at least 5 min duration occurred in 26% of the patients.on follow - up over the next 4 - 7 months, those patients experiencing hypoxemiaduring the first 24 h of hospitalization had more than a threefold highermortality than patients who did not desaturate . Although episodic desaturationmay simply be a marker of increased risk rather than the direct cause ofdecreased survival, an increased mortality rate was still observed in patientswith episodic hypoxemia when the investigators corrected for severity ofillness . Whether or not the early detection and treatment of episodic hypoxemiacan affect patient outcome remains unknown . Pulse oximetry is based on two physical principles: (a) the presenceof a pulsatile signal generated by arterial blood, which is relativelyindependent of non - pulsatile arterial blood, venous and capillary blood, andother tissues; and (b) the fact that oxyhemoglobin (o2hb) andreduced hemoglobin (hb) have different absorption spectra . Currently available oximeters use two light - emitting diodes (leds) that emit light at the 660 nm (red) and the 940 nm (infrared) wavelengths.these two wavelengths are used because o2hb and hb have differentabsorption spectra at these particular wavelengths . In the red region, o2hb absorbs less light than hb, while the reverse occurs in theinfrared region . The ratio of absorbencies at these two wavelengths iscalibrated empirically against direct measurements of arterial blood oxygensaturation (sao2) in volunteers, and theresulting calibration algorithm is stored in a digital microprocessor withinthe pulse oximeter . During subsequent use, the calibration curve is used togenerate the pulse oximeter's estimate of arterial saturation(spo2) (fig . In addition to the digitalreadout of o2 saturation, most pulse oximeters display aplethysmographic waveform which can help clinicians distinguish an artifactualsignal from the true signal (fig . The accuracy of commercially available oximeters differ widely, probably because of the different algorithms employed in signal processing . These algorithms are limited by the range ofsaturations that can be safely obtained in volunteers, and also the accuracy ofthe measurement standard (co - oximeter). Comparisonof pulse oximetry with direct co - oximeter measurements should be reported interms of the mean difference between the two techniques (bias) and the standarddeviation of the differences (precision). In healthy volunteers, oximeters commonly have a mean difference(bias) of <2% and a standard deviation (precision) of <3% whensao2 is 90% or above . Inhealthy volunteers under hypoxic conditions, bias of pulse oximetry varies from-15.0 to 13.1 while the precision ranges from 1.0 to 16.0 . In a study in critically ill patients, eight out of 13oximeters had a bias 5% when sao2was <80% . In a study of 54 ventilator - dependentpatients,> 90%, and it increased to 5.1 2.7% when sao2 was 90% . Different probes that are used with a pulse oximeter can also affectthe accuracy of spo2 measurements . Inpatientswith poor peripheral perfusion as a consequence of car - diopulmonarybypass surgery, finger probes had lower precision and more readings within 3%of the reference (co - oximeter) than the other probes . Overall rankings weresignificantly better for the finger probes than probes on other sites (fig.4). The response time ofoximeter probes was assessed by severinghaus and naifeh who induced 30 - 60s hypoxic plateaus between ansao2 of 40 and 70% in healthy volunteers.oximeter probes placed on the ear generally had a much faster response to asudden decrease in fractional inspired oxygen concentration(fio2) than did the finger probes (10 - 20 versus24 - 35s, repectively). Also observedthat the response time of the finger probes were slower than the ear probes inresponse to either a decrease or increase in o2 saturation . Oximeters have a number of limitations which may lead to inaccuratereadings (table 1). Pulse oximeters measuresao2 that is physiologically related to arterialoxygen tension (pao2) according to the o2hbdissociation curve . Because the o2hb dissociation curve has asigmoid shape, oximetry is relatively insensitive in detecting the developmentof hypoxemia in patients with high baseline levels ofpao2 . Pulse oximeters employ only two wavelengths of light and, thus, candistinguish only two substances, hb and o2hb . When carboxyhemoglobin(cohb) and methemoglobin (methb) are also present, four wavelengths arerequired to determine the' fractional sao2': i.e.,(o2hb 100)/(hb + o2hb + cohb + methb). In the presence ofelevated cohb levels, oximetry consistently over- estimated the truesao2 by the amount of cohb present . Anemia does not appear to affect the accuracy of pulse oximetry: in non - hypoxemic patients with acute anemia (mean hb, 5.2 0.3(se) g / dl), pulse oximetry was accurate in measuring o2 saturationwith abias of only 0.53% . In patients withsickle cell anemia presenting with acute vaso - occlusive crisis, mean bias of pulse oximetry was 4.5% (in some patients it was as high as 8%), which was significantly greater than in a control group ofpatients without sickle cell anemia . Severe hyperbilirubinemia (mean bilirubin,30.6 mg / dl) does not effect the accuracy of pulse oximetry . Intravenous dyes such as methylene blue, indocyaninegreen, and indigocarmine can cause falsely low spo2 readings, an effect that persists for up to 20 min . Nail polish, if blue, green or black, causes inaccurate spo2 readings, whereas acrylic nails do not interfere with pulse oximetry readings . Motion artifact continues to be a significant source of error andfalse alarms . In a recent, prospective study in an intensive care unit (icu) setting, spo2signals accounted for almost half of a total of 2525 false alarms (fig . Recoveringfrom general or spinal - epidural anesthesia, 77% of pulse oximeter alarms werefalse in nature, which the investigators attributed to sensor displacement, motion artifact, and a decrease in skin perfusion .in this study, the alarm threshold was set at anspo2 of 90% and it is not clear if a minimumduration was specified . A recent study in 647 patients in the recovery roomcompared the influence of two pulse oximeter lower alarm limit settings(spo2 90% = group 90 andspo2 85% = group 85) on the incidence ofhypoxemia . Although the number of audible alarms waslower in group 85, hypoxic episodes (defined asspo2 90% lasting> 1 min) weremore common in group 90 than in group 85 (11 versus 6%, respectively). Theinvestigators concluded that decreasing the alarm limit to reduce false alarmsmay lead to increase in more relevant episodes of hypoxemia . An innovative technological approach, termed masimo signal extraction technology (set;masimo corporation, mission viejo, california, usa), was recently introduced toextract the true signal from artifact due to noise and low perfusion .this technique incorporates new algorithms for processing the pulseoximeter's red and infrared light signals that enable the noisecomponent, which is common to the two wavelengths, to be measured andsubtracted . When tested in healthy volunteers during standardized motion, masimo set exhibited much lower error rates (defined aspercentage of time that the oximeter error exceeded 5%, 7%, and 10%) anddropout rates (defined as the percentage of time that the oximeter provided nospo2 data) than did the nellcor n-200 andnellcor n-3000 oximeters (nellcor puritan bennett, pleasanton, california, usa)for all test conditions . The lowest performanceindex (defined as the percentage of time that the oximeter's value waswithin 7% of the control spo2 value) was 97% formasimo set comparedwith 47% for the n-3000 and 68% for then-200 . In 50 postoperative patients, dumas et al observed that a pulse oximeter's alarm frequency wasdecreased twofold with a masimo set system versus aconventional oximeter (nellcor n-200). Improved performance was particularlystriking during conditions of gross (non - rhythmic) motion and tremor, when a22-fold reduction in signal loss over time was observed (fig . Inaccurate oximetry readings have been observed in pigmented patients, but not by all investigators . In 33 healthy blacksubjects during normoxia and hypoxia, the correlation betweenspo2 and sao2 wasinferior with a biox iia oximeter (ohmeda, boulder, colorado, usa) (r= 0.80) than with the older hewlett - packard (waltham, massachusetts, usa)(non - pulse) oximeter (r = 0.94). Incritically ill patients, bias precision wasgreater in black patients, 3.3 2.7%, than in white patients, 2.2 1.8%; also, a bias> 4% occurred more frequently in black patients (27%) thanin white patients (11%). Low perfusion states, such as low cardiac output, vasoconstriction andhypothermia, may impair peripheral perfusion and may make it difficult for asensor to distinguish a true signal from background noise . In cardiac surgerypatients experiencing hypothermia and poor perfusion, only two of 20 oximeters(criticare csi 503, criticare systems, inc ., milwaukee, wisconsin, usa; datexsatlite, datex instrumentarium corp ., helsinki, finland) provided measurementswithin 4% of the co - oximeter value .measurements of spo2 with a biox 3700 oximeter had a bias> 4% in 37% of patients receiving vasoactive therapy . An under - recognized and worrisome problem with pulse oximetry is thatmany users have a limited understanding of how it functions and theimplications of its measurements . In a recent survey some clinicians alsohave a limited knowledge of the o2-dissociation curve, andthey do not recognize that spo2values in the high 80s represent seriously low values ofpao2 . In the above survey, some doctors andnurses were not especially worried about patients withspo2 values as low as 80% (equivalent topao2 45 torr). Cullen et al . Demonstrated thatthe introduction of pulse oximetry to areas where anesthesia was administereddecreased the overall rate of unanticipated admissions to the icu . Conducted the first prospective, randomized study of pulse oximetry on the outcome of anesthesia care in 20 802surgical patients . A 19-fold increase in the detection of hypoxemia (defined asan spo2 <90%) myocardial ischemia was more common in the controlgroup versus the oximetry group (26 and 12 patients, respectively); however, pulse oximetry did not decrease the rate of postoperative complications ormortality . In general care units of a university hospital, bowton etal . Reported that 75% of patients had at leastone episode of desaturation with spo2 <90%,and 58% had at least one episode with spo2 <85% . Despite these events, few nurses, and even fewerphysicians, made mention of these hypoxemic episodes in their clinical notes.moreover, the decrease in spo2 values rarelyresulted in a change in respiratory care orders . Pulse oximetry can assist with titration offio2 in ventilator - dependent patients, althoughthe appropriate spo2 target depends on apatient's pigmentation . In whitepatients, anspo2 target value of 92% predicts asatisfactorylevel of oxygenation whereas, in black patients, this target mayresult in significant hypoxemia . While a higher targetspo2 value (95%) avoids hypoxemia in blackpatients, some will have pao2 values as high as198torr (fig . 7) and, if receiving a highfio2 to achieve thespo2 target of 95%, o2 toxicity mayresult the potential usefulness of pulse oximetry as a screening tool thatcould supplement or supplant respiratory rate as a' pulmonary vitalsign' was investigated . Paired measurementsof respiratory rate (counted while auscultating breath sounds for 1 min) andspo2 were obtained in over 12000 adult patientsin the triage area of an emergency department . Therelationship between spo2 and respiratory raterevealed correlation coefficients of 0.378 to -0.454 with a weighted mean of-0.160, in other words, a weak inverse relationship betweenspo2 and respiratory rate . Overall, only 33% ofpatients with an spo2 below 90% exhibited anincrease in respiratory rate (defined as any rate in the upper five percentileby age). The study confirmed previous observations that respiratory rate alone isnot accurate in detecting hypoxemia . The usefulness of pulse oximetry as a means of screening forrespiratory failure defined as pao2 <60 mmhgand paco2> 45 mmhg in patients with severeasthma was examined . Respiratory failure occurredin six patients out of 82 (7.3%) with an sao2> 90% versus only three out of 72 (4.2%) patients with ansao2> 92% (p <0.005). Theinvestigators concluded that an spo2> 92%suggests that respiratory failure is unlikely and therefore arterial blood gasmeasurements are unnecessary when evaluating patients with acute severe asthma.interestingly, this threshold value of 92% is the same target value thatpredicted reliably a satisfactory level of oxygenation during titration offio2 in ventilator - dependent patients . Bierman et al . Reported that fewerarterial blood gas (abg) samples were obtained in cardiac surgery patientsifspo2 data were available to the caregivers.interestingly,the availability of oximetry data had no effect on the durationof icu stay, duration of mechanical ventilation, or the need for supplementalo2 . In an emergency department, a recent report showed that thenumber of unjustified abgs (as determined by independent experts) over a2-month period decreased from 29% when pulse oximetry was unavailable to 12%when oximetry was available; the number of justified abgs did not change . Solsona et al . Measured thenumber of blood gas measurements in 417 patients admitted to a medical - surgicalicu during a 12-month period in which only two pulse oximeters were available(i.e . They then studied 306 patients admitted over a 9-monthperiod when 12 pulse oximeters were available for the same number of beds(i.e . Less frequent use of mechanical ventilation and aslightly lower number of arterial blood samples were observed when pulseoximetry was fully available . Inman et al . Examined the effect of implementing pulse oximetry without any specificalgorithm for its appropriate use . They studied 148 patients before theimplementation of oximetry in their icu and 141 patients after itsimplementation . The number of abg samples decreased from 7.2 to 6.4 per patientper day, a reduction of only 10.3% compared with average reductions of 39% inthe previous studies . Thissuggests that, without explicit guidelines, the pulse oximeter was used inaddition to, rather than instead of, abg samples . Pulse oximetry is probably one of the most important advances inrespiratory monitoring . Over the last 15 years, numerous studies have focusedon the technical aspects of pulse oximeters and found that these instrumentshave a reasonable degree of accuracy . This degree of accuracy, coupled with theease of operation of most instruments, has led to the widespread use of pulseoximetry for monitoring patients in the icu . Perhaps the major challenge facingpulse oximetry is whether this technology can be incorporated effectively intodiagnostic and management algorithms that can improve the efficiency ofclinical management in the intensive care unit . Sequential distribution plots of oxygen saturation at intervals of2 min over a 3-h period in a stable patient (a) and unstable patient (b). Red (r) and infrared (ir) scaled alternating current (ac) signals at arterial oxygen saturation (sao2) of 0%, 85% and 100% . The numeric value of the red - to - infrared (r / ir) ratio can be easily converted to sao2 . Published with permission . (top panel) normal signal showing the sharp waveform with a clear dicrotic notch . (lowest panel) pulsatile signal during motion artifact showing an erratic waveform . Published with permission . Pulse oximeter probes placed on the finger, ear, nose or foreheadranked for accuracy in terms of bias under conditions of poor perfusion . Biasof pulse oximeters ranged from 0.2 to 1.7 for finger probes and 0.1 to 8.1 forother probes . Number of false alarms for devices used to monitor respiratoryrate, mean systemic blood pressure from an arterial catheter, heart rate froman electrocardiogram (ekg), heart rate measured by pulse oximetry (pox) ando2 saturation measured by a pulse oximetry(spo2)., gross arm motion; top panel) and during parkinsonian tremor (lowerpanel). Solid line denotes masimo signal extraction technology(set), aimedat minimizing spurious pulse oximetry readingsdue to motion artifact; dashed line denotes conventional pulse oximetry.spurious changes in spo2 were less with masimoset than with conventional pulse oximetry arterial oxygen tension (pao2) values atpulse oximetry o2 saturation (spo2) value of90, 92, 94, and 95% . The inspired o2 concentration(fio2) was adjusted until the desired steady - statespo2 value was achieved . The closed and open circles represent valuesobtained in black and white patients, respectively . In whitepatients, anspo2 target of 92% resulted in a satisfactory level ofoxygenation, whereas a higher spo2 target, 95%, wasrequired in black patients . Published with permission
|
A 38-year - old woman was admitted to our outpatient clinic with accelerating back pain and fatigue following a kick to her back by her husband two days previously . On her physical examination, an ecchymotic area on her back between the scapulae was observed . She had pallor, her blood pressure was 80/60 mmhg in both arms, and was tachycardic on auscultation . St segment elevations were observed in the d1, avl, and v2 leads, along with accelerated idioventricular rhythm (fig . 1). Transthoracic echocardiography demonstrated akinesia of the anterior septal, apical, basal - mid septal, and basal - mid anterior walls, and her ejection fraction was 20% . We detected a dissection of the left main artery, the left anterior descending artery (lad), and the circumflex artery, originating from the middle portion of the left main coronary artery (lmca) (figs . 2 and 3, supplemental videos 1, 2). A saphenous vein was grafted to the distal lad . During the intraoperative evaluation of the epicardial vessels, our team of cardiac surgeons did not plan to place a bypass graft to the circumflex artery because it was thin and non - dominant . Since the patient was hypotensive under noradrenaline and dopamine infusions, she was transferred to the cardiovascular surgery intensive care unit on an extracorporeal membrane oxygenator (ecmo) and intra - aortic balloon pump (iabp). During follow - up, her blood pressure remained low, at approximately 60/40 mmhg, despite aggressive inotropic and mechanical support . On the second postoperative day, although the patient s lad artery had been revascularized by a saphenous vein graft, her left ventricular ejection fraction remained as low as 10%15%, leading to ventricular failure . Asystole and cardiovascular arrest then quickly developed, and despite aggressive cardiopulmonary resuscitation, she died . Coronary artery dissection after blunt chest trauma is an extremely rare condition that can be fatal, and some cases are detected in postmortem examinations . Left main coronary artery dissection is even rarer . Multiple mechanisms exist leading to coronary artery dissection, including intimal tears due to a deceleration injury, compression of the artery between the heart and sternum, coronary spasm, and impairment of the coronary flow by a dissection flap or a superimposed thrombosis . Coronary artery dissection is detected most commonly in the lad (76%), the right coronary artery (12%), and the circumflex artery (6%) [4, 5]. In necropsy series, the most common cause of acquired non - atherosclerotic coronary artery disease is spontaneous coronary artery dissection, and the lad is the artery in which this condition is most commonly detected . The risk factors for spontaneous coronary artery dissection are exercise, arteriosclerosis, cardiovascular disease, oral contraceptive use, marfan syndrome, systemic lupus erythematosis, and connective tissue disorders . However, the time from injury to coronary artery occlusion may vary, ranging from immediately after the trauma to five weeks later . Bedside electrocardiography (ecg) provides important clues about coronary artery dissection after blunt anterior chest and back trauma . It has been previously reported that patients with baseline ecg changes on admission should be monitored for 24 hours . The ecg may be normal on presentation, but was found to demonstrate st abnormalities in 63% of patients who are admitted for blunt thoracic trauma within 24 hours of observation . Our patient presented with st segment elevation the in d1, avl, and v2 leads, along with accelerated idioventricular rhythm . Some emergency therapeutic options exist for patients with spontaneous coronary artery dissection . In some previous case reports, the patients were managed by primary percutaneous coronary angioplasty, especially patients without lmca lesions . However, surgical treatment remains most common treatment and is associated with the best outcomes . An internal mammary artery graft was not utilized in our patient, because she was in cardiogenic shock, and the surgical team tried to save time by using a saphenous vein graft . In order to prevent death, all available measures were used by our surgical team, including intravenous inotropic agents, iabp, and ecmo . Nevertheless, using the internal mammary artery for revascularization may have been more effective for restoring left ventricular function . Additionally, complete revascularization, including the circumflex artery, spontaneous coronary artery dissection is a rare and potentially fatal complication of blunt chest trauma in younger patients, and early diagnosis and prompt treatment can be life - saving . Physicians should be aware of this possibility when evaluating patients in emergency conditions after blunt trauma of any kind . The 12-lead ecg, cardiac bio - markers, and transthoracic echocardiography, along with other imaging modalities, are important in the management of suspected coronary artery dissection in trauma patients.
|
A fit and healthy 25-year - old male presented with palpitations from a regular broad complex tachycardia (bct) of left bundle branch block (lbbb)-like morphology, 330-ms cycle length, and no discernible p waves . Intravenous adenosine terminated the bct, resting electrocardiogram (ecg) was not pre - excited, and echocardiogram was normal . During the electrophysiologic study, retrograde conduction was central and decremental . 1a shows that conduction switched from nodal (narrow qrs complexes with distinct his potential solid blue arrow) to the atriofascicular pathway (lbbb - like complexes and interpolation of the his potential dotted blue arrow) during wenckebach - pacing maneuver . 1b shows that the intellatip mifi ablation catheter was used to map the atriofascicular pathway potential in sinus rhythm . The 8-mm tip ablation catheter has three radially positioned, equally spaced mini - electrodes, 2 mm from the tip . Bipolar recordings were made between these mini - electrodes: m12, m23, and m31 . This enabled precise signal localization and clearer identification of the pathway potential than the conventional ablation distal bipolar recording (blue dotted box, fig . The gains on the catheter mini - electrodes were 5000-fold greater, and those in the conventional distal pole of the ablation catheter were 10,000-fold greater . Radiofrequency ablation (rfa) at this site (60 c, 70 w, and 120 s) resulted in no pathway conduction and non - inducible bct . Atriofascicular pathways consist of fibers arising from the right atrial free wall and insert at or adjacent to the distal right bundle . They usually only conduct in an anterograde manner, participating in the anterograde limb of an antidromic atrioventricular reciprocating tachycardia (avrt) with lbbb - like morphology and decremental properties . Standard therapy involves targeted rfa around the tricuspid annulus as guided by pathway potentials also known as mahaim (m) potentials, . These m potentials can be as large as the his deflection or can be small and narrow with a low amplitude . Furthermore, unintentional mechanical trauma by catheter manipulation can result in transient abolition of these potentials from a few minutes to a few hours, . These m potentials are also recorded only in close proximity to the atrial insertion site, and thus, accurate localization and ablation at this site result in successful abolition of this pathway,, . The intellatip mifi catheter has been shown to delineate local electograms better in the isthmus than the conventional bipolar electrode during atrial flutter ablation . The signal amplitude in the mini - electrodes has been noted to be higher than that of the conventional bipolar catheter . We used the same principle in this case to make use of the special characteristics of the novel ablation catheter to localize the atrial insertion point accurately . Furthermore, the m potentials, as recorded from the distal mini - electrodes, were clearer and of higher amplitude compared with those recorded using the conventional distal pole of the ablation catheter . We did not compare this with a standard ablation catheter in the same patient to keep the costs of the procedure within reasonable limits and to avoid causing unintentional mechanical trauma to the atrial insertion site.
|
We included only first strokes between 2005 and 2007 in persons with a period free from stroke of at least 1 year . Strokes (cerebral ischemia, intracerebral hemorrhage, subarachnoid hemorrhage, and stroke of uncertain cause, but no transient ischemic attacks) were defined following the world health organization definition (16), using specific icd-10 codes of hospital admissions (i60i61, i63i64, and including i62 to avoid missing unspecific cases). Diabetes status was assessed according to an established algorithm that has been used in several studies analyzing claims data of german statutory health insurance funds (13,17). A person was identified as having diabetes if at least one of the following characteristics was fulfilled within 12 months in the observation period between 2004 and 2007: 1) diabetes diagnosis (icd e10e14) in at least three of four consecutive quarters in outpatient care, 2) at least two prescriptions of antihyperglycemic medication (anatomical therapeutic chemical code a10) within 12 months, or 3) at least one prescription of an antihyperglycemic medication and one diabetes diagnosis or one measurement of blood glucose or hba1c within 12 months . In the previously published analysis, data of 6,160 patients (n = 1,932 had diabetes) with an incident stroke between 2005 and 2007 were available . For this study, only persons aged 30 years were included (n = 6,100). We further excluded all persons coinsured as a dependent and members who left the gek for reasons other than death within the study period (n = 343). Both criteria were applied to avoid informative censoring in the survival analysis (e.g., an insurance period ends because of death, but this reason might not be documented in these cases). Our final cohort, therefore, consisted of 5,757 patients with a first stroke during 2005 to 2007 and follow - up until the end of 2009 . From the claims data, we assessed reimbursed medications as well as services of the long - term care insurance for the year preceding the index event (the first stroke). Treatment with cardiovascular drugs (-blockers, ace inhibitors / sartans, and calcium antagonists) and antihyperglycemic drugs (insulin and oral antihyperglycemic agents) was assessed . We determined the number of distinct medications prescribed within this period as a comorbidity measure because it has been shown to be a good predictor of mortality (18). Services from the german long - term care insurance are provided to those who require support in the activities of daily living, including personal hygiene, eating, mobility, and there are three levels of care dependency related to the estimated time required for assistance and indicating considerable (level 1), severe (level 2), and extreme (level 3) care dependency (19). The highest level of dependency within the year before the index date was included as a proxy for functional and cognitive impairments . Furthermore, we assessed the following outpatient diagnoses: hypertension (icd-10: i10i15), chronic ischemic heart diseases (icd-10: i20i21, i25), and renal failure (icd-10: n18n19) coded according to a previous study using german claims data (20). At least one of these diagnoses had to be recorded in a 1-year period (including the quarter of the index date and the preceding three quarters). Quarters had to be chosen because this is the basic time period for coding diagnoses in outpatient care in germany . The outcome of interest was the time from the first stroke up to death or the end of the study period (31 december 2009), whichever came first . We assessed crude survival with the kaplan - meier estimator, stratified for diabetes as well as for sex . The appropriateness of the cox proportional hazards assumption was further visualized using log - log survival plots, that is, plotting log (-log(s(t)) against log(t). Furthermore, we tested the proportional hazards assumption via the test proposed by grambsch and therneau (21). Because we expected that the interaction between diabetes and time was statistically significant, which means that the proportional hazards assumption was violated, we performed cox regression using discrete time intervals to model the time dependency of diabetes (22). We estimated time - dependent hazard ratios (hrs) and 95% cis in multivariate analyses . As predictors, we included diabetes, interaction of diabetes with the discrete time intervals (30 days and 6, 12, 24, 36, and 60 months), and age (as continuous variable). We chose the time intervals in line with previous studies to be able to compare our results and on the basis of clinical experience . In a second model, type of stroke (ischemic, hemorrhagic, and not specified), number of prescribed medications (as continuous variable), level of care dependency (four categories), and the above - stated outpatient diagnoses for hypertension, coronary heart diseases, and renal failure were added as further independent variables . The results of the cox models were verified using r (a language and environment for statistical computing, release 2.12.1, r foundation for statistical computing, http://www.r-project.org). We considered the strobe statement (strengthening the reporting of observational studies in epidemiology) and the criteria of a national good practice guideline (23,24). The use of health insurance claims data for scientific research is regulated by the german code of social law (sgb x). Because our study was based on pseudonymous data, we did not have to obtain informed consent . According to the good practice of secondary data analysis, a national guideline for the use of administrative databases, no approval of an ethical committee we used data of a cohort of patients with incident stroke, for which analyses on incidence and attributable risks have been published elsewhere (2). In brief, these patients were derived from a statutory health insurance company, the gmnder ersatzkasse (gek), which insures 1.6 million people located in all regions of germany (1.9% of the german population). We included only first strokes between 2005 and 2007 in persons with a period free from stroke of at least 1 year . Strokes (cerebral ischemia, intracerebral hemorrhage, subarachnoid hemorrhage, and stroke of uncertain cause, but no transient ischemic attacks) were defined following the world health organization definition (16), using specific icd-10 codes of hospital admissions (i60i61, i63i64, and including i62 to avoid missing unspecific cases). Diabetes status was assessed according to an established algorithm that has been used in several studies analyzing claims data of german statutory health insurance funds (13,17). A person was identified as having diabetes if at least one of the following characteristics was fulfilled within 12 months in the observation period between 2004 and 2007: 1) diabetes diagnosis (icd e10e14) in at least three of four consecutive quarters in outpatient care, 2) at least two prescriptions of antihyperglycemic medication (anatomical therapeutic chemical code a10) within 12 months, or 3) at least one prescription of an antihyperglycemic medication and one diabetes diagnosis or one measurement of blood glucose or hba1c within 12 months . In the previously published analysis, data of 6,160 patients (n = 1,932 had diabetes) with an incident stroke between 2005 and 2007 were available . For this study, only persons aged 30 years were included (n = 6,100). We further excluded all persons coinsured as a dependent and members who left the gek for reasons other than death within the study period (n = 343). Both criteria were applied to avoid informative censoring in the survival analysis (e.g., an insurance period ends because of death, but this reason might not be documented in these cases). Our final cohort, therefore, consisted of 5,757 patients with a first stroke during 2005 to 2007 and follow - up until the end of 2009 . From the claims data, we assessed reimbursed medications as well as services of the long - term care insurance for the year preceding the index event (the first stroke). Treatment with cardiovascular drugs (-blockers, ace inhibitors / sartans, and calcium antagonists) and antihyperglycemic drugs (insulin and oral antihyperglycemic agents) was assessed . We determined the number of distinct medications prescribed within this period as a comorbidity measure because it has been shown to be a good predictor of mortality (18). Services from the german long - term care insurance are provided to those who require support in the activities of daily living, including personal hygiene, eating, mobility, and there are three levels of care dependency related to the estimated time required for assistance and indicating considerable (level 1), severe (level 2), and extreme (level 3) care dependency (19). The highest level of dependency within the year before the index date was included as a proxy for functional and cognitive impairments . Furthermore, we assessed the following outpatient diagnoses: hypertension (icd-10: i10i15), chronic ischemic heart diseases (icd-10: i20i21, i25), and renal failure (icd-10: n18n19) coded according to a previous study using german claims data (20). At least one of these diagnoses had to be recorded in a 1-year period (including the quarter of the index date and the preceding three quarters). Quarters had to be chosen because this is the basic time period for coding diagnoses in outpatient care in germany . The main analyses were performed stratified for men and women . The outcome of interest was the time from the first stroke up to death or the end of the study period (31 december 2009), whichever came first . We assessed crude survival with the kaplan - meier estimator, stratified for diabetes as well as for sex . The appropriateness of the cox proportional hazards assumption was further visualized using log - log survival plots, that is, plotting log (-log(s(t)) against log(t). Furthermore, we tested the proportional hazards assumption via the test proposed by grambsch and therneau (21). Because we expected that the interaction between diabetes and time was statistically significant, which means that the proportional hazards assumption was violated, we performed cox regression using discrete time intervals to model the time dependency of diabetes (22). We estimated time - dependent hazard ratios (hrs) and 95% cis in multivariate analyses . As predictors, we included diabetes, interaction of diabetes with the discrete time intervals (30 days and 6, 12, 24, 36, and 60 months), and age (as continuous variable). We chose the time intervals in line with previous studies to be able to compare our results and on the basis of clinical experience . In a second model, type of stroke (ischemic, hemorrhagic, and not specified), number of prescribed medications (as continuous variable), level of care dependency (four categories), and the above - stated outpatient diagnoses for hypertension, coronary heart diseases, and renal failure were added as further independent variables . The results of the cox models were verified using r (a language and environment for statistical computing, release 2.12.1, r foundation for statistical computing, http://www.r-project.org). We considered the strobe statement (strengthening the reporting of observational studies in epidemiology) and the criteria of a national good practice guideline (23,24). The use of health insurance claims data for scientific research is regulated by the german code of social law (sgb x). Because our study was based on pseudonymous data, we did not have to obtain informed consent . According to the good practice of secondary data analysis, a national guideline for the use of administrative databases, no approval of an ethical committee table 1 shows the characteristics of the 5,757 individuals with a first stroke between 2005 and 2007 in total as well as stratified for diabetes and sex . Cerebral infarction was by far the most common type of stroke, followed by cerebral hemorrhage and subarachnoid hemorrhage . Comorbidities, such as hypertension and coronary heart disease, were predominant among these individuals . Approximately 13% were at least considerable (level 1) care dependent . On average, description of the study population: gek insurants with first incident stroke during 2005 to 2007, stratified for diabetes and sex for both sexes, diabetic patients were older than nondiabetic individuals and had more ischemic strokes compared with nondiabetic patients . Persons with diabetes were more likely to have a diagnosis of hypertension, coronary heart disease, or renal failure, with similar differences among men and women . They also had a higher level of care dependency, which was particularly pronounced in the female population . Likewise, diabetic patients had an almost twofold higher intake of drug prescriptions for both sexes, which was also true for cardiovascular medications, such as -blockers, ace inhibitors / sartans, and calcium antagonists . The mean observation time was 2.66 years (25 and 75% quartiles 1.98 and 3.83, respectively). Overall, 1.828 individuals died within the study period of up to 5 years, including 470 and 698 men as well as 264 and 396 women with and without diabetes, respectively . The cumulative mortalities, including the population at risk, are presented in table 2 . Crude mortality estimates after first incident stroke: gek insurants during 20052007, stratified for sex and diabetes figure 1 shows the kaplan - meier curves and the log - log survival plots . We present both since in the log - log plots, the early period after stroke event can be seen, whereas the kaplan - meier curves give a better picture of the later period . In men, the crude relative mortality risk as a result of diabetes was significantly time dependent (p = 0.002): in the first month after stroke, diabetic individuals had better survival, but thereafter, mortality risk in diabetic men increased, resulting in a higher survival in nondiabetic men . After 3 years, the curves seem to become more convergent again, which means that the difference between diabetic and nondiabetic men is no longer visible . A significant time dependency of diabetes on mortality could also be seen in multivariate analysis (p = 0.008). Adjusted for age, it yields a significant decreased mortality risk in diabetes within the first month and an increased mortality risk in diabetes between 1 month and 3 years of follow - up, which was no longer the case after 3 to 5 years (model 1, table 3). After further adjustment for comorbidities, level of care dependency, number of prescribed medications, and subtype of stroke, relative risks decreased somewhat but remained significantly increased between 1 and 3 years (model 2, table 3). A: kaplan - meier estimates of crude survival after first incident stroke for male gek insurants, germany, 20052007 . B: kaplan - meier estimates of crude survival after first incident stroke for female gek insurants, germany, 20052007 . C: crude log - log survival curves after first incident stroke for male gek insurants, germany, 20052007 . D: crude log - log survival curves after first incident stroke for female gek insurants, germany, 20052007 . Predictors for mortality after first incident stroke, cox regression gek insurants during 20052007, stratified for sex in women, there is a quite similar pattern . However, time dependency was not statistically significant in crude (p = 0.08) or multivariate analysis (p = 0.89). The curves do cross, albeit only slightly, in the first week of follow - up, and the cox model shows no significant decreased hr in the first months after stroke . Nevertheless, the relative risk of mortality in the fully adjusted model was significantly increased for diabetic women between 6 months and 1 year as well as between 2 and 3 years, with an almost twofold increased risk of diabetic women for the first time interval . Again, we found no significant differences between 3 and 5 years of follow - up . Increasing age, renal failure (only in men), levels of care dependency, number of prescribed medications, and hemorrhage stroke were positively associated with mortality in the fully adjusted model (model 2, table 3). In contrast, mortality was significantly lower in patients with a diagnosis of hypertension for both sexes . Table 1 shows the characteristics of the 5,757 individuals with a first stroke between 2005 and 2007 in total as well as stratified for diabetes and sex . Cerebral infarction was by far the most common type of stroke, followed by cerebral hemorrhage and subarachnoid hemorrhage . Comorbidities, such as hypertension and coronary heart disease, were predominant among these individuals . Approximately 13% were at least considerable (level 1) care dependent . On average, description of the study population: gek insurants with first incident stroke during 2005 to 2007, stratified for diabetes and sex for both sexes, diabetic patients were older than nondiabetic individuals and had more ischemic strokes compared with nondiabetic patients . Persons with diabetes were more likely to have a diagnosis of hypertension, coronary heart disease, or renal failure, with similar differences among men and women . They also had a higher level of care dependency, which was particularly pronounced in the female population . Likewise, diabetic patients had an almost twofold higher intake of drug prescriptions for both sexes, which was also true for cardiovascular medications, such as -blockers, ace inhibitors / sartans, and calcium antagonists . The mean observation time was 2.66 years (25 and 75% quartiles 1.98 and 3.83, respectively). Overall, 1.828 individuals died within the study period of up to 5 years, including 470 and 698 men as well as 264 and 396 women with and without diabetes, respectively . The cumulative mortalities, including the population at risk, are presented in table 2 . Crude mortality estimates after first incident stroke: gek insurants during 20052007, stratified for sex and diabetes figure 1 shows the kaplan - meier curves and the log - log survival plots . We present both since in the log - log plots, the early period after stroke event can be seen, whereas the kaplan - meier curves give a better picture of the later period . In men, the crude relative mortality risk as a result of diabetes was significantly time dependent (p = 0.002): in the first month after stroke, diabetic individuals had better survival, but thereafter, mortality risk in diabetic men increased, resulting in a higher survival in nondiabetic men . After 3 years, the curves seem to become more convergent again, which means that the difference between diabetic and nondiabetic men is no longer visible . A significant time dependency of diabetes on mortality could also be seen in multivariate analysis (p = 0.008). Adjusted for age, it yields a significant decreased mortality risk in diabetes within the first month and an increased mortality risk in diabetes between 1 month and 3 years of follow - up, which was no longer the case after 3 to 5 years (model 1, table 3). After further adjustment for comorbidities, level of care dependency, number of prescribed medications, and subtype of stroke, relative risks decreased somewhat but remained significantly increased between 1 and 3 years (model 2, table 3). A: kaplan - meier estimates of crude survival after first incident stroke for male gek insurants, germany, 20052007 . B: kaplan - meier estimates of crude survival after first incident stroke for female gek insurants, germany, 20052007 . C: crude log - log survival curves after first incident stroke for male gek insurants, germany, 20052007 . D: crude log - log survival curves after first incident stroke for female gek insurants, germany, 20052007 . Predictors for mortality after first incident stroke, cox regression gek insurants during 20052007, stratified for sex in women, there is a quite similar pattern . However, time dependency was not statistically significant in crude (p = 0.08) or multivariate analysis (p = 0.89). The curves do cross, albeit only slightly, in the first week of follow - up, and the cox model shows no significant decreased hr in the first months after stroke . Nevertheless, the relative risk of mortality in the fully adjusted model was significantly increased for diabetic women between 6 months and 1 year as well as between 2 and 3 years, with an almost twofold increased risk of diabetic women for the first time interval . Again, we found no significant differences between 3 and 5 years of follow - up . Increasing age, renal failure (only in men), levels of care dependency, number of prescribed medications, and hemorrhage stroke were positively associated with mortality in the fully adjusted model (model 2, table 3). In contrast, mortality was significantly lower in patients with a diagnosis of hypertension for both sexes . In this study based on data of a nationwide health insurance fund, we analyzed survival in patients with incident stroke in germany during a period of up to 5 years (20052009), with a focus on diabetes as a predictor . After 5 years of follow - up, more than one - third of the patients in our cohort had died . It is interesting that the influence of diabetes in our study was significantly time dependent in men: in the first 30 days after incident stroke, mortality was lower in diabetic than in nondiabetic individuals . Thereafter, there was an increasing trend of diabetes risk during observation time, and after approximately a quarter of a year, diabetic individuals had a higher mortality than nondiabetic individuals . Age, renal failure (only in men), level of care dependency, number of prescribed drugs, and hemorrhagic stroke were significantly associated with mortality; however, they did not alter the association between diabetes and mortality . Our results remained almost unchanged in several sensitivity analyses, for example, using logistic regression models with the variable log(time) as well as time as a linear predictor (data not shown). Looking for an explanation for our finding that mortality in the first 30 days after stroke was lower in diabetic men, one may find several possible hypotheses . First, one could argue that diabetic patients are more closely monitored by several specialists because of their chronic disease . They have more comorbidities, as indicated by medications and outpatient diagnoses; however, if problems arise, they might be identified and treated earlier . In germany, these programs define contents and time frames for the treatment of diabetes and its complications, as well as the associated cardiovascular risk factors . In this context, the observed larger number of prescribed medications in diabetic patients also might hint at more aggressive management of cardiovascular risk factors . The question remains, however, as to why diabetic men should have a higher benefit than diabetic women . Women with a stroke event are older than men and more likely to be in long - term care; hence, they might be less likely to be included in a disease management program (25). Furthermore, it might be that cardiovascular diseases in particular are treated earlier in younger patients and in men . Increasing age and female sex have been found to be related to a prolonged delay of emergency care in acute stroke events (26,27). This hypothesis is further supported by our observation that the impact of diabetes seems to differ between younger and older individuals . In stratified models, there was a more pronounced time dependency in individuals aged 70 compared with those> 70 years . In individuals aged 70 years, mortality during the first 30 days was lower in diabetic compared with nondiabetic individuals in both men and women (even though the time dependency was significant only in men), while this was not the case in patients> 70 (data not shown). In previously published series, patients with ischemic strokes had a lower case fatality (mortality during the first 28 or 30 days after stroke) than patients with a hemorrhage stroke, whereas in the period after 30 days, mortality was higher after ischemic strokes (28). Third, hypertension was more prevalent in diabetic persons . Previously known hypertension at the time of the stroke event has been reported to be significantly associated with a decreased mortality for both sexes, possibly explained by a better tolerance toward higher admission blood pressure in those individuals, which might be clinically more relevant for the early outcome of hemorrhagic than ischemic strokes (29,30). Fourth, other factors may play a role, such as a higher prevalence of obesity among diabetic individuals undergoing stroke compared with their nondiabetic counterparts (31). It is known that obesity and overweight have a potential protective effect in elderly stroke patients (32,33). These phenomena should have larger effects in men since the degree of obesity is commonly higher in men than in women . However, in our data, we have no information about detailed clinical or lifestyle variables and only limited information about history of coronary events, chronic heart failure, and renal function . Also, exact causes of deaths cannot be determined by our data . In the period after 30 days, the mortality risk in individuals with diabetes compared with those without diabetes increased . The observation that in the 3 to 5 years after stroke there was no longer a difference in mortality between individuals with and without diabetes in both men and women may be due to a lack of power resulting from lower case numbers . However, it might be explained by the fact that individuals who survive 3 years are healthier, independent from their diabetes status . The mortality after 30 days (case fatality, 10.5%) as well as the 1-, 2-, and 5-year mortalities in our study (19.5, 25.1, and 37.3%, respectively) were well in line with the findings of other more recent studies and, as expected, lower than earlier studies . Case fatalities in the literature ranged between 10 and 22% (7,9,34), and 1- and 5-year mortalities were 27 and 53%, respectively (10). Only a few studies investigated mortality after stroke in diabetic and nondiabetic patients (1012,35,36); however, these studies were in part clinic based and analyzed only single subtypes of stroke or shorter periods of follow - up . In the study by rautio et al . (10), only case fatality was investigated . During the period from 1985 to 1987, case fatality was 18% in diabetic and 15% in nondiabetic patients, with higher mortality in women than in men . However, in the period from 2000 to 2003, case fatality was 15% in diabetic women, whereas in diabetic men as well as in nondiabetic men and women, it was 10% . In our study, case fatality was 9% in diabetic as well as nondiabetic men, and 16 and 13% in diabetic and nondiabetic women, respectively . (10) study that finds diabetic women have a higher excess risk to die within the first course after stroke; on the other hand, rautio et al . A further study analyzes the 3-month mortality in a clinic - based sample and finds diabetes to be a significant predictor of mortality (35). To the best of our knowledge, there is no population- or insurance - based study that analyzes diabetic and nondiabetic patients for longer periods . Several studies evaluate diabetes as a predictor of mortality after incident stroke, but with conflicting results . (8) found diabetes to be associated with mortality after stroke, benatru et al . (11) analyzed the mortality after discharge from the hospital for a longer period, but only ischemic strokes were included in their study . Kaplan - meier survival plots did not show a difference between individuals with and without diabetes during the first 60 days after discharge but did reflect lower survival in individuals with diabetes compared with those without diabetes after 1 year (11). Likewise, gunarathne et al . (36) analyzed the 5-year mortality in individuals with and without diabetes after ischemic strokes . The 5-year mortality was significantly increased 1.6-fold in individuals with diabetes compared with those without diabetes; however, the study subjects were a clinic - based sample of migrant south asian patients (36). (12) analyzed the 28-day and 1-year fatality after stroke, yet they included only ischemic strokes in their study . Both were significantly increased in individuals with diabetes compared with those without diabetes, without differences between men and women (12). Thus, study results remain conflicting, and further studies are warranted to confirm and explain our findings . First, in particular during the last years of observation and especially in women, the case numbers are low, leading to a lack of power to detect statistically significant differences between patients with and without diabetes . Second, we cannot exclude misclassification when we define patients with diabetes because our identification criteria had to be fulfilled within 12 months in the observation period between 2004 and 2007 and not solely before the first stroke . On the other hand, diabetes is often identified for the first time in hospital stays as a result of typical complications, such as strokes, and these patients would not be classified as patients with diabetes if we used only the period before the event . However, we performed a sensitivity analysis, defining a person as having diabetes when our criteria were fulfilled within the 12 months before the first stroke . We found that approximately 9 of 10 diabetic patients already fulfilled our criteria before their index stroke . Third, we studied stroke survivors, and the number of fatal strokes may differ among those with and without diabetes . This may be an explanation for the reduced mortality seen within the first 30 days among patients with diabetes . However, on the basis of data from the german stroke registry as well as from several other countries, it can be assumed that the number of fatal strokes and strokes that are treated outside the hospital are small . Approximately 95% of stroke patients are hospitalized in clinics and, thus, identified by our data (40). Fourth, information about clinical variables (e.g., blood glucose and diabetes duration) and patient lifestyle (e.g., smoking and physical activities) is not available in the database . However, we included number of prescribed drugs as well as outpatient diagnoses of relevant comorbidities and level of care dependency . Fifth, a translation of our results to other populations should be performed with caution since it is known that differences in morbidity as well as demographic and socioeconomic variables exist between health insurance funds (37,38). However, the incidence of stroke in our population was well in line with the incidence of stroke in a well - designed regional register - based study (2,39,40). Furthermore, the population has been used for several analyses regarding comorbidities in diabetes (2,13,14). The main strength of our study is that we were able to analyze a large dataset without selection with respect to diabetes complications that could be followed up to 5 years . In conclusion, in our german study, based on data from a nationwide health insurance fund, we found a high mortality in patients with a first stroke . It is interesting that the influence of diabetes was time dependent in men: in approximately the first quarter of a year after incident stroke, mortality was lower in diabetic than in nondiabetic individuals . Thereafter, diabetic patients had a higher mortality than nondiabetic patients, and after 3 years, there was a convergence . In women, the pattern seems to be similar: no significant time dependency was found . Our observation is in line with findings for mortality in diabetic compared with nondiabetic patients after beginning renal replacement therapy and amputation . Possible explanations may be differences in the type of stroke or in earlier and more intensive treatment of distinct cardiovascular risk factors in diabetic patients, in particular men . Patients that survive up to 3 years after stroke might be healthier, independent of their diabetes status . However, results remain conflicting, and further studies are warranted to confirm and explain the results . In this study based on data of a nationwide health insurance fund, we analyzed survival in patients with incident stroke in germany during a period of up to 5 years (20052009), with a focus on diabetes as a predictor . After 5 years of follow - up, more than one - third of the patients in our cohort had died . It is interesting that the influence of diabetes in our study was significantly time dependent in men: in the first 30 days after incident stroke, mortality was lower in diabetic than in nondiabetic individuals . Thereafter, there was an increasing trend of diabetes risk during observation time, and after approximately a quarter of a year, diabetic individuals had a higher mortality than nondiabetic individuals . Age, renal failure (only in men), level of care dependency, number of prescribed drugs, and hemorrhagic stroke were significantly associated with mortality; however, they did not alter the association between diabetes and mortality . Our results remained almost unchanged in several sensitivity analyses, for example, using logistic regression models with the variable log(time) as well as time as a linear predictor (data not shown). Looking for an explanation for our finding that mortality in the first 30 days after stroke was lower in diabetic men, one may find several possible hypotheses . First, one could argue that diabetic patients are more closely monitored by several specialists because of their chronic disease . They have more comorbidities, as indicated by medications and outpatient diagnoses; however, if problems arise, they might be identified and treated earlier . In germany, these programs define contents and time frames for the treatment of diabetes and its complications, as well as the associated cardiovascular risk factors . In this context, the observed larger number of prescribed medications in diabetic patients also might hint at more aggressive management of cardiovascular risk factors . The question remains, however, as to why diabetic men should have a higher benefit than diabetic women . Women with a stroke event are older than men and more likely to be in long - term care; hence, they might be less likely to be included in a disease management program (25). Furthermore, it might be that cardiovascular diseases in particular are treated earlier in younger patients and in men . Increasing age and female sex have been found to be related to a prolonged delay of emergency care in acute stroke events (26,27). This hypothesis is further supported by our observation that the impact of diabetes seems to differ between younger and older individuals . In stratified models, there was a more pronounced time dependency in individuals aged 70 compared with those> 70 years . In individuals aged 70 years, mortality during the first 30 days was lower in diabetic compared with nondiabetic individuals in both men and women (even though the time dependency was significant only in men), while this was not the case in patients> 70 (data not shown). In previously published series, patients with ischemic strokes had a lower case fatality (mortality during the first 28 or 30 days after stroke) than patients with a hemorrhage stroke, whereas in the period after 30 days, mortality was higher after ischemic strokes (28). Third, hypertension was more prevalent in diabetic persons . Previously known hypertension at the time of the stroke event has been reported to be significantly associated with a decreased mortality for both sexes, possibly explained by a better tolerance toward higher admission blood pressure in those individuals, which might be clinically more relevant for the early outcome of hemorrhagic than ischemic strokes (29,30). Fourth, other factors may play a role, such as a higher prevalence of obesity among diabetic individuals undergoing stroke compared with their nondiabetic counterparts (31). It is known that obesity and overweight have a potential protective effect in elderly stroke patients (32,33). These phenomena should have larger effects in men since the degree of obesity is commonly higher in men than in women . However, in our data, we have no information about detailed clinical or lifestyle variables and only limited information about history of coronary events, chronic heart failure, and renal function . Also, exact causes of deaths cannot be determined by our data . In the period after 30 days, the mortality risk in individuals with diabetes compared with those without diabetes increased . The observation that in the 3 to 5 years after stroke there was no longer a difference in mortality between individuals with and without diabetes in both men and women may be due to a lack of power resulting from lower case numbers . However, it might be explained by the fact that individuals who survive 3 years are healthier, independent from their diabetes status . The mortality after 30 days (case fatality, 10.5%) as well as the 1-, 2-, and 5-year mortalities in our study (19.5, 25.1, and 37.3%, respectively) were well in line with the findings of other more recent studies and, as expected, lower than earlier studies . Case fatalities in the literature ranged between 10 and 22% (7,9,34), and 1- and 5-year mortalities were 27 and 53%, respectively (10). Only a few studies investigated mortality after stroke in diabetic and nondiabetic patients (1012,35,36); however, these studies were in part clinic based and analyzed only single subtypes of stroke or shorter periods of follow - up . In the study by rautio et al . (10), only case fatality was investigated . During the period from 1985 to 1987, case fatality was 18% in diabetic and 15% in nondiabetic patients, with higher mortality in women than in men . However, in the period from 2000 to 2003, case fatality was 15% in diabetic women, whereas in diabetic men as well as in nondiabetic men and women, it was 10% . In our study, case fatality was 9% in diabetic as well as nondiabetic men, and 16 and 13% in diabetic and nondiabetic women, respectively . (10) study that finds diabetic women have a higher excess risk to die within the first course after stroke; on the other hand, rautio et al . A further study analyzes the 3-month mortality in a clinic - based sample and finds diabetes to be a significant predictor of mortality (35). To the best of our knowledge, there is no population- or insurance - based study that analyzes diabetic and nondiabetic patients for longer periods . Several studies evaluate diabetes as a predictor of mortality after incident stroke, but with conflicting results . (8) found diabetes to be associated with mortality after stroke, benatru et al . (11) analyzed the mortality after discharge from the hospital for a longer period, but only ischemic strokes were included in their study . Kaplan - meier survival plots did not show a difference between individuals with and without diabetes during the first 60 days after discharge but did reflect lower survival in individuals with diabetes compared with those without diabetes after 1 year (11). (36) analyzed the 5-year mortality in individuals with and without diabetes after ischemic strokes . The 5-year mortality was significantly increased 1.6-fold in individuals with diabetes compared with those without diabetes; however, the study subjects were a clinic - based sample of migrant south asian patients (36). (12) analyzed the 28-day and 1-year fatality after stroke, yet they included only ischemic strokes in their study . Both were significantly increased in individuals with diabetes compared with those without diabetes, without differences between men and women (12). Thus, study results remain conflicting, and further studies are warranted to confirm and explain our findings . Several limitations have to be considered . First, in particular during the last years of observation and especially in women, the case numbers are low, leading to a lack of power to detect statistically significant differences between patients with and without diabetes . Second, we cannot exclude misclassification when we define patients with diabetes because our identification criteria had to be fulfilled within 12 months in the observation period between 2004 and 2007 and not solely before the first stroke . On the other hand, diabetes is often identified for the first time in hospital stays as a result of typical complications, such as strokes, and these patients would not be classified as patients with diabetes if we used only the period before the event . However, we performed a sensitivity analysis, defining a person as having diabetes when our criteria were fulfilled within the 12 months before the first stroke . We found that approximately 9 of 10 diabetic patients already fulfilled our criteria before their index stroke . Third, we studied stroke survivors, and the number of fatal strokes may differ among those with and without diabetes . This may be an explanation for the reduced mortality seen within the first 30 days among patients with diabetes . However, on the basis of data from the german stroke registry as well as from several other countries, it can be assumed that the number of fatal strokes and strokes that are treated outside the hospital are small . Approximately 95% of stroke patients are hospitalized in clinics and, thus, identified by our data (40). Fourth, information about clinical variables (e.g., blood glucose and diabetes duration) and patient lifestyle (e.g., smoking and physical activities) is not available in the database . However, we included number of prescribed drugs as well as outpatient diagnoses of relevant comorbidities and level of care dependency . Fifth, a translation of our results to other populations should be performed with caution since it is known that differences in morbidity as well as demographic and socioeconomic variables exist between health insurance funds (37,38). However, the incidence of stroke in our population was well in line with the incidence of stroke in a well - designed regional register - based study (2,39,40). Furthermore, the population has been used for several analyses regarding comorbidities in diabetes (2,13,14). The main strength of our study is that we were able to analyze a large dataset without selection with respect to diabetes complications that could be followed up to 5 years . In conclusion, in our german study, based on data from a nationwide health insurance fund, we found a high mortality in patients with a first stroke . It is interesting that the influence of diabetes was time dependent in men: in approximately the first quarter of a year after incident stroke, mortality was lower in diabetic than in nondiabetic individuals . Thereafter, diabetic patients had a higher mortality than nondiabetic patients, and after 3 years, there was a convergence . In women, the pattern seems to be similar: no significant time dependency was found . Our observation is in line with findings for mortality in diabetic compared with nondiabetic patients after beginning renal replacement therapy and amputation . Possible explanations may be differences in the type of stroke or in earlier and more intensive treatment of distinct cardiovascular risk factors in diabetic patients, in particular men . Patients that survive up to 3 years after stroke might be healthier, independent of their diabetes status . However, results remain conflicting, and further studies are warranted to confirm and explain the results.
|
In late february 2006, a 27-year - old pregnant woman, at 6 weeks gestation, was referred to grenoble university hospital, grenoble, france, for investigation of persistent left cervical lymphadenopathy with fever . The lymphadenopathy occurred 3 weeks earlier, along with a sore throat, and persisted despite 10 days treatment with amoxicillin (3 g daily). At admission, the patient was febrile (38c) and had a tender, swollen, submaxillary cervical lymph node on the left side . Magnetic resonance imaging of the left cervical region showed a large mass extending from the parotid region to the submandibular region, with hypo- and hypersignals in t1- and t2-weighted imaging, respectively . Lymph node tissue was obtained by needle aspiration, and examination revealed nonspecific lesions of lymphadenitis . Laboratory test results showed moderate inflammatory syndrome (c - reactive protein 67 mg / l). Serologic results were negative for hiv, hepatitis b and c viruses, rubella virus, treponema pallidum (syphilis), coxiella burnetii (q fever), and borrelia, bartonella, brucella, and legionella spp . And showed only residual igg - type antibodies against cytomegalovirus, epstein - barr virus, herpes simplex virus, parvovirus, mycoplasma pneumoniae, and chlamydophila pneumoniae . Because we suspected severe streptococcus pyogenes infection, a second course of amoxicillin was administered for 13 additional days . After initial clinical improvement, the patient relapsed, and the lymphadenopathy evolved to suppuration and necrosis . In march 2006, a large amount of pus was surgically drained from the site, and inflamed lymph nodes were removed . The patient denied receiving any tick bite but reported that she regularly fed domestic rabbits in cages . Her symptoms began a few days after she killed and skinned rabbits, which were then kept frozen but not eaten by the family . Routine cultures and mycobacterial cultures remained sterile, but f. tularensis dna was detected in lymph node samples by using specific real - time pcr targeting the gene encoding a 23-kda surface protein (5). Pcr amplification and sequencing of the 16s rdna23s rdna intergenic spacer region (5) directly from lymph node tissue confirmed the presence of dna from f. tularensis subsp . Serum samples collected in late february and on march 15, 2006, were tested by using an immunofluorescence assay and a homemade f. tularensis antigen (5). Both samples were positive, with igm and igg titers of 320 (cutoff titers of> 160). Because the patient was pregnant and tularemia in france holarctica (2), which are naturally susceptible to macrolides, she was treated with azithromycin (500 mg / d for 6 weeks). The patient reported that the rabbits were reared outdoors, in floor - level, wire - mesh cages; therefore, we suspected the animals were infected with f. tularensis through contact with wild fauna . Attempts to detect f. tularensis in pieces of the frozen rabbits by culture and pcr tests were unsuccessful . Clinical symptoms of tularemia are primarily related to the portal of entry of bacteria, the f. tularensis strain virulence, and the immune status of the patient . The incubation period is usually 13 days but may last up to 15 days (3,6). The primary clinical forms are glandular and ulceroglandular (skin inoculation), oculoglandular (conjunctival inoculation), oropharyngeal (oral contamination), pneumonic (inhalation of an infected aerosol), and typhoidal (various modes of infection) (2,7). Tularemia may be severe and even fatal; patients with lymphadenopathy may experience lymph node suppuration in 30% of cases (1). Our patient s symptoms were fever, pharyngitis, and cervical lymphadenopathy; treatment with a -lactam drug did not improve her condition, which rapidly evolved to local suppuration, a sign that should prompt physicians to consider oropharyngeal tularemia in disease - endemic regions . Because tularemia cases remain extremely rare in the french alps, where the patient lived, this diagnosis was not considered at the time of the first medical consultation . Because the rabbit meat was not consumed by the patient, we suspected she became infected at the time she skinned these animals however, the animals had no overt disease at the time they were killed, so low bacterial inoculum may explain why the tests performed on rabbit meat had negative results . Diagnosis of tularemia is often made by serologic tests (3), although these are negative during the first 2 weeks following the onset of symptoms (1). In the case described here, the same high antibody titers were obtained in 2 serum samples taken 2 weeks apart, which indicates that the peak secretion of specific antibodies was achieved . Culture of f. tularensis from clinical samples remains poorly sensitive, but pcr - based testing of pharyngeal swab specimens or lymph node suppurations or biopsy specimens enables rapid diagnosis of oropharyngeal tularemia and identification of the f. tularensis subspecies involved (1,4,5,7,8). A few tularemia cases occurring in pregnant women have been reported (9). Severe illness or death caused by infection with f. tularensis could be a risk for a pregnant woman or her fetus; the role of f. tularensis as an agent of abortion and intrauterine death is well recognized in sheep (10) but not in pregnant women . A major difficulty in this instance was the choice of the antimicrobial drug regimen, because first - line antibiotics currently recommended for treatment of tularemia, including the aminoglycoside gentamicin, fluoroquinolones, and tetracyclines (1,2,7,8), may be toxic for pregnant women or fetuses . No treatment recommendation for tularemia during pregnancy is available (9). The pregnancy outcome was favorable, and the patient and the infant were healthy at 12-month follow up . Macrolides are usually not recommended for treatment of tularemia patients (2,7,8), especially because f. tularensis subsp . Holarctica biovar 2 strains, mainly found in eastern europe and asia, are naturally resistant to macrolides (1114). The ketolides (e.g., telithromycin) are highly active against f. tularensis in vitro (11,15), but their use in pregnant women is currently discouraged . This case emphasizes the usefulness of azithromycin as a first - line treatment for tularemia in pregnant women in areas where infections caused by biovar 2 strains of f. tularensis subsp.
|
The phlebotomine sand fly phlebotomus sergenti s.l . Parrot, 1917 originally described from algeria in 1917, has a broad range of distribution which covers areas of the southern mediterranean (morocco, algeria, tunisia), the western mediterranean (portugal, spain, sicily), middle east, arabia, iran, afghanistan, pakistan, and northern parts of india . Flies of this species have been incriminated as the main vector of cutaneous leishmaniasis due to leishmania tropica (clt) throughout iran and other clt foci in the world (nadim et al . The presence of this sand fly in l. tropica free areas and the differences in the transmission patterns of the clt could be related to the existence of intraspecific variability or even cryptic vector species (depaquit et al . 2002, yahyia et al . 2004). Intraspecific variability has been shown in some morphological and molecular characteristics of various populations from different countries . (2002) studied the intraspecific variability of the internal transcribed spacer 2 (its2) of 12 populations of p. sergenti s.l . From ten different countries accordingly, two branches could be identified: one was related to the northeastern mediterranean area (cyprus, syria, and turkey) and pakistan, and the second related to southwestern of the first one including north africa, egypt, and morocco . These branches are in accordance with postulated migration routes of p. sergenti s.l . Along the tethys sea during the miocene era . The sand flies belonging to these two different branches seem to differ in ecology, host preferences, and possibly also in vectorial capacity (depaquit et al . Were also studied on 28 iranian populations and a few samples from greece, morocco, lebanon, turkey, pakistan, and syria (moin - vaziri et al . According to this study, based on the number of setae and the width of basal lobe of coxite, three morphotypes were identified as a, b and c, with some intermediate forms . Morphotype a was considered as p. sergenti sergenti, morphotype b, was identified as p. sergenti similis, and morphotype c had an elongated style in comparison with p. sergenti sergenti . In another molecular study on p. sergenti s.l . Populations of spain and morocco using rdna its2 and mtdna cytb sequences (baron et al . 2008), a high genetic diversity including five ribosomal and 16 mitochondrial haplotypes was found within 25 specimens . Based on these studies, it is judicious to consider the potential existence of sibling species within this taxon . If sibling species within p. sergenti s.l . Were proven, it would have important implications in epidemiology as well as in experimental studies . However, having found several intermediate morphotypes as well as sympatric ecological niche of morphotypes a, b and c, postulated us to test the gene flow between the sympatric morphotypes of this taxon using sequence analysis of the its2 region of rdna gene . This multicopy gene involves homogeneization processes usually called molecular drive (dover 1982) and has provided resolution in several studies at the taxonomic level for the larroussius and paraphlebotomus subgenera (dover 1982, depaquit et al . The use of its2 region has many advantages including, high and low mutation rhythm at interspecies and intraspecies level respectively, speed and ease of use, multiple target sites, predefined marker systems, known pcr primers, pre - existing knowledge of them in some sand fly species (di muccio 2000, depaquit et al . This study was conducted to verify the molecular variation of iranian p. sergenti populations, and to compare them with available data in genbank . The geographical locations from which p. sergenti s.l . Was sampled are shown in fig . Specimens caught by sticky papers were washed in a bath of acetone before being stored . Only male specimens were selected for the study of morphological and molecular variability since their characters tends to be more reliable . (1) tehran, (2) mashhad, (3) neishabur, (4) izeh, (5) bushehr, (6) bandar - e - abbas, (7) bam, (8) iranshahr . Dark spots represent clt endemic foci in iran the head and genitalia of individual male sand flies were cut off within a drop of ethanol, cleared in boiling marc - andr solution, and mounted between slide and cover slide in berlese fluid and morphologically identified based on external and internal characters of the head and genitalia according to the known identification keys (theodor and mesghali 1964). Morphometric measurements were performed to determine morphotypes of specimens as explained by moin - vaziri et al . The body related to the specimen was stored dried in a vial at 20 c before dna extraction . Based on ecological conditions of the location where specimens were collected and on the morphological differences noted by means of morphometric analysis, a few specimens from each morphotype (moin - vaziri et al . Genomic dna was extracted from the thorax, wings, legs and abdomen of either individual sand flies using the qiamp dna mini kit (qiagen, germany) (depaquit et al . 2002) or ish - horowicz with small modification as described by rassi et al . Pcr was used to amplify a fragment of 480516 bp containing the its2 of sand fly rdna (depaquit et al . Pcr were performed in a 50 l volume using 5 l of extracted dna solution and 50 pmol of each of the two primers of c1a: 5-cct ggt tag ttt ctt ttc ctc cgc t-3 and jts3: 5-cgc agc taa ctg tgt gaa atc-3. The pcr mix contained (final concentrations) 10 mm tris hcl, ph 8.3, 1.5 mm mgcl2, kcl 50 mm, triton x 100 0.01%, 200 m dntp each, and 0.25 l (1.25 units) of taq dna polymerase (eurobio). Initial denaturation at 94 c for 5 min was followed by 35 cycles of denaturation at 94 c for 30 sec, annealing at 62 c for 1 min . And extension at 72 c for 1 min with a final elongation time of 10 min at 72 c . Purification of the pcr product was made by agarose - gel fractionation, using the perfect prep gel cleanup (eppendorf, germany). Direct sequencing of both dna strands was performed by qiagen (hilden, germany) and the department of parasitology (university of valencia, spain) using the primers used for dna amplification . Sequences were edited and aligned to identify haplotypes (= unique sequences) by means of the clustalw software package (www.ebi.ac.uk/clustalw) and manually adjusted, if necessary . They were analyzed using the neighbor - joining (nj) method provided in the must software package (philippe 1993). Available sequences of p. sergenti and p. similis were retrieved from genbank and used for phylogenetic analysis (table 1). The geographical locations from which p. sergenti s.l . Was sampled are shown in fig . Specimens caught by sticky papers were washed in a bath of acetone before being stored . Only male specimens were selected for the study of morphological and molecular variability since their characters tends to be more reliable . (1) tehran, (2) mashhad, (3) neishabur, (4) izeh, (5) bushehr, (6) bandar - e - abbas, (7) bam, (8) iranshahr . Dark spots represent clt endemic foci in iran the head and genitalia of individual male sand flies were cut off within a drop of ethanol, cleared in boiling marc - andr solution, and mounted between slide and cover slide in berlese fluid and morphologically identified based on external and internal characters of the head and genitalia according to the known identification keys (theodor and mesghali 1964). Morphometric measurements were performed to determine morphotypes of specimens as explained by moin - vaziri et al . The body related to the specimen was stored dried in a vial at 20 c before dna extraction . Based on ecological conditions of the location where specimens were collected and on the morphological differences noted by means of morphometric analysis, a few specimens from each morphotype (moin - vaziri et al . Genomic dna was extracted from the thorax, wings, legs and abdomen of either individual sand flies using the qiamp dna mini kit (qiagen, germany) (depaquit et al . 2002) or ish - horowicz with small modification as described by rassi et al . Pcr was used to amplify a fragment of 480516 bp containing the its2 of sand fly rdna (depaquit et al . Pcr were performed in a 50 l volume using 5 l of extracted dna solution and 50 pmol of each of the two primers of c1a: 5-cct ggt tag ttt ctt ttc ctc cgc t-3 and jts3: 5-cgc agc taa ctg tgt gaa atc-3. The pcr mix contained (final concentrations) 10 mm tris hcl, ph 8.3, 1.5 mm mgcl2, kcl 50 mm, triton x 100 0.01%, 200 m dntp each, and 0.25 l (1.25 units) of taq dna polymerase (eurobio). Initial denaturation at 94 c for 5 min was followed by 35 cycles of denaturation at 94 c for 30 sec, annealing at 62 c for 1 min . And extension at 72 c for 1 min with a final elongation time of 10 min at 72 c . Purification of the pcr product was made by agarose - gel fractionation, using the perfect prep gel cleanup (eppendorf, germany). Direct sequencing of both dna strands was performed by qiagen (hilden, germany) and the department of parasitology (university of valencia, spain) using the primers used for dna amplification . Sequences were edited and aligned to identify haplotypes (= unique sequences) by means of the clustalw software package (www.ebi.ac.uk/clustalw) and manually adjusted, if necessary . They were analyzed using the neighbor - joining (nj) method provided in the must software package (philippe 1993). Available sequences of p. sergenti and p. similis were retrieved from genbank and used for phylogenetic analysis (table 1). Morphological investigation on specimens of 28 populations of p. sergenti sl collected from 11 provinces of iran revealed three main morphotypes (a, b and c) and a few intermediate forms (moin - vaziri et al . Morphotype b was related to p. cf similis, according to perfiliev (1968) and morphotype c represents specimens with a curved basal lobe of coxite without a style as globulous as that of p. sergenti . In addition to the three main morphotypes, some intermediate forms were identified among the collected samples . These morphotypes were found sympatric in several provinces of the country . However, the proportion of each morphotype varied within each region . The size of rdna - its2 fragments amplified was about 480 bp, from which 278 nucleotides were attributed to its2, 130 bp to 5.8s and 72 bp to 28s genes . However, in addition to the main pcr product, there was an additional product, close to the main amplicon, in all amplification . Despite of gel purification before sequencing, these unwanted products affected the results of sequencing . By the way, we could obtain a total of 249253 bp length, including most part (218222 bp) of its2 and 31 bp of 28s, from 11 specimens comprising 5, 4, and 2 individuals of morphotype a, b and intermediate form a / c respectively . The sequences were submitted to genbank (accession numbers: ef434818-ef434828). Comparative its2 sequence analysis of 11 p. sergenti s.l . Individuals showed 13 (5.2%) polymorphic sites, from which 46% was due to indel (insertion / deletion) and 54% was due to substitutions . All polymorphic sites were located in its2 region and 28s region was identical in all specimens . There were two variable microsatellite regions in the its2 aligned part of the specimens, which showed variable repeats of a poly (at) microsatellite . In the first variable microsatellite sites, two specimens contain 9, three specimens had 8, and six other specimens comprised 7 repeats of at (fig . Other entries from genbank also showed variable number of at repeats in this site . In the second polymorphic microsatellite, variations between the iranian specimens were due to two single nucleotide insertions / deletions (indels) and one variable repeats of a poly (at) microsatellite . Lower degrees of variation in the poly (at) microsatellite also have been observed in non - iranian entries (fig . 2). Microsatellite region in its2-rdna sequences of different populations of phlebotomus sergenti s.l . From iran and other parts of world retrieved from genbank (marked with *). A: morphotype a, b: morphotype b, a / c: intermediate forms of morphotype a and c, ser, p. sergenti, sim, p. similis based on the sequence alignment, nine different haplotypes (nominated as haplotype i ix) have been identified, three haplotypes (vii ix) in the south west (sw), two haplotypes (i and vi) in the north east (ne) and 5 haplotypes (i v) in the north - center - south (ncs) populations (table 1, 2, fig . 3). A sample of haplotype i was positioned in ne lineage . These haplotypes differed in 111 nucleotide positions . The its2 ribosomal haplotype i with three repeats was the most frequently haplotype among all haplotypes . Neighbor - joining tree inferred from 253 bp of its2-rdna sequences including 222bp of its2 and 31 bp of 28s of phlebotomus sergenti s.l . Populations from iran . A, morphotype a, b, morphotype b, a / c, intermediate form of morphotype a and c, ser, p. sergenti, sim, p. similis comparison of nucleotide characters of phlebotomus sergenti of iran at polymorphic sites of the rdna its2 . Gap (indel) shows by distance analysis of the its2 sequences indicated three main lineages (fig . These lineages were so - called sw, ne, ncs lineages which included haplotypes of south - west, north - east and a mix of haplotypes extended from northwest to the central to the southeast of the country, respectively . When we added the its2 sequence data of other p. sergenti and p. similis populations as representatives of other parts of the world for phylogenetic analysis, except for one iranian sample which was close to the european samples, all of the iranian haplotypes were associated with the north - eastern mediterranean populations including turkey, cyprus, syria, and also pakistan (fig . The p. similis populations from iran were associated with other p. sergenti populations, however, the european p. similis populations clustered separately . This might be due to independent accumulated mutations in distinct geographical populations resulted in separate branch in the tree . The phylogenetic tree deduced from combination of the its2 and the 381 bp of mtdna cytb - nadh1 sequences obtained in our previous study (moin - vaziri et al . 2007) revealed similar tree and did not resolve p. segenti from p. similis (data are not shown). Neighbor - joining tree inferred from 253 bp of its2-rdna sequences including 222 bp of its2 and 31 bp of 28s of phlebotomus sergenti s.l . Populations from iran and other available data from genbank originated from europe, africa, and asia . A, morphotype a, b, morphotype b, a / c, intermediate form of morphotype a and c, ser, p. sergenti, sim, p. similis, p. papatasi (an: ef408801) has been used as an outgroup morphological investigation on specimens of 28 populations of p. sergenti sl collected from 11 provinces of iran revealed three main morphotypes (a, b and c) and a few intermediate forms (moin - vaziri et al . Morphotype b was related to p. cf similis, according to perfiliev (1968) and morphotype c represents specimens with a curved basal lobe of coxite without a style as globulous as that of p. sergenti . In addition to the three main morphotypes, some intermediate forms were identified among the collected samples . These morphotypes were found sympatric in several provinces of the country . However, the proportion of each morphotype varied within each region . The size of rdna - its2 fragments amplified was about 480 bp, from which 278 nucleotides were attributed to its2, 130 bp to 5.8s and 72 bp to 28s genes . However, in addition to the main pcr product, there was an additional product, close to the main amplicon, in all amplification . Despite of gel purification before sequencing, these unwanted products affected the results of sequencing . By the way, we could obtain a total of 249253 bp length, including most part (218222 bp) of its2 and 31 bp of 28s, from 11 specimens comprising 5, 4, and 2 individuals of morphotype a, b and intermediate form a / c respectively . Individuals showed 13 (5.2%) polymorphic sites, from which 46% was due to indel (insertion / deletion) and 54% was due to substitutions . All polymorphic sites were located in its2 region and 28s region was identical in all specimens . There were two variable microsatellite regions in the its2 aligned part of the specimens, which showed variable repeats of a poly (at) microsatellite . In the first variable microsatellite sites, two specimens contain 9, three specimens had 8, and six other specimens comprised 7 repeats of at (fig . 2). Other entries from genbank also showed variable number of at repeats in this site . In the second polymorphic microsatellite, variations between the iranian specimens were due to two single nucleotide insertions / deletions (indels) and one variable repeats of a poly (at) microsatellite . Lower degrees of variation in the poly (at) microsatellite also have been observed in non - iranian entries (fig . 2). Microsatellite region in its2-rdna sequences of different populations of phlebotomus sergenti s.l . From iran and other parts of world retrieved from genbank (marked with *). A: morphotype a, b: morphotype b, a / c: intermediate forms of morphotype a and c, ser, p. sergenti, sim, p. similis based on the sequence alignment, nine different haplotypes (nominated as haplotype i ix) have been identified, three haplotypes (vii ix) in the south west (sw), two haplotypes (i and vi) in the north east (ne) and 5 haplotypes (i v) in the north - center - south (ncs) populations (table 1, 2, fig . 3). A sample of haplotype i was positioned in ne lineage . These haplotypes differed in 111 nucleotide positions . The its2 ribosomal haplotype i with three repeats was the most frequently haplotype among all haplotypes . Neighbor - joining tree inferred from 253 bp of its2-rdna sequences including 222bp of its2 and 31 bp of 28s of phlebotomus sergenti s.l . Populations from iran . A, morphotype a, b, morphotype b, a / c, intermediate form of morphotype a and c, ser, p. sergenti, sim, p. similis comparison of nucleotide characters of phlebotomus sergenti of iran at polymorphic sites of the rdna its2 . Gap (indel) shows by distance analysis of the its2 sequences indicated three main lineages (fig . These lineages were so - called sw, ne, ncs lineages which included haplotypes of south - west, north - east and a mix of haplotypes extended from northwest to the central to the southeast of the country, respectively . When we added the its2 sequence data of other p. sergenti and p. similis populations as representatives of other parts of the world for phylogenetic analysis, except for one iranian sample which was close to the european samples, all of the iranian haplotypes were associated with the north - eastern mediterranean populations including turkey, cyprus, syria, and also pakistan (fig . The p. similis populations from iran were associated with other p. sergenti populations, however, the european p. similis populations clustered separately . This might be due to independent accumulated mutations in distinct geographical populations resulted in separate branch in the tree . The phylogenetic tree deduced from combination of the its2 and the 381 bp of mtdna cytb - nadh1 sequences obtained in our previous study (moin - vaziri et al . 2007) revealed similar tree and did not resolve p. segenti from p. similis (data are not shown). Neighbor - joining tree inferred from 253 bp of its2-rdna sequences including 222 bp of its2 and 31 bp of 28s of phlebotomus sergenti s.l . Populations from iran and other available data from genbank originated from europe, africa, and asia . A, morphotype a, b, morphotype b, a / c, intermediate form of morphotype a and c, ser, p. sergenti, sim, p. similis, p. papatasi (an: ef408801) has been used as an outgroup results of this study showed a high diversity between specimens of p. sergenti s.l . In iran where we found nine ribosomal haplotypes . However, the rate of genetic variation (3%) within the its2 locus between the iranian populations is half of the rate (6%) that was previously observed in the mitochondrial (cytb - nadh1) sequences (moin - vaziri et al . It seems that mtdna sequences are more appropriate for the study of the intraspecific variability of p. sergenti s.l . In a more limited geographical environment . The high diversity observed in its2 is in agreement with previous studies indicating different ecological, morphological, and molecular variation among iranian p. sergenti populations . Previous studies revealed at least three morphotypes (a, b and c). However, in this study like previous study, we have not found any correlation between genotypes, ecotypes, or morphotypes and the results obtained here do not support the presence of sibling species (p. sergenti and p. similis) within the taxon . However, results of this study are in conflict with a phylogenetic analysis of nuclear ribosomal dna of depaquit et al . (2002) that showed the monophyly both of p. sergenti s.l . And p. similis and they were not sister species . Their result confirmed a study previously carried out, using morphological and morphometric approaches for examining the status of the two species by the same investigators (depaquit et al . Their worldwide attempt showed allopatric situation of the two taxa at that time . According to our findings, it seems that this group (morphotypes) cannot be considered as two different species because firstly we have found many intermediate morphological and genetically forms among the specimens, which indicate possible gene flow and lack of reproductive isolation between them . Secondly, finding identical genotypes (100%) among different morphotypes that are p. sergenti and that p. similis in mtdna (moin - vaziri et al . The molecular drive characteristic of its2-rdna strongly indicated that these two morphotypes are not isolated reproductively . Moreover having found these two taxa sympatric in most provinces of iran differs from taxonomic conception of subspecies . According to our data, we encountered with different morphologically populations of p. sergenti s.l . In iran . In light of our results, it is too early to come to a final decision on taxonomic status of the species . More molecular, morphological, and hybridization studies between the two taxa, particularly between geographically distinct populations is necessary . In iran, clt is endemic in 14 foci located in 8 provinces restricted to large and medium sized cities in different parts of the country (yaghoobi - ershadi 2012).we identified three its2 lineages of p. sergenti which is in agreement to the ones identified using the mtdna loci (moin - vaziri et al . Lineage ne and ncs correspond to the main foci of clt in the country which included both typical and intermediate morphotypes of p. cf sergenti and p. cf similis . These findings warrant studies to examine if clt is due to differences in the vectorial capacity of the p. sergenti s.l . Lineages (ne / ncs versus sw) or other ecological and epidemiological factors are involved . Phylogenetic sequence analysis revealed that most iranian haplotypes were associated with the northeastern mediterranean populations . Similar to the sequences of mtdna coi gene, its2 sequences could not resolve p. sergenti from p. similis and did not support the possible existence of sibling species or subspecies within p. sergenti s.l .. more molecular studies on other genes or hybridization should be done to clarify the status of different morphotypes of mentioned species in iran . Moreover, with regard to the importance of the epidemiology of leishmaniasis, further studies need to be performed on the possible role of these three morphotypes in the transmission of l. tropica.
|
Cancer is a leading cause of death worldwide . Colorectal cancer (crc) ranks fourth in the most common cause of death due to cancers worldwide . In usa, crc is the second leading cause of cancer - related deaths in cancers affecting both male and female . According to the saudi cancer registry, crc ranks second in the most common cancers among saudis after breast cancer and first among saudi males with constant rise in the incidence for the past few years . Furthermore, the overall survival rate (44.6%) is generally lower than the typically reported survival rates all over the world . Crc in saudi arabia present late with metastasis and obstruction in percentages more than what is reported in western communities . Nearly, 40% of crc diagnosed were equal to or below 50 years of age with mean age of 58 years, which is lower than developed countries [48]. Crc is an ideal tumor for screening, with its high incidence and long time between adenomatous polyp and carcinoma, and early diagnosis by screening can reduce incidence and mortality of the disease [914]. Family physicians have a key role in screening practice due to frequent contact with large proportion of population . Furthermore, the involvement of family physicians in screening program implementation has been recommended by several guidelines . However, physicians - reported crc screening recommendation rates in the literature are still below the demand if a major impact on crc mortality is targeted . Moreover, this is the first local study in saudi arabia that is looking for data regarding crc screening practice . The aim of this study is to explore the current knowledge, attitude and practice of family physicians toward crc screening and to identify the barriers of conducting such a screening . A cross - sectional descriptive study was carried out among family physicians working in family medicine clinics in national guard health affairs (ngha), riyadh, saudi arabia between february and march 2013 . A validated questionnaire was adopted from the national cancer institute in usa, customized by adding and eliminating questions to be in line with the institution (ngha) characteristics . A pilot study on 10 physicians was conducted to check understanding of the questionnaire, resulted in some vocabularies replacement and format enhancing to avoid confusion . Then the questionnaire was reviewed and approved by tow reviewers from king abdulla international medical research center (kaimrc). It contains demographic characteristics, questions to assess the knowledge, attitude, current practice and barriers of crc screening . Questionnaires were handed out to the physicians during working hours by the researcher or an assigned nurse and collected in the next following day . Data management and statistical analysis were carried out using statistical package for social sciences (spss) software version 20.0 . Categorical variables were presented as frequencies and percentages, chi - square test () used to explore the association between variables . Knowledge score was computed on the basis of 11 questionnaire items, the answers were evaluated according to the centers for disease control and prevention (cdc) and united states preventive services task force (uspstf) guidelines recommendations . Attitude score: answers with positive attitude marked with 1 and negative attitude marked with 0 . Means of scores were compared between groups using student t - test (t). Questionnaire cover sheet of the study explained that the participation of the physicians considered as a consent form of agreement . Out of 130 physicians, 68 (52.3%) were females, 51 (39%) were family medicine board certified, and 56 (43%) were mbbs holders (table 1). The mean value results for the knowledge score was founded to be 5.02 with standard deviation of 2.73 and ranged between 011 (table 2). Board certified physicians had higher knowledge score than other physicians (t = 2.6; p = 0.009). Physicians who reported practicing crc screening scored more on the knowledge score than those not practicing, (t = 2.7; p = 0.007). Physicians influenced by uspstf recommendations achieved better knowledge score compared to those who were not (t = 3.1; p = 0.002). Physicians influenced by their patients' preference for crc screening also achieved better knowledge score when compared to those who were not (t = 3.2; p = 0.002). Although not statistically significant, male physicians had higher knowledge score than female (t = 1.8; p = 0.071) (table 3). Among the sample studied, 123 physicians (94.6%) considered crc screening for asymptomatic average - risk patients to be effective and 106 (81.5%) prefer having structured screening program over opportunistic screening . Among the screening modalities, flexible sigmoidoscopy was considered very effective by 58 (44.6%) and 42 (32.3%) only for fecal occult blood testing (fobt). The mean value of the attitude score was found to be 6.95 with standard deviation of 1 and ranged between 4 and 8 (table 4). Male physicians scored better on attitude score than female (t = 2.3; p = 0.025). Physicians aged 40 years and above had better attitude than younger physicians (t = 2; p = 0.047). Physicians who reported having a reminder system for crc screening scored better attitude than those who did not, (t = 3.4, p = 0.001) (table 5). Around more than half (56.2%) of fobt is discussed more as screening modality (80.8%) in comparison with colonoscopy (76.90%) and flexible sigmoidoscopy (35.4%). Majority of the physicians conduct fobt by delivering stool samples to the labs (90%). Board certification contributed significantly to practicing crc screening when compared to other physicians (= 7.65; p = 0.005). Physicians whose practice was influenced by uspstf and and american cancer society recommendations reported practicing crc screening more than those who were not influenced by these resources (= 4.86; p = 0.025) and (= 3.92; p = 0.041), respectively . Although lacking statistical significance, male physicians reported practicing crc screening more than female physicians (= 2.9; p = 0.063) (table 6). When asked about barriers to practice crc screening, 80% of physicians who did not perform the screening believed time was a barrier compared to 56% of those who actually practice the crc screening (= 9.29; p = 0.002). Also, 70% of physicians who did not practice the screening thought that the patients do not want to discuss colorectal cancer . This belief was noted among only 49% of physicians who practiced crc screening (= 5.78; p = 0.013). Nearly, 77% of physicians who did not practice the screening reported that patients are having difficulty understanding the information about crc screening, compared to only 45.6% of physicians who do practice the screening (= 13.29; p <0.00). Moreover, 60.3% of physicians not practicing crc screening believed that patients do not perceive crc as a serious health threat, compared to only 36.8% of physicians who practice crc screening (=7.03; p = 0.007). When asked about having a clear policy and procedure for crc screening in their workplace, 86.3% of physicians not practicing crc screening reported that there is no policy in their workplace compared to 65% of physicians who practice crc screening (= 8.25; p = 0.004). Also, physicians who do not practice the screening were more to report shortage of trained providers to conduct further screening procedures other than fobt (77.8% compared to 54.4% of those who practice crc screening) (= 7.93; p = 0.004) (table 7). Among patients related barriers for crc screening, 92.4% of physicians reported that patients are not aware of crc screening and (63.1%) commented that their patients have difficulties understanding the information about crc screening . In relation to workplace related barriers, not having a reminder system was the top barrier on the list with (86%) followed by unavailability of crc screening policy and procedure (76.9%). Seventy percent of physicians reported that they do not have enough time to discuss crc screening with their patients (table 8). Several studies have been done in saudi arabia in regard to crc, and all of them concluded and highlighted the importance of screening [4, 5, 7, 8]. This is the first study that looked at the current knowledge, attitude, practice, and perceived barriers of physicians in saudi arabia in order to close the gap for a better screening . The response rate was 76%, higher than that obtained from similar studies which involved more centers and more than one city [9, 16, 17]. This study found that crc screening is underutilized since around more than half of the study subjects were not practicing crc screening despite a strong evidence supporting the effectiveness of the screening, physicians' knowledge, and their positive attitude toward its effectiveness, which is in accordance with findings in the literature [9, 17, 18]. Knowledge of the physicians was acceptable, and main deficiency was on the age of stopping crc screening which is similar to klabunde et al . Board certified physicians achieved higher knowledge score than physicians with lower certification, which highlights their role in educating other physicians about the importance of screening . Physicians influenced by uspstf recommendations achieved better knowledge score compared to those who were not, and this goes in line with considering the cdc guideline as a reference for knowledge evaluation in this study and also reflects the importance of uspstf as a trusted source of information . Physicians with a higher knowledge score were more patient - oriented in considering their patients' preference for screening, which will increase patient involvement in decision making, and that will help build a stronger doctor patient relationship . Result showed significant positive association between knowledge score and practicing crc screening which reflects the importance of physicians' education for the improvement of crc screening, and this finding was in accordance with the literature . In general, physicians have a positive attitude toward crc screening reflected by the attitude score . Positive attitude toward crc screening was associated with being male; this might be due to the fact that, under the setting of ngha clinics, female physicians are almost exclusively treating female subjects . Among female patients, other cancers (e.g., breast and cervical cancers) are of greater significance than colorectal cancer which has higher incidence among male subjects . Also, physicians aged 40 years and above had better attitude than younger physicians, which may indicate the importance of experience; there is similar finding in greece . Physicians who reported having a reminder system for crc screening scored better attitude than those did not; this is supporting sarfaty and wender finding that crc screening can be improved by implementing a reminder system targeting both doctors and patients . In contrary to knowledge score, attitude score showed positive association with practicing crc screening but the result was statistically insignificant . Most of the physicians consider colonoscopy to be the most effective screening test, followed by flexible sigmoidoscopy . Only one - third of physicians found fobt to be very effective . In contrary, fobt is the most used test followed by colonoscopy, which is similar to what klabunde et al . And federici et al . This might be due to more patients' acceptance or the availability of the fobt in comparison to colonoscopy . The study found that barriers were cited in higher rates among physicians not practicing crc screening compared with practicing physicians . Lack of patients' awareness was the most cited barrier, which was also highlighted in the literature . Not having a reminder system was reported by 86% . Taking that into consideration, tackling and finding solutions to overcome these barriers might improve the crc screening . Miles et al . Concluded that the most effective way of achieving reduction in cancer related mortality is by providing screening as part of an organized program . The study was carried out in one institution in one city of saudi arabia and does not necessarily reflect general population.study design (cross - sectional) has limited internal validity and it is sensitive to a variety of biases.data collection tool was self - administered and lack of observation carries a risk of recalling bias or contamination by the participants . The study was carried out in one institution in one city of saudi arabia and does not necessarily reflect general population . Study design (cross - sectional) has limited internal validity and it is sensitive to a variety of biases . Data collection tool was self - administered and lack of observation carries a risk of recalling bias or contamination by the participants . Unfortunately, large percentage of family physicians in this study do not recommend crc screening, despite the knowledge level and the positive attitude . This is pointing to the importance of increasing the attention of the doctors about their crucial rule in crc prevention . Moreover, patients' involvement and awareness are of a good impact on the crc screening process . Therefore, we recommend increasing the patients' awareness by the mass media and health campaigns and considering a national organized screening program that will help the physicians to achieve a desirable outcome.
|
Messenger rna (mrna) has achieved great success in an increasing number of biological applications . Apropos, the notion of nonviral genetic vaccination is also increasingly associated with mrna instead of dna . Given a mature drug and gene delivery field, mrna nanoparticle delivery science is often deferred or closely compared with dna and sirna systems [1, 2]. However, as various reports have shown, unique properties of mrna delivery exist [3, 4] and continue to be a relevant research focus today . Mrna delivery science has made significant progress since the first demonstration of cell based mrna tumor vaccine delivery via rna loaded dcs . They include the optimization of the mrna molecular structure [6, 7], direct in vivo administration of mrna [8, 9], delivery routes [3, 4], evaluation of rationally designed gene carriers [1014], and, recently, self - replicating rna . Along this developmental trajectory, dc - targeted nanoparticle gene delivery systems may be an imminent next step forward for nonviral tumor vaccine delivery . In this brief report, this will be followed by a brief discussion on three promising dc receptors that are suitable for targeted delivery of mrna nanoparticles for tumor vaccination . Ligands targeting surface receptors on dcs are molecules grafted onto surfaces of formulated nanoparticles, recognizable by dc - specific uptake mechanisms, and endow nanoparticles with the ability to be taken up exclusively by them . This has the benefit of reducing effective doses of vaccine required through nonspecific uptake by other cell types . In the case of vaccines, which typically contains proinflammatory adjuvant molecules since a wide variety of nanoparticle delivery systems exist, different ligand conjugation strategies have been developed . In this section, we will discuss three conjugation strategies that are most often applied to gene delivery systems . First, nanoparticles with solid cores such as poly(lactic - co - glycolic acid) (plga) and inorganic nanoparticles (e.g., gold nanospheres, calcium phosphate) possess excellent colloidal stability such that ligands can be covalently conjugated directly onto particles surfaces without aggregation . In plga systems, nanoparticles are formulated by emulsion techniques [1618] using plga - peg - cooh copolymer, which can be synthesized by grafting peg - cooh onto the ends of plga . The resultant mrna infused plga nanoparticles bearing surface carboxylate groups (cooh) can be further functionalized with any ligands bearing amine groups (e.g., peptides, antibodies, nanobodies, and aptamers) via n - hydroxysuccinimide (nhs) chemistry, which proceeds with good efficiencies under physiological conditions if nhs bearing ligands are applied in excess (figure 1(a), top). However, this conjugation strategy will require the colloidal nanoparticles to remain stable through every step of the conjugation process (surface chemistry modifications, purification and lyophilization). Ligand conjugated nanoparticles are normally purified from the reaction mixture via centrifugation, and hence this strategy is compatible with formulations bearing a solid core because they can withstand compression without aggregation . However, dialysis is not compatible with plga (as well as other polyesters, e.g., poly--amino esters) as ester bonds in these polyesters undergo hydrolysis . Conversely, formulations that are chemically inert (e.g., gold nanoparticles, immunoliposomes, and polyamide - based nanoparticles) but aggregate upon centrifugation can be purified by dialysis (figure 1(b)). A similar approach uses functionalized amphiphilic surfactants commonly used to stabilize the plga nanoparticles in colloidal suspension (figure 1(a), bottom). These surfactants, which bear reactive chemical moieties (e.g., cooh, nh2, and oh), are optimally incorporated on particle surfaces and amenable for subsequent conjugation with targeting ligands bearing compatible linkers . In particular, avidin - fatty acid surfactants have been applied to stabilize plga nanoparticles [22, 23]. The resulting nanoparticles can be subsequently functionalized with biotinylated ligands such as antibodies, which are easily available, to render user defined dc surface receptor targets such as dec-205 and dc - sign [22, 24, 25]. However, notwithstanding the immunological consequences of antibodies, the sheer size of antibodies may result in low surface coverage due to steric hindrance . This can be mitigated with more advanced ligands such as single chain fragment variable (scfv) [26, 27] or aptamers, making this an attractive conjugation method . Second, targeting moieties can instead be incorporated as part of the carrier molecule (polymer or lipid). The ligand conjugated carrier is directly used to formulate the nanoparticles via coacervation between positively charged gene carriers and negatively charged mrna, and hence no additional step is needed to affix the ligands . This strategy is typically applicable for electrostatically neutral, low molecular weight ligands to ensure that they do not interfere with the carrier molecule during nanoparticle formulation (figure 1(c)). Mannan / mannose, a sugar that interacts with c - type lectin / lectin - like receptors, is the most commonly applied dc - targeting ligand incorporated into nanoparticles using this approach . A large number of mannosylated lipids and polymers have been developed hitherto for the purpose of vaccination [2936]. For liposomal systems, mannose are grafted onto the head groups of lipids [2931], while, for polymeric systems, they are normally covalently attached along the backbone of polymeric carriers [3236]. Most of these systems are tested for delivery of different vaccine molecules including peptides, dna, and sirna with a consistent improvement in uptake efficiencies over nonmannosylated nanoparticles, which translates to an improved immunization outcome . Notably, midoux group elegantly demonstrated, as a proof - of - concept, that mrna - loaded mannosylated lipophosphoramides target dcs in vivo and translate into a better survival outcome based on a b16-f10 prophylactic tumor model [31, 37]. Third, another tried and tested strategy for ligand conjugation primarily in liposomal systems exploits the use of hydrophobic interaction (figure 1(d)). It is well known that liposomes / lipopolyplexes are not thermodynamically stable colloids that aggregate slowly over time [3840]. Aggregation is a fusion process when hydrophobic interactions between the lipid tails are stronger than the repulsive forces on the surfaces of the liposomes . Factors determining this balance include temperature, ionic concentration of the buffer, and amphiphilic property (surface charge of the lipids versus length and number of the lipid tails). Exploiting effects of temperature on lipid fusion, liposomes or lipopolyplexes encapsulated with mrna or other payloads can be incubated with ligand - micelles (e.g., dspe - peg-2000-x, where x = ligand) at a temperature of 55c for at least 15 minutes . Due to increased hydrophobic interaction at a higher temperature, ligand conjugated lipids from these micelles can be transferred to the liposomes, effectively decorating them with the desired targeting ligands . These ligand conjugated micelles can be prepared by reacting thiol (sh-) or amine (nh2-) bearing ligands with dspe - peg - nhs or dspe - peg - maleimide (dspe: 1,2-distearoyl - sn - glycero-3-phosphoethanolamine) available commercially with different peg molecular weight . This so - called postinsertion strategy is a facile approach to functionalizing liposomes with any desired ligands . Unlike plga system, dspe - peg - ligand can be prepared separately and conveniently incorporated into formulated liposomes on demand [4143]. The amount of peg coverage over a 100 nm liposome needed to prevent aggregation in serum is determined to be> 8 mole% (based on total lipid content) in the liposome formulation [44, 45]. A caveat to postinsertion strategy is that if the amphiphilicity of the micelles is significantly affected by an excessively hydrophilic head (e.g., highly charged aptamer, long peg chain), postinsertion method may fail because the increased hydrophobic interaction induced at a higher temperature may not be sufficient to trigger micelle fusion with the liposomes / lipopolyplexes . When particles are administered into the body, unless the injected site is already the lymph node (e.g., intranodal administration) or has a high density of antigen presenting cells (e.g., intradermal or intranasal administration), nanoparticles need to be passively transported from the site of administration to the lymph nodes via the body's circulatory system such as the lymphatics or the systemic circulation [28, 4648]. During passive transport from the site administration to the lymphoid tissues, nanoparticles may be taken up nonspecifically by bystander cells based on a range of physiochemical factors such as size, surface charge, and chemical structure of surface molecules . Targeting ligands may reduce such occurrences due to incompatible surface chemistries while increasing uptake efficiencies of nanoparticles when reaching the target site [4951]. While generally it means selective delivery of the vaccine to dcs bearing specific surface receptors, direct outcome of receptor binding depends on what receptors are being targeted . Targeting ligands can, amongst other functions, help increase the uptake by binding to receptors designed to endocytose larger particles, mitigate repulsive forces, or improve surface compatibility between the particles and the cell membrane . Since intracellular fate of the particles taken up by endocytosis is determined largely by the mechanism through which they are being taken up, targeting ligands may help direct endosomes into specific intracellular trafficking pathways that are less degradative so that gene delivery efficiencies are increased . Dcs, unlike other somatic cells, possess unique endocytic receptors catered to antigen uptake and processing . These receptors are special because they not only trigger particle uptake, but also mediate cross presentation and the development of the immune response . Although cross presentation in dcs influenced the development of subunit nanoparticle vaccines, its impact on genetic vaccination is less conclusive . The genetic vaccination delivery model has been described as a process where both bystander and antigen presenting cells are transfected [53, 54]. According to this model, as illustrated in figure 2, antigen presentation occurs through direct transfection of dcs and also through indirect transfer by transfected bystander cells . When the mrna nanoparticles are targeted to dcs directly, those that escape the endosomes will have a higher chance of being expressed . In dcs, endosome escape not only depends on the efficiency of the gene carrier, but also depends on the trafficking mechanisms . For example, cross presentation mechanisms in dcs can disrupt lysosome trafficking pathways via mediation of endosomal ph leading to higher delivery efficiencies . But, on the other hand, intracellular trafficking pathways of nonprofessional antigen presenting cells often terminate at the lysosomes . When mrna nanoparticles are delivered without specific dc - targeting ligands, they will also transfect bystander cells . The latter provide an alternative source of antigens by secreting them (if the antigens are secretory in nature or designed with a secretory signal) into the extracellular space for capture by dcs . Finally, according to the consensus genetic vaccination, the other indirect delivery mechanism occurs when transfected bystander cells become apoptotic due to significant stress caused by viral or tumor infection . Sufficient literature exists to suggest that indirect delivery mechanisms via bystander cells does not play a significant role in targeted delivery systems since targeted genetic nanoparticle vaccines consistently improve immunization outcomes [3234, 5658]. Dc - specific receptors that not only increase uptake but also enhance transfection via less degradative intracellular trafficking pathways will be attractive for mrna nanoparticle tumor vaccination . While a long list of dc receptors has been discovered to possess immune modulating function, only a few may benefit mrna delivery beyond uptake enhancements because they are also targeted towards less degradative intracellular trafficking pathways . They are type i c - type lectins such as cd205 (dec-205) and cd206 (macrophage mannose receptor) and type ii c - type lectins such as cd370 (clec9a / dngr-1). It is a type i c - type lectin - like molecule consisting of a single polypeptide chain that functions as recycling endocytic receptor and caters for a wide range of cargos that include, notwithstanding lectin - like molecules, apoptotic cells, necrotic cells, and cpg . Dec-205 is an attractive target receptor because antigens delivered via this receptor are presented on both mhc - i and mhc - ii molecules . Furthermore, engagement of dec-205 does not lead to proinflammatory response, making it an attractive receptor target for tolerance immunization . The anti - dec-205 ligand is one of the most developed ligands in immunotherapy . While ligands targeting most of the other dc - specific receptors continue to manifest in antibody molecules, anti - dec-205 ligands in form of scfv [26, 27] and aptamer for example, being a cognate endocytic receptor for apoptotic cells, dec-205 will efficiently uptake both nano- and microparticles it comes into close contact with . Hence, given mrna nanoparticles tendency to aggregate in vivo (increased particle sizes), administered dose will have higher bioavailability when targeted towards dec-205 . In addition, cross presenting properties of dec-205, thought to be results of leaky endosomes or less degradative endocytic pathway, will facilitate endosome escape of mrna nanoparticles into the cytoplasm and avoid the lysosomes . The mannose receptor, another type i c - type lectin receptor with a well - established role in tissue homeostasis, recognizes sulfated carbohydrates, collagen, and oligosaccharides through its cysteine - rich domain [68, 69], fibronectin domain, and c - type lectin domains [71, 72], respectively . The mannose receptors have been well - known endocytic receptors for decades in part because they are extensively studied as scavenging receptors in macrophages, which were initially thought to be the major antigen presenting cells before dcs were discovered . The ligand for this receptor is mannose residue grafted on the gene carrier [2936] as previously described . Its role in antigen presentation was conclusively determined through the use of dcs derived from mannose receptor negative transgenic mice . This study confirmed that dcs' mannose receptors not only serve as uptake receptors [7477], but also mediate cross presentation of soluble mannosylated antigens [7880]. Since payload taken up via mannose receptor stably accumulates in the early endosome and is excluded from lysosomes for up to 6 hours [78, 79], this intracellular trafficking pathway is expected to be less degradative and highly attractive for mrna nanoparticle delivery . Dngr-1 or cd370) is a recently discovered endocytic receptor that is implicated in the clearance of damaged and dead [82, 83] cells . This receptor, currently targeted via antibody, is restricted to a very small population of blood bdca3 dcs (in humans) and its equivalent in mice models is cd8 dcs . Due to its endocytic nature, antigen delivery properties of clec9a are rapidly investigated [85, 86]. Recent reports show that clec9a are effective in cross presenting antigens for cell mediated immunity [83, 87] and can be as effective t cell activators compared to langerin and dec-205 . Similar to other receptors capable of cross presenting soluble antigens, nanoparticles targeted to clec9a are expected to enter a less degradative intracellular trafficking pathway, leading to higher transfection efficiency . Restricted expression of clec9a to blood dcs may limit it as a practical receptor compared to the mannose receptor and dec-205 for targeted delivery to conventional dcs . As a late bloomer, development of mrna therapeutics benefits from a plethora of related knowledge on similar delivery systems . Advancing from passive targeting strategies employed for most mrna nanoparticle tumor vaccine to date, active targeting of mrna nanoparticles to dcs will further improve current therapeutic outcome for the treatment of cancer . Practicable conjugation strategies as well as target receptors reviewed in this paper will provide a convenient reference to facilitate future development of targeted mrna nanoparticle vaccine.
|
Rheumatoid arthritis (ra) is one of the most common and severe autoimmune diseases affecting the joints . This chronic inflammatory disease, in which the immune system attacks healthy tissue lining the joints, leads to functional disability and reduced quality of life, as a result of bone and cartilage destruction, joint swelling, and pain . Ra is a widely prevalent systemic disease and affects 1% of the population around the globe.13 since the ra inflammatory process remains unclear, finding effective therapies and tools for early diagnosis has been extremely challenging and remain non - existent or with limited efficacy.13 diagnosis of ra can be a demanding task, considering that the disease may occur even before symptoms start to manifest themselves . Additionally, confirmation of the presence of this autoimmune disease requires use of several different criteria to establish a definite diagnosis, leading to a high risk of overtreatment.4 magnetic resonance imaging (mri) has been attracting considerable medical interest for early disease detection and drug therapy monitoring.5,6 superparamagnetic iron oxide nanoparticles (spions) have emerged as highly effective contrast agents for mri,6 but active targeting strategies are required in order to increase their accumulation at tissues of interest while decreasing nonspecific biodistribution in order to reduce background interference.7 currently, the gold standard for ra therapy is methotrexate (mtx), a drug approved by us food and drug administration.8 this drug is usually administered together with other disease - modifying antirheumatic drugs, and sometimes in combination with short - term, low - dose glucocorticoids or tumor necrosis factor inhibitors.9 however, due to the lack of targeting ability using the intravenous formulations available, this therapeutic strategy does not allow specific distribution of mtx to the affected joints, and leads to drug accumulation in healthy tissues, causing harmful side effects.1,3,10 therefore, additional research is required in order to develop novel strategies for achieving effective and major long - term approaches for ra therapies, aiming to prevent joint destruction and associated comorbidities . In the particular case of ra, recent studies have proposed that insufficient apoptosis of synovial inflammatory cells, especially macrophages, may contribute to persistence of the disease . Since macrophages play a pivotal role in progression of the disease, effective imaging and therapy systems may rely on the ability to target these cells.3 bearing this in mind, a new approach for ra theranostics may take advantage of the vast potential of nanomedicine . A new wave of medical innovation is emerging due to the possibility of multifunctionalization in nanomedicine - based strategies, since nanoparticles (nps) may have the ability to: carry therapeutic agents; be conjugated to specific ligands, namely antibodies, to target a specific tissue or organ; and amplify imaging signals, by coencapsulating contrast enhancers; among other possibilities.10,11 this study aimed to develop a nanoparticulate system that can actively target macrophages for ra imaging and therapy by intravenous administration . The work consists of the association of spions as a contrast agent for mri, and mtx for ra therapy into poly(lactic - co - glycolic acid) (plga) nps . Combining these two agents in a single platform in addition, the work comprises functionalization of plga nps with a monoclonal antibody against the macrophage - specific cell surface receptor, cd64, which is overexpressed in ra.12 different plga - based nps were prepared in order to compare the effects of each component (ie, mtx, spions, and anti - cd64 antibody) on the properties of the nps . Acid - terminated plga copolymer (50:50 purasorb pdlg 5002a) was a kind gift from purac biomaterials (gorinchem, the netherlands). Iron oxide nanocrystals (10 nm, coated with oleic acid and dispersed in chloroform 25 mg / ml), were provided by ocean nano - tech llc (springdale, ar, usa). Anti - human cd64 (fc gamma receptor 1) antibody solution (1.0 mg / ml) was purchased from ebioscience (san diego, ca, usa). A thermo - scientific coomassie plus (bradford) assay kit was sourced from pierce biotechnology (rock - ford, il, usa). Poly(vinyl alcohol) (87%90% hydrolyzed, with an average molecular weight of 3070 kda), ethyl acetate, 2-morpholinoethanesulfonic acid (mes), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (edc), n - hydroxysulfosuccinimide (nhs), sodium phosphate dibasic dihydrate, citric acid, dimethyl sulfoxide, thiazolyl blue tetrazolium bromide 98% (mtt), and triton x-100 (for molecular biology) were purchased from sigma - aldrich (st louis, mo, usa). Dulbecco s phosphate - buffered saline 10 (ph 7.4), dulbecco s modified eagle s medium (dmem, high glucose, glutamax supplement, pyruvate), fetal bovine serum, penicillin - streptomycin (10,000 u / ml) and fungi - zone antimycotic were purchased from gibco (invitrogen corporation, paisley, uk). Aqueous solutions were prepared with double - deionized water (arium pro, sartorius ag, gttingen, germany). Formulations containing plga were prepared using a solvent emulsification - evaporation method based on an oil in water (o / w) single emulsion technique.13 following the standard procedure, 200 mg of plga were dissolved in 2 ml of ethyl acetate, and then added to 8 ml of a 2% (w / v) poly(vinyl alcohol) aqueous solution . The emulsion formed was homogenized using a sonicator (vibracell vcx 130 equipped with a vc 18 probe, sonics & materials inc ., the previous emulsion was then added to 15 ml of a 0.2% (w / v) poly(vinyl alcohol) aqueous solution and the organic solvent was removed by evaporation using a rotavapor for 90 minutes (300 hpa, 35c). Nps were then recovered by centrifugation (21,000 g, 10 minutes, 4c) and washed three times with 20 ml of water . After final redispersion, the nps were transferred to 20 ml aluminum - sealed screw - neck vials (la - pha - pack gmbh, langerwehe, germany) and stored at 4c until further analysis . The same method was used to associate mtx and spions into plga nps, both separate and simultaneously, by adding the previous components (40 l of spions and/or 20 mg of mtx) to the organic phase.14 a schematic of the preparation process is shown in figure 1 . Considering that cd64 is a fcri receptor,12 the anti - cd64 antibody should be linked through the fab fragment, leaving the fc region available for macrophage recognition . Therefore, the coupling reaction was carried out in the presence of edc and nhs, allowing the carboxyl - terminated nps to react with the primary amine of the antibody present at the fab region, and yielding an amide bond . Figure 2 summarizes the conjugation process . Following an adapted protocol,15 10 ml of purified nps were centrifuged (21,000 g, 10 minutes, 4c) and redispersed in 10 ml of mes buffer (ph 5.0). The ph was maintained at 5.0 in order to maximize the attachment of edc to the plga carboxyl groups.15 activation was achieved by adding 1 ml of 0.1 m edc and 1 ml of 0.7 m nhs (both dissolved in mes buffer, ph 5.0) to the np suspension, which was kept at room temperature under moderate stirring for 1 hour . To remove the remaining reagents, the activated nps were centrifuged (21,000 g, 10 minutes, 4c) and redispersed in phosphate - buffered saline, yielding a final concentration of 1.0 mg / ml . To conjugate the antibody to the activated nps, 10 l of the anti - cd64 antibody solution were added to 1 ml of activated np suspension . After homogenizing with a vortex mixer, the suspensions were incubated at 4c for 24 hours . The conjugated nps were again centrifuged (21,000 g, 10 minutes, 4c) to remove excess unconjugated antibody and remaining reagents . The produced nps were characterized for particle size, size distribution (polydispersity index), and zeta potential . Mean hydrodynamic diameter and polydispersity index were assessed by dynamic light scattering using a 90 plus particle size analyzer (brookhaven instruments corporation, holtsville, ny, usa) and zeta potential was determined by phase analysis light scattering using a zetapals zeta potential analyzer (brookhaven) at 660 nm, with a detection angle of 90 at 25c . All samples were diluted in water to a suitable scattering intensity and measurements were performed with three independent batches of nps (six runs, ten cycles each). In order to evaluate the surface morphology of the nps, scanning electron microscopy was performed using a high resolution quanta 400 scanning electron microscope (fei company, hillsboro, or, usa). Samples were mounted on metal stubs and coated with a gold / palladium thin film by sputtering for 60 seconds, with a 15 ma current, using a spi module sputter coater system . The morphological features of the developed nps and the presence of spions were assessed by transmission electron microscopy . Samples were prepared by placing 10 l of np dispersion on a copper - mesh grid and, after 2 minutes, excess water was removed by capillarity using filter paper . For contrasting, 10 l of 0.75% uranyl acetate solution was added and left at room temperature for 30 seconds . The grids were then observed using a jem-1400 transmission electron microscope (jeol ltd ., the association efficiency of mtx on nps was determined by calculating the ratio between the amount of mtx measured in the nps and the total amount of mtx, both quantified in the nps and in the three supernatants collected during the purification protocol, as follows: the quantification was performed by high - performance liquid chromatography (hplc) with ultraviolet detection . The hplc system comprised a md-2015 multi - wavelength detector (jasco, easton, md, usa) programmed for peak detection at 302 nm, a high - pressure pump (pu-2089), an autosampler (as-2057), and a controller (lc - net ii / adc) mastered by chromnav software . A reversed - phase monolithic column chromolith rp-18e (1004.6 mm internal diameter; merck) connected to a guard column of the same material (54.6 mm internal diameter) was used as stationary phase . Separation conditions were adapted16 and conducted by isocratic mode (mobile phase containing 10% [v / v] acetonitrile and 90% [v / v] of ph 6.0 phosphate / citrate buffer) at 1.0 ml / min . The phosphate / citrate buffer was composed by 0.2 m dipotassium phosphate and 0.1 m citric acid (63:37; v / v). Standard mtx solutions were prepared at 1, 3, 6, 10, 25, 50, and 100 g / ml in mobile phase . To prepare the np samples for hplc analyses, 100 l of np dispersion or 100 l of supernatant were added to 900 l of mobile phase, to a final concentration of 10% (v / v). Mtx - free plga nps, namely plga nps and spions - loaded plga nps, were also analyzed and no interference was observed on the chromatograms . The bradford assay was performed using the coomassie plus (bradford) assay kit to assess the efficiency of anti - cd64 antibody conjugation to multifunctional nps,17 following the instructions . Diluted bovine serum albumin standards were prepared (2.525 g / ml) using the same solvent as used for the samples (supernatant of the centrifugation of activated plga nps). Coomassie plus reagent was added to the supernatant of the centrifugation of anti - cd64 conjugated nps . The protein concentration for each unknown sample was determined and the conjugation efficiency (%) was assessed, as follows: where a is the initial amount of antibody and p is the protein in supernatant . Nps were characterized by fourier transform infrared spectroscopy (ft - ir), using a frontier ft - ir spectrometer with universal attenuated total reflectance sampling accessory (perkinelmer, waltham, ma, usa). For each np spectrum, a 50-scan was collected with 4 cm resolution in the mid - infrared region (3,600600 cm). For these analyses, the np dispersions were lyophilized using a virtis advantage 2.0 benchtop freeze dryer (sp scientific, gardiner, ny, usa). Samples were frozen at 60c for 12 hours, and primary drying was performed at 20c and 150 mtorr for 20 hours . Additionally, secondary drying was performed at 25c and 100 mtorr for 20 hours, for complete sublimation . Murine macrophage raw 264.7 cells (passages 3138) from the american type culture collection (atcc tib-71, rockville, md, usa) were cultured in dmem supplemented with 10% (v / v) fetal bovine serum, 1% (v / v) penicillin - streptomycin, and 1% (v / v) fungizone antimycotic . The cells were maintained in a humidified chamber at 37c and 5% co 2, and the culture medium was changed every 23 days . Following exposure to the developed nps, mtt and ldh assays were performed to measure cell viability and cytotoxicity, respectively.18 briefly, raw 264.7 cells were cultured in 96-well plates at a density of 2.510 cells / ml and cultured for 24 hours before use . The following day, the culture medium was removed, and the np dispersions or free mtx were added at different concentrations (corresponding to 0.01100 g / ml mtx). Mtx - free nps were added at concentrations corresponding to the polymer concentration of the mtx - loaded nps (approximately 0.151,500 g / ml). Two controls, ie, cells treated with culture medium and cells treated with triton x-100 2% (w / v) in culture medium, were also included . For the mtt assay,18 after 24 hours of incubation, the culture medium was removed and replaced by 200 l of mtt diluted in fresh dmem at 0.5 mg / ml . The mtt solution was discarded and formazan crystals were solubilized using 200 l of dimethyl sulfoxide . The plate was shaken for 10 minutes at room temperature, and absorbance (590 nm, 630 nm) was measured using a synergy ht multi - mode microplate reader (biotek instruments inc ., winooski, vt, usa). The ldh assay was performed following the instructions . Briefly, after 24 hours of incubation, the plate was centrifuged (250 g, 10 minutes, at room temperature) and 100 l were collected and transferred to a new 96-well plate . The ldh cytotoxicity detection kit reaction mixture was added, and absorbance (490 nm, 630 nm) was read after 20 minutes of incubation at room temperature in the dark . Cell viability and cytotoxicity were assessed and expressed as a percentage in relation to both controls . Statistical analysis was performed using ibm spss statistics version 21.0 (ibm corporation, armonk, ny, usa). The results are reported as the mean standard deviation for a minimum of three independent experiments . The two - tailed student s t - test and one - way analysis of variance were performed to compare two or multiple independent groups, respectively . When the group was significantly different (p<0.01), differences between groups were compared with the tukey s post hoc test . Paired samples were analyzed with the paired - samples two - tailed student s t - test . Acid - terminated plga copolymer (50:50 purasorb pdlg 5002a) was a kind gift from purac biomaterials (gorinchem, the netherlands). Iron oxide nanocrystals (10 nm, coated with oleic acid and dispersed in chloroform 25 mg / ml), were provided by ocean nano - tech llc (springdale, ar, usa). Anti - human cd64 (fc gamma receptor 1) antibody solution (1.0 mg / ml) was purchased from ebioscience (san diego, ca, usa). A thermo - scientific coomassie plus (bradford) assay kit was sourced from pierce biotechnology (rock - ford, il, usa). Poly(vinyl alcohol) (87%90% hydrolyzed, with an average molecular weight of 3070 kda), ethyl acetate, 2-morpholinoethanesulfonic acid (mes), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (edc), n - hydroxysulfosuccinimide (nhs), sodium phosphate dibasic dihydrate, citric acid, dimethyl sulfoxide, thiazolyl blue tetrazolium bromide 98% (mtt), and triton x-100 (for molecular biology) were purchased from sigma - aldrich (st louis, mo, usa). Dulbecco s phosphate - buffered saline 10 (ph 7.4), dulbecco s modified eagle s medium (dmem, high glucose, glutamax supplement, pyruvate), fetal bovine serum, penicillin - streptomycin (10,000 u / ml) and fungi - zone antimycotic were purchased from gibco (invitrogen corporation, paisley, uk). Aqueous solutions were prepared with double - deionized water (arium pro, sartorius ag, gttingen, germany). Formulations containing plga were prepared using a solvent emulsification - evaporation method based on an oil in water (o / w) single emulsion technique.13 following the standard procedure, 200 mg of plga were dissolved in 2 ml of ethyl acetate, and then added to 8 ml of a 2% (w / v) poly(vinyl alcohol) aqueous solution . The emulsion formed was homogenized using a sonicator (vibracell vcx 130 equipped with a vc 18 probe, sonics & materials inc ., the previous emulsion was then added to 15 ml of a 0.2% (w / v) poly(vinyl alcohol) aqueous solution and the organic solvent was removed by evaporation using a rotavapor for 90 minutes (300 hpa, 35c). Nps were then recovered by centrifugation (21,000 g, 10 minutes, 4c) and washed three times with 20 ml of water . After final redispersion, the nps were transferred to 20 ml aluminum - sealed screw - neck vials (la - pha - pack gmbh, langerwehe, germany) and stored at 4c until further analysis . The same method was used to associate mtx and spions into plga nps, both separate and simultaneously, by adding the previous components (40 l of spions and/or 20 mg of mtx) to the organic phase.14 a schematic of the preparation process is shown in figure 1 . Considering that cd64 is a fcri receptor,12 the anti - cd64 antibody should be linked through the fab fragment, leaving the fc region available for macrophage recognition . Therefore, the coupling reaction was carried out in the presence of edc and nhs, allowing the carboxyl - terminated nps to react with the primary amine of the antibody present at the fab region, and yielding an amide bond . Figure 2 summarizes the conjugation process . Following an adapted protocol,15 10 ml of purified nps were centrifuged (21,000 g, 10 minutes, 4c) and redispersed in 10 ml of mes buffer (ph 5.0). The ph was maintained at 5.0 in order to maximize the attachment of edc to the plga carboxyl groups.15 activation was achieved by adding 1 ml of 0.1 m edc and 1 ml of 0.7 m nhs (both dissolved in mes buffer, ph 5.0) to the np suspension, which was kept at room temperature under moderate stirring for 1 hour . To remove the remaining reagents, the activated nps were centrifuged (21,000 g, 10 minutes, 4c) and redispersed in phosphate - buffered saline, yielding a final concentration of 1.0 mg / ml . To conjugate the antibody to the activated nps, 10 l of the anti - cd64 antibody solution were added to 1 ml of activated np suspension . After homogenizing with a vortex mixer, the suspensions were incubated at 4c for 24 hours . The conjugated nps were again centrifuged (21,000 g, 10 minutes, 4c) to remove excess unconjugated antibody and remaining reagents . The produced nps were characterized for particle size, size distribution (polydispersity index), and zeta potential . Mean hydrodynamic diameter and polydispersity index were assessed by dynamic light scattering using a 90 plus particle size analyzer (brookhaven instruments corporation, holtsville, ny, usa) and zeta potential was determined by phase analysis light scattering using a zetapals zeta potential analyzer (brookhaven) at 660 nm, with a detection angle of 90 at 25c . All samples were diluted in water to a suitable scattering intensity and measurements were performed with three independent batches of nps (six runs, ten cycles each). In order to evaluate the surface morphology of the nps, scanning electron microscopy was performed using a high resolution quanta 400 scanning electron microscope (fei company, hillsboro, or, usa). Samples were mounted on metal stubs and coated with a gold / palladium thin film by sputtering for 60 seconds, with a 15 ma current, using a spi module sputter coater system . The morphological features of the developed nps and the presence of spions were assessed by transmission electron microscopy . Samples were prepared by placing 10 l of np dispersion on a copper - mesh grid and, after 2 minutes, excess water was removed by capillarity using filter paper . For contrasting, 10 l of 0.75% uranyl acetate solution was added and left at room temperature for 30 seconds . The grids were then observed using a jem-1400 transmission electron microscope (jeol ltd ., tokyo, japan), with an acceleration voltage of 80 kv . The association efficiency of mtx on nps was determined by calculating the ratio between the amount of mtx measured in the nps and the total amount of mtx, both quantified in the nps and in the three supernatants collected during the purification protocol, as follows: the quantification was performed by high - performance liquid chromatography (hplc) with ultraviolet detection . The hplc system comprised a md-2015 multi - wavelength detector (jasco, easton, md, usa) programmed for peak detection at 302 nm, a high - pressure pump (pu-2089), an autosampler (as-2057), and a controller (lc - net ii / adc) mastered by chromnav software . A reversed - phase monolithic column chromolith rp-18e (1004.6 mm internal diameter; merck) connected to a guard column of the same material (54.6 mm internal diameter) was used as stationary phase . Separation conditions were adapted16 and conducted by isocratic mode (mobile phase containing 10% [v / v] acetonitrile and 90% [v / v] of ph 6.0 phosphate / citrate buffer) at 1.0 ml / min . The phosphate / citrate buffer was composed by 0.2 m dipotassium phosphate and 0.1 m citric acid (63:37; v / v). Standard mtx solutions were prepared at 1, 3, 6, 10, 25, 50, and 100 g / ml in mobile phase . To prepare the np samples for hplc analyses, 100 l of np dispersion or 100 l of supernatant were added to 900 l of mobile phase, to a final concentration of 10% (v / v). Mtx - free plga nps, namely plga nps and spions - loaded plga nps, were also analyzed and no interference was observed on the chromatograms . The bradford assay was performed using the coomassie plus (bradford) assay kit to assess the efficiency of anti - cd64 antibody conjugation to multifunctional nps,17 following the instructions . Diluted bovine serum albumin standards were prepared (2.525 g / ml) using the same solvent as used for the samples (supernatant of the centrifugation of activated plga nps). Coomassie plus reagent was added to the supernatant of the centrifugation of anti - cd64 conjugated nps . The protein concentration for each unknown sample was determined and the conjugation efficiency (%) was assessed, as follows: where a is the initial amount of antibody and p is the protein in supernatant . Nps were characterized by fourier transform infrared spectroscopy (ft - ir), using a frontier ft - ir spectrometer with universal attenuated total reflectance sampling accessory (perkinelmer, waltham, ma, usa). For each np spectrum, a 50-scan was collected with 4 cm resolution in the mid - infrared region (3,600600 cm). For these analyses, the np dispersions were lyophilized using a virtis advantage 2.0 benchtop freeze dryer (sp scientific, gardiner, ny, usa). Samples were frozen at 60c for 12 hours, and primary drying was performed at 20c and 150 mtorr for 20 hours . Additionally, secondary drying was performed at 25c and 100 mtorr for 20 hours, for complete sublimation . Murine macrophage raw 264.7 cells (passages 3138) from the american type culture collection (atcc tib-71, rockville, md, usa) were cultured in dmem supplemented with 10% (v / v) fetal bovine serum, 1% (v / v) penicillin - streptomycin, and 1% (v / v) fungizone antimycotic . The cells were maintained in a humidified chamber at 37c and 5% co 2, and the culture medium was changed every 23 days . Following exposure to the developed nps, mtt and ldh assays were performed to measure cell viability and cytotoxicity, respectively.18 briefly, raw 264.7 cells were cultured in 96-well plates at a density of 2.510 cells / ml and cultured for 24 hours before use . The following day, the culture medium was removed, and the np dispersions or free mtx were added at different concentrations (corresponding to 0.01100 g / ml mtx). Mtx - free nps were added at concentrations corresponding to the polymer concentration of the mtx - loaded nps (approximately 0.151,500 g / ml). Two controls, ie, cells treated with culture medium and cells treated with triton x-100 2% (w / v) in culture medium, were also included . For the mtt assay,18 after 24 hours of incubation, the culture medium was removed and replaced by 200 l of mtt diluted in fresh dmem at 0.5 mg / ml . The mtt solution was discarded and formazan crystals were solubilized using 200 l of dimethyl sulfoxide . The plate was shaken for 10 minutes at room temperature, and absorbance (590 nm, 630 nm) was measured using a synergy ht multi - mode microplate reader (biotek instruments inc ., the ldh assay was performed following the instructions . Briefly, after 24 hours of incubation, the plate was centrifuged (250 g, 10 minutes, at room temperature) and 100 l were collected and transferred to a new 96-well plate . The ldh cytotoxicity detection kit reaction mixture was added, and absorbance (490 nm, 630 nm) was read after 20 minutes of incubation at room temperature in the dark . Cell viability and cytotoxicity were assessed and expressed as a percentage in relation to both controls . Statistical analysis was performed using ibm spss statistics version 21.0 (ibm corporation, armonk, ny, usa). The results are reported as the mean standard deviation for a minimum of three independent experiments . The two - tailed student s t - test and one - way analysis of variance were performed to compare two or multiple independent groups, respectively . When the group was significantly different (p<0.01), differences between groups were compared with the tukey s post hoc test . Paired samples were analyzed with the paired - samples two - tailed student s t - test . For a successful ra - targeted theranostic approach, it was paramount that all components in the devised plga nps, ie, spions (for imaging diagnosis), mtx (therapeutic drug), and the anti - cd64 antibody (for specific ra macrophage targeting), were effectively integrated in the nanoparticulate system, without significantly altering their known drug delivery characteristics . The nps were designed as part of an intravenous administration strategy for ra - targeted therapy and imaging . Therefore, the physicochemical properties of the developed nps, which influence their physical stability and interaction with biological tissues, deserved detailed attention . The nps were characterized in terms of their particle size, polydispersity index, zeta potential, association with spions and mtx, anti - cd64 antibody conjugation, and their effect on cell viability and cytotoxicity . The mean particle size of the developed nps, measured by dynamic light scattering, is presented in table 1 . All formulations tested in this work were within the size range of 130200 nm, and showed a relatively homogeneous size distribution as revealed by polydispersity index values . In order to avoid sequestration of nps in spleen sinusoids and liver fenestrae further, nps with a diameter smaller than 6 nm can be excreted by the kidneys, so are rapidly eliminated from the bloodstream.19 consequently, the sizes obtained for the developed nps are suitable for intravenous administration . Focusing on the non - conjugated nps, spions and mtx association did not significantly affect particle size for any of the formulations, suggesting that they do not considerably interfere with np formation, and that it is possible to create a complex multifunctional nanoparticulate system maintaining the primary properties plga nps . However, conjugation with the anti - cd64 antibody interfered slightly with particle size . Nps underwent a shift in mean particle size, increasing in the order of 3050 nm (table 1), which could be explained by the presence of the antibody on the surface of the particles, as well as by a higher water content on hydration of the conjugate.20 the polydispersity indexes obtained were between 0.1 and 0.3 (table 1), indicating that well defined and monodispersed nanoparticulate populations were produced with uniform and consistent sizes, and without suffering aggregation . Regarding surface charge, a negative charge is typical of carboxyl - terminated nps owing to the contribution of the carboxyl groups, which are deprotonated at physiological ph or, in this particular case, at the ph of double - deionized water.21 zeta potential values around 30 mv contribute to the stability of hydrophobic particles in aqueous dispersion, avoiding formation of aggregates.22 the zeta potential values decreased significantly in all formulations after conjugation of anti - cd64 (table 1). The principle behind the antibody conjugation relies on establishment of a covalent amide bond between the amine and carboxyl termini of the antibody and plga, respectively . Consequently, it is expected that partial surface charge shielding occurs due to the depletion of carboxyl groups at the surface as they were involved in the reaction with the amine termini of the antibody . The images show the spherical shape of the nps as well as their smooth surface, which is devoid of pores (figure 3). The nps had sizes below 200 nm, confirming the results previously obtained by dynamic light scattering . The homogenous and flat surface, not varying between different formulations, suggests that both mtx and spions are entrapped within the plga polymeric matrix . Images of the anti - cd64-conjugated plga nps show a more aggregated and gel - like state, possibly due to the functionalization protocol and the presence of the antibody on the np surface (figure 3aii, bii, cii and dii). Transmission electron micrographs (figure 4) allowed further confirmation of the results obtained by dynamic light scattering and scanning electron microscopy with regard to particle size . The micrographs show a monodispersed population of individual, smooth, and spherical particles with well defined sizes . Association with mtx and spions, as well as anti - cd64 antibody conjugation, did not considerably affect particle shape or overall size . Figure 4bi bii shows spions - loaded nps in which the spions are evident inside the plga nps as smaller and electronically denser, well dispersed spots, confirming their efficient association, both alone and when coassociated with mtx (figure 4di dii). Figure 4aii, bii, cii and dii shows micrographs of the np formulations after conjugation with the anti - cd64 antibody . A denser and thicker corona is apparent surrounding the lighter np core (figure 4aii), which may indicate the presence of the antibody on the surface of the np . Similar observations using transmission electron microscopy were reported by thamake et al for similar antibody - conjugated plga nps.23 a previously described hplc method was used to quantify the association of mtx in the devised nps.16 given that mtx has a solubility of 0.01 mg / ml in water at 20c,8 the mtx - loaded plga nps were prepared using a single emulsion technique in order to achieve elevated values of association efficiency . High efficiency was demonstrated for both mtx - loaded and mtx- and spions - loaded nps, being 79.1% and 75.5%, respectively (table 1). These values did not differ significantly between the two different formulations (p=0.32), indicating that a co - association of both agents is possible in a plga - based theranostic approach . Mtx is a very effective drug against ra but is extremely toxic and has serious side effects, limiting the dose that can be administered, thereby compromising ra therapy.3,8 a high association between mtx and plga in a targeted nanosystem could provide a new opportunity for ra therapy, since the bioavailability, safety, and efficacy of mtx would be improved . The amount of anti - cd64 present in the nanoparticulate system was shown to be approximately 3.5 g per mg of nps . Different antibody / nps ratios were obtained in previous works when conjugating plga nps with different monoclonal antibodies.15,21,24 however, the conjugation efficiency obtained in this work was significantly higher (31%37%), because considerably lower amounts of antibody (at least 20-fold less) were used for functionalization of the plga nps . Statistical analysis of the conjugation efficiencies did not show significant differences between the formulations (p=0.26), indicating that association with both mtx and spions did not considerably affect the main features of the nps and their interaction with the antibody . Ft - ir spectra of the samples were obtained to confirm the association of mtx into plga nps and the functionalization of the nps with the anti - cd64 antibody . The ft - ir spectra of plga nps and mtx - loaded plga nps were compared to the ft - ir spectrum of free mtx (figure 5a). At 1,750 cm, a marked peak indicates the presence of a carbonyl bond (c = o stretching vibration), which is characteristic of plga.25 further, in the mtx spectrum, it is possible to observe a different peak at 1,638 cm (c = c stretching vibration), which is characteristic of the drug molecule but not of plga.26 the characteristic peak from plga was not altered in the mtx - loaded nps spectrum, and the carbon - carbon double bond typical of mtx is evident in the spectrum, confirming that mtx was successfully associated into the plga nps . The ft - ir spectra of plga nps and anti - cd64 conjugated plga nps were compared with the spectrum of the anti - cd64 antibody (figure 5b). At 1,750 cm, the plga - characteristic peak is also present in the antibody (c = o stretching bond).25 near 1,640 cm, an amide bond (c = n stretching vibration) should be recognized, identifying conjugation of the antibody to plga nps . However, at this wavenumber, both plga nps and anti - cd64 antibody spectra show again a clear peak, which corresponds to the c = o stretching bond present on both components, not allowing us to immediately draw conclusions regarding conjugation of the nps.26,27 despite this, in the anti - cd64 spectrum, an additional peak at 1,560 cm stands out, corresponding to the amine groups of the antibody (n - h bending vibrations).27 in the case of the anti - cd64 conjugated nps spectrum, this characteristic peak emerges, confirming that anti - cd64 was present in the functionalized plga nps, either covalently linked or physically adsorbed . The effect of nps on cell viability and cytotoxicity after 24 hours of incubation was studied in vitro on raw 264.7 cells, performing mtt and ldh assays, respectively, as a function of the devised formulations and the different concentrations of mtx (0.01100 g / ml). Both mtt and all formulations displayed a concentration - dependent effect, with toxicity increasing proportional to the concentration (figure 6). The results also demonstrated that the toxicity of mtx - loaded nps was greater than of the free drug, suggesting that a future approach for ra therapy based on these nps may enhance the therapeutic efficiency of mtx . Additionally, with the exception of the highest concentration, mtx - free nps did not significantly affect cell viability, confirming the safety profile of the devised nanosystem (figure 6). Anti - cd64-conjugated nps did not originate higher cytotoxicity when compared with non - conjugated nps . This is justified by the fact that raw 264.7 macrophages, being a mouse cell line, do not express the human cd64 receptor, and these cells were used in this assay as a cell model that does not express this receptor . In figure 6, it is apparent that anti - cd64-conjugated mtx - loaded nps are not as toxic as non - conjugated mtx - loaded nps, demonstrating that this system may work as a targeted approach . Future work using cell models that express or are modified for overexpressing the cd64 receptor will allow studying of the targeting ability of the anti - cd64-conjugated nps, aiming for the envisioned theranostic application . In this work, mtx, spions, and anti - cd64 antibody were successfully co - associated into plga nps for the management of ra . The physicochemical features of the devised nps, ie, their size, zeta potential, morphology, high mtx association efficiency, association with spions, anti - cd64 functionalization, and in vitro safety profile are key elements for a future biomedical and pharmaceutical approach . This new design for a targeted ra theranostic strategy could be considered and studied in order to find new means for ra therapy and also work as an enhanced imaging tool for techniques such as mri . Multifunctional plga - based nanocarriers for drug targeting and in vivo imaging are of particular interest due to their biodegradability and biocompatibility . In this work, by effectively co - associating mtx and spions into plga nps, and successfully functionalizing them with an anti - cd64 antibody, a novel attempt was made to achieve targeted therapy and imaging for ra . Overall, the association of both mtx and spions did not significantly affect the properties of the plga nps . The nps had a reduced particle size and were stable in solution, which are paramount requisites for their application as drug delivery systems . Consequently, the proposed nanoparticulate system may potentiate the action of mtx without injuring healthy tissues and organs, simultaneously providing a non - invasive and specific imaging tool for ra . After their development and thorough characterization in this study, these nps are now ready for further in vitro studies aiming for the assessment of their performance in targeting ra macrophages and reducing inflammation at sites of ra.
|
Chronic obstructive pulmonary disease (copd) is a progressive disease with a significant burden to the individual and society . Management of the condition is complex, but one important aim is to promote physical activity and minimize the impact on day - to - day functioning . Management can include smoking cessation, optimization of inhaled drug therapies, pulmonary rehabilitation, and strategies centered on the identification and treatment of exacerbations . Self - care has been identified as a function that individuals must perform to maintain life, health, and well - being,1 and more specifically applied to chronic illness, it has been defined by riegel et al as a process of maintaining health through health promoting practices and managing illness.2 self - management is often referred to more formally as a range of behaviors and skills necessary for disease management.3 these behaviors and skills are often considered as taught activities, whereas self - care could be considered as a function that occurs more automatically.1 supporting these behaviors has also been identified in the chronic care model4 to improve outcomes for those with long - term conditions . Patients individual experiences with the disease are important for developing the strategies to cope with increasing levels of breathlessness associated with copd.5 a range of factors are considered important for influencing the adoption of self - management behaviors, including personal factors (for example, knowledge and social support) as well as contextual factors (for example, the physical environment, ie, air quality, mobility aids).5 previous research, largely involving patients with moderate to severe copd, suggests that patients experience a shift from an active to a more sedentary lifestyle and describes various strategies to adjust physically to the disease.6,7 planning, pacing and prioritizing are described as behaviors adopted to compensate for this shift6 and are not unique to copd.8 early education and support has also been identified as important when adjusting to a new diagnosis of copd, further stressing the importance of the health care relationship.9,10 however, much of this literature has focused on patients with moderate to severe copd rather than mild disease.7,911 with increasing emphasis on the need to target interventions for those newly diagnosed or at mild stages of the disease, it is important to determine if the self - care experiences of those with milder levels of the disease share the same priorities for disease management . It has been stated that self - management should focus on solving what the patient perceives to be problematic.12 however, what is less well known is how those with milder disease, who are primarily managed in the primary care setting, attempt to manage their disease and what the main challenges are to doing so . The literature currently lacks a description of self - care from the perspective of patients with less severe disease . If disease management strategies are to focus on this population, we need to have a better understanding of their perceptions of the problem . This paper aims to address this gap in the literature by drawing on qualitative interviews with patients with less severe disease . It describes their perceptions of the impact of the condition and their corresponding attempts to manage the symptoms . This qualitative study was nested within a larger quantitative trial examining the effectiveness of a self - management programme of activity, coping and education (space) for copd . Details of the program are reported elsewhere.14 patients were recruited from primary care copd registers . Patients were eligible for the study if they had: a diagnosis of copd confirmed by spirometry with a forced expiratory volume in 1 second / forced vital capacity (fev1/fvc) ratio of <70%; score 25 on the medical research council dyspnoea scale (mrc);13 were not affected by neurological, locomotor, or cognitive problems that would prevent participation in an exercise program; and had not undertaken pulmonary rehabilitation in the previous 12 months . Participants randomized to the self - management arm of the trial were invited to attend an interview, having previously consented to the qualitative study . This was an opportunistic sample, and recruitment for the interviews stopped once it was felt that saturation had been met (n=15). One participant who earlier consented to the qualitative study later declined to attend an interview due to a lack of time . Demographics of those interviewed can be found in table 1 . Face - to - face, semistructured interviews took place after randomization in the randomized controlled trial but before participants had started the self - management program . Interviews were carried out by one of two researchers (lda, slh) and took place either in the hospital or in the participant s own home as determined by the individual . The interview schedule was initially developed by researchers (lda, sjs) and then reviewed for validity following the initial five interviews in which minor changes were made . Topics included perceptions of copd, expectations, and understanding of self - management strategies . The data were analyzed using thematic analysis which allows for identification of patterns in the data and rich descriptions,15 supported by the use of nvivo software (version 8, qsr international, melbourne, australia). Two researchers (lda, slh) carried out the initial coding of all transcripts . Analysis followed six stages as outlined by braun and clarke,15 ie, familiarization with data, generating initial codes, searching for themes, reviewing themes, defining and naming themes, and producing the report . A third researcher (jeaw) familiar with the study aims analyzed a subgroup of transcripts and all researchers met to ensure agreement about themes . A reflective log was kept during this process to ensure that themes remained grounded in the data set . Two large meta - themes emerged from the analysis: the experience of copd and the emergence of informal self - care strategies and expectations and understanding of formal self - management . Due to space restraints and to allow for a richer description of self - care strategies, this paper focuses specifically on the first theme and describes patients experiences of living with copd in three related subthemes . A selection of illustrative, verbatim quotations are provided throughout in order to support the generalizations made from the data . Ethical approval was gained through the national health service research ethics committee (east midlands). Written informed consent was gained from participants at the point of entering the larger self - management trial (isrctn33482179). Patients were eligible for the study if they had: a diagnosis of copd confirmed by spirometry with a forced expiratory volume in 1 second / forced vital capacity (fev1/fvc) ratio of <70%; score 25 on the medical research council dyspnoea scale (mrc);13 were not affected by neurological, locomotor, or cognitive problems that would prevent participation in an exercise program; and had not undertaken pulmonary rehabilitation in the previous 12 months . Participants randomized to the self - management arm of the trial were invited to attend an interview, having previously consented to the qualitative study . This was an opportunistic sample, and recruitment for the interviews stopped once it was felt that saturation had been met (n=15). One participant who earlier consented to the qualitative study later declined to attend an interview due to a lack of time . Face - to - face, semistructured interviews took place after randomization in the randomized controlled trial but before participants had started the self - management program . Interviews were carried out by one of two researchers (lda, slh) and took place either in the hospital or in the participant s own home as determined by the individual . The interview schedule was initially developed by researchers (lda, sjs) and then reviewed for validity following the initial five interviews in which minor changes were made . Topics included perceptions of copd, expectations, and understanding of self - management strategies . The data were analyzed using thematic analysis which allows for identification of patterns in the data and rich descriptions,15 supported by the use of nvivo software (version 8, qsr international, melbourne, australia). Two researchers (lda, slh) carried out the initial coding of all transcripts . Analysis followed six stages as outlined by braun and clarke,15 ie, familiarization with data, generating initial codes, searching for themes, reviewing themes, defining and naming themes, and producing the report . A third researcher (jeaw) familiar with the study aims analyzed a subgroup of transcripts and all researchers met to ensure agreement about themes . A reflective log was kept during this process to ensure that themes remained grounded in the data set . Two large meta - themes emerged from the analysis: the experience of copd and the emergence of informal self - care strategies and expectations and understanding of formal self - management . Due to space restraints and to allow for a richer description of self - care strategies, this paper focuses specifically on the first theme and describes patients experiences of living with copd in three related subthemes . A selection of illustrative, verbatim quotations are provided throughout in order to support the generalizations made from the data . Ethical approval was gained through the national health service research ethics committee (east midlands). Written informed consent was gained from participants at the point of entering the larger self - management trial (isrctn33482179). The meta - theme the impact of early experiences of copd and informal self - management is the main focus of this paper . It is constituted by three subthemes, ie, experiencing and understanding symptoms of copd, current self - care activities, and the importance of family perceptions in managing copd . Shortness of breath was often the main symptom that disrupted daily tasks and prompted participants to make adaptations, plan more, or allow other family members to do tasks for them . Participants reported feeling frustrated and confused about breathlessness and their condition more generally; something that was exacerbated by the feeling that there had been inadequate information provided to them by health care professionals . Six participants reported feeling unsure about the condition itself, its progression, and what to expect in the future . In particular, these participants felt that they lacked specific information about what to do in the event of an exacerbation, how to monitor symptoms, and when to act (see table 2). Feeling that their knowledge level was insufficient also had implications for how confident participants felt about their medication use and what they should do in an acute event (see table 2). Only two of our 15 participants recalled receiving specific advice from health care professionals about management strategies such as increasing exercise; however, in these cases, a lack of detail about what kinds of exercise to do had not led to any behavior change . These participants described receiving no further support or having any concerns allayed about whether increasing shortness of breath was harmful (see table 2). Despite this reported lack of understanding or knowledge, in the course of the interviews, participants described several self - care strategies that they had developed to limit the impact of the symptoms on their day - to - day lives . Various strategies for limiting symptoms were described and included smoking cessation, adapting activities to accommodate breathlessness, pacing activities to conserve energy, taking more rest, and distraction from symptoms when they did occur . There was also some description of utilizing walking aids such as shopping trolleys or buggies as they were found to be effective (see table 3). All such strategies had been initiated by the participants without formal instruction in self - management techniques from professionals involved in their care . Often the adaptation of activities or strategies employed had evolved through the experiences they had of their illness so far . These strategies were introduced on a trial and error basis to adapt activities and try out different ways of managing symptoms, the end result being self - care activities that they felt confident worked . As part of this self - care strategizing and informal management of their symptoms, participants also reported decisions about the use of medications and varying their use in response to symptoms . Inhalers were viewed as core to managing symptoms and participants would generally increase their use if shortness of breath felt worse . Participants also reported cutting down the use of their regular inhaler or nebulizer doses for fear of reliance or having nowhere else to go if their condition worsened . Participants were worried that their medications might not work as their disease progressed so they withheld doses in order that they had them to control their symptoms in the future (see table 3). These views were limited to the regular use of inhalers: participants were more positive about using antibiotics and steroids in the event of an exacerbation if they felt that they had previously been beneficial . This discrepancy between the use of medicines during stable and acute episodes of the disease highlights how attitudes to medicines may differ over the course of the disease . Participants differed in how much control they felt over the need to make changes in their lives . For half of the participants, changes were viewed as a necessity, that they had had no choice but to carry out certain activities less frequently or give them up entirely, ie, that the disease had forced the change . For other participants, the decision to adapt certain tasks was perceived as a proactive choice made in order to help themselves (see table 3). Loss of strength and fitness or the presence of fatigue, for some, could be attributed to getting older and not seen as a direct consequence of copd . Participants felt that they would experience some of these symptoms without having been diagnosed with copd . A reduced ability to engage in more physical activities was often attributed to the aging process regardless of whether they had developed a long - term health condition . The final theme in the paper relates to the involvement of family members or partners in patients self - care . This was described as a challenge to self - care as participants report a dichotomy to social support whereby it was experienced as both creating conflict and being a positive source of support . For most participants, the invisibility of their lung disease was an important consideration within their social environment . Whilst one participant made attempts to hide her symptoms from her family when she was first diagnosed, many participants instead described the struggle of convincing their friends and family of the seriousness of their symptoms (see table 4). Friends and family were portrayed by participants as expecting more, in terms of household or caring duties, than they felt able to cope with . This often led to feelings of frustration and sometimes triggered conflict within relationships as participants attempted to restrict their activities whilst meeting the expectations of others around them . As described above, whilst participants would limit and avoid certain activities as a self - care strategy, such behavior could sometimes make it difficult to perform or live up to a certain role they themselves and others would have previously expected of them . In contrast, some participants described receiving a degree of social support, which aided disease management strategies . One married participant, for example, described a successful working partnership with her husband where jobs were shared, allowing for more quality time to be spent together . For others, there was often a need for a more extensive transfer of jobs / roles to a partner or another family member . Whilst this transfer of responsibilities helped with symptom management, it was not always easy to tolerate as shifting roles again were experienced as having an impact on relationships . One male participant described a scenario where his wife had to dig a trench to prevent flooding (see table 4). The change in roles in this case was emphasized by the sex of the participant and the transfer of a manual, and traditionally masculine, task to his female partner . Three participants who lived alone also reported sharing jobs with family members, though there was less conflict reported in these accounts . Shortness of breath was often the main symptom that disrupted daily tasks and prompted participants to make adaptations, plan more, or allow other family members to do tasks for them . Participants reported feeling frustrated and confused about breathlessness and their condition more generally; something that was exacerbated by the feeling that there had been inadequate information provided to them by health care professionals . Six participants reported feeling unsure about the condition itself, its progression, and what to expect in the future . In particular, these participants felt that they lacked specific information about what to do in the event of an exacerbation, how to monitor symptoms, and when to act (see table 2). Feeling that their knowledge level was insufficient also had implications for how confident participants felt about their medication use and what they should do in an acute event (see table 2). Only two of our 15 participants recalled receiving specific advice from health care professionals about management strategies such as increasing exercise; however, in these cases, a lack of detail about what kinds of exercise to do had not led to any behavior change . These participants described receiving no further support or having any concerns allayed about whether increasing shortness of breath was harmful (see table 2). Despite this reported lack of understanding or knowledge, in the course of the interviews, participants described several self - care strategies that they had developed to limit the impact of the symptoms on their day - to - day lives . Various strategies for limiting symptoms were described and included smoking cessation, adapting activities to accommodate breathlessness, pacing activities to conserve energy, taking more rest, and distraction from symptoms when they did occur . There was also some description of utilizing walking aids such as shopping trolleys or buggies as they were found to be effective (see table 3). All such strategies had been initiated by the participants without formal instruction in self - management techniques from professionals involved in their care . Often the adaptation of activities or strategies employed had evolved through the experiences they had of their illness so far . These strategies were introduced on a trial and error basis to adapt activities and try out different ways of managing symptoms, the end result being self - care activities that they felt confident worked . As part of this self - care strategizing and informal management of their symptoms, participants also reported decisions about the use of medications and varying their use in response to symptoms . Inhalers were viewed as core to managing symptoms and participants would generally increase their use if shortness of breath felt worse . Participants also reported cutting down the use of their regular inhaler or nebulizer doses for fear of reliance or having nowhere else to go if their condition worsened . Participants were worried that their medications might not work as their disease progressed so they withheld doses in order that they had them to control their symptoms in the future (see table 3). These views were limited to the regular use of inhalers: participants were more positive about using antibiotics and steroids in the event of an exacerbation if they felt that they had previously been beneficial . This discrepancy between the use of medicines during stable and acute episodes of the disease highlights how attitudes to medicines may differ over the course of the disease . Participants differed in how much control they felt over the need to make changes in their lives . For half of the participants, changes were viewed as a necessity, that they had had no choice but to carry out certain activities less frequently or give them up entirely, ie, that the disease had forced the change . For other participants, the decision to adapt certain tasks was perceived as a proactive choice made in order to help themselves (see table 3). Loss of strength and fitness or the presence of fatigue, for some, could be attributed to getting older and not seen as a direct consequence of copd . Participants felt that they would experience some of these symptoms without having been diagnosed with copd . A reduced ability to engage in more physical activities was often attributed to the aging process regardless of whether they had developed a long - term health condition . The final theme in the paper relates to the involvement of family members or partners in patients self - care . This was described as a challenge to self - care as participants report a dichotomy to social support whereby it was experienced as both creating conflict and being a positive source of support . For most participants, the invisibility of their lung disease was an important consideration within their social environment . Whilst one participant made attempts to hide her symptoms from her family when she was first diagnosed, many participants instead described the struggle of convincing their friends and family of the seriousness of their symptoms (see table 4). Friends and family were portrayed by participants as expecting more, in terms of household or caring duties, than they felt able to cope with . This often led to feelings of frustration and sometimes triggered conflict within relationships as participants attempted to restrict their activities whilst meeting the expectations of others around them . As described above, whilst participants would limit and avoid certain activities as a self - care strategy, such behavior could sometimes make it difficult to perform or live up to a certain role they themselves and others would have previously expected of them . In contrast, some participants described receiving a degree of social support, which aided disease management strategies . One married participant, for example, described a successful working partnership with her husband where jobs were shared, allowing for more quality time to be spent together . For others, there was often a need for a more extensive transfer of jobs / roles to a partner or another family member . Whilst this transfer of responsibilities helped with symptom management, it was not always easy to tolerate as shifting roles again were experienced as having an impact on relationships . One male participant described a scenario where his wife had to dig a trench to prevent flooding (see table 4). The change in roles in this case was emphasized by the sex of the participant and the transfer of a manual, and traditionally masculine, task to his female partner . Three participants who lived alone also reported sharing jobs with family members, though there was less conflict reported in these accounts . This is the first study to focus on the experience of dyspnea and its resulting impact on activity and self - care in patients with predominantly mild to moderate copd . There are clear parallels with studies exploring these issues in patients with severe copd,9,10,16 showing that such losses and the associated distress may not be unique to the severe population and may be a core concern for anyone living with copd who experiences reduction in activities of daily living due to breathlessness . Participants independently initiated a range of self - management behaviors such as pacing, adapting activities, distraction, and utilizing social support, which has been described elsewhere.17 in this study, patients appeared to be unaware that they were performing self - management behaviors and instead described a lack of confidence about managing their symptoms as they did not feel confident about their current level of disease knowledge . Figure 1 outlines some of these behaviors, demonstrating the overlap between what may evolve naturally and what may require more support . Adaptive behaviors that occur more spontaneously should be further enhanced with taught skills to increase patients confidence to manage their condition . This highlights the importance of health care professionals and the need for them to be clear with patients about how they can best manage their condition and what benefits they can expect from these techniques . Discussing patients prior experiences with their condition and considering what self - care activities they have already engaged in may enable health care professionals to better capitalize on disease management activities in these patients . This may speed up the learning cycle that patients go through as they test and find actions to promote well - being . Such actions and the importance of disease experiences have previously been discussed in relation to becoming an expert in the self - management of chronic dyspnea.5 although only time can provide patients with such experiences, ensuring effective and timely learning may help patients with this process . The role of health care professionals in instilling patient faith in treatment has been shown to aid participation in a disease management intervention.11 this may be especially important in relation to adherence with medication since these findings demonstrate that patients may limit use of inhalers as a way of managing future progression of disease . Health care professionals are key, not only to maximizing self - care activity, but to ensuring that strategies invested in are likely to result in maximum benefit for the patient . Although many patients reported a reliance on family members to help with the management of their disease, there was also some conflict, as the invisibility of the disease challenged its acceptance by family members . This is a particularly interesting finding as research often focuses on the benefits of social support,7,10,11 although williams highlights the issues of legitimacy and understanding of others when living with chronic lung disease.17 participants interviewed here felt the burden of their symptoms and a need to engage in self - care, but felt this had not been acknowledged by family members around them . This seemingly unsympathetic response to patients symptoms expressed by those closest to them perhaps in part stems from a general lack of knowledge regarding the seriousness of the disease.18 the patients taking part in these interviews were predominantly mild to moderate (with ten participants at mrc grades 23 and only one at mrc grade 5) and are unlikely to have experienced hospital admissions or severe exacerbations of their disease . As a result, family members may have yet to realize the significance of the disease.18 this study provides a description of living with and managing copd from the perspective of a less severe population . The presence of an illness that affects activities of daily living may lead to a reappraisal of important activities for patients, in which autonomy is preserved as expectations are reframed according to current circumstances.19 it seems plausible to suggest that if patients feel confident and empowered about what they are doing to cope with their illness, this will encourage further positive behavior and buffer against distress as a result of the changes made . Participants spontaneously engage in self - care activities when living with a condition that limits activities of daily living, and health care professionals should utilize these earlier experiences to better promote self - management . There needs to be awareness among practitioners, not only of how copd impacts on patients activities of daily living and the family network, but also on the acceptance they feel of the adaptations they make . Early experiences of managing their condition are an important part of this picture and should be considered when designing or implementing self - management programs . Health care professionals should give advice, information, and guidance and should help patients reframe their expectations, acknowledging their current situation and enhancing autonomy . This paper highlights the self - care behaviors and decision - making that patients actively and independently engage in when faced with limiting symptoms . Playing an active role or having what remains unknown, and is beyond the scope of this paper, is an understanding of which factors may influence self - management behaviors in copd patients.
|
The authors report the case of a 17-week intact abdominal pregnancy diagnosed in the course of an investigation of lower abdominal pain . Subsequent angiography was performed to occlude the supportive artery of the pregnancy by selective embolization . The placenta was left in the abdominal cavity because of the high risk of massive and often uncontrollable bleeding, and treatment with methotrexate was applied postoperatively . Preoperative embolization and the postoperative methotrexate therapy facilitate the safe surgical treatment of abdominal pregnancy . Reports on its frequency vary, ranging from 1 in 3371 deliveries to greater than 1 in 10 200 deliveries [13]. The maternal mortality risk from abdominal pregnancy is 7.7 times greater than that of tubal ectopic pregnancy . The treatment of these cases requires attention due to the risk of life - threatening bleeding arising from the rupture of the fetus . Maternal morbidity can also be substantial, with high incidence of pelvic abscess, peritonitis, and sepsis caused by retained placental remnants . A 28-year - old patient (gravida 3, para 2, with 1 caesarean section in the history) was referred to our department by her general practitioner, with lower abdominal pain developed 3 weeks before . The physical examination verified abdominal tenderness, principally at the right side of the lower abdomen and around the umbilicus . Transvaginal sonography described the uterus 7089 mm in size with no visible fetus in the cavity . Above the uterus at the right side an amniotic cavity was seen, in which the fetus was visible in an oblique - breech presentation . Biometric data (biparietal diameter (bpd): 44 mm, head circumference (hc): 145 mm, abdominal circumference (ac): 104 mm, femur length (fl): 24 mm) corresponded to a 1718-week - old fetus without any signs of malformation . The quantity of the amniotic fluid was normal, the placenta was attached to the right back side, and both ovaries were normal . Laboratory evaluation (red blood cell count, hemoglobin, hematocrit, white blood cell count, platelet count, westergren) did not show any clinically significant abnormality . Pelvic mri (with native multiplane, multisequential imaging) showed that above the urinary bladder in the pelvis minor a 60 mm thick uterus was visible with stir (short tau inversion recovery) sequence and t2 enhancement . Above the uterus risen from the right iliac region there was a 5060 mm thin walled cystic object with embryonic elements inside (skull, orbit, lambda suture could be observed) which were also visible with long - period sequences . Without contrast material the exact location of the placentation was not precisely determinable, but it was found that the outer surface of the uterus did not communicate with the fetus . The patient was referred to the department of cardiovascular surgery, semmelweis university, for a selective angiography and embolization . Selective angiography verified a normal upper and lower as well as internal iliac artery . In the height of the right renal artery a winding artery was noticed running towards the pelvis minor, which was suspected to be the ovarian artery . This artery was hypertrophic, thicker than usual, with a winding shape, and seemed to supply a parenchymal bulge situated to the right of the uterus . A selective embolization of this artery was performed with pva (polyvinyl alcohol) particles . During the control angiography, the control ultrasound examination showed no signs of fetal life . In the perioperative period, antibiotic treatment was administered: 31.5 g cefuroxime intravenously, and 2250 mg metronidazole orally . A day after the selective angiography and embolizations, after adhesiolysis, it became visible that the fetus was situated in the lower right region of the abdominal cavity, adhered to the colon descendens and to the small intestine . The membranes were separated until the edge of the placenta . As the complete removal of the whole fetus without the risk of life - threatening bleeding was not possible, in the postoperative course, intramuscular methotrexate therapy was administered (20 mg / m twice a week, a total of 5 times). Ablactation was started with bromocriptine therapy and the antibiotic treatment was completed with intramuscular gentamicin with the dose of 1240 mg each day . On the 7 postoperative day ultrasound examination was performed . At the right side of the lower abdomen a 5949 mm solid bulge the retained placenta the majority of pregnancies located in the abdominal cavity result from reimplantation of tubal abortions; thus they cannot be considered as real (primary) abdominal pregnancies . The reported case matches the criteria defined by studdiford in 1942 as being a primary abdominal pregnancy . To be considered as such, the pregnancy must meet the following criteria: intact tubes and ovaries must be normal, without evidence and signs of recent or remote injury; no evidence of uteroperitoneal fistula should be found; the gestation must be related exclusively to the peritoneal surface and be early enough to eliminate the possibility that it is a secondary implantation . Early diagnosis of an abdominal pregnancy is critical because a catastrophic hemorrhage can result from separation of the placenta . Ultrasonography is considered to be the criterion standard for obtaining exact information about the location of the pregnancy and its relation to the surrounding organs . In cases where ultrasonography is equivocal selective catheterisation and subsequent embolization of the pelvic blood vessels was introduced in the early 1980 s in gynecological practice . The main indication was massive pelvic hemorrhage (eg, in certain cases of advanced, bulky cervical cancers). In our case, embolization of the main supporting vessel leading to the placenta was performed in order to avoid a massive intraoperative bleeding and to promote the absorption of the retained placenta . Retaining the placenta in its original place is a common method during operative procedures of abdominal pregnancies . The drainage of the placental bed is highly recommended in order to detect bleeding in the postoperative stage . Methotrexate therapy is a well - known alternative treatment in certain cases of ectopic pregnancies in order to hasten trophoblastic degeneration . In this case, it was administered in the postoperative interval to promote the absorption of the retained placenta . Involution of the gestational tissue can be controlled by monitoring postoperative beta - hcg serum levels . Reviewing the literature regarding the management of genuine abdominal pregnancies, it is hard to find a publication explaining the same method reported by us, perhaps due to the rarity of the cases and the limited accessibility of angiography . Numerous cases are recognized only when symptoms of abdominal bleeding are present; therefore, laparotomy must be performed instantly . As in the majority of cases in the literature, the outcome of our case was successful . Therefore, preoperative selective angiography and embolization followed by laparotomy and adjuvant methotrexate therapy can be a safe and effective method in the treatment of abdominal pregnancies in the second trimester.
|
Asthma is a chronic syndrome characterized by reversible airway obstruction, airway inflammation, and airway hyperresponsiveness; estimates have indicated that 8% (18.7 million) of adults in the united states currently have asthma, and more than 300 million people are affected by asthma worldwide . Moreover, its prevalence has increased considerably in the past 20 years . Patients with asthma may experience recurrent episodes of chest tightness, shortness of breath, coughing, and wheezing; consequently, people with this condition are at reportedly higher risks of nonrespiratory disorders such as depression, anxiety disorder, and dementia development compared with people without asthma . The goal of current asthma management is to focus on managing rather than curing the disease . Migraine is a highly prevalent and disabling neurological disorder characterized by episodic unilateral headache attacks that are often accompanied by photophobia, phonophobia, nausea, and vomiting, and the estimated cumulative lifetime incidence of migraine is 43% in women and 18% in men . Although various factors have been identified to be associated with migraine attacks, such as stress, auditory hypersensitivity, and hormone imbalance, the pathogenesis of migraine is only partially understood . Asthma has been described as an acephalic migraine and pulmonary migraine, and several epidemiologic studies have reported an association between migraine and respiratory disorders . However, some of these studies have used a relatively small sample, and all of them have adopted a case - control design . No study has investigated the temporal frequency of migraine development in patients with asthma, and the results of previous studies may be difficult to generalize . Therefore, we conducted this retrospective nationwide cohort study by using data retrieved from taiwan's national health insurance research database (nhird) to test the hypothesis that patients with asthma have a higher risk of migraine . The nhird was created by the national health research institutes (nhri) and contains claims data from the taiwan national health insurance (nhi) program . The nhi program, implemented in 1995, is a compulsory single - payer health care system with over 99.9% coverage of the population of taiwan at the end of 2014 . This study used the longitudinal health insurance database 2000 (lhid2000), which contains data for 1 million enrollees derived from the medical claims records of the nhi program between 1996 and 2011 . The nhird contains beneficiary demographics, clinical visit dates, prescription details, and diagnostic codes based on the international classification of diseases, ninth revision, clinical modification (icd-9-cm). The nhri manages the claims data and provides scrambled random identification numbers for insured patients to secure patient privacy . This study was approved by the institutional review board of china medical university (cmuh-104-rec2 - 115). We conducted a nationwide retrospective population - based cohort study involving 2 groups: an asthma group and a nonasthma group . From outpatient and inpatient care dates in the lhid2000, we identified 41,011 patients newly diagnosed with asthma (icd-9-cm 493) between january 1, 2000 and december 31, 2005 . We excluded patients from the analyses if they were younger than 12 years (n = 13,813), were previously diagnosed with migraine (icd-9-cm-346) (n = 1635), or had missing information on age or sex (n = 3). The date of asthma diagnosis was considered the index date . To ensure the accuracy of the asthma diagnosis, we selected patients who had received treatment involving inhaled corticosteroids, systemic (oral or intravenous) corticosteroids, or inhaled beta-2 agonists (short - acting beta-2 agonists [sabas] or long - acting beta-2 agonists [labas]) as the asthma group . For each patient with asthma, 4 insured persons without a diagnosis of asthma were selected from the lhid2000 as the nonasthma group and were frequency - matched according to sex, age (every 5-year span), and index year by using the same inclusion criteria as those of the asthma cohort . The considered demographic factors were sex and age (1219, 2044, and 45 years). Comorbidities were evaluated using the charlson comorbidity index (cci) and were considered confounding factors . According to each subject's inpatient diagnosis, we calculated the cci scores as the sum of the weighted score of 17 comorbid conditions . A weight was assigned to each indicated diagnosis and added together to provide a total cci score . For the cci score comorbidities such as myocardial infarction, congestive heart failure, peripheral vascular disease, cerebrovascular disease, dementia, chronic pulmonary disease, rheumatologic disease, peptic ulcer disease, mild liver disease, and diabetes mellitus were weighted 1; moderate to severe diabetes with chronic complications, hemiplegia or paraplegia, renal disease, leukemia, tumor of any type, and malignant lymphoma were weighted 2; moderate - to - severe liver diseases were weighted 3; and aids and metastatic solid tumor were weighted 6 . Medications that could affect the progression of migraine, such as inhaled corticosteroids, systemic corticosteroids, and beta-2 agonists (including sabas and labas), were included as analysis variables . Prescribed medications were defined as those prescribed for 30 successive days within 1 year of the index date . Moreover, the number of annual outpatient department (opd) visits was also included as an analysis variable . The outcome measure of interest, which was determined at least thrice by outpatient services or inpatient hospitalization claims, was based on the icd-9-cm diagnosis code for migraine (icd-9-cm 346). Both groups were observed from the index date until the date of migraine diagnosis, withdrawal from the nhi program, or the end of 2011 . The continuous variables are expressed as the mean and standard deviation (sd), whereas the categorical variables are expressed as frequencies and percentages . This study used the student t test for continuous variables and the pearson chi - square test for categorical variables to compare the differences in the demographic factors, cci scores, medications used, and annual opd visits between the asthma and nonasthma groups . The sex, age, and cci score - specific incidence density rates (per 1000 person - years) of migraine were calculated using the number of migraine incidents divided by the person - years at risk in both groups . The cumulative incidence curves of migraine in the asthma and nonasthma groups were estimated using kaplan meier analysis, and the difference between the groups was compared using the log - rank test . Univariate and multivariate cox proportional - hazards regression models were used to assess the risk of migraine and migraine - associated risk factors . The multivariate model was adjusted for sex, age, the cci score, medications used, and annual opd visits . We also compared the hazard ratio (hr) of migraine between the asthma and nonasthma groups after stratification by sex, age, and the cci score . A p value less than 0.05 was considered statistically significant, and sas version 9.3 software (sas institute, inc ., cary, nc) was used to perform all the statistical analyses . The nhird was created by the national health research institutes (nhri) and contains claims data from the taiwan national health insurance (nhi) program . The nhi program, implemented in 1995, is a compulsory single - payer health care system with over 99.9% coverage of the population of taiwan at the end of 2014 . This study used the longitudinal health insurance database 2000 (lhid2000), which contains data for 1 million enrollees derived from the medical claims records of the nhi program between 1996 and 2011 . The nhird contains beneficiary demographics, clinical visit dates, prescription details, and diagnostic codes based on the international classification of diseases, ninth revision, clinical modification (icd-9-cm). The nhri manages the claims data and provides scrambled random identification numbers for insured patients to secure patient privacy . This study was approved by the institutional review board of china medical university (cmuh-104-rec2 - 115). We conducted a nationwide retrospective population - based cohort study involving 2 groups: an asthma group and a nonasthma group . From outpatient and inpatient care dates in the lhid2000, we identified 41,011 patients newly diagnosed with asthma (icd-9-cm 493) between january 1, 2000 and december 31, 2005 . We excluded patients from the analyses if they were younger than 12 years (n = 13,813), were previously diagnosed with migraine (icd-9-cm-346) (n = 1635), or had missing information on age or sex (n = 3). The date of asthma diagnosis was considered the index date . To ensure the accuracy of the asthma diagnosis, we selected patients who had received treatment involving inhaled corticosteroids, systemic (oral or intravenous) corticosteroids, or inhaled beta-2 agonists (short - acting beta-2 agonists [sabas] or long - acting beta-2 agonists [labas]) as the asthma group . For each patient with asthma, 4 insured persons without a diagnosis of asthma were selected from the lhid2000 as the nonasthma group and were frequency - matched according to sex, age (every 5-year span), and index year by using the same inclusion criteria as those of the asthma cohort . The considered demographic factors were sex and age (1219, 2044, and 45 years). Comorbidities were evaluated using the charlson comorbidity index (cci) and were considered confounding factors . According to each subject's inpatient diagnosis, we calculated the cci scores as the sum of the weighted score of 17 comorbid conditions . A weight was assigned to each indicated diagnosis and added together to provide a total cci score . For the cci score comorbidities such as myocardial infarction, congestive heart failure, peripheral vascular disease, cerebrovascular disease, dementia, chronic pulmonary disease, rheumatologic disease, peptic ulcer disease, mild liver disease, and diabetes mellitus were weighted 1; moderate to severe diabetes with chronic complications, hemiplegia or paraplegia, renal disease, leukemia, tumor of any type, and malignant lymphoma were weighted 2; moderate - to - severe liver diseases were weighted 3; and aids and metastatic solid tumor were weighted 6 . Medications that could affect the progression of migraine, such as inhaled corticosteroids, systemic corticosteroids, and beta-2 agonists (including sabas and labas), were included as analysis variables . Prescribed medications were defined as those prescribed for 30 successive days within 1 year of the index date . Moreover, the number of annual outpatient department (opd) visits was also included as an analysis variable . The outcome measure of interest, which was determined at least thrice by outpatient services or inpatient hospitalization claims, was based on the icd-9-cm diagnosis code for migraine (icd-9-cm 346). Both groups were observed from the index date until the date of migraine diagnosis, withdrawal from the nhi program, or the end of 2011 . The continuous variables are expressed as the mean and standard deviation (sd), whereas the categorical variables are expressed as frequencies and percentages . This study used the student t test for continuous variables and the pearson chi - square test for categorical variables to compare the differences in the demographic factors, cci scores, medications used, and annual opd visits between the asthma and nonasthma groups . The sex, age, and cci score - specific incidence density rates (per 1000 person - years) of migraine were calculated using the number of migraine incidents divided by the person - years at risk in both groups . The cumulative incidence curves of migraine in the asthma and nonasthma groups were estimated using kaplan meier analysis, and the difference between the groups was compared using the log - rank test . Univariate and multivariate cox proportional - hazards regression models were used to assess the risk of migraine and migraine - associated risk factors . The multivariate model was adjusted for sex, age, the cci score, medications used, and annual opd visits . We also compared the hazard ratio (hr) of migraine between the asthma and nonasthma groups after stratification by sex, age, and the cci score . A p value less than 0.05 was considered statistically significant, and sas version 9.3 software (sas institute, inc . Our study population comprised 25,560 and 102,238 participants in the asthma and nonasthma groups, respectively . Table 1 presents the demographic characteristics, cci scores, medications used, and annual opd visits in both groups . The study groups were predominantly female (52.3%), and 63.0% of the study participants were older than 45 years . No significant differences in the distributions of sex and age were observed between the asthma and nonasthma groups . The mean ages of the asthma and nonasthma groups were 51.4 (sd = 19.6) and 51.1 (sd = 19.6) years, respectively . A higher cci score was found in the asthma group than in the nonasthma group (0.38 0.94 vs 0.23 0.77). The asthma group exhibited a higher prevalence of using beta-2 agonists, systemic corticosteroids, inhaled corticosteroids, and opd visits compared with the nonasthma group . Baseline demographic factors and comorbidity of study participants according to asthma status the mean follow - ups were 8.40 years (sd = 2.79 years) for the asthma group and 8.42 years (sd = 2.72 years) for the nonasthma group . We used the log - rank test to examine the cumulative incidence of migraine between the groups with and without asthma . We found that the cumulative incidence of migraine was significantly higher in the asthma group than in the nonasthma group (p <0.001). . The incidence density rate of migraine was 3.70 per 1000 person - years in the asthma group, 1.77-fold higher than that in the nonasthma group (2.10 per 1000 person - years), with an adjusted hr of 1.45 (95% confidence interval [ci] 1.331.59) (table 2). Multivariate analysis showed that men (adjusted hr 0.42, 95% ci 0.390.46) and cci scores of 2 and higher (adjusted hr 0.74, 95% ci 0.610.90) were significantly associated with a lower risk of migraine . By contrast, the results showed that patients aged 2044 years and those with more than 30 annual opd visits had an increased risk of migraine (adjusted hr 1.52, 95% ci 1.271.83; adjusted hr 2.57, 95% ci 2.352.81, respectively). Cox model measured hazard ratios and 95% confidence intervals of migraine associated with asthma and covariates in the analysis stratified by sex, the risk of migraine in the asthma patients was significantly higher in both sexes compared with the patients without asthma, with adjusted hrs of 1.22 (95% ci 1.101.36) and 1.51 (95% ci 1.271.80) for women and men, respectively . Stratified by age group, the patients with asthma had a significantly higher risk of migraine compared with those without asthma in all age groups, except for the 12 to 19-year - old age group . The adjusted hrs of migraine were 1.30 (95% ci 1.121.51) among the 20 to 44-year - olds and 1.28 (95% ci 1.141.44) among the 45-year - olds . In study participants with a cci score of 0, patients with asthma had a higher risk of migraine than that of the patients without asthma (adjusted hr 1.29, 95% ci 1.171.42) (table 3). Incidence density rates and hazard ratios of migraine according to asthma status stratified by sex, age, and cci score in this large nationwide cohort study, after adjusting for sex, age, the cci score, medications used, and annual opd visits, we observed that adult patients with asthma were 1.45-fold more likely to develop migraine than those without asthma . Although the mean cci score, prevalence of beta-2 agonists and corticosteroids used, and number of annual opd visits were significantly higher in the asthmatic patients than in the participants without asthma, the risk of migraine remained significantly higher after adjustment for these confounding factors . An additional stratified analysis revealed that the risk of migraine in asthmatic patients remained significantly higher between both sexes and among all age groups older than 20 years . Recently, davey et al reported that the relative risk of asthma in migraineurs was 1.59 compared with nonmigraineurs in a case - control study that used 64,678 case - control pairs from the british general practice research database . Ozge et al reported in a clinical study that 41.4% of migraineurs have at least 1 atopic disorder, including asthma, which was higher than in the general population . A large questionnaire - based study by aamodt et al reported that both migraine and nonmigrainous headaches were approximately 1.5-fold more likely in people with asthma than in those without asthma moreover, kaleagasi et al reported that a positive bronchial provocation test, a key feature of asthma, was more prevalent for migraineurs than for controls . Our data are consistent with these studies, indicating an association between asthma and migraine . However, the definitions of asthma and migraine in the study by aamodt et al were based on participant - report questionnaires, which might not be as valid as our data source . In addition, the relatively small samples in the studies by ozge et al and kaleagasi et al might render the study results difficult to generalize . Finally, the temporal relationship between asthma and migraine risk has been poorly defined in all of these studies . On the basis of our research, the present study is the first large population - based nationwide cohort study demonstrating that adult patients with asthma have a significantly higher subsequent risk of migraine than those without asthma do . However, several lines of evidence from previous studies have suggested that asthma and migraine have a shared pathophysiology . First, a nonselective cation channel expressed in the cell membranes of afferent sensory fibers, named transient receptor potential vanilloid subfamily member 1 (trpv1), has been shown to play a significant role in the generation and pathophysiology of both asthma and migraine . Trpv1 in the airway c - fiber sensory nerves activated by an endogenous or inhaled irritant can result in the release of various neuropeptides, which are believed to contribute to the manifestation of pathophysiological features of asthma such as bronchoconstriction, hypersecretion, and coughing . Similarly, activation of tprv1 by various chemical substances, low ph, and noxious temperature may result in the release of various neuropeptides at the peripheral termini of the trigeminal nociceptors, and these neuropeptides can exert a vasodilatory effect and initiate neurogenic inflammation, both of which are crucial in the generation of migraine headache . Second, although it is well established that mast cells are involved in the pathogenesis of asthma by infiltrating the airway smooth muscle and inducing airway remodeling by releasing various inflammatory mediators, emerging evidence has shown that meningeal and brain mast cells are closely associated with neurons particularly in the dura and these mast cells are believed to be the potent modulators of meningeal nociceptor activity and the genesis of migraine headache . Finally, production of the platelet activating factor, a proinflammatory mediator that has been implicated as being responsible for airway hyperresponsiveness and airway inflammation in asthma, has been reported to increase and potentially result in persistent platelet activation and hyperfunction in the cerebral circulation during a migraine attack . In our study, women displayed a significantly higher risk of migraine than men did, and people aged 20 to 44 years had a significantly higher risk of migraine compared with their younger and older counterparts, consistent with several studies indicating that the prevalence of migraine is generally higher in women than in men, and varies considerably with age, increasing from adolescence to approximately 40 to 45 years of age, and declining thereafter in both sexes . This was why we stratified adult asthmatic patients into 2 subgroups: aged 20 to 44 years and 45 years, to observe the influences of asthma on migraine risk . Although the prevalence of migraine is higher in women than in men in the general population, as indicated by both previous studies and ours, the hr of migraine among asthmatic patients seems to be no different between the sexes . Because this study was observational and, thus far, no comparative studies have explored the sex differences of migraine risk in patients with asthma, future study is warranted to explore this issue . Because inhaled beta-2 agonists, and oral and systemic corticosteroids are common medications prescribed to patients with asthma, and because corticosteroids are also known to be prescribed either as monotherapy or as adjuvant therapy in aborting migraines when other acute care medications have failed, we evaluated whether these medications prescribed for 30 successive days after asthma diagnosis affect migraine risk . Our data show that beta-2 agonists did not significantly affect migraine risk, consistent with wilkinson et al, who reported that in schoolchildren aged 5 to 15 years, no association was observed between frequent headache and use of bronchodilators . Our data show that systemic / inhaled corticosteroids did not significantly affect migraine risk either . Because no comparative study exists, further study examining whether corticosteroids affect migraine risk is necessary for corroborating or refuting our findings . The strengths of this study are its nationwide population - based design, relatively long follow - up (up to 12 years), and the representativeness of the cohorts . First, detailed information on the lifestyle of the patients, such as cigarette smoking, alcohol consumption, dietary habits, and environmental effects, is not provided in the nhird, all of which might have been confounding factors . Second, the diagnoses were based on icd-9 codes obtained from the administrative data . Each patient's clinical information, such as imaging results, serum laboratory data, lung function tests, migraine frequency, and the presence or absence of auras, was not available in the nhird . Therefore, it was difficult to distinguish between allergic and nonallergic asthma, and also different types of migraine . Third, because the information about pain medications was incomplete in the nhird, we could not determine whether these medications affected migraine development in our study . Finally, despite our meticulous study design with adequate control for confounding factors, unknown or unmeasured confounders were present and may have biased the study results . Nevertheless, in the nhi program, from which the nhird is derived, all the insurance claims must be reviewed and audited by medical reimbursement specialists, upholding the validity and accuracy of the asthma and migraine diagnoses . Furthermore, because of the validity of the database, and also the large sample and long follow - up period, we believe that our finding regarding the association between asthma and migraine is reliable . In summary, our study revealed that adult patients with asthma exhibited a significantly higher risk of migraine than did those without asthma . This increased risk was significantly higher in both sexes and in adults of all ages . We suggest that clinicians be aware that asthma is a potential risk factor for migraine.
|
Cutaneous amyloidosis were classified into primary cutaneous amyloidosis (pca), secondary cutaneous amyloidosis, and systemic cutaneous amyloidosis . Pca is a rare, chronic progressive skin disease, defined as deposition of amyloid in previously apparent normal skin without systemic involvement . Its prevalence were rarely reported . Until now, there are fewer than 40 published cases worldwide . Etiological factors associated with pca is still unknown, but its striking familial occurence suggests the role of genetic . We report a case with peculiar mottled pigmentation originally referred for vitiligo, but later proved as acd by histopathological examination . The disorder was thought to be familial as his siblings were affected with the similar condition . A 12-year - old boy presented with asymptomatic, generalized mottled hypo- and hyper - pigmented lesions of 6-year duration . The hypopigmented macules were first noticed on his lower extremities and had been slowly progressing to involve almost the entire body (figure 1a). He did not have any history of systemic or cutaneous disease before the onset of the lesions . History of trauma, rubbing of the skin with any material, and extensive sun exposure was denied . His 11-year - old sister and 6-year - old brother experienced similar yet milder symptoms . Physical examination revealed an extensive, discrete, pigmented macules distributed nearly all over the body in symmetrical pattern . He was referred to our hospital for skin biopsy with the initial differential diagnoses of vitiligo and pityriasis alba . Histopathological examination (figure 2) showed uneven distribution of melanin in the epidermis and deposits of pale pink amorphous material in the papillary dermis . Based on clinical presentation and the histopathology examination, we made the diagnosis of primary cutaneous amyloidosis, presented as amyloid cutis dyschromica . The patient was treated with oral acitretin 25 mg per day . By the third month, some improvement was observed as the pigmented macules were slightly lightened (figure 1b). The patient could tolerate the treatment, as there was no significant increase of transaminases and the lipid profile . The diagnosis of acd in our patient was not readily recognized as it mimicked, to some degree, other relatively common disorders with pigmentation feature . That, combined with its low prevalence, had eluded previous attempts at the correct diagnosis and treatment . The peculiar asymptomatic mottled pigmentation is much likely seen in poikiloderma, but as our case showed, without the corresponding signs such as telangiectasia or atrophy . Furthermore, our patient was otherwise healthy, showing no signs that might indicate systemic disorders or photosensitivity, e.g. Dermatomyositis, lupus erythematosus, or xeroderma pigmentosum . As defined by morishima, acd characterized by diffuse speckled hyperpigmentation with hypopigmented spots without papulation, atrophy, and telagiectasia, mild or no itching, onset before puberty, and focal amyloid deposition in the papillary dermis . Duh begins in infancy or childhood, and unlike acropigmentation of dohi, might encompass the whole skin surface with exception of face . For definite diagnosis, histopathologic examination should be shought . The most common epidermal findings of pca were hyperkeratosis, irregular acanthosis with thinning of rete ridges, and expansion of dermal papillae by amyloid deposition . The finding of amyloid bodies in the papillary dermis was crucial in establishing the diagnosis of acd in our patient and disproved duh, in which such deposition was absent . Its visualization under polarized light, showing apple - green birefrigence, confirmed the presence of amyloid . Special histochemical stains were helpful for confirming the existence of amyloid . In our case, we tried to obtain histopathological examination of the siblings, but the parents denied the request to perform biopsy on the grounds that their clinical appearances were quite similar that the histopathological findings would likely be the same . Multiple factors such as race, genetic, and enviromental may play collective roles, making variable degrees of cutaneous amyloidosis . Although most cases of acd were sporadic, many have also reported positive family history of pca, suggesting that the important role of genetic factors in its pathogenesis . Our patient s siblings experienced similar yet milder symptoms that the disorder was thought to be familial ., familial relationship was found in 5 of 10 patients, and consanguinity was denied in all . Amyloidosis cutis dyschromica is assumed to be a congenital disorder and exposure to sunlight is thought to be the major causal factor . The lesions in our patient were more pronounced on exposed parts of body, that we thought sun exposure might be an important cause . Acitretin was given and seems to be effective because it may act by minimizing keratinization defect that causes keratin degeneration to amyloid as proposed by some to be the pathogenesis of this disorder . Qiao et al . In his series has convincingly shown 100% positivity to immunohistochemical staining for keratin, ck34 e12 and ck5/6, that the amyloid is thought to be of epidermal origin . That points at a disturbance of keratinocyte repairs after irradiation with ultraviolet . At the third month his serum transaminase and lipid profile were only slightly increased, that we think the drug was safe to be continued . However, it is interesting to note that some authors consider certain populations of asia, e.g. Chinese, japanese and thai, particularly susceptible . Therefore, it is possible that several cases of this elusive disorders exist, but are often misdiagnosed . On the other hand, the treatment of acd still remains a challenge.
|
Eccentric exercise was shown to provoke skeletal muscle damage leading to delayed onset of muscle soreness (doms)1,2,3 . Acute muscle damage can cause discomfort at the site of injury, inflammation, oxidative stress, edema, and loss of muscular function and strength4, 5 . Many researchers have attempted to ameliorate symptoms by various treatments, such as nutritional interventions3, 6, pharmacological interventions7, massage8,9,10, low - intensity exercise10, ultrasound application11, or cryotherapy12 . Even though some positive effects have been reported, several studies did not find any reductions in the symptoms of muscle injuries . Beside these treatments, static or pulsed electromagnetic field therapy have been used for the treatment of doms13,14,15 . Again, some authors reported positive effects on doms symptoms, whereas others were not able to find benefits13, 14 . Application of a pulsating electrostatic field (pesf), which is a comparable but not identical technique, might be a further method capable of reducing the symptoms of muscle soreness . A pesf was shown to increase vasomotion and as a consequence blood flow16, to reduce the formation of rouleaux, and to increase metabolic activity16, 17 . Furthermore, a pesf may function as an ion pump able to move calcium ions, the concentration of which was shown to be increased after eccentric exercise with detrimental effects2, 18 . Due to these effects, it might be speculated that a pesf could prevent the development of doms and muscle strength loss after muscle damaging exercise . Therefore, the aim of the present study was to investigate the effect of pesf application on sensation of muscle soreness and muscle strength after eccentric exercise . Nine sport students (5 males, 4 females) without any medical problems and not adapted to eccentric exercise were recruited for the study . All of them were informed about the study aims and gave written informed consent for participation . Two participants (one male and one female) had to be excluded from the final analysis, one because of a pre - start muscle soreness score of already 4 and one because of a pre - start ck value of 333 u / l . Therefore, 7 participants (4 males and 3 females; weight, height, and age: 74.35.7 kg and 71.05.3 kg, 183.09.9 cm and 172.36.4 cm, and 26.52.4 years and 24.31.2 years, respectively) were included in the final analysis . The study was carried out in conformity with the ethical standards of the declaration of helsinki and was approved by institutional review board of the department of sport science, university of innsbruck . The study was designed as a single - blinded, placebo - controlled cross - over intervention study . All participants performed 2 downhill running sessions on a treadmill separated by at least 4 weeks . Before and at different time points after the sessions, muscle soreness score, creatine kinase (ck), and jump ability were assessed as described in detail below . The first running session was carried out in a slightly modified form according to kingsley et al5 . Running sessions started with a 3 min stage of horizontal running at 5 km / h (warm - up). Thereafter, the inclination was set at 17.5%, and the speed was increased every minute by 1 km / h until reaching approximately 70% of the age - predicted maximal heart rate (hr) (calculated as 220 age). Overall, the running duration was 30 minutes, not including the warm - up time . During the second test, the speed was regulated in the same way as during test one but independent of hr in order to have identical test speeds and a comparable muscular stimulus . Immediately after each run, half of the subjects were treated in a sitting position with a pesf for 45 min, and half of the subjects were exposed to a sham treatment . After at least 4 weeks, in which participants were advised not to change their physical activity habits, the subjects that were treated with a pesf during the first session received the sham treatment and those that were treated with the sham treatment during the first session received the pesf treatment . The (negative) pesf was generated with a new health 9000 device (akern srl, pontassieve, italy) and was applied through a mat with a field intensity of 9000 v, a current of <9 ma, and a pulsatile frequency of 50 hz . The electrostatic field was confined to the ion mat surface, and it has been proven to be safe17 . The general rating of muscle soreness was assessed as described by trombold et al.3 before and immediately after the runs, immediately after the 45 min treatment or sham treatment, and 48 hours after treatment . Participants subjectively rated the degree of soreness using a visual analog scale of 0 to 10, with 0 describing no soreness and 10 describing unbearable soreness . Jump ability (jump and reach test) was assessed before running, after the 45 min session, and 48 hours after the running . Ck activity was assessed before running and 48 hours after running from capillary blood samples with a reflotron sprint device (reflotron sprint, mannheim, germany; reference range for men: 24195 u / l at a measurement temperature of 37 c). Delta values () were calculated as post values (at different time points) minus pre values for both the pesf application and sham setting . Differences at baseline, changes in the course of pesf or sham treatment, and differences between conditions (values for the pesf and sham treatment) were assessed by paired student s t - tests or the wilcoxon test as appropriate . Spearman correlation analyses were used to calculate associations between ck and cmj changes and muscle soreness score . When compared with the pre values, the muscle soreness score was increased at all time points and under both conditions (p<0.05). Jump performance was reduced immediately after the sham application (p=0.038) and tended to be reduced immediately after the pesf application (p=0.054). Table 1table 1.values for jump height, creatine kinase, and muscle soreness score in the courses of the pesf and sham treatmentspreafterrunafter45 min 24 hafter48 hafterjump and reach (cm)pesf276.28.7273.07.03.33.9273.010.53.33.6sham276.521.6272.023.54.55.3272.023.24.56.2creatine kinase (u / l)pesf133.632.1171.971.338.358.7sham100.141.0271.7274.5171.6285.8muscle soreness scorepesf1.41.43.61.72.12.04.02.02.61.65.10.93.71.64.61.53.12.0sham1.01.04.01.43.01.74.11.73.12.26.71.75.72.26.42.45.43.2pesf: pulsating electrostatic field . The table represents values of the same participants receiving the pesf and sham treatments (n=7, crossover design). There was a trend toward significant differences in delta values (= after values (i.e. 45 min, 24 h, and 48 h after) minus pre value) between treatments (pesf vs. sham). Shows values for ck, muscle soreness score, and jump height in the courses of the pesf and sham treatments . Twenty - four and 48 hours after downhill running, the muscle soreness score (mss) tended to be less after pesf administration when compared with the sham treatment (mss: 3.71.6 vs. 5.72.2 after 24 h and 3.12.0 vs. 5.43.2 after 48 h, respectively; 0.05<p<0.01). Furthermore, the highest muscle soreness scores were found after the sham session, with one subject having a score of 9 and two subjects having a score of 8 after 24 hours . After 48 h, one subject had a score of 10, one subject had a score of 9, and one subject had a score of 7 . After 48 hours, one subject had a score of 7, whereas all others had lower values . The table represents values of the same participants receiving the pesf and sham treatments (n=7, crossover design). There was a trend toward significant differences in delta values (= after values (i.e. 45 min, 24 h, and 48 h after) minus pre value) between treatments (pesf vs. sham). The main finding of the present investigation was that pesf application tended to reduce muscle soreness scores without significantly affecting ck increase and jump performance decrease . To the best of our knowledge, this is the first study to use a pesf after downhill running for the prevention of muscle soreness . In the literature, different methods, including cold or infrared therapy, massage, pharmacological intervention, etc ., have been applied to reduce muscle pain after eccentric exercise7, 8, 10, 12 . The mechanisms causing beneficial effects are as diverse as the methods applied . For cold application, the main beneficial effect is thought to be the cold - related vasoconstriction, which may limit vessel permeability and thus inflammatory processes12 . The mechanism by which infrared therapies might induce potential positive effects is mainly based on the increased peripheral flow due to warmth - related vasodilatation, which could enhance the evacuation of edema, limiting inflammation and perceived pain12 . The mechanisms mediating the biological benefits of nutritional interventions are not clear but may be linked to the attenuation of oxidative stress and inflammation3 . With respect to electromagnetic field therapy, it was suggested that the treatment influences muscle immune and inflammatory responses by an unknown mechanism, may disrupt afferent input from free nerve endings of nociceptors, and may influence excitation - contraction coupling processes13, 15, 19 . In the present study additionally, the improvement of vasomotor performance might have provoked some beneficial effects16, 17 . Comparable to the effects of infrared application, the increased blood flow might have enhanced the removal of edema, limited the inflammatory response, and reduced perception of pain12 . A further mechanism might be the function of a pesf as an ion pump moving positively charged ions, e.g., calcium and hydrogen16 . The calcium concentration was shown to be elevated in skeletal muscle cells following eccentric contractions18 . Increased calcium concentrations within the sarcoplasm have the potential to activate many different molecular pathways in skeletal muscle, including the phospholipase - prostaglandin pathway and the calpain proteolytic pathway, which might lead to breakdown of the cell membrane and myofibrils2 . However, whether or not such mechanisms might be involved in processes of recovery from exercise - induced muscle damage still needs to be determined . It has to be mentioned that although there was a trend toward a decrease in muscle soreness score, ck and jump performance did not seem to be affected by pesf application . In this regard, the small sample size certainly is a main limitation of the present study and outlines its nature as a pilot study . Nonetheless, when looking at the mss and ck values in table 1, some beneficial effects clearly seem to be apparent . This is supported by the finding that the highest mss values were found after the sham treatment . Additionally, pesf application for only 45 min may be considered a short exposure time, and it is not known if a longer pesf application time would have been more effective . In conclusion, application of a pesf might be a promising treatment to reduce muscle soreness after exercise - induced muscle damage . However, further studies are needed to confirm the present outcomes and to establish the mechanism by which a pesf may reduce muscle pain.
|
A 74 year old woman visited our clinic, who had been suffering from pain in the low back and right lower limbs and right calf hypertrophy . Six years ago, the patient had experienced low back pain and right 5 lumbar (l5) radiating pain . At that time, the patient had been diagnosed as spinal stenosis between the 4 lumbar (l4) and 5 lumbar (l5) intervertebral space by lumbar spine magnetic resonance imaging (mri) and a hemi - laminectomy with interspinal device insertion had been done at another hospital . 5 years before the present visit, low back pain and right l5 and s1 radiating pain were developed . And she described her pain as a continuous dull pain in the low back area and along the right l5 and s1 dermatomes . This pain worsened while walking and was alleviated with rest . At the time of presentation, the patient's pain level was 70/100 mm on visual analogue scale (vas) from 0/100 mm (no pain) to 100/100 mm (worst pain imaginable). A physical examination showed 50% sensory loss along the right l5 and s1 dermatomes, using pin pricking and light touching . The 6-scale muscle strength test that goes from grade 0 (no evidence of muscle contractility) to grade 5 (normal: complete range of motion against gravity with full resistance) revealed decreasing power of ankle plantar flexion at grade 4 (good: complete range of motion against gravity with some resistance) out of 5 on the right side . In deep tendon reflex (dtr), the knee jerk was normal (2+/2 +), however, ankle jerk on the right site was decreased (1+/2 +). The straight leg raising test showed a positive result at 45 in the right lower limb . The pulsation of the dorsalis pedis artery was normal . When we measured calf girth, the left side was 37 cm, while the right side was 42 cm . Lumbar mri, lower extremity mri, electromyography (emg) and a nerve conduction velocity (ncv) test and laboratory test was done for diagnosis . In a recent lumbar mri, central and lateral canal narrowing was revealed central and lateral canal narrowing at the l4-l5 intervertebral space (fig . 1). A lower extremity mri that was done to confirm the reason for the right lower limb hypertrophy revealed a complete fatty change accompanying the right medial head of the gastrocnemius, and hypertrophy of the soleus (fig . 2). Also, following electrodiagnostic examinations including electromyography (emg) and nerve conduction velocity (ncv) testing, the chronic state of right l5 and s1 polyradiculopathy was evident . The levels of creatinine kinase (ck), ck - mb and alkaline phosphatase (alp) were normal respectively . We performed right an l5, s1 selective transforaminal epidural block using, in each instance, 5 mg of triamcinolone and 2.5 ml of 0.5% mepivacaine for the low back pain and the right l5, s1 radiating pain . Pregabalin, a non - steroidal anti - inflammatory drug, and muscle relaxant were prescribed for 2 weeks . The low back pain and right lower limb radiating pain the low back pain and right leg pain were increased, as judged by a vas score of 50/100 mm . Two weeks later, her pain was decreased to a vas score of 30/100 mm . We are still constantly observing the diameter of the right calf, which has not changed in the past 6 months . It is mostly caused by nerve root compression . Among the body regions deformed by hypertrophy, calf hypertrophy can be caused by l5, s1 nerve root compression, as in the present case . One posits that the hypertrophy is produced as a secondary result of chronic muscle stimulation . In a case that features chronic muscle stimulation, emg will reveal constant and abnormal complex repetitive discharges, such as myokymia, which is caused by the involuntary trembling of muscle fibers, and neuromyotonia, which involves peripheral nerve muscle fiber activity as a result of abnormal nerve stimulation . The second theory posits that hypertrophy of muscle fiber results from excessive exercise loading in the innervated muscle fiber . In the present case, the muscle weakness or decreased dtr the patient had mild muscle weakness consistent with a rank of grade 4 in ankle plantar flexion . The gastrocnemius and soleus are the muscles related with ankle plantar flexion, in which part of the innervation is from the tibial nerve from the s1 - 2 nerve root . We also discovered a sensory decrease from the dermatome, which is managed by l5 and s1 . According to the deep tendon reflex, there was decreased right ankle jerk with a 1 + lesion . To identify the result clearly, we performed lumbar spine mri which showed lateral canal narrowing central and lateral canal narrowing at the l4-l5 intervertebral space . The congruity of the imaging findings and the physical examination results indicate a high reliability of the final result . Myopathic muscle hypertrophy is evident with a diffuse lesion, which is centralized on the muscle, not a nerve innervated lesion . Conversely, neurogenic muscle hypertrophy is mostly an isolated muscle innervated by an abnormal nerve . To confirm the lesion on the right calf muscle hypertrophy characterized by fatty change and pseudohypertrophy was focused on the gastrocnemius and soleus . Myopathic muscle hypertrophy increases muscle enzymes (ck, ck - mb, alp), but in neurogenic hypertrophy the increase is mild, or the levels remains normal . In this case, there was a normal level from muscle enzyme test . According to the history of the patient, there was no particular muscular and metabolic disease, or excessive exercise history . According to the emg and nerve conduction velocity results, we could not confirm any decreased duration of motor unit potential, which is the most sensitive and specific parameter in myopathic muscle hypertrophy . In this case, right l5 and s1 polyradiculopathy was noted . The collective observations supported the diagnosis as neurogenic muscle hypertrophy . Among the various causes of neurogenic muscle hypertrophy, such as anterior horn cell disease, plexopathy, sciatic nerve injury, and polyneuropathy, we can confidently conclude that l5, s1 nerve roots compression caused the sustained and exacerbated symptoms in this patient . Neurogenic muscle hypertrophy shows increased size and number of muscle fibers, internal nucleation, fiber splitting and disruption of intermyofibrillar architecture in muscle biopsies on the affected muscles . Treatment can involve surgery or can be nonsurgical . In nonsurgical treatment, an injection of botulinum toxin and steroid medication are effective . In surgical treatment, gibson and waddell reported that microdiscectomy is the effective way to reduce radiculopathy and muscle hypertrophy . In the present case, we performed a right l5, s1 selective transforaminal epidural block to relieve right l5, s1 radiating pain . After that, radiating pain was relieved and patient was informed about the disease and further management . We found that the selective transforaminal epidural block decreased pain, but was not enough to improve the calf muscle hypertrophy . In conclusion, muscular hypertrophy has a myopathic cause and a neurogenic cause . To discriminate between these, a proper neurologic examination and laboratory tests such as muscle enzyme test, emg, mri, and muscle biopsy are needed . Neurogenic muscle hypertrophy is very rare, but we recommend clinicians consider this problem when patients complain of lower limb hypertrophy and pain.
|
Inactivation of the mismatch repair (mmr) genes mlh1, msh2, msh6 and pms2 causes lynch syndrome (ls), an autosomal dominant predisposition for colorectal and endometrial cancer . Inactivation of the mismatch repair pathway can also occur sporadically, through somatic mlh1 hypermethylation or by acquired biallelic somatic inactivation (variant affecting function or loss of heterozygosity (loh)) of the mmr genes . Inaccurate dna repair leads to a high frequency of somatic variants, with loss of mmr leading to hypermutated' tumors with 10100 variants / mb . Ls tumors are characterized by microsatellite instability (msi) and immunohistochemical loss of expression of mmr proteins . However, germline variants affecting function cannot be detected in up to 59% of patients displaying msi and/or loss of mmr, referred to as suspected ls' (sls). Recently, germline and somatic variants in the exonuclease domains (edms) of dna polymerase (pole) and polymerase d (pold1) were described . These pole / pold1 variants affect proofreading function and lead to an ultramutated phenotype with a variant incidence exceeding 100 variants / mb . Germline pole - edm variants can result in a ls phenotype and microsatellite instable colorectal cancers (crcs). The exact role of somatic pole / pold1 variants in tumors with high microsatellite instability (msi - h) remains unclear . The aim of our study was to identify the underlying genetic cause of disease in a cohort of 64 sls cases selected on the basis of msi, loss of mmr, young onset and often a family history for ls by screening the mmr, pole and pold1 genes in both leukocyte and tumor dna . This study included 64 patients with lynch - associated tumors recruited in four academic centers in the netherlands between 1997 and 2014: leiden university medical centre (n=37), maastricht university medical centre (n=11), erasmus medical centre (n=9) and university medical centre utrecht (n=7). Demographic and clinical data, as well as informed consent, were obtained at the time of diagnosis . This study was approved by the local medical ethical committee of the lumc (p01 - 019e). Patients were selected based on loss of mmr (as indicated by immunohistochemical staining) and/or msi . Unexplained tumors with low msi or tumors with inconclusive ihc results were also included in this study (see supplementary table 1 and supplementary methods). Fifty - eight (91%) patients fulfilled bethesda criteria, and families of 24 (38%) patients also fulfilled amsterdam ii criteria . Patients were previously screened in a diagnostic setting for germline mmr variants . Whereas 57 patients showed no disease - causing germline variants, 7 patients were found to have a germline variant of unknown significance (vus). Of the total cohort, 75% of patients presented with crc (n=48), 14% with endometrial cancer (ec, n=9) and 11% with another ls - associated tumor two patients were excluded from the analysis because of poor dna quality . Of the remaining 62 tumors, tumor and leukocyte dna was sequenced for variants in the exonic regions of mlh1, msh2, msh6, pms2, pole and pold1 using the ion pgm system (life technologies, carlsbad, ca, usa). Raw data analysis, alignments and variant calling was carried out using the default parameters in torrent suite v4.0 (thermo fisher scientific, waltham, ma, usa; see supplementary methods). The full data set was filtered and prioritized by variant frequency (> 10%) and coverage (> 50). The in silico prediction programs were used to predict effect on function (see supplementary methods). All variants (likely) affecting function, including two variants with a 9% variant frequency, were validated with sanger sequencing . For all pms2 variants, pms2-specific primers were created to confirm that the variant is present in pms2 and not in a pms2 pseudogene . Loh was determined for every heterozygous snp by comparing the ratio of allele a with allele b in leukocyte and tumor dna samples . Furthermore, for every heterozygous snp, the allelic imbalance factor (aif) was calculated and fisher's exact test was performed to determine whether the difference between normal and tumor is significant . If all heterozygous snps of one gene showed loh with an aif> 2 and fisher's exact p - value <0.05, loh was called (supplementary table 1). Seven patients with a germline mmr vus (class 3) were included in this study (see supplementary table 1). In all cases, the variant was detected with ngs in leukocyte and tumor dna . During the course of the study, three of these germline variants were reclassified as class 4 or 5 ((probably) affects function) by the international society for gastrointestinal hereditary tumors incorporated (insight). Six cases with somatic mlh1 hypermethylation fulfilling revised bethesda criteria (three from families fulfilling amsterdam ii criteria) were sequenced for underlying hereditary defects explaining the family history, but no germline variants were found . One of these mlh1 hypermethylated tumors carried a somatic mlh1 variant likely to affect function and one displayed mlh1 loh (supplementary table 1). One (n=27, 44%) or two (n=13, 21%) somatic aberrations (variant or loh) in a mmr gene were found in a total of 62 tumors (see supplementary table 1). Of the 13 tumors with two somatic aberrations, 12 had variants in mlh1 or msh2 and were msi - h . Although the majority (83%) of tumors showed <10 somatic variants in the genomic region analyzed, 10 cases displayed a larger number of somatic variants, ranging from 16 to 375 somatic variants within the sequenced area of 31 kb . Out of 10 tumors, 9 showed a pole or pold1 variant that (probably) affects function (table 1). Of the highly mutated tumors, two carried novel germline heterozygous pole / pold1 variants that are predicted to affect proofreading (supplementary table 2). Of these two germline cases, tumor sls-67 was also found to carry two somatic mlh1 variants, explaining the tumor phenotype (loss of mlh1 and pms2 expression and msi - h). The second tumor, sls-16, was msi - l, showed positive mlh1 and msh2 staining (msh6 and pms2 were not tested) and had no somatic mmr variants . Seven of the highly mutated tumors showed somatic pole / pold1 variants likely to affect function . Six tumors carried a somatic pole / pold1-edm hot spot variant (pole: c.857c> g, c.856c> t, c.1231g> t, c.1366g> c, c.1367c> t or c.1376c> t and pold1 c.1433g> a) that has previously been described to impair proofreading . In the seventh tumor (sls-105), a novel pole c.846_847delinstt variant was detected . This variant lies close to a known pole hot spot site (pole c.857) and is predicted to affect function by two out of three prediction programs (supplementary table 2). Four pole / pold1-edm mutated tumors displayed additional somatic nonsense pole variants outside the exonuclease domain (see supplementary table 3). Eight of the nine ultramutated tumors with a pole / pold1-edm variant found in our study showed msi (3 msi - h and 5 msi - l). In six of these ultramutated tumors, ihc detected loss of at least one of the mmr proteins and all six tumors displayed somatic variants in the affected mmr gene likely to affect function . Tumor sls-19 with two pole variants was found to have two somatic mlh1 aberrations, as well as two somatic pms2 aberrations, and ihc showed solitary loss of pms2 expression . Reanalysis of staining also showed ambiguous mlh1 staining (cytoplasmic enhancement and vague, focal nuclear staining). Tumor sls-09 displayed a nonsense msh6 variant and missense msh2 variant predicted to affect function, and ihc showed loss of msh6 expression and weak positive msh2 expression . In three pole / pold1-edm mutated tumors with positive or inconclusive mmr expression and msi - l phenotype, no somatic mmr variants (likely) to affect function were found . However, in one of those three tumors (sls-05), solitary mlh1 loh and pms2 loh without variants was found (see table 1). In three non - ultramutated tumors, a pole / pold1-edm variant was found (sls-80, sls-87 and sls-101, see table 1). These variants have not been described before, but are predicted to affect function (see supplementary table 2). Two variants co - occur with a germline msh6 vus and a somatic msh6 variant (sls-80 and sls-87), whereas one (sls-101) co - occurs with a somatic pms2 variant . Four additional non - ultramutated tumors showed pole (sls-18, sls-21) or pold1 loh (sls-12, sls-49) in all heterozygous snps (supplementary table 1), without germline or somatic pole variants . Pole - edm variants are reported to be the mutagenic factor driving ultramutation in tumors . The same report also noted that the increased mutational load seen in pole mutated tumors exceeds that expected because of loss of exonuclease activity . The number of variants detected in the sequenced area in the present study implicates an ultramutated phenotype, with> 100 variants / mb in all pole / pold1-edm mutated tumors in this cohort . As only a limited region (31 kb) was sequenced, we can only extrapolate the total number of variants per mb . In our cohort, the pole / pold1 mutated mmr - deficient tumors display two deficient pathways increasing the mutational load . Comparing frequencies of the different variants found in these tumors, it might be concluded that faulty proofreading may be the initiating event in some of these tumors, possibly resulting in loss of mmr and thereby in msi . Interestingly, four tumors show pole / pold1 loh without germline or somatic pole / pold1 variants . These tumors however do not show the typical ultramutated phenotype, whereas single variants without loh do show that . This phenomenon of loh without variants affecting the exonuclease domain has not yet been described . Furthermore, three tumors show somatic pole / pold1 variants, without the ultramutated phenotype . All three variants are missense, but are predicted to affect function (supplementary table 2). As these variants are not found in ultramutated tumors, evidence of deleterious functional effect is lacking . In conclusion, targeted next - generation sequencing of 62 sls cases led to the detection of 9 highly mutated tumors with a germline (n=2) or somatic (n=7) pole / pold1-edm variant . Even though pole germline variants have previously been shown to co - occur with somatic mmr variants, in this study we found germline and somatic pole / pold1 variants in a cohort selected for sls characteristics . Importantly, although current literature mainly addresses pole / pold1 variants in mss tumors, somatic pole / pold1 variants in sls patients are likely to be overlooked . However, a very recent recommendation for genetic testing and surveillance states that mmr deficiency should not be an exclusion criterion for genetic testing of germline pole / pold1 variants . Our results further emphasize the importance of pole / pold1 germline and somatic screening in unexplained msi - h and mmr - deficient tumors.
|
Heat stroke is defined clinically as a core body temperature above 40c and accompanied by central nervous system (cns) abnormalities such as delirium, convulsions, or coma . Heat stroke, the extreme form of hri is associated with significant mortality and morbidity, mortality in these patients range between 10% to 50% . Heat stroke is often associated with multiple organ system involvement and survivors may sustain permanent neurologic damage . Heat stroke may be divided into exertional and non - exertional (classical) heat stroke . While exertional heat stroke is associated with exercise, classic heat stroke is typically seen in debilitated patients during high ambient temperature and humidity . Risk factors for classical heat stroke include - old age, alcoholics, antihypertensive or psychiatric medications, neurological disease and dehydrating illness . The year 2012 recorded the highest temperatures in last 10 years in chennai [figure 1]. Due to the unusually high temperatures recorded in the summer of 2012, there was spurt in such patients admitted to our intensive care unit (icu). Arrow indicates admission time frame of patients to intensive care unit with heat - related illness the aim of the study was to identify the profile of patients admitted to our multidisciplinary icu with clinical features of hri and analyse their clinical outcomes . This was a retrospective case series, of patients admitted with features of hri during the period from april to june 2012 in our 25 bedded multidisciplinary icu . After obtaining institutional ethics committee approval, we included all patients admitted with complaints of fever with cns abnormalities such as delirium, convulsions, acute alteration of consciousness, or coma and absence of any other evident cause for the fever or acute onset of cns dysfunction . We excluded patients showing features of cns infection, stroke and other causes of encephalopathy . All the patients were managed in the icu in a similar manner as mandated by their severity of illness which included appropriate organ support, fluid resuscitation, electrolyte management and active cooling as required . Patients also received other supportive measures such as, stress ulcer, deep vein thrombosis prophylaxis, glycaemic control, nutritional support and analgesia / sedation . The patients were evaluated to rule out any underlying infections including cns infection, stroke or encephalopathy as deemed necessary . Data collected from patient's medical records, included demographics, co - existing illness, current medications, admission vitals and glasgow coma scale (gcs) score . Other laboratory parameters required to calculate acute physiology and chronic health evaluation ii (apache ii) and sequential organ failure assessment (sofa) scores were also captured . These two scores were calculated using the worst parameters recorded during the first 24h of icu admission . Details regarding cerebrospinal fluid analysis, echocardiography and neuroimaging studies obtained wherever indicated were also captured . Outcome data on mortality, icu length of stay (los), number of ventilator days, discharge gcs and sofa score were recorded . Continuous variables with normal distribution were compared using student's t - test whereas those not normally distributed were analysed using mann - whitney u - test . Categorical data were analysed using pearson chi - square test or fischer's exact test . During the study period, 30 patients presented with features of hri as described above, of whom two patients had features suggestive of cns infection and another two patients had features of acute stroke in neuroimaging studies . Excluding these patients, data from the remaining 26 patients were analysed . Demographic characteristics of patients are presented in [table 1]. 20 patients (77%) had at least one co - existing illness, of which hypertension and diabetes mellitus were the most common [table 1]. 18 (69.2%) demographics, pre - existing medical conditions and prior medications of patients with hri patients admitted to icu with hri were critically ill as indicated by high admission apache ii (19.62 7.7, mean sd) and sofa (7.5 2.64, mean sd) scores [table 2]. Organ system involvement includes cns (100%), renal (57%), liver (34%), coagulation (26%), respiratory (26%) and heart (23%). Characteristics of patients with hri in line with our inclusion criteria, all our patients were febrile (temperature 102.7 2.7 f, mean sd). More than 30% of patients presented with seizures and more than a third were comatose on admission [figure 2]. Apart from cns involvement, 30% of patients also had gastrointestinal (gi) symptoms such as nausea, vomiting (8/26) and diarrhoea (4/26). Spectrum of neurological involvement in heat - related illness seventy - three percentage (19/26) of our patients had hyponatraemia (serum sodium <135 meq / l) on admission (mean serum sodium 124.38 13.33 meq / l [sd]) and most responded to resuscitation with normal saline [figure 3]. However, hypertonic saline (3%) was used in seven patients who had persistent neurological symptoms (gcs 11.2 1.79, mean sd) and hyponatraemia (serum sodium 111 4.0 meq / l, mean sd). Meq / l) (mean serum potassium 3.5 0.9 meq / l [sd]). Creatinine phosphokinase (cpk) levels were elevated in all patients (1856 2372 iu / l, mean sd). L on admission; this patient had a previous history of heat stroke, had myoglobinuria and renal failure requiring haemodialysis . 15 (57%) patients had elevated serum creatinine; five of these patients had progressively worsening renal function requiring haemodialysis . Liver enzymes were raised in 50%(13/26) patients (aspartate aminotransferase [ast] 198.50 395.6 iu / l and alanine aminotransferase [alt] 97.5 110 iu / l, mean sd) with mildly deranged coagulation profile (international normalised ratio 1.40 0.55) with no obvious bleeding tendencies . Serum sodium levels in patients with heat stroke neuroimaging was performed in patients who were comatose or had focal neurological deficit post - resuscitation and stabilisation . Computerised tomography scan of brain was done in 11 (11/26) patients, while magnetic resonance imaging (mri) of brain was done in 12 (12/26) patients . Diffusion - weighted mri features suggestive of heat stroke [figure 4] were seen in five patients . These included diffusion restriction involving cerebellum (4/5), basal ganglia and thalamus (3/5), hippocampus (2/5), cerebral cortex and subcortical white matter (2/5). There were corresponding increased t2/t2 fluid - attenuated inversion recovery signal intensities in these regions . Features of diffuse cerebral oedema (3/5) and diffuse cerebellar oedema (4/5) were also seen in these patients . Diffusion - weighted magnetic resonance imaging showing restricted diffusion involving (a) cerebellum (b) thalamus (c) basal ganglia and (d) hippocampus sixteen patients (61.5%) required mechanical ventilation to protect airway due to low gcs . Six patients (23%) who remained in shock (mean arterial pressure <70 mmhg) despite adequate fluid resuscitation required vasopressor support . Mean icu los was 16.0 17.05 days (sd)(range 2 - 60 days). Mean ventilator days were 10.16 12.46 days (sd) [table 2]. The mean discharge gcs of our patients was 13.17 1.8, (sd). Nine out of 17 patients were discharged with normal gcs . Of the remaining eight patients, these four patients had a prolonged icu stay (28 5 days, mean sd). The remaining four patients had a discharge gcs of 14, of whom two had pre - existing, parkinsonism, one had dementia and another had previous stroke . Raised serum lactate, high admission sofa scores and prolonged ventilator days were risk factors for mortality in univariate analysis; however, multivariate analysis by logistic regression did not identify any risk factors of mortality . Hri following periods of sustained heat wave have been reported previously; however, in our study, we had cases admitted over a period of 2 months corresponding to high ambient temperature . Risk factors for hri include old age, alcohol abuse, neurological disease, psychiatric illness and patients on long - term medications . In our study, this was a strikingly younger group as compared to the patients in the studies by argaud et al . Nearly 30 - 50% of patients had significant functional limitations and in our study, most of the patients were functionally active prior to admission . Hence, our patients may have been exposed to high ambient temperatures in an open environment making them more prone to hri . Exposure to high temperatures lead to elevation of body temperature, which causes activation of various physiological compensatory mechanisms which include increased cutaneous circulation and concomitant vasoconstriction of the renal and splanchnic circulation . Heat stress causes cell damage by apoptosis, release of inflammatory mediators and endothelial damage leading to multi organ failure . . Most common sites of cns involvement of heat stroke include cerebellum, basal ganglia, thalamus, hippocampus and fronto - parietal cortex all our patients had neurological involvement of varying degrees . Mri features were due to cerebral ischaemia (uk english) resulting from auto regulatory mechanisms that divert blood flow towards periphery to dissipate excessive heat . Apart from cns symptoms, 30% of patients also presented with gi symptoms such as nausea, vomiting and diarrhoea . Studies have shown that even though thermal stress results in an increased cardiac output, splanchnic flow is reduced because of increased splanchnic vascular resistance, resulting in gut ischaemia with focal necrosis, resulting in gi symptoms . A high prevalence of elevated liver aminotransferase levels has been reported in exertional heat stroke and has been attributed to ischaemia and direct thermal injury . The primary event seems to be related to direct thermal injury of vascular endothelium, initiating platelet aggregation and activation of the clotting cascade along with increased fibrinolysis and disseminated intravascular coagulation . Even though our patients had mildly elevated liver enzymes and slightly deranged coagulation parameters, they didn't show any obvious bleeding tendencies . The french heat wave reported 40% incidence of raised alt levels in their study compared to the 50% in our study . All but 5 of the 15 patients with elevated serum creatinine improved their renal function with fluid resuscitation, this points to volume depletion as a cause for renal involvement . Three of the five patients requiring haemodialysis had myoglobinuria . Suggested mechanisms of renal injury in patients with heat stroke include direct thermal injury, renal hypoperfusion secondary to compensatory renal artery vasoconstriction and rhabdomyolysis . Haemodynamic failure requiring vasopressor support was seen in 23% of our patients compared to 43% reported by argaud et al . Shock in heat stroke is due to the inability of the cardiovascular system to increase cardiac output in the context of heat - related dehydration and vasoplegia enhanced by proinflammatory response . Cardiovascular dysfunction could be exacerbated in patients with pre - existing cardiovascular disease and patients on long - term antihypertensive medications . Reported a mortality rate of 62% in 345 patients following 2003 heat stroke in france . Compared to ours, their patients were elderly (67.2 14.1 vs. 53.11 20.59 years), were sicker as reflected by lower gcs (5.7 vs. 8.6) and higher rate of mechanical ventilation (99% vs. 64%). Various predictors of mortality have been previously described in heat stroke which includes long - term use of antihypertensive medication, anuria at admission, coma, cardiovascular failure, high simplified acute physiology score (saps ii), high body temperature and prolonged prothrombin time . Described 33% of their survivors having moderate to severe neurological impairment which was higher than the rate of 23.52% (4 of 17 with gcs <10) observed in our study . This difference may be due to the greater sickness of their patients (apache ii 28 6 vs. 19.62 7.7 mean sd) as compared to our patients . However, survivors regained normal function of other organ systems in our study . Due to the retrospective nature of our study we faced certain limitations . History of intensity of heat exposure, or exertion prior to admission and information regarding cooling methods employed were not reliable . Similarly there was a lack of clarity on the degree of neurological involvement in patients with pre - existing neurological disease . We also did not have information on long - term follow - up of patients after hospital discharge . Patients admitted to icus with heat related illness had multiple organ system dysfunction and were associated with significant neurological morbidity, and mortality.
|
There are several observations indicating that podocyte injury is at the center of the development of fsgs . First, podocyte injury is the earliest morphologic feature of fsgs . In recurrent fsgs in the allograft kidney, podocyte injury is detected by electron microscopy prior to the development of overt sclerosis [14 - 16]. Injection of immunotoxin which binds to human cd25 induced podocyte - specific injury and fsgs occurred a few weeks later . Third, histologic appearance of fsgs and clinical symptoms were in proportion with the number of injured podocytes . Therefore, the pathogenesis of podocyte injury is a key to understand the characteristics of fsgs . As for the pathophysiology of podocyte injury, several mechanisms have been proposed with supporting evidences . Circulating permeability factors have been reckoned as the initiating factor of podocyte injury in primary fsgs and its recurrence after transplantation . The presence of serum factors that can cause podocyte injury was suggested from the therapeutic effect of immunoadsorption therapy and observations that plasmapheresis could decrease the glomerular injury induced by patients serum . Further, serum of recurrent fsgs patients significantly increased albumin permeability of glomeruli in an in vitro test . Among the proposed circulating permeability factors, soluble urokinase receptor (supar) wei et al . Presented data in a mouse model suggesting that the urokinase receptor of podocytes contributed to podocyte loss and proteinuria . Serum levels of supar were increased in about two - thirds of primary fsgs patients and were also associated with recurrent fsgs after transplantation . They also demonstrated that supar was increased in two different cohorts of biopsyproven fsgs patients . However, supar was inversely correlated with estimated glomerular filtration rate (egfr) and treatment response . Other authors found that plasma supar level was significantly increased in fsgs patients versus patients with minimal change disease, membranous nephropathy, or normal control . However, supar level was not useful in distinguishing primary and secondary fsgs . Several contradicting reports on supar importantly, egfr affects plasma levels of supar in patients with non - fsgs glomerular lesions, and a supar cut off value could not be determined even in fsgs patients due to the effect of egfr . Plasma levels of supar were also increased in lupus nephritis patients compared to lupus patients without renal involvement . In iga nephropathy patients, the plasma level of supar was related to the development of secondary segmental sclerosis . Therefore, whether supar plays a role in the development of focal segmental lesions and its specificity to the primary fsgs are still open to further investigation . Cardiotrophin - like cytokine-1 (clc-1 or cardiotrophin - like cytokine factor 1 [clcf-1]) is another candidate circulating permeability factor for primary fsgs . Savin et al . Have published on a serum factor purified from fsgs patients, which increased albumin permeability in isolated rat glomeruli . This factor had affinity for galactose and its molecular weight was less than 30 kda . They identified this factor as clc-1 by proteomic analysis and also found that the activity of clc-1 was decreased by several factors such as heterodimer formation with cosecreted cytokine receptor - like factor 1 (crlf1), janus kinase 2 (jak2) inhibitor, and signal transducer and activator of transcription 3 (stat3) inhibitor . A phase ii clinical trial on therapeutic effect of galactose in patients with steroid - resistant fsgs was performed with inconclusive results due to small sample size . This study design is interesting, considering that clc-1 has high affinity for galactose and the clc-1galacotse complex can be easily removed in the liver . Though known to be related to minimal change disease rather than fsgs, angiopoietin - like-4 (angptl4) is also of interest . While proteinuria was induced by hyposialylated angptl4 located within the glomerulus, normosialylated angptl4 was present in the peripheral circulation and mediated hypertriglyceridemia, indicating that these two symptoms of nephrotic syndrome could be linked through a common circulating factor . Fsgs, as a podocytopathy, may be caused by mutation in several genes, which are important in maintaining podocyte morphology and function . Most of these genes can be categorized as those which are related with slit diaphragm structure, actin cytoskeleton of podocytes, or podocyte - glomerular basement membrane interaction through foot processes [37 - 39]. In addition, a specific channel mutation (see below) has also been identified as a cause of fsgs (table 1). Alteration of these genes results in autosomal dominant or recessive congenital, infantile, or late onset nephrotic syndrome, some of which presents as fsgs histologically . Mutation in the nphs1 gene and resulting loss of its product nephrin, are responsible for congenital nephrotic syndrome of finnish type . The locus of nphs1 was identified at 19q13.1 in 1998, which was the first identification of a podocytopathy - related gene . After this discovery, nphs2, plce1 (phospholipase c1, nphs3), wt1 (wilms tumor 1, nphs4), lamb2 (laminin 2, nphs5), ptpro (protein tyrosine phosphatase receptor type o, nphs6), arhgdia (rho gdp dissociation inhibitor, nphs8), adck4 (aarf domain containing kinase 4, nphs9), and emp2 (epithelial membrane protein 2, nphs10) were identified and related to autosomal recessive nephrotic syndrome . Many other genes related to nephrotic syndrome have been identified including actn4 (actinin 4, fsgs1), trpc6 (transient receptor potential cation channel 6, fsgs2), cd2ap (cd2-associated protein, fsgs3), apol1 (apolipoprotein l1, fsgs4), inf2 (inverted formin, fsgs5), myo1e (myosin 1e, fsgs6), pax2 (paired box gene 2, fsgs7), anln (anillin, fsgs8), and crb2 (crumbs homolog 2, fsgs9). For example, wt1 transcriptionally regulates nephrin encoding of nphs1, therefore, wt1 mutations influence nphs1 function . A study in a european cohort reported that two thirds of nephrotic syndrome within 1 year of life are related to alteration of nphs1, nphs2, wt1, or lamb2 . Another study in a non - finnish ethnic group also reported that nphs1 and nphs2 mutations were the most common genetic alterations in congenital nephrotic syndrome . In contrast to these western studies, a genetic analysis of 30 korean congenital and infantile nephrotic syndrome patients revealed that wt1 and nphs1 mutations were the most frequent alterations, while nphs2 mutations were the lowest frequency genetic alteration . Podocytes are terminally differentiated cells having very limited ability of regeneration or proliferation . Therefore, the mechanism of repopulation of podocytes after podocyte injury has been of great interest . Recently, it has been suggested that parietal epithelial cells (pecs) lining bowman s capsule play an important role in this process by migrating from their original site to replace injured podocytes . During glomerulogenesis, pecs and podocytes originate from common mesenchymal progenitors and finally have different phenotypes . Although little is known about the function of terminally differentiated pecs, they express tight junction molecules such as claudin-1, zonula occludens-1, and occludin and have barrier function against protein . Some pecs express both cd133 and cd24, which are known to be stem cell markers, and these cells have regenerative ability . Pecs located at the urinary pole express cd133 and cd24 without the expression of podocyte markers (nestin, complement receptor-1, and podocalyxin). Pecs of the vascular pole express podocyte markers without the expression of cd133 or cd24 . In other areas, pecs express both cd133/cd24 and podocyte markers . Cd133 and cd24-expressing pecs have the ability to ameliorate kidney injury by potentiating tubular regeneration and podocyte replacement, however animal models and human posttransplant biopsies demonstrated that invasion of activated pecs through the adhesion sites of the capillary tuft contributed to the development of fsgs . The adhesion of the glomerular tuft to the bowman s capsule as a bridge of pec migration appears to occur at early stages of fsgs development . Therefore, detecting activated pecs on bowman s capsule or on the glomerular tuft could be an adjunctive diagnostic tool for early fsgs . In support of this concept, cd44 as a marker of activated pecs successfully distinguished early primary fsgs and early post - transplant recurrence of fsgs from minimal change disease . Interestingly, mutation of arhgdia, which is responsible for nephrotic syndrome, increased migration activity of cultured podocytes . Current research is focusing on the role of podocytes and interaction with pecs . Understanding the mechanism of podocyte injury, its progression and possible recovery is important not only for basic research but also for daily diagnostic pathology practice.
|
The spinal cord injury (sci) often leads to serious neurological sequelae and various complications such as neurogenic bladder, lower extremities muscle atrophy, etc . Electrostimulation on sacral nerve roots is currently being tried with some promising results as a treatment for a wide spectrum of voiding dysfunctions . Besides, several investigators reported additional effects of the sacral nerve stimulation such as change of spasticity and spasm, increased motility of intestinal tract and defecation, and improvement of locomotor function in experimental and clinical studies3,6,11,12,14). Furthermore, there is a case report about additional improvement of lower extremity motor6). They reported that one patient with a long history of progressive spinal multiple sclerosis was able to stand and transfer, but not walk, before the sacral nerve stimulation6).another paraplegic patient was showed improvement of motor power slowly, and after 2 years the patient could stand as confident as before6). The purpose of this study is (1) to establish a rat thoracic spinal cord stimulation model and (2) to compare effects of thoracic cord neuromodulation to that of sacral nerve neuromodulation on locomotor function, bladder and lower extremities muscles . Twenty sprague dawley rats of 6 months old female, weighing 200 to 250 gm, were randomly divided into 4 groups: a normal control group (n=5), sci with sham stimulation group (sci, n=5), and sci with electrical stimulation at thoracic spinal cord (sci+tes, n=5), and sci with electrical stimulation at sacral nerve (sci + ses, n=5). The sci group included rats that were injured with electrodes implanted but did not receive stimulation . The sci+tes and sci+ses group included rats that received epidural electrical stimulation at the thoracic spinal cord or s2/s3 nerve root using needle electrode, respectively . Rats in the each group were kept in separate cages under the same living conditions for a week before receiving sci . The animals were anesthetized with an intramuscular injection of zoletil 15 mg and xylazine 3 mg . Under general anesthesia, rats were placed in a prone position . After routine disinfection, a surgical incision was made through the skin, subcutaneous tissue, and the t8 - 12 vertebral lamina to the spinal canal . The scis were induced in rats using an nyu - mascis (new york university - multicenter animal spinal cord injury study) impactor . The impact height of 25 mm was considered for severe cord injury based on a prior investigation1). For sacral neuromodulation, a surgical incision at the s1 - 3 level was made through the skin, subcutaneous tissue, and the s2 - 3 vertebral lamina (fig . Needle electrodes (0.527 g) were implanted at bilateral s2 or s3 neural foramina . Wounds were cleaned and sutured with wires inserted through the subcutaneous tissue extruding from the neck . For thoracic spinal cord stimulation, we put the needle electrode on the thoracic epidural space through the laminectomy site (fig . The sci+tes and sci+ses groups received electrical stimulation 7 days after sci with the following protocol: stimulation time 30 minutes; pulse duration, 0.1 ms; and frequency, 20hz . Epidural low frequency electric stimulation was applied 30 minutes per day for 4 weeks . Post - injury motor behavior is assessed by the basso, beattie, and bresnahan (bbb) locomotor scale method4,5). The scale (0 - 21) represents sequential recovery stages and categorizes combinations of rat joint movement, hindlimb movements, stepping, forelimb and hindlimb coordination, trunk position and stability, paw placement, and tail position . To evaluate the bladder contracting function, urodynamic study using cystometrogram was performed with a serial fashion . All rats were sacrificed at 4 weeks after sci . Under general anesthesia, the gastrocnemius muscle was harvested, weighed, and stained with hematoxylin and eosin (h&e) to identify the muscle fiber cross - sectional area (csa). The animals were anesthetized with an intramuscular injection of zoletil 15 mg and xylazine 3 mg . Under general anesthesia after routine disinfection, a surgical incision was made through the skin, subcutaneous tissue, and the t8 - 12 vertebral lamina to the spinal canal . The scis were induced in rats using an nyu - mascis (new york university - multicenter animal spinal cord injury study) impactor . The impact height of 25 mm was considered for severe cord injury based on a prior investigation1). For sacral neuromodulation, a surgical incision at the s1 - 3 level was made through the skin, subcutaneous tissue, and the s2 - 3 vertebral lamina (fig . Needle electrodes (0.527 g) were implanted at bilateral s2 or s3 neural foramina . Wounds were cleaned and sutured with wires inserted through the subcutaneous tissue extruding from the neck . For thoracic spinal cord stimulation, we put the needle electrode on the thoracic epidural space through the laminectomy site (fig . The sci+tes and sci+ses groups received electrical stimulation 7 days after sci with the following protocol: stimulation time 30 minutes; pulse duration, 0.1 ms; and frequency, 20hz . Epidural low frequency electric stimulation was applied 30 minutes per day for 4 weeks . Post - injury motor behavior is assessed by the basso, beattie, and bresnahan (bbb) locomotor scale method4,5). The scale (0 - 21) represents sequential recovery stages and categorizes combinations of rat joint movement, hindlimb movements, stepping, forelimb and hindlimb coordination, trunk position and stability, paw placement, and tail position . To evaluate the bladder contracting function, the gastrocnemius muscle was harvested, weighed, and stained with hematoxylin and eosin (h&e) to identify the muscle fiber cross - sectional area (csa). Three of the five suddenly died after the first stimulation on the thoracic spinal cord . All rats but the sci+tes group survived through the entire 4 weeks . Before the injury, all rats showed normal function on the bbb score . All injured rats exhibited a severe bbb score at 1 day after sci . At day 1 of sci, the bbb scores of the sci and the sci+ses group were 2.80.7 and 2.70.8, respectively . The difference between two groups was not significant . During subsequent scoring periods, bbb scores increased and plateaued around 14 days after sci . The locomotor function improved significantly at days 3, 7, and 14 after sci (3 days: 7.31.2 and 7.31.5, 7 days: 13.10.9 and 13.21.1, and 14 days: 14.60.8 and 14.70.6). The all survived rats improved over time but there was no significant difference at any point in time between sci and sci + ses groups . All rats experienced urinary retention after the injury and recovered self - voiding after 3 - 9 days . Voiding contraction interval was 25.57.5 minutes in the sci group, 16.55.3 minutes in the sci+ses group, and 12.54.2 minutes in the control (fig . The recovery of voiding contraction interval was significant in the sci+ses group comparing to the sci group (p<0.05). The gastrocnemious muscle weight decreased after the sci, whereas the weight increased by 7.9% after sacral neuromodulation . The csa of the muscle was slightly greater in the sci+ses than in the sci group, however, the difference was not significant . Cross - sectional shape of the muscle fiber was markedly turned into round by sci . After sacral neuromodulation, the shape was turned into angled again (fig . 3 and 4). This study was designed to establish a rat thoracic spinal cord stimulation model and to compare effects of thoracic cord neuromodulation to that of sacral nerve neuromodulation on locomotor function, bladder and lower extremities muscles . However, we failed to accomplish the comparative analysis, because all five rats of the sci+tes group expired within 3 days after the injury . Three of the five suddenly died after the first stimulation on the thoracic spinal cord . We suggest that the cause of death is associated with epidural spinal cord stimulation in the thoracic spine which is located near the heart . Nevertheless, the present study showed that sacral neuromodulation has a therapeutic potential to improve neurogenic bladder and muscle atrophy . The urodynamic study using cystometrogram revealed that the recovery of voiding contraction interval was significant in the sci+ses group comparing to the sci group . Furthermore, lower extremities muscle such as the gastrocnemious muscle was relatively better preserved after sacral neuromodulation . Electrical stimulation has been reported to cause neurobiological effects, such as relieving pain in the area of head and face and restoring movement and function in individuals with spinal cord injury6,8). It has also been indicated that electrical stimulation can directly influence on regenerating neural tissues observed under a well - controlled experimental environment8). Several investigators have reported that a low - frequency electrical stimulation is a promising approach to accelerate nerve regeneration after injury2,10). There are a few studies on neuroprotective effects of sacral nerve stimulation in the sci patients2,3,6,9,12,14). The additional effect of sacral neuromodulation was improvement of neurogenic bladder, intestinal motility, penile erection, pain discrimination, and motor power of leg6). However, the current study showed that sacral neuromodulation to rat injured thoracic spinal cord did not improve the locomotor function . Several investigators have reported that a low - frequency electrical stimulation could accelerate nerve regeneration after injury2,10). However, a high frequency of electrical stimulation may increase failure rate of nerve regeneration13). Hence, these results cannot exclude potential beneficial effects of sacral nerve stimulation in other models of sci with different treatment protocols, the disappointing outcomes in this study should be limited by the treatment protocol . In a rat thoracic spinal cord contusional model, we failed to establish a rat spinal cord stimulation model . However, sacral neuromodulation has a therapeutic potential to improve neurogenic bladder and muscle atrophy . Further experimental studies are needed to establish a rat spinal cord stimulation model and to determine the most effective protocol of stimulation.
|
A 9-month - old, 10-kg girl without any relevant medical history was presented at our emergency room because she had aspirated a peanut 5 hours previously . Chest computed tomography showed a foreign body at the carina and the proximal right main bronchus, and emphysema in both lung fields (fig . Anesthesia was induced intravenously with thiopental 60 mg and rocuronium 6 mg after administering atropine 0.2 mg . Approximately 25 minutes after the start of the operation, spo2 gradually decreased to 78% from 100% . Accordingly, the surgeon was asked to stop the procedure, and an uncuffed endotracheal tube (i d 4.0, euromedical, denmark) was intubated and fixed at 10 cm . Left lung sounds were heard clearly, but no right lung sounds were evident on auscultation . Spo2 did not improve despite manual ventilation with 100% o2 and an increased inspiratory pressure airway was detected . Accordingly, a simple chest radiograph was taken . While waiting for the result of the chest radiograph, the otolaryngologist extubated the endotracheal tube and attempted to re - insert the rigid bronchoscope in order to check for possible causes of the airway obstruction, such as, residual foreign body . Meanwhile, the heart rate decreased to 50 beats / min, and spo2 decreased to 57% (blood pressure was not measured). Atropine 0.2 mg and epinephrine 1 g were injected intravenously, but vital signs did not improve and no carotid artery pulse was palpable . External cardiac compression was performed and epinephrine 10 g was injected intravenously three times . Heart rate and blood pressure then recovered, and thus, cardiac compression was stopped . A chest tube was immediately inserted into the right thorax and a massive air leak was observed . At this time, the sounds of both lungs were clearly heard and spo2 improved to 100% . Follow up chest radiography showed a significant reduction of the right tension pneumothorax and a scattering of subcutaneous emphysema at the left upper chest and around the neck (fig . The tension pneumothorax suggested major tracheobronchial injury and a subsequent bronchoscopic examination revealed three major sites of laceration, one in the mid trachea of length 1.5 cm, a second in the distal trachea of length 2 cm (immediately above the carina) (fig . 4), and a third in the right main bronchus of length 1 cm . An arterial line was placed in the left radial artery to monitor blood pressure continuously . Arterial blood gas analysis showed; ph 7.10, paco2 63 mmhg, pao2 77 mmhg, hco3 19.6 mmol / l at fio2 1.0, etco2 24 mmhg, and spo2 100% . After a rigid bronchoscope examination confirmed the absence of remaining foreign body, an uncuffed i d 4.5 endotracheal tube was inserted into the left main bronchus for single lung ventilation under fiberoptic bronchoscopic guidance . However, the patient did not tolerate single lung ventilation well and her spo2 decreased to 79% . In view of the facilitation of surgical approach and the risks of hypoxemia and hypoventilation, after 1,000 units of heparin was injected as a bolus, a 10 fr catheter (fem - flex ii femoral cannula, edwards lifesciences, irvine, ca, usa) was placed in the right internal jugular vein for drainage, and a 12 fr catheter was placed in the right femoral vein for perfusion using a sono - guided seldinger technique (fig . 5). Ecmo flow was maintained at 0.7 - 0.8 l / min, and spo2 remained at 98 - 100% without mechanical ventilation . At the initiation of ecmo, anesthesia was maintained with continuous intravenous infusions of sufentanil 5 g / kg / h, midazolam 0.1 mg / kg / h, and vecuronium 0.1 mg / kg / h during ecmo . Dopamine was infused at 5 g / kg / min and vital signs were stabilized . An activated clotting time (act) of approximately 200 seconds was maintained and arterial blood gas analysis under maximal ecmo support showed; ph 7.34, paco2 41 mmhg, pao2 81 mmhg, and hco3 22.1 mmol / l at fio2 1.0 . When the ecmo flow rate reduced, it was recovered by pulling the cannula in the internal jugular vein slightly . Body temperature, measured using an oral thermometer, fell to as low as 35.0 after ecmo was initiated, and thus, a forced air warmer was applied to prevent further hypothermia . Total anesthesia time was 8 hr 25 min, estimated blood loss was 300 ml, and urine output was 120 ml; 250 ml of crystalloid solution and 1 unit of packed red blood cells were administered . Ecmo was maintained postoperatively to minimize airway pressure . In the intensive care unit (icu), the mechanical ventilation was performed using pressure control mode (inspiratory airway pressure 10 cmh2o, pressure support 11 cmh2o, respiratory rate 20/min, fio2 0.5 and peep 3 cmh2o), which resulted in the expiratory tidal volume of 49 ml . Vital signs were stable in the icu, and the patient awoke with no signs of respiratory or neurologic deficit on the first postoperative day . The simple chest radiograph taken in the icu showed reduced subcutaneous emphysema and no residual pneumothorax . Ventilating bronchoscopy has the risk of severe complications, such as, infection, laryngeal edema, laryngospasm, bronchospasm, bleeding, hypoxia, pneumomediastinum, and pneumothorax . Rothmann and boeckman reported that the probability of pneumothorax during rigid bronchoscopy for the removal of a foreign body is approximately 1% . Therefore, the possibility of pneumothorax should be suspected when ventilation worsens during ventilating bronchoscopy . However, an early diagnosis of pneumothorax during anesthesia is not easy because the symptom is masked by anesthesia . Tension pneumothorax during surgery may manifest as a reduction in oxygen saturation, a rapid increase in airway pressure, hypotension, and/or tachycardia . In this case, the reasons for the delayed diagnosis in the present case were that the reduction in oxygen saturation and the diminished right lung sounds were mistakenly attributed to airway obstruction by a foreign body . In addition, if lung sounds had been checked more carefully, it might have been easier to determine whether the respiratory failure was due to foreign body obstruction or tension pneumothorax . Furthermore, it would have been safer to evaluate airway with a fiberoptic bronchoscope inserted through the endotracheal tube under ventilation rather than attempt airway assessment using a ventilating bronchoscope after extubation . During surgery on the trachea and bronchus, a double lumen endotracheal tube or however, in small children, because conventional single lung ventilation methods are likely to fail to maintain adequate ventilation and oxygenation, the elective application of ecmo may be a good option . Ecmo was also useful during the postoperative course in the described case because it can minimize inspiratory airway pressure during mechanical ventilation . During ecmo, heparin was administered as a single bolus of 100 unit / kg followed by continuous intravenous infusion at 20 - 60 unit / kg / min . Anticoagulation was accomplished by titrating heparin administration to maintain an act of between 180 and 220 seconds . Furthermore, levels of blood anticoagulation were monitored hourly, and the amount of heparin administered was carefully controlled to prevent bleeding complications . In addition, because the ecmo machine was not equipped with a sevoflurane vaporizer and almost no mechanical ventilation was performed during ecmo, intravenous opioids and midazolam were added for anesthesia . Summarizing, the tension pneumothorax should be considered when inspiratory pressure is increased and oxygen saturation is decreased during ventilating bronchoscopy . Effective communication between the operator and anesthesiologist is essential when unexpected complications are encountered to facilitate the prompt planning of coping strategies.
|
The ludwigshafen risk and cardiovascular health (luric) study is an ongoing prospective study of currently more than 3000 individuals of german ancestry in whom the cardiovascular and metabolic phenotypes cad, myocardial infarction (mi), dyslipidemia, hypertension, metabolic syndrome, and diabetes mellitus have been defined or ruled out using standardized methodologies (8). All the patients were in secondary prevention, ie, had known cad . Patients with german ancestry were recruited at the cardiac center in ludwigshafen from 1997 to 2002 . After obtaining a written informed consent, baseline examination was done consisting of a standardized individual and family history questionnaire and extensive sampling of fasted venous blood in the early morning . We first selected male cad patients (n = 258) from the luric cohort who died due to cardiovascular disease (cvd) reasons within the first 3 years of follow - up . The control group consisted of male cad patients who did not die during the follow - up . Frequency matching was used for matching case and control groups for age, body mass index (bmi), statin use, and smoking . The total number of controls (n = 187) remained smaller than the total number of cases . This was due to the exclusion of numerous stable diabetic patients who had had events indicative of plaque vulnerability such as mi and/or stroke prior to the study entry . Coronary angiography was used to verify cad (stenosis> 20% in one or more coronary arteries) in both study groups . Cardiovascular deaths were defined as sudden cardiac death, fatal mi, death due to congestive heart failure, death immediately after intervention to treat cad, fatal stroke, and other causes of deaths due to cardiac disease . Two experienced clinicians blinded of the study data independently went through death certificates to classify deaths to cvd and non - cvd causes (9). Patient characteristics abbreviation: apo, apolipoprotein; crp, c - reactive protein; dm2, diabetes mellitus type 2; n.s ., not significant; tc, total cholesterol; tg, triglycerides . Framingham score of coronary heart disease (10 y risk) (17). Plasma samples of healthy males from a single - center, randomized, parallel, three - group study performed at the university of cologne (10) were analyzed to evaluate the potential of ezetimibe (10 mg; n = 24), simvastatin (40 mg; n = 24), or their combination (n = 24) to reduce plasma concentrations of molecular lipids species that had been identified to be related to cardiovascular death in the luric cohort . Furthermore, we used the luric study genome - wide association studies database and identified subjects who were carrying the previously described loss - of - function mutation r46l (rs11591147) (11) of the pcsk9 gene to reveal the possible lipidomic effect of pcsk9 deficiency in men . Altogether 19 heterozygous male mutation carriers and 868 male homozygous major allele carriers were identified from our database with both genetic information and serum lipidomic data available . Known amounts of internal standards were added to the samples before extraction and the final lipid extracts were dried under nitrogen . Sphingolipids were analyzed as described elsewhere (13) on a 4000 qtrap mass spectrometer (applied biosystems / mds analytical technologies) equipped with an ultrahigh pressure liquid chromatography system, ctc pal autosampler (leap technologies), and rheos allegro ultrahigh pressure liquid chromatography (flux instruments) using multiple reaction monitoring . Shotgun lipidomics was performed by multiple precursor ion and neutral loss scanning as described elsewhere (14) on a qtrap 5500 mass spectrometer (applied biosystems / mds analytical technologies) equipped with a robotic nanoflow ion source nanomate hd (advion). Mass spectrometry data files were processed using multiquant 1.1.0.26 or lipid profiler (15) (applied biosystems / mds analytical technologies). Identified lipids were quantified by normalizing against their respective internal standard and volume for plasma or serum . Quality control (qc) samples were used to monitor the overall quality of the lipid extraction and mass spectrometry analyses (16). The qc samples were mainly used to remove technical outliers and lipid species that were detected below the lipid class - based lower limit of quantification . In total the average coefficient of variation of all the lipids detected in the study samples was 20% . Lipid class concentrations were calculated by summing up the concentrations of corresponding molecular lipids . For analysis of cad mortality and pcsk9 mutation data, an unpaired student t test was performed on log - transformed concentrations and lipid to lipid ratios because variables were approximately log normal . Equality of variance was tested, and pooled t test was used in case of equal variance and satterthwaite t test was used in case of unequal variance . Cad mortality case and controls groups were frequency matched; therefore, an unpaired t test was used . The t test results are presented as volcano plots and heat maps (figures 13). In volcano plots, the magnitude of relative difference between groups (horizontal axis) is plotted against the statistical significance (vertical axis). Odds ratios were calculated using logistic regression model with and without adjustment for age, bmi, fasting glucose, hdl - c, ldl - c, c - reactive protein, and triglycerides . A paired t test was conducted for comparing baseline and after - treatment lipid concentrations in subjects receiving ezetemibe, simvastatin, or their combination . The false discovery rate q values were calculated to correct the multiple hypotheses testing results . Volcano heat map indicating molecular lipid species difference (percentage) between stable and high - risk cad patients . Right panel (red color) indicates lipids that associate with cvd outcome risk in cad patients . Lipid concentrations in controls are taken as a reference; thus, positive values correspond to higher values in cases vs controls . B, heat map of sphingolipid differences . Volcano heat map indicating molecular lipids and ceramide to ceramide ratio differences between all studied stable cad patients and high - risk cad patients (a), differences between stable cad patients and vulnerable cad patients with diabetes (b), and patients without diabetes (c). Lipid concentrations in controls are taken as a reference; thus, positive values correspond to higher values in cases vs controls . The ludwigshafen risk and cardiovascular health (luric) study is an ongoing prospective study of currently more than 3000 individuals of german ancestry in whom the cardiovascular and metabolic phenotypes cad, myocardial infarction (mi), dyslipidemia, hypertension, metabolic syndrome, and diabetes mellitus have been defined or ruled out using standardized methodologies (8). All the patients were in secondary prevention, ie, had known cad . Patients with german ancestry were recruited at the cardiac center in ludwigshafen from 1997 to 2002 . After obtaining a written informed consent, baseline examination was done consisting of a standardized individual and family history questionnaire and extensive sampling of fasted venous blood in the early morning . We first selected male cad patients (n = 258) from the luric cohort who died due to cardiovascular disease (cvd) reasons within the first 3 years of follow - up . The control group consisted of male cad patients who did not die during the follow - up . Frequency matching was used for matching case and control groups for age, body mass index (bmi), statin use, and smoking . The total number of controls (n = 187) remained smaller than the total number of cases . This was due to the exclusion of numerous stable diabetic patients who had had events indicative of plaque vulnerability such as mi and/or stroke prior to the study entry . Coronary angiography was used to verify cad (stenosis> 20% in one or more coronary arteries) in both study groups . Cardiovascular deaths were defined as sudden cardiac death, fatal mi, death due to congestive heart failure, death immediately after intervention to treat cad, fatal stroke, and other causes of deaths due to cardiac disease . Two experienced clinicians blinded of the study data independently went through death certificates to classify deaths to cvd and non - cvd causes (9). Patient characteristics abbreviation: apo, apolipoprotein; crp, c - reactive protein; dm2, diabetes mellitus type 2; n.s ., not significant; tc, total cholesterol; tg, triglycerides . Framingham score of coronary heart disease (10 y risk) (17). Plasma samples of healthy males from a single - center, randomized, parallel, three - group study performed at the university of cologne (10) were analyzed to evaluate the potential of ezetimibe (10 mg; n = 24), simvastatin (40 mg; n = 24), or their combination (n = 24) to reduce plasma concentrations of molecular lipids species that had been identified to be related to cardiovascular death in the luric cohort . Furthermore, we used the luric study genome - wide association studies database and identified subjects who were carrying the previously described loss - of - function mutation r46l (rs11591147) (11) of the pcsk9 gene to reveal the possible lipidomic effect of pcsk9 deficiency in men . Altogether 19 heterozygous male mutation carriers and 868 male homozygous major allele carriers were identified from our database with both genetic information and serum lipidomic data available . Amounts of internal standards were added to the samples before extraction and the final lipid extracts were dried under nitrogen . Sphingolipids were analyzed as described elsewhere (13) on a 4000 qtrap mass spectrometer (applied biosystems / mds analytical technologies) equipped with an ultrahigh pressure liquid chromatography system, ctc pal autosampler (leap technologies), and rheos allegro ultrahigh pressure liquid chromatography (flux instruments) using multiple reaction monitoring . Shotgun lipidomics was performed by multiple precursor ion and neutral loss scanning as described elsewhere (14) on a qtrap 5500 mass spectrometer (applied biosystems / mds analytical technologies) equipped with a robotic nanoflow ion source nanomate hd (advion). Mass spectrometry data files were processed using multiquant 1.1.0.26 or lipid profiler (15) (applied biosystems / mds analytical technologies). Identified lipids were quantified by normalizing against their respective internal standard and volume for plasma or serum . Quality control (qc) samples were used to monitor the overall quality of the lipid extraction and mass spectrometry analyses (16). The qc samples were mainly used to remove technical outliers and lipid species that were detected below the lipid class - based lower limit of quantification . In total, 14 qc samples evenly distributed along analytical runs of the study were analyzed . The average coefficient of variation of all the lipids detected in the study samples was 20% . Lipid class concentrations were calculated by summing up the concentrations of corresponding molecular lipids . For analysis of cad mortality and pcsk9 mutation data, an unpaired student t test was performed on log - transformed concentrations and lipid to lipid ratios because variables were approximately log normal . Equality of variance was tested, and pooled t test was used in case of equal variance and satterthwaite t test was used in case of unequal variance . Cad mortality case and controls groups were frequency matched; therefore, an unpaired t test was used . The t test results are presented as volcano plots and heat maps (figures 13). In volcano plots, the magnitude of relative difference between groups (horizontal axis) is plotted against the statistical significance (vertical axis). Odds ratios were calculated using logistic regression model with and without adjustment for age, bmi, fasting glucose, hdl - c, ldl - c, c - reactive protein, and triglycerides . A paired t test was conducted for comparing baseline and after - treatment lipid concentrations in subjects receiving ezetemibe, simvastatin, or their combination . The false discovery rate q values were calculated to correct the multiple hypotheses testing results . Volcano heat map indicating molecular lipid species difference (percentage) between stable and high - risk cad patients . Right panel (red color) indicates lipids that associate with cvd outcome risk in cad patients . Lipid concentrations in controls are taken as a reference; thus, positive values correspond to higher values in cases vs controls . B, heat map of sphingolipid differences . Volcano heat map indicating molecular lipids and ceramide to ceramide ratio differences between all studied stable cad patients and high - risk cad patients (a), differences between stable cad patients and vulnerable cad patients with diabetes (b), and patients without diabetes (c). Lipid concentrations in controls are taken as a reference; thus, positive values correspond to higher values in cases vs controls . The classical risk markers were unable to distinguish cad patients who died during the follow - up from stable cad patients who had structural coronary disease at baseline . Modestly higher total cholesterol concentrations (6%, p = .002) were recorded in the case groups and hdl - c and apoa1 were slightly lower in cases compared with controls (7.5%, p = .001, and 7.5%, p = .001, respectively) (table 1). Both case and control groups had median framingham 10-year risk of coronary heart disease (17) of 20% . The estimation of the 10-year risk of fatal cardiovascular disease in europe (systematic coronary risk evaluation, score) (18) was 7.1% in cases and 6.7% in controls, and the slight difference was not statistically significant . More pronounced differences between groups were observed through lipidomic analyses (figure 1 and supplemental table 1, published on the endocrine society's journals online web site at http://jcem.endojournals.org). Lactosylceramides laccer (18:1/18:0) and laccer (18:1/20:0) were among the risk - associated lipids together with cer (18:1/16:0) and cer (18:1/18:0). Several molecular lipid species such as ce20:5 (17.4%, p = .0001) and ce18:3 (15.1%, p = .000037) displayed reduced concentrations in cases as compared with the control group . Abundance of several ceramide species containing specific fatty acids was higher along the axis of the glycosphingolipid pathway (figure 2) in which ceramide is converted to galactosylceramide and glucosylceramide, glucosylceramide is converted to lactosylceramide, and lactosylceramide is metabolized to globotriaosylceramide (gb3). For example, d18:1/22:0-containing species were higher in ceramide, lactosylceramide, and globotriaosylceramide classes . A similar pattern is demonstrated by other long - chain containing ceramides (d18:1/16:0 and d18:1/18:0). However, cer(d18:1/24:0) lipid species concentration was lower in cases reflected in lower concentration of sphingomyelin containing the same fatty acid sm(d18:1/24:0) (figure 2b). To account for lipid concentration differences due to metabolome influencing factors interestingly, when these patients were studied separately, very distinct lipidomic profiles were recorded for the two groups . In nondiabetic cad patients, the risk of fatal outcome was associated with elevated sphingolipids, whereas in diabetic cad patients, the risk was mainly associated with reduced cholesteryl ester species (figure 3). Because we noted that factors like diabetes, smoking, and lipid - lowering treatment among others, could influence the expression of the risk - associated lipids, we decided to calculate ceramide and cerebroside ratios to check whether these would be less dependent on different patient characteristics while still indicative of risk . It turned out that the ratios of cer(18:1/16:0)/cer(18:1/24:0) and cer(18:1/22:0)/cer(18:1/24:0) were significantly related to increased risk of cvd death in all subjects and subgroups . Cer(18:1/24:0)/cer(18:1/24:1) was indicative of a reduced risk of cvd death, regardless of diabetes status (figure 3). Importantly, odds ratios for cvd death of these ratios remained significant after adjustment for traditional cvd death risk factors, such as ldl - c (table 2). Unadjusted and adjusted odds ratios for cvd death of ceramide ratios abbreviation: or, odds ratio . Models adjusted for age, bmi, fasting glucose, hdl - c, ldl - c, c - reactive protein, triglycerides, and systolic and diastolic blood pressure . Predictive potential for cad mortality was tested by receiver - operating characteristic analysis for the ceramide ratios and compared with ldl - c . The area under curve and 95% confidence limits were 0.67 (range 0.620.72) for cer(d18:1/16:0)/cer(d18:1/24:0), 0.65 (range 0.600.71) for cer(d18:1/20:0)/cer(d18:1/24:0), and 0.68 (range 0.630.73) for cer(d18:1/24:0)/cer(d18:1/24:1), whereas for ldl - c, these values were 0.55 (range 0.500.61). Finally, the effect of lipid - lowering treatments on ceramides and cerebrosides associated with increased risk of cvd death in statin - nave subjects as well as on ldl - c was evaluated (figure 4). As a comparison basis, we used the most significant lipids that separate the high - risk cad patients from stable cad patients (figure 4a). The effect of different ldl - c - lowering methods on cad outcome - related lipidomic markers . Results shown for top ranked cad risk lipids, ldl - c, and ceramide (18:1/24:0). Effect of ezetimibe (10 mg) (b), simvastatin (40 mg) (c), simvastatin + ezetimibe (40 mg+10 mg), (d) and pcsk9 loss - of - function mutation (r46l) (e) on cad mortality risk lipids . For evaluation of ezetimibe and simvastatin effects on these putative risk lipids we used plasma samples from a randomized clinical trial comparing simvastatin 40 mg, ezetimibe 10 mg, and their combination (supplemental table 2). As expected, simvastatin 40 mg lowered ldl - c significantly by 40%, and this reduction was accompanied by a significant reduction (25%) also in all ceramides and cerebrosides recorded in this study (figure 4c). Ezetimibe treatment (10 mg) in turn lowered ldl - c by 21% but affected only very modestly the plasma levels of cvd death - related lipid species (figure 4b). In fact, cer18:1/18:0 and cer18:1/20:0 concentrations were slightly elevated in ezetimibe - treated subjects by 7.6% and 8.8%, respectively, whereas cer18:1/16:0 levels remained unchanged during this treatment . There was no statistically significant difference between treatment effects of simvastatin alone or in combination with ezetimibe on molecular lipids . However, combined treatment resulted in significantly lower ldl - c levels than simvastatin or ezetimibe alone (figure 4d). Drugs that inhibit pcsk9 are being developed as an alternative to statin lipid - lowering therapy, and we studied the effects of pcsk9 inhibition on the risk lipids (supplemental table 3). The effect was investigated by using subjects with the pcsk9 allelic variants from the luric patient population (figure 4e). The pcsk9 loss - of - function mutation (r46l) resulted in a significant reduction of ceramides indicative of cad outcome risk comparable with that observed in simvastatin - treated subjects . Interestingly, the mutation carriers had only modestly lower ldl - c levels (12.9%) compared with major allele carriers while being still efficient in risk lipid reduction . Furthermore, pcsk9 deficiency lowered the reduced cad outcome risk - associated cer(18:1/24:0) nonsignificantly by 6.4%, whereas simvastatin and simvastatin - ezetimibe combination both resulted in significant (> 25%) reductions . The classical risk markers were unable to distinguish cad patients who died during the follow - up from stable cad patients who had structural coronary disease at baseline . Modestly higher total cholesterol concentrations (6%, p = .002) were recorded in the case groups and hdl - c and apoa1 were slightly lower in cases compared with controls (7.5%, p = .001, and 7.5%, p = .001, respectively) (table 1). Both case and control groups had median framingham 10-year risk of coronary heart disease (17) of 20% . The estimation of the 10-year risk of fatal cardiovascular disease in europe (systematic coronary risk evaluation, score) (18) was 7.1% in cases and 6.7% in controls, and the slight difference was not statistically significant . More pronounced differences between groups were observed through lipidomic analyses (figure 1 and supplemental table 1, published on the endocrine society's journals online web site at http://jcem.endojournals.org). Lactosylceramides laccer (18:1/18:0) and laccer (18:1/20:0) were among the risk - associated lipids together with cer (18:1/16:0) and cer (18:1/18:0). Several molecular lipid species such as ce20:5 (17.4%, p = .0001) and ce18:3 (15.1%, p = .000037) displayed reduced concentrations in cases as compared with the control group . Abundance of several ceramide species containing specific fatty acids was higher along the axis of the glycosphingolipid pathway (figure 2) in which ceramide is converted to galactosylceramide and glucosylceramide, glucosylceramide is converted to lactosylceramide, and lactosylceramide is metabolized to globotriaosylceramide (gb3). For example, d18:1/22:0-containing species were higher in ceramide, lactosylceramide, and globotriaosylceramide classes . A similar pattern is demonstrated by other long - chain containing ceramides (d18:1/16:0 and d18:1/18:0). However, cer(d18:1/24:0) lipid species concentration was lower in cases reflected in lower concentration of sphingomyelin containing the same fatty acid sm(d18:1/24:0) (figure 2b). To account for lipid concentration differences due to metabolome influencing factors interestingly, when these patients were studied separately, very distinct lipidomic profiles were recorded for the two groups . In nondiabetic cad patients, the risk of fatal outcome was associated with elevated sphingolipids, whereas in diabetic cad patients, the risk was mainly associated with reduced cholesteryl ester species (figure 3). Because we noted that factors like diabetes, smoking, and lipid - lowering treatment among others, could influence the expression of the risk - associated lipids, we decided to calculate ceramide and cerebroside ratios to check whether these would be less dependent on different patient characteristics while still indicative of risk . It turned out that the ratios of cer(18:1/16:0)/cer(18:1/24:0) and cer(18:1/22:0)/cer(18:1/24:0) were significantly related to increased risk of cvd death in all subjects and subgroups . Cer(18:1/24:0)/cer(18:1/24:1) was indicative of a reduced risk of cvd death, regardless of diabetes status (figure 3). Importantly, odds ratios for cvd death of these ratios remained significant after adjustment for traditional cvd death risk factors, such as ldl - c (table 2). Unadjusted and adjusted odds ratios for cvd death of ceramide ratios abbreviation: or, odds ratio . Models adjusted for age, bmi, fasting glucose, hdl - c, ldl - c, c - reactive protein, triglycerides, and systolic and diastolic blood pressure . Predictive potential for cad mortality was tested by receiver - operating characteristic analysis for the ceramide ratios and compared with ldl - c . The area under curve and 95% confidence limits were 0.67 (range 0.620.72) for cer(d18:1/16:0)/cer(d18:1/24:0), 0.65 (range 0.600.71) for cer(d18:1/20:0)/cer(d18:1/24:0), and 0.68 (range 0.630.73) for cer(d18:1/24:0)/cer(d18:1/24:1), whereas for ldl - c, these values were 0.55 (range 0.500.61). Finally, the effect of lipid - lowering treatments on ceramides and cerebrosides associated with increased risk of cvd death in statin - nave subjects as well as on ldl - c was evaluated (figure 4). As a comparison basis, we used the most significant lipids that separate the high - risk cad patients from stable cad patients (figure 4a). The effect of different ldl - c - lowering methods on cad outcome - related lipidomic markers . Results shown for top ranked cad risk lipids, ldl - c, and ceramide (18:1/24:0). Effect of ezetimibe (10 mg) (b), simvastatin (40 mg) (c), simvastatin + ezetimibe (40 mg+10 mg), (d) and pcsk9 loss - of - function mutation (r46l) (e) on cad mortality risk lipids . For evaluation of ezetimibe and simvastatin effects on these putative risk lipids we used plasma samples from a randomized clinical trial comparing simvastatin 40 mg, ezetimibe 10 mg, and their combination (supplemental table 2). As expected, simvastatin 40 mg lowered ldl - c significantly by 40%, and this reduction was accompanied by a significant reduction (25%) also in all ceramides and cerebrosides recorded in this study (figure 4c). Ezetimibe treatment (10 mg) in turn lowered ldl - c by 21% but affected only very modestly the plasma levels of cvd death - related lipid species (figure 4b). In fact, cer18:1/18:0 and cer18:1/20:0 concentrations were slightly elevated in ezetimibe - treated subjects by 7.6% and 8.8%, respectively, whereas cer18:1/16:0 levels remained unchanged during this treatment . There was no statistically significant difference between treatment effects of simvastatin alone or in combination with ezetimibe on molecular lipids . However, combined treatment resulted in significantly lower ldl - c levels than simvastatin or ezetimibe alone (figure 4d). Drugs that inhibit pcsk9 are being developed as an alternative to statin lipid - lowering therapy, and we studied the effects of pcsk9 inhibition on the risk lipids (supplemental table 3). The effect was investigated by using subjects with the pcsk9 allelic variants from the luric patient population (figure 4e). The pcsk9 loss - of - function mutation (r46l) resulted in a significant reduction of ceramides indicative of cad outcome risk comparable with that observed in simvastatin - treated subjects . Interestingly, the mutation carriers had only modestly lower ldl - c levels (12.9%) compared with major allele carriers while being still efficient in risk lipid reduction . Furthermore, pcsk9 deficiency lowered the reduced cad outcome risk - associated cer(18:1/24:0) nonsignificantly by 6.4%, whereas simvastatin and simvastatin - ezetimibe combination both resulted in significant (> 25%) reductions . The present study demonstrates that specific molecular lipid species are significantly associated with mortality in cad . Importantly, the predictive potential of distinct ceramides was superior to the currently used standard ldl - c measurement, underscoring the value of molecular lipidomic analyses . Moreover, we show that these molecular lipid species are highly influenced by the choice of lipid - lowering treatment and that simple numerical ldl - c lowering per se is not necessarily translated to modulation of all risk - associated lipid species . In earlier experimental studies, ceramides and other sphingolipids have been associated with the development of atherosclerosis, and several enzymes in the ceramide synthetic pathway have been tested as potential drug targets in animal models (1921). The present study suggests that different molecular ceramides associate with cad outcome risk, and thus, they may play a significant role also in plaque vulnerability in addition to development of atherosclerotic disease . Notably, for some ceramide species containing specific fatty acids, higher concentrations were observed along the axis of glycosphingolipid pathway (figure 2) with no significant differences observed in corresponding sphingomyelin species, although lower concentrations of other ceramide species with longer - chain fatty acids were reflected in lower concentrations of the corresponding sphingomyelin . These findings together with the fact that total sphingolipid levels remained unchanged indicate that alteration in sphingolipid balance is species dependent . A role of the glycosphingolipid pathway in atherosclerosis was previously suspected based on the following observations: lactosylceramide and glucosylceramide accumulate in the atherosclerotic plaque (22), and both lactosylceramide and glucosylceramide suppress production of macrophage apoe and lead to an accumulation of cholesterol in macrophage foam cells (23). Importantly, inhibition of the glycosphingolipid pathway was previously shown to decrease atherosclerosis in mice (24). To the best of our knowledge, our data for the first time link specific molecular lipid species from the glucosphingolipid pathway measured in human serum to cad mortality . On the other hand, a reverse association of long - chain ceramide and sphingomyelin d18:1/24:0 with cad mortality demonstrates the complexity of this disease, which is not seen by analyzing the lipid classes alone . Further studies are needed to evaluate the potential connections between the ceramide species and thrombogenesis, fibrinolysis, and plaque instability that may explain the observed linkage between death from cvd reasons and molecular ceramides . Another main finding of this study is that two different lipid - lowering drugs display distinct effects on the cvd risk - associated plasma ceramides, beyond their ldl - c - lowering effects . Simvastatin 40 mg resulted in a broad lowering of all recorded ceramide and cerebroside species; however, the magnitude of reduction was clearly lower than that for ldl - c . On the other hand, we demonstrated in this study that a very modest ldl - c effect due to pcsk9 deficiency resulted in a larger proportional reduction in cad outcome - related plasma ceramides . This suggests that there is an interaction between the means of ldl receptor (ldlr) up - regulation and the levels of circulating ceramides . Both statins and pcsk9 inhibition increase ldlr - mediated hepatic lipid uptake, and it could thus be expected that they result in similar lipid - lowering profiles . However, statins are acting indirectly via inhibition of 3-hydroxy-3-methylglutaryl coenzyme a reductase and inhibition of this rate - limiting enzyme of the mevalonate pathway has multiple effects on a number of genes related to lipid metabolism in hepatocytes . This is due to subsequent activation of the sterol regulatory element binding protein (srebp)-1- and -2-mediated gene transcription (25, 26). Pcsk9 inhibition, in turn, may act more precisely on ldlr in hepatocytes and thus may lead to more targeted lipid lowering without more systemic effects on lipid metabolism . Therefore, pcsk9 inhibition seems a promising lipid - lowering method of choice from the plasma lipidomic point of view . In particular, this is the case if this inhibition leads to similar plasma lipidomic composition as the well - characterized loss - of - function mutation . However, these data are based on a 2-week intervention, and thus, the long - term effect must await confirmation in longer studies . On the other hand, taylor et al demonstrated recently that in the arterial biology for the investigation of the treatment effects of reducing cholesterol-6 trial, ezetimibe treatment resulted in a paradoxical progression of carotid intima - media thickness in association with both greater ldl - c reduction and cumulative drug exposure (27). The authors concluded that their findings may suggest the presence of off - target actions of ezetimibe . Based on our current results, one could hypothesize that the paradoxical effect on carotid intima - media thickness is due to the minimal effects on ceramides despite the substantial ldl - c - lowering effects . This study suggests that ldl - c and traditional risk factors provide only limited predictive information on fatal cvd complications in patients with established cad . However, molecular lipid assays can be used to improve the identification of cad patients at high risk, and such assay results may also serve as better efficacy indicators for lipid - modifying drugs . It is important to note that although traditional risk scores are developed for the general population, for the purpose of this study, we selected only diseased patients and investigated association of lipids with stable or unstable disease . As a result, both patient groups were at high risk from the start of the follow - up as indicated by framingham and european risk score values . A limitation of our study is that only male subjects from only one cohort were evaluated . We selected men for reducing variability in the data because atherosclerotic development is gender specific (28), and traditionally risk scores are evaluated separately for men and women (17, 18). Thus, our results should be validated in an independent large cohort and extended to women . In the present work, longer exposure time, greater subject numbers, and possibly study in patients with cad will be needed to further understand the dynamics of modulation of the risk lipids.
|
Data on tree canopy and impervious cover provide important information on the extent and variation of these characteristics across a region . Measurements of tree canopy cover provide basic structural data used to model tree services, such as air pollution mitigation and carbon dioxide sequestration (nowak and crane 2002; nowak and others 2006), while impervious surface data are important for assessing development impacts on urban temperatures, precipitation runoff, and water quality (heisler and others 2007; theobald and others 2009). Tree canopy and impervious cover data provide essential information related to natural resources and development planning and policies at the local to national scale . The 2001 national land cover database (nlcd) provides free, easily accessible, 30-m resolution percentage tree canopy and percentage impervious cover values for the conterminous united states created from a consistent peer - reviewed methodology (mrlc 2009). Several studies have used nlcd data for assessing the urban tree canopy cover (bridges 2008; nowak and greenfield 2008), urban temperature modeling (heisler and others 2007), estimates of canopy height (walker and others 2007), distribution of constructed manmade surfaces (elvidge and others 2007), non - point source nitrogen export into water systems (shields and others 2008), and wildlife habitat distribution (martinuzzi and others 2009). While a formal accuracy assessment of nlcd land cover estimates has been conducted (wickham and others 2010), a formal accuracy assessment of nlcd tree canopy and impervious cover data has yet to be completed (stehman and others 2008; us epa 2010). In 2007, the 2001 nlcd was made publicly available by the multi - resolution land characteristics consortium (mrlc) (mrlc 2009; homer and others 2007). The 2001 nlcd provides 30-m resolution classified land cover and percentage tree canopy and impervious cover estimates for the conterminous united states derived from circa 2001 landsat 7 imagery . Twelve mapping teams employed by the mrlc used standardized data preparation, classification, and quality control to process the landsat imagery within 65 distinct mapping zones (huang and others 2001; yang and others 2003; homer and others 2004, 2007). Mapping zones were delimited to represent relative geographic homogeneity with consideration of economy (cost), physiography, land - cover distribution, spectral uniformity, and optimal edge - matching (homer and gallant 2001). High resolution tree canopy and impervious cover maps, derived from 1-m resolution digital orthoimagery quarter quadrangles, were used to develop unique algorithms for each mapping zone to estimate percentage tree canopy and impervious cover from raw landsat 7 imagery (c. 2001). Each cover layer is accompanied by metadata documenting error estimates based on a cross - validation technique utilizing the algorithms and training data for each mapping zone (mrlc 2009; homer and others 2007). According to these preliminary error estimates, the tree canopy cover values have an average error ranging from 6 to 17% and impervious cover has an average error ranging from 4 to 17% (mrlc 2009; homer and others 2007). With an early and limited release of the 2001 nlcd, walton (2008) found potential underestimation of tree canopy cover in 36 cities and villages in nlcd mapping zone 63 (western new york state). A later study was developed to compare 2001 nlcd cover estimates with photo - interpreted estimates of google earth imagery from randomly sampled and geographically dispersed census - designated places (e.g., cities, villages; hereafter referred to as places) and counties in the united states (greenfield and others 2009). Results of this comparison revealed that 2001 nlcd underestimates tree canopy cover by an average of 9.7% and underestimates impervious cover by an average of 5.7% within places and 1.3% in counties . The underestimate appeared to be consistent across the country with no statistical differences among physiographic regions . However, there were statistical differences in the degree of underestimation of tree canopy cover among mapping zones and of impervious cover by population density class . The study reported here continues this work by expanding the analysis to the entire conterminous united states to further explore the differences between nlcd - derived and photo - interpreted percentage tree canopy and impervious cover among all 65 mapping zones . Google earth imagery is used as a reference data source for tree canopy and impervious cover estimates because of its national aerial imagery coverage . Google earth imagery has been used to augment existing geographic data and when other data sources specific to a particular application are incomplete, inconsistent, or nonexistent . For example, google earth has been used to evaluate the spatial distribution of insurance risk and natural disaster mapping and crisis management (slingsby and others 2008; nourbakhsh and others 2006), as reference data to validate land cover maps (cha and park 2007), to enable the use of volunteered geographic information to post, reference and verify geographic data (goodchild 2007; wood and others 2007), for nlcd land cover accuracy assessments when other media were unavailable (wickham and others 2010), and to make applications of geographic visualization and decision - making support available to the public (sieber 2006; butler 2006; goodchild 2007; sheppard and cizek 2009). Stehman and others (2008) designed of a formal accuracy assessment of the 2001 nlcd, which includes recommended evaluation protocols to meet six defined objectives . However, only the first objective, which assesses the per - class thematic accuracy of the classified land cover, has been completed (wickham and others 2010). The protocol set out by stehman and others (2008) establishes a pixel - by - pixel assessment of the nlcd percentage tree canopy and impervious data that meets several mrlc objectives . Results reported here differ in that the analysis was not designed to be a pixel - by - pixel accuracy assessment, rather it was designed to test differences between nlcd - derived and photo - interpreted estimates of overall percentage tree canopy and impervious cover for each of the 65 mapping zones . This assessment was conducted to provide a better understanding of the potential limitations of nlcd tree canopy and impervious cover estimates for each mapping zone . The comparison between nlcd - derived and photo - interpreted tree canopy and impervious cover percentages was conducted within the boundaries of the 65 nlcd mapping zones (mrlc 2009). The nlcd 2001 percentages for tree canopy and impervious cover for each zone were derived from zone boundary maps registered with the nlcd 2001 layers in a u.s . The nlcd percentage tree canopy and impervious cover for the entire mapping zone polygon was extracted using gis software (zonal statistics). Overall percentage cover in each zone was calculated as the total nlcd cover in the zone divided by the total area in each zone . These same mapping zone boundaries were used to randomly draw a sample of 1,000 points within each zone . These points then were converted and transformed into a google earth compatible format (google inc . Each random point was interpreted as to its cover type to statistically estimate the percentage tree canopy and impervious cover within each mapping zone . Despite its widespread and growing use, past editions of google earth and its content have been known to have issues regarding unknown dates of imagery (dates of imagery currently are provided) and erroneous content (goodchild 2007; potere 2008; sheppard and cizek 2009). Potere (2008) specifically found that the horizontal positional accuracy of google earth imagery for several developed countries, including the united states, had a root mean squares error of 22.6 m and had a mean error of 19 m. however, the positional accuracy will have a negligible effect on results in this study as the cover estimates are based on random samples within large geographic areas (mapping zones). Sample points that are off from a given coordinate will still produce a valid random sample of points within the mapping zone area for the cover analysis . Inaccurate horizontal positions would only affect the sample for points near the boundary of the map zones as some points may actually represent areas outside of the mapping zone . Given the large zone area relative to the mapping zone boundary, the potential number or effect of points interpreted outside the mapping zone is negligible . There are other aerial sources of data to compare nlcd - derived values (e.g., digital orthoimagery quarter quadrangles), however, google earth imagery provides one of the best means to assess overall tree canopy and impervious cover as it offers nearly complete coverage of the conterminous united states with interpretable images . Trained photo - interpreters with experience interpreting leaf - off and leaf - on imagery classified each point as trees (yes / no), impervious surface (yes / no), or as a non - interpretable image . As reflected in the 2001 nlcd, tree canopy and impervious cover designations are not mutually exclusive (e.g., tree cover over sidewalk or road), and the photo interpreters were instructed to determine if the tree canopy covered an impervious surface, in which case it was classified as both tree and impervious . Most points (99.6%) fell on images that were readily interpretable (high - resolution imagery). Points falling on imagery with medium to coarse resolution (e.g., 30-m resolution) or with atmospheric obstructions (clouds) were considered non - interpretable and not included in the final analysis . Four photo - interpreters were used, with each mapping zone being assessed by one photo - interpreter . Photo - interpretation results were verified by having 100 points within each zone reinterpreted by another photo - interpreter . Some disagreements with the audit values were due to changes in google imagery between the original interpretation and the audit . Zones with less than 90% agreement were reinterpreted and rechecked until at least 95% agreement was attained . Overall, the audit control checks resulted in a 95% average agreement between the original interpretation and the audit values . To help understand how differences within zones might differ by land - cover classes, interpreted points in each zone were stratified into 4 groups based on general nlcd land - cover classes (general lc class): (1) trees / shrubs (nlcd classes: deciduous forest, evergreen forest, mixed forest, scrub / shrub, and woody wetland); (2) agriculture / grassland (classes: grassland / herbaceous, pasture / hay, and cultivated crops); (3) developed (classes: developed, open space, low intensity, medium intensity, and high intensity); and (4) other (classes: barren land and emergent herbaceous wetland) (mrlc 2010). General nlcd classes with small areas within a zone would have a relatively small sample size . For general classes with a sample size of less than 20 interpretable points, additional random points were interpreted to ensure a minimum sample size of 20 . Within each general lc class in each zone, the percentage of tree canopy or impervious cover (p) was calculated as the number of sample points (x) hitting the cover attribute divided by the total number of interpretable sample points (n) within the general lc class (p = x / n). The standard error of the estimate (se) was calculated as \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$se = \sqrt {{\frac{{p*\left ({1 - p} \right)}}{n}}} $$\end{document} (lindgren and mcelrath 1969). This method has been used to assess canopy cover in many cities (e.g., nowak and others 1996). Total cover and se for each mapping zone was calculated by weighting the general lc class cover estimates by nlcd general lc class area in each zone . A 95 and 99% confidence interval of the photo - interpreted cover values was used to test for differences between the photo - interpreted and nlcd predicted cover values for each zone . That is, the nlcd estimate was determined to be significantly different from a photo - interpreted value if the nlcd value was outside the 95% confidence interval bounds of the interpreted value . In some cases, the number of points falling on tree canopy or impervious cover within an nlcd general lc class would be zero, and thus the standard error and confidence interval estimate of the percentage cover would also be zero . In this case, any non - zero nlcd cover estimate would be considered significantly different from the interpreted values, no matter how small the difference . To avoid these minor differences being considered significantly different from cover values of zero, the standard error of the zero cover estimates was calculated using a sample size of one for the cover estimate (i.e., x = 1 instead of x = 0). These adjustments for the test of significance are noted in the appropriate tables (tables 1, 2). Spearman correlations were also used to determine if differences between photo - interpretation and nlcd values were correlated with the amount of photo - interpreted cover.table 1difference between photo - interpreted and nlcd 2001 derived tree canopy cover values by generalized nlcd land cover classes within each mapping zonezonedeveloped land coverforest land coveragriculture grassland land coverother land covernpisenlcddiffnpisenlcddiffnpisenlcddiffnpisenlcddiff16347.66.331.715.9 * 79980.01.469.410.6**8417.94.20.417.4**4613.05.00.412.6 * 29348.45.229.019.4**69587.51.370.716.8**17915.12.70.314.8**2917.27.00.516.7 * 34139.07.626.013.083882.71.350.831.9**11811.02.91.29.9**200.04.61.91.9416621.73.24.117.5**50861.02.221.539.6**28011.11.90.710.4**468.74.20.38.4 * 59310.83.23.17.7 * 5341.56.816.025.6**8159.01.05.33.7**3915.45.81.813.6 * 62536.09.621.114.981168.81.642.626.2**12215.63.33.512.0**378.14.50.87.372035.010.715.419.681071.41.649.521.9**13319.53.40.918.6**3619.46.61.817.7**84314.05.31.112.9 * 4684.91.02.12.8**4641.30.50.50.82317.47.911.16.392119.08.65.813.278720.81.414.36.5**1015.02.20.34.7 * 214.84.60.24.5102040.011.06.733.3**85978.61.453.325.2**12421.03.76.914.1**2015.08.05.010.0122010.06.70.59.589612.11.16.65.5**585.22.90.34.9390.02.50.00.0132010.06.71.98.18798.60.90.87.9**2213.67.31.612.0813.72.10.13.6144316.35.61.115.2**85617.31.31.016.3**725.62.70.74.8293.43.40.52.9152634.69.312.921.7 * 94534.91.624.610.3**358.64.70.67.92520.08.00.919.1 * 16210.04.68.68.691837.51.629.58.0**410.02.42.82.8230.04.30.40.4172714.86.810.14.775719.81.48.611.2**1037.82.60.37.4**1130.00.90.00.0182020.08.91.618.459910.51.33.37.3**3594.51.10.34.1**2920.77.50.520.2**19205.04.92.62.466462.31.947.115.3**3056.91.41.95.0**2015.08.01.713.3202010.06.71.18.915851.34.023.228.1**81814.91.20.214.7**2025.09.72.122.9 * 21205.04.95.30.378550.21.842.87.4**1877.01.92.05.0**200.04.94.14.1222015.08.02.212.88007.80.91.95.9**1659.12.21.18.0**2611.56.35.46.123205.04.93.31.781221.21.413.77.5**1160.90.90.80.0653.12.10.13.024219.56.46.13.477916.31.314.12.21884.81.62.32.5273.73.61.12.6252123.89.32.421.4 * 81716.91.37.39.6**1323.01.50.82.2290.03.40.10.1262010.06.70.59.58789.61.00.88.7**999.12.90.19.0**2114.37.60.114.227205.04.92.62.426621.42.512.29.2**7113.70.70.03.6**205.04.91.63.428200.04.911.311.3 * 74961.51.848.413.1**19910.12.12.97.1**365.63.83.52.029205.04.92.82.244821.92.014.17.8**5280.80.40.10.7200.04.91.11.130210.04.60.40.45217.35.218.51.29161.00.30.10.8 * 210.04.60.80.831240.04.10.90.93455.98.543.812.19192.50.50.52.0**319.75.31.18.53210214.73.53.511.2**21372.83.159.813.0**67610.81.20.810.0**2010.06.75.54.533360.02.91.31.32626.98.712.914.09401.20.40.01.1**224.54.41.72.934340.02.70.40.430024.02.54.020.0**6592.10.60.02.1**205.04.90.54.5353912.85.48.54.465424.21.719.05.2**2956.11.41.34.8**205.04.91.33.7367514.74.18.26.541952.02.422.129.9**4648.01.30.87.2**424.83.34.30.4376432.85.919.813.0 * 66782.21.567.714.4**20213.42.40.612.7**659.23.60.78.5 * 38498.23.93.64.62676.98.359.717.2 * 9223.00.60.92.1**2114.37.64.89.539596.83.30.86.02470.89.345.125.8 * 8901.20.40.11.2**352.92.80.12.740464.33.01.03.33262.58.666.84.38632.40.50.32.1**561.81.82.10.3415918.65.19.49.258779.21.778.90.326414.42.23.910.5**8625.64.724.70.942869.33.14.64.77483.84.380.13.78313.10.61.61.6**2619.27.713.26.1436414.14.33.710.3 * 14381.83.270.711.1**7879.41.01.57.9**2025.09.713.411.6445630.46.122.77.662289.11.377.511.5**32210.91.73.57.4**2030.010.213.416.6455420.45.59.610.8 * 23286.62.274.712.0**7103.90.71.22.7**2030.010.227.72.3467549.35.828.520.8**71585.71.373.911.8**20122.93.01.921.0**2227.39.524.52.8476716.44.52.114.3**48784.01.765.019.0**44110.21.40.59.7**2240.910.523.917.0489435.14.98.226.9**59586.41.476.79.7**30619.62.30.119.5**2010.06.70.59.54912715.03.25.59.5**15172.23.667.74.57182.80.60.22.6**2119.08.64.914.1506939.15.912.626.5**46783.11.772.610.5**43110.71.50.99.8**3218.86.95.513.35110738.34.714.523.8**53488.21.470.218.0**32516.32.04.711.6**3450.08.619.930.1**5213010.02.66.13.99189.03.376.812.2**7747.10.91.35.8**2114.37.626.612.3537154.95.932.922.0**74794.10.985.28.9**17230.83.59.021.8**2920.77.52.218.5 * 5413040.04.330.69.4 * 60989.71.281.08.7**25335.23.01.933.3**2030.010.20.329.7**556647.06.128.118.9**62789.31.278.710.6**26725.12.72.023.1**4015.05.62.812.2 * 5619926.13.18.018.2**36469.22.455.114.1**24917.32.42.414.9**18531.43.43.827.5**575935.66.229.36.374893.70.986.86.9**19314.52.51.313.2**2035.010.70.234.8**587737.75.516.221.5**54984.71.566.817.9**34423.82.32.421.4**3013.36.23.110.25910945.94.817.528.4**61189.51.282.37.2**28027.52.71.625.9**2055.011.11.153.9**6013829.73.910.819.0**43985.91.780.05.8**36922.52.20.422.1**5226.96.21.225.7**619145.15.224.021.0**69092.91.074.718.1**20717.42.64.213.2**267.75.26.01.76214344.14.217.027.1**58987.41.482.15.4**26319.02.43.415.6**2020.08.96.613.4637234.75.617.816.9**60687.31.478.39.0**31218.32.29.58.8**2317.47.929.812.4647538.75.633.65.171690.51.187.13.5**16821.43.20.720.8**2729.68.81.628.0**6518242.93.729.313.5**68688.91.285.93.0 * 11422.83.90.422.4**219.56.40.68.9663842.18.07.434.7**88591.50.978.013.5**5928.85.94.224.6**2733.39.114.119.2*n number of photo interpreted points, pi photo - interpreted value, se standard error of photo - interpretation estimate, nlcd nlcd derived cover value, diff pi value minus nlcd value * significant difference at 95% confidence level; * * significant difference at 99% confidence levelnumber of points hitting the cover type was 0, so se = 0 . Number of points hitting cover type was set to one to calculate displayed se and that adjusted value was used in the significance test to avoid testing using a zero standard error and confidence intervaltable 2difference between photo - interpreted and nlcd 2001 derived impervious cover values by generalized nlcd land cover classes within each mapping zonezonedeveloped land coverforest land coveragriculture grassland land coverother land covernpisenlcddiffnpisenlcddiffnpisenlcddiffnpisenlcddiff16323.85.429.45.67991.30.40.01.2**842.41.70.12.3462.22.20.02.129328.04.726.81.26950.60.30.00.6 * 1792.81.20.02.7 * 290.03.40.00.03419.84.614.95.18380.00.10.00.01181.71.20.01.71130.90.90.00.8416640.43.835.94.55080.60.30.00.62802.91.00.02.8**460.02.20.00.059325.84.529.63.8531.91.90.01.98151.50.40.01.5**390.02.50.10.162532.09.324.17.98110.40.20.10.21220.00.80.20.2370.02.70.00.072015.08.017.42.48100.40.20.10.21330.80.70.10.6360.02.70.10.184332.67.116.615.9 * 4680.20.20.10.24640.60.40.00.6230.04.30.10.19214.84.613.68.87870.30.20.10.11010.01.00.10.1210.04.60.10.1102025.09.726.71.78590.80.30.10.7 * 1240.80.80.20.6200.04.90.20.2122015.08.015.70.78960.20.20.10.1580.01.70.10.1390.03.50.10.1132035.010.733.71.38790.60.30.10.5220.04.40.10.1813.72.10.33.4144346.57.632.014.68561.10.30.11.0**722.81.90.02.7293.43.40.03.4152615.47.119.23.89450.20.10.10.1352.92.80.12.7250.03.90.10.1162123.89.314.89.09180.20.20.00.2412.42.40.02.4230.04.30.00.0172744.49.634.79.77570.50.30.20.31033.91.90.23.71130.00.90.10.1182030.010.214.315.75990.80.40.10.7 * 3591.40.60.01.4 * 290.03.40.00.019200.04.919.719.7**6640.80.30.10.7 * 3050.70.50.20.5200.04.90.00.0202015.08.012.52.51581.30.90.11.18180.40.20.20.2200.04.90.20.2212025.09.712.312.77850.00.10.00.01870.00.00.10.1200.04.90.00.0222010.06.718.18.18000.60.30.20.41650.60.60.20.4260.03.80.20.2232035.010.720.914.18120.40.20.10.21160.00.90.10.1650.01.50.00.024219.56.416.77.27790.60.30.20.51880.50.50.10.4270.03.60.10.1252152.410.923.429.0 * 8170.60.30.10.51320.80.80.10.7293.43.40.13.4262025.09.714.210.88780.60.30.10.4991.01.00.11.0210.04.60.30.3272010.06.714.44.42660.80.50.10.77110.80.30.10.8 * 205.04.90.14.9282045.011.118.426.6 * 7490.90.40.10.8 * 1992.01.00.21.8360.02.70.00.0292025.09.719.45.64480.40.30.10.35280.20.20.20.0200.04.90.40.430219.56.49.40.2520.01.90.00.09160.70.30.10.6 * 210.04.60.00.0312420.88.313.96.9340.02.90.00.09190.70.30.10.6 * 310.03.20.10.13210228.44.522.65.82131.40.80.01.46761.00.40.01.0**200.04.90.10.1333641.78.215.126.5**260.03.80.00.09400.70.30.00.7**224.54.40.04.5343423.57.311.212.33001.00.60.10.96590.80.30.10.7 * 205.04.90.24.8353917.96.120.52.56541.70.50.11.5**2951.40.70.11.3205.04.90.44.6367524.04.926.42.44190.70.40.00.74640.60.40.00.6420.02.40.00.0376412.54.126.113.6**6671.60.50.01.6**2022.01.00.11.9651.51.50.01.5384918.45.516.12.2260.03.80.00.09221.40.40.01.4**210.04.60.00.0395927.15.810.616.5**240.04.10.00.08900.90.30.00.9**350.02.80.00.0404615.25.311.04.2323.13.10.03.18630.60.30.00.6 * 561.81.80.01.8415937.36.322.414.9 * 5870.90.40.10.8 * 2643.01.10.03.0**861.21.20.01.1428619.84.314.35.4741.41.30.01.38311.30.40.01.3**263.83.80.03.8436425.05.420.05.01430.70.70.00.77871.40.40.01.4**205.04.90.14.9445626.85.918.78.16221.10.40.01.1**3222.80.90.02.8**2010.06.70.19.9455425.96.010.715.2 * 2320.40.40.00.47102.50.60.02.5**200.04.90.00.0467522.74.817.15.57151.40.40.01.4**2014.01.40.04.0**224.54.40.04.5476723.95.218.05.94871.00.50.01.0 * 4412.50.70.02.5**220.04.43.63.6489431.94.819.512.4**5951.70.50.01.7**3064.61.20.04.5**2010.06.70.69.44912726.03.928.32.31510.70.70.00.77181.70.50.01.7**214.84.60.24.6506917.44.621.23.84671.10.50.01.1 * 4312.10.70.02.0**320.03.10.00.05110729.04.425.33.75341.10.50.01.1 * 3251.50.70.01.5 * 345.94.00.05.95213033.14.121.112.0**912.21.50.02.27742.10.50.02.1**219.56.40.29.353718.53.321.713.2**7470.80.30.00.8 * 1725.21.70.05.2**2910.35.70.010.35413024.63.820.74.06091.30.50.01.3**2534.71.30.04.7**2010.06.70.29.8556621.25.017.53.76271.60.50.01.6**2673.01.00.03.0**402.52.50.02.55619943.23.526.416.8**3642.50.80.02.4**2495.21.40.05.2**1851.60.90.01.6575925.45.713.112.4 * 7481.90.50.01.9**1934.71.50.04.6**200.04.90.10.1587726.05.019.56.55492.40.60.02.4**3444.41.10.04.3**300.03.30.00.05910939.44.719.819.6**6111.50.50.01.5**2808.61.70.08.6**205.04.90.05.06013844.94.233.311.6**4391.10.50.01.1 * 3693.30.90.03.2**521.91.90.11.9619117.64.018.91.36900.60.30.00.6 * 2073.91.30.03.9**267.75.21.66.16214328.73.821.76.95890.80.40.00.8 * 2634.21.20.04.2**2015.08.00.714.3637227.85.320.57.36062.10.60.02.1**3122.60.90.02.5**230.04.30.10.1647534.75.522.212.5 * 7161.80.50.01.8**1683.61.40.03.6 * 270.03.60.00.06518239.63.634.65.06863.10.70.03.1**1147.92.50.07.9**219.56.40.09.5663826.37.121.15.28851.80.40.01.8**593.42.40.03.4277.45.00.07.4n number of photo interpreted points, pi photo - interpreted value, se standard error of photo - interpretation estimate, nlcd nlcd derived cover value, diff pi value minus nlcd value * significant difference at 95% confidence level; * * significant difference at 99% confidence levelnumber of points hitting the cover type was 0, so se = 0 . Number of points hitting cover type was set to one to calculate displayed se and that adjusted value was used in the significance test to avoid testing using a zero standard error and confidence interval difference between photo - interpreted and nlcd 2001 derived tree canopy cover values by generalized nlcd land cover classes within each mapping zone n number of photo interpreted points, pi photo - interpreted value, se standard error of photo - interpretation estimate, nlcd nlcd derived cover value, diff pi value minus nlcd value * significant difference at 95% confidence level; * * significant difference at 99% confidence level number of points hitting the cover type was 0, so se = 0 . Number of points hitting cover type was set to one to calculate displayed se and that adjusted value was used in the significance test to avoid testing using a zero standard error and confidence interval difference between photo - interpreted and nlcd 2001 derived impervious cover values by generalized nlcd land cover classes within each mapping zone n number of photo interpreted points, pi photo - interpreted value, se standard error of photo - interpretation estimate, nlcd nlcd derived cover value, diff pi value minus nlcd value * significant difference at 95% confidence level; * * significant difference at 99% confidence level number of points hitting the cover type was 0, so se = 0 . Number of points hitting cover type was set to one to calculate displayed se and that adjusted value was used in the significance test to avoid testing using a zero standard error and confidence interval comparisons of photo - interpreted and nlcd - derived values reveal that nlcd underestimates tree canopy cover by a national average of 9.7% (standard error [se] = 1.0%) and underestimates impervious cover by 1.4% (se = 0.4%). Results varied by mapping zone with a maximum underestimation of tree canopy cover of 28.4% (zone 3) and a maximum underestimation of impervious cover by 5.7% (zone 56) (tables 3, 4; figs . 1, 2). Overall, nlcd significantly underestimated tree canopy cover in 64 of the 65 zones (98%) and impervious cover in 44 zones (68%) compared to photo - interpreted cover values.table 3difference between photo - interpreted and nlcd 2001 derived tree canopy cover values by mapping zonezonenoverall percent tree canopy coverphotosenlcddifference199269.71.358.111.6**299669.71.153.016.7**3101772.31.243.928.4**4100037.41.311.525.9**5100011.01.05.55.5**699559.01.435.623.4**799962.01.440.721.3**89983.60.61.32.3**993019.01.312.66.4**10102371.91.347.824.1**12101311.11.05.95.2**1310028.40.90.77.7**14100015.91.11.014.9**15103133.81.523.410.4**16100334.71.527.57.2**17100016.11.16.89.3**1810079.00.92.26.8**19100942.81.331.011.8**20101620.71.23.916.8**21101241.01.434.36.7**2210118.20.91.86.4**23101317.51.211.26.3**24101513.61.111.42.2 * 2599914.91.16.28.7**2610189.60.90.88.8**2710178.30.83.35.0**28100449.51.438.011.5**29101610.10.96.33.8**3010101.70.41.10.6**3110084.40.61.92.5**32101124.11.113.410.7**3310241.60.40.31.3**3410138.30.81.27.1**35100818.21.213.25.0**36100027.51.310.816.7**3799856.91.243.413.5**3810186.20.63.32.9**3910082.40.40.71.7**409974.40.62.51.9**4199653.81.250.13.7**4210179.80.67.82.0**43101421.41.012.88.6**44102059.91.050.09.9**45101623.00.817.85.2**46101370.11.255.914.2**47101744.91.130.514.4**48101559.51.245.014.5**49101715.80.812.13.7**5099947.51.136.111.4**51100058.61.241.816.8**52101615.30.89.36.0**53101979.11.066.812.3**54101270.41.255.714.7**55100065.91.251.114.8**5699740.41.422.817.6**57102075.00.966.98.1**58100058.91.239.919.0**59102067.41.252.514.9**6099852.11.337.514.6**61101472.31.055.017.3**62101561.41.250.011.4**63101362.61.153.29.4**6498675.01.168.56.5**65100370.11.262.67.5**66100984.71.069.615.1**n number of photo interpreted points, photo photo - interpreted value, se standard error of photo - interpretation estimate, nlcd nlcd derived cover value, difference photo value minus nlcd value * significant difference at 95% confidence level; * * significant difference at 99% confidence leveltable 4difference between photo - interpreted and nlcd 2001 derived impervious cover values by mapping zonezonenoverall percent impervious coverphotosenlcddifference19923.10.52.30.829963.40.52.41.0 * 310170.70.30.70.0410008.10.76.21.9 * 510003.80.62.90.969950.90.30.60.379990.60.20.30.389981.80.40.71.0**99300.30.20.20.01010231.10.30.40.7 * 1210130.30.10.20.11310021.50.40.80.7 * 1410003.20.51.41.8**1510310.40.20.30.21610030.60.20.20.41710001.60.30.80.8 * 1810071.80.40.51.3**1910090.70.30.40.32010160.70.20.30.42110120.10.00.10.02210110.70.30.40.32310130.60.20.30.32410150.70.20.30.4259991.80.30.61.2**2610180.80.30.20.6 * 2710171.00.30.30.7 * 2810041.50.30.31.2**2910160.50.20.30.23010100.80.30.30.53110081.00.30.40.7 * 3210113.60.52.01.5**3310242.50.40.71.9**3410131.60.40.41.2**3510082.20.40.91.3**3610002.60.52.20.4379982.50.51.90.63810182.30.50.91.4**3910082.20.40.51.6**409971.40.40.50.9 * 419963.30.51.12.1**4210172.70.51.11.6**4310143.00.51.41.5**4410203.30.51.12.1**4510163.40.50.62.8**4610133.40.51.22.2**4710173.40.61.42.0**4810155.60.71.93.7**4910174.40.63.41.0509992.60.51.51.1 * 5110004.50.62.81.6**5210165.90.72.63.3**5310192.30.51.80.55410125.00.62.52.5**5510003.50.61.32.2**5699710.50.84.85.7**5710204.00.60.93.1**5810004.70.61.53.2**5910207.80.82.35.5**609987.80.74.43.3**6110142.80.51.71.1 * 6210155.80.73.12.7**6310134.10.61.52.6**649864.60.61.72.9**65100311.30.97.24.1**6610093.00.50.92.1**n number of photo interpreted points, photo photo - interpreted value, se standard error of photo - interpretation estimate, nlcd nlcd derived cover value, difference photo value minus nlcd value * significant difference at 95% confidence level; * * significant difference at 99% confidence levelfig . 1differences in tree canopy cover estimates between photo - interpreted (pi) values and nlcd 2001 by mapping zone (pi minus nlcd value). 2differences in impervious cover estimates between photo - interpreted (pi) values and nlcd 2001 by mapping zone (pi minus nlcd value). Differences of 0% indicate no statistical difference difference between photo - interpreted and nlcd 2001 derived tree canopy cover values by mapping zone n number of photo interpreted points, photo photo - interpreted value, se standard error of photo - interpretation estimate, nlcd nlcd derived cover value, difference photo value minus nlcd value * significant difference at 95% confidence level; * * significant difference at 99% confidence level difference between photo - interpreted and nlcd 2001 derived impervious cover values by mapping zone n number of photo interpreted points, photo photo - interpreted value, se standard error of photo - interpretation estimate, nlcd nlcd derived cover value, difference photo value minus nlcd value * significant difference at 95% confidence level; * * significant difference at 99% confidence level differences in tree canopy cover estimates between photo - interpreted (pi) values and nlcd 2001 by mapping zone (pi minus nlcd value). Differences of 0% indicate no statistical difference differences in impervious cover estimates between photo - interpreted (pi) values and nlcd 2001 by mapping zone (pi minus nlcd value). Differences of 0% indicate no statistical difference based on photo - interpretation, tree canopy cover varied by mapping zone from a low of 1.6% in zone 33 to a high of 84.7% in zone 66 (table 3). Impervious cover varied by mapping zone from a low of 0.1% in zone 21 to a high of 11.3% in zone 65 (table 3). Within developed land, nlcd significantly underestimated tree canopy cover in 31 mapping zones, overestimated tree cover in one zone, and had an overall underestimation of 13.7% (se = 4.6%). Nlcd estimates in developed land also significantly underestimated impervious cover in 14 mapping zones, overestimated impervious cover in three zones, and had an overall impervious cover underestimation of 5.2% (se = 4.8%) (tables 1, 2). Within forest lands, nlcd significantly underestimated tree canopy cover in 57 mapping zones with an overall underestimation of 11.7% (se = 1.4%). It also significantly underestimated impervious cover in 30 mapping zones with overall impervious cover underestimation of 0.9% (se = 0.4%) (tables 1, 2). Within agricultural and grass lands, nlcd significantly underestimated tree canopy cover in 55 mapping zones with an overall underestimation of 6.7% (se = 1.0%). It also significantly underestimated impervious cover in 38 mapping zones with overall impervious cover underestimation of 1.5% (se = 0.5%) (tables 1, 2). Within other lands, nlcd significantly underestimated tree canopy cover in 19 mapping zones with an overall underestimation of 8.0% (se = 3.6%). It did not significantly underestimate impervious cover in any mapping zones; overall impervious cover in other lands was underestimated by 1.3% (se = 1.4%) (tables 1, 2). Differences between photo - interpreted and nlcd tree canopy cover were significantly correlated with the amount of photo - interpreted tree cover (spearman correlation coefficient (rs) = 0.70). Differences between photo - interpreted and nlcd impervious cover were also significantly correlated with the amount of photo - interpreted impervious cover (rs = 0.89). Thus, differences between photo - interpretation and nlcd cover values tended to increase with increased amounts of tree canopy or impervious cover . Differences between cover estimates generated by computer - classified nlcd and photo - interpreted images are not surprising due to methodological differences (dougherty and others 2004) and the reported accuracy of the nlcd (mrlc 2009). However, the overall and variable underestimation of tree canopy cover by nlcd relative to photo - interpretation is important to understand because of the increasing use of nlcd products in environmental management and planning applications (e.g., use of nlcd canopy cover in evaluating habitat distribution and conservation (martinuzzi and others 2009) or hydrologic modeling and monitoring using estimates of impervious surfaces (journal of hydrologic engineering 2009). The average differences found in this analysis of all mapping zones were similar to differences projected from the sampling of geographically dispersed areas within varying population density classes in a preliminary analysis (greenfield and others 2009). The preliminary analysis estimated an average nlcd underestimation in tree canopy cover of 9.7%, which is the same overall difference exhibited by the analysis of all 65 mapping zones . The preliminary analysis found an average underestimation in impervious cover of 5.7% within places (e.g., cities villages) and 1.3% in counties . The analysis of all 65 mapping zones found an average difference of 1.4%, which is similar to the difference from the county analysis . The preliminary county estimates are more representative of the entire mapping zones because of the lower density of populations and impervious surfaces in the counties compared to places . The preliminary study underestimation of impervious surfaces in places (5.7%) is comparable the underestimation exhibited by developed lands (5.4%), as places contain significant amounts of developed land . In comparing nlcd tree canopy cover data with various other estimates of tree canopy cover in syracuse, ny, nlcd produced the lowest tree cover estimate (12.7%), which was much lower than the other canopy estimates that ranged between 21.4 and 26.6% tree cover (walton and others 2008). Contrasting nlcd impervious and canopy estimates with cover estimates derived from higher resolution imagery within a sampled sub - watershed in urban and suburban baltimore, md, revealed that nlcd - derived tree canopy and impervious cover estimates were 10 and 7%, respectively, below the higher resolution estimates (smith and others 2010). This difference was attributed to fine - scale variations in canopy (small patches of trees) and impervious cover (smaller buildings and noncontiguous pavement) that were not detected by the nlcd method . Smoothing of fine scale variation within a coarser resolution datasets has been noted in many other studies (wickham and others 2010; maxwell and others 2008). The limitations of photo - interpretation methods can contribute to the differences found between nlcd and photo - interpreted estimates of tree canopy and impervious cover . Nlcd cover maps were based on circa 2001 imagery, while google earth imagery tended to be from the mid 2000s . When image interpretation began, dates of google earth imagery were not available . When dates became available, imagery dates ranged from the early to late 2000s, with the plurality of image dates tending to be around 20052006 . Thus, the google earth images are from dates subsequent to nlcd images, and with varying temporal differences . Later imagery dates would tend to lead to increased impervious cover due to urban development, which would tend to enhance underestimation by nlcd estimates . The same development factors that increase impervious cover would potentially decrease tree canopy cover over time as trees may be cleared to make space for impervious surfaces . However, increases in tree canopy cover could also be occurring through time through tree planting or natural regeneration . In the northeastern united states, forest land has increased by nearly 7% since 1953, mainly due to agricultural lands that have reverted to forests (smith and others 2009). Thus in some areas, underestimation of nlcd tree canopy cover may be exacerbated by tree growth between the dates of imagery; in other areas the underestimation may be reduced . The overall influence of potential changes in tree canopy cover due to the differing imagery dates is unknown, but believed to be minimal . If differing dates of imagery have a significant effect, this would indicate significant landscape change within the 2000 s, which would signify that the nlcd cover maps are currently obsolete due to rapid landscape change . Wickham and others (2010) found that time lags between reference and map image acquisition dates have little effect on agreement and discuss that land - cover change is rare . Landscape change is not likely the dominant factor in the differences exhibited between nlcd and photo - interpretation estimates . It is more likely that the nlcd cover maps are underestimating tree canopy cover and to a lesser extent, impervious cover . Another photo - interpretation limitation may be photo - interpreter error . As various quality control tests were conducted based on paired comparisons of image interpretation among photo interpreters, the issue of the horizontal positional accuracy of google earth imagery is also not an issue as the mapping zones are large . Google earth was only used to estimate the overall proportion of cover in each mapping zone, not the accuracy of individual pixels . For tree canopy cover, the greatest difference between photo - interpretation and nlcd values tended to be in the zones with the greatest tree cover (figs . 1 and 3). The underestimation of tree cover was greatest in developed land (13.7%), followed by forest (11.7%), other (8.0%) and agriculture and grass lands (6.7%). One potential reason for the underestimation in tree canopy cover may be related to the tree cover structure and distribution . As tree canopy cover tends to become less contiguous (more individual tree crowns) due to development patterns this pattern may partly explain why developed lands have greater overall underestimation of tree canopy cover than forest lands . The potential for greater underestimation would tend to increase with greater tree cover and this underestimation may be exacerbated as the canopy cover becomes more fragmented or unevenly distributed.fig . 3tree canopy cover by mapping zone based on aerial photo - interpretation tree canopy cover by mapping zone based on aerial photo - interpretation as the overall difference between photo - interpretation and nlcd impervious cover is relatively small (1.4%) and impervious cover has likely increased between the nlcd and google earth images, the nlcd impervious cover estimates at the zone scale are reasonable . However, the underestimation of impervious cover tends to be greatest in the eastern united states (fig . 2), which is the most urbanized portion of the country (nowak and others 2005) and likely has had some of the greatest urban development in the 2000s (nowak and walton 2005). These results are comparable to impervious surface area assessment of the mid - atlantic region that showed that nlcd underestimated impervious cover by approximately 5%, with the underestimation occurring regardless of development intensity (jones and jarnagin 2009). The underestimation for the mid - atlantic region (zone 60) in this study was 3.3% . Geographic differences in accuracy have also been shown for nlcd land - cover classes (e.g., shrubland, grassland, deciduous forests) (wickham and others 2010). Other factors that may affect the impervious and canopy underestimation are varying geographies (e.g., topography, vegetation types), different processing or training methods, and varied interpretations and applications of the nlcd protocols used among the 12 different teams within their assigned mapping zones . One example of an interpretation that may result in the varied underestimation of surface cover is the selection of training sites and data used in developing the sub - pixel classification and algorithm used to process the landsat imagery . The image processing and classification derived from rural training sites may vary considerably from training data obtained from urban areas, or from more homogeneous land cover to more heterogeneous land cover sites (greenfield and others 2009; walton and others 2008). Underestimation of tree and impervious cover may also be partly due to masking during map development, including the varied selection of ancillary data used for masking (homer and others 2007; homer and others 2004; huang and others 2001; mrlc 2009; yang and others 2003). While masking was generally used to decrease overestimation resulting from the nlcd regression application, it may have overcompensated and produced this underestimation . While the nlcd tree canopy cover and impervious surface data are a free and easily accessible data set created with consistent methodology that may be used effectively in comparisons across the united states, users of the nlcd tree canopy cover maps should be aware of the overall and variable underestimation of tree canopy and impervious cover . Utilizing nlcd cover estimates for secondary analysis (e.g., tree biomass, rainfall interception) can lead to regional to national underestimation of these cover - dependent secondary estimates . Future research should investigate the apparent widespread underestimation to help improve national cover mapping . A formal accuracy assessment would help in this regard (e.g., stehman and others 2008; stehman and czaplewski 1998, 2003). Nlcd 2001 cover estimates appear to be underestimating tree canopy and impervious cover across the conterminous united states to varying degrees . The absolute underestimation of tree canopy cover (9.7%) is much higher than that exhibited for impervious cover (1.4%). These results indicate that underestimation of tree canopy and impervious cover was related to the amount of tree and impervious cover, with developed lands exhibiting the greatest underestimation of both tree canopy and impervious cover . A better understanding of the differences between nlcd and photo - interpreted cover values can be used to produce more accurate cover maps across the united states.
|
Sheldon - hall syndrome (shs, mim #601680), or distal arthrogryposis (da) type 2b, is a rare autosomal dominant, inherited arthrogryposis syndrome characterized by congenital contractures of two or more different body areas, with no primary neurological disease . The common clinical features of shs include contractures of the distal limb joints causing camptodactyly, and clubfeet, triangular face, downslanting palpebral fissures, prominent nasolabial folds, small mouth, and high arched palate (1). Shs is distinguished as a separate entity from freeman - sheldon syndrome (fss, da type 2a, mim #193700), another da type 2 syndrome, because fss includes striking contractures of the orofacial muscles and is a more severe da than shs (2). Da is a genetically heterogeneous disorder, with at least 10 different da types characterized to date (3). Although the prevalence of arthrogryposis is known to be 1/3,000 (4), there are no epidemiological data for da and fewer than 100 cases of shs have been reported in the literature (5). To date, five genes that encode the skeletal muscle contractile fiber complex have been confirmed as the causative genes of da types 1 and 2 . Among these, mutations in tnni2, tnnt3, tpm2, and myh3 have been shown to cause shs (6). In this study, we present for the first time a korean family with two generations of shs, resulting from a rare tpm2 mutation, who manifested the facial features, short stature, and da typical of shs . We studied a family in which shs affected two individuals, a mother and her daughter . A one - month - old korean girl was referred to the seoul national university children's hospital, for the evaluation of multiple congenital contractures of both hands and feet on december 22th, 2011 . The patient was delivered by cesarean section, because of her mother's narrow pelvic bones, at the 38th week of gestation; her birth weight was 2.56 kg (3rd-10th percentile) and her birth length was 43.0 cm (<3rd percentile). On prenatal ultrasonography, bilateral clenched hands and bilateral talipes equinovarus were suspected . At the initial examination after birth, camptodactyly, overlapping fingers, and adducted thumbs were identified in both her hands . A calcaneovalgus deformity with congenital vertical talus in the right foot and an equinovarus deformity in the left foot she also displayed subtle facial dysmorphism, including a triangular face, downslanting palpebral fissures, and a small mouth . Ten days after discharge from the neonatal unit of our hospital, the infant was admitted to a regional hospital for pneumonia, presumed to be aspiration pneumonia, for one week . At the age of one month, she was on frequent bottle feeding with small amounts of whole milk, because her micrognathia and small mouth caused feeding difficulties . One month later, she suddenly showed difficulty breathing one hour after feeding at home and was transferred to the emergency room of a regional hospital . Although cardiopulmonary resuscitation was applied on arrival at the emergency room, she did not recover and died . She was born in the 34th week of gestation with a birth weight of 1.8 kg (25th-50th percentile). She had difficulty breast - feeding because of her retrognathia and small mouth, so she was bottle fed . Her height was 153 cm (5th-10th percentile) and her weight was 53 kg (25th-50th percentile). Her facial characteristics include a triangular face with downslanting palpebral fissures, low set ears with attached earlobes, small mouth, high arched palate, receding chin, prominent nasolabial folds, broad and long nasal bridge and root, and long philtrum (fig . She was of normal intelligence, but mild bilateral sensorineural hearing impairment was detected with pure - tone audiometry . This was accompanied by camptodactyly with ulnar deviation of all 10 fingers on both hands, and bilateral talocalcaneal coalition, with left foot clubbing . Contracture release operations of fingers had been performed five times, and the talocalcaneal coalition had been excised . However, she did not complain of gait disturbance and the motion in neither hip joint was limited . Familial shs was suspected based on the family's medical history and the findings and the findings of physical examinations . A direct sequencing analysis of the myh3, tnni2, tnnt3, and tpm2 genes was performed to confirm shs at the molecular genetic level . A previously reported mutation in exon 4 of the tpm2 gene, c.397c> t (p.r133w), was detected in the daughter (fig . A genetic analysis of her parents revealed that the mother carried the same mutant allele . The tpm2 gene is also known to be one of the causative genes of nemaline myopathy . Therefore, we examined the mother with electromyography (emg), although there was no subjective muscle weakness in her daily activities . The emg findings revealed generalized myopathy, with relative sparing of the slow - twitch muscle fibers . We provided genetic counseling for the family, and the mother is planning a prenatal genetic diagnosis in her next pregnancy . Da is clinically defined as a group of inherited limb malformation syndromes, with contractures primarily involving the distal limbs, and skeletal muscle weakness (7). Da shows marked clinical and genetic heterogeneity, with at least 10 different forms of da documented to date (3), the classification of the different types of da syndromes is difficult because of the reduced penetrance and variable expression of the disease . Fss and shs are the most distinctive da subtypes because they include additional facial characteristics . Recently, mutations in the genes encoding the skeletal muscle contractile fiber complex (tnni2, tnnt3, tpm2, myh3, and mybpc1) have been identified as the causes of da types 1 and 2 (6). Among these genes, mutated myh3 is thought to be the most common cause of shs, with myh3 mutations found to account for 32% of shs (8). Therefore, we sequenced the tnni2, tnnt3, and tpm2 genes directly and identified the p.r133w mutation in tpm2 . Tropomyosin is central to the control of calcium - regulated striated muscle contractions via its interaction with actin and the troponin complex (9). Tropomyosin has three isoforms,,, and -tropomyosins, and -tropomyosin is encoded by the tpm2 gene . Tpm2 is mainly expressed in slow - twitch muscle fibers, but -tropomyosin protein expression is higher in fast - twitch muscle fibers than in slow - twitch fibers (10). Different tpm2 mutations have been identified in association with a wide range of skeletal myopathies besides das, including congenital myopathy, nemaline myopathy, and cap disease (11, 12). However, das associated with tpm2 mutations are quite rare: only two tpm2 mutations have been reported as causes of da, each in only one family . The p.r91 g mutation was found in one family with da type 1 (13), and the p.r133w was identified in one family with shs with muscle weakness . However, the muscle weakness associated with this mutation was not accompanied by progressive muscle wasting or histopathological abnormalities, except slow - twitch fiber predominance (14). Although we did not take a muscle biopsy and our patient did not complain of motor weakness during her daily activities, her emg results suggested generalized myopathy, with relative sparing of slow - twitch muscle fibers . The previously reported patients with the p.r133w mutation were 28 and 65 yr old at the time, and had prominent muscle weakness, mainly in the hands and feet . Mild progression of this muscle weakness was described only by the 65-yr - old patient . Our patient is 26 yr old now, and further long - term follow - up with regular monitoring of her motor function will be necessary . The mechanism by which certain mutations in tpm2 cause multiple congenital contractures is still unclear . One hypothesis is that mutations in the tropomyosin genes cause changes in the actin - myosin interaction and modify the contractile speed (11). A previous in vitro study of the p.r133w mutant tropomyosin also showed that this mutation caused a slower actin - myosin attachment rate and a faster detachment rate (15, 16). Hence, this mutant protein may cause a reduction in the number of myosin molecules in the strong actin - binding state, resulting in overall muscle weakness and da (15, 17). Here, we have reported a family with shs resulting from a tpm2 mutation, who showed typical clinical phenotypes . Although shs is a rare disease, it can be transmitted as an autosomal dominant condition and familial recurrence has been reported for approximately 50% of cases (18). Progressive motor weakness is also possible, and associated feeding problems may lead to a sudden deterioration of the clinical condition, as in our infant patient . An accurate diagnosis followed by appropriate management and genetic counseling should be provided to these patients.
|
A 45-year - old man presented to our outpatient department with painless progressive diminution of vision both eyes for the past 10 days . Medical history included xdr - tb on treatment with linezolid (600 mg / day), ethambutol (800 mg / day), moxifloxacin (400 mg / day), cycloserine (500 mg / day), ethionamide (500 mg / day), and kanamycin (750 mg / day) for the past 6 months . On examination, his visual acuity was 20/200 (ou), not improving with pin hole . Anterior segment examination was unremarkable and pupils were 3 mm, round, regular, and reacting to light in both eyes (direct and indirect). Fundus examination revealed hyperemic disc with blurred margins (ou) [figs . 1 and 2]. Visual field evaluation by humphrey field analyzer showed peripheral constriction and quadrantanopia in the right eye [fig . 3] and low reliable fields in the left eye [fig . 4]. Optical coherence tomography (oct) revealed increased retinal nerve fiber layer (rnfl) thickness in both eyes [fig . 5]. Fundus photograph showing disc edema in right eye fundus photograph showing disc edema in left eye visual field showing peripheral constriction and superotemporal quadrantanopia in right eye visual field left eye showing quadrantanopia (low reliability) stratus oct showing increase in retinal nerve fiber layer thickness involving superior and inferior quadrants in right eye and superior, inferior, and nasal quadrants in left eye (arrows) ethambutol - induced toxic optic neuropathy was initially suspected and tablet ethambutol was discontinued after discussing with the treating physician . After two weeks, the patient's visual acuity had dropped to 20/400 in both eyes, and the fundus picture remained unchanged . Hence, the possibility of toxic optic neuropathy due to linezolid was considered as reported in the literature and linezolid was discontinued (a total cumulative dose of 126 g had been already consumed by the patient). Color vision was restored to normal and patient's vision was restored to 20/20 after one month . Fundus examination revealed resolved optic disc edema with setting in of temporal pallor in both eyes [figs . The patient is under regular follow - up and no toxic effects have been noted at three months of follow - up . Fundus photograph showing resolved disc edema with temporal pallor of optic disc in right eye fundus photograph left eye showing temporal pallor of optic disc follow - up visual field after discontinuation of linezolid showing partial recovery in right eye follow - up visual field in left eye after discontinuation of linezolid stratus oct showing reduction in retinal nerve fiber layer edema in both eyes toxic optic neuropathies are characterized by gradual, progressive, painless, bilaterally symmetric visual loss affecting central vision, and causing central or centrocecal scotoma . Xdr - tb is defined as resistance to at least rifampicin and isoniazid among the first - line anti - tb drugs (which is the definition of multidrug - resistant tb) in addition to resistance to any fluoroquinolone and at least one of the three injectable second - line anti - tb drugs (capreomycin, amikacin, kanamycin). Linezolid inhibits protein synthesis by preventing formation of the ribosome complex that initiates protein synthesis . Its unique binding site located on 23s ribosomal rna of the 50s subunit results in no cross resistance with other drug classes . Hence, linezolid is being increasingly used for the treatment of infections caused by multidrug - resistant gram - positive bacteria . Long - term linezolid interferes with bacterial ribosomes and also with mammalian ribosomes, thereby disrupting mitochondrial oxidative phosphorylation and protein synthesis . Serious adverse reactions demanding withdrawal of the drug include myelosuppression, peripheral and optic neuropathy, lactic acidosis, and serotonin syndrome . The safety of linezolid treatment has been established for use only up to 28 days . There are several case reports of linezolid - induced optic or peripheral neuropathy in patients treated for a time period beyond 28 days . Only two cases of toxic optic neuropathy have been reported following short - term linezolid treatment of 16 days . Fundus picture can be varied, showing temporal pallor, disc edema, or essentially normal . The most common indication for long - term linezolid therapy in these patients has been infection with methicillin - resistant staphylococcus aureus . In our case, neuropathy occurred after linezolid had been used for six months at a dose of 600 mg per day for infection with mycobacterium tuberculosis . We attribute toxic optic neuropathy to linezolid in our patient because even two weeks after stopping ethambutol there was deterioration of vision, and it was only after withdrawal of linezolid that visual improvement started . Linezolid is recommended by the world health organization (who) to treat drug - resistant tuberculosis as a medicine with unclear efficacy . Linezolid, approved for gram - positive infections in 2000 has been used off - label for drug - resistant tuberculosis . There is limited data available regarding the efficacy and safety of linezolid in multidrug - resistant tb since it is always administered as part of combination therapy . Linezolid may improve the chance of bacteriological cure only in the most complicated xdr - tb cases . Its safety profile precludes its use in cases for which there are other alternatives . With increasing emergence of xdr - tb, for which treatment options are limited, physicians are compelled to resort to new drug therapies . Although ethambutol is the most common antitubercular drug implicated to cause toxic optic neuropathy, it is pertinent to be aware that if withdrawal of one drug does not show visual recovery or there is further deterioration of vision, the possibility of toxicity due to other drugs should be thought of . With our country bearing the brunt of tuberculosis, ophthalmologists and physicians must be aware that monitoring of visual function is important in patients on long - term linezolid therapy and that early recognition of toxicity and discontinuation of drug results in complete visual recovery.
|
Atrial fibrillation (af), the most common clinically significant cardiac arrhythmia, has an estimated diagnosed prevalence of about 1.4% in the uk and is rising.1 2 patients with af have a fivefold increased risk of stroke compared with those in sinus rhythm.3 stroke events result in substantial management and follow - up care, and the incurred costs tend to be higher in stroke patients with af than those without.3 4 additionally, af is associated with an increased risk of non - cranial systemic embolism (se).5 despite the substantial clinical risks and economic costs, many patients with af receive inadequate stroke prevention treatment resulting in a rising burden of stroke in patients with af in the uk.2 3 one of the principal aims of af treatment is to avoid thromboembolic events by instituting antithrombotic therapy.3 vitamin k antagonists such as warfarin may prevent up to 64% of strokes in patients with non - valvular af.6 however, warfarin treatment is complex; it has many important interactions with food and drugs, requires frequent laboratory monitoring of the international normalised ratio (inr), and has potential to cause serious haemorrhagic events that can be catastrophic.7 8 due to these concerns, many patients in the uk are under - treated with aspirin or remain untreated . There is a need, therefore, for safer, more efficacious and less complex stroke prevention therapy.3 9 dabigatran etexilate (dabigatran) is the first new oral anticoagulant to become available for the prevention of stroke and se in patients with af in over 50 years.10 it is a reversible direct thrombin inhibitor with stable pharmacokinetic and pharmacodynamic properties and a wide therapeutic margin.11 unlike warfarin, it does not require inr monitoring or frequent dose adjustments . In the randomised evaluation of long - term anticoagulation therapy (re - ly) comparative trial, the 150 mg twice daily dose of dabigatran was superior to warfarin in preventing stroke and se, and the 110 mg twice daily dose was non - inferior (rr vs warfarin 0.65, 95% ci 0.52 to 0.81 for dabigatran 150 mg twice daily; rr 0.90, 95% ci 0.74 to 1.10 for dabigatran 110 mg twice daily). Similar rates of major bleeding occurred with dabigatran 150 mg twice daily and warfarin (rr vs warfarin 0.93, 95% ci 0.81 to 1.07), but the 110 mg twice daily dose had fewer major bleeds (rr vs warfarin 0.80, 95% ci 0.70 to 0.93). The most important haemorrhagic complications associated with anticoagulation therapy (intracranial haemorrhage (ich) and haemorrhagic stroke) were significantly less likely with both doses of dabigatran compared with warfarin.12 13 based on the benefits of dabigatran demonstrated in the re - ly trial, clinical guidelines in europe and north america now include dabigatran as an alternative to warfarin for stroke prevention in patients with af.8 10 14 besides an assessment of relative efficacy and safety, adoption of new treatments is also influenced by cost - effectiveness; the decision whether the added value is worth the added cost . The present study used an economic model to systematically assess the costs and consequences of dabigatran treatment used per its european indication, in which the 150 mg dose is recommended for patients under age 80 and the 110 mg dose for those aged 80 and over . The analyses compare dabigatran with warfarin (with trial - like inr control), aspirin and no treatment . The overall cost - effectiveness of dabigatran was quantified as incremental cost incurred per quality - adjusted year of life (qaly) gained with dabigatran treatment . A markov model was developed to estimate the cost - effectiveness of dabigatran in eligible patients with af, which has been described in detail previously.15 16 in brief, the model assumed that patients received dabigatran 150 mg twice daily until age 80 and 110 mg twice daily thereafter (sequential dabigatran), which reflects the intended clinical use of dabigatran based on the approved european label.17 comparators relevant for the uk setting and evaluated in the model were warfarin treatment, aspirin monotherapy, and no treatment . Patients receiving warfarin were assumed to maintain a level of inr control consistent with that observed in re - ly (mean of 64% time in therapeutic range), which compares favourably with that observed in routine uk practice.18 19 therefore it can be regarded that dabigatran was compared to trial - like warfarin in the uk setting . The model followed patients with af through the natural course of disease in 3-month cycles, included all relevant clinical outcomes and incorporated health states stratified by treatment history, stroke history and disability level.16 major clinical events included in the model were primary and recurrent strokes (ischaemic (is) and haemorrhagic (hs)), se, transient ischaemic attack (tia), acute myocardial infarction (ami), ich excluding hs, major extracranial haemorrhage (ech), minor bleeding and death . Each event was defined in accordance with clinical definitions from the re - ly trial.12 13 is, hs and ich could be disabling or non - disabling, with disabling events resulting in permanent functional deficits characterised by modified rankin score (mrs) for is, and by glasgow outcomes scale (gos) for ich and hs . Patients in each of the four treatment groups in the left figure proceed through the markov process designated by m. patients can be in any of the health states defined by disability and stroke history (illustrated in the middle figure) and by treatment line (not depicted). All surviving patients can have any of the events noted by the square (right diagram) occurring in a given model cycle, including no event . Based on these events, patients may change their health state (eg, developing stroke history or greater disability) and treatment regimen . Stroke history refers to history of those events that increase the risk of subsequent stroke as per the chads2 score (ie, ischaemic stroke or tia). * 150 mg twice daily for patients <80 years; 110 mg twice daily for patients 80 years . Ami, acute myocardial infarction; ech, extracranial haemorrhage; ich, intracranial haemorrhage; tia, transient ischaemic attack . Model outcomes included number of clinical events normalised to 100 patient - years, qalys, total and disaggregated costs (drug, clinical event and follow - up costs) and incremental cost per qaly gained . Qalys are computed by multiplying the time a patient survives by a weight representing their quality of life during that time, with weights ranging from 1 (perfect health) to 0 (death). Because the consequences of stroke and haemorrhage could be life long, the model assumed a lifetime horizon (up to 100 years of age) in the base case . The model assumed patients not discontinuing remain adherent to antithrombotic treatment, and the relative treatment effect remained constant over time . Patients discontinuing treatment received no further clinical benefit . The intention was that all aspects of the analysis were conducted in line with the principles of the national institute for health and clinical excellence (nice) reference case wherever possible.20 the model implementation used microsoft excel . The primary source of clinical input data was the re - ly trial,12 13 and an adaptation of a published mixed treatment comparison based on a network meta - analysis to synthesise efficacy and safety data of treatments frequently used in prevention of stroke and se in patients with af.21 baseline characteristics of the patient population in the model matched those randomised to the re - ly trial . Patients were diagnosed with af plus at least one additional risk factor for stroke or se, as defined by the chads2 risk stratification scheme, or impaired left ventricular ejection fraction . The mean chads2 score in re - ly was 2.1, with roughly two - thirds of patients having a chads2 score of 2 or higher . At baseline as a result, the stroke risk profile of included patients aligns with that expected to be observed in uk practice . The re - ly population was stratified into those aged <80 years and those aged 80 years using a post - hoc subgroup analysis . A sensitivity analysis using the full re - ly dataset for each dose utility values for each disability level and utility decrements due to clinical events were taken from published literature.22 23 these parameters have been described in detail previously and are summarised in the online appendix.16 table 1 summarises drug, event and follow - up costs used in this analysis . Drug costs for warfarin and aspirin were 0.04 and 0.09 per day, respectively, while dabigatran costs were 2.52 per day.24 the average annual cost of inr monitoring for warfarin patients was based on an analysis undertaken by nice25 in the base case, with a plausible range tested in sensitivity analysis due to regional variation within the uk . Acute management costs following se, ami, tia and ech were assessed based on national health service reference costs.26 costs for management of minor bleeds were based on the nice clinical guideline for af.25 acute management costs for all other events (is, ich and hs) were calculated from a recent study of af patient data from a uk stroke registry, as were costs for long - term management of disability resulting from is, ich or hs.27 all cost inputs were inflated to 2010 when necessary.28 major cost parameters (2010 prices in) the base - case analysis compared sequential dabigatran to trial - like warfarin, that is, in patients initiating treatment before 80 years of age, who may receive the full treatment sequence, and in patients starting at or after 80 years of age, who receive only the 110 mg twice daily dabigatran dose . Deterministic sensitivity analyses were used to identify key determinants of cost effectiveness by varying parameters individually . Finally, probabilistic sensitivity analyses (psas) assessed the uncertainty associated with the cost - effectiveness results by performing 5000 simulations for each comparison in which clinical, cost and utility parameters were simultaneously varied randomly within their statistical distributions, based on their means and 95% cis . When 95% cis were not available, standard errors were assumed to be 20% of the mean . For the psas, baseline risks of clinical events were assumed to have beta distributions, while rrs were assumed to be log - normally distributed . A markov model was developed to estimate the cost - effectiveness of dabigatran in eligible patients with af, which has been described in detail previously.15 16 in brief, the model assumed that patients received dabigatran 150 mg twice daily until age 80 and 110 mg twice daily thereafter (sequential dabigatran), which reflects the intended clinical use of dabigatran based on the approved european label.17 comparators relevant for the uk setting and evaluated in the model were warfarin treatment, aspirin monotherapy, and no treatment . Patients receiving warfarin were assumed to maintain a level of inr control consistent with that observed in re - ly (mean of 64% time in therapeutic range), which compares favourably with that observed in routine uk practice.18 19 therefore it can be regarded that dabigatran was compared to trial - like warfarin in the uk setting . The model followed patients with af through the natural course of disease in 3-month cycles, included all relevant clinical outcomes and incorporated health states stratified by treatment history, stroke history and disability level.16 major clinical events included in the model were primary and recurrent strokes (ischaemic (is) and haemorrhagic (hs)), se, transient ischaemic attack (tia), acute myocardial infarction (ami), ich excluding hs, major extracranial haemorrhage (ech), minor bleeding and death . Each event was defined in accordance with clinical definitions from the re - ly trial.12 13 is, hs and ich could be disabling or non - disabling, with disabling events resulting in permanent functional deficits characterised by modified rankin score (mrs) for is, and by glasgow outcomes scale (gos) for ich and hs . Patients in each of the four treatment groups in the left figure proceed through the markov process designated by m. patients can be in any of the health states defined by disability and stroke history (illustrated in the middle figure) and by treatment line (not depicted). All surviving patients can have any of the events noted by the square (right diagram) occurring in a given model cycle, including no event . Based on these events, patients may change their health state (eg, developing stroke history or greater disability) and treatment regimen . Note that stroke history refers to history of those events that increase the risk of subsequent stroke as per the chads2 score (ie, ischaemic stroke or tia). * 150 mg twice daily for patients <80 years; 110 mg twice daily for patients 80 years . Ami, acute myocardial infarction; ech, extracranial haemorrhage; ich, intracranial haemorrhage; tia, transient ischaemic attack . Model outcomes included number of clinical events normalised to 100 patient - years, qalys, total and disaggregated costs (drug, clinical event and follow - up costs) and incremental cost per qaly gained . Qalys are computed by multiplying the time a patient survives by a weight representing their quality of life during that time, with weights ranging from 1 (perfect health) to 0 (death). Because the consequences of stroke and haemorrhage could be life long, the model assumed a lifetime horizon (up to 100 years of age) in the base case . The model assumed patients not discontinuing remain adherent to antithrombotic treatment, and the relative treatment effect remained constant over time . Patients discontinuing treatment received no further clinical benefit . The intention was that all aspects of the analysis were conducted in line with the principles of the national institute for health and clinical excellence (nice) reference case wherever possible.20 the model implementation used microsoft excel . The primary source of clinical input data was the re - ly trial,12 13 and an adaptation of a published mixed treatment comparison based on a network meta - analysis to synthesise efficacy and safety data of treatments frequently used in prevention of stroke and se in patients with af.21 baseline characteristics of the patient population in the model matched those randomised to the re - ly trial . Patients were diagnosed with af plus at least one additional risk factor for stroke or se, as defined by the chads2 risk stratification scheme, or impaired left ventricular ejection fraction . . The mean chads2 score in re - ly was 2.1, with roughly two - thirds of patients having a chads2 score of 2 or higher . At baseline as a result, the stroke risk profile of included patients aligns with that expected to be observed in uk practice . The re - ly population was stratified into those aged <80 years and those aged 80 years using a post - hoc subgroup analysis . A sensitivity analysis using the full re - ly dataset for each dose utility values for each disability level and utility decrements due to clinical events were taken from published literature.22 23 these parameters have been described in detail previously and are summarised in the online appendix.16 table 1 summarises drug, event and follow - up costs used in this analysis . Drug costs for warfarin and aspirin were 0.04 and 0.09 per day, respectively, while dabigatran costs were 2.52 per day.24 the average annual cost of inr monitoring for warfarin patients was based on an analysis undertaken by nice25 in the base case, with a plausible range tested in sensitivity analysis due to regional variation within the uk . Acute management costs following se, ami, tia and ech were assessed based on national health service reference costs.26 costs for management of minor bleeds were based on the nice clinical guideline for af.25 acute management costs for all other events (is, ich and hs) were calculated from a recent study of af patient data from a uk stroke registry, as were costs for long - term management of disability resulting from is, ich or hs.27 all cost inputs were inflated to 2010 when necessary.28 major cost parameters (2010 prices in) sequential dabigatran to trial - like warfarin, that is, in patients initiating treatment before 80 years of age, who may receive the full treatment sequence, and in patients starting at or after 80 years of age, who receive only the 110 mg twice daily dabigatran dose . Deterministic sensitivity analyses were used to identify key determinants of cost effectiveness by varying parameters individually . Finally, probabilistic sensitivity analyses (psas) assessed the uncertainty associated with the cost - effectiveness results by performing 5000 simulations for each comparison in which clinical, cost and utility parameters were simultaneously varied randomly within their statistical distributions, based on their means and 95% cis . When 95% cis were not available, standard errors were assumed to be 20% of the mean . For the psas, baseline risks of clinical events were assumed to have beta distributions, while rrs were assumed to be log - normally distributed . Compared with warfarin, patients treated sequentially over their remaining lifetime with dabigatran experienced fewer total ich and hs (0.43 vs 0.99) and is (3.74 vs 3.97) events per 100 patient - years, but more ech (3.88 vs 3.57) and ami (1.27 vs 1.06) events per 100 patient - years . Predicted number of fatal ich and hs events was also lower with dabigatran (0.18 vs 0.47), whereas the number of fatal is events was similar (1.40 vs 1.43). Clinical event differences were found to be somewhat smaller for is and larger for ich and hs in patients initiating treatment at age 80 or above . In this population, the dabigatran and warfarin groups experienced similar numbers of is (4.19 vs 4.13) but total ich and hs events were more than halved in the dabigatran - treated group (0.58 vs 1.32). These differences in clinical event rates resulted in an increase in qalys for dabigatran - treated patients versus warfarin (8.06 vs 7.82). This was accompanied by higher lifetime cost per patient for disease management with dabigatran (19 645 vs 18 474), due to the higher drug costs (35% of total dabigatran costs vs 17% of total warfarin costs, including inr monitoring). In both treatment groups, follow - up costs represented the largest share of costs (47% for dabigatran vs 61% for warfarin), with the remaining fraction attributed to acute event management . In the scenario analyses, aspirin and no treatment provided fewer qalys (7.59 and 7.12, respectively) than dabigatran . Aspirin resulted in lower overall costs than dabigatran, but the higher event rate in the no treatment group resulted in higher total management costs, despite the absence of drug costs (18 561 for aspirin and 20 475 for no treatment). Note that aspirin and no treatment were compared with dabigatran without a second - line treatment (7.99 qalys and 19 961 management costs). In the population initiating treatment at age 80 or above, total qalys and costs were reduced (4.11 qalys and 10 424 management costs for dabigatran vs 4.04 qalys and 9919 management costs for warfarin). Costs had a similar breakdown of drug, acute event and follow - up costs as the population initiating treatment before age 80 . In the population initiating treatment before age 80, the incremental cost - effectiveness ratio (icer) was 4831/qaly gained, while in the population initiating treatment at 80 the icer was 7090/qaly gained . In the scenario analyses which compared initiating dabigatran before age 80 with treatment with aspirin, the icer was found to be 3457/qaly gained, while dabigatran dominated receiving no thrombophylaxis (ie, more effective and less costly). Deterministic sensitivity analyses for the base - case model showed cost - effectiveness of dabigatran versus warfarin was robust to variations in the majority of parameters, including changes in underlying clinical event rates, costs, utilities and discounting . Key parameters that affected the cost - effectiveness were the degree of inr control attained by patients on warfarin, the rr and overall rates of is, ich and hs for dabigatran versus warfarin, the cost of long - term follow - up care for patients with disability, and time horizon analysed (figure 2). Significant differences in the cost of inr monitoring while on warfarin also had an effect on the icer . To reach a willingness - to - pay threshold of 20 000 and 30 000/qaly gained required an average time in therapeutic range for the whole cohort of approximately 91% and 97% for the population starting therapy at age <80 years, and 80% and 83% in the population starting therapy at age 80 years, respectively . Deterministic sensitivity analysis showing the effect of varying key parameters over a plausible range on incremental cost - effectiveness ratios (icers) of dabigatran initiated prior to age 80 versus warfarin . The black bar shows the results with the first parameter variation indicated in the parentheses (eg, the upper ci limit for rr of ischaemic stroke with dabigatran yields an icer of 13 353/quality - adjusted life year (qaly)), while the grey bar shows the results using the second variation in the parentheses (eg, the lower ci limit for rr of ischaemic stroke with dabigatran yields an icer of 2124/qaly). The base case icer is 4831/qaly . Over a time horizon of only 10 years, the mean survival of the modelled patient population, the icer was 11 898/qaly gained . Using the upper limit of the 95% ci for the rr of is, ich or hs for dabigatran versus warfarin increased the icers to 13 353, 10 013 and 8420/qaly gained, respectively . Using full re - ly clinical results instead of age - stratified results yielded an icer of 4985/qaly and 13 645/qaly in the <80 and 80 populations, respectively . Psa simulation of dabigatran versus warfarin treatment for patients initiating treatment at age 80 or above showed that dabigatran increased qalys in all simulation runs, with most, but not all, showing increased costs . Similarly, dabigatran resulted in an increase in qalys in simulations versus aspirin or no treatment when patients initiated treatment before age 80, and in 82% of simulations versus warfarin when patients initiated treatment at age 80 or above . These curves show the fraction of simulations that resulted in cost - effectiveness below a specific willingness - to - pay (wtp) threshold . For example, the probability that dabigatran is cost effective for patients under the age of 80 years at the commonly cited wtp threshold of 20 000/qaly gained was 98% against warfarin, and 100% against aspirin and no treatment . In patients initiating treatment at age 80, the probability of cost - effectiveness versus warfarin was 63% at the same wtp threshold . Cost - effectiveness acceptability curves showing the fraction of probability sensitivity analysis simulations that yield incremental cost - effectiveness ratios below a specified threshold for dabigatran initiated prior to age 80 versus warfarin (solid black), aspirin (dashed grey), and no treatment (dashed black). Compared with warfarin, patients treated sequentially over their remaining lifetime with dabigatran experienced fewer total ich and hs (0.43 vs 0.99) and is (3.74 vs 3.97) events per 100 patient - years, but more ech (3.88 vs 3.57) and ami (1.27 vs 1.06) events per 100 patient - years . Predicted number of fatal ich and hs events was also lower with dabigatran (0.18 vs 0.47), whereas the number of fatal is events was similar (1.40 vs 1.43). Clinical event differences were found to be somewhat smaller for is and larger for ich and hs in patients initiating treatment at age 80 or above . In this population, the dabigatran and warfarin groups experienced similar numbers of is (4.19 vs 4.13) but total ich and hs events were more than halved in the dabigatran - treated group (0.58 vs 1.32). These differences in clinical event rates resulted in an increase in qalys for dabigatran - treated patients versus warfarin (8.06 vs 7.82). This was accompanied by higher lifetime cost per patient for disease management with dabigatran (19 645 vs 18 474), due to the higher drug costs (35% of total dabigatran costs vs 17% of total warfarin costs, including inr monitoring). In both treatment groups, follow - up costs represented the largest share of costs (47% for dabigatran vs 61% for warfarin), with the remaining fraction attributed to acute event management . In the scenario analyses, aspirin and no treatment provided fewer qalys (7.59 and 7.12, respectively) than dabigatran . Aspirin resulted in lower overall costs than dabigatran, but the higher event rate in the no treatment group resulted in higher total management costs, despite the absence of drug costs (18 561 for aspirin and 20 475 for no treatment). Note that aspirin and no treatment were compared with dabigatran without a second - line treatment (7.99 qalys and 19 961 management costs). In the population initiating treatment at age 80 or above, total qalys and costs were reduced (4.11 qalys and 10 424 management costs for dabigatran vs 4.04 qalys and 9919 management costs for warfarin). Costs had a similar breakdown of drug, acute event and follow - up costs as the population initiating treatment before age 80 . In the population initiating treatment before age 80, the incremental cost - effectiveness ratio (icer) was 4831/qaly gained, while in the population initiating treatment at 80 the icer was 7090/qaly gained . In the scenario analyses which compared initiating dabigatran before age 80 with treatment with aspirin, the icer was found to be 3457/qaly gained, while dabigatran dominated receiving no thrombophylaxis (ie, more effective and less costly). Deterministic sensitivity analyses for the base - case model showed cost - effectiveness of dabigatran versus warfarin was robust to variations in the majority of parameters, including changes in underlying clinical event rates, costs, utilities and discounting . Key parameters that affected the cost - effectiveness were the degree of inr control attained by patients on warfarin, the rr and overall rates of is, ich and hs for dabigatran versus warfarin, the cost of long - term follow - up care for patients with disability, and time horizon analysed (figure 2). Significant differences in the cost of inr monitoring while on warfarin also had an effect on the icer . To reach a willingness - to - pay threshold of 20 000 and 30 000/qaly gained required an average time in therapeutic range for the whole cohort of approximately 91% and 97% for the population starting therapy at age <80 years, and 80% and 83% in the population starting therapy at age 80 years, respectively . Deterministic sensitivity analysis showing the effect of varying key parameters over a plausible range on incremental cost - effectiveness ratios (icers) of dabigatran initiated prior to age 80 versus warfarin . The black bar shows the results with the first parameter variation indicated in the parentheses (eg, the upper ci limit for rr of ischaemic stroke with dabigatran yields an icer of 13 353/quality - adjusted life year (qaly)), while the grey bar shows the results using the second variation in the parentheses (eg, the lower ci limit for rr of ischaemic stroke with dabigatran yields an icer of 2124/qaly). The base case icer is 4831/qaly . Over a time horizon of only 10 years, the mean survival of the modelled patient population, the icer was 11 898/qaly gained . Using the upper limit of the 95% ci for the rr of is, ich or hs for dabigatran versus warfarin increased the icers to 13 353, 10 013 and 8420/qaly gained, respectively . Using full re - ly clinical results instead of age - stratified results yielded an icer of 4985/qaly and 13 645/qaly in the <80 and 80 populations, respectively . Psa simulation of dabigatran versus warfarin treatment for patients initiating treatment at age 80 or above showed that dabigatran increased qalys in all simulation runs, with most, but not all, showing increased costs . Similarly, dabigatran resulted in an increase in qalys in simulations versus aspirin or no treatment when patients initiated treatment before age 80, and in 82% of simulations versus warfarin when patients initiated treatment at age 80 or above . These curves show the fraction of simulations that resulted in cost - effectiveness below a specific willingness - to - pay (wtp) threshold . For example, the probability that dabigatran is cost effective for patients under the age of 80 years at the commonly cited wtp threshold of 20 000/qaly gained was 98% against warfarin, and 100% against aspirin and no treatment . In patients initiating treatment at age 80, the probability of cost - effectiveness versus warfarin was 63% at the same wtp threshold . Cost - effectiveness acceptability curves showing the fraction of probability sensitivity analysis simulations that yield incremental cost - effectiveness ratios below a specified threshold for dabigatran initiated prior to age 80 versus warfarin (solid black), aspirin (dashed grey), and no treatment (dashed black). This economic evaluation estimated the cost - effectiveness of dabigatran compared with warfarin, aspirin and no treatment for prevention of stroke and se in patients with af . The modelled evaluation estimated that use of dabigatran was likely to be cost effective in all comparisons and analyses conducted . That is, for all comparisons, the icers for dabigatran were well below the benchmark wtp threshold of 20 000/qaly gained . The low icers for patients receiving dabigatran reflect the significant reduction in catastrophic events (is, ich and hs) and the substantial savings that were achieved through the reduction in long - term disability as a consequence . Cost effectiveness for dabigatran treatment versus warfarin was demonstrated for patients initiating treatment at age 80 years despite similar clinical benefit for warfarin and dabigatran treatment in terms of is . In this population, which receives only the 110 mg twice daily dose of dabigatran, cost - effectiveness is driven specifically by the reduction in ich and hs and associated reductions in mortality and disability . Deterministic and probabilistic sensitivity analyses showed that these icers were robust to uncertainty and variability in the model parameters . It was demonstrated that average population warfarin control would need to be raised to levels not observed in routine practice for 20 000/qaly gained to be exceeded . These consistent cost - effectiveness results are in line with the improved efficacy and safety outcomes demonstrated in re - ly . These results are also consistent with analyses in the us29 30 and canadian settings,16 though, using a higher us dabigatran price, shah and gage found low risk subpopulations in which dabigatran was less cost effective . The key modelling assumption is that of continued benefit with ongoing anticoagulation treatment . The decision to anticoagulate patients with af should be life long; therefore, it was appropriate to model costs and outcomes over the lifetime without arbitrarily truncating the model time horizon, especially as post - stroke disability continues over patients' remaining life . To be conservative (given the higher discontinuation rate with dabigatran versus warfarin), the model included clinical event risks based on the intent - to - treat population, while also explicitly including discontinuation of treatment . A major driver in the model is cost of long - term disability management . As systematic follow - up of patients in re - ly suffering an event was limited to 36 months, there were limited data available . However, it was possible to stratify stroke outcomes by patient - level data (mrs), and thereby, assign different cost estimates for different disability levels . This should have resulted in more accurate estimates of total costs than assigning overall stroke follow - up costs.31 this model and evaluation offer a number of strengths . Foremost is the use of clinical parameters estimated directly from individual patient - level data in the re - ly study . Second, the main comparator, warfarin as studied in the re - ly trial, can be considered as conservative because the inr control observed in routine uk practice is likely to be inferior in comparison . Third, the model allowed the approved stratification of the two dabigatran doses to be reflected in the correct populations . Fourth, it provided the flexibility to investigate the cost effectiveness of dabigatran compared with other treatment strategies provided to uk patients . Finally, the model has been populated with uk - relevant data, and the stroke risk profiles of the patient populations are representative of those that would be expected in uk patients . In conclusion, treatment with dabigatran reduced the risk of stroke and intracranial haemorrhage compared with warfarin, aspirin and those patients remaining untreated . These clinical benefits offset a substantial portion of the additional drug cost associated with dabigatran, yielding favourable cost - effectiveness ratios well below standard wtp thresholds . Overall, this economic evaluation supports the use of dabigatran as a cost - effective first - line treatment for the prevention of stroke and se in eligible uk patients with af . Using data from the randomised evaluation of long - term anticoagulation therapy (re - ly) trial, and relevant uk costs and resource use, this analysis suggests that the use of dabigatran as a first - line treatment for the prevention of stroke and systemic embolism is likely to be cost - effective in eligible uk patients with atrial fibrillation compared with well controlled warfarin, and
|
Parkinson's disease (pd) is an age - related chronic neurodegenerative disorder with an estimated prevalence of 160 per 100,000 affecting 2 - 3% of people aged 55 and above [1, 2]. The clinical diagnosis is based on the presence of the motor symptoms bradykinesia, resting tremor, rigidity, and postural instability, while the definitive diagnosis can only be made post mortem by detection of -synuclein containing lewy bodies (lb) in the substantia nigra (sn). A number of therapies are available to alleviate the motor symptoms including l - dopa (as gold standard), dopamine agonists, mao - b - inhibitors, the nmda - receptor - antagonist amantadine, and neuromodulation by deep brain stimulation . However, none of them has proven disease - modifying effects and the clinical benefits of the therapy may wear off as the disease progresses . Cell replacement strategies to replace lost dopaminergic input in the striatum of pd patients have led to the proof of principle that fetal mesencephalic transplantations into the striatum increase striatal dopamine levels [6, 7] and reinnervate the striatum [6, 813] but came to a first stop when severe graft induced dyskinesias were found as a major complication [14, 15]. Moreover, detailed analysis of the tissue revealed signs of host - to - graft propagation of lb pathology [10, 12]. Recently, a new trial has been instated that aims at reviving and refining the technique and is funded by the eu as the multicenter project transeuro . Nonmotor symptoms of pd have gained increasing interest due to their major impact on the patients' quality of life and due to the limited availability of symptomatic treatments [1719]. Indeed, the variety of nonmotor symptoms reflects the multisystemic nature of pd, according to the current concept of disease propagation in an ascending pattern [3, 20]. Affected brain regions are identified first by -synuclein - positive lbs and dystrophic lewy neurites (ln). Lb deposition is accompanied and followed by neurodegeneration, but the processes that precede this stage are unclear as yet . According to the staging proposed by braak and colleagues, pd pathology is only detected in two basal cranial nerves nuclei, namely, the glossopharyngeal and the vagal nerve and in the olfactory bulb at an early disease stage (ob, stage 1). This corresponds to the premotor symptoms hyposmia and autonomic dysfunction, including obstipation [2225]. At stage 2, pathology is detectable also in the pontine areas of the locus coeruleus, the raphe nuclei, and the reticular formation . This brainstem affection may cause rapid - eye - movement sleep behavior disorder which is one of the most specific indicators for the future development of pd and occurs in 3050% of pd patients [26, 27]. Depression is a nonspecific but frequent nonmotor symptom of pd that often begins in the prodromal phase and severely affects the quality of life in pd [28, 29]. Stage 3 of pd histopathology marks the involvement of the sn and the anterior olfactory nucleus, whereas significant rates of degenerating neurons in the pars compacta of the sn are only seen in stage 4 . Motor symptoms of pd emerge at stage 4 or later, when disease pathology is already widespread and a substantial proportion of sn neurons degenerated . Thus, disease - modifying therapies should be much more promising when instated in early premotor stages of pd . To this end, pd risk scores have been introduced [30, 31]. Stages 5 and 6, finally, are characterized by the involvement of the basal forebrain and cortical regions, including the entorhinal cortex and the cornu ammonis regions of the hippocampus . This advanced stage of pd is clinically dominated by complicated control of motor symptoms (e.g., fluctuations, dyskinesias, and dysphagia) and severe nonmotor symptoms like parkinson's disease dementia (pdd), psychosis, and sleep - wake disorders . Dementia with lewy bodies (dlb) is characterized clinically by a predominant dementia syndrome preceding motor symptoms and pathologically by neocortical accentuation of lb pathology [3234]. In light of the chronically progressive disease pattern of pd involving olfactory and hippocampal systems, the presence of neural stem cells and active neurogenesis throughout life serves as an attractive model to study pd disease pathology and to test neuroprotective and neuroregenerative treatment approaches . Therefore, in the following review, we will elaborate current knowledge about adult neurogenesis in pd patients and pd models, and we will discuss how these findings may help to understand and to treat pd . It is accepted today that neurogenesis persists in humans in the dentate gyrus (dg) of the hippocampus and in the subventricular zone (svz) beyond embryonic neurogenesis [3538]. Few studies have addressed adult neurogenesis in pd patients, mainly in the svz / ob system . Small and heterogeneous sample groups, post - mortem delay, and the availability of immunohistochemical markers have limited the direct investigation of alterations of adult neurogenesis in the two neurogenic zones of pd patients . The number of cells positive for proliferating cell nuclear antigen (pcna) was reduced in the svz of 4 pd patients when compared to 4 controls . This proliferation defect was related to reduced dopaminergic innervation from the sn pars compacta as seen in animal models of dopaminergic deafferentation [3942]. In line with the hypothesis of dopaminergic control of svz proliferation, a decreased number of epidermal growth factor (egf) receptor positive cells were found in the svz of 6 pd patients as compared to 6 age- and sex - matched controls . In a clinicohistological study of a cohort of 32 pd patients, the number of musashi - positive cells within the svz (representing neural stem and progenitor cells within this area) was positively correlated with the extent of dopaminergic treatment whereas disease duration showed a negative correlation . A similar reduction of musashi - positive cells within the svz was noted in specimen of 5 dlb and 6 pdd cases as compared to 5 controls . The observed decrease in svz proliferation of patients with lb disease may thus be due to a reduction of the number of putative stem cells as a consequence of dopamine depletion . However, a recent study did not detect changes in svz proliferation (as determined by expression of pcna and phh3) in post - mortem tissue of 10 pd patients when compared to 10 controls . No changes in the number of gfap-positive cells as another putative marker of svz stem cells were observed . This study controlled for age, sex, and post - mortem delay and included additional specimen from 5 nondemented controls with incidental lb pathology to take dopaminergic treatment into account . The authors did not detect differences of svz proliferation in the presence of high intragroup variability . The ability to generate svz - derived cultures from pd post - mortem tissue provides a hint for its intact proliferative capacity but currently does not allow quantitative conclusions [46, 48]. Within the authors' explanations for the discrepancies between these human svz studies, it became clear that consent about the optimal methodology concerning tissue sampling, anatomical definition of sampling area, choice of markers for svz stem and progenitor cells, and quantification still needs to be defined [49, 50]. Future studies with new methods thus need to be designed and carefully conducted to resolve these conflicting data . There is a strong correlation between nigral dopaminergic degeneration, cholinergic deficits within the limbic system, and the premotor symptom hyposmia as shown by imaging data [51, 52]. Odor discrimination is a hippocampus - dependent task and hippocampal dopaminergic hypoactivity correlates with hyposmia in pd . However, in addition to the early neuropathological involvement of the ob during the course of pd, direct studies about alterations of the ob in pd suggest that pathogenesis of hyposmia may also take place within the ob . The ob volume was found to be reduced in a post - mortem study of 7 pd patients and 7 controls . Other studies, however, found unchanged ob volumes in pd patients on mr - imaging and histopathologically along with an increased number of dopaminergic ob neurons [57, 58]. In summary, currently available data suggest a complex pathogenesis of hyposmia in pd involving the ob and potentially secondary brain structures . With regard to hippocampal neurogenesis, the density of nestin- and beta3-tubulin - positive cells was found to be reduced in the dg of 3 patients with pd and in the dg of 5 patients with pdd when compared to 3 controls . Similarly, in the dg of 6 patients with dlb, the number of sox2-positive putative stem cells was decreased, as compared to 6 controls . Fatigue and depression have been related to hippocampal serotonergic dysfunction by positron emission tomography with specific metabolites of serotonergic metabolism [60, 61]. Besides, the hippocampus is modulated by dopaminergic input from the ventral tegmental area and the olfactory bulb and by noradrenergic input from the locus coeruleus and may thus be involved in drive and mood regulation . Cognitive deficits in pd are heterogeneous and have mainly been implicated in cholinergic and noradrenergic dysfunction involving hippocampal functions (reviewed in). The extent of hippocampal lb pathology correlated with the degree of dementia in pd patients . Significant hippocampal atrophy is seen on magnetic resonance imaging of patients with pdd when compared to nondemented pd patients (reviewed in). Alterations of hippocampal connectivity by diffusion tensor imaging in pd patients predicted the emergence of declarative memory deficits indicating that altered plasticity may be one of the reasons for structural changes . In summary, hippocampal dysfunction is common in pd patients and likely contributes to depression and cognitive impairment . As both of these nonmotor symptoms have been related to defects in adult neurogenesis, more research about human hippocampal neurogenesis in pd is needed to prove a causal role . In contrast to the limited amount of data and material from human pd brains, many studies have been conducted in pd animal models, mainly in rodents . Stereotactic delivery of 6-hydroxydopamine (6-ohda) into the sn or the medial forebrain bundle leads to lesions of the striatonigral pathway and thus replicates the striatal dopaminergic deficit . Different studies have shown a negative impact of dopaminergic deafferentation on neural progenitor cell (npc) proliferation, probably due to decreased input via d2l - receptors [39, 40, 42, 43, 68]. Despite a decrease in svz proliferation in the 6-ohda lesion model, the number of newly generated dopaminergic neurons in the glomerular layer of the ob is increased, paralleling the finding of higher numbers of dopaminergic glomerular neurons in the ob of pd patients . Local application of the growth factors egf and fgf-2 not only enhances svz proliferation but also induces striatal migration of npcs [7072]. In contrast to the aforementioned results, two studies found increased svz proliferation upon 6-ohda lesion [73, 74]. Systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (mptp) is another way of ablating dopaminergic neurons via mitochondrial damage . Acute administration of high doses results in decreased svz proliferation along with an increased rate of apoptosis of migrating neuroblasts [39, 7577] which was confirmed in a study in nonhuman primates . In contrast, another group reported increased rates of proliferation in the acute mptp model [78, 79] and chronic mptp - treatment at lower doses did not alter svz proliferation . In summary, a definite statement about the precise effect of dopaminergic lesions on svz proliferation cannot be made, but a negative effect of dopaminergic depletion on svz proliferation was a common finding . As mentioned in the previous chapter, it remains disputable whether this also holds true for pd patients . Lesion models acutely damage dopaminergic structures and result in a pronounced motor phenotype . Transgenic models of pd exhibit milder but chronically progressive deficits including nonmotor symptoms (reviewed by [8083]). In addition, transgenic models provide an insight into the disease mechanism of relevant genes and proteins . It is present in lb and ln [21, 84, 85] and different -synuclein mutations and duplications and triplications cause genetic pd [8692]. Common single nucleotide polymorphisms in the -synuclein locus are significantly associated with pd [93, 94]. Adult neurogenesis has been studied in different -synuclein transgenic animal models . A transgenic mouse model overexpressing human wild - type -synuclein under control of the pdgf gene promoter exhibits widespread accumulation within the central nervous system including the hippocampus along with age - dependent memory deficits [95, 96]. In these mice, the survival of newborn neurons is compromised both in the hippocampus and in the olfactory bulb, paralleled by increased levels of cell death in these regions . In mice overexpressing the familial -synuclein mutant a53 t under the same promoter, the neurogenesis deficit is even higher indicating increased toxicity due to the mutation [98, 99]. In a different transgenic mouse model of synucleinopathies, -synuclein is overexpressed under tetracycline - regulatable control of the camkii promoter which led to neurodegeneration within the sn and the hippocampus . Similar to the pdgf promoter transgenic animals, the survival of newborn neurons is impaired in the hippocampus and in the olfactory bulb of these animals and in conditional overexpressers of a30p - mutant human -synuclein [100103]. Interestingly, transgene repression reversed the neurogenic deficit in overexpressers of wild - type -synuclein in the hippocampus, but only partially in the ob . In contrast, transgene repression in a30p - mutant -synuclein transgenic mice reversed the neurogenic deficit in the olfactory bulb, but not in the hippocampus . In principle, the partial restoration of the neurogenic deficit indicates a survival deficit at the integration site rather than a persisting developmental defect and proves in principal that -synuclein pathology is reversible . The persisting deficit of hippocampal neurogenesis despite transgene repression in a30p - mutant -synuclein transgenic animals was related to the uptake of -synuclein from neurons into glial cells . -synuclein is released into the extracellular space and can be taken up by neurons, npcs, and astrocytes [105107]. This may explain the continuous spread of pathology in pd and has to be kept in mind when planning transplantation strategies [108, 109]. The finding that glial a30p--synuclein is not cleared upon transgene repression suggests that -synuclein propagated into glial cells persistently impairs the integration of newborn neurons independent of cell - autonomous expression of -synuclein within the neuron itself . In a bac - transgenic rat model, human -synuclein gene was expressed with its whole genomic locus . These rats exhibited early behavioral changes and a subsequent progressive motor phenotype along with a marked decrease of striatal dopamine content and nigral degeneration . The number of newborn neurons in the glomerular layer of the ob (mostly dopaminergic neurons) was increased in these rats, paralleling preliminary results in humans and the 6-ohda lesion mouse model . The mechanisms that lead to defective neurogenesis in -synuclein transgenic animals are still not well understood . Transgenic overexpression of -synuclein is accompanied by decreased levels of notch which may be mediated by increased p53 signaling [111, 112]. There is growing evidence that oligomeric forms of -synuclein rather than lb and ln constitute the toxic species in the process of -synuclein aggregation . Interestingly, an artificial mutant of -synuclein that is highly prone to form oligomers causes increased dopaminergic toxicity within the sn and synaptic loss in a transgenic mouse model [114, 115]. Therefore, the specific effect of oligomeric species on newborn neurons may be of interest to study pathogenic events of synaptic integration in the future . When studying survival of adult newborn neurons, one has to keep in mind that a complex process of migration, phenotypic transition, lineage determination, outgrowth, and synaptic integration is involved, modulated by many different stimuli . The survival of newborn neurons depends on their proper integration and on a certain degree of synaptic input activity (reviewed in). The outgrowth of dendrites and the formation of synaptic spines are prerequisites for synaptic input . Indeed, dendritic morphology of adult newborn neurons is significantly reduced in -synuclein transgenic animals with an example shown in figure 1 . In addition, the density of mushroom spines reflecting stable synaptic input onto newborn neurons is reduced in these animals . Therefore, increased cell death and reduced survival of newborn neurons in -synuclein transgenic animals may be due to defects in outgrowth and synaptic integration . Upon cell - specific overexpression of -synuclein in newborn neurons, dendrite outgrowth but not mushroom spine formation this led to the conclusion that the dendritic outgrowth defect is due to cell - autonomous effects of -synuclein . -synuclein may, for example, interfere with dendritic outgrowth by direct interaction with microtubule associated proteins and thereby disrupt microtubule assembly and transport . The camp response element - binding protein (creb) pathway could also play a causal role since its activation by the phosphodiesterase inhibitor rolipram rescued the outgrowth defect . Dystrophic lns are a common feature of pd pathology, but they represent a final stage of neuritic degeneration and -synuclein aggregation [20, 119]. The defect of dendrite growth and spine formation rather represents an early feature of pd pathology . Indeed, synaptic dysfunction is an early feature in synucleinopathies and is accompanied by loss of dendritic spines [120122]. Transgenic overexpression of -synuclein alters the vesicle composition of the synapse (vacant synapse) and leads to neurotransmitter release deficits . Thus, the neurogenic system in -synuclein transgenic animal models represents certain features of pd pathology and therefore constitutes a model to study the effect of drugs on synaptic pathology and cellular survival in pd . It is still unclear whether the physiological function of -synuclein overlaps with its pathogenic effects in pd . Knockout of -synuclein in mice does not lead to an overt phenotype, but rather to minor changes of dopaminergic neurotransmission, especially when -synuclein is also deleted [126, 127]. In line with this, -synuclein knockdown by rna - interference in hippocampal neurons reduces the presynaptic vesicle pool size . Physiologically, -synuclein also exerts neuroprotective functions, since deletion leads to increased vulnerability to cysteine - string protein- (csp) deletion and to nigral cell death [129, 130]. Therefore, it is not surprising that neurogenesis is altered in -/-synuclein double - knockout mice . Neuronal differentiation of newborn neurons was increased, which may be caused by altered dopaminergic signaling inputs from the perforant path . Overexpression of human -synuclein in newborn neurons in the -/-synuclein - null background does not impair dendrite outgrowth . This suggests that a certain amount of -synuclein may be necessary to exert these pathological effects, as indicated by the genetic pd forms due to gene duplication and triplication . Mutations in leucine - rich repeat kinase 2 (lrrk2) are the most frequent cause of genetic pd [132, 133]. Clinical and neuropathological features of lrrk2-related pd are mostly indistinguishable from idiopathic pd [134, 135]. Transgenic overexpression of the entire human lrrk2 gene carrying the mutant gly2019ser in a mouse model results in abnormal dopamine signaling and increased levels of phosphorylated tau . In this model, lrrk2 was expressed at high levels within the svz, ob, and the hippocampus which led to a significant reduction of proliferation and survival of newborn neurons within both neurogenic regions . The morphology of newborn hippocampal neurons was markedly impaired with reduced dendrite length and spine density . It remains to be determined in the future whether common mechanisms are involved in the -synuclein and the lrrk2 models . Lrrk2, for example, has been shown to directly impair neurite outgrowth and dendritogenesis in c. elegans and in mouse neurons [138, 139]. Proliferation and survival of newborn neurons were not altered by deletion of endogenous lrrk2, but there was a significant increase of doublecortin - positive (dcx) neuroblasts with higher dendritic complexity in the knockouts . This pro - outgrowth effect may be either due to the direct effects of lrrk2 on neurite outgrowth or due to enhanced integration into the molecular layer of the dentate gyrus . Adult newborn neurons have been proposed to exert different functions that partly overlap with premotor symptoms observed in pd . Data from rodent studies indicate a function of newborn neurons in the adult hippocampus in depression . Depression is a frequent symptom in pd patients that often predates the onset of motor symptoms and was shown to have a high impact on quality of life in pd . Serotonergic inputs to the hippocampus are decreased in pd - related depression (reviewed by). There are indications that adult neurogenesis, on the other hand, is impaired in depression and that the effect of antidepressant therapy relies upon adult - generated neurons which led to the neurogenic hypothesis of depression a number of important studies have shown that alterations in adult neurogenesis are not the one single cause of depressive - like behavior; rather, the dentate gyrus, including the adult generation of newborn neurons, represents one part of a mood - network with other hippocampal subregions, amygdala, thalamus, the anterior pituitary, and other cortical and subcortical areas [145148]. Data are mostly from preclinical models due to the methodological constraints of the investigation of adult neurogenesis in humans, but both mri and post - mortem studies have shown reduced hippocampal volumes in major depressive disorder [149, 150]. Epidemiological studies and the presence of neurotransmitter imbalances in pd suggest depression as a specific nonmotor symptom in pd rather than a reactive pathogenesis due to impaired mobility . Likewise, cognitive disturbances in pd (which are also related to pathology within the limbic system including the hippocampus) may be partly caused by alterations in adult neurogenesis . In fact, the most important function of adult hippocampal neurogenesis in rodents is the ability of memorizing two temporally related events (pattern separation [152, 153]). In light of this overlap with pd premotor symptoms and the known involvement of the hippocampus, changes in the plasticity of newborn neurons may contribute to the pathogenesis of depression and of cognitive decline but certainly need more investigation . Regardless of the causal role of adult newborn neurons in pd pathology, strategies to reverse the observed neurogenesis defects might have therapeutic implications . In a recent paper, adult neurogenesis was rescued by chronic oral treatment with the selective serotonin reuptake inhibitor (ssri) fluoxetine which is in routine use as antidepressant . In fluoxetine - treated transgenic animals, proliferation, number of sox2-positive progenitor cells, the number of dcx - positive neuroblasts, and the number of surviving newborn neurons were all restored to the level of fluoxetine - treated nontransgenic animals . As the levels of transgenic -synuclein were unchanged upon treatment, the effects of fluoxetine rather made newborn neurons resistant to the deleterious effects of -synuclein . This effect was paralleled by increased levels of the growth factors bdnf and gdnf which are both investigated in preclinical models of pd [155157]. Fluoxetine treatment also showed marked benefits in a transgenic animal model of atypical pd expressing human -synuclein under control of the myelin basic protein promoter . Ssris are often prescribed in depression including depression in pd; however, detailed studies about the clinical effect in pd patients are lacking . Dual modulation of the serotonergic pathway has been shown to accelerate the onset of antidepressant action on adult neurogenesis and may therefore also be tested in pd models . Pharmacological screens have identified small molecules with a strong impact on adult neurogenesis, but the application in pd models has not been tested so far . Physical activity, a known strong inducer of adult neurogenesis, was found to be another strategy to reverse pd - related alterations of adult neurogenesis as observed in the lrrk2-transgenic mouse model of pd . Interestingly, deletion of the serotonin gene abolishes the proneurogenic effects of running indicating overlapping mechanisms and a causal role of serotonin in exercise - induced neurogenesis . Different kinds of physical activity had positive effects on executive function and, to a limited degree, on cognition in pd patients and in lesion models of pd (reviewed by). The activity - related rescue of adult neurogenesis may also be affected by disease pathology, as observed in a mouse model of huntington's disease . Therefore, investigation of the effects of physical activity on adult hippocampal neurogenesis in more pd models will be necessary . In addition, there are speculations that counteracting inflammatory processes in pd may halt disease pathology . While epidemiological clinical studies may indicate a reduced pd risk after use of nonsteroidal anti - inflammatory drugs (nsaids) but have not been conclusive due to methodological difficulties [164, 165], it is widely accepted that neuroinflammation is involved in pd pathogenesis . Neuroinflammatory activation is not confined to the substantia nigra but is found along with the progressing pathology of the disease . This holds especially true for the olfactory bulb, where microgliosis is found in the olfactory bulb of pd patients and mouse models . Notably, neuroinflammation induced by irradiation or cortical injection of lipopolysaccharides negatively regulates adult hippocampal neurogenesis . Moreover, the proinflammatory cytokine tnf- impairs proliferation of neural progenitor cells in vitro . Levels of tnf- were found to be elevated in the serum of pd patients and were associated with the presence of the nonmotor symptoms depression and anxiety in pd . Thus, inflammatory processes in the neurogenic regions may contribute to the decline of neurogenesis in different transgenic animal models . A detailed analysis of inflammatory changes in the neurogenic regions of these models is still lacking but may represent one of the mechanisms contributing to the neurogenesis deficits . Adult neurogenesis itself is regulated by inflammatory activation and sophisticated studies showed both pro- and anti - inflammatory effects for different subtypes of microglia [176178]. Interestingly, the modulation of adult neurogenesis by physical activity and enriched environment also seems to be dependent on microglial function [179, 180] which again underlines the need for a better understanding of microglial activation in the neurogenic niche in pd . Although the precise contribution of microglial activation to pd pathology is still elusive and may function as a multiplier of pd - associated neurodegeneration, an interaction between microglia and adult neurogenesis in the pd brain is likely . In summary, many studies in animal models have shown effects of pd pathology on the adult generation of newborn neurons, in part with conflicting results owing to different experimental conditions . Data on adult neurogenesis in human pd are still scarce but will be important to validate experimental findings . In the future, novel techniques will facilitate analysis of adult neurogenesis in animals and in patients [181, 182]. In addition, the discussed models of impaired neurogenesis in pd will serve as drug screening platform to validate drugs aimed at modifying the course of pd.
|
The community health environmental scan survey (chess) is an empirical tool developed by the cih evaluation team to systematically document, map (via gps), and assess the environments in which people, shop, live, work, and play as they relate to diet, physical activity, and tobacco use . The main objective of chess is to improve our understanding of the environment's that we live in that promote healthy eating, physical activity, and tobacco use and the link between this and a population's health behaviors and resulting health outcomes . We first performed a literature search of available tools that assess community environments related to diet, physical activity, and tobacco use . Prior to chess, there were no tools addressing all three risk factors simultaneously; however, separate tools were found that assess stores (20), restaurants (21), farmer's markets, schools, workplaces, and the built environment supporting physical activity (2224). We also consulted with international experts in the fields of diet, physical activity, and tobacco use in order to develop a framework for assessing each risk factor . Some aspects of accessibility are measured using gis mapping as well as other key attributes (e.g. Hours of operation). Affordability could not be systematically measured, although some aspects of cost data are collected . Chess includes eight brief assessment tools that inventory streets, stores, restaurants, street vendors, recreational facilities, parks / gardens, vending machines, and the information environment . Table 1 includes the main items of chess that are used in the analysis for this paper (the complete listing of chess components can be found in appendix 1). The assessment of a community assessment using chess is conducted via a neighborhood walk, which initiates from selected schools within each community and extends in a 400 m radius . We used schools as the main focal point of interest because it is a common urban planning practice to define neighborhood units beginning with schools and other civic facilities (25, 26). Furthermore, schools tend to be more than just places of education for a narrow segment of the population; they are typically integral centers of communities (25, 27), and places of community growth and vitality . Using schools also allows for the collection of comparable data across the cih pilot sites and provides a representative glimpse of the community . Small non - chain grocery chain convenience store non - chain convenience kiosk / fixed stall / mobile stall fresh fruits and/or vegetables high - fat / salt / sugar options (such as sweets, chips, and sugar - sweetened drinks) low - fat / salt / sugar options variety of high - fat / salt / sugar, low- fat / salt / sugar items, fresh fruits, and/or fresh vegetables fast food chain (global) fast food chain (country) fast food chain (local) mixed (fast food and fresh) restaurant chain (global) restaurant chain (country) restaurant chain (local) fig . 2 depicts the overall strategy for the neighborhood walk, beginning with schools in each neighborhood . The neighborhood environmental scan includes walking a 400 m radius around each school and identifying and/or surveying all stores, vending machines, restaurants, recreational facilities, vendors, and so on . The design of the cih project within each community includes administering surveys to children (approximately 2,000 children between 12 and 14 years of age per intervention community).1 maps of the areas to be scanned were created using google earth pro, and the 400 m radii were created using a circular ruler program . Chess data were collected using a personal digital device (pda) with integrated gps and camera (magellan mobile mapper 6). Chess was conducted around a minimum of 50% and a maximum of 100% of all sampled schools (with a minimum of 10 schools and a maximum of 20 schools in the intervention and control areas). To ensure reliability between raters, standard definitions were developed for categorization of the key features of each of the eight scan components and a training manual was created . Members of the evaluation team went to each site and worked with the local research teams to collect data over a 1421 day period . For the first 3 days of each visit, the local researchers from each cih site were trained on the scan and participated in country level adaptations with the evaluation team . Prior to the formal scanning of each community, all raters were trained using the training guidebook, practiced in the field in teams, and then one radius was completed by all raters to ensure reliability . A preliminary inter - rater reliability study of the environmental scan was conducted in one community setting using four raters . In general, the agreement was consistently high overall for the main variables including number of stores, restaurants, and parks (kappas and ac1 close to 1.0), as well as for the presence of fruits and/or vegetables (kappa=0.707, p<.049 and ac1=.901, p<.00) (2) and tobacco (kappa=1.000, p<.008 and ac1=1.000). In order to demonstrate the scope and impact of the chess tool, we present the results along with baseline school level data from the cih mexico site . This data illustrates two important functions of chess: (1) to define the availability and accessibility of healthy food options, and (2) to guide the development and planning of interventions . Similar strategies and analyses can also be conducted to examine physical inactivity and tobacco use, but they are beyond the scope of this illustration . Figure 3 presents a map of the area assessed by chess in both the intervention and control areas . Each of the school radii are mapped, numbered, and noted as intervention or control area . Using the schools as our focal point gives us an understanding of the types of environments in which students interface in their daily lives as well as a representative sample of the entire community as schools are distributed throughout the area, and it allows us to capture the different types of settings rural, semi - urban, and urban in both the intervention and control areas . Overview of the control and intervention area and distribution of the selected schools in both areas . In addition to the school - centric, community - level data collected with chess, the individual - level survey data from students attending each of these schools allows for comparisons to be drawn between the community - level data and student - level behavioral data . These comparisons provide a comprehensive picture of the built environment including facilitators and barriers . In our mexico cih site example, 15 neighborhoods were scanned in the intervention and control areas . Examining only the information collected relating to the food environment and the availability of cigarettes, a total of 583 stores / kiosk / fix care / mobile carts, and 168 restaurants were scanned and gis mapped . The school surveys conducted on students living in the scanned radii included a total of 4,608 youth aged 1214 years . For the modeling of environmental data and student behavior, student data was merged with environmental data that had 2,733 observations from 16 schools . For a summary of indicators used in this analysis and their descriptive statistics, refer to table 2 . More than half of the students reported eating at a fast food restaurant in the last week . The proportion of students smoking cigarettes was 14% and overall tobacco use was 17%; it was not surprising that the overwhelming majority of tobacco use was cigarette use . Summary of fast food and tobacco consumption indicators in table 3, the results of log - linear regression models are presented that explore the association between the types of restaurants scanned and the frequency of students eating at fast food2 restaurants in the past 7 days . Similar associations were explored between the availability of tobacco products in stores and student's tobacco use behavior . Food environment and food behavior the number of days of eating at fast food restaurants during the past 7 days was significantly associated with the percentage of restaurants providing fast foods, as was the percentage of restaurants providing both fast foods and healthier items (mixed restaurant) (table 3). There was not a significant association between the number of days of fast food and the total number of restaurants . When the students were dichotomized into those who had and those who had not eaten in fast food restaurants in the last week, a different pattern of association was observed . There was a significant association between not eating fast food and the total number of restaurants and the proportion of restaurants serving fast foods . These negative associations mean that persons will be more likely to fast food where there are more restaurants and where a higher proportion of them sell fast food . The relationship between the availability of tobacco products and student tobacco use behaviors is presented in table 4 . The odds of a student being a current cigarette smoker, smoking tobacco user, overall tobacco user, or ever having tried smoking cigarettes was greater in radii with a higher percentage of stores selling tobacco . However, a greater proportion of stores having no sales to minor signs was significantly negatively associated with current smokeless tobacco use but not any other type of tobacco use . Tobacco use and tobacco environment by comparing and contrasting the results from the environmental scan (chess) with student food consumption patterns and tobacco use behaviors from their surveys, we have a better understanding of the environments in which they are living . We observe that the types of restaurants and the availability of tobacco products does influence their consumption behavior . Moreover, we can accurately identify the specific communities to design targeted interventions to address tobacco use and unhealthy diet . Results from the chess tool highlight the importance of capturing data about the community environment . As additional layers of data are added, a more complete picture of the community can be developed in order to improve the understanding of the environmental determinates for unhealthy diet, physical inactivity, and tobacco use . The results of the information presented here can serve as a guideline for intervention development around healthy eating and tobacco use among youth . Specific examples include but are not limited to: (1) improving availability of fruits / vegetables by working with local food vendors including school canteens, creating farmers markets, encouraging fast food restaurants to provide fruits and vegetables; (2) providing educational interventions for youth about healthy food choices in restaurants and/or unhealthy aspects of tobacco use; and (3) instituting fines / penalties for selling tobacco to minors . This is one example of many to illustrate the contribution of chess to understanding how a community's attributes affect health behaviors in order to design effective intervention programs . Other examples include but are not limited to: (1) locations of parks and recreational centers and reported physical activity, (2) types of retailers selling single cigarettes and tobacco use and (3) availability of low fat / salt / sugar food options and eating habits . There are few empirical tools available that systematically and simultaneously assess opportunities for healthy eating, physical activity, or reduced tobacco use in neighborhood environments . Tools that do exist focus on physical activity levels (22, 24, 28) or specific aspects of food habits (20, 21) and have been developed for use in developed countries . Chess is the first tool to address all three risk factors for chronic disease simultaneously . The multifactorial nature of chronic disease and its risk factors warrants the development of tools to address proximal factors that influence unhealthy diet, physical inactivity, and tobacco use . Many of the components of these tools are context specific and do not easily translate to non - western and developing country settings . The chess tool and overall methodology was created to address the gap in our current knowledge regarding community context . It is possible to identify secondary data sources and map some information about communities such as parks, stores, and restaurants . However, it is not possible to obtain reliable data on what stores sell, what restaurants sell, what vendors are selling surrounding schools, and/or type of recreational facilities available without actually physically walking and assessing the community . Moreover, the availability of community gis data is limited to developing countries and they do not take into consideration the sometimes rapid changes in communities (e.g. Closing of stores, restaurants). One of the advantages of applying chess in the community is that it enriches one's understanding and engagement of the community environment, which is an important factor in developing successful interventions . All western countries have recognized that the strain on health systems attributed to chronic diseases cannot be sustained if the rates of chronic disease remain unabated . One of the most cost - effective investments is to implement effective prevention programs (29). The environments in which people live, play, and work are important agents in determining their diet, physical activity, and tobacco use . In order to develop effective interventions for the future and scaling up of successful programs, an improved understanding of the complex interplay between environmental determinates and individual behavior is needed . A potential limitation of the environmental scan is that it provides a community snapshot and may not capture the dynamic nature of communities . The street markets and mobile vendors are important sources for where people buy their foods in some communities and may not be captured at the time or on the day the scan is conducted . Another limitation is the ability to measure affordability, given the variability of units in how fruits and vegetables were sold, it was difficult to collect cost data that can be compared across different types of food vendors . However, the pricing data collected is informative in understanding cost of fruits and vegetable for communities where the chess was applied . The process of conducting the neighborhood walk is labor intensive; however, once completed it serves as an important resource to the community as long as data sharing strategies are presented and discussed . Although chess has face validity, it is clear that it will need to be tested in a variety of settings and further reliability and validity studies are needed . Chess is the first environmental assessment tool of its kind to simultaneously assess the three key primary risk factors for chronic disease . The results of the cih research project using the chess will provide the first set of evidence of its kind on the complex interplay between behavior and environmental determinants on food consumption patterns, tobacco use, and physical activity levels . Chronic disease is emerging as the greatest public health challenge of the twenty - first century . Although much of the burden could be prevented through known interventions eating a healthy diet and increasing physical activity (30) little is known about how to address the causes within a complex web of behavioral and societal factors . Despite some successes in intervention programs, there remains limited evidence available on how to translate best and promising practices for chronic disease prevention into different settings . Research in north america and europe has demonstrated the correlation between environmental determinants and physical activity levels and food consumption patterns (13). The evidence generated by this research is now being translated into the development of policies around urban planning, education, health care, and social services . However, research on environmental determinants of physical activity levels and food consumption patterns in developing countries is absent . Finite resources for tackling today's pressing global health challenges mean that it is important for policy- and decision - makers to be armed with the most up - to - date data in order to efficiently and effectively allocate resources . As developing countries continue rapid modernization, a greater understanding of how the health and well - being of their citizens are affected by these changes is required.
|
Clinical practice gives students the opportunity to make decisions, apply theories, prioritize those decisions, learn time management, and provides them with a chance to practice their clinical skills (2, 3). In a supportive learning environment, clinical teachers encourage students to be independent in their learning and they advise them to be self - reliant (4, 5). However, some earlier studies conducted in iran have shown that clinical learning is usually ineffective for nursing students (6), students experience high levels of anxiety (6), nursing instructors are mostly inexperienced, and clinical learning fails to integrate theory and practice (7). However, few studies are available on the reasons behind the ineffectiveness of clinical learning in nursing education . This study aimed to find out the factors that can impede nursing students clinical learning . Participants were purposively selected among the nursing students who were studying in their seventh semester of the baccalaureate program in 2014 and who had clinical rotations in the hospitals affiliated to bushehr university of medical sciences . After the researchers explained the objectives of the study to the participants, 12 students, out of 30, volunteered to participate and to keep reflective journals . In a briefing session, the first researcher gave them a notebook and presented them with a verbal description of how to complete the journaling task . The students were asked to write in their journals about their own experiences, important learning events, attitudes, feelings, and their own opinions on their clinical learning experiences in after passing a week of their practical course in each ward . They were asked to return these anonymous journals to the researcher at the end of each week . 46327) and review board of bushehr university of medical sciences approved and granted the proposal of this study . All of the participants signed a written informed consent form and were assured that their written texts would not be shown to any persons other than the researchers and would not affect their clinical evaluation scores . Thus, the researchers received each journal from the participants, and then they considered them carefully . Analyses consisted of identifying, coding, and summarizing themes according the approach suggested by graneheim and lundman (8). In order to evaluate the data s trustworthiness, the researchers gave summarized results to a few participants to confirm the credibility of the data analysis . The established relationship between the researchers and participating students facilitated a trust - based relationship between the parties in relation to data generation and treatment . Credibility was also enhanced because of the prolonged engagement of the researchers with participants and having experience in the practical field as an instructor . Participants were purposively selected among the nursing students who were studying in their seventh semester of the baccalaureate program in 2014 and who had clinical rotations in the hospitals affiliated to bushehr university of medical sciences . After the researchers explained the objectives of the study to the participants, 12 students, out of 30, volunteered to participate and to keep reflective journals . In a briefing session, the first researcher gave them a notebook and presented them with a verbal description of how to complete the journaling task . The students were asked to write in their journals about their own experiences, important learning events, attitudes, feelings, and their own opinions on their clinical learning experiences in after passing a week of their practical course in each ward . They were asked to return these anonymous journals to the researcher at the end of each week . 46327) and review board of bushehr university of medical sciences approved and granted the proposal of this study . All of the participants signed a written informed consent form and were assured that their written texts would not be shown to any persons other than the researchers and would not affect their clinical evaluation scores . Thus, the researchers received each journal from the participants, and then they considered them carefully . Analyses consisted of identifying, coding, and summarizing themes according the approach suggested by graneheim and lundman (8). In order to evaluate the data s trustworthiness, the researchers gave summarized results to a few participants to confirm the credibility of the data analysis . The established relationship between the researchers and participating students facilitated a trust - based relationship between the parties in relation to data generation and treatment . Credibility was also enhanced because of the prolonged engagement of the researchers with participants and having experience in the practical field as an instructor . The participants believed that interpersonal communication between students, nurses, teachers, physicians, and patients was one of the most important factors affecting clinical learning . From the students point of view, a positive and friendly relationship between students, nurses, and instructors affects the clinical learning environment and increases their self - confidence and learning . Students described that communication with their classmates in the educational environment was positive and satisfying . My friend and i always are together, and ask each other questions that we have, and this is good . However, relationships were not respectful from the side of the qualified nurses, and this affected the students motivation to learn . Some nurses frowned at me, when i asked them a question . From the participants point of view, some nurses did not trusted the students' work, and they blamed them when anything bad happened in the ward . Participants emphasized that the competence of the clinical instructors and staff nurses -as a role models- was a crucial factor affecting their learning in the clinical field . To the students, their instructors kindness, knowledge, clinical skills, expertise, and self - efficacy were among the main characteristics that made them good role models . Some instructors have sufficient knowledge and skills, so they have a higher self confidence level, and they help us (students) to understand how to do our duties . Some of them say to follow whatever staff do . In this however, the majority of the students believed that the lack of clinical experience among instructors was the main barrier that decreased their competency to train students effectively . Nonetheless, although they were technically skillful, they did not always have a caring attitude and sometimes performed procedures without upholding any standards . Participants revealed that there is a theory - practice gap in the clinical setting, and this hindered their clinical learning . The participants indicated that they confronted problems such as confusion about how to do procedures based on theoretical knowledge or like nurses in real situations . One student wrote: we did nt see many things that we have learnt in theoretical class . If we follow standards, the nurses may laugh us and i dread this . Participants wrote in their journals that the nurses were often unaware of educational objectives and the students learning needs; further, their expectations of the students were not the same as the teachers. One of them wrote: the staff nurses demand that we do their routine tasks, such as checking vital signs and changing linens . These are not useful for us, because these are repeated each day and we learnt them well, but we did nt learn some special procedures, like cpr . The participants believed that interpersonal communication between students, nurses, teachers, physicians, and patients was one of the most important factors affecting clinical learning . From the students point of view, a positive and friendly relationship between students, nurses, and instructors affects the clinical learning environment and increases their self - confidence and learning . Students described that communication with their classmates in the educational environment was positive and satisfying . My friend and i always are together, and ask each other questions that we have, and this is good . However, relationships were not respectful from the side of the qualified nurses, and this affected the students motivation to learn . Some nurses frowned at me, when i asked them a question . From the participants point of view, some nurses did not trusted the students' work, and they blamed them when anything bad happened in the ward . Participants emphasized that the competence of the clinical instructors and staff nurses -as a role models- was a crucial factor affecting their learning in the clinical field . To the students, their instructors kindness, knowledge, clinical skills, expertise, and self - efficacy were among the main characteristics that made them good role models . Some instructors have sufficient knowledge and skills, so they have a higher self confidence level, and they help us (students) to understand how to do our duties . Some of them say to follow whatever staff do . In this however, the majority of the students believed that the lack of clinical experience among instructors was the main barrier that decreased their competency to train students effectively . Although they were technically skillful, they did not always have a caring attitude and sometimes performed procedures without upholding any standards . Participants revealed that there is a theory - practice gap in the clinical setting, and this hindered their clinical learning . The participants indicated that they confronted problems such as confusion about how to do procedures based on theoretical knowledge or like nurses in real situations . One student wrote: we did nt see many things that we have learnt in theoretical class . If we follow standards, the nurses may laugh us and i dread this . Participants wrote in their journals that the nurses were often unaware of educational objectives and the students learning needs; further, their expectations of the students were not the same as the teachers. One of them wrote: the staff nurses demand that we do their routine tasks, such as checking vital signs and changing linens . These are not useful for us, because these are repeated each day and we learnt them well, but we did nt learn some special procedures, like cpr . The current study has shown that, despite the crucial role of appropriate relationships in students clinical learning experiences, it is not enough . This finding was in line with findings of jahanpour et al ., who studied clinical decisionmaking in senior nursing students (2). According the participants in this study, the competence of their clinical instructors and the theory - practice gap were among the most important factors affecting them and their learning in clinical settings . Some earlier studies have also pointed out the role of clinical instructors and staff nurses as role models for nursing students learning in clinical setting (6, 9, 10). In iran, many experienced nursing instructors who hold master s or phd degrees are reluctant to participate in clinical education, and so this role is mostly assigned to inexperienced, newly graduated nurses with master s degrees, or those who are still master s degree students . These people are not ready to fulfill their role as competent role models . On the other hand, as cheraghi et al . (7) reported, due to the domination of task - oriented work in clinical settings, nurses clinical behaviors cannot support nursing students efforts to integrate theory and practice . This study was only conducted in one nursing school, and this might limit the generalizability of the results . However, based on out findings, we can say that nursing students lack sufficient clinical learning due to unsupportive and inappropriate communication running throughout clinical settings, they lack effective role models, and they are harmed by the theory - practice gap . Restructuring the healthcare setting so that experienced nursing instructors are encouraged to enter and participate in clinical settings, establishing appropriate workshops for preparing inexperienced nursing instructors for their roles as clinical instructors, and implementing appropriate strategies to motivate staff nurses to take part in the education of nursing students might help in decreasing the barriers to effective learning in clinical settings.
|
Nonsteroidal anti - inflammatory drugs (nsaids) are the most commonly used medications during the dental treatment for their creditable efficacy in reducing pain and inflammation.1,2 however, the burden of their unwanted side effects is high particularly with traditional nsaids.1,2 this is more likely due to chronic use and reflecting the fact that nsaids are used extensively in the more vulnerable elderly population.1 the reported adverse effects include gastrointestinal and cardiovascular events, alterations in renal function, effects on blood pressure, hepatic injury, and platelet inhibition which can lead to increased bleeding.1 this case report describes an unusual rare adverse event of the use of ibuprofen for pain control post restorative treatment . A 26-year - old, otherwise healthy male has reported to the restorative clinic at al - farabi college, riyadh for investigation and management of pain affecting the posterior left region of the maxilla . His history of chief complaint showed typical presentation of severe pain due to advanced buccal cervical carious lesion . Based on clinical findings, radiographic interpretation, and vitality tests, the carious lesion was removed and replaced by composite material (filtek p90, 3 m). In addition, cleaning and shaping were performed using crown down technique with protaper rotary instruments (dentsply). The patient reported to has an increased erectile function and libido after 2 h from taking the ibuprofen tablet . He has reported that has done three sexual intercourses with his wife at that day . On the next day, he was curious, and he repeated taking the ibuprofen tablet . Surprisingly, he had the same experience of his first use of ibuprofen of having increased libido and erectile function that has lasted for at nearly 6 h. he contacted us to question this unusual experience . His medical history was re - reviewed thoroughly with an endocrinologist, and nothing was mentioned . Furthermore, tests for complete blood count, testosterone, thyroid stimulating hormone, cholesterol pre and post ibuprofen intake were assessed, and all results came up as normal . The unwanted side effects are well - reported . To our knowledge, this is the first report to present an unusual side effect of increased libido and erectile function post use of ibuprofen . We searched the literature using medline, accessed via the national library of medicinepubmed interface (http://www.ncbi.nlm.nih.gov/pubmed), for articles relating to the existence of reports of adverse events of erectile dysfunction in relation to the use of ibuprofen or nsaids written in english from 1966 to september 2014 . We used the following mesh terms (nsaids, ibuprofen, erectile dysfunction, libido, and adverse event). Two relevant reports were found and presented in table 1 . These included studies reported sporadic erectile or sexual dysfunction associated with the use of different types of nsaids . In an animal model study, uqochukwu et al . (2011) found that the treatment with nimesulide has an impact on the testosterone and estradiol levels . However, at the doses studied, there were no significant changes in testicular architecture except for mild degenerative changes.5 the most recent study showed that the aspirin was effective in improving lithium - related sexual dysfunction in men with stable bipolar affective disorder.6 with no doubt, we cannot speculate that ibuprofen can improve sexual dysfunction, but nsaids could have a role in treating such patients.
|
As an uncommon injury, laryngeal trauma accounts for less than 1% of blunt trauma in adult patients . Possible sequelae of laryngeal trauma include soft - tissue edema, ruptures of ligaments, fractures of laryngeal cartilage, and dislocation of arytenoid cartilage . Dislocation of the arytenoid cartilage results in reduced mobility of the vocal fold and incomplete glottic closure that mimic vocal fold paralysis . It has been rarely reported to be caused by external blunt forces [15]. Diagnosis of arytenoid dislocation with indirect laryngoscopy, flexible fiberoptic laryngoscopy, videostrobolaryngoscopy, high - resolution ct, and lemg is generally accepted . Lemg is recommended as a major diagnostic tool, although it still has some limitations in distinguishing arytenoid dislocation from laryngeal nerve injury . However, as lemg requires special equipment and skills as well as considerable experience, it is still rarely used in many clinics and hospitals . As such, we investigated whether it is possible to definitively diagnose and effectively treat arytenoid dislocation due to external laryngeal trauma without using lemg . This retrospective study was performed over a period of 15 years from january 1997 to december 2011 . From all patients who visited our department during this period, 97 patients in total were considered to have arytenoid cartilage dislocation, of which 85 (87.6%, 85/97) had received tracheal intubations . In the current study, we re - visited the records of the 12 patients who had a known histology of external neck trauma . Written informed consent was obtained from each patient, and the study was approved by the institutional review board of second affiliated hospital, school of medicine, zhejiang university . They were divided into the satisfied group (n=9) and dissatisfied group (n=3) based on patients self - satisfied with voice qualities at 1 week after the last closed reduction manipulation . The 12 patients underwent a complete head and neck examination and a thorough voice evaluation using the vhi scale, indirect laryngoscope, flexible fiberoptic laryngoscope, video strobolaryngoscope, and/or high - resolution ct . The vhi scale, including functional (f), physiological (p), emotion (e), and the sum denoted by t, was applied in self - assessment before initial closed reduction and at 1 week after the last closed reduction, and mpt was measured as well . The laryngoscopic examinations were selectively performed before initial closed reduction, immediately after the last closed reduction, and 1 week after the last closed reduction . Laryngoscopic findings in arytenoid dislocation include impaired or absent vocal fold mobility and the asymmetric position and different length of vocal cords with poor glottic closure . In addition, arytenoid dislocation should be highly suspected if video strobolaryngoscopy further shows shortened mpt, normal vocal fold vibration, discrepancy between vocal cord heights, and absence of a jostle phenomenon (a symptom of vocal cord paralysis characterized by lateral movement of the arytenoid on the immobile side, caused by contact with the mobile side during adduction). Ct scan of the larynx was used to reveal arytenoid dislocation by interarytenoid asymmetry and clouding or obliteration of cricoarytenoid joint space . Attempted closed reduction, as a diagnostic and therapeutic method, anterior dislocations were reduced with posterior - upward push on the arytenoids during phonation and posterior dislocations with anterior - upward push during inspiration . Arytenoid dislocation was thought to be confirmed in patients who experienced immediate improvement in laryngeal symptoms such as hoarseness after attempted closed reduction . After each closed reduction manipulation, patients were encouraged to speak as much as they could, and taught to hold the larynx gently with fingers and shake it side to side while making a strong sound as if coughing up phlegm from the throat . Attempted closed reduction the means were compared using paired t - test, wilcoxon match - pair signed - ranks test, or mann - whitney u - test . This retrospective study was performed over a period of 15 years from january 1997 to december 2011 . From all patients who visited our department during this period, 97 patients in total were considered to have arytenoid cartilage dislocation, of which 85 (87.6%, 85/97) had received tracheal intubations . In the current study, we re - visited the records of the 12 patients who had a known histology of external neck trauma . Written informed consent was obtained from each patient, and the study was approved by the institutional review board of second affiliated hospital, school of medicine, zhejiang university . They were divided into the satisfied group (n=9) and dissatisfied group (n=3) based on patients self - satisfied with voice qualities at 1 week after the last closed reduction manipulation . The 12 patients underwent a complete head and neck examination and a thorough voice evaluation using the vhi scale, indirect laryngoscope, flexible fiberoptic laryngoscope, video strobolaryngoscope, and/or high - resolution ct . The vhi scale, including functional (f), physiological (p), emotion (e), and the sum denoted by t, was applied in self - assessment before initial closed reduction and at 1 week after the last closed reduction, and mpt was measured as well . The laryngoscopic examinations were selectively performed before initial closed reduction, immediately after the last closed reduction, and 1 week after the last closed reduction . Laryngoscopic findings in arytenoid dislocation include impaired or absent vocal fold mobility and the asymmetric position and different length of vocal cords with poor glottic closure . In addition, arytenoid dislocation should be highly suspected if video strobolaryngoscopy further shows shortened mpt, normal vocal fold vibration, discrepancy between vocal cord heights, and absence of a jostle phenomenon (a symptom of vocal cord paralysis characterized by lateral movement of the arytenoid on the immobile side, caused by contact with the mobile side during adduction). Ct scan of the larynx was used to reveal arytenoid dislocation by interarytenoid asymmetry and clouding or obliteration of cricoarytenoid joint space . Attempted closed reduction, as a diagnostic and therapeutic method, was performed by indirect laryngoscope and laryngeal forceps under local anesthesia . Anterior dislocations were reduced with posterior - upward push on the arytenoids during phonation and posterior dislocations with anterior - upward push during inspiration . Arytenoid dislocation was thought to be confirmed in patients who experienced immediate improvement in laryngeal symptoms such as hoarseness after attempted closed reduction . After each closed reduction manipulation, patients were encouraged to speak as much as they could, and taught to hold the larynx gently with fingers and shake it side to side while making a strong sound as if coughing up phlegm from the throat . Attempted closed reduction the means were compared using paired t - test, wilcoxon match - pair signed - ranks test, or mann - whitney u - test . All analyses were performed using spss 16.0 statistical software (spss inc ., chicago, il). Voice change occurred immediately after injury in 10 cases, and in the other cases at 4 and 7 days after injury . There were no obvious external signs of laryngeal trauma, neither subcutaneous emphysema nor signs of hematoma and fractures, in these patients . Examination with indirect laryngoscope, flexible fiberoptic laryngoscope, and video strobolaryngoscope revealed immobility of vocal cords in 8 patients and impaired movement of vocal cords in 4 patients . The typical observations were the asymmetric position and different length of vocal cords with poor glottic closure (figures 1a, 1b and 2a, 2b). Arytenoid cartilage was anteriorly displaced in 11 patients (5 on the left side and 6 on the right side). One patient had both left - sided anterior and right - sided posterior displacement of the arytenoid cartilages (table 1). Vocal cords were located at the intermediate, paramedian, and lateral positions in 7, 1, and 3 patients, respectively . In the patient with affected bilateral vocal cords, the left vocal cord was located at the intermediate position and the right one at the lateral position . Video strobolaryngoscopic examination demonstrated that mpt was longer than 5 s in 8 patients with normal vocal fold vibration, and mpt for the other 4 patients was too short for us to observe vibration of vocal folds during phonation . The sensibility of the endolaryngeal mucosa was unaffected in all patients, indicating a normal function of the internal branch of the superior laryngeal nerves . Jostle sign was absent, and discrepancy between vocal cord heights was noticeable in these patients (table 2). Typically, the level of the affected vocal cord was lower than that of the normal one during phonation in 9 of the 11 patients with unilateral lesion . The different length between both vocal cords was observed in 12 patients . The affected vocal cord was shorter than the normal one in the cases with anterior malposition of arytenoid cartilage . Seven patients underwent high - resolution ct scans of the larynx . Among them, anterior displacement of the arytenoid cartilage was confirmed in 4 patients (figure 3). As illustrated in table 3, vhi scores at 1 week after the last closed reduction were lower than those before initial reduction in the satisfied group (p<0.05), but these scores were not statistically significantly different from those in the dissatisfied group . Among the 9 patients in the satisfied group, fiberoptic laryngoscopic and video strobolaryngoscopic examination demonstrated that 5 patients obtained normal movement of the vocal cord and the symmetric position and equal length of both vocal cords, with tight closure of the glottis during phonation instantly after closed reduction (figure 1c, 1d). Impaired movement of the vocal cord was still visible and vocal cords were located almost at the same level, with improved glottic closure in the other 4 patients (figure 2c, 2d). Mpt (20.116.11s) in the satisfied group was significantly lengthened at 1 week after the last closed reduction (p<0.05). Hoarseness, together with immobility of the vocal cord and incomplete glottic closure, remained in the dissatisfied group . The time interval between injury and closed reduction in this group was 157.6776.07 days, which was significantly longer than that in the satisfied group (p<0.05). Laryngeal trauma caused by external forces is relatively rare, with an incidence of 1 in every 22 900 emergency room visits [911]. In this report, the finding of 12 out of 97 (12.4%) patients with arytenoid dislocation caused by external trauma is similar to an earlier finding by rubin that 15.9% of their arytenoid dislocation cases were attributed to external neck trauma . It is worth noting that vocal cord immobility may originate either from neurological paralysis or joint abnormalities, or even from both . Lemg could be useful in differentiating these 2 causes by indicating the presence of an innervated vocal cord . However, as pointed out by rubin et al ., lemg has its limitations, with 39.7% of their patients with arytenoid cartilage dislocation displaying abnormalities on lemg . This is possibly due to co - occurrence of denervation and a joint dislocation, nerve susceptibility to inflammatory mediators infiltrating after trauma, and/or hematoma and scarring in the posterior portion of the thyroarytenoid muscle, leading to the appearance of denervation on lemg . Importantly, lemg alone may fail to establish the diagnosis of laryngeal paralysis, because cross - innervation may occur in a paralyzed vocal fold [1214]. The time interval between injury and initial medical examination in our patients was 14240 days (median time 72.0067.59 days), which might be long enough for the occurrence of reinnervation . Furthermore, lemg is unavailable in the majority of the hospitals in china, including our hospital . Therefore, combined with our video strobolaryngoscopic observation showing good muscle tension with vibration of vocal folds in our patients, lemg was not included in the current study . Except forin addition to lemg, flexible fiberoptic laryngoscopy, videostrobolaryngoscopy, and computed tomography are also helpful in the diagnosis of arytenoid cartilage dislocation . According to rubin et al ., the most useful examination is video strobolaryngoscopy, which allows evaluation of jostle sign, flickering of muscle activity associated with an intact nerve supply, and submucosal hematoma that can result in scarring and vocal cord stiffness . Most of the studies investigating the difference in level between paralyzed and innervated vocal cords reported that the paralyzed vocal fold presented at a higher level than a normally innervated one [1519]. In our cases with arytenoid dislocation, conversely, the level of affected vocal cords was lower than that of normal ones in 9 of the 11 patients with a unilateral lesion . As suchtherefore, we would suggest thate an obvious discrepancy in height between vocal cords is an important feature in the differential diagnosis between arytenoid dislocation and laryngeal paralysis . During the 15-year period, we preferpreferred to perform attempted closed reduction by indirect laryngoscope and laryngeal forceps manipulation under local anesthesia . Firstly, it is a feasible method to facilitate diagnosis when patients are highly suspected of arytenoid dislocation, especially in the medical institutions without lemg . The diagnosis of arytenoid dislocation can be confirmed in patients who experience instantaneous improvement in laryngeal symptoms such as hoarseness after attempted closed reduction . More importantly, it can shorten the time interval between the diagnosis and treatment of arytenoid dislocation, which is helpful to achieve a successful reduction . Secondly, it prevents the larynx from being further injured by tracheal intubation under general anesthesia . Thirdly, it is impossible to judge the position of arytenoid cartilage after reduction and to monitor the patient s voice under general anesthesia . Fourthly, most of patients can tolerate the procedure by indirect laryngoscope and laryngeal forceps under local anesthesia, and high cure or improvement rate (78.4%, 76/97) was achieved during our 15-year experience . Rubin et al . Reported that 7 out of 10 patients (70%) with arytenoid dislocation due to external trauma regained normal or improved voice only by closed reduction using direct laryngoscope under local anesthesia and/or voice therapy . In our series, the patients obtained similar outcomes (75%, 9/12). Therefore, closed reduction for patients with arytenoid cartilage dislocation under local anesthesia is practicable and effective . Early treatment of arytenoid cartilage dislocation is crucial for good clinical recovery of voice quality . We have found that vhi scores and mpt were significantly improved in the satisfied group, who had a median time interval between injury and treatment of 43.4434.13 days, whereas the dissatisfied group showing no voice improvement had a median time interval of 157.6776.07 days . A critical difference between the 2 groups was immobility of the vocal cord in the latter, probably related to scarring and fixation of the cricoarytenoid joint that occurred due to the delayed treatment . Similar results were also found by sataloff, who reported that the average time interval between injury and surgical treatment for patients who regained their normal voice was 10 weeks and an average time interval of 29 weeks for patients who had no response to treatment, although the causes of laryngeal trauma were different . Nevertheless, it is obvious that recovery will become more difficult if clinical intervention is delayed, regardless of whether arytenoid cartilage dislocation is caused by internal or external forces . In addition, in our study, the 3 patients had no voice improvement by using a diagnostic reposition approach . There may be another possibility that arytenoid cartilage dislocation and associated vocal fold paralysis could occur among them . Arytenoid dislocation after external blunt laryngeal trauma does occur, although rarely, and is often associated with impaired mobility of the vocal cord . Attempted closed reduction should be considered as a diagnostic approach for highly suspected arytenoid dislocation, especially in the clinics or hospitals without lemg . Furthermore, early closed reduction under local anesthesia is also a practicable and effective treatment option for this kind of disorders as well.
|
The reverse shoulder arthroplasty was approved by the fda in 2004 and has proven a very effective prosthesis for treatment of cuff tear arthropathy and other shoulder conditions requiring arthroplasty in the setting of a deficient rotator cuff . However, in some series, the overall revision rate for the reverse shoulder is approximately 10%, with instability and infection being the most common precipitating causes. [13] studies have also shown that aseptic loosening may contribute to 7% of failures, but as indications expand and long - term follow - up increases, the incidence of aseptic loosening will likely increase . Have further shown that pain increases with longer term follow - up of reverse replacements (510 years). Therefore, in the future, surgeons will be faced with difficult choices when revising the reverse prosthesis, and optimal management of the failed reverse replacement is not known . When instability or loosening of a reverse prosthesis does occur, retention of the reverse prosthesis can be performed by (1) increasing deltoid tension with additional polyethylene or metallic spacers, (2) increasing constraint with a retentive cup or (3) revision of the humeral or glenoid components. [514] however, due to poor bone stock, infection or refractory instability, removal of a reverse replacement (explant) or conversion to hemiarthoplasty can be required . We present a series of failed reverse replacements that were converted back to hemiarthroplasty with either a metallic prosthesis or preformed antibiotic shoulder spacer for infection, instability or glenosphere loosening . To date, there are few reports of reverse replacement conversion to hemiarthroplasty, and none specifically examining the shoulder function in this unique group . We examine a group of patients after conversion of failed or infected reverse shoulder replacement to hemiarthroplasty; the level of function and pain in this group is important for surgeons and patients considering a revision reverse shoulder replacement . From 2008 to 2010, the senior author performed 115 primary and revision shoulder arthroplasty procedures . As part of the shoulder registry (irb#10 - 000859), seven patients were identified that had presented to the senior author's clinic with a reverse replacement that was dislocated (3), infected (4) or loose (3). All of the failed reverse replacements in this study were referred from outside hospitals for tertiary care; revision surgery was performed an average of 9.2 months after implantation of the reverse prosthesis . Patients with known infection were treated with explant of the reverse prosthesis using a slot osteotomy, cement extraction with an oscar device (orthosonics, chatham, nj, usa) and conversion mean size of the defect from 273 (n = 3) (exactech, interspace, gainesville, fl, usa). This antibiotic spacer was cemented in place with additional antibiotic - loaded cement (tobramycin and vancomycin). In three cases of gross loosening of the glenosphere these patients were treated with removal of glenosphere only, bone grafting of glenoid with allograft including cancellous bone chips and demineralized bone matrix, and retention of the humeral stem by converting it via the manufacturer's guidelines to a hemiarthroplasty (n = 4). At the time of reverse removal in this elderly population, the bone stock of the glenoid was cavitary and uncontained, not deemed of sufficient quality to support immediate replacement of a single - stage revision glenosphere . Preoperative computated tomography (ct) was also used in four patients to evaluate the glenoid bone stock, but was not clinically very useful due to metal artifact . This group of seven patients status post removal of reverse replacement were contacted for reevaluation with physical exam and outcome scores specifically for this study . One patient who had been converted from a dislocated, infected reverse replacement to preformed antibiotic spacer chose not to participate, but has not had any further surgery . The case details of the remaining six patients who consented for examination and inclusion in this study are contained in table 1 . Details of six cases of failed reverse shoulder arthroplasty converted to hemiarthroplasty using either a metallic head and retention of the reverse stem or an explant and implantation of a preformed antibiotic - loaded cement hemiarthroplasty there were three males and three females (average age 74.5 years) who had had an average of three previous shoulder surgeries including the index reverse replacement . The reverse replacement had been implanted an average of 9.2 months before failure of the prosthesis required removal . Radiographic evidence of technical problems with the index reverse arthroplasty surgery was present in five of six cases, with superior implantation of the glenosphere (n = 4) being the most common . Average follow - up after removal of reverse replacement was 26.5 months (range 1041 months). The decision to include patients with less than 2-year follow - up was made to maximize inclusion of patients that have been offered conversion back to reverse replacement . Shoulder function was very poor postoperatively (average forward elevation of 42.5 degrees and average external rotation near zero), but the pain level was generally low . Sf-12 scores were similar to us averages in the 75 years - plus age group . Anterosuperior escape was present in five patients (vas instability score averaged 4.7 [range 08]). In the sixth patient, there was static radiographic anterior subluxation (case 1). In the four cases where bone graft was used for glenoid deficiency, radiographic follow - up showed good incorporation of graft and reimplantation with a reverse prosthesis seemed feasible, although none of the patients elected to have this performed . A peripherally inserted central catheter (picc) line was inserted and culture - specific intravenous antibiotics were administered for 6 weeks postoperatively . There were no reoperations and, to date, no patient has accepted an offer to be converted back to a reverse replacement . A 58-year - old male (case 2) presented 6 months after reverse arthroplasty for failed fracture fixation with anterior dislocation of the prosthesis . Notice the superior placement of the glenosphere with slight superior tilt . At the time of revision surgery, no combination of glenosphere or reverse humeral component could result in stability as the prosthesis levered out with adduction past 40 degrees . Removal of a well - fixed glenosphere and baseplate was performed and glenoid bone stock was not sufficient for immediate revision of the baseplate to an improved position . (c and d) conversion to large head hemiarthroplasty and bone grafting of glenoid defect . Cultures were positive for p. acnes at the time of revision a 74-year - old female (case 4) underwent reverse replacement for severe cuff tear arthropathy (a). The glenosphere loosened at 8 months postoperatively with breakage of the inferior screw (c). (d and e) ap and axillary views after conversion to hemiarthroplasty an 80-year - old man (case 5) presented with a draining sinus and a dislocated, infected reverse replacement 6 months after primary reverse replacement at an outside institution that was complicated with reoperation the same day after a recovery room dislocation . Glenosphere position is acceptable, but the entire humeral head was essentially resected, likely compromising stability . (c and d) postoperative radiographs of preformed antibiotic spacer preoperative draining sinus an intraoperative photograph after irrigation and debridement, removal of glenosphere, slot osteotomy and cementation of preformed antibiotic spacer (exactech interspace, gainesville, fl, usa) with additional antibiotic - loaded cement . Notice how the humeral head is devoid of any soft tissue attachments creating a shoulder with profound anterosuperior escape in farshad and gerber's review of reverse arthroplasty complications, they state that the optimal management for a failed reverse arthroplasty is not known and that complications that require removal of the prosthesis result in poor shoulder function . Once a severe complication in reverse arthroplasty is established, retention of a stable reverse prosthesis is difficult and multiple reoperations are common . Gallo et al . Reviewed a series of nine cases of reverse replacement requiring revision for instability in the setting of subscapularis deficiency . In these nine patients, three eventually required explant, three remained chronically dislocated and three were concentrically intact . In a retrospective review of describe their experience with dislocation and infection of reverse prosthesis . In a series of 284 reverse replacements, the rate of instability was similar in patients with primary (11 of 212 [5%]) and revision (six of 72 [8%]) reverse arthroplasty . The rate of infection was higher in the revision (five of 72 [7%]) than in the primary (three of 212 [1%]) group . In this series, the authors reported that a stable noninfected prosthesis was present in only 12 of 25 shoulders revised for infection or instability, and multiple reoperations for both persistent infection and recurrent instability were common in this series . In 2008, norris presented a series of 26 revision reverse replacements for various causes . Not all series of revision reverse arthroplasty have reported such a high rate of rerevision and reoperation . If retention of the reverse shoulder prosthesis in the setting of infection, dislocation or loosening is to be performed, the reoperation rate could be as high as 50%, and there is a potential for multiple reoperations and eventual resection arthroplasty . Because of poor bone stock, persistent infection or persistent instability, conversion back to reverse replacement is not always possible in the immediate revision setting . In addition, due to the very elderly nature of this population and the often presence of severe medical comorbidities, a surgical strategy that could result in multiple reoperations can be unwise . For this reason, conversion of the reverse replacement to metallic or cement hemiarthroplasty is an attractive option, especially in the multiply operated patient unwilling to harbor further surgical risk . There seems to be less risk of reoperation using this surgical strategy, and the option remains to revise back to reverse replacement at a later date . However, successful single - stage treatment of infected and loose reverse replacements have been reported in the literature . Grammont revised one patient who was revised on postoperative day 1 due to an intraoperative glenoid fracture, and this patient maintained only 40 of active elevation and had persistent shoulder pain . Frankle reported on two patients who were converted to a hemiarthroplasty because of insufficient bone stock and a deep infection; these patients rated the outcome of their revision as good and satisfactory . In another large series of reverse replacements, steinmann reported on a subset of four patients who were converted to a hemiarthroplasty as a result of loosening of the glenoid component in three and recurrent instability in the other . Three of these patients had moderate pain and one had severe persistent pain, and patient satisfaction was low . This case series can be criticized for the small number of patients included and only very short - term follow - up . While the short - term follow - up may be used to predict future shoulder function, pain has been shown to increase with time . Therefore, it is possible that shoulder pain after hemiarthroplasty will become more significant over time, compelling patients to seek further treatment such as reimplantation of a reverse prosthesis . Despite these limitations, the literature is very sparse on what comes next? After reverse replacement . We felt that this is the optimal time to report this series as it provides a snapshot of patients that are now candidates for reimplantation of reverse replacement to restore shoulder function . In addition, we feel that this series does provide the shoulder arthroplasty surgeon with several pieces of useful information: (1) in five of six patients, technical error was evident on postoperative radiographs with superior placement of the glenosphere often with superior tilt accounting for three cases of glenosphere loosening and one case of dislocation . These technical errors likely resulted in the average failure of the index reverse replacement at 9.2 months . . The reoperation rate of this series will certainly increase if patients choose to have conversion back to reverse replacement . (3) pain level was acceptable in most patients after conversion of reverse to hemiarthroplasty, with an average vas pain score of 2.4 . (4) shoulder function was poor, with anterosuperior escape present in five of six patients . This escape is likely accentuated by reversion to a state where the deltoid is again detensioned . Failure of reverse replacement will increase in the future, with expansion of indications to younger patients and longer term follow - up . The optimal treatment for a failed reverse replacement is unknown and often very unique to each individual patient and each mode of failure . When counseling a patient with an infected, dislocated or loose reverse replacement, the patient should understand that attempts at retention and salvage of the reverse configuration of their shoulder is not always successful and can be associated with a high rate of reoperation . This small series of patients shows that safe removal of a reverse replacement and conversion to hemicement spacer or hemiarthroplasty can provide the shoulder with a relatively low pain level, but that the shoulder will be nearly devoid of meaningful function postoperatively . Every effort should be made to meticulously assure proper initial implant placement and avoid infection in primary reverse replacement . In addition, when complications do occur, retention of the reverse replacement is necessary to maintain shoulder function as conversion to hemiarthroplasty results in poor function . The failed reverse replacement can result in extraordinarily difficult salvage situations and therefore we echo the sentiment published by rockwood that the reverse replacement should be implanted most frequently by shoulder arthroplasty specialists prepared to treat the severe complications encountered too frequently by even the world's experts . Removal of the reverse shoulder replacement and conversion to hemiarthroplasty results in a shoulder with minimal meaningful function but with an acceptably low level of pain . This information can be helpful when discussing preoperatively the relative risk of revision reverse arthroplasty versus conversion to hemiarthroplasty in patients with a failed reverse replacement.
|
The metabolic syndrome (mes) is defined as a cluster of interrelated metabolic abnormalities that doubles the risk of type 2 diabetes mellitus and cardiovascular disease (cvd). The major features of mes are insulin resistance, central obesity, hypertension, and dyslipidaemia . This condition is often associated with suppression of adiponectin and elevation of leptin and various inflammatory markers (e.g., interleukin-6 [il-6], monocyte chemoattractant protein-1 [mcp-1], c - reactive protein [crp], and regulated on activation, normal t cell expressed and secreted [rantes]) [2, 3] that may play a causal role in insulin resistance . Also, insufficient physical activity accompanied with increased or inappropriate fat accumulation [4, 5] may potentially increase the risk of cvd . The prevalence of mes in gulf cooperation council (gcc) countries is ranked amongst the highest in the world . Furthermore, the risk of mes is higher amongst arab women (1355%) compared to arab men; it is 18% higher in women in oman [8, 9], 55% in qatar, 22% in united arab emirates, and 13% in saudi arabia . On the other hand, data from caucasian populations either point to men and women being equally at risk of developing mes [1315] or to men being more predisposed to developing mes than women [1621] this difference in mes risk is thought to be, at least partly, explained by men having a greater propensity for abdominal obesity compared to premenopausal women . It is unclear as to what factors confer the reported higher risk of developing mes in arab women compared to men . Gender differences in fat accumulation and/or the secretory function of adipose tissue may explain some of this disparity . Poor aerobic fitness, in addition to low grade inflammation, contributes to the development of insulin resistance and is often a better predictor for cvd risk factors than self - reported physical activity . Furthermore, muscular strength and aerobic fitness combined or independently are inversely related to mes . Therefore, this study investigated gender differences in body composition, systemic levels of adipokines and inflammatory markers, and aerobic fitness in a cohort of healthy qatari adults matched for age and body mass index (bmi). A matched case - control study was conducted at aspetar, qatar orthopaedic and sports medicine hospital between february 2009 and december 2009 . This study was approved by the institutional research ethics committee and all subjects provided written consent prior to participation . Healthy qatari men (n = 29) and women (n = 29) were matched for age and bmi . Subjects with diabetes mellitus, those who were pregnant or postmenopausal, or receiving medical treatment for any chronic disease were excluded from the study . After a 10-hour overnight fast, subjects underwent a detailed clinical assessment, including body composition, fat distribution, anthropometry measurements, and blood pressure . Subjects also underwent a series of tests for aerobic fitness and indices of muscular strength . Height was measured to the nearest 0.1 cm (seca 242, germany), and weight was measured to the nearest 0.1 kg using a portable stadiometer (detecto, usa). Both height and weight measurements waist circumference was measured to the nearest 0.1 cm at the smallest girth horizontally around the trunk underneath the subject's clothing . Central obesity was defined as> 80 cm for women and> 90 cm for men . Two blood pressure readings were taken 5 minutes apart with the subject at rest in a relaxed sitting position . The average systolic and diastolic blood pressures were calculated and used in subsequent analyses . Dxa (ge medical system lunar, madison, wisconsin, usa) using the encore software (version 12.10) was used to quantify fat mass (g), tissue (g), lean mass (g) and percentage of body fat . Ct scans were performed to obtain 5 axial images of each of the following regions: heart, liver, abdomen, and midthigh . For the abdominal region, omental adipose tissue was differentiated from subcutaneous adipose tissue by manual drawing, and subcutaneous adipose tissue was further classified as superficial and deep . Two cross - sectional axial images of the left and right thigh at the femoral midpoint region were obtained . Intramuscular adipose tissue and subcutaneous adipose tissue in the thigh were distinguished by manual drawing using the right thigh image . An upper limit of 30 hounsfield units (hu) and a lower limit of 190 hu were used to differentiate adipose tissue from other tissue types on the ct images . All volumetric analyses were performed by an experienced radiologist using the somaris/5 syngo ct2006a system (siemens, germany). A complete blood count was performed, and serum levels of iron, ferritin, iron - binding capacity, transferrin, and lipids, including high - density lipoprotein (hdl), low - density lipoprotein (ldl), total cholesterol, and triglycerides, determined by the pathology laboratory at aspetar . Fasting plasma glucose (beckman, ca, usa) insulin (mercodia, uppsala, sweden) was also assessed, and the homa - ir (homeostasis model of assessment - insulin resistance) was calculated using the following formula: (fasting insulin in miu / l fasting glucose in mmol / l)/22.5 . Fasting serum levels of crp, adiponectin, leptin, rantes, mcp-1, and il-6 were measured using human 2-site elisas (r&d systems, oxon, uk). Il-6 concentrations were assayed with the high sensitivity elisa with a limit of detection of 0.09 pg / ml . All inter- and intraassay cvs were less than 10% . Starting speed was set to 2.74 km / hr, and the intensity (speed and incline) was increased by 2% every 3 minutes until volitional exhaustion . Peak heart rate (hr), percentage of predicted maximal hr, test duration, and peak oxygen uptake were measured . The hand grip strength was measured with the lode dynamometer (lode bv, groningen, the netherlands). Leg strength was assessed by recording isokinetic knee flexion and extension concentrically (at 30/second and 120/second) and isometric extension at 90 on the dominant leg (biodex 3.0 system, version 3.4, shirley, data were analysed using the spss (statistical package for the social sciences, version 15.0) software . Descriptive statistics included mean sd for normally distributed data and median (interquartile range, iqr) for skewed data . An independent sample t - test was used to compare continuous data between two groups and the nonparametric equivalent mann - whitney u test was used when appropriate . Log transformations were applied to normalize the distribution of adipokines and cytokines before computing spearman's correlation coefficients against metabolic markers . Data from this study population was compared to the published age- and gender - specific percentiles for aerobic fitness from the aerobic center longitudinal study (acls). A total of 29 men and 29 women matched for age and bmi were included in the study, with a mean age of 33.4 10.3 years (table 1). Despite the similarity in bmi and waist circumference, body fat percentage was higher in women (women: 43.4 6.3% versus men: 32.7 8.8%, p <0.01). Women had significantly lower levels of triglycerides (p <0.01) and ldl (p = 0.05) and higher levels of hdl (p <0.01), compared to men . Women had lower values for various iron indices compared with men: serum iron (p = 0.05), serum ferritin (p <0.01), and haemoglobin level (p <0.01). Indices of insulin sensitivity and glucose handling (insulin, homa - ir, fasting glucose, and hba1c levels) were similar in men and women . Total fat in the abdominal region was higher in women compared to men, especially in the deep subcutaneous abdominal region (table 2). Adiposity was also significantly higher in women than men in the heart intra- (p = 0.04) and overall heart (p <0.01) regions, as well as in the thigh region (p <0.01). Gender differences in the correlations between serum metabolic markers, adipokines / cytokines, and vo2 max were investigated (table 3). In men, adiponectin and rantes were not associated with any of the metabolic markers studied, whereas in women, adiponectin was negatively correlated with triglycerides (r = 0.45, p = 0.02) and insulin (r = 0.39, p = 0.05). In both men and women, crp was negatively correlated with aerobic fitness (r = 0.43, p = 0.05 and r = 0.54, p <0.01 resp . ). Il-6, leptin, and crp were strongly correlated with waist circumference and systolic bp only in men (table 3). Serum adiponectin and leptin levels were significantly higher in women than men, whereas rantes, crp, mcp-1, and il-6 were not different (figure 1). Tests of aerobic fitness showed that both men and women reached at least 95% of their age - predicted maximum hr (table 4). On average, women ran on the treadmill for almost 7 minutes, whereas men completed 9.5 minutes (p <0.01). Calculated aerobic fitness (vo2 max) was markedly higher in men than women (p <0.01) as were all indices of leg and hand grip strength (p <0.01). Using multiple regression analysis, we found that gender was the strongest determinant for poor aerobic fitness (7.5, 95% ci: 10.3 to 4.8) followed by waist circumference (0.22, 95% ci: 0.34 to 0.11), after adjusting for bp . Reports suggest that prevalence of coronary heart disease is higher in men and postmenopausal women . [17, 28, 29]. However, in the arab population, coronary heart disease - associated risk of morbidity and mortality are elevated even in younger women compared to other ethnic groups [30, 31]. This study sought to clarify the gender differences in fat distribution, serum markers of metabolism and inflammation, and measures of aerobic fitness in an age- and bmi - matched population of healthy qatari men and women . The relatively young premenopausal women in this study had significantly greater fat content in the heart, abdominal, and thigh regions compared with age - matched men (table 2). However, this fat distribution pattern was also accompanied by a favourable lipid profile (high hdl and low ldl) and elevated adiponectin and leptin in women, compared with men . Subcutaneous fat alone is responsible for 80% of leptin production in the body, and thus leptin levels appear to be a good marker for this type of fat deposition in women . The positive correlation of femoral fat with adiponectin (r = 0.44, p = 0.01) in the present study (table 3) is consistent with previous findings that gluteofemoral deposition of fat is cardioprotective . Adiponectin was negatively correlated with homa - ir (r = 0.38, p = 0.056) and serum triglycerides (r = 0.46, p = 0.02) in women, but these relationships were surprisingly absent in men (homa - ir; r = 0.08, p = 0.72 and tg; r = 0.04, p = 0.84). Adiponectin acts as an endogenous insulin sensitizer, both directly on muscle cells and indirectly through insulin, and circulating concentrations of this adipokine are strongly and positively correlated with hdl concentration and negatively correlated with triglyceride levels . While adiponectin may be related to the favourable lipid profile seen in the women in this study, it does not appear to be anti - inflammatory, as both women and men had comparable levels of il-6, mcp-1, crp, and rantes (figure 1). The comparable levels of circulating adipokines / cytokines may reflect similar levels of omental adipose tissue amongst the study subjects . Adipose tissue - derived il-6 and mcp-1 account for approximately 15%30% of systemic levels of these cytokines in obese individuals . Crp plays a role in inflammation and insulin resistance [3, 36] and may be derived from adipose tissue or elevated as a consequence of higher il-6 and mcp-1 levels . Adipose tissue also releases rantes, another putative mediator of impaired glucose tolerance and type 2 diabetes . Among patients with elevated risk of type 2 diabetes, both men and women in the present study had extremely low levels of aerobic fitness . Vo2 max for half of the men (44.8%) and the majority of women (87.5%) was below the 20th percentile reported by acls for a caucasian population (figure 2). However, for measures of muscular strength, approximately 87.0% of men and 75.9% of women were above the 75th percentile of values reported by acls . Despite higher levels of abdominal body fat compared with arab men, premenopausal arab women have elevated serum adiponectin that may maintain normolipidemia and insulin sensitivity . The accumulation of omental adipose tissue, combined with extremely low aerobic fitness, in arab women may abrogate the protective effect of adiponectin, leading to a greater risk of developing mes . Further research is needed to understand the gender - specific causal factors that increase the risk of mes among arab women.
|
The advance medial pivot knee (wright medical technology inc ., memphis, tn, usa) was designed to reproduce the kinematics of the medial pivot motion in the normal knee1,2,3,4,5). The medial compartment of the prosthesis has ball - in - socket geometry to improve joint congruity for stability and the lateral compartment allows for rolling and gliding during knee flexion as in the intact knee6,7,8,9). Other major benefits of the prosthesis include reduced incidences of condylar lift - off and polyethylene wear due to the improved conformity of the polyethylene insert6,10). However, the influence of such biomechanical characteristics of the advance medial pivot knee on patient satisfaction and implant survivorship has not been sufficiently investigated in studies . Long - term studies have never been published and short - term or mid - term follow - up studies are rare11,12,13). In this study, we investigated the clinical and radiological results and complications of total knee arthroplasty (tka) using the advance medial pivot knee for a minimum follow - up of 5 years under the hypothesis that the posterior cruciate ligament sacrificing, non - substituting (ps) implant designed to replicate the medial pivot motion in the non - arthritic knee would yield satisfying functional improvement without significant complications . Of the patients who had undergone tka using the ps advance medial pivot knee between october 2004 and december 2006, 80 patients (120 knees) who were available for more than 5-year follow - up were retrospectively reviewed . There were 9 males (15 knees) and 71 females (105 knees). Their mean age at the time of surgery was 66.4 years (range, 42 to 83 years). The mean follow - up period was 64.7 months (range, 60 to 86 months). The preoperative diagnosis was osteoarthritis in 108 knees (90.0%), rheumatoid arthritis in 4 knees (3.3%), and osteonecrosis in 8 knees (6.7%). The indications for surgery were defined as severe pain or difficulty in performing daily living activities without any improvement with conservative therapy, kellgren - lawrence grade 3 or 4 in cases of patients 65 years of age, and kellgren - lawrence grade 4 in cases of patients with <65 years of age . The number of patients aged 60 years was 24, 20 of which had osteoarthritis and the remaining 4 had rheumatoid arthritis . All the operations were performed by the same surgeon . After a midline skin incision, limited medial parapatellar arthrotomy was performed and traditional sequential medial release was carried out . The distal femoral cut was performed using an intramedullary alignment guide and the proximal tibial cut was done using an extramedullary alignment guide . The femoral component was placed in a 3 externally rotated position with reference to the posterior condylar axis . In order to maintain the medial pivot motion during flexion and extension, care was taken not to cause excessive release of the medial collateral ligament (mcl). However, if an unavoidable excessive ligament release in the case of varus knee results in a 3 mm medial gap, the ligament was sutured to the pes anserinus attachment site after implant insertion to maintain proper tension for <2 - 3 mm medial gap during flexion . The mcl suturing was required in 11 knees in whom the postoperative rehabilitation regimen was identical to that in the other patients . The medial / lateral ligament balance and flexion / extension gap balance were adjusted to confirm the absence of abnormal findings on the valgus / varus stress test, the ability to get full knee extension, and absence of medial / lateral laxity or lift off of a trial component during flexion . Patellar resurfacing was selectively performed: the patella was resurfaced in all knees with rheumatoid arthritis and in all of the remaining knees except for 16 knees with relatively good articular surface . Immediately after surgery, patients were allowed to undertake quadriceps strengthening exercises and active straight leg - raising as instructed prior to surgery . On the first postoperative day, continuous passive motion exercises were initiated . At 3 - 4 days after surgery, when straight leg - raising could be performed without difficulty, progressive weight bearing was started with an assistance of a walker or a cane . Clinical evaluation was based on the pre- and postoperative range of motion (rom), knee society (ks) knee score and function score, western ontario and mcmaster universities osteoarthritis index (womac) score, and postoperative complications . The maximum further flexion without pain was measured in supine position using a goniometer . On the radiological evaluation, the standing anteroposterior view and the 30 flexion lateral view taken at the last follow - up were assessed according to the american knee society roentgenographic evaluation system . In particular, fluoroscopy was used to determine the appearance of radiolucency around the femoral and tbial components . The femorotibial angle () change was measured . To assess the implant position, the valgus angle of the femoral component () and the varus angle of the tibial component () were measured on the anteroposterior view and the flexion angle of the femoral component () and the posterior slope of the tibial component () were measured on the lateral view . The kaplan - meier method was used for survival analysis with revision or the need for revision defined as failure . The paired t - test was used to determine the significance of clinical results and changes in the femorotibial angle . Of the patients who had undergone tka using the ps advance medial pivot knee between october 2004 and december 2006, 80 patients (120 knees) who were available for more than 5-year follow - up were retrospectively reviewed . There were 9 males (15 knees) and 71 females (105 knees). Their mean age at the time of surgery was 66.4 years (range, 42 to 83 years). The mean follow - up period was 64.7 months (range, 60 to 86 months). The preoperative diagnosis was osteoarthritis in 108 knees (90.0%), rheumatoid arthritis in 4 knees (3.3%), and osteonecrosis in 8 knees (6.7%). The indications for surgery were defined as severe pain or difficulty in performing daily living activities without any improvement with conservative therapy, kellgren - lawrence grade 3 or 4 in cases of patients 65 years of age, and kellgren - lawrence grade 4 in cases of patients with <65 years of age . The number of patients aged 60 years was 24, 20 of which had osteoarthritis and the remaining 4 had rheumatoid arthritis ., limited medial parapatellar arthrotomy was performed and traditional sequential medial release was carried out . The distal femoral cut was performed using an intramedullary alignment guide and the proximal tibial cut was done using an extramedullary alignment guide . The femoral component was placed in a 3 externally rotated position with reference to the posterior condylar axis . In order to maintain the medial pivot motion during flexion and extension, care was taken not to cause excessive release of the medial collateral ligament (mcl). However, if an unavoidable excessive ligament release in the case of varus knee results in a 3 mm medial gap, the ligament was sutured to the pes anserinus attachment site after implant insertion to maintain proper tension for <2 - 3 mm medial gap during flexion . The mcl suturing was required in 11 knees in whom the postoperative rehabilitation regimen was identical to that in the other patients . The medial / lateral ligament balance and flexion / extension gap balance were adjusted to confirm the absence of abnormal findings on the valgus / varus stress test, the ability to get full knee extension, and absence of medial / lateral laxity or lift off of a trial component during flexion . Patellar resurfacing was selectively performed: the patella was resurfaced in all knees with rheumatoid arthritis and in all of the remaining knees except for 16 knees with relatively good articular surface . Immediately after surgery, patients were allowed to undertake quadriceps strengthening exercises and active straight leg - raising as instructed prior to surgery . On the first postoperative day, continuous passive motion exercises were initiated . At 3 - 4 days after surgery, when straight leg - raising could be performed without difficulty, progressive weight bearing was started with an assistance of a walker or a cane . Clinical evaluation was based on the pre- and postoperative range of motion (rom), knee society (ks) knee score and function score, western ontario and mcmaster universities osteoarthritis index (womac) score, and postoperative complications . The maximum further flexion without pain was measured in supine position using a goniometer . On the radiological evaluation, the standing anteroposterior view and the 30 flexion lateral view taken at the last follow - up were assessed according to the american knee society roentgenographic evaluation system . In particular, fluoroscopy was used to determine the appearance of radiolucency around the femoral and tbial components . The femorotibial angle () change was measured . To assess the implant position, the valgus angle of the femoral component () and the varus angle of the tibial component () were measured on the anteroposterior view and the flexion angle of the femoral component () and the posterior slope of the tibial component () the kaplan - meier method was used for survival analysis with revision or the need for revision defined as failure . The paired t - test was used to determine the significance of clinical results and changes in the femorotibial angle . There was significant improvement in the rom between the preoperative and last follow - up assessments: the mean flexion contracture decreased from 7.6 to 1.5 and the mean range of further flexion increased from 115.1 (rom, 107.5) to 120.5 (rom, 119) (p<0.05). The ks knee score and function score, and the womac score for pain and stiffness and function were significantly improved (p<0.05) (table 1). The femorotibial angle was improved from a mean of 4.64.5 varus preoperatively to a mean of 5.82.4 valgus postoperatively . The mean,, and angle were 96.22.1, 89.11.7, 2.51.5 and 84.42.7, respectively . Except for 2 cases of aseptic loosening, 2 mm radiolucency was not observed in any case when assessed according to the american knee society roentgenographic evaluation system (table 2). Complications occurred in 4 knees (3.3%): periprosthetic patellar fracture in 2 (1.7%) and aseptic loosening in 2 (1.7%). There was no case of infection . Periprosthetic patellar fractures were caused by aggressive flexion exercises in one and by trauma in the other . Conservative treatment for the former with no displacement and open reduction and internal fixation using tension band wiring for the latter resulted in satisfactory outcomes . Aseptic tibial component loosening was observed at 1 year and 9 months after surgery in one knee, which was revised using structural allograft and stemmed tibial component (fig . Aseptic loosening in the other case was observed at 5 years and 7 months after surgery, and the plain radiography revealed osteolysis in the femur and aseptic loosening in the tibia . The 7-year survival rate according to the kaplan - meier method was 98.1% (95% confidence interval, 0.96 to 1.00) (fig . The clinical and radiological results of tka using the ps advance medial pivot knee were satisfactory without significant complications after the minimum 5-year mid - term follow - up . The significance of this study can be found in the fact that the mid - term results of advance medial pivot knee in tka have never been addressed in domestic studies and our results were comparable to those described in international mid - term follow - up studies11,12,13). Fan et al.11) reported minimum 5-year follow - up results of 58 medial pivot tkas: the mean rom was improved from 103.5 preoperatively to 115.4 at the last follow - up; non - progressive radiolucent lines were observed in only 2 cases; and there was no case of revision for loosening . In a mean 6.7-year follow - up study by karachalios et al.12), the mean rom was improved from 101 preoperatively to 117 at the last follow - up and the 5-year survival rate and 9-year survival rate were 99.1% and 97.5%, respectively . Vecchini et al.13) followed 160 patients (172 knees) for a mean of 7 years after medial pivot tka: the mean rom was improved from 97.7 preoperatively to 112.5 at the last follow - up and the survival rate was 98.6% . In our mean 6.1-year follow - up study, the mean rom was improved from 107.5 preoperatively to 119 at the last follow - up, showing more favorable results than the above - mentioned studies . The 7-year survival rate (98.1%), significant clinical improvement was noted in all patients after tka using the ps advance medial pivot knee . Although we used the ps type implant in all knees during tka, there are some studies providing comparisons with other types of implants . Bae et al.14) reported there was no notable difference in the clinical and radiological results of tka between the cruciate retaining (cr) type advance medial pivot knee group (67 cases) and the ps type group (70 cases). Pritchett15) investigated patient preferences in knee prostheses for a mean of 7 years after bilateral tka: 77% of the 344 patients preferred the medial pivot knee prosthesis to other ps type prostheses . Youm et al.16) showed that the minimum 2-year follow - up results of tka using the ps type advance medial pivot knee were comparable to those of tka using the nexgen lps system . On the other hand, kim et al.17) reported that the satisfaction rate in the cr type advance medial pivot knee group was significantly low compared to that in the pfc sigma mobile - bearing prosthesis group and the complication rate was remarkably higher in the former group during the mean 2.6-year follow - up . However, we believe their results should be interpreted with caution because it is difficult to associate the complications occurred in their patients (infection, persistent flexion contracture, and constant swelling) with the design features of the implant . Most ps type implants necessitate an extensive box cutting to enable the cam - post mechanism . However, the ps type advance medial pivot knee does not require box cutting because the ultracongruent polyethylene insert that has no post provides anteroposterior stability . Accordingly, considering that more bone stock preservation would be of benefit for possible revisions, we preferred to use the advance medial pivot knee in young patients with a relatively high likelihood of revision, if they met the indications . This is why relatively young patients 60 years of age (19 patients, 24 knees) were included in the study . However, we believe long - term follow - up studies should be conducted to assess the influence of bone stock preservation in the box of the femur on future revision surgery . Of the 2 cases of aseptic loosening, the early loosening of the tibial component in one knee can be attributed to surgical errors: 2 of varus cutting of the proximal tibia . In addition, the use of a 10-mm metal augment without a stem for proximal tibial bone loss may have resulted in increased stress in the medial aspect of the proximal tibia, eventually resulting in subsidence of the medial tibia and tibial component loosening . Regarding the other case in which osteolysis of the femur and aseptic loosening of the tibia were found at 5 years and 7 months after surgery, it is difficult to identify whether the complications were associated with the ultracongruent design of the advance medical pivot knee . Rather, minoda et al.10) reported that the medial pivot knee prosthesis caused significantly less wear particles than other ps type prostheses . In the other aforementioned mid - term follow - up studies, the incidences of osteolysis and loosening were not significantly high in the knees with medial pivot knee prostheses . Thus, we believe this should be elucidated in further long - term follow - up biomechanical studies . One of the limitations of this study is that due to the retrospective study design, the level of evidence was relatively low compared to prospective studies . In addition, the number of cases was small and the follow - up was not maintained for the long term . Finally, the study did not examine whether replication of medial pivoting, the biomechanical advantage of the medial pivot knee, was successfully realized after tka . The clinical and radiological results of tka using the ps advance medial pivot knee designed to reproduce normal knee kinematics were satisfactory after the minimum 5-year mid - term follow - up . And the incidence of tibial component loosening after using this ultracongruent implant was not remarkable.
|
In the last decade, the parathyroid hormone (pth) has become more important as a possible alternative for the treatment of postmenopausal osteoporosis . Most of the studies with pth have focused mainly on the dose - dependent effects of this hormone or its combination with other drugs [14]. Since researchers found that the intermittent substitution of pth, in contrast to its continuous application, was able to prevent postmenopausal bone loss, the question has been which application frequency would deliver the best anabolic result? The time interval between the anabolic und catabolic effects of intermittent application of pth (anabolic window) is the key to respond and to understand such behavior of this hormone . In addition, another unanswered question is how pth affects different skeletal sites like the wrist, proximal tibia, vertebral bodies and, especially, proximal femur . It is known that the trochanteric fracture of the femur is one of the most common fracture types in menopausal women . Therefore, investigations of the strength of this skeletal site after treatment with antiosteoporotic agents seem to be important and have a high clinical relevance . Such investigations, however, are rare because of the difficulty in producing a reliable trochanteric fracture in animal models . The ovariectomized (ovx) rat is a well - proven animal model for osteoporosis studies [8, 9]. There are many similarities between the human and rat femur, both at the microstructural and macrostructural levels [10, 11]. In the present study, we investigated the region - specific influence of two different application frequencies of pth on femoral trochanteric strength of ovariectomized rats . The experiments were carried out using forty - three - month - old female sprague - dawley rats fed a standard diet ad libitum . The animals were randomized by weight into four experimental groups (n = 10 in each group): ovx soy - free group (ovx), pth group receiving 5x / w), pth group receiving subcutaneous injections of 0.040 mg / kg parathyroid hormone (1 - 34) every 2 days (pth e2d), and a sham group . The experimental procedures were approved by the local ethics commission under german animal protection law (permission from 11.03.1998, az: 509.42502/01 - 02.98 . Eight weeks after bilateral ovx, we started the drug treatments, which were continued for the next five weeks . After five weeks of drug therapy, the rats were euthanized, and both femurs were dissected free of soft tissue to be used for biomechanical and histomorphometric tests . During the treatment, the animals were subcutaneously injected with four fluorescent substances (merck, darmstadt, germany) to mark the process of bone formation, especially in the cortical surface . The following fluorescent agents injected were xylenol orange (90 mg / kg) on day 13, calcein green (10 mg / kg) on day 18, alizarin red (30 mg / kg) on day 24/26, and tetracycline (25 mg / kg) on day 35 . It is well known that tetracyclines are able rapidly to bind to new formatted bones immediately after application . The results of the fluorochrome labeling were analyzed in cross sections of femurs 15 mm distal from the femoral head in the subtrochanteric region . The biomechanical test was performed with our new breaking test as previously described . In a deepening (4 mm diameter), the femoral head (left femurs) was fixed at proximal end of the breaking machine . Force was applied with a zwick - testing machine, type 145660 z020/tnd (zwick / roell, ulm, germany), from the lateral side of the bone (vertically to the trochanter tertius in rat) to the greater (major) trochanter using a metallic stamp . The range of assessment was from 2 n to 500 n. during the mechanical test, the bone had the possibility to slide between the roller clamps . The slope of our breaking curve (load - displacement - curve) corresponds to the tangent of the strength - strain - curve . This means that in our study the slope of the breaking curves shows the elasticity of the bones (femurs). Load and displacement were recorded, and ultimate maximal breaking strength (maximal load, fmax (n)) and stiffness (elasticity, slope of the linear part of the curve, n / mm) were calculated . The measurement of yield point (yield load) of bone is not easy . In the opinion of many researches, it is only possible to measure the yield area and not a point . The mean reason for this is the inhomogeneity of bone (consist of mineral, organic material and many other elements and water). Using the curve of f(max) (maximal load) and the load displacement, we can only measure the yield area . The work of brzo'ska et al . Confirmed the fact that after breaking the femoral neck they could see different curves . In their study, this fact made the measurement of yield load of femoral neck more difficult . Our previous experiments could, however, show that the yield area also in our femurs corresponds approximately to both standard deviations . We defined the yield point (load) as a decrease in elasticity (stiffness) of more than twice the standard deviation (sd). X - ray radiography (in the anterior - posterior and lateral view) of all left femurs was performed in the study (figures 1(a) and 1(b)). For this, we used a special kodak - film (kodak sr type 45) and a faxitron fine - focus cabinet x - ray system (model 43855a; faxitron x - ray system) with 40 kv . The left femurs were fixed, after the biomechanical test, in 70% ethanol for 2 days (48 hours), dehydrated through an alcohol gradient, and at least embedded in methyl methacrylate gel . Sagittal, sections (150 m thick) of the embedded proximal femur were prepared using a microtome (leica, sawmicrotom 1600). The target region of embedded femur for the histomorphometry analysis was the frame between the epiphyseal zone and the femoral intertrochanteric line (2 mm distally). These microradiographs of the femoral sections were used to analyze the histomorphometric changes in the trabecular surfaces (figure 1(c)). The system consisted of a microscope (leica - system mz 7.5), a digitizing pad coupled to a personal computer with an additional morphometry program (qwin software). We measured trabecular area (tb.ar), the number of trabecular nodes (n.nd), trabecular connectivity (n.nd/mm), and mean trabecular width (tb.wi). A problem is the very difficult measurement of the cortical changes at cortical surface in medial proximal femoral neck . The proximal (medial) part of the femoral neck in rats and other large animals seems not to be covered by periosteal tissue . This is an important factor to consider, especially when anabolic agents are tested with pronounced periosteal stimulation . In contrast, the trochanteric region contains a cortical surface covered by a sufficient periosteum . Because measurable changes in the cortical area occur first long time after ovx, the real early changes of thickness in this region remain difficult to measure . We measured the ratio between diameter of femoral bone (b.dm) and marrow diameter (ma.dm) in the cross sections 15 mm distal of the capitis femoris (femoral head) in the subtrochanteric area . We assessed the b.dm of the cross sections in the midline of the cross sections (dorso - ventral - axis) and on the same line as the ma.dm as previously described (figure 2). It is here important to mention that this area in rat is a region between major trochanter, minor trochanter, and tertius trochanter . This means that this part (in rat) belongs to the trochanteric region . The further advantage of using the proximal femur (15 mm distal to the femoral head) is the opportunity to have a relatively homogenous width of cortical surface and its independence to the technical problems during providing sufficient cross sections . Blood samples (5 ml) were collected from the sacrificed animals and centrifuged at 3000 g for 10 minutes . The serum was stored at 20c until the electrochemiluminescence immunoassay (eclia, roche diagnostics, mannheim, germany) was performed . A further marker of bone remodeling, alkaline phosphatase (ap), was also quantitatively determined . To determine the amount of mineralized bone, the right femurs were mineralized at 750c and weighed to the nearest 10 g. the femurs were weighed (dry) before and after ashing . At the end of the experiment, the mineral content of each bone was indicated as a percentage of the total weight of the same femur (weight after ashing / weight before ashing). The mean values of the differences between the study groups in all of the comparative bioassays were assessed using one - way anova test with tuckey_kramer post hoc test . As demonstrated in table 1, there were no significant differences in body weight between the groups at the beginning of the study . At the end of experiment, we observed a significant weight gain in all groups compared to the sham group (table 1). After ashing of the left femurs, both pth 5x / w and pth e2d groups (49.54% and 48.30%, resp .) Showed significantly higher mineral content and similar mineral content than that of the sham group compared to the ovx (45.80%) group . Although the mean value in the pth 5x / w rats was higher than the pth e2d animals, the difference in mineral content was not statistically significant . In addition, the ovx animals clearly had less mineral content in comparison to the sham (49.68%) rats . The mean values of maximal load (fmax), stiffness, and yield load (yl) showed higher results after treatment with parathyroid hormone 5x / w (fmax = 194.1 n, stiffness = 347.6 n / mm, yl = 134.6 n) in comparison to the e2d group (fmax = 176.3 n, stiffness = 250.9 n / mm, yl = 110.3 n). Concerning biomechanical parameters, the results of the pth 5x / w animals showed a significant improvement compared to the ovx rats, but there were no significant differences between the ovx group (fmax = 169.3 n, stiffness = 230.2 n / mm, yl = 86.96 n) and the pth e2d group . Concerning stiffness and yield load, the sham animals had higher results (fmax = 187.0 n, stiffness = 294.8 n / mm, yl = 120.6 n) than ovx rats (table 1). Concerning biomechanical parameters, the results of the pth 5x / w animals showed a significant improvement compared to the ovx rats, but there were no significant differences between the ovx group (fmax = 169.3 n, stiffness = 230.2 n / mm, yl = 86.96 n) and the pth e2d group . The mean values of maximal load (fmax), stiffness, and yield load (yl) showed higher results after treatment with parathyroid hormone 5x / w (fmax = 194.1 n, stiffness = 347.6 n / mm, yl = 134.6 n) in comparison to the e2d group (fmax = 176.3 n, stiffness = 250.9 n / mm, yl = 110.3 n). Serum osteocalcin levels differed between both pth - treated groups (p <.05) in comparison to the sham and ovx animals . There were no significant differences in serum oc levels between the pth 5x / w (37.43 ng / ml) and pth e2d groups (32.55 ng / ml). In sham animals (14.59 ng / ml), oc levels were lower compared to ovx rats (17.83 ng / ml), but the results were not significant . The concentration of ap in the pth e2d group (74.27 ng / ml) was increased, but this result was only significant compared to the sham group (40.90 ng / ml) (table 1). The number of trabecular nodes / mm (n.nd/mm = connectivity) was significantly higher in sham (21.34) and pth e2d (19.10) groups compared to ovx rats (11.91). Conversely, there were no significant differences in connectivity between the pth 5x / w and pth e2d groups . The pth 5x / w rats demonstrated better results concerning tb.ar and tb.wi in comparison to pth e2d animals . Both pth - treated groups (5x / w and e2d) presented improved results for tb.ar (81.54% versus 73.38%) and tb.wi (17.59 versus 14.65 m) compared to ovx rats (tb.ar = 41.15%, tb.wi = 11.51 m). Concerning tb.ar and tb.wi, the pth 5x / w showed significantly better results than the sham group (tb.ar = 66.85%, tb.wi = 12.24 m). To determine the changes in the cortical surface of the femurs, we measured the diameters of subtrochanteric bone cross sections (b.dm) and the marrows (ma.dm) in the ventro - dorsal axis in all groups . The b.dm/ma.dm ratio was able to compare even minimal changes in the cortical width of the subtrochanteric region of the rat femur among all groups . Although we did not see any significant changes between any of the groups concerning b.dm, the mean values of ma.dm were significantly lower in both pth - treated groups compared to ovx animals . The mean values of the b.dm/ma.dm ratio were significantly higher in both pth 5x / w rats (1.843) and pth e2d rats (1.805) compared to the ovx group (1.652). The pth 5x / w rats also showed a significantly higher b.dm/ma.dm ratio compared to the sham animals (1.726) (table 2). These results in addition to the results of fluorescence microscopy could show useful information about endosteal and periosteal bone remodeling (apposition bands) within the cortical surface . The increased bone formation rate was observed under pth treatment in both groups mainly at the endosteal side by fluorescent microscopic analysis of the cross sections from the proximal femur . The endosteum seems here to be one of the targets of pth with an accelerate bone formation and a pronounced filling - in of intracortical cavities with higher intensity for the pth 5x / w in comparison to pth e2d rats (figure 3). He observed that continuous intravenous administration of pth was able to elevate predominantly the bone resorption . In contrast, the intermittent administration of pth mainly resulted in a stimulation of bone formation, especially in the trabecular area . In the last several years, studies have emphasized the importance of evaluating the effects of this hormone in cortical areas [16, 17]. Although many studies could confirm the anabolic effect of pth on bone, the question of which application frequencies produce the best results is still controversial . Another important question is in which skeletal sites (proximal tibia, vertebral bodies, etc .) The trochanteric region of rat femur contains major trochanter, minor trochanter, and tertius trochanter . The area between these three trochanters was the region of interest in our studies . Next to the femoral neck fracture, the trochanteric fracture of the femur is one of the most common fracture types in elderly women . This part of the rat femur contains both trabecular and cortical bone, in contrast to the femoral shaft . Therefore, the intertrochanteric part of the femur seems to be an important region to investigate the biomechanical changes after therapy with antiosteoporotic substances like the parathyroid hormone because pth appears to influence both cortical and trabecular bone surfaces [6, 19]. Hence, it is important to look for therapy options and drugs that prevent such fractures in postmenopausal osteoporotic bone . The intermittent administration of pth gained more importance in the last decade as a possible alternative for the treatment of osteoporosis and prophylaxis of fractures . In the present study, we investigated two therapy options with pth . We compared the anabolic effect of pth on the proximal femur of ovx rats after five weeks of subcutaneous injections (0.040/kg), five days a week, or subcutaneous injections every 2 days . At the end of experiment, we observed a significant weight gain in ovx group compared to the sham group . Neither the pth 5x / w nor the pth e2d treatment could prevent the weight gain caused by ovx . Indeed, the sham animals had the lowest weight gain of all of the rats . When assessed by the breaking test, femurs of the pth 5x / w treatment reached the strength level of sham rats . Although the treatment with pth e2d also led to better mean values in the biomechanical test, the results were not statistically significant in comparison to ovx rats . The changes observed in the biomechanical test were partially evident when bones from pth 5x / w- and pth e2d - treated rats were examined by histomorphometry . In our opinion, the main reason for significantly better results of tb.ar in pth 5x / w rats was the improvement of trabecular thickness in these animals ., however, showed an increase in trabecular number in younger rats, whereas older rats demonstrated increases in trabecular thickness . Therapy with bone anabolic agents causes serum levels of the bone formation markers oc and ap to increase, and we observed similar results after pth treatment . However, we did not observe any statistically significant differences between the two pth application paradigms in our study . In case of ap, this could be due to the little sensitivity and specificity of this bone formation marker . Some studies have shown that after pth treatment, the addition of bone to the periosteal surface seems to be responsible for a much greater contribution to bone strength than bone added to the endosteal surface . In contrast, the present work demonstrated increases in the b.dm/ma.dm ratio in both pth - treated animals . This finding was caused by a decrease in ma.dm rather than an increase in b.dm . In our study, the fluorescence apposition bands in the endosteal side of femoral cross sections after pth treatments underline this effect . Although the b.dm/ma.dm-ratio was in the pth 5x / w higher than pth e2d rats, the results were not statistically significant . But in our opinion, the higher dosages of pth seem to have more intensive anabolic effect on the endosteal side of the cortical surface . Komatsu et al . Also showed that pth induced new bone formation at endocortical (endosteal) surfaces . Interestingly, in a fracture healing rat model, the same authors found that pth dose dependently (30 g / kg) stimulated bone formation within the intramedullary cavity . In the ash test, higher total doses of 134 pth induce a better but not statistically significant improvement on bmc, reversing the effects that the ovx has on this quantitative determinant of bone strength . Many of the measured parameters in our work did not show any statistically significant differences between the pth 5x / w and pth e2d groups, but the mean values in most of the tests were slightly higher in the pth 5x / w animals compared to the pth e2d animals . In our opinion, the antiosteoporotic effects of pth 5x / w seem to be slightly stronger than pth e2d treatment although we saw only in tb.wi and in stiffness statistically significant differences . The exact signaling pathways of the anabolic effect of pth are not clear, but the pathways activated after pth treatment determine whether this hormone has catabolic or anabolic actions . The parathyroid hormone (pth) stimulates the processes that lead to bone formation prior to stimulating the pathways associated with bone resorption . Rubin and bilezikian showed that the bone formation markers reached a maximum level within a few days after pth treatment, whereas the bone resorption parameters had the maximum level after approximately 3 weeks [21, 22]. We believe that during these three weeks between the maximal anabolic and maximal catabolic phases of pth, named anabolic window by rubin and bilezikian, different pth application frequencies could lead to an improvement of bone strength; however, this would probably occur with different intensities . It is important to mention that the half - life of pth after a single application as well as the dose and skeletal site where the anabolic effect of pth is investigated are important aspects that play critical roles in determining the effects of pth . Therefore, future investigations should evaluate the time- and dose - dependent changes of the trochanteric region after pth therapy . Furthermore, additional experiments should be performed in animals of different ages and in male rats . The trochanteric region of the rat femur is an important skeletal site for osteoporosis studies because of its high clinical relevance . The present study showed that treatment with pth 5x / w and pth 2ed both improved the biomechanical and histomorphometric properties and partially reversed the effects of ovx in the trochanteric region of the rat femur . Under conditions presented in our study, the anabolic effect of pth 5x / w seems to be slightly stronger than pth e2d therapy, but the main significant changes were observed concerning elasticity and tb.wi . Further experiments are needed to determine which form of pth therapy options is able to effectively prevent trochanteric fractures . Thus, future studies related to dose- and time - related investigations should be conducted.
|
Ischemic heart disease is one of the major leading causes of death for both men and women . Depriving the organ from its blood supply has long been documented as a critical factor in the clinical outcome of stroke, hemorrhagic shock, myocardial infarction, and organ transplantation . Although the restoration of blood flow to an ischemic organ is essential to prevent permanent tissue damage, reperfusion may increase tissue injury in excess of that produced by ischemia alone . Restoration of blood flow to ischemic myocardium results in the ischemia reperfusion (i / r) injury . Cellular damage after reperfusion of previously viable ischemic tissue is defined as ischemia reperfusion (i / r) injury . I / r injury may occur in a variety of clinical situations, including reperfusion after thrombolytic therapy, coronary angioplasty, organ transplantation, and cardiopulmonary bypass . Reperfusion of ischemic tissue results in both a local and systemic inflammatory responses that, in turn, may give rise to wide spread microvascular dysfunction and changed tissue barrier and function . If sever enough, the inflammatory response after i / r may even lead to systemic inflammatory response or multiple organ dysfunction syndrome, which account for up to 3040% of intensive care unit mortality . Gender differences have been distinguished in i / r injury [410], with several studies connecting the sex hormone estrogen in the cardioprotection found in females [1115]. In contrast, testosterone has received little attention . Presently the majority of evidence points toward the detrimental effects of testosterone on myocardium, possibly by adverse effects on lipoproteins, thrombosis, and cardiac hypertrophy [1619]. Indeed, some studies established that chronic endogenous testosterone has a deleterious effect in the isolated rat heart subjected to i / r . Recent advances in our understanding of cell death during ischemia reperfusion implicate two forms of cell death in the pathology of myocardial infarction that is to say necrosis and apoptosis . Apoptosis cell death in rat heart has been established to be induced by prolonged episode of ischemia alone in absence of reperfusion [22, 23]. Some studies have suggested that reperfusion accelerates the apoptotic cell death process initiated during ischemia [22, 2426]. In contrast, several studies suggest that the apoptotic component of cell death is triggered at time of reperfusion and does not manifest during the ischemic period . Therefore, evidence suggests that the apoptotic component of cell death is either generated or accelerated during the reperfusion phase . The fact that the apoptosis is an energy dependent process and atp levels are depleted during ischemia and replenished on reperfusion may explain why the apoptotic component of cell death is associated with reperfusion . A total number of 24 adult male albino rats weighting (200250 g) were purchased from animal resource center, national center for drug control and research . They were housed in the animal house of kufa university / college of medicine in a temperature - controlled (25 1 c) room (humidity was kept at 6065%) with alternating 12-hr light/12-hr dark cycles and were allowed free access to water and chow diet until the start of experiment . After the 1st week of acclimatization, the rats were randomized into 4 groups (6 rats in each group) as follow.in sham group, rats underwent the same anesthetic and surgical procedures (for an identical period of time for regional myocardial ischemia and reperfusion) but without lad ligation.in control group (induced untreated), rats underwent surgical operation for lad ligation and were subjected to 25 min of ischemia and 40 min of reperfusion.in castrated group, surgically castrated rats were left 4wks for recovery and then underwent surgical lad ligation and were subjected to 25 min of ischemia and 40 min of reperfusion.in treated group (goserelin treated), rats of this group take a s.c injection of goserelin acetate (lhrh analogue) 3 wks before the surgery and then were subjected to surgical lad ligation with 25 min ischemia and 40 min of reperfusion . In sham group, rats underwent the same anesthetic and surgical procedures (for an identical period of time for regional myocardial ischemia and reperfusion) but without lad ligation . In control group (induced untreated), rats underwent surgical operation for lad ligation and were subjected to 25 min of ischemia and 40 min of reperfusion . In castrated group, surgically castrated rats were left 4wks for recovery and then underwent surgical lad ligation and were subjected to 25 min of ischemia and 40 min of reperfusion . In treated group (goserelin treated), rats of this group take a s.c injection of goserelin acetate (lhrh analogue) 3 wks before the surgery and then were subjected to surgical lad ligation with 25 min ischemia and 40 min of reperfusion . The procedure of lad ligation in rats is modified from that mentioned in previous study with modification . The rats were anesthetized by intraperitoneal (ip) injection of 100 mg / kg ketamine and 5 mg / kg xylazine . When the rat anesthetized (within 510 min), we placed it in supine position, then, fix the four limbs, tail and extended the head . The trachea is intubated with a cannula sized either 22 fg or 20 fg according to the weight of animal as the small catheter is reserved for the smaller animal . And the tube is connected tightly to the ventilation machine . With scissors and fine pickup, (2 - 3) cm long skin incision was made over the left thorax area starting from midline, just 30 mm above the xiphoid process to the left anterior axillary fold . Zoom in the microscope on the heart and then the pericardium is opened using two pairs of small micropickup . The left ventricle (lv) with its partly overlying left auricle (atrium) is now visible . The left anterior descending coronary artery (lad) is ligated just distal to the left auricle, the distance from the ligation site to the left auricle being the length of both tips of a pair of rounded forceps as this is equal to (2 - 3) mm . The ligation continues for 25 min; then the ligation is terminated by cutting the ligature by using microscissor . At this time the reperfusion time is started to be calculated, then closing the chest wall by enclosing the ribs with figure eight of 4 zero silk suture . The pectoral muscles should be moisturized and returned back into the original position (first the minor and then the major partly overlying it); after that, the skin is closed with 4 zero silk suture, watching for the spontaneous breathing, and when it is sufficient, then the decision is made for gentle and careful extubation after freeing the mouse from tapes . Finally, the mouse should be transferred into clean cage oxygenated with 100% oxygen and placed near fair heating lamp . The reperfusion time is calculated from the moment of removing the ligature and the reperfusion is continued for 40 min . At the end of reperfusion period, the animal is reanesthetized and placed in the supine position with its limbs immobilized, the chest reopened, and the heart exposed, and then a needle of the syringe is introduced into right ventricle to aspirate around (58) ml of blood for later blood analysis . The blood sample was placed in a tube containing disodium edta (22 mg / ml) as anticoagulant and mixed thoroughly and then centrifuged at 3000 rpm for 15 min . Then it was used for determination of plasma ctn i by elisa with a commercially available elisa kit (literature of kit by life diagnostic, usa) according to the manufacturer's instructions . The excised heart tissues were rinsed with ice cold saline to remove any red blood cells or clots and then homogenized with a high intensity ultrasonic liquid processor in 1: 10 (w / v) phosphate buffered saline that contained 1% triton x-100 and a protease inhibitor cocktail . The supernatant was collected for determination of tnf-, il-1, and icam-1 by elisa with a commercially available elisa kit (literature of kit by life diagnostic, usa) according to the manufacturer's instructions . The cardiac sections for histopathological study were fixed in 10% formalin and embedded in paraffin . Evaluate scores were performed by an investigator who was blinded to the experimental treatment groups . The following morphological criteria were used to assess the histopathological damage: score 0, no damage; score 1 (mild), interstitial edema and focal necrosis; score 2 (moderate), diffuse myocardial cell swelling and necrosis; score 3 (severe), necrosis with the presence of contraction bands, neutrophil infiltration, and the capillaries were compressed; and score 4 (highly severe), widespread necrosis with the presence of contraction bands, neutrophil infiltration, capillaries compressing, and hemorrhage . The part of ventricular samples that were used for measurement of apoptosis level underwent lyses by trypsin . Trypsin is a serine protease commonly used for detachment of adherent cell lines and dissociation of tissues . Incubating cells with too high trypsin concentration for too long time period will damage cell membranes and kill the cells . For the dissociation of tissues, trypsin has been used alone or as a supplement to other enzymes.transfer 1,000 to 10,000 nonfixed cells to each well.centrifuge plate at 200 g for 5 minutes . Remove medium, add 200 l of fixative (80% methanol in pbs), and incubate at room temperature for 30 minutes . Then the measurement is done by elisa with a commercially available elisa kit (ssdna apoptosis elisa kit, chemicon international, inc . The part of ventricular samples that were used for measurement of apoptosis level underwent lyses by trypsin . Trypsin is a serine protease commonly used for detachment of adherent cell lines and dissociation of tissues . The concentration of trypsin can range from 0.025% to 0.5% . Incubating cells with too high trypsin concentration for too long time period will damage cell membranes and kill the cells . For the dissociation of tissues, trypsin has been used alone or as a supplement to other enzymes . Transfer 1,000 to 10,000 nonfixed cells to each well . Remove medium, add 200 l of fixative (80% methanol in pbs), and incubate at room temperature for 30 minutes . Then the measurement is done by elisa with a commercially available elisa kit (ssdna apoptosis elisa kit, chemicon international, inc . Quantitative variables were tested for statistical significance of difference between more than 2 groups by one - way anova test followed by post hoc . The statistical significance of difference between more than 2 groups was assessed by kruskal - wallis test, while mann - whitney u test was used for the difference between 2 groups . In all tests, compared with the sham group, levels of myocardial tnf-, il-1, and icam-1 were increased (p <0.05) in control group . Both castration and goserelin acetate injection significantly counteract the increase in myocardial levels of tnf-, il-1, and icam-1 (p <0.05), but there was no significant difference between castrated group and goserelin treated group as shown in figures 1, 2, and 3 . Compared with the sham group, levels of plasma ctn both surgical castration and goserelin injection significantly counteract the increase in level of plasma ctn i (p <0.05), but there was no significant difference between castrated group and goserelin treated group as shown in figure 4 . Compared with the sham group, myocardial apoptosis levels were increased (p <0.05) in control group . Both surgical castration and goserelin injection significantly counteract the increase in myocardial apoptosis level (p <0.05), but there was no significant difference between castrated group and goserelin treated group as shown in figure 5 . Histologically, all control group rats showed a significant myocardial injury (p <0.05) compared with sham group as shown in figures 6, 7, and 8 . Both castration and goserelin injection markedly reduced (p <0.05) the severity of heart injury in the rats which underwent the regional ischemia - reperfusion procedure, but there was no significant difference between castrated group and goserelin treated group as shown in figures 6, 8, 9, and 10 . The main findings of the present study were that after myocardial i / r, compared with control group, both castrated group and goserelin treated group (1) exhibited a lower histopathological damage, (2) had decreased proinflammatory cytokine production (tnf-, il-1, and icam-1), (3) exhibited a lower apoptotic injury, and (4) there were no significant differences between castrated group and goserelin treated group . The role of testosterone in cardiac injury may be very important because the heart can accumulate testosterone at higher concentrations than other androgen target organs and functional androgen receptors are found in isolated cardiac myocytes . Role of testosterone in cardiac injury is controversial; some studies indicated that testosterone was reported to confirm cardioprotection against ischemia reperfusion by upregulating the cardiac alpha(1)-adrenoceptor and enhancing the effects of stimulation of this adrenoceptor . On the contrary, other studies hypothesized that testosterone may exert deleterious effects on myocardial proinflammatory cytokine production through proinflammatory and/or the proapoptotic properties of endogenous testosterone in ischemia reperfusion . Furthermore, exogenous testosterone supplementation increased apoptosis in adult rat ventricular myocytes during i / r injury . The relationship between apoptotic and necrotic cell death during ischemia reperfusion injury is unresolved with some authors showing that there may be significant overlap in terms of early signaling steps between these two pathways, this observation may be useful in development of future therapeutic targets for clinical use . Zhao et al . Have used a canine model of ischemia reperfusion injury to demonstrate the contribution of necrotic and apoptotic cell death . They established that both forms of cell death occur together during the reperfusion phase with necrotic cell death peaking after 24 hr . Of reperfusion and apoptotic cell death increasing up to 72 hr . Other studies have showed that the pharmacological inhibition of the apoptotic signaling cascade during the reperfusion phase is able to attenuate both the apoptotic and the necrotic cell death [4043]. They suggested that the apoptotic death can evolve into necrotic cell death . To the best of our knowledge, there is no previous study that used goserelin acetate to produce a chemical castration for male rats but there are many studies that used the testosterone receptor blockers as flutamide to block the testosterone receptors . Wang et al . Demonstrated that male rats of both (1) treated group with testosterone receptor blocker (flutamide) and other groups (2) with surgical castration when were subjected to i / r show a decrease in production of proinflammatory cytokines (tnf-, il1, and icam-1) compared with untreated control group, explaining the possible role of testosterone in promoting the myocardial inflammatory response . Other studies report protected cardiac performance in animals with testosterone depletion or testosterone receptor blockade after trauma and hemorrhage and suggest that endogenous testosterone may have a negative effect on the heart subjected to i / r . Wang et al . Demonstrated that both male rats treated with testosterone receptor blocker (flutamide) and male rats underwent surgical castration when subjected to i / r show a decrease in the activation of proapoptotic signaling cascade (caspase-3, caspase-11) and increase antiapoptotic bcl2 compared with control untreated group so the net result is decrease in apoptosis . Showed that female rat hearts had better functional recovery after acute ischemia than male rat and expressed less myocardial tnf-, il1- (mrna and protein) than male hearts subjected to the same i / r insult . The present study showed that there was a significant lowering (p <0.05) in plasma level of ctni in both castrated group and goserelin group compared to control group . Crisostomo et al . Also demonstrated that in rat hearts devoid from chronic exposure to testosterone, a single dose of exogenous testosterone increases apoptosis level . Findings in the present study according to apoptosis level are in agreement with the above 2 studies . The present study showed a marked decrease in ischemic injury in both castrated and goserelin treated groups (p <0.05) compared with the control group but there was no significant difference between castrated group and goserelin treated group . 50% of castrated group show mild cardiac damage (interstitial edema and focal necrosis), 33.33% of castrated group show moderate cardiac damage (diffuse myocardial cell swelling and necrosis), and 16.67% of castrated group show normal tissue appearance . Showed that castrated male rats when subjected to the ischemia have significantly decreased in the severity of histological finding of ischemia so that there are less ischemic expansion and less neutrophil infiltration at the infarction border than the noncastrated male control group . Histopathological finding in goserelin treated group explains that 33.33% of treated group show mild myocardial damage (interstitial edema with focal necrosis), 50% of treated group show moderate myocardial damage (diffuse myocardial cell swelling and necrosis), and 16.67% of treated group show normal myocardial appearance . Regarding goserelin treated group, to the best of our knowledge there is no study yet available to compare our data . This study is further supporting the role of inflammatory cytokine (il-1, tnf-, and icam-1) in the pathology of regional myocardial i / r and myocardial apoptosis during regional i / r injury . Both surgical castration and goserelin administration reduce the level of inflammatory mediators in myocardial tissues, which may provide mechanistic answer for their protective effect . Both surgical castration and goserelin administration ameliorate myocardial injury and apoptosis in regional i / r induced by lad ligation in rats as evidenced by reduction in the release of cardiac specific enzyme troponin i. the findings of the present study support the idea that endogenous testosterone may have deleterious effect on myocardial injury and apoptosis during regional i / r.
|
Healthcare has been described as a complex adaptive system (cas) that involves multiple, interdependent entities and organizational levels [13]. Such complexity poses a challenge for transformative change as relationships within a cas are nonlinear and unpredictable [4, 5]. This challenge is echoed by leaders in the field of quality improvement who have identified shortcomings, such as a lack of rigorous research methods, a failure to study contextual variables, and weak evaluation designs [68]. These shortcomings are an indication that the interwoven processes of healthcare delivery are difficult for a single investigator to tease apart . Thus, a collaborative model that integrates multiple perspectives from several disciplines may help advance the field of improvement science and facilitate dissemination and implementation strategies . Transdisciplinary collaboration is a potentially effective model as it brings together a diverse group of individuals who fully integrate theories, methodologies, and frameworks from their respective fields to work as a cohesive unit on complex issues . This differs from multidisciplinary and interdisciplinary collaboration where multiple individuals work together but remain grounded in their respective ideologies [911]. Recently, a few studies have addressed the potential of transdisciplinary collaboration to develop effective interventions in healthcare [1214]. Despite these efforts, research in healthcare improvement has typically involved a single researcher studying an intervention at a single hospital or clinical unit . This approach is ineffective as the complexity of organizational change and contextual influence can impact the implementation and effectiveness of an intervention [7, 15]. The current consensus in the field of improvement science is that new methodologies are needed to address complex issues associated with healthcare delivery [6, 16]. Specifically, researchers and clinicians need to adopt strategies that redefine relationships and establish new ways of communicating [6, 1618]. In fact, implementation science frameworks suggest that transdisciplinary interaction promotes effective and sustainable intervention programs . The shift from a single investigator / single site model to a transdisciplinary investigative team / multisite model calls for unique interactions between clinicians and researchers . Currently, there are few competencies and models for team performance; especially in the context of building transdisciplinary teams for improvement . This paper describes the adoption of a transdisciplinary model in a healthcare improvement research network and the impact of collaboration on the conduct of a national improvement study . The results from this study show promise for enhancing research on improvement and uptake of best practices using transdisciplinary collaboration . The guiding framework to implement and evaluate a transdisciplinary collaborative model in this project was grounded in principles from the science of team science (scits). Scits is a hybrid scientific field that incorporates factors that facilitate and hinder scientific collaboration . This field has generated the evidence to support the claim that transdisciplinary collaboration spurs innovation and accelerates scientific discovery [2224]. Additionally, scits is founded on evidence from human factors engineering and offers guidance on how investigative teams can be effective in studying healthcare improvement . The scits framework for this study was adapted from a previous review of factors contributing to collaborative success . Concepts were adopted by a healthcare research network based on the number of times they were referenced, how often they were used for an evaluation of collaborative work, and their relevance to long - distance collaboration . The four concepts in the framework are as follows: (1) readiness for collaboration, (2) creating a shared mental model, (3) management and planning, and (4) virtual readiness . These concepts are briefly described below . In order for a collaboration to be successful, scientific teams need to be ready to collaborate on an individual, group, and organizational level . This readiness comes in the form of one's adaptability and flexibility, openness to diverse perspectives, communication skills, conflict resolution, respect for others, institutional support, and availability of reliable technology [27, 28]. A shared mental model is the organized knowledge that people use to interpret, explain, analyze, and predict what is happening around them . A shared mental model helps collaborating researchers to coordinate with teammates, form accurate expectations about tasks, and understand and anticipate each other's actions and needs . Teams make fewer mistakes than individuals, especially when each team member knows his or her responsibilities, as well as the responsibilities of other team members . Organizational researchers have observed that when team members have a shared mental model, it increases the overall team engagement and performance . The success of a project is dependent on the way the work is organized and carried out in a scientific team . Structuring and monitoring are necessary to maintain scientific rigor and protocol fidelity especially in multisite studies . Without proper management, the project may not meet its objectives, or results generated from a study may not be reliable due to variations in study implementation and data collection . Virtual collaboration requires a high quality, well - functioning technical infrastructure that is designed to fit the nature of the work [22, 27]. Research suggests that user - centered technology, such as access to email, web space for sharing documents, and a centralized databases, is essential features for long distance collaboration [25, 33]. Without a stable technical infrastructure to support virtual collaboration, readiness to collaborate, shared mental models, and management planning are ineffective in multisite, transdisciplinary teams . The main research questions for this study were (1) does an scits framework facilitate transdisciplinary collaboration in a healthcare improvement research network? And (2) what impact does a transdisciplinary model have on the conduct of a national, multisite improvement study? This project was approved by the university of texas health science center san antonio (uthscsa) institutional review board (irb). The objectives of this project focused on transdisciplinary collaboration within a multisite study conducted by the improvement science research network (isrn). The isrn is a national hospital - based research network comprised of approximately 200 partners from academic and practice settings across the nation that have an interest in studying quality improvement . Demographic information for the isrn membership at the time of this study is presented in table 1 . The majority of the membership consisted of nurses working in various fields in acute care settings or at academic institutions . Doctorate prepared members represented a variety of fields including nursing research, health services research, public health, translational science, quality improvement, and implementation science . From the isrn membership, each network study is supported by a research infrastructure modeled after practice - based research networks (pbrns) and multisite clinical trials . The research infrastructure supports a virtual collaboratory, or center without walls, that allows a team of scientists to work on common problems regardless of location [25, 34]. The cornerstone of this infrastructure is a set of national research priorities that serve as a common rallying point to attract diverse perspectives and integrate paradigms from multiple disciplines in order to study improvement . These priorities were developed through a national stakeholder survey and include transitions of care, high - performing clinical systems and microsystems approaches to improvement, evidence - based quality improvement and best practice, and learning organizations and culture of quality and safety . Isrn studies are supported through a cyber infrastructure equipped with appropriate communications technology (virtual meeting platforms and teleconference lines), a dedicated web space for sharing information and a central database for data entry . This technology platform is backed with sufficient bandwidth, electronic networking capabilities, and technical support . Adequate capacity for data security, integrity, privacy, rapid retrieval, and long - term archiving are also integrated into the isrn infrastructure to support data entry and storage . For this study, the average hospital size was 425 beds (range: 120665) with an average daily census of 74.7% (range: 60%100%). Each site had at least one principal investigator (pi) and one research coordinator . Both pis and research coordinators had nursing backgrounds with educational experiences that ranged from bachelor's to doctorate levels . Although the collaborative team also consisted of two study pis (a doctorate - prepared nurse and a physician, both with extensive research backgrounds) and two research scientists (both phd prepared with backgrounds in psychology and physiology), they were not included in the data collection due to their proximity to the evaluation and interpretation of results . Scits principles were used to develop the key isrn resources to facilitate the conduct of network studies . These resources included training meetings to build readiness and a shared mental model, a protocol implementation kit to facilitate study management, and a robust technical infrastructure to support long distance collaboration . Sites on the investigative team were required to complete capacity building exercises, including a training session on participating in a research collaborative . This 2-hour training session consisted of an overview of the isrn mission and research priorities, a review of the study objectives, an introduction to the protocol, an explanation of data entry procedures, and a presentation of how to work as a collaborative team . Sites were also asked to review recorded isrn presentations from experts in the fields of team science and virtual collaboration . Concepts from these presentations were discussed during study meetings . Finally, sites were given a resource guidebook on building successful research collaboration . This guidebook synthesized essential qualities for succeeding in research collaboratives for healthcare improvement . To facilitate the study implementation and ensure the protocol fidelity, sites were given a protocol implementation kit (pik). The pik was designed with two goals in mind: (1) systematic implementation of the study protocol across multiple sites to yield analyzable / reliable data and valid study outcomes; (2) guidance for site pis to facilitate the conduct of the study . The pik provided a structured overview of various topics related to the study including: forming the project team, preparing for irb submission, establishing project timelines, identifying participating staff members, using data collection materials, submitting data to the isrn, and understanding results from the study . Finally, sites were given access to a variety of technical resources, including a shared web space, access to conference lines, and a centralized database . ; santa barbara, ca) and a teleconferencing line were used for real - time meetings and presentations . Lastly, a centralized database was created and maintained by the uthscsa department of epidemiology and biostatistics using the informatics data exchange and acquisition system (ideas), a robust web - based human studies research informatics data management framework . Variables that best represented measures of collaboration readiness, study management, and the presence of a strong mental model were selected for analysis . These variables included (1) the number of protocol deviations (study management), (2) meeting attendance percentage (shared mental model), (3) number of study timeline adjustments (readiness), and (4) number of questions received from pis and coordinators regarding study objectives (study management, shared mental model, and readiness) which were all collected from regulatory documents (e.g., protocol deviation logs), study meeting notes, and email communication . Additionally, site pis (n = 16) and research coordinators (n = 14) were surveyed on isrn coordinating center services and resources . The survey was designed in survey monkey (surveymonkey.com, llc; palo alto, ca), and a link to access the survey was emailed to site pis and research coordinators . Respondents were asked to rate the importance and quality of isrn features and services on 6-point likert scale . The number of protocol deviations, number of email correspondence, number of timeline adjustments, and meeting attendance percentage is broken down by site in table 2 . On average, collaborating sites reported six protocol deviations . Examples of deviations reported were study instruments turned in after data collection period ended and data collected from unconsented participants . A review of the study notes revealed that a total of ten sites (70%) successfully adhered to study timelines . Study timeline delays were due to late irb approvals, accreditation visits, or a change in health information technology systems . The average number of email correspondences per site was 55 emails (range: 20101 emails). The majority of questions from site pis and coordinators pertained to the conduct of the study, including irb regulations, participant recruitment, data collection, and data entry . Few questions (<1%) were directed towards the study's goals or the function of the collaboration . Finally, the attendance on conference calls averaged 94% with sites being absent due to scheduling conflicts or unexpected emergencies . A survey on the quality and importance of isrn coordinating center services and resources was sent to a total 30 site pis and coordinators . Each respondent had previous experience participating in studies as a part of a formal investigative team (mean: 6 studies per respondent, range: 120 studies). On average, half of these studies involved professions other than nursing (mean: 3 studies per respondent, range: 015 studies). Results from survey items specific to providing a collaborative environment are presented in table 4 . Most services and resources were positively rated in terms of quality, but those related to organizational structure, study objectives, and communication were rated highest by respondents (> 90%). The present study described the adoption of a transdisciplinary collaborative model in a multisite improvement study . Resources developed using best practices from scits facilitated the study's conduct as evident by consistent protocol implementation, active engagement, and focused task completion . The impact on productivity could have implications on identifying effective improvement strategies that lead to rapid uptake and spread . Improvement scientists and clinicians in the field need to engage in systems change to support team science and adopt resources, such as research collaborative guidebooks and training modules on scits principles, to build capacity for transdisciplinary collaboration . In the context of collaboration readiness, individuals who demonstrate high levels of readiness are less likely to make mistakes, communicate effectively, and complete objectives in a timely manner . This level of readiness can create a fertile environment for multiple disciplines to come together, blend knowledge, and create a new intellectual space . Both researchers and clinicians the importance of this concept is further validated by the development of standardized evaluations that directly assess readiness in scientific teams, including the national cancer institute's transdisciplinary research on energetics and cancer (trec) survey and the collaboration success wizard [29, 37]. Currently resources do exist such as national research priorities in improvement science, frameworks for implementation science, and national methodology conferences [6, 15, 3841]; yet, more is needed to help build transdisciplinary scientific teams for research . For example, experts have called for the establishment of professional organization for improvement and implementation science . Attention has also been called to taxonomy development for an overarching language in hybrid fields . In the present study, the isrn research priorities and mission were intended to help create a shared mental model that kept research partners engaged in conducting the study . Without a mental model, the observed level of engagement would have decreased and impacted the quality of study outcomes . The present study also demonstrated the effectiveness of the isrn research infrastructure, in particular, the management of a multisite study with standardized approaches . A protocol implementation kit was shown to be an effective tool for ensuring protocol fidelity . The use of such a tool helps meet a need in the field for rigorous research and standardized implementation methods [6, 16, 39]. A failure to ensure consistent implementation and protocol fidelity can decrease the reliability of research findings and thus impact translation into multiple clinical settings . The move from research to clinical practice requires scaling - up quality improvement initiatives to large - scale network studies . Doing so may yield more effective improvement and implementation strategies, improve dissemination of knowledge, and ultimately change policy . An example of transdisciplinary collaboration is in the numbers associated with the isrn study evaluated for this paper . The dataset consisted of 24,014 data points reported by 716 acute care medical - surgical nurses across 14 hospitals . A team - based approach enabled this particular study to capture a representative national sample to enhance the quality of research and raise scientific rigor . This is an indication of a need for established evaluation tools for transdisciplinary collaboration in improvement and implementation research . In fact, there is a nonhealthcare specific tool that is designed to identify potential barriers to collaboration and provide recommendations for improvement . The collaboration success wizard (csw) was developed to evaluate virtual collaborations based on 15 years of evidence that has identified factors that predict collaborative success . An external evaluation of the same isrn study described in the present paper revealed that the project was well positioned for successful collaboration using the csw . The present study was also limited in its ability to measure the impact of transdisciplinary collaboration on dissemination and implementation . Thus, more research and funding are needed to demonstrate the impact of transdisciplinary research on spread and uptake of evidence - based quality improvement uptake . Given the magnitude of data collected from improvement studies using this approach, it can be inferred that rigors methods and generalizable results may speed uptake and spread; however, data are still needed to justify this claim . This project demonstrated the effectiveness of a transdisciplinary model for academic and clinical scientists that are interested in studying improvement . Indications are that national improvement studies are positively assisted by the guidance gleaned from scits . Further research is needed on the causal relationship between team - based research and dissemination and implementation of evidence - based quality improvement . With advances in team research and increased funding opportunities to investigate dissemination, implementation, and improvement strategies in healthcare, the gap between what works and what is actually practiced will narrow to raise the quality of care delivery.
|
Hospitalizations for mental illness and alcohol and drug addiction represent a substantial cost to the united states health care system . One recent estimate indicates that roughly two - thirds of the $29.8 billion spent by mental health organizations in this country in 1992 were expenditures for hospitalization (redick et al ., 1996). Moreover, this estimate likely understates actual expenditures associated with such hospitalizations, because it excludes both the costs attributable to inpatient treatment of alcohol and drug disorders, as well as hospital expenditures associated with the treatment of medical conditions caused or exacerbated by substance abuse . Despite the efforts of deinstitutionalization over the last several decades, expenditures for hospitalization continue to represent a majority of the costs associated with treatment of individuals with mental and addictive disorders . This study focuses on medicare inpatient psychiatric care since there has been limited information to date regarding diagnoses, use, and expenditures for inpatient treatment of psychiatric disorders among medicare beneficiaries . In addition, the percentage of revenues collected by mental health organizations (such as psychiatric hospitals and psychiatric units in general hospitals) from federal government sources (mostly medicare and medicaid) has grown from 25 percent in 1990 to 31 percent in 1992 . In contrast, funding to these facilities from all other major sources was proportionately smaller in 1992 than in 1990 (redick et al ., 1994; 1996). The relative growth in expenditures for institutional psychiatric services suggests the need for an updated analysis of service utilization and cost under programs funded by the federal government . The goals of the current study are thus threefold: (1) to provide an overview of medicare's current coverage and payment policies regarding hospitalization for psychiatric disorders; (2) to present descriptive statistics that update some of the data provided in earlier reports on psychiatric hospitalizations of medicare beneficiaries (freiman, goldman, and taube, 1990; lave and goldman, 1990); and (3) to provide new information on demographic, diagnostic, utilization, and expenditure characteristics associated with 1995 inpatient psychiatric care among medicare beneficiaries . Enhancing our understanding of the types of persons being treated and their service utilization and expenditure patterns may enable decisionmakers to monitor (and perhaps modify) medicare policies and programs to assure the most efficient and effective care delivery to beneficiaries in need of psychiatric services . The hospital insurance (part a) portion of the medicare program helps beneficiaries pay for hospital and skilled nursing facility (snf) services and for home health and hospice care . In general, medicare part a benefits for mental and addictive disorders are similar to part a benefits for physical disorders (health care financing administration, 1995b). Thus, the benefit policies regarding deductibles, copayments, covered days, and reserve days for acute care in most hospital settings are the same for physical conditions as they are for mental and addictive conditions . However, part a places a lifetime limit on the amount of services covered in psychiatric hospitals . Once an individual reaches a lifetime limit of 190 total days in a psychiatric facility, part a ceases to cover the cost of additional days of hospitalization, although medicare may continue to receive information about that person's hospitalization . Hcfa uses various methods to pay for inpatient psychiatric care under fee - for - service (ffs) coverage . For psychiatric hospitals and distinct psychiatric units of general hospitals facilities for the most part exempted from the prospective payment system (pps)hcfa uses a reasonable - cost reimbursement methodology . Payment is on a per discharge basis and is limited by a target amount determined on a facility - specific base year that is adjusted on an annual basis by an inflation factor . Regular beds in short - stay facilities are paid under the diagnosis - related - groups (drg) method of pps . The specific drg weight assigned to psychiatric diagnostic groups is converted to a dollar amount that is then adjusted for factors such as hospital teaching status, local wage rates, and urban - rural location . Units in general hospitals specializing in the treatment of alcohol or drug addiction disorders that meet certain staffing requirements may qualify as distinct psychiatric units . Otherwise, they are not considered pps - exempted and are paid under the drg methodology . Snfs are paid reasonable per diem costs up to a limit based on the area wage index . The limit also varies if the snf is freestanding or a hospital - based facility . Average routine costs were computed in a base year, adjusted for inflation until 1993, and then frozen . Ancillary services, therapies, and drugs are paid in addition at reasonable costs (ingber, 1996). Obtaining accurate and reasonably current expenditure data for psychiatric hospitals and units can be difficult given the complexity and the delays associated with cost - based reimbursement . Final payment amounts are based on facility - specific cost reports that may take 2 or more years to be settled . Interim payments are calculated by fiscal intermediaries based on processed claims and cost reports from previous years . These program payments do not include deductible and coinsurance amounts billed by facilities to beneficiaries . Data for these analyses were obtained from the medicare provider analysis and review (medpar) file for 1995, maintained by hcfa . Medpar data contain a summarized record for 100 percent of all admissions to acute and long - stay hospitals, as well as snfs . The hospital inpatient records include only completed hospitalizations, while snf records may include both completed and ongoing stays since discharge dates from these facilities are not always received by hcfa . Thus the medpar file for 1995 contains completed hospitalizations and both completed and ongoing snf stays taking place in that year . Beneficiaries enrolled in health maintenance organizations (hmos) were excluded from our study as most hmos do not submit bills to medicare for hospital services . In 1995, these individuals represented approximately 11 percent of all medicare beneficiaries (health care financing administration, 1995a). Beneficiaries residing outside of the 50 states and the district of columbia also were excluded from the study . For this analysis, records with a principal diagnosis of mental illness or addiction were identified using diagnostic codes from the international classification of diseases, 9th revision, clinical modification (icd-9-cm) (public health service and the health care financing administration, 1994). Psychiatric diagnoses were grouped in the following 7 categories: (1) delirium, dementia, amnestic and other acquired cognitive disorders, e.g., alzheimer's disease (codes 290.00, 290.99, 293.00, and 294.00 to 294.99); (2) substance - related disorders, such as alcohol or cocaine dependence (codes 291.00 to 292.99, and 303.00 to 305.99); (3) schizophrenia and other psychotic disorders (codes 293.81, 293.82, 295.00 to 295.99, 297.1, 297.3, 298.8, and 298.9); (4) affective disorders, such as major depression or manic - depressive illness (codes 293.83, 296.00 to 296.99, 300.4, 301.13, and 311); (5) anxiety disorders, such as panic disorder and obsessive compulsive disorder (codes 293.89, 300.00 to 300.02, 300.21 to 300.3, 308.3, 309.21, and 309.81); (6) adjustment disorders, which comprise stress - related, clinically significant, and time - limited emotional disturbances (codes 309.0, 309.24, 309.28, 309.3, 309.4, and 309.9); and (7) other psychiatric disorders (all other psychiatric codes not included above). Identified cases were divided into those occurring in the following settings: psychiatric hospitals; psychiatric units in general hospitals; regular beds in general hospitals (i.e., not within a designated psychiatric unit); and snfs . To calculate the rate of hospitalization for psychiatric diagnoses in the medicare population, we created a denominator file that included all persons eligible for part a coverage as of july 1, 1995, who resided in one of the 50 states or the district of columbia and who were not enrolled in an hmo . The first was the length of stay reported for a hospitalization, the second was the length of stay only for those days of care covered by medicare . As noted earlier, we could not calculate length of stay for snf admissions, as the date that the person is discharged or no longer covered by medicare is frequently not reported . The interim payment amounts represent the best payment estimates by fiscal intermediaries before a final settlement . The number of hospitals receiving bonus payments is likely substantial, since target amounts for a large proportion of providers were set at a time when psychiatric stays were generally much longer than they are at present . Second, a number of hospitals request and obtain exemption payments after their cost reports are settled . Table 1 presents the number of hospital discharges and snf stays in 1995 for medicare beneficiaries with a primary psychiatric diagnosis and the rate per 1,000 beneficiaries by type of treatment facility and by age group . Hospital discharges for psychiatric illnesses represented approximately 5.6 percent of all medicare - covered hospital discharges in 1995 . Psychiatric units of general hospitals accounted for approximately 43 percent of the total of hospital discharges and snf stays, followed by general hospitals and long - stay or specialty psychiatric hospitals at 26 percent and 24 percent respectively . Table 2 presents the number and rate of hospital discharges and snf stays related to psychiatric disorders in 1995 for medicare beneficiaries by age group, race, and sex . The data indicate that the rate of hospitalization for these disorders varied greatly by age, from a low of 8.5/1,000 for those 65 - 74 years of age, to a high of 195.8/1,000 for those 25 - 34 years of age . The rates of hospitalizations for psychotic, affective, and alcohol and drug disorders were particularly high among disabled beneficiaries in the 25 - 34 and 35 - 44 year age groups . Rates of hospitalizations or snf stays for acquired cognitive disorders were highest for beneficiaries 85 years of age or over . The hospitalization rate for psychiatric disorders among males (25.98 per 1,000) was higher than for females (19.38 per 1,000). Males had higher rates of hospitalizations than females for alcohol and drug disorders across all age groups . Among the disabled through 44 years of age, males had greater rates of hospitalizations for psychotic disorders . Females had higher hospitalization rates for affective disorders, which accounted for over 40 percent of psychiatric stays for all female beneficiaries . Black beneficiaries were hospitalized for psychiatric disorders at a considerably higher rate (38.12) than white beneficiaries (20.21). The rates were notably higher in the categories of alcohol and drug disorders, psychotic disorders, and, in the case of males, affective disorders . Tables 3 and 4 present the number and percentage distribution of hospital discharges and snf stays for mental and addictive disorders by age group and type of facility . Regardless of age, the largest percentage of discharges for these disorders (43 percent) occurred from psychiatric units in general hospitals . In fact, when all psychiatric and regular beds within general hospitals were included, the data indicate that almost 7 out of 10 hospital discharges and snf stays for psychiatric conditions occurred in such settings . The likelihood of admission to a snf, was quite small for beneficiaries under 65 years of age, but increased substantially with age thereafter . For example, almost 4 out of 5 snf stays for psychiatric disorders (79 percent) were attributable to beneficiaries 75 years of age or over . The number of hospital discharges and snf stays for mental and addictive disorders by diagnosis and type of facility, and the percentage distributions for this data are presented in tables 5 and 6, respectively . The data suggest that there is substantial variability in the diagnostic mix across different types of facilities . More than one - third (34 percent) of all psychiatric hospitalizations and snf stays involved a diagnosis of affective disorder . The overwhelming majority of these episodes (86 percent) occurred in specialty psychiatric hospitals (40 percent) or psychiatric units of general hospitals (46 percent). Approximately one - fourth (26 percent) of hospitalizations and snf stays for mental or addictive disorders involved treatment of some form of psychosis . More than four - fifths of this treatment occurred in either psychiatric units of general hospitals (51 percent) or specialty psychiatric hospitals (30 percent). In contrast, hospitalizations for alcohol and drug disorders were much more likely to occur in non - psychiatric wards of general hospitals (61 percent) than in any other type of facility . In fact, more than one - third (35 percent) of all psychiatric discharges from regular beds in general hospitals were related to treatment for substance abuse, compared with approximately 15 percent of similar discharges from specialty psychiatric hospitals, and only 5 percent of similar discharges from psychiatric units of general hospitals . While more than one - half (55 percent) of all psychiatric - related snf stays and almost one - fourth (24 percent) of all psychiatric discharges from regular beds in general hospitals involved a primary diagnosis of dementia or other acquired cognitive disorder, relatively few discharges from specialty psychiatric hospitals or psychiatric units in general hospitals involved these diagnoses (7 percent and 12 percent respectively). Table 7 presents the average length of stay (alos) for psychiatric hospitalizations when all days were included, as well as when only medicare covered days were included, by type of facility and diagnostic group . The data suggest that when all days were included, the alos for these disorders ranged from approximately 1 week (7.9 days) in regular beds of general hospitals, to almost 2 weeks (13.7 days) in psychiatric units of general hospitals, to more than 3 weeks (24.6 days) in specialty psychiatric hospitals . When only medicare covered days were included, there was a decline of alos to 19.9 days for psychiatric hospitals . Examining the data by diagnostic group reveals that the alos for both affective disorders and dementia was almost twice as long as the alos for adjustment, anxiety, or alcohol and drug disorders . The alos for psychotic disorders was almost 3 weeks (20.9 days), but was somewhat shorter (17.7 days) when only medicare - covered days were included . Interestingly, the large standard deviations related to alos for psychiatric hospitals, especially for persons admitted with a diagnosis of psychosis, suggest that the length of hospitalization in such facilities is quite variable, and may be rather long (e.g., 2 months or more) for certain patients . Total interim payments and average interim payments for 1995 medicare psychiatric hospitalizations and snf stays by age group were calculated per all medicare beneficiaries in the same age group (table 8). Interim payments to hospitals and snfs for inpatient treatment of mental and addictive disorders totaled almost $3.5 billion in 1995 . This figure represents more than 4 percent of the approximately $85.7 billion in medicare interim payments made to hospitals and snfs in 1995 for treatment of all physical and mental disorders (see footnote 3). Notably, almost one - half (46.9 percent) of all payments went to reimburse hospitals and snfs for psychiatric care for beneficiaries under 65 years of age, and approximately one - third of that sum ($597 million) was associated with care for individuals 35 - 44 years of age . Beneficiaries between 25 - 34 years of age were associated with the largest per beneficiary interim payments ($834), which were almost 18 times higher than those made on behalf of beneficiaries with the smallest average interim payments ($47 for beneficiaries between 65 - 74 years of age). Moreover, the average interim payment per beneficiary for those under 65 years of age ($397) was almost 6 times higher than for those age 65 or over ($67), and even 3 times larger than average interim payments for the oldest group of medicare beneficiaries ($119 for those 85 years of age or over). Table 9 provides total and per discharge interim payments for 1995 medicare psychiatric services by diagnostic group and type of facility . Discharges of beneficiaries with a primary diagnosis of alcohol or drug - related disorders constituted 17 percent of total discharges but accounted only for slightly more than 10 percent of total interim payments to facilities . The data also indicate fairly wide variations by facility type in both the total amount of interim payments and the per discharge payments . More than one - half (51.1 percent) of all medicare part a interim payments for psychiatric services went to psychiatric units in general hospitals, with approximately one - fourth to specialty psychiatric hospitals (24.6 percent), almost one - fifth to regular beds in general hospitals (18.6 percent), and less than one - tenth (5.6 percent) to snfs . When treatment for all disorders are considered together, per discharge interim payments are lowest to regular beds in general hospitals ($3,535), followed by snfs ($4,265), psychiatric hospitals ($5,073), and psychiatric units in general hospitals ($5,898). Comparing these data with alos data from table 7 suggests that, while the average length of hospitalization (including only medicare - covered days) in specialty psychiatric facilities is almost 50 percent longer than in psychiatric units of general hospitals, the per discharge interim payments to psychiatric units in general hospitals are 16 percent higher than similar per discharge payments to specialty psychiatric hospitals . Wide variations in per discharge interim payments are also noted among different types of facilities by diagnostic group . Per discharge payments related to treatment of psychotic disorders, affective disorders, or dementia in psychiatric units of general hospitals were substantially more than per discharge payments for treatment of similar disorders in any of the three other facility types . Similarly, per discharge payments related to treatment of alcohol and drug disorders in either psychiatric hospitals or snfs were substantially higher than per discharge payments for treatment of similar disorders in either psychiatric units or non - psychiatric beds within general hospitals . The considerable variation in per discharge interim payments by facility type is consistent with freiman, goldman, and taube's (1990) examination of 1985 medicare data for average covered costs of psychiatric hospitalizations . Table 1 presents the number of hospital discharges and snf stays in 1995 for medicare beneficiaries with a primary psychiatric diagnosis and the rate per 1,000 beneficiaries by type of treatment facility and by age group . Hospital discharges for psychiatric illnesses represented approximately 5.6 percent of all medicare - covered hospital discharges in 1995 . Psychiatric units of general hospitals accounted for approximately 43 percent of the total of hospital discharges and snf stays, followed by general hospitals and long - stay or specialty psychiatric hospitals at 26 percent and 24 percent respectively . Table 2 presents the number and rate of hospital discharges and snf stays related to psychiatric disorders in 1995 for medicare beneficiaries by age group, race, and sex . The data indicate that the rate of hospitalization for these disorders varied greatly by age, from a low of 8.5/1,000 for those 65 - 74 years of age, to a high of 195.8/1,000 for those 25 - 34 years of age . The rates of hospitalizations for psychotic, affective, and alcohol and drug disorders were particularly high among disabled beneficiaries in the 25 - 34 and 35 - 44 year age groups . Rates of hospitalizations or snf stays for acquired cognitive disorders were highest for beneficiaries 85 years of age or over . The hospitalization rate for psychiatric disorders among males (25.98 per 1,000) was higher than for females (19.38 per 1,000). Males had higher rates of hospitalizations than females for alcohol and drug disorders across all age groups . Among the disabled through 44 years of age, males had greater rates of hospitalizations for psychotic disorders . Females had higher hospitalization rates for affective disorders, which accounted for over 40 percent of psychiatric stays for all female beneficiaries . Black beneficiaries were hospitalized for psychiatric disorders at a considerably higher rate (38.12) than white beneficiaries (20.21). The rates were notably higher in the categories of alcohol and drug disorders, psychotic disorders, and, in the case of males, affective disorders . Tables 3 and 4 present the number and percentage distribution of hospital discharges and snf stays for mental and addictive disorders by age group and type of facility . Regardless of age, the largest percentage of discharges for these disorders (43 percent) occurred from psychiatric units in general hospitals . In fact, when all psychiatric and regular beds within general hospitals were included, the data indicate that almost 7 out of 10 hospital discharges and snf stays for psychiatric conditions occurred in such settings . The likelihood of admission to a snf, was quite small for beneficiaries under 65 years of age, but increased substantially with age thereafter . For example, almost 4 out of 5 snf stays for psychiatric disorders (79 percent) were attributable to beneficiaries 75 years of age or over . The number of hospital discharges and snf stays for mental and addictive disorders by diagnosis and type of facility, and the percentage distributions for this data are presented in tables 5 and 6, respectively . The data suggest that there is substantial variability in the diagnostic mix across different types of facilities . More than one - third (34 percent) of all psychiatric hospitalizations and snf stays involved a diagnosis of affective disorder . The overwhelming majority of these episodes (86 percent) occurred in specialty psychiatric hospitals (40 percent) or psychiatric units of general hospitals (46 percent). Approximately one - fourth (26 percent) of hospitalizations and snf stays for mental or addictive disorders involved treatment of some form of psychosis . More than four - fifths of this treatment occurred in either psychiatric units of general hospitals (51 percent) or specialty psychiatric hospitals (30 percent). In contrast, hospitalizations for alcohol and drug disorders were much more likely to occur in non - psychiatric wards of general hospitals (61 percent) than in any other type of facility . In fact, more than one - third (35 percent) of all psychiatric discharges from regular beds in general hospitals were related to treatment for substance abuse, compared with approximately 15 percent of similar discharges from specialty psychiatric hospitals, and only 5 percent of similar discharges from psychiatric units of general hospitals . While more than one - half (55 percent) of all psychiatric - related snf stays and almost one - fourth (24 percent) of all psychiatric discharges from regular beds in general hospitals involved a primary diagnosis of dementia or other acquired cognitive disorder, relatively few discharges from specialty psychiatric hospitals or psychiatric units in general hospitals involved these diagnoses (7 percent and 12 percent respectively). Table 7 presents the average length of stay (alos) for psychiatric hospitalizations when all days were included, as well as when only medicare covered days were included, by type of facility and diagnostic group . The data suggest that when all days were included, the alos for these disorders ranged from approximately 1 week (7.9 days) in regular beds of general hospitals, to almost 2 weeks (13.7 days) in psychiatric units of general hospitals, to more than 3 weeks (24.6 days) in specialty psychiatric hospitals . When only medicare covered days were included, there was a decline of alos to 19.9 days for psychiatric hospitals . Examining the data by diagnostic group reveals that the alos for both affective disorders and dementia was almost twice as long as the alos for adjustment, anxiety, or alcohol and drug disorders . The alos for psychotic disorders was almost 3 weeks (20.9 days), but was somewhat shorter (17.7 days) when only medicare - covered days were included . Interestingly, the large standard deviations related to alos for psychiatric hospitals, especially for persons admitted with a diagnosis of psychosis, suggest that the length of hospitalization in such facilities is quite variable, and may be rather long (e.g., 2 months or more) for certain patients . Total interim payments and average interim payments for 1995 medicare psychiatric hospitalizations and snf stays by age group were calculated per all medicare beneficiaries in the same age group (table 8). Interim payments to hospitals and snfs for inpatient treatment of mental and addictive disorders totaled almost $3.5 billion in 1995 . This figure represents more than 4 percent of the approximately $85.7 billion in medicare interim payments made to hospitals and snfs in 1995 for treatment of all physical and mental disorders (see footnote 3). Notably, almost one - half (46.9 percent) of all payments went to reimburse hospitals and snfs for psychiatric care for beneficiaries under 65 years of age, and approximately one - third of that sum ($597 million) was associated with care for individuals 35 - 44 years of age . Beneficiaries between 25 - 34 years of age were associated with the largest per beneficiary interim payments ($834), which were almost 18 times higher than those made on behalf of beneficiaries with the smallest average interim payments ($47 for beneficiaries between 65 - 74 years of age). Moreover, the average interim payment per beneficiary for those under 65 years of age ($397) was almost 6 times higher than for those age 65 or over ($67), and even 3 times larger than average interim payments for the oldest group of medicare beneficiaries ($119 for those 85 years of age or over). Table 9 provides total and per discharge interim payments for 1995 medicare psychiatric services by diagnostic group and type of facility . Discharges of beneficiaries with a primary diagnosis of alcohol or drug - related disorders constituted 17 percent of total discharges but accounted only for slightly more than 10 percent of total interim payments to facilities . The data also indicate fairly wide variations by facility type in both the total amount of interim payments and the per discharge payments . More than one - half (51.1 percent) of all medicare part a interim payments for psychiatric services went to psychiatric units in general hospitals, with approximately one - fourth to specialty psychiatric hospitals (24.6 percent), almost one - fifth to regular beds in general hospitals (18.6 percent), and less than one - tenth (5.6 percent) to snfs . When treatment for all disorders are considered together, per discharge interim payments are lowest to regular beds in general hospitals ($3,535), followed by snfs ($4,265), psychiatric hospitals ($5,073), and psychiatric units in general hospitals ($5,898). Comparing these data with alos data from table 7 suggests that, while the average length of hospitalization (including only medicare - covered days) in specialty psychiatric facilities is almost 50 percent longer than in psychiatric units of general hospitals, the per discharge interim payments to psychiatric units in general hospitals are 16 percent higher than similar per discharge payments to specialty psychiatric hospitals . Wide variations in per discharge interim payments are also noted among different types of facilities by diagnostic group . Per discharge payments related to treatment of psychotic disorders, affective disorders, or dementia in psychiatric units of general hospitals were substantially more than per discharge payments for treatment of similar disorders in any of the three other facility types . Similarly, per discharge payments related to treatment of alcohol and drug disorders in either psychiatric hospitals or snfs were substantially higher than per discharge payments for treatment of similar disorders in either psychiatric units or non - psychiatric beds within general hospitals . The considerable variation in per discharge interim payments by facility type is consistent with freiman, goldman, and taube's (1990) examination of 1985 medicare data for average covered costs of psychiatric hospitalizations . The data presented in this article provide an overview of demographic, diagnostic, utilization, and expenditure characteristics associated with the delivery of medicare part a services to beneficiaries with mental and addictive disorders in 1995 . Comparison with previous studies (freiman, goldman, and taube, 1990; lave and goldman, 1990) suggests that the rate of hospitalizations among medicare beneficiaries for treatment of mental or addictive disorders almost doubled in the past decade, from approximately 11 to 21 discharges per 1,000 beneficiaries . Although the rate of discharges is quite variable across age group and race, the overall rate would appear to be substantially higher than recent epidemiological estimates (9.0/1,000) of the use of inpatient psychiatric services by the general population (bourdon et al . The growth in the rate of inpatient psychiatric hospitalization for medicare beneficiaries also contrasts with the decrease in the overall rate of inpatient psychiatric admissions among the general population nationwide between 1990 and 1992, the last year for which published data are available (redick et al ., 1996). A variety of factors affecting the demand and supply of services may explain the significant increase in utilization of inpatient psychiatric care for medicare beneficiaries in recent years . On the demand side, the rise may be related in part to the substantial growth since 1986 in the number of medicare beneficiaries under 65 years of age, in particular those disabled by mental impairments (kennedy and manderscheid, 1992). Medicare beneficiaries under 65 years of age (i.e., individuals who qualified for medicare due to disability) represented only 12 percent of all beneficiaries in 1995 (health care financing administration, 1995a) but accounted for more than one - half (53.1 percent) of all hospital discharges and snf stays attributable to mental or addictive disorders . On the supply side, increased capacity nationwide in private psychiatric facilities may also account in part for the rise in medicare psychiatric hospitalizations (redick et al ., 1996). Bed rates per 100,000 population for private psychiatric hospitals and non - federal general hospital psychiatric inpatient services experienced moderate growth between 1980 and 1990 (redick et al ., 1994). A certain degree of supplier - induced demand for medicare beneficiaries may have occurred also because medicare payments to pps - exempted facilities have been more generous compared with those made by other payors during the early 1990s (u.s . Moreover, third - party authorization for admissions and extended stays are not required for medicare beneficiaries under fee - for - service, whereas these utilization management practices have become commonplace during this period in both private and (to a lesser extent) public programs . In the future, changing attitudes among the elderly that have resulted in less stigma associated with the use of counseling and psychiatric care (borinstein, 1992) may reinforce the trend toward greater use of psychiatric services . The influx of younger, more disabled beneficiaries, who tend to remain enrolled in the medicare program for longer periods, is also likely to increase utilization and expenditures for a range of psychiatric and non - psychiatric medicare services (kennedy and manderscheid, 1992). An ongoing concern for program administrators is the risk that beneficiaries with mental illness and addictive disorders (a relatively vulnerable group) may be subject in an unmanaged fee - for - service environment to provider - induced demand for better reimbursed (but not necessarily more appropriate) services . The data on treatment settings show that general hospitals, and particularly psychiatric units within these facilities, continue to provide the majority of inpatient treatment services to medicare beneficiaries with psychiatric disorders . Yet that pattern may be changing . Comparisons with 1985 data (freiman, goldman, and taube, 1990) reveal that during the past decade psychiatric hospitals have assumed a greater role delivering inpatient services to medicare beneficiaries . In 1985, almost four - fifths (79.6 percent) of discharges occurred from general hospitals (including psychiatric units), while one - fifth (19.4 percent) occurred from specialty psychiatric hospitals (freiman, goldman, and taube, 1990). In 1995, the percentage of discharges from general hospitals (including psychiatric units) had declined to 74.2 percent, while the percentage of those from psychiatric hospitals had increased to 25.8 percent . Part of the explanation for this change may be related to the growth of disabled medicare beneficiaries, who accounted for more than two - thirds (70.7 percent) of all medicare discharges for psychiatric and addictive disorders from psychiatric hospitals . The finding that inpatient care for addictive disorders represents 17 percent of all discharges and only 10 percent of payments may indicate underdiagnosis and relatively low utilization for these disorders since they have been associated with roughly 30 percent of persons receiving inpatient services for mental illness and addictions in the general population (bourdon et al ., 1994). Data were not broken down by other categories such as hispanic, native - american, or asian - american because the accuracy of these codes in the medicare data system has not yet been verified . With respect to psychiatric hospital settings, we were unable to distinguish hospitals that are state - administered from those that are private . These two types of facilities differ in the populations they serve, the intensity of services provided, and the average length of patients' stay . Internal hcfa data show, for instance, that alos in 1993 for both proprietary and not - for - profit private psychiatric hospitals was less than 17 days whereas government - run facilities had an alos of over 77 days . In addition, the data presented in this study include only inpatient psychiatric hospitalizations in the fee - for - service component of the medicare program, but do not include payments to individual practitioners for inpatient care or payments for any outpatient (part b) services . Recent programmatic changes to expand coverage for other benefits (i.e., partial hospitalization and outpatient benefits enacted through the omnibus budget reconciliation acts, 1987, 1989, 1990) have increased overall utilization of psychiatric services by medicare beneficiaries . To understand fully the sociodemographic, epidemiological, and service patterns of psychiatric utilization will require joint study of inpatient, partial hospitalization, and other outpatient services in both the fee - for - service and hmo sectors of the medicare program . Our understanding of the impact of mental illness on the medicare program will still be limited, however, unless the use of other forms of medical care by beneficiaries receiving psychiatric services is also taken into account persons with mental illnesses and addictions tend to use more medical care than the average beneficiary often as a substitute for less readily available behavioral health services (fuller, 1995). Conversely, certain subgroups of persons with severe mental illnesses and addictions may, in fact, need more medical care than the average beneficiary, a need at times unmet . The literature suggests, for example, that individuals with schizophrenia have high mortality rates from non - psychiatric medical causes at a younger age than individuals without the disorder (massachusetts critical incident reporting task force, 1995). Better data on the use of psychiatric and general medical services for these populations would improve our understanding of the relationship between virtually separate systems of care: how use of behavioral care affects use of general medical care and vice versa, and the likely increase in future use of medicare inpatient psychiatric services may be altered by the influence of managed care . In recent years, the private sector has witnessed a revolution in the provision of mental health and substance abuse services (iglehart, 1996). In the late 1980s, corporate purchasers of health insurance saw the cost of coverage for mental health and substance abuse services increase at a faster pace than general medical services, primarily driven by high utilization of inpatient services (frank, salkever, and sharfstein, 1991). An industry almost non - existent a decade ago emerged to meet the need for cost containment of these private purchasers of care . Among various management practices, these programs have substituted less costly forms of treatment for more costly health services, primarily inpatient care . As a consequence, the patterns of utilization for psychiatric conditions including the service settings utilized have changed dramatically . In the case of inpatient psychiatric care, management has generally reduced the number of hospital admissions as well as shortened the average length of hospital stays . Questions about the appropriateness of current inpatient utilization rates as well as the quality, mix, and coordination of inpatient and outpatient behavioral health services under medicare may warrant consideration of a variety of potential changes in the delivery of these services . For example, hcfa could seek authority to adopt some of the third party management practices used in the private sector (e.g., concurrent review of psychiatric services). Another possibility is to consider perhaps through a carve - out in fee - for - service medicare a demonstration on the use of case management for behavioral health services with the goal of providing care in the least restrictive setting and improving coordination of needed services for beneficiaries with the most severe and persistent mental disorders . This type of demonstration might also examine the benefit of including expanded coverage for psychotropic drugs in certain complex and costly to treat cases . A common basis for setting payment rates in managed behavioral health care contracting is the use of past utilization by the covered population in question (frank, mcguire, and newhouse, 1995). This study provides evidence about variations in spending for different subpopulations, particularly age - groups, documenting significant differences in cost between the under the 65 years of age and the 65 years of age or over categories and within these groups as well . These age - based distinctions may provide information necessary to assist in setting actuarial or capitation rates for behavioral health services . With respect to medicare hmos, little is known specifically about access and cost of psychiatric services for the increasing proportion of medicare beneficiaries already enrolled in managed care . The findings reported in this study also suggest the need to explore the adequacy and types of subcapitation rates presently paid by the large proportion of medicare hmos that subcontract for the provision of behavioral health services . In addition, the data illustrate the fact that high cost users of psychiatric care may be readily identifiable just by age, creating an opportunity for health care plans given their current responsibility over beneficiary enrollment to favorably select lower - cost beneficiaries . Given this and other potential concerns, there is need to monitor the evolution of medicare risk hmos in this service sector . At a time when the debate to reform medicare is gaining momentum, a question in the fee - for - service medicare behavioral health care area for program administrators is whether medicare should adopt and adapt management techniques broadly utilized in other public and private programs . Research that documents medicare demographic, diagnostic, utilization, and expenditure trends in behavioral health care, and studies comparing these data with trends in both the private and public sectors should prove useful for policymakers charged with the responsibility of reforming and improving the medicare program . The likely increase in future use of medicare inpatient psychiatric services may be altered by the influence of managed care . In recent years, the private sector has witnessed a revolution in the provision of mental health and substance abuse services (iglehart, 1996). In the late 1980s, corporate purchasers of health insurance saw the cost of coverage for mental health and substance abuse services increase at a faster pace than general medical services, primarily driven by high utilization of inpatient services (frank, salkever, and sharfstein, 1991). An industry almost non - existent a decade ago emerged to meet the need for cost containment of these private purchasers of care . Among various management practices, these programs have substituted less costly forms of treatment for more costly health services, primarily inpatient care . As a consequence, the patterns of utilization for psychiatric conditions including the service settings utilized have changed dramatically . In the case of inpatient psychiatric care, management has generally reduced the number of hospital admissions as well as shortened the average length of hospital stays . Questions about the appropriateness of current inpatient utilization rates as well as the quality, mix, and coordination of inpatient and outpatient behavioral health services under medicare may warrant consideration of a variety of potential changes in the delivery of these services . For example, hcfa could seek authority to adopt some of the third party management practices used in the private sector (e.g., concurrent review of psychiatric services). Another possibility is to consider perhaps through a carve - out in fee - for - service medicare a demonstration on the use of case management for behavioral health services with the goal of providing care in the least restrictive setting and improving coordination of needed services for beneficiaries with the most severe and persistent mental disorders . This type of demonstration might also examine the benefit of including expanded coverage for psychotropic drugs in certain complex and costly to treat cases . A common basis for setting payment rates in managed behavioral health care contracting is the use of past utilization by the covered population in question (frank, mcguire, and newhouse, 1995). This study provides evidence about variations in spending for different subpopulations, particularly age - groups, documenting significant differences in cost between the under the 65 years of age and the 65 years of age or over categories and within these groups as well . These age - based distinctions may provide information necessary to assist in setting actuarial or capitation rates for behavioral health services . With respect to medicare hmos, little is known specifically about access and cost of psychiatric services for the increasing proportion of medicare beneficiaries already enrolled in managed care . The findings reported in this study also suggest the need to explore the adequacy and types of subcapitation rates presently paid by the large proportion of medicare hmos that subcontract for the provision of behavioral health services . In addition, the data illustrate the fact that high cost users of psychiatric care may be readily identifiable just by age, creating an opportunity for health care plans given their current responsibility over beneficiary enrollment to favorably select lower - cost beneficiaries . Given this and other potential concerns, there is need to monitor the evolution of medicare risk hmos in this service sector . At a time when the debate to reform medicare is gaining momentum, a question in the fee - for - service medicare behavioral health care area for program administrators is whether medicare should adopt and adapt management techniques broadly utilized in other public and private programs . Research that documents medicare demographic, diagnostic, utilization, and expenditure trends in behavioral health care, and studies comparing these data with trends in both the private and public sectors should prove useful for policymakers charged with the responsibility of reforming and improving the medicare program.
|
Maxillofacial injuries have the potential to cause airway compromise and are associated with pain and swelling causing difficulty in mouth opening, chewing and deglutition . It helps in intraoperative pain relief and also helps in early post operative rehabilitation of maxilla facial trauma patients mandibular nerve block is often performed for diagnostic, therapeutic and anesthetic purposes for surgery involving mandibular region . We report a case where intraoperative and post - operative pain in a case of unilateral fracture mandible was effectively managed through intermittent mandibular nerve block via a catheter . A 30-year - old male with right sided parasymphyseal fracture mandible was scheduled for open reduction and internal fixation . The patient had difficulty in opening the mouth due to pain (3 cm). Informed written consent for the nerve block was obtained and visual analogue scale (vas) of 0 - 10, was explained to patient . In the operation theater, neuromuscular blockade was achieved with vecuronium and anesthesia was maintained with o2 and n2o using controlled ventilation . The right side of the face was prepared for mandibular nerve block with lateral extraoral approach [figure 1]. Arrow showing skin site of epidural needle insertion for mandibular nerve block an 18-gauge i.v . Cannula was inserted at midpoint of lower border of the zygomatic arch and was advanced perpendicular to face until it contacted the lateral pterygoid plate . The length of the cannula outside the skin was marked and cannula was redirected slightly posterior to reach behind the posterior border and was advanced further by 0.5 cm . Catheter was tunnelled subcutaneously and the filter was attached to its other end [figure 2]. For surgical analgesia, a bolus dose of 4 ml of 0.25% bupivicaine was given through the catheter . Fentanyl 1mg/ kg i.v . Was given only when there was more than 20% increase in heart rate or blood pressure above base line . The surgery lasted for 2 h. at the end of surgery, neuromuscular blockade was reversed and the trachea extubated . Following extubation, the patient was conscious and pain free and then shifted to ward . Post operatively, he received 4 ml of 0.25% bupivacaine through the epidural catheter every 12 h for two days . Vas score was measured immediately after surgery and thereafter at 1, 2, 4, 6, 12, 24, and 48 h respectively . Patient was observed for numbness at the surgical site, need for rescue analgesia, complications (nausea, vomiting). Numbness in area of lower jaw line was present throughout the period but subsided after discontinuation of local anesthetic through epidural catheter . He was discharged on fourth post operative day with advice for follow up in o.p.d . Mandibular nerve block can be used to manage intra as well as post operative pain in cases of fracture mandible . We performed lateral extra oral approach because of restricted mouth opening and the need to retain the catheter for post operative analgesia . 18 g cannula instead of epidural needle was used to minimize the bleeding from pterygoid plexus of veins . The cannula was advanced further after contacting lateral pterygiod plate so that the catheter comes in vicinity of mandibular nerve and there is no displacement during jaw movements . The catheter was further tunnelled subcutaneously to prevent dislodgement and filter was used to prevent infection . There was excellent post operative analgesia achieved with this technique as shown in decreased pain scores, both static as well as dynamic.
|
Elevated serum uric acid levels are associated with a variety of adverse health outcomes, including gout, hypertension, diabetes mellitus, metabolic syndrome, and cardiovascular diseases . Several genome - wide association studies (gwass) have identified genes that may be causally associated with uric acid levels, including the atp - binding cassette, subfamily g, member 2 (abcg2) gene (mim 603756) [2, 3, 4, 5, 6, 7]. Replication studies of abcg2 variants have been performed in western and asian countries [8, 9]. Identified a significant association between the rs2725220 single - nucleotide polymorphism (snp) on chromosome 4q22 and uric acid levels . The abcg2 protein is a high - capacity transporter for uric acid excretion in the kidney, liver, and gut [10, 11]. In this study, we analyzed the relationship between uric acid levels and the abcg2 snp rs2725220 using a group of volunteers from the korean metabolic syndrome research initiative study in seoul . We also analyzed the association of the abcg2 snp with body mass index and waist circumference levels . Subjects for the gwas were recruited from the korean metabolic syndrome research initiative study in seoul city, which was initiated in december 2005 . A total of 9,128 individuals were recruited in 2006, and an additional 17,569 individuals were recruited in 2007 [12, 13, 14]. Volunteers from the first round underwent routine health examinations at the health promotion centers in university hospitals between january 2006 and december 2007 . From this the subject characteristics were described in a previous study . In brief, of the 6,563 individuals whose adiponectin was measured, 1,004 individuals were genotyped . The 1,004 subjects were all healthy individuals and were not undergoing any treatment for hyperuricemia . The institutional review board of human research of yonsei university approved the protocols of this study, and written informed consent was obtained from all subjects prior to enrollment . Participants were interviewed using a structured questionnaire to collect a personal history of cigarette smoking (never smoked, ex - smoker, or current smoker) and demographic characteristics (age, gender, etc . ). Waist circumference was measured midway between the lower rib and iliac crest . For measurements of weight and height, body mass indices were calculated as the subject's weight (kg) divided by the square of the subject's height (m). For clinical chemistry assays, serum was separated from peripheral venous blood samples obtained from each participant after a 12-hour fast and stored at -70. biomarkers of metabolic syndrome, including fasting blood glucose, total cholesterol, triglycerides, and high - density lipoprotein cholesterol, and uric acid levels were measured . Quality control of the data was conducted in accordance with the procedures recommended by the korean association of laboratory quality control . Samples from the seoul city cohort were genotyped on the affymetrix genome - wide human snp array 5.0 (affymetrix inc ., santa clara, ca, usa) at dnalink . For the data obtained from this chip, internal quality control (qc) measures were used: the qc call rate (dynamic model algorithm) always exceeded 86% . Ten of the 1,004 individuals were removed due to low genotyping call rates (<95%). Three individuals were excluded due to failed genotyping of rs2725220, leaving a total of 991 individuals for the study . Most statistical analyses were performed using plink and sas (version 9.2; sas institute, cary, nc, usa). The abcg2 gene snp rs2725220 was selected using the affymetrix genomewide human snp array 5.0 . Each snp was tested for possible effects on uric acid levels under an additive model . The multivariate linear regression models used in the study incorporated covariates (age and sex). Odds ratios (ors) with 95% confidence intervals (cis) were calculated to examine the association of the abcg2 snp with abnormal uric acid levels (7 mg / dl for men and 6 mg / dl for women). All statistical tests were two - sided, and the statistical significance was determined as p <0.05 . Subjects for the gwas were recruited from the korean metabolic syndrome research initiative study in seoul city, which was initiated in december 2005 . A total of 9,128 individuals were recruited in 2006, and an additional 17,569 individuals were recruited in 2007 [12, 13, 14]. Volunteers from the first round underwent routine health examinations at the health promotion centers in university hospitals between january 2006 and december 2007 . From this the subject characteristics were described in a previous study . In brief, of the 6,563 individuals whose adiponectin was measured, 1,004 individuals were genotyped . The 1,004 subjects were all healthy individuals and were not undergoing any treatment for hyperuricemia . The institutional review board of human research of yonsei university approved the protocols of this study, and written informed consent was obtained from all subjects prior to enrollment . Participants were interviewed using a structured questionnaire to collect a personal history of cigarette smoking (never smoked, ex - smoker, or current smoker) and demographic characteristics (age, gender, etc . ). Waist circumference was measured midway between the lower rib and iliac crest . For measurements of weight and height, body mass indices were calculated as the subject's weight (kg) divided by the square of the subject's height (m). For clinical chemistry assays, serum was separated from peripheral venous blood samples obtained from each participant after a 12-hour fast and stored at -70. biomarkers of metabolic syndrome, including fasting blood glucose, total cholesterol, triglycerides, and high - density lipoprotein cholesterol, and uric acid levels were measured . Quality control of the data was conducted in accordance with the procedures recommended by the korean association of laboratory quality control . Samples from the seoul city cohort were genotyped on the affymetrix genome - wide human snp array 5.0 (affymetrix inc ., santa clara, ca, usa) at dnalink . For the data obtained from this chip, internal quality control (qc) measures were used: the qc call rate (dynamic model algorithm) always exceeded 86% . Ten of the 1,004 individuals were removed due to low genotyping call rates (<95%). Three individuals were excluded due to failed genotyping of rs2725220, leaving a total of 991 individuals for the study . Most statistical analyses were performed using plink and sas (version 9.2; sas institute, cary, nc, usa). The abcg2 gene snp rs2725220 was selected using the affymetrix genomewide human snp array 5.0 . Each snp was tested for possible effects on uric acid levels under an additive model . The multivariate linear regression models used in the study incorporated covariates (age and sex). Odds ratios (ors) with 95% confidence intervals (cis) were calculated to examine the association of the abcg2 snp with abnormal uric acid levels (7 mg / dl for men and 6 mg / dl for women). All statistical tests were two - sided, and the statistical significance was determined as p <0.05 . The majority of individuals in this study was middle - aged (table 1). Mean levels of uric acid in the dataset were higher in males (6.23 mg / dl) than in females (4.21 mg / dl). The percentage of individuals with abnormal uric acid levels (7.0 mg / dl) was 14.7% for the seoul city sample . The percentage of current smokers in the seoul city dataset was 46.4% among male subjects and 3.9% among female subjects . Table 2 shows the p - values from a linear regression model for uric acid levels in the cohort sample, with age and sex included as covariates in the model . The rs2725220 snp in the abcg2 gene was found to be associated with mean uric acid levels (effect per allele 0.25 mg / dl, p <0.0001), which was significant after bonferroni correction (p = 0.00025). The minor allele frequency of rs2725220 was 0.222, and hardy - weinberg equilibrium was satisfied (p = 0.7079). We also analyzed the association of rs2725220 with abnormal uric acid levels (table 3). Subjects with the gc / cc genotype had a 1.78-fold (range, 1.22- to 2.62-fold) higher risk of having abnormal uric acid levels (7.0 mg / dl) than subjects with the gg genotype . When analyzed by gender, the association with abcg2 was stronger in men than in women . In table 4, we analyzed the association of abcg2 by body mass index (bmi) quartile levels and waist circumference quartile levels in male subjects . The association with abcg2 was much stronger in male subjects with bmi 26.4 (or, 5.09; 95% ci, 2.41 to 10.8) than in male subjects with bmi <26.4 . The association with abcg2 was also a little bit stronger in male subjects with waist circumference (wc) 91 (or, 2.10; 95% ci, 1.08 to 4.10) than in male subjects with wc <91 . In a cohort study of 991 subjects, an abcg2 gene snp was associated with increased uric acid, consistent with previous studies . Several gwass have reported that abcg2 exerts a major influence on uric acid levels [2, 3, 6, 7]. Conducted a genomewide study in which an snp in the abcg2 gene, rs2231142, displayed strong evidence of an association with uric acid levels (p <10). Another gwas also reported that rs2231142 showed a strong association with uric acid levels (p = 5.1 10). More recently, a gwas in european - american obesity cases and controls reported significant genome - wide association of two snps with uric acid levels: rs2622605 and rs1481017 . Another recent gwas also reported a strong association between rs2231142 and uric acid levels (p = 3.34.1 10). In the present study, rs2725220 was found to have a strong association with uric acid levels . In agreement with these findings, a recent study reported that rs2725220 was strongly associated with uric acid levels (effect per allele -0.135 mg / dl, p = 4.2 10). Several recent studies reported that the association between serum uric acid and the allelic effects of snps in abcg2 was sex - specific [8, 9]. In a recent study of 4 us populations, the association between rs2231142 and serum uric acid was significantly stronger in men and postmenopausal women compared to their premenopausal counterparts . In the present study, the association of the abcg2 gene snp with uric acid was stronger in men than in women . A modification of the effect of the associations between abcg2 snps and serum uric acid concentrations by interactions with obesity has been reported [3, 9]. In a recent study of 4 us populations, the association between rs2231142 and serum uric acid was stronger in normal - weight subjects than in obese subjects . In the present study, we also examined the association of the abcg2 snp with uric acid levels according to bmi level and waist circumference . The association was stronger in subjects with a high bmi and high wc levels than in subjects with a low bmi and low wc levels . The abcg2 gene encodes a membrane transporter belonging to the atp - binding cassette superfamily of membrane transporters, a group involved in the trafficking of biological molecules across cell membranes . . Allikmets et al . Mapped the abcg2 gene to human chromosome 4q22 using radiation hybrid analysis . We estimated the identity by state over all snps, and only four individuals were shown to be biological relatives . Genetic studies in asian populations may not identify the same set of genes as those in european populations . Nevertheless, the seoul city cohort study in korean populations indicates that the abcg2 gene on chromosome 4 is associated with serum uric acid levels, as is the case in several other populations.
|
Anatomists have observed that the length of the esophagus, i.e. The distance from the incisors to the esophagogastric junction, varies in different individuals from 32 to 50 cm . By endoscopy, the usual length of on adult essphagus is measured as 40 cm from the incisor to the point where mucosal change occurs, but the esophageal length varies from one person to another according to physical status . This variability is often experienced when trying to insert a ph meter, manometer or even nasogastric tube, or when placing the prosthesis for benign or malignant stenosis . We measured various lengths of esophagi in korean adults using a flexible fibercope and measured the external body intervals . Then, we analyzed these data and extrapolated the various esophageal lengths with measurable external body intervals to obtain the correlations . The study was conducted for the measurement of esophageal length in 196 persons, 97 males and 99 females, with normal esophagogastric endoscopic findings . The distances from the upper incisors to 5 anatomical levels of esophagus (cricopharyngeus narrowing, aortic narrowing, left main bronchus narrowing, hiatus and gastroesophageal junction) were measured by an endoscope with 1 cm markings (olympus co. q20 foreward view) (fig . 1). Each length was the mean value of double measurements during full inspiration and expiration respectively . The external body interval lengths studied were standing height, sitting height, the distance from the 7th vertebra to the coccyx and the distance from the upper incisors to the external occipital protuberance . The standing height and sitting height the distance from the 7th cervical vertebra to the coccyx was measured with a tape measure . A specially designed measure was used for the distance from the incisors to the external occipital protuberance (fig . Data analysis and correlation of esophageal lengths to external body intervals were done using spss / pc (statistical package for social science / pc). Observations have shown great variation in the length of the esophagus in adults but good correlation between esophageal lengths and external parameters . The measured lengths of external body intervals and various anatomical esophageal levels are depicted in table 1 . The mean standing - height and sitting - height of 196 persons studied were 160.808.64 cm and 86.605.91 cm, respectively . The interval from the upper incisors to each anatomical landmark of the esophagus (cricopharyngeal narrowing, aortic arch compression, left main bronchus compression, hiatus, and esophagogastric junction) were 15.70 cm, respectively . The true esophageal length, which is the distance from the cricopharyngeal narrowing to the esophagogastric junction, was 24.932.76 cm . The correlation of each esophageal anatomical level with external body intervals using the multiple stepwise reggression method is shown in table 2 . Each of these 4 external variables was statistically significant, but better correlations were demonstrated by the h1 and h3 . Although we employed the measurable distance (h4) instead of the unmeasurable distance (l1), the equations using h4 and h2 did not express the esophageal lengths more accurately than the equation using h1 and h3 only . For example, the distance from the incisors to the esophagogastric junction (l5) could be neatly expressed in simple equations as the following: l5a=0.178 h1 + 0.176 h3 + 2.219 (r2=0.58, p<0.01)l5b=0.242 h1 + 2.076 (r2=0.55, p<0.01) likewise, all other esophageal lengths could be approximated with the equations using h1 and h3 or with the equation using h1 only . The development of improved capability in medical and surgical managment of esophageal disease processes has been significant . To understand and appropriately use current technologic advancements, the anatomic properties of the esophagus available information about esophageal length, however, has been based on cadaveric measurements, because it is not feasible to measure esophageal length in vivo . In adults, the distances from the incisor teeth to the esophagogastric junction have been described to be in the range of 3250 cm by various studies, and the described distance from the cricopharyngeus narrowing to the esophagogastric junction usually varies from 20 to 30 cm . On the other hand, jackson described esophageal lengths according to various age groups . But such wide ranges of esophageal values and different physical status even in the same age group hamper the application of these data to individual patients in esophageal procedures . The best approximation of esophageal length would be useful for various clinical procedures, such as placement of a ph meter, manometer and nasogastric tubes, insertion of an endoprosthesis and colonic interpostition for both benign and malignant stenotic segments . We conducted this study to determine the mean esophageal length of various anatomical levels and to derive a method approximating true esophageal lengths . The mean esophageal lengths from the upper incisors to the esophagogastric junction were 42.612.42 cm in men and 39.472.24 cm in women . These values are not smaller than western data despite the fact that koreans are shorter . This is possible because previous data were based on cadaveric examination and koreans have a different racial stature . We correlated several external body intervals with esophageal lengths by the multiple stepwise regression method: standing height, sitting height, the distance from the 7th cervical vertebra to the coccyx and the distance from the upper incisors to the occiput . All external parameters significantly correlated with the true esophageal lengths . Based on our results, the distance from the upper incisors to the esophagogastric junction (l5) can be best approximated by employing following equations: l5=0.178 h1 + 0.176 h3 + 2.219 (r2=0.58, p<0.001)l5=0.242 h1 + 2.078 (r2=0.55, p<0.01) but accuracy determined by the other equations using more variables other than height did not increase remarkably despite their complexities . Likewise, other anatomical levels of the esophagus can be predicted accurately as shown in the previous results . In conclusion, we consider that an accurate appraisal of the true length of an adult esophagus can be made by measuring the height and the other external measurable parameters.
|
Lead (pb) exposures have decreased with the removal of pb from gasoline . However, pb exposure and toxicity remains an important public health issue . Certain populations in the usa as well as in many developing countries still experience high exposures . An inverse association between blood pb level and cognitive abilities is observed at very low blood pb concentrations, and the pb associated intellectual decrement was steeper at low blood pb levels than at higher blood pb levels [13]. In adults, it has been shown that even low pb exposures are associated with significant health effects among nonoccupationally exposed populations [49]. Traditionally, blood pb is used as a biomarker to determine pb exposures, but blood pb has a half - life of 30 days and therefore correlates less well with long - term exposure than does bone pb, for which the half - life is several years to decades [10, 11]. Cd-109 induced k x - ray fluorescence (kxrf) technology has been used to measure pb in bone for over two decades and has made significant contributions to the study of associations between long - term cumulative pb exposure and adverse health outcomes [4, 5, 7, 1214]. However, the system requirement of a radioactive source, long acquisition times, and a sizeable space for the equipment limits this research to very few groups who possess this technology . In a previous study, we demonstrated the validity of a portable xrf system that made use of pb l x - rays to quantify pb in bone . Improvements to this portable system's geometry and detector have been made, which decrease the minimum detection limit and make the device more compatible for use in vivo . The new system was tested with phantoms to determine the minimum detection limit of the device . Tests with phantom, goat bone, and cadaver bone samples were used to determine the accuracy of the device in determining bone pb concentrations . Pb l x - rays, which have relatively low energies, have greater soft tissue attenuation for the signals and hence the correction for this is a significant issue . To this end, new calibration methods are being explored in this study to establish a more accurate approach to quantify the pb in bone in vivo . Kxrf technology is used in this study to validate the results found with the portable xrf device . The setup of the device is the same as that used in previous studies [16, 17]. The system uses four 16 mm diameter high - purified germanium (hpge) detectors with 10 mm thickness, four feedback resistance preamplifiers, four digital signal processing systems, and a computer . A 135 mci cd source is used to irradiate tibia bone or bone equivalent samples to produce the pb k x - rays . The bone pb measurements were taken for 30 minutes with the hpge detector and then processed with digital electronics . The spectra were analyzed using an in - house peak fitting program and the final pb concentrations were calculated [1719]. The whole body effective dose from this system was measured to be 0.26 sv for adults . Two customized portable xrf devices were used in this project (xl3 t and xl3 t goldd+, thermo fisher scientific inc ., billerica, ma). The xl3 t device used in our previous study is used in this study for a comparison to determine how the improvements in the device technology impact the measurements . Previously the device was equipped with a thermoelectric cooled si pin diode with 8 mm area and 1 mm thickness . The device also has a tube voltage of up to 50 kv, a current of up to 40 a, and various filter combinations . The new device (xl3t - goldd+) has a more compact and optimized geometry . It uses a thermoelectric cooled silicon drift detector with a 25 mm area and 1 mm thickness . The devices were customized so that the voltage of the x - ray tube, the current of the tube, and the filter combinations could be selected to allow the best performance for in vivo measurement of pb in bone . In our experiment, we used a measurement time of 3 minutes . Based on our previous study, by adjusting values for increased measurement time and tube current, we estimated the entrance skin dose of the system was 31 msv to a 1 cm area and the whole body effective dose was 3.6 sv . This can be compared to the whole body effective dose for a standard ap chest x - ray of about 100 sv . Soft tissue and bone equivalent phantoms were used in this study to determine the sensitivity of the device and to calibrate the system . Lucite plate phantoms were used to simulate soft tissue over bone by placing the lucite over the flat surface of the bone phantoms in increments of 1 mm up to 5 mm of lucite thickness . Cylindrical pb doped phantoms made of plaster - of - paris were used to simulate bone with pb concentrations ranging from 0 to 100 ppm (0, 5, 10, 15, 20, 30, 50, 75, and 100 ppm). These measurements were made from the flat base of the phantom . In our new calibration method (i.e., background subtraction), the compton scattering peak was used to determine the background under the pb l x - ray peak and the attenuation of the pb signal . Hence, mc simulations were performed to test the differences between plaster - of - paris and bone and lucite and soft tissue in terms of pb over compton signal . No significant differences in xrf spectra were found between plaster - of - paris with lucite and bone with soft tissue . Thus, the phantom measurements were used to accurately calibrate the system, correlate the compton peak counts with soft tissue thickness, and calculate the detection limit of the system . Four goat bone samples and ten human cadaver tibia bone samples were measured with the device as well . The bones were all vacuum - sealed in plastic bags and labeled for ease of measurements . For goat bone, measurements were made at 0, 1, 2, 3, 4, and 5 mm of lucite and for bare cadaver bone, measurements were made using 0, 1, 2, and 3 mm of lucite for comparison between portable xrf devices and kxrf . The cadaver bones measurements did not include 4 and 5 mm of lucite due to the difficulty of adjusting the geometry in these situations . Three cadaver bone samples had intact soft tissue over them and were measured through the soft tissue . The cadaver and goat bone samples were taken to give a more realistic sense of the device capabilities for in vivo use by attempting to replicate the difficulties from increased attenuation with lxrf energies and soft tissue thickness . The spectrum was analyzed using a background subtraction method described in detail in our previous study . In summary, the method is focused on deriving two functions that will enable us to estimate the pb concentration . First, we define the relation of the background in the pb peak areas to compton peak area counts for 0 to 5 mm of lucite . Second, we define the relation between pb l - x ray signal and compton peak counts for 0 to 5 mm of lucite . The background at 0 ppm will relate to scatter events, which will be the main contribution to background in the spectrum . The compton scattering peak will give a correlation to the amount of scatter events in the spectrum and the background throughout the spectrum . Thus, defining a function that will relate the compton peak counts and the 0 ppm background can be feasibly used to determine the background under the pb l - x ray peaks . The net signal will decrease with increasing lucite thickness because of an increase in attenuation of the signal as well as distance from the bone . The compton peak has been shown to accurately correlate with lucite thickness through the increase in scatter events created by additional lucite . Since the attenuation and distance will increase directly with lucite thickness, we can correct each pb peak by relating it with compton peak counts . This function can then be used to accurately determine the signal attenuation that occurs in each spectrum . Then, one can determine from the spectrum the net counts coming from pb in the sample and relate that to a known signal concentration value to compute the final sample concentration . In our study, as a modification to this method for better applicability for use in vivo, we tried two modifications in addition to the original method . Our second method is similar to the background subtraction method . Instead of making an adjustment to match the phantom calibration for this calibration we used four goat bones with concentrations of 1, 13, 16, and 31 ppm of pb at varying lucite thicknesses as our calibration standards for the background subtraction method . For our 0 ppm data, we extrapolated from these values for each lucite thickness from the 1 ppm bone and used our highest concentration bone at 31 ppm to replace the 100 ppm phantom in the background subtraction method . For this calibration method, we found the difference in compton peak between actual bone samples and our calibration phantoms for varying lucite thicknesses . These peaks change both with bone versus phantom and with varying lucite thicknesses because of the densities and effective z values of the materials . We were able to apply a fit to this change and using this fit, we can apply the change between phantom and bone to any bone data we take, thus correcting it for use with phantoms . We implemented a traditional peak fitting method primarily for comparisons between our novel calibration methods and the calibration methods used in previous studies of lxrf bone pb measurement systems . The fitting was performed on the pb l and l peaks for phantoms at different lucite thicknesses to determine the signal associated with each concentration . Then the same function was used to fit the l and l peaks associated with our cadaver bone and goat bone samples and the corresponding concentration was determined based on the net counts in those peaks corrected for lucite or soft tissue attenuation . In previous studies of lxrf technology, it was concluded that the technology was not suitable for in vivo bone pb measurement due to the significant soft tissue attenuation . We included traditional peak fitting results from our goat bone data to show the comparison to our novel calibration methods . Figure 2 shows the resultant portable xrf spectrum from a measurement of an intact human cadaver bone with 1.3 mm soft tissue . As shown in the spectrum, the compton scattering peak comes from the x - ray tube silver characteristic x - rays undergoing compton scattering in our sample . This peak is a significant spectral feature and can be related to the background scattering events throughout the x - ray spectrum as we will demonstrate with our background subtraction calibration method in later results . This spectrum also demonstrates the difficulty in using traditional peak fitting methods, since with more soft tissue, there will be more background and the peaks will become increasingly noisier . The measurements of the lucite covered pb doped phantoms were used to calculate the detection limit . The detection limit was calculated as (1)dl=20 ppm=211/,0 ppm2 + 1/,0 ppm2, where (2),0 ppm=100 ppmbkg0 ppm/180 sgross100 ppmbkg0 ppm, where bkg0 ppm is the background count rate under the l or l peak for the 0 ppm phantom and gross100 ppm is the total count rate under the l or l peak for the 100 ppm phantom . Table 1 lists the detection limit of the portable xl3 t goldd+ system and the older portable xl3 t system . This comparison was taken at the same x - ray tube settings and filter on each device in order to demonstrate the improvements of the new system . This data was taken for 3 minutes at the same settings used for other measurements . Measurements were made to validate the portable xrf system against the standard kxrf systems for in vivo bone pb measurements . Phantoms, goat bones, and cadaver bones with 0, 1, 2, and 3 mm of lucite were measured by both systems, with goat bone also being measured at 4 and 5 mm of lucite thickness . Table 2 shows the measured phantom pb concentrations at different lucite thicknesses . The correlation (r - squared) between the expected concentrations and those measured with portable xrf system ranges from 0.991 to 0.999 for soft tissue thicknesses of 0 to 3 mm, demonstrating a good agreement of pb concentrations determined by kxrf and portable xrf for bare and lucite covered phantoms . Tables 3 and 4 demonstrate the ability of the three calibration methods to quantify bare cadaver bone pb values . Table 3 shows the pb concentration in bare cadaver bone calculated using the three calibration methods . Table 4 shows the bone pb concentrations for cadaver bone covered with 3 mm of lucite . Without lucite the calibration methods tend to be fairly similar, but with the introduction of more lucite the bone adjustment method tends to get further from kxrf values by overestimating background levels . Bone calibration has a similar correlation, but with only 4 points on the calibration line and the highest point at 30 ppm the actual values tend to deviate from kxrf especially for higher pb concentrations . Higher concentration standards are necessary to get visible signal while defining our function to correct for the inverse square and attenuation signal degradation as soft tissue increases . Background subtraction was the most reliable calibration method for higher lucite thicknesses and lower pb concentrations, which was determined using the correlation values for the cadaver bone evaluated at different lucite thicknesses . Figures 3(a)3(f) show the comparison of the correlations between goat bone pb concentrations calculated by kxrf and portable xrf at lucite thicknesses of 05 mm, with the pb concentrations for portable xrf being calculated using traditional peak fitting or background subtraction . From the correlations, one can see that traditional peak fitting does fairly well for bare bone or at lower lucite thicknesses, but with higher lucite thicknesses the correlation falls off quickly due to the high background leading to the pb peak being highly distorted especially at low concentrations . The chi - squared values for all the spectral fittings are close to 1, with the average chi - squared and standard deviation of the chi - squared value for these fits being 1.1 0.4, which demonstrates that even for poor results the data is accurately represented by fitted function . The data for cadaver bones with different lucite thicknesses analyzed using the background subtraction method is presented in table 5 . The correlations (r - squared) between the concentrations obtained from kxrf and those from the portable xrf system range from 0.58 to 0.94 with lucite thicknesses of 0 to 3 mm . The correlations between the pb concentrations obtained from kxrf and portable xrf are worse for the cadaver bones than for the goat bones . This is mainly due to the lack of pb concentration variation among the cadaver bones and geometry stability . Also, cadaver bone 6918 is an outlier (see discussion). To test the reliability and reproducibility of the technology for in vivo measurement, only three such cadaver bones were available in our lab, so there are limited data for this test . Table 6 shows the pb concentrations from lxrf and kxrf for the three cadaver bones with intact soft tissue . The comparison in table 6 demonstrates the abilities of the device in use through actual soft tissue . Using the compton peak to determine soft tissue thickness, which was shown to be comparable to an ultrasound measurement in our previous paper, we found the intact soft tissue thicknesses for our three cadaver bone samples to be 1.3 mm for cadaver bone 7042, 4.1 mm for cadaver bone 7031, and 5.6 mm for cadaver bone 7168 . The higher errors for individual measurements and higher standard deviation for grouped measurements are associated with larger soft tissue thicknesses, which is what we expect . This study investigated the detection limit of an improved portable xrf system for in vivo bone pb quantification and validated the system using phantoms, goat bones, and human cadaver bones . The improved system geometry and detector size greatly enhanced the detection limit of the device and the ability of the device to accurately determine the concentration of pb in bone especially at the in vivo situation . The detection limit for the portable xrf device is improved from the previous portable xrf device by a factor of about 2 . Through soft tissue thickness of 4 mm the device has the capability of a detection limit of 8 ppm, which is comparable to detection limit of 610 ppm with kxrf bone pb measurement systems in most labs . It is also relevant to point out that this was with a 3-minute measurement time and that time could be increased by a factor of 2 or 3 to lower the detection limit further, while maintaining a reasonable radiation exposure . The main disadvantage of lxrf systems is the lack of penetration of the low energy x - rays and thus at depth, the ability of the system to determine concentration becomes limited . With this system, it is shown that even at depth of 4 mm the portable xrf device now has the capability of obtaining measurements in 3 minutes, which would be equivalent to a kxrf device with a 30-minute measurement . In studies it has been shown that tibia measurement sites with tissue thickness of less than 4 mm can be found on most seniors and about half of the general population [15, 22]. One of our target populations for this device is the senior people whose mobility might be confined by their health conditions . Another point of clarification is the fact that, with the low penetration depth of lxrf, the kxrf and portable xrf systems are sampling different sites of the bone . Kxrf would be sampling the whole bone and lxrf would be sampling the superficial 0.51 mm of the bone . It is not very clear how pb distributes over the layer of tibia bone and the literature on this topic is limited . Todd et al . Showed higher concentrations of lead at 1 - 2 mm to the surface in bone, while bellis et al . While the bone pb concentrations from kxrf and lxrf are highly correlated in our study, further investigation with larger amount of samples is needed on the comparison for the absolute bone pb concentrations from these two methods . It is also relevant to point out that with measurements of bone lead the goal is a correlation with health effect, which should be reflected in both surface and depth bone measurement sites . The pb concentrations found through kxrf and portable xrf measurements of bare bone show good correlation . In order for our device to determine the in vivo pb concentration bone has different density and effective atomic number compared to our calibration phantoms, which led to differences in its resultant xrf spectrum . Our results with cadaver and goat bones show that our calibration methods adequately address these differences as the results are well correlated with kxrf data . In comparison, the traditional peak fitting calibration method results are shown with goat bones, and at larger lucite thicknesses this method is worse than the background subtraction method . It is relevant to point out that other studies exploring the validity of lxrf for pb studies used traditional peak fitting methods and showed that the results were not reliable especially at higher soft tissue thicknesses . The bone adjustment method did correct the compton peak to phantom values for a more equal comparison, but it fails to take into account the balance between the compton peak, background, and signal, and because of this, at higher lucite thicknesses, it exaggerates the problems seen with background subtraction . Bone calibration should be the best calibration method in theory, but due to the lack of standard bones with higher pb concentrations, the calibration line for this method tended to produce results that were less accurate than the background subtraction method . The correlation of bone pb concentrations between kxrf and portable xrf is very good for phantoms and goat bones with lucite thickness up to 5 mm, while the r - squared degraded a little for cadaver bones . This is mainly due to the small variation of the pb concentration for these cadaver bones, as well as the difficulty to adjust the geometry of the bare bones . In measuring cadaver bones, the geometry presented issues if not strictly monitored . We found that the cadaver bones were prone to air gaps in the geometry, which led to significant changes in the spectrum caused by the increased distance without significant attenuation . Although this effect was visible in the bare bone data, given the geometry of in vivo measurements this effect would not be present as the bone is covered in soft tissue, so there will not be air gaps between the soft tissue and bone in vivo . Attenuation by soft tissue is accounted for with our calibration by determining the soft tissue thickness from the compton peak, which in turn corrects for distance, as the gap between the detector and bone is filled with soft tissue . Although only three intact cadaver bones were used to test the reproducibility of the system for bone pb quantification and to validate the system in a real in vivo situation, several conclusions can be drawn from these limited data . First, this set of data confirmed the validity of the system for in vivo measurements, especially for the measurements with soft tissue thicknesses less than 5 mm . Second, the data confirmed that the thicknesses of the soft tissue significantly affect the uncertainties of the resultant concentrations . The standard deviations from the repeat measurements are lower than the uncertainties for individual measurements, which indicate that the uncertainties for individual measurements may be overestimated . In addition, the detection limit of the measurements calculated from the pb concentration uncertainties (dl = 2 sigma) for cadaver bones listed in table 6 would be higher than those listed in table 1 for corresponding soft tissue thicknesses . This is because the uncertainty calculated in table 6 includes the error on the gross count and net count of the signal under the pb l x - ray peak, while the dl calculated in table 2 only includes error associated with the background of a blank phantom covered with the corresponding thicknesses of lucite . Nonetheless, this data set shows an excellent agreement of bone pb concentrations for cadaver bones at thickness of 1.3 and 4.1 mm, while the agreement deteriorates at 5.6 mm . In the cadaver bone measurements, there is one bone (cadaver bone 6918), which we considered an outlier in our dataset . This bone came from a 100-year - old female and presents further challenges with the lxrf device . The spectrum from the bone had a much higher than normal compton peak, which we attributed to the bone appearing more like soft tissue with respect to the spectral features . In general the background subtraction method should overcome slight variations in bone between individuals, as the compton peak will also relate back to the material of the bone . The relationship we had derived between the compton peak and the pb signal broke down for this particular bone . We have not isolated the characteristic that causes this issue but plan to look into bone density effects on the lxrf spectrum through simulation as well as our cadaver bone samples . Although bone 6918 has a compton peak that is significantly different, other spectral features show differences that may be able to be exploited to correct the issues with the compton peak in this spectrum . The portable xrf system, now with a significantly lower detection limit, has its main advantages over kxrf with its portability, acquisition times, and ease of use . The new system can achieve a minimum detection limit equivalent to a kxrf measurement even through tissue thicknesses up to 5 mm . The portable xrf system also has the advantage of using an x - ray tube, which can be turned off when it is not in use and is less complicated for radiation license than a radioisotope source . The portable device lends itself for use in epidemiologic studies because of its quick measurement times and portability . The device allows for on - site pb surveys and risk assessments of the environment, while performing exposure assessment of the community members . In the future, the device can be improved by perfecting the data analysis algorithms for pb as well as other metals . Monte carlo methods could be used to accurately model the device and the spectrum of in vivo situations . This would help decrease the variability of measurements over different bone densities while also accounting for the tissue thickness over the bone . A main goal for the future of portable xrf technology would be applying it for the detection of other metals in vivo . The device can be used in collaboration with metal epidemiologists and toxicologists to study exposures and health effects of metals . We have validated an advanced portable xrf system for in vivo bone pb measurement and demonstrated the validity of using such a system to accurately quantify pb in bone with soft tissue thickness up to 4 - 5 mm . The detection limit of the device with 4 mm of soft tissue is approximately the same as the detection limit of kxrf systems, and the novel analysis methods provide a better correlation for pb quantification in bone samples . This device now has vast applicability in pb exposure assessment in clinical and research settings.
|
Currently, gonadotropin - releasing hormone (gnrh) agonist is commonly used for various sex - hormone - related diseases (uterine myoma, endometriosis, precocious puberty, dysfunctional uterine bleeding, assisted reproductive technology, etc . ). The main mechanism of action is the change in the secretion of gonadotropin and sex hormones . In the initial administration, they are associated with an increase in estrogen due to the increase of follicle stimulating hormone and luteinizing hormone, and with the prolonged administration, they are associated with estrogen reduction due to desensitization by down - regulation of gnrh receptor . The main side effects of these drugs are hypoestrogenism - induced symptoms, such as facial flushing, headache, depression, anxiety, insomnia, colpoxerosis, and osteoporosis . Meanwhile, the autoimmune thyroid diseases, including graves' disease and painless thyroiditis, are affected by various genetic and environmental factors . Of environmental factors, the typical example that can infer from the relation between sex hormones and thyroid immune tolerance and autoimmunity is postpartum thyroid dysfunction [3 - 5]. In addition, some case reports have shown that gnrh agonist - induced relative estrogen reduction may cause autoimmune thyroid disease by immune rebound [6 - 10], but there have been no reports in the korean literature . Here, we report three cases of thyroid dysfunction related to the prolonged administration of gnrh agonist . A 30-year - old female came to the department of obstetrics and gynecology at cheil general hospital & women's healthcare center with dysmenorrhea 3 months ago . She received subcutaneous injections of leuprorelin acetate 3.75 mg per month, three times in all, with a diagnosis of uterine myoma and endometriosis . She was examined for thyroid function prior to laparoscopic surgery 1 week ago and she was sent to the department of internal medicine because of thyroid dysfunction . Her blood pressure was 116/76 mm hg, pulse was 60 per minute, and body mass index (bmi) was 21.9 kg / m 160 cm, 56 kg . She had no past medical history, but her mother had previously taken medication for hypothyroidism . She had no special symptoms, such as fatigue, weight fluctuation, edema, and gastrointestinal problems . However, she complained of recently occurred sweating and facial flushing . On physical examination, she had slightly diffuse thyroid enlargement and the electrocardiography (ecg) was normal . Serum free t4 (ft4, 6.9 pmol / l) was decreased and thyroid stimulating hormone (tsh, 154.4 u / ml) was elevated . Antithyroglobulin antibody (anti - tg ab, 100 u / ml) and anti thyroid peroxidase antibody (anti - tpo ab, 58 u / ml) were both positive, whereas tsh binding inhibitory immunoglobulin (tbii, 4%) was negative . Thus, she started thyroid hormone replacement therapy (levothyroxine 0.1 mg) with a diagnosis of hypothyroidism . One month later, her thyroid function was normalized (ft4, 21.9 pmol / l; tsh, 2.91 u / ml) and 2 months later, she underwent laparoscopic myomectomy . After the operation, she received injections of leuprorelin acetate three more times; thereafter, her thyroid function was maintained within the normal range . She is currently maintaining levothyroxine 0.1 mg under serial assessment by the department of internal medicine (table 1). A 35-year - old female came to the department of obstetrics and gynecology at cheil general hospital & women's healthcare center with sterility 6 months ago . In the past medical history, she had been treated with medication for hyperthyroidism and had underwent surgery for endometriosis . She had received subcutaneous injections of leuprorelin acetate of 3.75 mg per month, three times in all, as a pretreatment for in vitro fertilization from 2 months ago . Since she had a past history of hyperthyroidism, she was refered to the department of internal medicine for further evaluation . Her blood pressure was 118/79 mm hg, pulse was 79 per minute, and bmi was 18.6 kg / m (159 cm/47 kg). She had been diagnosed with hyperthyroidism a decade ago and had taken methimazole 10 to 2.5 mg until 3 months before she came to our hospital because her thyroid function was normalized . She complained of sweating, heat sensation intolerance, and facial flushing . On physical examination, she had slightly diffuse thyroid enlargement and the ecg was normal . Serum t3 (120.6 ng / dl), ft4 (21.8 pmol / l), and tsh (0.51 u / ml) were all whinin the normal range, but anti - tg ab (13 u / ml), anti - tpo ab (> 100 u / ml), and tbii (22%) were all positive . One month later, she complained of palpitation and fatigue, while t3 (259.5 ng / dl) and ft4 (60.6 pmol / l) were elevated, tsh (<0.01 u / ml) was suppressed, and anti - tg ab (17 u / ml), anti - tpo ab (> 100 u / ml), and tbii was elevated to 27% . Two months later, t3 (304.0 ng / dl), ft4 (57.0 pmol / l), tsh (<0.01 u / ml), and tbii was elevated to 37% and medication was changed to propylthiouracil 200 mg . Three months later, she is currently maintaining propylthiouracil 100 mg under serial assessment by the department of internal medicine (table 2). A 24-year - old female came to the department of obstetrics and gynecology at cheil general hospital & women's healthcare center with right lower abdominal pain 2 months ago and she underwent laparoscopic ovarian cystectomy . Subsequently, she had received subcutaneous injections of leuprorelin acetate of 3.75 mg per month, twice in all . It was accidentally found by another hospital that she had thyroid dysfunction and thyroid enlargement verified by a sonogram 2 days ago . Her blood pressure was 111/67 mm hg, pulse was 80 per minute, and bmi was 18.4 kg / m (165 cm/50 kg). Nothing was noteworthy in her medical histoy or family history . She complained of weight loss of 4 kg over 1 year, cold sensation intolerance, and facial flushing . On physical examination, she had diffuse thyroid enlargement and the ecg was normal . Pmol / l) were elevated and tsh (<0.01 u / ml) was suppressed . Anti - tg ab (17 u / ml) and anti - tpo ab (> 100 u / ml) were all positive and tbii was 4% . One month later, tsh (0.01 u / ml) still had been suppressed, but both t3 (112.3 ng / dl) and ft4 (14.3 pmol / l) were normalized; thereafter, leuprorelin acetate was injected subcutaneously four more times . Two months later, ft4 (9.1 pmol / l) was decreased and tsh (9.81 u / ml) was elevated without symptoms of hypothyroidism, and observation was continued . Four months later, both ft4 (11.5 pmol / l) and tsh (2.33 u / ml) were normalized and she is currently under serial assessment (table 3). A 30-year - old female came to the department of obstetrics and gynecology at cheil general hospital & women's healthcare center with dysmenorrhea 3 months ago . She received subcutaneous injections of leuprorelin acetate 3.75 mg per month, three times in all, with a diagnosis of uterine myoma and endometriosis . She was examined for thyroid function prior to laparoscopic surgery 1 week ago and she was sent to the department of internal medicine because of thyroid dysfunction . Her blood pressure was 116/76 mm hg, pulse was 60 per minute, and body mass index (bmi) was 21.9 kg / m 160 cm, 56 kg . She had no past medical history, but her mother had previously taken medication for hypothyroidism . She had no special symptoms, such as fatigue, weight fluctuation, edema, and gastrointestinal problems . However, she complained of recently occurred sweating and facial flushing . On physical examination, she had slightly diffuse thyroid enlargement and the electrocardiography (ecg) was normal . Serum free t4 (ft4, 6.9 pmol / l) was decreased and thyroid stimulating hormone (tsh, 154.4 u / ml) was elevated . Antithyroglobulin antibody (anti - tg ab, 100 u / ml) and anti thyroid peroxidase antibody (anti - tpo ab, 58 u / ml) were both positive, whereas tsh binding inhibitory immunoglobulin (tbii, 4%) was negative . Thus, she started thyroid hormone replacement therapy (levothyroxine 0.1 mg) with a diagnosis of hypothyroidism . One month later, her thyroid function was normalized (ft4, 21.9 pmol / l; tsh, 2.91 u / ml) and 2 months later, she underwent laparoscopic myomectomy . After the operation, she received injections of leuprorelin acetate three more times; thereafter, her thyroid function was maintained within the normal range . She is currently maintaining levothyroxine 0.1 mg under serial assessment by the department of internal medicine (table 1). A 35-year - old female came to the department of obstetrics and gynecology at cheil general hospital & women's healthcare center with sterility 6 months ago . In the past medical history, she had been treated with medication for hyperthyroidism and had underwent surgery for endometriosis . She had received subcutaneous injections of leuprorelin acetate of 3.75 mg per month, three times in all, as a pretreatment for in vitro fertilization from 2 months ago . Since she had a past history of hyperthyroidism, she was refered to the department of internal medicine for further evaluation . Her blood pressure was 118/79 mm hg, pulse was 79 per minute, and bmi was 18.6 kg / m (159 cm/47 kg). She had been diagnosed with hyperthyroidism a decade ago and had taken methimazole 10 to 2.5 mg until 3 months before she came to our hospital because her thyroid function was normalized . She complained of sweating, heat sensation intolerance, and facial flushing . On physical examination, she had slightly diffuse thyroid enlargement and the ecg was normal . Serum t3 (120.6 ng / dl), ft4 (21.8 pmol / l), and tsh (0.51 u / ml) were all whinin the normal range, but anti - tg ab (13 u / ml), anti - tpo ab (> 100 u / ml), and tbii (22%) were all positive . One month later, she complained of palpitation and fatigue, while t3 (259.5 ng / dl) and ft4 (60.6 pmol / l) were elevated, tsh (<0.01 u / ml) was suppressed, and anti - tg ab (17 u / ml), anti - tpo ab (> 100 u / ml), and tbii was elevated to 27% . Two months later, t3 (304.0 ng / dl), ft4 (57.0 pmol / l), tsh (<0.01 u / ml), and tbii was elevated to 37% and medication was changed to propylthiouracil 200 mg . Three months later, she is currently maintaining propylthiouracil 100 mg under serial assessment by the department of internal medicine (table 2). A 24-year - old female came to the department of obstetrics and gynecology at cheil general hospital & women's healthcare center with right lower abdominal pain 2 months ago and she underwent laparoscopic ovarian cystectomy . Subsequently, she had received subcutaneous injections of leuprorelin acetate of 3.75 mg per month, twice in all . It was accidentally found by another hospital that she had thyroid dysfunction and thyroid enlargement verified by a sonogram 2 days ago . Her blood pressure was 111/67 mm hg, pulse was 80 per minute, and bmi was 18.4 kg / m (165 cm/50 kg). She complained of weight loss of 4 kg over 1 year, cold sensation intolerance, and facial flushing . On physical examination, she had diffuse thyroid enlargement and the ecg was normal . Both serum t3 (211.6 ng / dl) and ft4 (38.6 pmol / l) were elevated and tsh (<0.01 u / ml) was suppressed . Anti - tg ab (17 u / ml) and anti - tpo ab (> 100 u / ml) were all positive and tbii was 4% . One month later, tsh (0.01 u / ml) still had been suppressed, but both t3 (112.3 ng / dl) and ft4 (14.3 pmol / l) were normalized; thereafter, leuprorelin acetate was injected subcutaneously four more times . Pmol / l) was decreased and tsh (9.81 u / ml) was elevated without symptoms of hypothyroidism, and observation was continued . Four months later, both ft4 (11.5 pmol / l) and tsh (2.33 u / ml) were normalized and she is currently under serial assessment (table 3). One of them, gnrh agonist has a strong affinity to the gnrh receptor with a long half - life; therefore, it has almost the same effect as when gonadotropin has been continuously exposed to gnrh . After the initial flare effect, hormones administered repeatedly reveal the paradoxical antagonistic effect by down - regulation and desensitization, resulting in a hypogonadotrophic hypogonadism state . Accordingly, this agonist has been used for various sex - hormone - related diseases, but it also has side effects inducing pseudomenopause state with long - term use . Many environmental factors have been reported to induce autoimmune thyroid disease, and the fluctuation of sex hormones is one of the important factors of thyroid autoimmunity . Therefore, gnrh gonist - induced alteration in serum levels of gonadotropin and sex hormones may trigger thyroid autoimmunity . The increase of estrogen inhibits the immune reaction of autoimmune disease, which can be explained by the spontaneous remission of autoimmune disease during pregnancy . On the other hand, the reduction of estrogen induces immune rebound, as in the aggravation of autoimmune disease during postpartum period . It is assumed that the gnrh agonist - derived relative estrogen reduction induced immune rebound and aggravated the preexisting subclinical or mild autoimmune thyroid disease . In relation to the use of leuprolide acetate, published a report on transient thyrotoxicosis and hypothyroidism, amino et al . Published a report on graves' disease and painless thyroiditis, and van bon et al reported a case of transient thyrotoxicosis after using goserelin acetate in a hypothyroid patient, but nothing has been reported in the korean literature . Furthermore, gnrh agonist - induced aggravation of autoimmune disease is not limited to thyroid disease and there is a case report that leuprolide acetate - associated lupus nephritis was also aggravated . In the above three cases, we found hypothyroidism, hyperthyroidism, and both transient thyrotoxicosis and hypothyroidism respectively in sequence, and this indicates that there may be various kinds of thyroid dysfunction after gnrh agonist administration, like postpartum thyroid dysfunction . Considering that there was a change in thyroid function at 1 to 3 months after subcutaneous injections of leuprorelin acetate in the above cases, we assume that the main mechanism of disease was caused by the hypoestrogenic state, rather than by the initial gonadotropin surge . The findings resembled previous cases in which thyroid dysfunction began after 3 to 4 months [6 - 9]. Moreover, since most patients who had been treated with gnrh agonist complained of symptoms derived from hypoestrogenism, like sweating and facial flushing, this most likely rules out the possibility of thyroid dysfunction . In these cases, most patients also complained of sweating and facial flushing rather than typical symptoms of thyroid dysfunction . Under these circumstances, we think that thyroid function test and autoantibody test are needed in addition to medical examination by interview and physical examination . The gnrh agonist itself has no influence on thyroid hormone or tsh for healthy women or endometriosis patients, but it has been reported that preexisting diseases appear to be aggravated in patients with subclinical or mild autoimmune disease . Given that the present cases were positive for anti - tg ab and anti - tpo ab, it is assumed that the treatment of prolonged gnrh agonist in the presence of subclinical autoimmune thyroid disease generated hypothyroidism, transient thyrotoxicosis and the aggravation of hyperthyroidism . The above cases verified a possible association between the alteration of gonadotropin and sex hormones and thyroid dysfunction, and in the future, we suggest that clinicians pay attention to the thyroid state when starting the treatment of gnrh agonist for patients at risk of autoimmune thyroid disease . Likewise, it is very important to check the past history or family history of autoimmune diseases for all patients who will receive gnrh agonist treatment . However, the above cases are not sufficient to establish a cause - and - effect relationship and further study of the association between the gnrh agonist and autoimmune thyroid disease is needed . In summary, gnrh agonist is currently used in the treatment of various sex - hormone - related diseases and the mechanism is associated with estrogen reduction from desensitization by down - regulation of gnrh receptor caused by the prolonged administration . However, the gnrh agonist - derived relative estrogen reduction may induce immune rebound, resulting in autoimmune thyroid disease . A few cases have been reported, but none have been reported in the korean literature . Therefore, in the present case report, we describe three cases of thyroid dysfunction after the use of gnrh agonist . In the above cases, the administration of gnrh agonist showed the possibility of thyroid dysfunction, and therefore attention should be paid to the thyroid in patients vulnerable to autoimmune thyroid disease when beginning the treatement of gnrh agonist.
|
Psoriasis is a chronic, recurrent, inflammatory cutaneous disease that affects 1%3% of the general population . It is characterized by an abnormal cycle of epidermal development, with epidermal hyperproliferation, altered maturation of skin cells, vascular changes, and marked inflammation . Histologically, there is marked acanthosis, accompanied by parakeratosis and a mixed dermal infiltrate, including cd4 + t - cells, dendritic cells, macrophages, and mast cells . In the epidermis, dermal papillary blood vessels are dilated and tortuous.1 psoriasis is associated with significant impairment of health - related quality of life, especially in the more severe and nonresponder forms.2 the severity of psoriasis is graded as mild, moderate, or severe, mainly in accordance with the area of skin surface affected, the degree of scaling, and the type of infiltration.3 disease severity usually reflects the extent of skin involvement and is often associated with poor quality of life . The pathogenesis of psoriasis was initially described as an abnormal proliferation of epidermal keratinocytes, resulting from epidermal keratinocyte hyperplasia, para keratosis, leukocyte infiltration, and neoangiogenesis . Later, t - cells were identified as crucial to the initiation and maintenance of psoriasis.4 nowadays, it is accepted that genetic, environmental, and immunologic factors are involved in the disease onset and course . Moreover, different cells are important key players at different stages, and the onset of psoriasis is similar to an immune reaction.5 the t helper (th)1 pathway is believed to be important in the pathogenesis of psoriasis, as suggested by the increase in cytokines of this axis, such as interferon (ifn)-, tumor necrosis factor (tnf)-, interleukin (il)-2, and il-12, observed in plaques and in the serum of patients with psoriasis.68 levels of these cytokines have been correlated with severity of psoriasis,6,9 and the success of some therapies was associated with deviation of th1 to a th2 response.10 currently, psoriasis is known to be a complex disease in which the cytokine network is disturbed, and the il-23/th17 axis, as well as the th1 pathway, appear to be crucial for its pathogenic mechanisms.11,12 t - cell cytokines induce proliferation and activation of keratinocytes, also producing several proinflammatory cytokines and chemokines that sustain and amplify the inflammatory response by recruiting, retaining, and activating immune cells in the skin . The relevance of the il-23/th17 axis (figure 1) was highlighted by the successful treatment of moderate - to - severe psoriasis with drugs targeting the p40 subunit of il-12 and il-23.13 il-17 produced by th17 cells has a crucial role in the inflammatory process, by stimulating production of several proinflammatory cytokines and chemokines by keratinocytes, dendritic cells, and other immune cells . Moreover, both il-17 and th17 cells have been found to have a role in the pathology of several other autoimmune disorders.14,15 therefore, the th17 pathway is considered to be an attractive potential target for the treatment of several pathologic conditions, including psoriasis . Th17-type t - cells produce il-17 and are a distinct th cell population that plays a crucial role in cd4 + t - cell - mediated adaptive immunity . The transcription factors retinoid - related orphan receptor (ror)c and t - bet contribute to both phenotypes, and both phenotypes may produce il-17a and ifn-.16 il-12 or il-23 mediates the differentiation of these cells into th1 or th17, respectively.16 mediators of the th17 immune system include il-1, il-6, il-21, il-23, and transforming growth factor (tgf)-, which stimulate the differentiation of nave cd4 + t - cells into activated memory th17 cells.17,18 il-1 is involved in the activation of nave t - cells from peripheral blood, and seems to be the strongest inducer of il-17 production . Il-6 and il-21 further enhance il-17 production.19 in opposition, development of th17 cells from nave precursors is inhibited by ifn- and il-4 . Il-23, another th17 system mediator, drives proliferation of th17 cells.20 in humans, the lineage - specific transcription factor rorc leads to the developmental program of th17 cells . In psoriasis, at least in the severe form of the disease, cd4 + regulatory t - cells also showed a capacity to differentiate into th17 cells.21 the differentiation of cd4 + t - cells into regulatory t - cells is stimulated by tgf- in the absence of il-6 . However, regulatory t - cells may be induced to differentiate into il-17-producing th17 cells in the presence of an inflammatory stimulus . Apart from th17 cells, il-17-secreting cd8 + t - cells (tc) are also potent producers of il-17.22 il-6 and tgf-, which promote expression of the th17 lineage - specific transcription factor ror, are also required for induction of tc17 cells.23 moreover, il-23 supports the development of tc17 cells, which is inhibited by ifn-.24 t cells appear to be another source of il-17, particularly the early source of il-17 in response to infection . Natural killer t - cells are also another population of innate t lymphocytes known to produce il-17 . T and natural killer t - cells induce secretion of il-17 through stimulation of il-23.25,26 however, the activities and functions of these cells as a source of il-17 are not entirely understood . Il-17a is a proinflammatory cytokine, and il-17f seems to act in parallel with il-17a, but is less potent.27 apparently, in certain circumstances, il-17a may also be expressed by neutrophils, mast cells, and macrophages.28 mast cells and neutrophils discharge il-17 during formation of specialized structures, ie, extracellular traps, by the classical degranulation pathways.29 in opposition, il-17e (also known as il-25) is an anti - inflammatory cytokine, inducing a deviation to a th2 response.30 il-17b, il-17c, and il-17d are poorly characterized . Il-23 stimulates the production of il-17a and other th17 cytokines associated with the th17 pathway, namely il-17f.31 the il-17 receptor family includes il-17 receptors a e (il-17ra, il-17rb, il-17rc, il-17rd, and il-17re). The il-17 cytokines seem to bind to their receptors with different affinities, inducing production of proinflammatory cytokines and chemokines . The proinflammatory cytokines il-17a and il-17f, as well as the 17a / f heterodimer ligands, stimulate a receptor complex consisting of il-17ra and il-17rc subunits.27,32 the il-17ra / il-17rc complex induces signaling through a pathway that depends on the similar expression to fgf genes (sef)/il-17r containing the adaptor protein act1 (nuclear factor b activator 1 [nf-b1]).32 th17 and tc17 cells produce il-17a and il-22, but subsets of th17 and tc17 cells also secret ifn- and tnf-.33,34 recently, another t - cell population was described, ie, th22 cells that produce il-22 and tnf-.35 th17 cells also produce il-21, which has a crucial role in the th17 pathway, amplifying th17 differentiation.36 associations between psoriasis and gene regions involved in the th17 pathway have been established . In genes encoding for the il-12b, il-23a, and il-23 receptor, single nucleotide polymorphisms have been identified in psoriasis.37,38 the genes encoding for tnf--induced protein 3 (tnfip3), tnfip3-interacting protein 1, and tnf receptor - associated factor 3 interacting protein 2 (traf3ip2) are associated with risk of psoriasis.37,38 the gene traf3ip2 codes for the adaptor protein act1 (nf-b1), a regulator of the nf-b pathway that is involved in il-17 signaling . Il-17, namely il-17a, has an important role in host defense, inducing il-6 production to enhance acute - phase responses and differentiation of additional th17 cells, thereby intensifying the response against pathogens.40 however, regardless of its protective effects, in some autoimmune and immunoinflammatory diseases il-17 can be deregulated, contributing to the pathogenesis and/or maintenance of these disorders . Indeed, deregulation of il-17a favors chronic inflammation and tumor development.41 il-17 activates keratinocytes to produce interleukins and chemokines, such as il-8, which provides a strong chemotactic signal for neutrophil recruitment.42 it was reported that administration of il-17f to mouse skin increases the expression of il-8,43 which is known to be elevated in psoriasis.12 il-17 also upregulates keratinocyte expression of other chemo kines (eg, c - x - c motif ligand [cxcl]1, cxcl3, cxcl5, cxcl5, and cxcl6), which have been associated with recruitment of neutrophils.44 il-17a exerts its effects in multiple cell types, namely in macrophages, dendritic cells, neutrophils, fibroblasts, endothelial cells, epithelial cells, keratinocytes, and lymphocytes, leading to production of several cytokines and chemokines.45 using a human monolayer model, th17 cytokines (eg, il-17a, il-22, tnf-) stimulated the upregulation of chemokine (c - c motif) ligand (ccl)20.46 in psoriasis, il-17a induces keratinocytes to express ccl20, recruiting th17 cells and dendritic cells to the skin,44 which may contribute to maintain both cells in psoriatic lesions . A study of psoriatic dermal dendritic cells cultured with allogenic cd4 + t - cells showed that these cells induced a higher number of cd4 + t - cells to produce il-17 than normal dendritic cells.47 moreover, in keratinocytes, il-17a upregulates antimicrobial peptides such as -defensins and s100a family members, providing a stimulus for the innate immune system,44,46 downregulates filaggrin and other factors involved in cell adhesion, contributing to skin barrier disruption,48 and increases expression of keratin 17, contributing to epidermal hyperproliferation.49 il-17a also stimulates keratinocytes to express il-36 that, by acting synergistically with il-17a, promotes expression of the antimicrobial peptides cxcl8, il-6, and tnf-.50 il-17a stimulates fibroblasts and dendritic cells to produce il-6, which favors the commitment of more t - cells to the th17 phenotype (figure 1). Dendritic cells and macrophages are stimulated to produce il-1 and tnf- by il-17a.36 in summary, il-17 and th17-related cytokines, such as il-23 and il-22, contribute to the pathologic alterations found in psoriasis (figure 1). Il-17 is a critical component in the establishment and perpetuation of inflammation, inducing production of proinflammatory cytokines such as il-6, il-8, and prostaglandin e2,51 and also stimulates secretion of proinflammatory cytokines by other cells, namely endothelial cells and macrophages.36 il-22 induces epidermal hyperplasia and hypogranulosis; it also induces proinflammatory responses, such as the production of cytokines, chemokines, and acute - phase proteins from many cell types, and regulates the differentiation and migration of keratinocytes . Production of il-22 is directly induced by il-23, and il-22 can mediate il-23-induced acanthosis and dermal inflammation.52,53 il-17 also seems to promote angiogenesis . Il-17 indirectly enhances the proliferation of endothelial cells via induction of vascular endothelial growth factor and il-8 by fibroblasts.54 these cytokines can also induce production of chemokines and subsequently increase the recruitment of endothelial progenitor cells to support angiogenesis . Tnf- act synergistically or additively on keratinocytes to upregulate several genes, many of which are expressed significantly in psoriatic skin, such as s100a7, -defensin, il-23, ccl20, and cxcl1.55 il-17a also acts together with ifn- to increase production of il-6 and cxcl8,56 and acts in synergy with other proinflammatory cytokines, such as il-1 and il-6 . Circulating th17 cells are increased in psoriasis, as well as th22 and th1 cells, although to a lesser extent.57 as referred, increased levels of il-17 were found in skin lesions and in the blood of patients with psoriasis, and were correlated with disease severity;6,5860 moreover, the levels decreased after successful treatment,12,58 confirming the role of il-17 in the pathogenesis of psoriasis . Expression of il-17e, il-17b, and il-17d does not seem to increase in psoriatic skin lesions, while expression of il-17a, il-17c, and il-17f increases.61 apparently, il-17e, il-17b, and il-17d do not have a significant role in the development of psoriasis . However, il-17a in particular has been demonstrated to have a key role in the chronic inflammatory process of several immune - mediated diseases, including psoriasis . Therefore, blocking il-17a appears to be a sustainable approach for the treatment of such pathologies . The activity of il-17 and the effect of its inhibition have been evaluated in animal models of psoriasis . Induction of inflammation with tpa (12-o - tetradecanoylphorbol-13-acetate) seems to lead to a th17-like response in transgenic keratin 14/vascular endothelial growth factor mice, a potential animal model for psoriasis, and an increase in the ear tissue of tnf-, il-1, il-6, il-12/23p40, il-12p70, il-22, and il-17 levels was reported.62 imiquimod - induced psoriasis - like skin inflammation was almost blocked in mice deficient in il-17ra and il-23.63 in k5.htgf-1 transgenic mice, presenting a skin phenotype and cytokine alterations analogous to those found in psoriasis, administration of anti - il-17a blocked progression of disease.64 k5.stat3c mice treated with tpa developed psoriasis - like skin lesions, and treatment with anti - il-17a reduced epidermal hyperplasia.65 in mice, administration of il-23 induced epidermal hyperplasia mediated by il-17 and il-22 . In mice deficient in these cytokines or in il-17ra, epidermal hyperplasia is minor, while antibodies against these cytokines completely inhibit il-23-induced hyperplasia.66,67 inhibition of il-23, acting upstream from il-17, has been also studied, both in animals and in clinical studies . Mice deficient in il-23 showed defects in terminal differentiation and function of th17, namely a decrease in production of il-17.68 in clinical studies, agents that target il-23 have been shown to inhibit il-17, eg, ustekinumab, which is used to treat moderate and severe forms of psoriasis.13 successful conventional therapies, such as psoralen plus ultraviolet (uv) light a, narrow - band uvb (nb - uvb), and nb - uvb combined with calcipotriol, lead to a decrease in circulating levels of il-17 and th17-related cytokines, but acitretin had no such effect.12,59,69 treatment with cyclosporin normalized the il-17 and il-22 mrna levels that were increased in psoriatic skin.33 nb - uvb inhibited the il-23/th17 axis, with a decrease observed in cd11c+ dendritic cells and their products, namely il-20, inducible nitric oxide synthase, il-12/23p40, and il-23p19, as well as suppression of il-17 and il-22 mrna.70 concerning treatment with biologics, infliximab decreased the high th17 and th1 cell levels observed before treatment.57 moreover, early improvement was significantly correlated with a reduction in il-17 and its signaling pathway when anti - tnf agents were used as a treatment for psoriasis.59,71 according to a systematic review by tausend et al, biologic agents specifically targeting il-12, il-17, and il-23 are efficacious and safe for the treatment of moderate - to - severe psoriasis in adults, although long - term data are still needed.72 agents that target il-17 are under clinical investigation, namely brodalumab (amg-827), secukinumab (ain457), and ixekizumab (ly2439821). Secukinumab and ixekizumab are human monoclonal antibodies against il-17a,73,74 whereas brodalumab is a fully human monoclonal antibody that targets il-17ra.75 as referred, brodalumab (previously designated as amg-827) targets il-17ra, preventing its activation by il-17a, il-17a / f heterodimer, il-17f, and il-17e (il-25).75 in a placebo - controlled phase i study (table 1), 25 patients with moderate - to - severe psoriasis were randomized to receive a single subcutaneous (sc) dose of brodalumab 140 mg (n=4), a single sc dose of 350 mg (n=8), a single intravenous (iv) dose of 700 mg (n=8), or placebo (n=5), and were followed for 85 days . Two weeks after receiving a single dose of 350 mg sc or 700 mg iv, the patients showed a dose - dependent improvement in pasi (psoriasis area and severity index) score and on the static physician global assessment . The eight subjects who received brodalumab 700 mg iv achieved a 50% improvement in pasi (pasi 50) by day 29; at day 43, 88% achieved pasi 75 and 38% reached pasi 90 . During the study, 75% and 62.5% of patients receiving 350 mg sc achieved pasi 50 and pasi 75, respectively . Pasi 50 was achieved by two of the four patients who received brodalumab 140 mg sc . None of the placebo subjects achieved pasi 50.76 the sc 350 mg and iv 700 mg cohorts also showed significant reductions in epidermal thickening, keratin 16 levels, and ki67-expressing cells . Skin lesions in these cohorts showed an improvement in mrna levels of several il-17-modulated keratinocyte - derived factors (eg, cathelicidin, keratin 16, ccl18, and ccl20) and cytokines known to be directly regulated by il-17r (il-22, il-23a, il-12b). Il-17a, il-17c, and il-17f mrna levels were reduced to nonlesional levels during 6 weeks of treatment . The safety profiles for the subjects who received brodalumab and placebo were similar.76 in this study, a single iv dose of brodalumab up to 700 mg produced substantial improvements in clinical and histopathologic variables, with an acceptable safety profile . In a randomized, double - blind, placebo - controlled, dose - ranging phase ii study (table 1), involving 198 adult patients with moderate - to - severe psoriasis, placebo or brodalumab 70, 140, or 210 mg was administered sc on day 1 and at weeks 1, 2, 4, 6, 8, and 10, or at a dose of 280 mg sc on day 1 and at weeks 4 and 8 . At week 12, brodalumab reduced the pasi scores by 45.0% (70 mg), 85.9% (140 mg), 86.3% (210 mg), and 76.0% (280 mg), while improvement in pasi on placebo was 16.0% (p<0.001 for all comparisons with placebo). At week 12, pasi 75 and pasi 90 was observed in 77% and 72% of patients receiving brodalumab 140 mg, respectively; 82% and 75% of those receiving 210 mg had pasi 75 and pasi 90, respectively; and the placebo group showed no improvement (p<0.001 for all comparisons).75 improved values for static physician global assessment, dermatology quality of life index, and the medical outcomes study 36-item short - form health survey (sf-36) were also reported for the 140 mg and 210 mg groups when compared with placebo.75 moreover, at week 12, subjects receiving brodalumab showed significant improvements in mean psoriasis symptom inventory total score when compared with placebo (8.5 [70 mg], 15.8 [140 mg], 16.2 [210 mg], 12.7 [280 mg], and 4.8 [placebo]), and all the eight item scores improved significantly in patients on brodalumab.77 these data confirm that brodalumab is associated with improvement of the disease from the patient point of view, with an increase in quality of life also reported . Endres et al studied the pharmacokinetics of brodalumab and assessed the effects of covariates at the doses used in the phase i and phase ii clinical studies . For doses between 140 mg and 210 mg, they reported that the area under the curve was more than two - fold higher in subjects weighing less than 75 kg as compared with reference subjects; age and diagnosis had a smaller influence on exposure, and were not clinically significant . Their data suggest that body weight is an important covariate, explaining some of the pharmacokinetic variability observed in human clinical trials with brodalumab.78 recent data show that blockade of il-17 signaling by brodalumab in psoriatic skin leads to rapid transcriptional changes, initially in keratinocyte - expressed genes, followed by normalization of leukocyte abnormalities, and also established the essential role of il-17r in keratinocytes by driving the pathogenesis of psoriasis.79 il-17a has an important role in host defense, so its inhibition raises concern about a potential risk of serious infection and other immune - mediated diseases . Studies in animal models showed that il-17a contributes to host defense and to the expression of antimicrobial peptides;46,80,81 however, absence of il-17a has not been associated with infection or autoimmune manifestations,80 suggesting that inhibition of il-17 has a low potential for adverse effects on the immune system . In the clinical trials with secukinumab, ixekizumab, and brodalumab, infection rates were only slightly higher than those seen with placebo or than the rates expected for a comparable patient population . Indeed, in the phase ii study reported by papp et al, two cases of grade 3 neutropenia were found in the 210 mg brodalumab group; the adverse events reported more frequently in the brodalumab groups were nasopharyngitis (8%), upper respiratory tract infection (8%), and injection site erythema (6%).75 the costs of treating psoriasis with biologics are high, but their contribution to resolution of lesions and improved quality of life, especially in patients who are currently undertreated, might reduce the need for inpatient care and improve the outcome for patients . Indeed, according to schmitt and ford, the indirect costs of productivity lost exceed the direct costs, which may justify the use of more expensive medications.82 it is important to note that psoriasis is often associated with several comorbidities, in particular cardiovascular disease . Several risk factors for cardiovascular events have been reported in psoriasis, and some treatments for the disease appear to enhance the cardiovascular risk profile . Il-17 has been reported to be associated with atherosclerosis by favoring chronic vascular inflammation,83,84 so might contribute to the increased risk of cardiovascular disease found in patients with psoriasis . Brodalumab, and eventually the other anti - il-17 agents, decrease vascular inflammation by blocking il-17 signaling, and may have a positive effect on the risk of cardiovascular events by reducing the development of atherosclerotic lesions . Nonetheless, the efficacy and safety profile of brodalumab needs to be evaluated in larger clinical trials of longer duration . Currently, phase iii clinical trials for brodalumab are ongoing . Advances in our understanding of the pathology of psoriasis have allowed the development of new therapeutic strategies and the prospect of new agents, especially for severe cases and nonresponders with psoriasis . For instance, anti - tnf agents are important therapeutic options for psoriasis, but for patients in whom response is lost or does not exist, other treatment options are required . These options must have a good safety profile, with few effects in the immune system . Considering that il-17 signaling has a crucial role in the pathogenesis of psoriasis, its inhibition has emerged as a critical target . Phase iii clinical trials for brodalumab, secukinumab and ixekizumab, agents that target il-17 signaling, are ongoing . While secukinumab and ixekizumab target il-17a, brodalumab targets il-17ra, raising the question as to whether there are differences in antagonizing il-17a or the il-17 receptor . So far, brodalumab has demonstrated a favorable safety and tolerability profile, robust clinical activity, significant improvements in pasi and other scores for psoriasis severity, and a great impact on patient quality of life . Data for its long - term efficacy and safety will be provided by the phase iii clinical trial that is ongoing . Considering the published data, brodalumab appears to be a potential tool for use in the treatment of moderate - to - severe psoriasis.
|
In an attempt to address some of the problems associated with the lack of tumor selectivity and stability of conventional cytostatic drugs, a variety of novel drug delivery systems has been developed . Of these, liposomal drug carrier systems represent a mature and versatile technology, and several liposomal formulations of anti - cancer drugs have been approved for cancer chemotherapy or are in advanced stages of clinical development . They were first described by bangham et al (1965) in the mid 1960s and were initially used as a model system to study biological membranes . The term according to lamellarity and size (perez - soler 1989): unilamellar vesicles comprising one lipid bilayer have diameters of 50 to 250 nm . They contain a large aqueous core and are eligible for the encapsulation of water - soluble drugs . Multilamellar vesicles composed of several concentric lipid bilayers in an onion - skin arrangement have diameters of 1 to 5 m . The high lipid content allows these multilamellar vesicles to entrap lipid - soluble drugs passively . According to a phylogenetic scheme: classical or conventional liposomes (ie, simple mixtures of phospholipids and cholesterol) target the reticulo - endothelial system (res) and are called res - targeted liposomes . Vesicle size in liposomes of similar lipid composition is usually inversely correlated with the amount of res uptake (senior and gregoriadis 1982). Liposomal formulations aim reduce the toxic side effects of conventional cytostatic drugs without hampering the efficacy . Theoretically, these goals may be reached in two ways: (i) the encapsulated drug is prevented from reaching healthy tissue (site avoidance) and/or (ii) drug concentrations are delivered mainly to neoplastic tissue (drug targeting). Meanwhile, several liposomal formulations have been approved for the treatment of different tumors and have become an established addition to the anti - cancer drug armamentarium (hofheinz et al 2005). In this review we briefly summarize the results of liposomal encapsulated cytarabine for the treatment of lymphomatous and leukemic meningitis . Neoplastic meningitis is characterized by the infiltration of cancer cells into the leptomeninges and associated with a poor prognosis . About 40% to 90% of the patients with neoplastic meningitis suffer from neurological symptoms (deangelis 1998; chamberlain 2005). More sensitive methods such as flow cytometry indicate that central nervous system (cns) involvement in patients with non - hodgkin s lymphoma (nhl) or leukemia has been underestimated so far . A recently reported series using flow cytometry detected positive cerebrospinal fluid in 73% of patients (bromberg et al 2007). Generally, the treatment of neoplastic meningitis is palliative and the goal is prolongation of survival, reduction of neurological symptoms, and improvement of qualitiy of life . The treatment of disseminated lymphomatous meningitis, which may compromise up to 25% of high - grade lymphoma patients, requires a long exposure of the malignant cells to a high concentration of antineoplastic agent to achieve a sufficiently cytostatic effect (bleyer 1999). As only a few cyctotoxic drugs pass through the blood brain barrier, effective levels cannot be achieved by systemic chemotherapy alone (benesch and urban 2008). Therefore, frequent intrathecal injection of chemotherapeutic drugs either by lumbar punctures or via ventricular access devices has become the mainstay of therapy to achieve effective levels of chemotherapeutic agents in the cerebrospinal fluid (csf). The antimetabolites methotrexate (mtx) and cytarabine (ara - c) are agents of choice for intrathecal chemotherapy . Given the low proliferation index of malignant cells in the cns, their susceptibility to antimetabolite treatment is theoretically increased by longer exposure (bleyer 1999). The removal of mtx or ara - c from the csf is slow, which gives the rationale for the intrathecal use of these drugs . Until recently, the application of mtx has been preferred to ara - c for its even lower csf clearance and deeper penetration into the meninges and cns parenchyma (bleyer 1999). Ara - c a cornerstone in the treatment of hematologic malignancies (johnson 2001) requires metabolic activation by the enzyme cytidine deaminase to exert its cytotoxic properties . Within the csf the activity of cytidine deaminase is low and metabolization almost negligible (zimm et al 1984). As the terminal half - life of ara - c in the csf is only 3.4 hours, 2 to 3 intrathecal applications per week are required to achieve adequate cytotoxic drug concentration over time (benesch and urban 2008). Liposomal encapsulated cytarabine (depocyte, mundipharma gmbh, limburg / lahn, germany) is an intrathecal injectable suspension of ara - c encapsulated in multivesicular lipid based particles . They consist of numerous non - concentric water - filled polyhedral compartments separated by bilayered liquid septa (called depofoam - technology) (angst and drover 2006). These particles have a diameter of approximately 3 to 30 m consisting of hundreds of aqueous chambers in a honeycomb arrangement containing ara - c . The chambers are separated from each other by lipid bilayers consisting of dioleylphosphatidycholine, dipalmitoyl - phosphatidylglycerol, cholesterol, and triolein . Depofoam particles are therefore much larger than conventional uni- or multilamellar liposomes bearing a high drug - loading capacity . At storage temperatures of 2 to 8 c after inthrathecal injection the biodegradation of the lipid membranes at body temperature leads to a gradual release of ara - c ensuring prolonged cytotoxic drug concentrations of cytarabine in cerebrospinal fluid . Depocyte has a mean half - life of 130 to 277 hours compared with 3 to 4 hours for conventional ara - c (zimm et al 1984). In a preclinical study even 16 days after injection the free - drug concentration was higher than the minimal cytotoxic concentration (kim et al 1987). No ara - c was found in blood plasma after intrathecal administration of 50 mg of depocyte . Most of the clinical data published thus far are derived from clinical trials or larger case series (goekbuget et al 2005; bjrgvinsdttir et al 2006; camera et al 2006; cascavilla et al 2006; garcia - marco et al 2006; rossi et al 2006; sancho et al 2006a, b, 2007; shapiro et al 2006; aichberger et al 2007; brion et al 2007). Few reports deal with the prophylactic use of depocyte (anaclerico et al 2006; mcclune et al 2007; neumeister et al 2007). For induction therapy a biweekly dosing schedule of 50 mg depocyte has been established (consolidation and maintainance therapy in 4-weekly intervals). Using this schedule, cytotoxic csf levels of ara - c were found up to 14 days regardless of the site of drug injection (ventricular or lumbar) (kim et al 1993). This phase iii study compared intrathecal liposomal ara - c with conventional (free) intra - thecal ara - c in 28 patients with lymphomatous meningitis (glantz et al 1999b). The experimental treatment arm consisted of biweekly 50 mg depocyte and the reference treatment was ara - c 50 mg twice weekly for a 1-month induction period . In case of response (csf clearing of lymphoblasts) consolidation and maintainance cycles with longer application intervals response rates (ie, clearing of csf and absence of neurological progression) were statistically significant higher in the depocyte arm (71% versus 15%; p = 0.006). Moreover, a strong trend in favor of depocyte in terms of time to neurological progression (78 versus 42 days; p> 0.5) and overall survival (99 versus 63 days; p> 0.5) was observed . Depocyte treatment was associated with an improvement of karnofsky status at the end of the induction treatment (p = 0.041). Consistent with the higher csf levels of ara - c in the depocyte group, headache (grades 13) ocurred in a higher amount of treatment cycles (27% versus 2%). Arachnoiditis (grades 13) was reported to occur in 22% of depocyte cycles (13% with ara - c). To prevent or mitigate this adverse event, concomitant oral dexamethasone treatment (4 mg bid days 15) another randomized trial compared biweekly depocyte 50 mg (up to 6 applications) with inthrathecal mtx 10 mg twice weekly (up to 16 applications) in patients with cytologically proven neoplastic meningitis deriving from solid tumors (glantz et al 1999a). A total of 61 patients were accrued to receive the drugs either via lumbar puncture or an intraventricular ommaya reservoir . Responses occurred in 26% of patients in the depocyte and 20% in the mtx group . Median survival was not significantly different (105 days with depocyte versus 78 days with mtx), but a longer median time to neurological progression was obtained with depocyte (58 versus 30 days; p = 0.007). The grades and extent of adverse events observed were comparable between both groups . Meanwhile, several phase ii studies or larger case series with depocyte in patients with lymphomatous or leukemic meningitis have been reported, and similarly to the data published by glantz et al response rates in the range of 50% to 70% were noted (for overview see benesch and urban 2008). In the prophylactic treatment setting a prospective clinical trial using depocyte as cns prophylaxis was recently published (jabbour et al 2007). Thirty - three patients with previously untreated acute lymphoblastic leukemia or lymphoma received liposomal cytarabine concurrently to systemic chemotherapy . Serious neurotoxicity occurred in five patients within 14 days from the last intrathecal therapy (seizure n = 1, encephalitis n = 1, cauda equina syndrome n = 2, pseudotumor cerebri n = 1). The authors concluded that prior administration of cytotoxic drugs passing the blood brain barrier such as high - dose mtx or cytarabine might have increased the risk of neurotoxicity . Similar observations have been made by other groups as well, eg, in pediatric patients (benesch et al 2007). Admittedly, it is difficult to distinguish symptoms by infiltration of cns from side effects observed after administration of any substance available for intrathecal use alone or in combination with systemic chemotherapy (weiss et al 1974; resar et al 1991; schiller et al 1992; benesch and urban 2008). Nevertheless, these observations have led to a discussion among clinicians about the safety of depocyte (chamberlain and glantz 2007; pui 2007). Depocyte has consistently shown high response rates in the treatment of patients with lymphomatous and leukemic meningitis in a randomized clinical trial as well as in several case series or phase ii studies . Moreover, in this randomized trial the superiority over conventional ara - c with respect to the improvement of neurological sympstoms was demonstrated . Depocyte is approved in the us and in europe for the intrathecal treatment of lymphomatous meningitis . Owing to the scarcity of this disease, it is expected that further randomized clinical trials for the treatment of malignant leukemic or lymphomatous meningitis will not be conducted and that the question of whether depocyte is truly superior to conventional cytostatics in the intrathecal treatment will remain unresolved . Moreover, cost - utility analysis indicates that depocyte is cost - effective (moeremans et al 2004). In view of the recently reported neurological toxicities, the use of depocyte as prophylactic treatment, eg, for acute lymphatic leukemia, should remain reserved to clinical trials . Further open questions are the optimal treatment duration after the initial clearing of csf from malignant cells, the best dosing regimen for consolidation therapy, and the interval between the administration of depocyte and other potential neurotoxic cytostatic drugs, especially high dose methotrexate or ara - c (pui 2007). Finally, the potential benefit of depocyte treatment in malignant meningitis of solid tumors remains a challenge to be explored in further clinical trials.
|
Diabetes, especially poorly controlled (glycated hemoglobin or hba1c> 8%) one, is a metabolic disease associated with a variety of micro- and macrovascular complications . Elevation of postprandial plasma glucose and insulin stimulation following ingestion of high carbohydrate diet are suggested to increase severity of diabetes and to be independent indicators of risk for atherosclerotic diseases . As such, interventions to alleviate postprandial plasma glucose and insulin secretion by diet and lifestyle changes are the essential therapeutic objectives for diabetics . One dietary strategy aimed at improving both diabetes control and control of cardiovascular risk factors is the use of low - glycemic index (gi) diets . These approaches include diets containing 5060% calories from carbohydrates and administration of low - glycemic load (gl) diet (100 g) (glucose equivalents per day) without elevating fat intake . Conventional high carbohydrate intake recommended in diabetes, results in suboptimal glycemic control and lipoprotein profile, gradually increasing insulin and/or oral hypoglycemic medication requirement and eventually weight gain . Some trials have produced supportive evidence of the benefits of substituting polyunsaturated fatty acids (pufas) for saturated fatty acids (sfas). A meta - analysis of randomized control trials (rcts) found a 10% reduction in chronic heart disease for each 5% of energy from sfas substituted for pufas, while no benefits have been found by substituting carbohydrates for sfas . Therefore, the aim of this study was to examine the effect of low - gl diet (gl = 6777, 36% energy as fat, and 42% as carbohydrate), with having higher percentage of fat and lower amount of carbohydrate than conventional diabetics diet on cardiovascular risk factors changes in poorly controlled type 2 diabetic patients . The study was a prospective observational study conducted among caucasian patients, without having control group . The patients biochemical data, weight, and body mass index (bmi) were compared before and after intervention . The inclusion criteria of the study were males and females of age 3060 years with poorly controlled type 2 diabetes and having hba1c> 8% . The exclusion criteria of the study were subjects with renal, heart, chronic, metabolic (except diabetes) disease, pregnant and nursing mothers . One hundred diabetes patients who were referred to endocrine clinic during 6 months and were receiving either insulin or oral medication were recruited for this study . Before commencement of the study, patients were asked to fill a consent form, and their 7-day food dietary records were collected to estimate their usual energy intake . The procedures were followed in accordance with the ethical standards of the international guideline for human study, and the study was approved by the human research ethics committee of the qazvin university of medical sciences . The energy intake varied between 1800 and 2200 kcal according to the patients needs, which was calculated based on the food dairy record . The gi of each food was extracted from international table of glycemic index and glycemic load and glycemic index of iranian foods . The gl of foods was estimated using carbohydrate content (grams) of each food multiplied by gi of that food . The gl of subjects daily diet was the sum of gl of foods consumed during the day . At baseline, patients were on high carbohydrate low fat (55 - 60% carbohydrate and 20% fat) conventional diabetes diet . A 10-week experimental diet consisted of ordinary food item having gi 55, and each main meal had gl 20 with overall daily gl = 6777 (42% carbohydrate, total fat 36%, fat derived from olive oil and nuts 15%, 22% protein) [table 1]. This was accomplished by providing a list to each individual of the recommended daily intake of commonly used foods and a substitution list allowing exchanges within food groups . The compliance with diet program and gl of consumed meals was assessed by regular fortnightly visit of a dietitian . Composition of low - glycemic load diet with 1800 kcal administered to diabetic patients at baseline and 2 weeks after the diet intervention, blood samples were drawn after an overnight fast for determination of plasma glucose, hba1c, triglyceride (tg), total cholesterol, low density lipoprotein cholesterol (ldl - c), and high density lipoprotein cholesterol (hdl - c). Plasma glucose concentrations were determined by the glucose oxidase method (using a hitachi 917 analyzer, roche diagnostics, biomedical lab . The ldl level was measured by a homogeneous enzymatic assay (genzyme corp ., cambridge, ma, usa), and hdl, triglycerides, and cholesterol concentrations were measured using a hitachi 911 analyzer (roche diagnostics, indianapolis, in, usa). Using g - power exe software, the sample size was calculated based on effect sizes and mean obtained for fasting blood glucose in similar studies powered at 90% and an alpha of 5% . We estimated that a sample size of 96 were enough to meet the considered powerfor our study . Data were analyzed for normality of distribution before use of parametric statistics with spss version 16 (spss inc ., data were reported as mean sd and were analyzed by using paired student's t - test and pearson correlation to compare weight, bmi, fasting blood sugar (fbs), hba1c, and lipid profile of patients before and after intervention . The study was a prospective observational study conducted among caucasian patients, without having control group . The patients biochemical data, weight, and body mass index (bmi) were compared before and after intervention . The inclusion criteria of the study were males and females of age 3060 years with poorly controlled type 2 diabetes and having hba1c> 8% . The exclusion criteria of the study were subjects with renal, heart, chronic, metabolic (except diabetes) disease, pregnant and nursing mothers . One hundred diabetes patients who were referred to endocrine clinic during 6 months and were receiving either insulin or oral medication were recruited for this study . Before commencement of the study, patients were asked to fill a consent form, and their 7-day food dietary records were collected to estimate their usual energy intake . The procedures were followed in accordance with the ethical standards of the international guideline for human study, and the study was approved by the human research ethics committee of the qazvin university of medical sciences . The energy intake varied between 1800 and 2200 kcal according to the patients needs, which was calculated based on the food dairy record . The gi of each food was extracted from international table of glycemic index and glycemic load and glycemic index of iranian foods . The gl of foods was estimated using carbohydrate content (grams) of each food multiplied by gi of that food . The gl of subjects daily diet was the sum of gl of foods consumed during the day . At baseline, patients were on high carbohydrate low fat (55 - 60% carbohydrate and 20% fat) conventional diabetes diet . A 10-week experimental diet consisted of ordinary food item having gi 55, and each main meal had gl 20 with overall daily gl = 6777 (42% carbohydrate, total fat 36%, fat derived from olive oil and nuts 15%, 22% protein) [table 1]. This was accomplished by providing a list to each individual of the recommended daily intake of commonly used foods and a substitution list allowing exchanges within food groups . The compliance with diet program and gl of consumed meals was assessed by regular fortnightly visit of a dietitian . At baseline and 2 weeks after the diet intervention, blood samples were drawn after an overnight fast for determination of plasma glucose, hba1c, triglyceride (tg), total cholesterol, low density lipoprotein cholesterol (ldl - c), and high density lipoprotein cholesterol (hdl - c). Plasma glucose concentrations were determined by the glucose oxidase method (using a hitachi 917 analyzer, roche diagnostics, biomedical lab . The ldl level was measured by a homogeneous enzymatic assay (genzyme corp ., cambridge, ma, usa), and hdl, triglycerides, and cholesterol concentrations were measured using a hitachi 911 analyzer (roche diagnostics, indianapolis, in, usa). Using g - power exe software, the sample size was calculated based on effect sizes and mean obtained for fasting blood glucose in similar studies powered at 90% and an alpha of 5% . We estimated that a sample size of 96 were enough to meet the considered powerfor our study . Data were analyzed for normality of distribution before use of parametric statistics with spss version 16 (spss inc ., data were reported as mean sd and were analyzed by using paired student's t - test and pearson correlation to compare weight, bmi, fasting blood sugar (fbs), hba1c, and lipid profile of patients before and after intervention . One hundred subjects (55 m, 45 f), aged 52.8 4.5 years, of weight 74.0 5 kg and bmi = 27.2 1.9 kg / m, who were under treatment for a period of 11.25 3 years were recruited for this study . Fbs concentration, hba1c percentage, weight, and bmi were significantly different between the values before and after intervention (p <0.001), which reduced as follows: fasting blood glucose by 28.1 12.5 mg / dl (16.6%), hba1c by 1.1 0.3%, weight by 3.3 1 kg, and bmi by 1.2 0.4 kg / m (p <0.001). Cholesterol and tg concentrations were 205.9 21.6 mg/ dl and 181.5 22.2 mg / dl and reduced to 182.6 18.2 and 161.6 16.7, respectively (p <0.001). This study showed a significant effect of low - gl diet on cardiovascular risk factors including total cholesterol, tg, ldl, hdl, fbs, and hba1c . In our study, as we hypothesized, the administered low - gl diet suppressed the hba1c of the patients to 7.8 0.3%, which is not considered as poorly controlled level and was our target in the present study . While there is widespread concern about increasing diabetes and obesity and related health care costs, development of an appropriate diet for cardiovascular risk factor reduction and weight management the reduction in cardiovascular risk factors in poorly controlled diabetic patients in our study was due to weight loss and also low gl of diet . Although the poorly controlled diabetes patients had similar isocaloric diet before and during intervention, the low - gi, low - gl diet caused significant weight reduction after 10 weeks of intervention . Several studies have examined the effect of gi on human appetite, and most of them demonstrated increased satiety, delayed return of hunger, or decreased ad libitum food intake after consumption of low compared to high - gi foods . In contrast, hyperinsulinemia resulting from high - gi food intake may cause weight gain by directing nutrients away from oxidation in muscle and toward storage in fat . In animal study it was shown that hyperinsulinemia elevates glucose utilization in fatty tissue, but decreases utilization in muscles, a process that results in increased food intake and weight gain . In epidemiological studies, it has been reported that pima indian children with increased fasting insulin levels gain more weight than those children having normal insulin concentration . Energy - restricted diet based on low - gi foods produced greater weight loss than did an equivalent diet based on high - gi foods, and among healthy pregnant women, high - gi diet resulted in greater weight gain at term than isocaloric low - gi diet . The weight changes found in adult rats fed isoenergic, nutrient - balanced diets based on high - gi or low - gi diet for 32 weeks were significantly different . The low - gi group had reduced weight, while the high - gi group demonstrated increased weight . These diets have been reported to be beneficial as they control diabetes, increase hdl - c, lower serum tg, and reduce glycated proteins . In contrast, consuming high - gi diet and consequently high - gl diet was 4 times greater among women with a higher bmi, which may lead to diabetes and cardiovascular disease (cvd). Also, the epidemiological studies such as the nurses health study and health professional follow - up study, and also framingham offspring study have demonstrated the association between gl and type 2 diabetes, cvd, and metabolic syndrome . All the above studies confirm weight reduction in diabetes subjects of our study, following low - gl diet . The present study also gains support from a study in which consumption of an ad libitum low - gl diet by obese adults during 6 months resulted in significant body weight reduction which was comparable with conventional restricted energy (250500 kcal / day deficit) diet group (7.8% and 8.4% weight reduction, respectively). In our study, diabetes subjects with low gl and sufficient energy intake had 4.4% weight reduction during 10 weeks intervention . The low - gl diet in our study may have increased oxidation of nutrients in muscles rather than storing them in white tissue . In addition, the low - gl diet may have elevated satiety and reduced the intake of foods . In epidemiologic studies, both gi and the gl of the overall diet were associated with a greater risk of type 2 diabetes in whole adult population and low - gi diet had significant effect on reducing glycosylated proteins . The low - gi and low - gl diets independent of weight loss have significant effect on improving cardiovascular risk factors . In a study, ad libitum intake of the low - gi diet resulted in a 10% decrease in ldl - c compared with isocaloric high - gi diet after 10 weeks intervention, and also ad libitum intake of low - gi diet showed a significantly greater mean decline in plasma triacylglycerols than did the conventional restricted diet . In our present study, the ldl - c reduced by 4%, while the hdl - c increased by 8% . Beneficial effect of low - gi diet in the management of diabetes is well documented . A meta - analysis showed that after average duration of 10 weeks, subjects with type 1 and 2 diabetes who were consuming low - gi diets had hba1c concentration of 0.4% points lower than those who were following a high - gi diet . Comparing low - gi versus high - gi diet, the low - gi diet significantly improved fasting blood glucose and hba1c of type 2 diabetes . The patients who followed low - gi diet demonstrated a reduced hba1c level and it was 0.39% points lower than the hba1c level of those who followed high - gi diet . In our study, after 10 weeks intervention, the fasting blood glucose reduced by 28.1 12.5 mg / dl (16.6%), and hba1c by 1.1 0.3% . The mechanism underlying improvement of fasting blood glucose and hba1c in the present study probably is the elevated whole - body glucose disposal . The low - fat, high - carbohydrate diet, which causes postprandial hyperglycemia and hyperinsulinemia, has a significantly less favorable effect on circulating triacylglycerol and pai-1 (plasminogen activator inhibitor-1; a marker of fibrinolytic capacity) concentration than does low - gl diet . In turn higher concentrations of triacylglycerol and pai-1 have direct association with cardiovascular events . In our study, the moderate carbohydrate diet with gl = 6777 g / day, including 42% carbohydrate as energy intake, and 15% of fat derived from olive oil and nuts sources was almost similar to ada's recommendation which is more appropriate and compelling for glycemic control for long period . The gl of diet in our study was even lower than maximum g / day recommendation for low - gl diet . The meal plan provided by us for glycemic control and control of cardiovascular risk factors of poorly controlled diabetes subjects is appropriate . The mechanism of low gl diet for weight loss is due to its effect on oxidation elevation of nutrients in muscles rather than storing them in white adipose tissue, a process that increases satiety, delayed return of hunger, or decreased ad libitum food intake and weight loss.
|
Hypertensive patients have impaired cognitive and cns function, and some of these changes may precede development of frank essential hypertension . Subtle cognitive changes in younger persons at risk for hypertension may become more readily apparent during the systemic stress of sleep deprivation . We hypothesize that young adults at risk for hypertension will show significant declines in cognitive function during a night of sleep deprivation . Cognitive decline in older persons with advanced hypertension is especially well documented and likely represents, at least in part, the damaging effects of sustained high blood pressure on cns microvasculature, and, hence, brain function [15]. For example, hypertension is associated with increased likelihood of dementia in older adults [69]. Some of the cognitive decline in older hypertensive patients can be reversed by pharmacological, dietary, and weight loss approaches to blood pressure reduction, indicating that chronic high blood pressure can have a damaging effect, either directly or indirectly, on brain function . However, some cognitive changes may precede the development of frank clinical hypertension, suggesting a more complex association between the cns and blood pressure in the development of essential hypertension . For example, decreased cognitive / cns function has been recently found in middle aged and younger hypertensives [11, 12], young people with mildly elevated blood pressure, and in normotensives with a positive family history of hypertension [14, 15]. These findings suggest that relatively minor preclinical changes in blood pressure are associated with subtle changes in brain function . Therefore, cns changes may parallel, precede, and/or contribute to blood pressure elevations, especially in young persons whose cerebral vasculature has not been exposed to the deleterious effects of significant and sustained blood pressure elevations . Thus, the study of cognitive changes in early stages of hypertension development may provide insight into the possible neurogenic precursors and/or etiologic mechanisms of essential hypertension . Interestingly, the systemic stress and fatigue of sleep deprivation may exacerbate both cognitive and circulatory changes in young adults at risk for development of hypertension . For example, sleep deprivation has been shown to disrupt executive attention, working memory and other higher cognitive functions . Moreover, neural systems that underlie executive function are especially vulnerable to the effects of sleep deprivation in some individuals . Young adults at risk for hypertension development later in life show a spectrum of neural, endocrine and circulatory changes during stress [1820], possibly including the systemic stress of sleep deprivation . For example, some of studies from our sleep laboratory recently showed that a night of sleep deprivation increased blood pressure in young adults with a positive family history of hypertension versus negative family history controls . The present study, part of that larger series of sleep deprivation studies, focuses on the effect of sleep deprivation on cognitive function in persons at risk for hypertension . We hypothesize that persons at risk for hypertension will show declines in higher cognitive performance during sleep deprivation . Participants were fifty - one volunteers (28 males and 23 females) with an average age of 22.9 years . Young adult study participants were recruited from campus and the surrounding community with ages ranging from 1932 years old . Average body weight was 148 25.7 lbs ., and average body mass index was 22.6 3.26 . The participants completed questionnaires about personal and family medical history, sleep habits, and alcohol and tobacco use . The final study population was a healthy, normal sample without sleep disorders or significant cardiovascular, neurological, endocrine, or psychiatric disease . All subjects refrained from use of caffeine and tobacco and exercise throughout the study period . The present investigation is part of a larger series of studies of sleep deprivation and sustained operations . Participants were recruited through posted flyers detailing the two - day sleep deprivation study . After screening, volunteers met with researchers three days before the study to discuss the consent form, study procedures, and instructions in completing the study . The participants were instructed to sleep for eight hours each night for the three days prior to the sleep deprivation study . Participants were also instructed not to drink alcohol the day before the study and were told not to consume any caffeine or substances high in sugar (e.g., candy bar) the morning of the study ., bend, or, usa) that they wore for the three days prior to the study . Actiwatches were worn on the nondominant arm to record wrist movement indices of normal sleep / wake patterns . Participants also received a sleep log to provide information on sleep habits prior to the onset of the study . These sleep logs were to be completed each morning of the three days prior to the study . The sleep logs included questions inquiring about sleep quality, time going to bed, time getting out of bed, and napping throughout the day . Participants reported at 9:30 am on day 1 and were transported to the residential sleep laboratory . The participants completed a series of tasks and questionnaires and were given scheduled breaks and meals throughout the study period . Four testing sessions were scheduled at approximately 8:30 pm, 1:00 am, 5:30 am, and 10:00 am across the sleep deprivation period . The study ended on day 2 when participants were transported back to their residences and instructed to sleep before operating heavy equipment or driving . Questionnaires were administered to obtain information on typical sleep patterns and individual and familial medical history for each participant . The pittsburgh sleep quality index (psqi) its reliability and validity has been verified by buysse and coworkers . Resting blood pressure (bp) was measured upon arrival and departure at approximately 11 pm each day and at approximately 8:30 pm, 1:00 am, 5:30 am, and 10:00 am over the study period . Electronic blood pressure measurements were taken using ge dinamap pro100 machines (medical solutions, minneapolis, mn . ). Dinamap performance was verified on a regular basis for zero offset, integral offset, and gain using a mercury manometer . Research assistants were trained on theory and application of blood pressure determination using both auscultatory and oscillometric techniques, including use of appropriate cuff sizes and other american heart association guidelines for blood pressure determination . At each blood pressure determination, participants sat quietly in a comfortable armchair for five minutes prior to taking five bp readings at one - minute intervals . The last three readings were averaged to create a single, stable resting bp index at each time period . The participants also completed five subtests from the automated neuropsychological assessment metrics (anam). This measure is a battery of cognitive tests developed by the office of military performance assessment (ompat; washington, dc). The anam tests have strong correlations with traditional measures of neuropsychological functioning, high test - retest reliability (typically = 0.80, 0.95 range), high differential stability, and a large database of studies providing construct validity . The measure includes five different tasks, all of which were used for the purposes of this study . The tasks were given in the following order for each administration (testing and training): code substitution learning (cds), code substitution immediate memory recall (cdi), sternberg memory recall (st6), continuous performance test (cpt), and code substitution delayed memory recall (cdd). Participants were given the battery 5 times during the training sessions to control for learning effects and then once during each of the 4 testing sessions . Apart from the fixed order required by anam components, task order was fixed within subjects, but counterbalance between subjects . Participants were presented with a set of symbols (,,,,,,,,) that corresponded to a number, 19 . With the key on the screen for reference, they were given a symbol and number and asked to respond if the pair correctly matched the given key . Each stimulus was displayed for 4000 milliseconds, and the time allowed for response was 4200 milliseconds before the next stimulus was displayed . Participants were asked to recall the previous key of numbers and symbols that was given during the code substitution practice task . They were presented with a symbol and number pair and indicated if the stimulus matched the previously memorized key . Each stimulus was displayed for 4000 milliseconds, and the time allowed for response was 4200 milliseconds before the next stimulus was displayed . After completing two additional tasks (see st6 and cpt below), participants were asked to recall the memorized key given during the code substitution practice task after approximately 10 minutes had elapsed since it was presented . Each stimulus was displayed for 4000 milliseconds, and the time allowed for response was 4200 milliseconds before the next stimulus was displayed . Participants completed a category recall (catrecall) task presented on a computer to test memory . The catrecall memory task was developed using e - prime at the university of maryland's center for advanced study of language and derived from previously formed category memory tasks . At the beginning of the task, the participant was presented with 6 different categories containing 8 examples within each category, for example, animal: cat, bird, dog, horse, lion, mouse, snake and wolf . Subjects were allowed to study the words and categories for an unlimited amount of time before continuing . Participants were presented with a memory list, which was a series of six words, one from each category . Each word in the memory list was displayed on the computer screen for 400 milliseconds . Participants were then presented with a probe category such as music from the 6 available category names . Participants were then asked to identify which word from the memory list was originally included in the category presented . The order recall task presented participants with a memory list of 6 words, one at a time . Participants were asked to remember the 6 words in order, and when presented with a cue word from the memory list, to correctly choose the word from the memory list that followed the cue word . Once a second number appeared, they indicated whether that number was the same as the previously presented number (a 1-back task). This task was divided into two sections: with feedback (1 minute) and without feedback (4 minutes). The probe was displayed on the screen for 1000 ms, and participants were given a maximum of 1500 milliseconds to respond . Risk for subsequent development of hypertension was determined in two different ways, by resting blood pressure levels and by reported parental history of hypertension . Classification of risk by resting blood pressure levels was based on criteria outlined in the seventh report of the joint national committee on the prevention, detection, evaluation, and treatment of high blood pressure (jnc-7). Briefly, subjects rested in a seated position while blood pressure determinations were made on two different days at approximately the same time each day . Participants were classified as normal if they had no blood pressure above 120 mmhg systolic and 80 mmhg diastolic on both days of measurement . Participants were classified as prehypertensive if they had blood pressure between 120139 systolic or 8089 diastolic on both days of measurement . Risk associated with moderately elevated pressures in young adults has been shown by several studies . For example, the level of pressure in young adults has been shown to predict both the level of pressure and incidence of essential hypertension in later life [27, 28]. Moreover, a recent meta - analysis showed that persons with sbp 130139 or dbp 8589 have up to 55% increased risk of stroke . Participants were classified with a positive family history of hypertension if one or both biological parents were identified as having been diagnosed with essential hypertension by a physician . Validity of self - reported parental hypertension has been consistently demonstrated through direct contact with parents and parents' physicians [3032]. All bp data were entered into microsoft excel and imported into spss (ibm, armonk, ny, usa) for statistical analyses . Cognitive performance measures were initially analyzed in a 2 2 4 (risk sex time) design using the spss general linear model with time as the within subjects variable, risk and sex as between subjects variables, and multivariate f tests for main effects and interactions with time . One set of risk analyses were conducted using jnc-7 grouping of blood pressure (prehypertensive versus normotensive). Additional analyses were conducted using family history of hypertension (positive versus negative family history) as an alternate risk variable . Means and standard errors for blood pressures and cognitive performance of all participants at the four test sessions are shown in table 1 . Repeated measures anovas on cognitive performance generally showed significant sleep deprivation - induced declines in performance for all subjects across testing periods . For example, when collapsed across risk status, significant declines across time were observed for code substitution simultaneous (f(3, 41) = 7.71, p <.001), code substitution immediate (f(3, 41) = 8.903, p <.001), code substitution delayed (f(3, 41) = 21.876, p <.001), and continuous performance (f(3, 41) = 11.955, p <.001). Using the jnc-7 criteria for classification of blood pressure, 19.6% of participants (10 of 51) were classified as prehypertensive . Average age was 21.9 years for the prehypertensive group and 23.0 years for the normal group . Using family history of hypertension as an index of risk, 23.5% of participants (12 of 51) reported a positive family history of essential hypertension in at least one biological parent . Average age was 23.0 years for the positive family history group and 22.7 years for the negative family history group . There were no significant group differences in age, weight, body mass index, or distribution of sex among risk groups (p>.05). Only 3.9% of participants (2 of 51) had both a prehypertensive classification and a positive family history of hypertension . Briefly, the prehypertensive groups showed significantly higher resting systolic (multivariate f(1, 46) = 20.839, p <.001, = .312) and diastolic [multivariate f(1, 48) = 4.638, p = .036, = .088] blood pressure across all time periods . Using family history of hypertension for risk categorization, there were no significant initial baseline differences in blood pressure between high- and low - risk groups, however the family history x time interaction for diastolic blood pressure was significant [multivariate f(3, 46) = 4.574, p = .007, = .230], indicating that diastolic blood pressure for the two family history groups significantly diverged across the night of sleep deprivation, with slight decreases across the night in subjects with negative family history and concomitant increases in subjects with positive family history of hypertension . Repeated measures anovas show significantly greater declines in cognitive performance of prehypertensives versus normotensives over the period of sleep deprivation . For example, figure 1 shows the significant risk group time interaction for immediate code substitution memory performance [f(3, 41) = 4.073, p = .013, = .230]. Unlike normotensives, prehypertensives showed a large decline in memory performance at the 5:00 am test (p = .03), but their performance accuracy recovered by the subsequent 10:00 am test . Figure 2 shows the significant risk group time interaction for delayed code substitution memory performance [f(3, 41) = 4.359, p = .009, = .242]. Relative to normotensives, prehypertensives showed a large decline in memory at the 5:00 am test (p = .009) that remained through the subsequent 10:00 am test (p = .017). A similar risk group time interaction was observed for category recall [f(3, 45) = 3.288, p = .029, = .180]. In this case, the performance decline in prehypertensives did not emerge until the 10:00 am test (p = .046). For the sternberg memory task, a significant risk group time sex interaction achieved statistical significance [f(3, 41) = 3.248, p = .031, = .192], indicating a decline in performance at the 10:00 am testing in prehypertensive women . Using family history as the risk variable, a significant risk group time interaction was observed for order recall [f(3, 21) = 4.061, p = .020, = .367]. In this case, persons with a positive family history showed a decline in order recall performance at 1:00 am and 5:00 am, with a relative recovery by the 10:00 am testing . No other risk group time interactions achieved statistical significance . Throughout the night of sleep deprivation, therefore, we also examined the correlations between sleep - deprivation - induced changes in blood pressure and cognitive function across the night . While most correlations were not statistically significant, we did observe a pattern of significant positive correlations (2-tailed probabilities) between blood pressure and corresponding cognitive function, especially at the 1:00 am test session . For example, at the 1:00 am test session, sbp was positively correlated with performance on simultaneous code substitution (r(49) = .526, p <.001), sternberg memory task (r(49) = .335, p = .023) and the continuous performance task (r(49) = .435, p = .003). Similarly, dbp was significantly correlated with performance on simultaneous code substitution (r(49) = .394, p = .007) and the sternberg memory task (r(49) = .412, p = .004) at the 1:00 am test session . Sleep deprivation has been shown to impair functioning of the distributed thalamoprefrontal cortical networks subserving attention and higher order cognitive processes [16, 17]. Consistent with these and other studies of sleep deprivation, measures of higher cognitive performance declined over time across all participants, regardless of risk status [22, 33]. However, our results show significantly greater cognitive declines in otherwise healthy young adults at risk for hypertension . No consistent differences between high- and low - risk groups in cognitive function were seen at the earlier times of testing, but the cognitive decline in high risk groups emerged most consistently at the 5:30 am and 10:00 am tests, after significant sleep deprivation . This suggests that subtle cognitive differences between high- and low - risk groups became apparent only after significant sleep deprivation had occurred . Persons at risk did not show exaggerated cognitive declines in relatively simple tasks, even after significant sleep deprivation . However, high - risk groups showed larger performance decrements across time in tasks requiring significant sustained attention and working memory . For example, there were no interactions of risk groups across time on code substitution learning with simultaneous display, but significant declines were seen for code substitution in both immediate and delayed recall tasks . Interestingly, memory performance of prehypertensives degraded on the more difficult delayed recall task by the 5:30 am testing and remained low through the final 10:30 am test session . In contrast, performance on the easier immediate recall task declined in prehypertensives at the 5:30 am tests, but showed recovery at the 10:00 am testing . This pattern likely reflects the partial recovery of alertness and cortical arousal associated with the rise in circadian rhythms entrained by sunrise in the light / dark cycle . In addition, the decline in sternberg recall performance in prehypertensives was confined primarily to women . Because of the small sample size in this group, this result should be interpreted with caution until confirmatory evidence is available . Interestingly, we also observed a decline in order recall performance in persons with a positive family history of hypertension . Because there were no initial differences in resting blood pressure between positive and negative family history groups, this finding suggests that even mild elevations in resting pressure are not necessary for expression of the association between risk for hypertension and cognitive performance decline . Sleep deprivation and/or disruption can influence the sympathetic nervous system and blood pressure acutely and via chronic sleep loss [34, 35]. However in the present study, scores on the pittsburgh sleep quality index global scale showed no group differences in chronic sleep quality, regardless of risk categorization (all ps>.1). Thus, it is unlikely that the differential effects of acute sleep deprivation on these risk groups resulted from lower chronic sleep quality in high - risk groups . Interestingly, sleep - deprivation - induced changes in acute blood pressure and cognition across the night showed a trend for positive correlations, especially at the 1:00 am testing . This suggests that individuals with the worst cognitive performance at this time of night also showed signs of decreased arousal as indexed by lower acute blood pressure at the time of testing . This may reflect the relationship between cortical and autonomic arousal and is consistent with reports of decreases in blood pressure and attentiveness with increasing fatigue . Overall, the cognitive decline in persons with mild elevations in resting blood pressure (jnc-7 prehypertensive) is consistent with results of ditto et al . Others . To our knowledge, this is the first study to report cognitive declines in persons with a positive family history of hypertension, without concomitant elevations in chronic resting blood pressure . The effect of family history was observed only for the order recall task, and additional research is needed to confirm the role of family history without associated elevations in resting blood pressure . Nevertheless, the present results clearly indicate that the systemic stress and fatigue of sleep deprivation exposes occult cognitive / cns changes in otherwise healthy young adults at risk for hypertension development later in life . Changes in cognitive function in younger persons without a history of significant and sustained high blood pressure may provide insight into the early etiological mechanisms in the developmental pathophysiology of hypertension . It is possible that even modest, subclinical resting blood pressure elevations could directly engender subtle cns functional damage . However, our finding of cognitive decline in a group of young adults with familial hypertension raises the possibility that functional cns changes may be occurring in persons at risk before the development of mild elevations in chronic resting blood pressure . Nevertheless, persons with familial hypertension have increased blood pressure and hpa reactivity to psychological stress, so it is possible that these periodic stress - associated blood pressure elevations may influence cns function, even in the absence of modest elevations in chronic resting blood pressure . The precise role of glucocorticoids and other stress hormones in the observed cognitive declines in groups at risk remains to be determined . Additional research is needed to further explore the potential effect of acute, stress - induced elevations in blood pressure and stress hormones on brain function . Notwithstanding the above, there are other possible links between blood pressure and cns function in otherwise healthy young adults at risk . For example, cns changes could be involved intimately with the progressive blood pressure dysregulation that eventuates in frank essential hypertension later in life . Firstly, changes in cns function and blood pressure control could be related indirectly with each other, but correlated with a heretofore unknown, underlying mechanism affecting both through distinct, but noninteractive pathways . Secondly, and even more intriguingly, cns changes could contribute directly to dysfunction of autonomic and neuroendocrine systems involved in regulation of blood pressure . Under the latter scenario, changes in higher cns function could cascade distally via central control of sympathoadrenomedullary, hypothalamic pituitary adrenocortical (hpa), and perhaps other neuroendocrine axes [38, 39] to provoke the blood pressure dysregulation observed in the early stages of hypertension development . In support of this notion, an accumulating body of evidence points to a broad spectrum of subtle cns changes in otherwise healthy persons with either mildly elevated blood pressure or normal resting blood pressure with other risk factors such as a positive history of hypertension . For example, prior work in our lab has shown abnormalities in opioidergic inhibition of both the hpa and the sympathoadrenomedullary axes in persons at risk for hypertension, suggesting alteration in brain mechanisms, either at or proximal to the paraventricular hypothalamus . Moreover, a large literature shows that hypertensive humans and animals as well as persons at risk for hypertension show reduced responsivity to pain [40, 41]. Recent findings suggest that changes in affective pain sensitivity may reflect a more generalized emotional dampening [42, 43] and may result in impaired perception of affective environmental cues . This raises the possibility that subtle changes in brain function and performance may actually contribute to the autonomic dysregulation of blood pressure in persons at risk . For example, it is likely that modest changes in appraisal of threatening stimuli and/or memory function could directly contribute to increased psychological and/or psychosocial distress, with its consequent autonomic disturbance and blood pressure dysregulation . A dampening in threat appraisal could reduce motivation and directly contribute to reduced performance on complex tasks, including the cognitive learning and memory tasks . The notion of cns changes preceding blood pressure dysregulation is consistent with a recent report of reduced cry response to painful vitamin k injections in newborn infants with hypertensive grandparents . Therefore, genetic and/or maternal stress hormone - based changes in fetal nervous system development [46, 47] could provoke subtle alterations in brain function, blood pressure control, and programming for adult disease . Thus, the notion of preexisting changes in cns function in persons at risk for hypertension provides explanation for a large body of data and suggests a novel new possibility for cns origins of blood pressure dysregulation in the developmental etiology of essential hypertension . First, the 28-hour sleep deprivation methodology requires significant costs, as well as intensive time burden on participants . Thus, modest samples sizes are typical of studies where sleep deprivation is experimentally manipulated . Although a larger sample size would have been preferable, the present study nevertheless produced several significant results in line with hypothesized outcomes . This suggests that the current sample size has sufficient statistical power to expose reliable links between brain function and hypertension risk . Moreover, the experimental manipulation of sleep deprivation in the current study avoids the many potential covariates that confound correlational epidemiological studies of chronic sleep loss . Nevertheless, risk group differences in aerobic fitness or other related variables may have affected these results, despite similarities in body weight and bmi . For example, the categorization by family history in our sample could favor a later developing blood pressure rise . An older study sample might show more persons with a family history of hypertension in the prehypertensive blood pressure range . Regardless of methodological limitations, the present results suggest that healthy young adults at risk for hypertension show decreased memory performance during a single night of sleep deprivation . The overall findings of this study indicate that young adults at risk for hypertension show decreases in higher cognitive function during sleep deprivation . The sleep deprivation - induced cognitive declines in persons at enhanced risk are significantly greater than those seen in their low - risk counterparts . These risk - related cognitive declines are seen primarily in tasks that require sustained attention and working memory and emerge only after significant sleep deprivation . These results suggest that some cognitive effects in hypertensive patients may reflect cns changes that occur prior to development of significant and sustained high blood pressure . Taken together with other findings, these results suggest that cns changes may parallel, precede, or even contribute to blood pressure dysregulation in the early stages of the development of hypertension . Better understanding of the potential cns origins of essential hypertension could lead to new strategies for treatment and prevention of this costly and widespread disease.
|
Vision 2020 is a joint initiative by the world health organization (who) and the international agency for the prevention of blindness, that aims to eliminate avoidable blindness by the year 2020 . The vision 2020 strategy depends on the development of district level plans for the prevention of avoidable blindness. [26] india was the first country in the world to initiate a public funded program for the prevention of blindness as a national priority health problem . Population - based surveys have been the main stay of information regarding the effective implementation and monitoring of such eye care programs . Are expensive and time consuming . Rapid assessment of avoidable blindness (raab) survey is cheap and easy means of getting population - based data on prevalence and causes of blindness in people aged more than 50 years . Raab also has the other utility of monitoring programs at the unit / district level. [911] kolar district in south karnataka, india, has an estimated population of around 1.5 million, 51% of the population being males and 29% being over 50 years . The aim of the study was to conduct a raab study in persons aged more than 50 years in kolar district in order to estimate the prevalence and causes of blindness . This was also to help the ophthalmology and community medicine departments of the medical institution to develop student capacity as well as expose them to scientific survey methods of generating evidence for planning of eye care services . The survey was carried out by a team consisting of trained personnel from the departments of ophthalmology and community medicine, sri devaraj urs medical college, kolar, karnataka, india . The survey was carried out between march and june 2011 (4 months), in accordance with the helsinki declaration . Written informed consent was obtained from all study participants, after explaining the purpose of the study in their local language . Sample size was determined using a prevalence estimate of 4% for blindness (who definition of presenting vision <20/400 in better eye) among those aged over 50 years . The prevalence estimate was assumed to be around 4% considering the recent raab survey conducted across india which gave prevalence of blindness using the same definition to be around 3.6% . Using 95% confidence interval, 22.5% precision, design effect of 1.5 and 10% nonresponse rate, sample size was calculated to be 3017, which would require 61 clusters of 50 people aged over 50 years . Using the population data from the last census (2001) and the growth rate for 9 years, the population was estimated for end of 2010 and used as a sampling frame . A list of wards and villages in urban and rural areas was prepared taluk (revenue division) wise . In each, the population size of people aged over 50 years was listed . Households within each cluster were selected by compact segment sampling . A map of the selected cluster was drawn and divided into equal segments that would give segments were numbered and one segment was chosen by draw of lots . To be eligible for inclusion an individual had to reside in that household for at least the previous 6 months . In each cluster, the people in the selected cluster were briefed about the survey 23 days in advance by the local health worker along with the public relations officer of the team . If an eligible person was not available during the survey, at least two more attempts were made to assess information . If after repeated visits, examination could not be done, information of his visual status was obtained from his relatives or neighbors . Standard raab protocol was used for gathering information and for eye examination . A survey form comprising seven sections the form consisted of general information; vision and pin hole examination; lens status; principal cause of visual impairment; history if not examined; and barriers to uptake of cataract surgery and details of cataract surgery if operated . Visual acuity (va) was measured using a tumbling snellen - e chart using optotype size 20/60 on one side and 20/200 on the other . All measurements were taken in full daylight with available correction . If visual acuity was less than 20/60 in either eye, pin hole vision was tested . If presenting vision was <20/60, then pupil was dilated and ophthalmoscopy done to assess the cause of blindness . The interobserver agreement between the two teams was good as observed by the kappa value (> 0.60). Double data entry and analysis was done using the raab software program version 4.02 . To check for errors made during data entry of the survey record forms, the data are entered twice by different data entry clerks in two separate databases and then compared . Sample size was determined using a prevalence estimate of 4% for blindness (who definition of presenting vision <20/400 in better eye) among those aged over 50 years . The prevalence estimate was assumed to be around 4% considering the recent raab survey conducted across india which gave prevalence of blindness using the same definition to be around 3.6% . Using 95% confidence interval, 22.5% precision, design effect of 1.5 and 10% nonresponse rate, sample size was calculated to be 3017, which would require 61 clusters of 50 people aged over 50 years . Using the population data from the last census (2001) and the growth rate for 9 years, the population was estimated for end of 2010 and used as a sampling frame . A list of wards and villages in urban and rural areas was prepared taluk (revenue division) wise . In each, the population size of people aged over 50 years was listed . Households within each cluster were selected by compact segment sampling . A map of the selected cluster was drawn and divided into equal segments that would give segments were numbered and one segment was chosen by draw of lots . To be eligible for inclusion an individual had to reside in that household for at least the previous 6 months . In each cluster, the survey team visited each household accompanied by local health worker to facilitate compliance . The people in the selected cluster were briefed about the survey 23 days in advance by the local health worker along with the public relations officer of the team . If an eligible person was not available during the survey, at least two more attempts were made to assess information . If after repeated visits, examination could not be done, information of his visual status was obtained from his relatives or neighbors . Standard raab protocol was used for gathering information and for eye examination . A survey form comprising seven sections vision and pin hole examination; lens status; principal cause of visual impairment; history if not examined; and barriers to uptake of cataract surgery and details of cataract surgery if operated . Visual acuity (va) was measured using a tumbling snellen - e chart using optotype size 20/60 on one side and 20/200 on the other . All measurements were taken in full daylight with available correction . If visual acuity was less than 20/60 in either eye, pin hole vision was tested . If presenting vision was <20/60, then pupil was dilated and ophthalmoscopy done to assess the cause of blindness . The interobserver agreement between the two teams was good as observed by the kappa value (> 0.60). Double data entry and analysis was done using the raab software program version 4.02 . To check for errors made during data entry of the survey record forms, the data are entered twice by different data entry clerks in two separate databases and then compared . A total of 3050 persons aged over 50 years were included in the study and 2907 (95.3%) of them were examined . Of them the sampled population was relatively representative of the district population in terms of age and sex distribution . The overall unadjusted prevalence of blindness from all causes in persons aged over 50 years was 3.9% (95% ci 2.75.1). Table 1 shows the data regarding prevalence of blindness (va <20/400 in better eye with available correction); severe visual impairment (va <20/200 20/400 in better eye with available correction) and moderate visual impairment in persons (va <20/60 20/200 in better eye with available correction). The age and sex adjusted prevalence of blindness, severe visual impairment, and moderate visual impairment was 3.4%, 3.1%, and 9.7%, respectively . Sample prevalence of blindness, severe visual impairment and moderate visual impairment all causes untreated cataract was the primary cause of bilateral blindness (75%) and severe visual impairment (73%). Refractive errors were the most common cause of moderate visual impairment (56%) and second most common cause of severe visual impairment (11%). Avoidable causes accounted for 91% of all cases of blindness and 95% of cases of severe visual impairment [table 2]. Causes of blindness (va <20/400), severe visual impairment (va 20/20020/400) and moderate visual impairment (va 20/6020/200) the main barriers to uptake of cataract surgery were no one to accompany (27%); waiting for maturity (27%); do not know how to get surgery done (10%); and old age no need (7%). The cataract surgical coverage (csc) in persons with va <20/400 was high, with 82% of those requiring surgery having received the same . For people with va <20/200 and va <20/60, 72% and 64%, respectively of those needing surgery had received it [table 3]. Cataract surgical coverage by persons and eyes sex wise of the 707 eyes, which had received surgery, 641 (90.7%) had an intra - ocular lens (iol) implantated . Among the patients with iol, 72.7% had va of 20/60 or more with available correction [table 4]. In patients operated less than 5 years back, 86% of patients with iol had best corrected va of more than or equal to 20/60, when compared with 79% in patients with iol operated more than 5 years back . Around 85% of patients were satisfied with the results of cataract surgery . Ocular comorbidities, operative complications and long - term complications were the principal reasons for poor outcome in eyes operated less than 3 years back . Postoperative visual acuity with available correction low vision (persons with va <20/60 in better eye with correction and not due to cataract, refractive error, or uncorrected aphakia) was seen in 46 persons (1.6%). In india, eye care planning and monitoring under the national program for control of blindness has been guided by population - based surveys . Rapid assessment techniques, which provide reliable estimates have been used for the past 16 years and have been the basis for district level programming . The prevalence of blindness (va <20/400) was 3.9% in our study, which is almost similar to the raab study conducted across various states in india, which gave a prevalence of 3.6% . The raab india study which covered gulbarga district in karnataka, gave a prevalence of 4.3% for that district . The prevalence of blindness in our study is on the higher side when compared with other studies from kenya, bangladesh, china, palestinian territories and malawi where blindness prevalence ranged from 2.0% to 3.7% . Consequently, even though there has been a sharp increase in outreach programs and service delivery, this has been offset by the increasing elderly population as a result of increased life expectancy . In our study, almost 91% of blindness was avoidable . Untreated cataract still continues to be the major cause of blindness and severe visual impairment . Refractive errors and uncorrected aphakia are the other leading avoidable causes . Despite the increased csc, there are an estimated 4700 people [table 5] who are having bilateral cataract in kolar district, extrapolating the survey findings . This cataract burden can only be reduced by proper taluk wise segmentation of the cataract blind and targeting the community outreach programs toward these areas . Spectacle provision to the needy at a peripheral level by means of mobile refraction units and spectacle dispensing outfits age and sex adjusted prevalence modified strategies in the form of extensive health awareness and health education campaigns through a decentralized approach involving all major health care providers at the grass root level, down to the remote rural level could be one of the positive steps in reducing this cataract burden . Waiting for maturity. This indirectly indicates that patients have been told to wait and sent back, which is an indicator of service delivery deficiency . Such people once turned back are unlikely to return back because of reasons such as lack of funds for conveyance to the hospital, loss of daily wages during the visit to the hospital, and relative lack of knowledge about when to return . Thus not only increasing the outreach programs but also concentrating on proper case selection, good surgical techniques, proper follow - up care and spectacle provision will definitely reduce the barriers and improve the outcome after cataract surgery . This could be due to the fact that increased percentage of males seeks eye care services as reflected in the increased csc among males . Also females are less likely to report a need for sight than males. [1622] the strengths of our study was ready availability of taluk wise population data and the survey teams being trained by a certified raab trainer . Even though, there is a decline in prevalence of blindness, modified strategies need to be implemented to tackle the burden of untreated cataract . Raab done at regular intervals is an effective tool to quantify the problem of blindness and monitor the implementation of eye care programs.
|
Advances in intensive care medicine have enormously improved treatment of seriously ill patients . However, intensive treatment and prolongation of life is not always in the patient s best interest . Thus, difficult decisions frequently need to be made about limitation of life - sustaining treatments (lst). Approximately 20% of patients die during or shortly after a stay in intensive care units (icus). According to most previous studies, over 50% of all icu deaths previous surveys demonstrated that the limitation of lst (e.g., do - not - resuscitate (dnr) order, withholding and withdrawing therapy) is a common practice when therapeutic measures are considered to be futile; however, substantial international differences in end - of - life (eol) practices are reported [820]. Beside other contributions, a highly important insight into eol decision making is provided by the ethicus study group, presenting important international comparisons on the issue and empirical assessments of the most important aspects of eol decision making [5,2123]. The limitation of lst is reported as being more common in northern european countries as compared to southern europe . Unresponsiveness to therapy is reported as the most common primary reason for eol decision making . In addition, a more paternalistic pattern of eol decision making is characteristic for southern europe, with less communication with patients and their families, and the best interests of the patient are less commonly considered [5,2125]. National and international recommendations and guidelines were developed regarding limitation of lst in icus [2628]. Currently, the national guidelines on eol decision making are being finalized in slovenia, because the issue is being increasingly recognized as important among physicians . Thus, the main aim of the present study was to assess icu physician experiences with eol decision making . A cross - sectional study was conducted involving physicians from all 35 icus in slovenia; we included all physicians who work regularly and/or perform overnight shifts in the icus . The study was approved by the slovene national medical ethics committee (decision no.163/10/11). The questionnaire was designed to assess the experiences of the participating icu physicians with eol decision making, focusing on limiting lst in the icus (the term limiting lst included both withholding and withdrawing lst . Withholding was defined as not introducing additional treatment or not intensifying existing treatment, while withdrawing was defined as discontinuing a treatment that a patient was already undergoing . The first part of the questionnaire included questions on demographic characteristics of the participants, along with 3 questions assessing participant experiences with ethical committees . Other questions were designed to assess participant experiences with discontinuation of treatment when it was deemed futile in icu patients . The questionnaires were personally distributed to all icus in slovenia . All icu heads and/or other available icu physicians the questionnaires were collected in sealed boxes placed in a room to which only icu staff had access . Subsequently, 3 weeks after distribution, we personally collected all the submitted questionnaires . The distribution and collection of the questionnaires took place from november 2011 to february 2012 . Fisher s exact test and fisher - freeman - halton test were used for analyzing contingency tables; the significance level was set at p0.001 due to the large number of tested hypotheses . The analyses were performed using ibm spss statistics 20 (ibm corp ., somers, ny, usa). A cross - sectional study was conducted involving physicians from all 35 icus in slovenia; we included all physicians who work regularly and/or perform overnight shifts in the icus . The study was approved by the slovene national medical ethics committee (decision no.163/10/11). The questionnaire was designed to assess the experiences of the participating icu physicians with eol decision making, focusing on limiting lst in the icus (the term limiting lst included both withholding and withdrawing lst . Withholding was defined as not introducing additional treatment or not intensifying existing treatment, while withdrawing was defined as discontinuing a treatment that a patient was already undergoing . The first part of the questionnaire included questions on demographic characteristics of the participants, along with 3 questions assessing participant experiences with ethical committees . Other questions were designed to assess participant experiences with discontinuation of treatment when it was deemed futile in icu patients . The questionnaires were personally distributed to all icus in slovenia . All icu heads and/or other available icu physicians the questionnaires were collected in sealed boxes placed in a room to which only icu staff had access . Subsequently, 3 weeks after distribution, we personally collected all the submitted questionnaires . The distribution and collection of the questionnaires took place from november 2011 to february 2012 where appropriate, the averages and standard deviations (sd) were calculated . Fisher s exact test and fisher - freeman - halton test were used for analyzing contingency tables; the significance level was set at p0.001 due to the large number of tested hypotheses . The analyses were performed using ibm spss statistics 20 (ibm corp ., somers, ny, usa). The responses from icu physicians from all 35 slovene icus are included in the study . Altogether, 370 questionnaires were distributed (the number approximately represented the total number of slovene icu physicians), and 267 questionnaires were returned (the response rate was 72%). Of the 260 total study participants, 134 (52%) were males . Participant age ranged from 27 to 68 years, with a median of 43.5 years (sd 9.6 years). Participants years of work as a physician ranged from 2 to 41 years, with a median of 17.8 years (sd 9.6 years), and their years of work in the icu ranged from 0 to 38 years, with a median of 12.1 years (sd 8.8 years). Only 60% of study participants indicated they knew how to proceed when facing an ethical dilemma and only 23% of all participants had consulted the ethics committee . Furthermore, 42% of the responders knew name of the ethics committee head in their institution, and 17% of them reported there was no ethics committee in their institution . Most participants (90%) had been involved in the decision making process regarding limitation of lst . In 2011, 47% of them were involved 25 times, 20% 510 times, 20% more than 10 times, and 13% never . In the period 20062011, 32% of them were involved more than 15 times, 25% 25 times, and 22% 510 times . The most common reasons for limitation of lst were terminal illness (62%), brain death (28%), and persistent vegetative state (7%). The decision making process regarding limitation of lst was in almost all cases (97%) initiated by the physician; in the remaining 3% the process was initiated by relatives of the patient . Participate in decision making in 39% of cases, and nurses were reported to be never or rarely most (63%) participants had never encountered advanced directives in practice . Not using any instructions regarding the limitation of lst was reported by 37% of participants, 32% used written instructions, and 31% used oral instructions . A special form to be used for limitation lst was used very rarely (less than 0.5% of participants). In most cases (51%) the decision to limit lst was implemented immediately after adopting it and communicating it to the relatives . Within 6 hours from making the decision, it was implemented in 25% cases; only rarely (4%) did implementation take more than 24 hours . The study participants reported deciding more frequently to use a dnr order as compared to withholding of treatment (67% used a dnr order frequently, whereas 48% used withholding of treatment frequently; p<0.001). Detailed data on how frequently responders made decisions about different types of limitation of lst are presented in table 3 . With regard to the type of icu, the responders from medical icus used dnr orders more frequently as compared to the responders from surgical and/or pediatric icus (p<0.001). Furthermore, board - certified icu physicians were more likely to use dnr orders frequently as compared to residents (71% vs. 52%; p<0.001). No other statistically significant differences among the compared groups were found when compared with their gender, working status, years of work, or knowledge about how to proceed when facing an ethical dilemma (p = ns for all comparisons). The study participants reported deciding less frequently to withdraw mechanical ventilation or extubating patients as compared to withdrawal of inotropes or antibiotics (12% reported using withdrawal of mechanical ventilation or extubation frequently, and 66.7% used withdrawal of inotropes or antibiotics frequently; p<0.001). Interestingly, only 3% of participants from surgical icus reported using withdrawal of mechanical ventilation or extubation frequently as compared to approximately 20% in medical and pediatric icus (p<0.001). No other statistically significant differences in the frequency of use of withdrawal of antibiotics or inotropes among the compared groups were found when compared with regard to their gender, working status, years of work, or knowledge about how to proceed when facing an ethical dilemma (p = ns for all comparisons). Termination of hydration was reported to be only rarely used in slovene icus, and 76% of participants reported never terminating it (table 3). Termination of hydration was most used by physicians from medical icus (9.9%) as compared to 3.1% at the surgical icus and to 0% at the pediatric icus; however, the differences were slightly above the threshold of statistical significance; p=0.018). The responses from icu physicians from all 35 slovene icus are included in the study . Altogether, 370 questionnaires were distributed (the number approximately represented the total number of slovene icu physicians), and 267 questionnaires were returned (the response rate was 72%). Of the 260 total study participants, 134 (52%) were males . Participant age ranged from 27 to 68 years, with a median of 43.5 years (sd 9.6 years). Participants years of work as a physician ranged from 2 to 41 years, with a median of 17.8 years (sd 9.6 years), and their years of work in the icu ranged from 0 to 38 years, with a median of 12.1 years (sd 8.8 years). Only 60% of study participants indicated they knew how to proceed when facing an ethical dilemma and only 23% of all participants had consulted the ethics committee . Furthermore, 42% of the responders knew name of the ethics committee head in their institution, and 17% of them reported there was no ethics committee in their institution . Most participants (90%) had been involved in the decision making process regarding limitation of lst . In 2011, 47% of them were involved 25 times, 20% 510 times, 20% more than 10 times, and 13% never . In the period 20062011, 32% of them were involved more than 15 times, 25% 25 times, and 22% 510 times . The most common reasons for limitation of lst were terminal illness (62%), brain death (28%), and persistent vegetative state (7%). The decision making process regarding limitation of lst was in almost all cases (97%) initiated by the physician; in the remaining 3% the process was initiated by relatives of the patient . Participate in decision making in 39% of cases, and nurses were reported to be never or rarely not using any instructions regarding the limitation of lst was reported by 37% of participants, 32% used written instructions, and 31% used oral instructions . A special form to be used for limitation lst was used very rarely (less than 0.5% of participants). In most cases (51%) the decision to limit lst was implemented immediately after adopting it and communicating it to the relatives . Within 6 hours from making the decision, it was implemented in 25% cases; only rarely (4%) did implementation take more than 24 hours . The study participants reported deciding more frequently to use a dnr order as compared to withholding of treatment (67% used a dnr order frequently, whereas 48% used withholding of treatment frequently; p<0.001). Detailed data on how frequently responders made decisions about different types of limitation of lst are presented in table 3 . With regard to the type of icu, the responders from medical icus used dnr orders more frequently as compared to the responders from surgical and/or pediatric icus (p<0.001). Furthermore, board - certified icu physicians were more likely to use dnr orders frequently as compared to residents (71% vs. 52%; p<0.001). No other statistically significant differences among the compared groups were found when compared with their gender, working status, years of work, or knowledge about how to proceed when facing an ethical dilemma (p = ns for all comparisons). The study participants reported deciding less frequently to withdraw mechanical ventilation or extubating patients as compared to withdrawal of inotropes or antibiotics (12% reported using withdrawal of mechanical ventilation or extubation frequently, and 66.7% used withdrawal of inotropes or antibiotics frequently; p<0.001). Interestingly, only 3% of participants from surgical icus reported using withdrawal of mechanical ventilation or extubation frequently as compared to approximately 20% in medical and pediatric icus (p<0.001). No other statistically significant differences in the frequency of use of withdrawal of antibiotics or inotropes among the compared groups were found when compared with regard to their gender, working status, years of work, or knowledge about how to proceed when facing an ethical dilemma (p = ns for all comparisons). Termination of hydration was reported to be only rarely used in slovene icus, and 76% of participants reported never terminating it (table 3). Termination of hydration was most used by physicians from medical icus (9.9%) as compared to 3.1% at the surgical icus and to 0% at the pediatric icus; however, the differences were slightly above the threshold of statistical significance; p=0.018). This is the first study to assess the experiences of slovene icu physicians with eol decision making . Prior to this study, slovenia had not taken part in any of the international studies on eol decision making . Completed questionnaires were obtained from all 35 slovene icus, which included pediatric, medical, and surgical icus . Altogether, responses from approximately three - quarters of all the slovene icu physicians were included, making the study fairly representative of the population . In most previous survey studies however, few previous studies include all the national icus or compared different types of icus . We are aware of different approaches towards defining the term futility as reported by schneiderman, pellegrino, and others [3033]. Since, according to the literature, no general agreement exists on the definition of the term futility, we left the definition of the term to the icu physicians own understanding of the term . Thus, in our study, we were interested about eol decision making from the point at which any further treatment was assessed as futile by involved clinicians . The proportions of physicians who reported using withholding (94%) or withdrawing (86% for antibiotics and 95% for inotropes) lst were comparable to or even higher than in most of the previous national and international surveys . However, we observed substantial differences in reported frequencies of using different measures of limitation of lst . The dnr order was reported to be even slightly more frequently used (by 97% icu physicians) than withholding of treatment . Furthermore, it was shown to be more frequently used in the medical icus as compared to surgical and pediatric icus . Similarly, a recent german survey shows the dnr order is the most common measure of limitation of lst, followed by withdrawal of inotropes, hemodialysis, and antibiotics . Not surprisingly, withdrawal of inotropes (95%) or of antibiotics (86%) was reported to be much more frequently used than withdrawal of mechanical ventilation (51%) or of extubation (27%). This is in line with some previous studies showing that extubation especially is quite rarely performed . (e.g., withdrawal of inotropes or antibiotics) and the hard (e.g., extubation or withdrawal of mechanical ventilation) measures of withdrawal of lst, in line with the fact that the first were reported to be much more commonly used than the later . The soft measures might thus be preferable for the icu physicians from a practical point of view, despite no difference or even some opposite claims with regard to the ethical point of view found in the literature . Nevertheless, also most previous surveys indicated that withdrawal of therapy is more difficult for icu physicians than withholding therapy . Interestingly, extubation or withdrawal of mechanical ventilation were reported to be more regularly used in pediatric and medical icus, but only very rarely in surgical icus, but the reason is not clear . Furthermore, when compared to the ethicus study despite its geographic positioning and its cultural roots in central europe slovenia seems to be more similar to southern european countries regarding extubation or the withdrawal of mechanical ventilation . The decision making process regarding limitation of lst was reported to be almost always initiated by the icu physician . The decision was reported to be usually taken at a meeting of the involved physicians . Furthermore, merely 17% of icu physicians reported always including patients and/or relatives in eol decision making (never or rarely involved in the eol decision making process by 84% of physician respondents, which is quite concerning . This all indicates a rather paternalistic pattern of eol decision making, which is more characteristic of southern european countries . Our study confirmed the previous impressions from icu clinical practice that advance directives were only very rarely used, and most of our participants had not encountered a single one, despite their potential clinical significance in decision making . This is in concordance with some previous studies, which showed that advance directives were available for fewer than 5% of patients in all countries apart from the netherlands . This could be due to the restrictiveness of current slovenian law on advance directives, which has several procedural preconditions for their use to be valid and are binding for physicians only in situations in which treatment is clearly futile . Termination of hydration was reported to be only rarely used in slovene icus, with 76% of participants never terminating it . However, differences were found among icus: hydration was most frequently terminated by participants from medical icus (9.9%), and more rarely by those from surgical icus (3.1%) and never by those from pediatric icus . Similarly, in a german study, termination of hydration was reported by 35% of physicians . The eureld study in 6 european countries showed that hydration is most frequently terminated in the netherlands (11%) and least frequently in italy (2.6%). Finally, merely 60% of the icu physicians reported knowing how to proceed when facing an ethical dilemma, which indicates a need to strengthen medical ethics education and infrastructure, which would be especially important for icus, in which ethical dilemmas appear on a daily basis . This main limitation of this study is that the questionnaire only indirectly measured the real situation in the icus regarding eol decision making . On the other hand, it presents the first national data on this very important issue in slovenia and could serve as a solid basis for preparation of national guidelines, as well as being the basis for developing further observational studies of eol decision making in icus . This was the first nation - wide study on experiences with eol decision making in slovene icu physicians . Firstly, we found the limitation of lst to be ethically acceptable to icu physicians . Most widely practiced was dnr order, followed by the withholding of treatment . In addition, we found differences in reported frequencies of using soft (e.g., withdrawal of inotropes or antibiotics) and hard (e.g., furthermore, termination of hydration was reported to be only rarely used, mostly in the medical icus . In addition, advance directives were almost non - existent in practice, and the patients relatives and nurses only infrequently participated in eol decision making . Finally, better medical ethics education and infrastructure should be developed for icus and hospitals.
|
Recurrent shunt malfunction is a known complication after ventriculoperitoneal (vp) shunt surgeries . In chronic meningitis with hydrocephalus, we present an interesting case of chronic meningitis with shunt malfunction, presenting with expanding central canal syrinx and quadriparesis . This case was investigated with a contrast ventriculogram to demonstrate a communicating central canal syrinx . The present case illustrates that a historical investigation like ventriculogram can still aid in diagnosis and management even in this modern era . Relevant english literature on shunt malfunction presenting with syringomyelia along with pathophysiology, its management and the role of contrast ventriculogram have been discussed . A 2-year - old toddler presented to us with chronic fever and raised icp without any deficits in march 1998 . He was evaluated with computed tomography (ct) of the head and was found to have mild communicating panventriculomegaly . A provisional diagnosis of tubercular meningitis was made and he was started on empirical antitubercular therapy . His symptoms resolved then, but he presented back after 6 months with headache due to raised icp . His repeat ct head revealed an increase in the size of ventricles for which he underwent a vp shunt . He had an episode of shunt malfunction after 3 months, and the vp shunt was revised . Nine months later he underwent laparoscopic repositioning of the abdominal end of the shunt due to loculated ascites (pseudocyst). He had multiple episodes of shunt malfunction in the next year, for which he underwent removal of peritoneal shunt and placement of a ventriculoatrial (va) shunt . Six years later, he presented to our institute again with quadriparesis and truncal ataxia . On examination he was noted to have hypotonic quadriparesis, c5-d4 segmental dissociative sensory loss and absent reflexes . He was investigated with a craniospinal magnetic resonance imaging (mri) [figure 1a], which showed a cervicodorsal expanding syrinx and dilated 4 ventricle . As he had no features of raised icp, shunt malfunction was not thought of, and he underwent a syringopleural shunt . He was lost to follow - up only to present back after 5 years with recurrence of quadriparesis . Craniospinal mri [figure 1b] showed reappearance of the syrinx with dilated 4 ventricle . The case was reviewed at this time, and the scans revealed that 4 ventricular size was seen progressively increasing, without a distinct communication between the central canal and 4 ventricle on comparison with previous mri . Hence, a contrast ventriculogram [figure 2] was performed through an external ventricular catheter to delineate the communication pattern . It demonstrated a mild ventriculomegaly with dilated 4 ventricle communicating with the dilated central canal of the spinal cord exceptionally well . Subsequently shunt malfunction was thought of, and he underwent removal of the va shunt and placement of a fresh ventriculopleural shunt (as he had an abdominal pseudocyst). The patient's weakness improved in the postoperative period, and he was doing well at the last follow - up (1-year). Preoperative magnetic resonance imaging of craniovertebral junction (a) done in 2004 showing - dilated fourth ventricle with syringomyelia for which he underwent a syringopleural shunt (b) done in 2009 showing - dilated fourth ventricle with reappearance of syrinx computed tomography contrast ventriculogram showing a fourth ventricular communication with the spinal canal (a) axial image at the level of the fourth ventricle with contrast (b) coronal image showing craniospinal canal communication (c) sagittal image showing contrast in the spinal canal follow - up magnetic resonance imaging with resolved syrinx (a) sagittal image with normal fourth ventricle and resolved syrinx (b) axial cuts showing normal fourth ventricle (c) sagittal spine image with resolved syrinx ventriculoperitoneal shunt malfunction is a commonly faced problem, and a number of cases undergo shunt revision . Shunts in tubercular meningitis with hydrocephalus tend to malfunction more than others, especially if csf proteins are high . In the pediatric age group have reported on uncommon presenting features of shunt malfunction, and it includes seizures, vision loss, parkinsonian rigidity and syringomyelia (2 cases). This includes common congenital anomalies such as chiari malformations, spinal cord injury, spinal arachnoiditis and sometimes tumors . Our patient had tubercular meningitis with arachnoiditis which causes arachnoids scarring at the basal cisterns leading to communicating hydrocephalus . Probable 4 ventricular outlet obstruction at foramina of luschka might have pushed the csf into a patent central spinal canal (5 or terminal ventricle), which had been in communication with the 4 ventricle . Progressive dilation of the 4 ventricle and altered csf flows across the foramen magnum would have caused the spinal syrinx to expand and propagate . If a shunt malfunctions, ventricular dilation is expected with symptoms of raised icp . The following mechanism may be proposed for this phenomenon . In long standing cases as in tubercular meningitis with arachnoiditis, the compliance of the ventricular system might have reduced, and it may not enlarge sufficiently . Furthermore, as spinal cords compliance is better - maintained, the central spinal canal may enlarge preferentially and cause the symptoms accordingly . Milhorat et al . In 2003 have reported a patient of chiari 1 malformation presenting with shunt malfunction causing acute quadriparesis and syringomyelia within 3 days . Beswick et al . In 2005 have described a similar case of postmeningitic hydrocephalus who presented 9 years after initial presentation with syringomyelia . Lee et al . In their article on unique clinical presentation of paediatric shunt malfunction have reported that 2 (of 70 consecutive cases) had syringomyelia . The review suggests that the primary pathology is mostly chiari malformation or long standing meningitis like tubercular / posthemorrhagic meningitis . Syrinx is a late complication of shunt - malfunction with an average duration of around 10 years (the range being 6 months to 19 years). Multiple shunt revisions and posterior fossa decompressions are also common before shunt malfunction is diagnosed . Cases of syringomyelia secondary to shunt malfunction all the cases in this review should have had ventricle communicate with syringomyelia as the syrinx resolved after shunt surgery . Absence of ventricular communication with syrinx makes the posterior fossa decompression and other syrinx surgeries a necessity . The communication could be demonstrated in many, but not in all cases . When the communication is not clearly demonstrable, they have staged hydrocephalus as well as postshunt isolation of compartments . Among their 9 patients, 4 had holocord dilatation and hydrocephalus, and syringomyelia did not improve in 2 of them after a vp shunt . Interestingly, the case reported by muthukumar had anatomical communication (as per mri), but shunt revision did not resolve the syrinx . Hence, their patient had to undergo posterior fossa decompression . This has been explained by the presence of an isolated compartment and hypothetical ventriculosyringeal valve at the proximal central canal . In the english literature, two other cases have been reported in which contrast ventriculogram was used in similar scenarios . Kudo et al . Used it to confirm the communication between the 3 and 4 ventricle in their case . Injected contrast through the shunt to see whether the ventricular or the peritoneal catheter got blocked . In our case too, the contrast was injected through an external ventricular catheter to confirm the communication between the syrinx and 4 ventricle, as mri was unclear . A number of these secondary syrinxes end up with repeat posterior fossa decompression surgery or syringosubarachnoid / pleural / atrial shunts, as in our case . Though csf flow studies may aid in the diagnoses, interpretation may be difficult at times . We believe that the diagnosis of communicating syringomyelia (hydromyelia) can be definitely established by demonstrating communication between the ventricles and the dilated central canal by positive contrast ventriculography . Patients with vp shunt with late onset syringomyelia need to be evaluated for shunt malfunction . There seems to be a 4 ventricular outlet obstruction and its communication with a central canal which can sometimes be demonstrated by a contrast ventriculogram . Recognizing this condition is very important as a simple shunt revision benefits the patient.
|
Macular edema (me) is a complication of branch retinal vein occlusion (brvo) with serious adverse effects on vision.1,2 increased intravascular pressure and reduced blood flow in the macular capillaries lead to dysfunction of the endothelial blood retinal barrier and to increased vascular permeability, resulting in me.3 although grid laser photocoagulation was previously the only established treatment for me secondary to brvo, visual recovery was slow and limited.1 vascular endothelial growth factor (vegf) has been reported to play an important role in the pathogenesis of me secondary to brvo,46 and the introduction of intravitreal anti - vegf treatment has improved the visual prognosis of brvo;710 this treatment has consequently been adopted as the standard treatment . The effect of anti - vegf treatment on the absorption of me is rapid, but most eyes need to be treated repeatedly . In the horizon trial, the mean number of injections of ranibizumab was 2.02.4 in the second year after the initiation of the treatment for me associated with brvo.11 previously, some investigators reported the efficacy of pars plana vitrectomy combined with internal limiting membrane peeling for me associated with brvo.1220 it was reported that the reduction in me was not rapid after the surgery but the effect was maintained for years . For eyes with me refractory to repeated intravitreal injections of anti - vegf agents, recently, yunoki et al reported the efficacy of pars plana vitrectomy with internal limiting membrane peeling for recurrent me associated with brvo after intravitreal injections of bevacizumab.21 so far, however, limited information is available on this surgical intervention for recurrent or persistent me after anti - vegf treatment . In the study described herein, we retrospectively investigated the anatomic and functional outcomes of eyes treated with pars plana vitrectomy combined with internal limiting membrane peeling for recurrent me due to brvo, in spite of anti - vegf treatment . For this retrospective study, we reviewed the medical records of 24 eyes of 24 consecutive patients who underwent pars plana vitrectomy combined with internal limiting membrane peeling for recurrent me due to brvo after anti - vegf treatment at kagawa university hospital from october 2009 through december 2012 . Patients were offered pars plana vitrectomy with internal limiting membrane peeling if they had visual loss caused by recurred me after intravitreal injections of anti - vegf agents . Exclusion criteria were proliferative diabetic retinopathy, vitreous hemorrhage, central retinal vein occlusion, dense cataract, or a short follow - up period of <6 months after the surgery . Eyes with previous focal scatter photocoagulation were included, but eyes with any previous treatments for me were excluded from the current study (eg, intravitreal injections of any anti - vegf agent or triamcinolone acetonide, or grid laser photocoagulation). This retrospective study was approved by the institutional review board of the faculty of medicine, kagawa university . The study adhered to the tenets of the declaration of helsinki . We did not obtain written informed consent from each participant, because according to the guidelines of the institutional review board of the faculty of medicine, kagawa university, it is not necessarily mandatory to obtain informed consent from the patients for a retrospective study in which the researchers reviewed only the patients medical records . The diagnoses of brvo and me were made by fundus examination and confirmed by fluorescein angiography and optical coherence tomography (oct). Each patient supplied a medical history and then underwent a complete ophthalmologic examination, including best - corrected visual acuity (va) measurement with a landolt chart, slit - lamp biomicroscopy, indirect fundus ophthalmoscopy, and oct examination . In each patient, digital fundus photographs and fluorescein angiography were obtained using a digital fundus camera (trc-50lx; topcon, tokyo, japan) after pupil dilatation . Eyes with brvo were classified as ischemic when the area of nonperfusion was> 5 disk diameters in size.22 macular perfusion status was also determined as complete or incomplete, according to the previous report of finkelstein.23 repeated fluorescein angiography was performed if necessary . To evaluate the condition of me, oct examination was performed (cirrus; carl zeiss meditec ag, jena, germany) at each visit . Central retinal thickness (crt) was determined as the average retinal thickness in a 1 mm diameter circular region at the fovea . In the current study, patients who suffered visual disturbances due to me associated with brvo were offered intravitreal injection of bevacizumab (avastin; genentech, inc ., south san francisco, ca, usa). The inclusion criterion was eyes with a crt> 300 m . In this study, pseudophakic eyes were included, but eyes that had undergone a prior vitrectomy were excluded . Off - label use of bevacizumab was approved by the institutional ethics committee; the study protocol adhered to the tenets of the declaration of helsinki, and written informed consent was obtained from each patient . Intravitreal injection of ranibizumab (lucentis; novartis international ag, basel, switzerland) was also used for the recurred me after its approval in japan . For the treatment for recurrent me, all eyes in the current study underwent a standard 25-g three - port pars plana vitrectomy . After core vitrectomy, posterior vitreous detachment was induced if the cortical vitreous was adherent to the retina . The internal limiting membrane was peeled ~3.0 disk diameters around the fovea with the use of brilliant blue g. during the surgery, no laser photocoagulation was performed on the nonperfusion area of brvo . Statistical analysis was performed using ibm spss statistics version 21.0 (ibm corporation, armonk, ny, usa)., va measured with a landolt chart was converted to the logarithm of the minimum angle of resolution (logmar). Repeated measurement of analysis of variance was used to analyze crt and va after the initiation of the treatment . Student s t - test was used for comparisons of the change in va during the treatment between eyes classified by the initial retinal features . In the current study, 24 eyes of 24 patients with brvo (ten women and 14 men) were included (table 1). At the initial visit, no eyes had previously been treated with intravitreal injections of any anti - vegf agent or triamcinolone acetonide, or grid laser photocoagulation . After the comprehensive ophthalmic examinations, each eye was treated with an intravitreal injection of bevacizumab . Immediately after treatment, a reduction in me was achieved . Va was also significantly improved at 1 month after the initial injection (p=0.0050). However, all eyes showed recurrence of me, and 14 eyes were received additional injection of anti - vegf agents (bevacizumab or ranibizumab). Mean number of injections of anti - vegf agent was 2.331.46 (table 2). One eye was also treated with a subtenon injection of triamcinolone acetonide for recurrent me . The duration of the initiation of anti - vegf treatment to undergoing pars plana vitrectomy was 232 months (mean, 10.89.0 months). In spite of the treatment for me, all eyes showed recurrent me with subjective visual disturbance . Crt was significantly reduced at 1 month (p=0.031) after the surgery, and the reduction increased with time (p=0.007 at the final visit). With the reduction in crt mean follow - up after the surgery was 13.810.8 months . At the final visit, however, improvement in va was statistically significant compared with baseline va (p=0.048), although not significant compared with va before the surgery (p=0.078). No serious complications were seen during or after the surgery . In the current study, mean follow - up after the initial treatment was 24.510.8 months . Table 3 shows the comparisons of the change in va and crt during the treatment between eyes classified by the initial retinal features . The presence of cystoid spaces, serous retinal detachment, or subretinal hemorrhage under the fovea had no significant association with the change in va . However, the presence of epiretinal membrane showed a significant association with the visual recovery . Although eyes without epiretinal membrane showed visual improvement (0.100.32) with the treatment, eyes with epiretinal membrane showed greater visual improvement (0.380.12, p=0.012, figure 2). Since the branch vein occlusion study group reported the efficacy of grid laser photocoagulation for chronic me associated with brvo,1 grid laser photocoagulation has been the only established treatment for me associated with brvo . However, the visual recovery is slow and limited because the average number of lines gained in treated eyes is limited to 1.33 . Now, anti - vegf treatment is generally accepted as the first choice for me associated with brvo.79,24 indeed, the effect of anti - vegf treatment is rapid and remarkable . In the bravo study, the mean improvement in va was 16.6 and 18.3 letters with 6 monthly injections of ranibizumab (0.3 mg and 0.5 mg, respectively).10 however, most eyes need to be treated repeatedly . In the horizon trial, the mean number of injections of ranibizumab was 2.02.4 in the second year after the initiation of the treatment for me associated with brvo.11 the retain study showed that long - term outcomes in brvo treated with ranibizumab were excellent but that approximately half of the cases still required occasional injections after 4 years.25 although the anti - vegf treatment for me is convenient and has a rapid effect, repeated injections may be a burden for patients . In the current study, an intravitreal injection of bevacizumab achieved rapid reduction in me . In spite of repeated treatment for me, however, all eyes showed recurrent me with subjective visual disturbance . All our patients treated with pars plana vitrectomy with internal limiting membrane peeling achieved reduction in me . To date, some investigators have reported the efficacy of this surgical intervention for me associated with brvo.1220 however, most reports show efficacy for treatment - naive me, and limited information is available on recurrent me . Recently, yunoki et al showed promising effects of pars plana vitrectomy with internal limiting membrane peeling for recurrent me due to brvo after intravitreal injections of bevacizumab.21 in their report, the improvement in va was achieved as early as 1 month after the surgery while our patients did not as long as 6 months . This surgical intervention may be a treatment option for me refractory to the anti - vegf treatment . Vitrectomy may have beneficial effects on retinal ischemia by allowing oxygenated fluid to circulate in the vitreous cavity.26 in addition, vitreomacular attachment is suggested to be involved in persistent me in eyes with brvo . Takahashi et al reported that the incidence of me was higher in eyes with no or partial posterior vitreous detachment.27 therefore, induction of posterior vitreous detachment may contribute primarily to the absorption of me associated with brvo . Internal limiting membrane peeling may contribute to the complete removal of traction in the macular area . Previous reports showed that fovea cystoid spaces, fovea serous retinal detachment, and subretinal hemorrhage are signs of poor visual prognosis in brvo.2831 in the current study, these features had no significant association with the change in va . Finkelstein reported incomplete macular perfusion as a sign of good va prognosis in ischemic me.23 in the current study, perfusion status in either the extramacular or the macular area showed no significant association with va improvement . However, the presence of epiretinal membrane showed a significant association with the visual recovery . Physicians sometimes see eyes with me due to brvo together with a fine epiretinal membrane . In case the epiretinal membrane is the primary cause of visual disturbance, surgical intervention is indicated . Because the treatment effect of anti - vegf agents is limited in vitrectomized eyes because of rapid clearance,32 physicians tend to choose anti - vegf agents as the initial treatment for me even if it is accompanied by a fine epiretinal membrane . Previously, marticorena et al reported that intravitreal bevacizumab may be associated with early development of epiretinal membrane in eyes with retinal vein occlusion.33 when such eyes show persistent me, surgical intervention may help . This study has several limitations, mainly the small sample size (especially eyes with epiretinal membrane) and retrospective study design . In the current study, a recent report by yunoki et al showed no favorable va change in eyes with epiretinal membrane or vitreomacular traction after surgery . Small sample size in the current study may account for the discrepancy.21 in addition, the noncomparative design of this study prevented determination of whether surgical intervention improved the visual prognosis . In the current study, however, as shown in figure 1, while the reduction in crt was already significant at 1 month, postoperative improvement in va was slow . In addition, mean va change at 1 month in eyes with combined cataract surgery (0.060.33) was not different, compared with eyes without (0.000.12, p=0.525; data not shown). In the retrospective study reported herein, pars plana vitrectomy combined with internal limiting membrane peeling showed efficacy for recurrent me associated with brvo after anti - vegf treatment . There is no doubt that anti - vegf treatment is the first choice for me associated with brvo . For recurrent me, this is a small case series; therefore, a prospective study with larger sample populations is necessary to evaluate the efficacy of surgical interventions in such eyes.
|
Overweight and obesity are chronic health illnesses affecting many children and adults in the united states [1, 2]. The health consequences of overweight and obesity are enormous, particularly the risk of developing chronic diseases such as hypertension, type 2 diabetes mellitus (t2 dm), and cardiovascular disease (cvd). Obesity disproportionately affects ethnic minorities, women and individuals from lower socioeconomic groups [3, 4]. In particular, african americans (aas) are disproportionately affected by obesity, diabetes, hypertension, and cardiovascular disease, and it is likely that a host of factors interact in complex, and yet unexplained, ways to contribute to these health disparities . The prevalence of overweight or obesity in african women (66%) is 1.4 times that in caucasian (ca) women (47%), and african american (aa) women may be at greatest risk for the health consequences of obesity and have an almost twofold greater risk of developing diabetes and experiencing hypertension at earlier ages; they also have significantly greater abdominal fat than ca women . The objective of the present paper is to highlight selected ethnic differences associated with obesity by focusing on factors that contribute to obesity: metabolic syndrome (ms) indicators, regulation of the hypothalamic pituitary adrenal (hpa) axis, glucocorticoid sensitivity (gs), insulin resistance (ir), and physical activity among aas and caucasians (cas). Metabolic syndrome (ms) is a constellation of factors used globally for identifying individuals at greatest risk for developing cvd and t2 dm . This cluster of interrelated risk factors for cvd and t2 dm [8, 9] include glucose intolerance (t2 dm, impaired glucose tolerance, impaired fasting glycaemia, or insulin resistance / ir), elevated blood pressure, high triglyceride (tg) and low high - density lipoprotein cholesterol (hdl - c) levels, and excessive waist circumference (central adiposity) [8, 10]. For use as a global tool various organizations formulated simple criteria for ms diagnosis as described by grundy et al . . The national cholesterol education program - adult treatment panel iii (ncep - atp iii) definition of the ms is the one most often used in the united states (usa). Table 1 shows how to confirm ms by two separate definitions: the ncep - atp - iii and the international diabetes federation (idf) on the basis of a nondiabetic population . However, despite the widespread use, the effectiveness of ms criteria in early detection or prediction of disease risk across ethnic groups is very much debated [1417]. Ms is on the rise in usa and more prevalent among mexican americans compared with non - hispanic whites and blacks and among non - hispanic white men than non - hispanic black men . The available literature strongly indicates that the criteria for ms should be modified according to racial / ethnic differences [14, 20]. For example, despite a higher prevalence and mortality from cvd, hypertension, and other related chronic diseases, aa women typically have comparable, if not lower, rates of ms than cas but this is only because of the current screening criteria . The blood pressure criterion is suitable for aa and ca as aa have high rates of hypertension and is usually higher than cas [2123]. Although this criterion may be biased for aa, no changes in cutoffs for the criteria are needed . Furthermore, fasting glucose levels appear comparable between aas and cas, indicating that glucose is not a biased criterion . However, one striking and clinically important racial difference across ms criteria relates to dyslipidemia . A large number of studies suggest that aas, both with and without ms, have much lower rates of dyslipidemia than cas; that is, aas usually have significantly lower triglyceride (tg) levels and higher high - density lipoprotein (hdl - c) levels than cas [1417]. Table 2 shows the anthropometric and metabolic information of aa and ca participants in one study, whereas fasting morning blood glucose, homa - ir (homeostasis model assessment: fasting glucose (mmol / l) fasting insulin (mu / l)/22.5), and blood pressure did not vary by ethnicity; hdl - c was significantly higher and tg was significantly lower in aas compared to cas . Thus, two of only five / six criteria for ms are biased such that ms might not be diagnosed in aa . Yu et al . Reported that the activity of lipoprotein lipase (lpl), the enzyme that clears tg - rich lipid particles from the blood, was significantly higher in aas than cas . They postulated that this higher lpl activity might minimize the release of free fatty acids (ffa) from peripheral adipose tissue into the circulation to result in normal tg in the presence of ir . Currently, genome - wide association studies (gwass) are being conducted to elucidate gene - gene and protein - protein interactions and how such interactions might affect levels of tg, hdl, ldl, and other lipid classes . Until then another criterion for ms is waist circumference (wc), which serves as a surrogate marker for abdominal obesity . Abdominal obesity is likely a stronger cvd risk factor than the more commonly used measure of bmi . However, studies using advanced techniques to quantify abdominal fat suggest that waist size is not an appropriate marker of abdominal obesity for aa [29, 30]. Overall body composition appears to differ between aa and ca, in addition to wc . The debate on whether wc cutoffs should be based on the relationship between wc and bmi or wc and visceral adipose tissue (wc - vat) presents another challenge . In particular, aas have comparable or slightly higher wc than cas [33, 34]. However, wc does appear to be a superior predictor of mortality risk, regardless of ethnicity . Several studies [24, 3638] have shown that wc is highly correlated with other components of ms (serum insulin, ir, tg, hdl - c, and systolic and diastolic blood pressure), although differences among racial / ethnic groups were noted . Overall, ethnic-/race - specific cutoffs for wc may be necessary for adequately assessing health risk within different ethnic / race groups . Currently, c reactive protein (crp), a marker of systemic inflammation, is not included in any current definition of ms . However, crp is independently associated with the risk of myocardial infarction and cardiovascular death [39, 40]. Non - hispanic african adolescents with ms have higher levels of high - sensitivity crp than ca adolescents . Ethnic differences in crp have also been noted, with aa having higher median crp levels than ca . Due to its predictive value for cvd and t2 dm, overall, if ms is to function as a valid, early screener for cardiovascular disease, clearly ethnic - specific criteria are needed . The hypothalamic pituitary adrenal (hpa) axis and the locus ceruleus norepinephrine (lc - ne) system represent the primary components of the stress - responsive neuroendocrine systems, and together they manage physiological adaptations to stress to maintain homeostasis [4345]. Obesity, and abdominal obesity in particular, has been associated with several interesting hpa axis disturbances: (1) hypersecretion of cortisol and/or acth in response to various stimuli; (2) heightened glucocorticoid sensitivity; and (3) increased glucocorticoid resistance to negative feedback . These particular pathways influence many systemic processes relevant to health, including metabolic, cardiovascular, and blood pressure regulation, as well as immune and inflammatory function . Studies on the hpa axis and obesity are complicated and controversial . Some studies report comparable basal cortisol levels in obese relative to normal weight individuals, yet others report elevated basal cortisol levels in obese individuals . Additionally, evidence suggests that diurnal rhythms of cortisol are abnormal in obese individuals, with higher afternoon / evening levels and lower than normal upon a wakening, to result in a flatter slope . Interestingly, overweight aas have been reported to have significantly lower awakening cortisol levels than overweight cas despite having similar bmi, and disrupted diurnal cortisol rhythms were found among aa, but not ca men and women, as indicated by both lower a wakeing and higher bedtime cortisol levels . Although the precise reasons for lower a wakeing cortisol levels and flatter diurnal slopes are unclear, chronic persistent stressors and environmental disadvantages have been proposed [51, 52]. Many studies have linked both acute and chronic stresses to hpa axis dysregulation: repeated episodes of stress can induce acute phase responses (apr) and chronic inflammatory processes, as indicated by elevations in crp: the end result is cvd, t2 dm, ms, and/or obesity [45, 47, 5359]. Initially the high levels or excess cortisol are associated with increased adiposity, and particularly in the visceral fat, but eventually, chronic stress may result in low cortisol levels due to adrenal exhaustion and/or heightened sensitivity to glucocorticoids (gc). Obesity is characterized by a spectrum of abnormal insulin secretion, insulin resistance (ir), and t2 dm . Gc, in particular cortisol, serves as a key physiologic modulator in maintaining energy balance and mobilization of energy substrate . However, the magnitude of gc effects on ir and other metabolic actions is likely determined by the density and affinity of glucocorticoid receptors (gr) in various regions of the brain and peripheral tissues . Gr may be under- or overexpressed, have altered binding affinities, interact with other ligands, and/or respond to other such factors, all of which may lead to a state of gc resistance or heightened gc sensitivity . Importantly, islam et al . Showed that obese ca and aa men and women have higher gr densities in leukocytes when compared to normal and overweight men and women and that gr density was strongly correlated with waist circumference . Increased gc action in liver and adipose tissue would likely enhance ir, impair glucose tolerance, and promote stress - induced obesity . Moreover, administration of gc such as dexamethasone, and therapeutic treatment with gc, frequently impair glucose tolerance and promote ir, although the mechanism is not fully understood [6164]. Also, persons who become glucose intolerant after treatment with gc are more likely to develop t2 dm in the future [65, 66]. In addition to gc actions and gr sensitivity, abnormal regulation of the hpa axis alone contributes to ir [67, 68]. Several studies have demonstrated that insulin and glucose concentrations are higher in obese individuals compared to normal weight controls . . Demonstrated that fasting insulin levels were significantly higher in aa compared to ca in both normal and overweight bmi categories . Also, aa women are more likely to be diagnosed with ir and t2 dm compared to ca women [71, 72]. Hypersensitivity to gc may help explain the higher prevalence of ir in aa . Following treatment with dexamethasone, aa maintained greater fasting ir, as determined by homa, and higher fasting insulin levels than ca . Additionally, aa displayed a significantly higher postmeal ir than ca, as measured by insulin areas under the curve (auc) and higher peak postprandial insulin levels under dexamethasone conditions . Table 3 presents the means for fasting glucose and insulin, calculated, homas and auc for glucose and insulin following the meal . Moreover, serum insulin 50 minutes after a standardized meal was significantly higher in aas than cas . In addition to adults, studies on prepubertal children have shown that despite having similar bmi, bf%, and visceral adiposity, insulin sensitivity is 20% lower and insulin secretion is higher in africans versus cas . Overall, this hyperinsulinemia, induced by stress or steroids, may reflect a prediabetic state, and having access to such information would be critical for preventing the continuation of the epidemic of obesity, ir, and t2 dm in aa . Of note, the tg / hdl - c ratio, which has been reported to be closely related to ir in ca individuals, is not diagnostic for ir in aas or africans [15, 38]. Others have shown that although more aas are likely to be ir, a significantly lower percentage of aa met the proposed cutoffs for the tg / hdl - c . Thus, predicting ir in aa from the tg / hdl - c is inappropriate unless different cutoff criteria are established . Additionally, although wc is an outstanding predictor of ir in ca, it is not as good predictor as in aa . Therefore, traditional measures of ir to identify aa at risk lack sensitivity and specificity; however, administration of gcs may be more effective in aa for uncovering predisposition for developing t2 dm and possibly cvd than current measures . Clearly promoting early identification of risk and halting the prevalence of t2 dm among aas are critical, so consensus on the refinement of criteria and tools for assessment is needed . Finally the role of psychosocial, socioeconomic, and environmental factors in the development of ir and obesity remains to be determined . Previous work has demonstrated a relationship between positive appraisal and lower homa - ir and the use of negative appraisal as a coping style with increased insulin auc following a meal . Physical inactivity serves as a major role in the rising prevalence of obesity, although other factors such as excess energy intake also contribute . In fact, lack of physical activity is a leading contributor to the rapid rise in obesity among aa and hispanic populations, particularly among women, and is the fourth leading cause of death worldwide . It is clear that a sedentary lifestyle contributes to cvd, hypertension, t2 dm, obesity, ms, ir, and hyperlipidemia . Although the beneficial health effects of physical activity are common knowledge and widely recognized, most individuals do not achieve the recommended levels, and many report no leisure - time physical activity . Unfortunately, aas are less likely to be physically active than cas, and aa women report less leisure - time activity, fewer hours spent standing and fewer flights of stairs climbed per day than ca women . Resting energy expenditure and resting fat oxidation have also been shown to be depressed with obesity and may be lower in aa than in ca women, which could lead to a greater weight gain among aa than ca . Interestingly, a study conducted by lee and arslanian showed significant difference in fat oxidation rates between african and ca girls, but not between african and ca boys in response to the multistage graded treadmill task . Thus aa women may be at highest risk for reasons above and beyond their level of activity . Aerobic fitness is important in the development of obesity, with a greater aerobic fitness being associated with a lower risk of obesity, ms, cvd, hypertension, and t2 dm [13, 15, 20, 24, 80, 81]. Aas have a lower mean maximal aerobic exercise capacity compared to european americans, and aa men have been reported to have a 7% lower exercise capacity than ca men . Other cross - sectional studies have shown aa to have a lower vo2max than do ca, even after adjusting for body composition . Importantly, aerobic fitness is significantly and negatively related to adiposity with a high initial fitness resulting in less adipose tissue gains over a period of time . Also, a strong inverse association between aerobic fitness and body fat has been reported among aas . Because of the important relation between fitness and cvd, zeno et al . Examined vo2max, the primary index of cardiovascular fitness, in association with cardiovascular risk factors in healthy aa and ca: they found that 57% of aas fell within the fair / low fitness group as compared to only 40% of cas (figure 1). Moreover, those with fair / low fitness were most likely to have multiple risk factors for cvd . Finally, gaillard et al . Showed that moderate aerobic fitness is associated with reduced atherogenic lipid and lipoproteins profile in overweight or obese aa women, which could potentially lead to a lower risk of cvd . Women in their very low and low aerobic fitness groups had higher glucose and insulin values, greater body weight, bmi, and% bf, and lower lean body mass when compared to the moderate fitness group . Thus, it is clear that aerobic capacity or fitness is protective, and aa men and women could benefit substantially from regular exercise . Of note, many studies have also shown a strong inverse association between crp and aerobic fitness [20, 80, 85, 86], which completes the circle: aerobic fitness, hpa axis function, and inflammation . Those who are regular exercisers and have moderate to high aerobic fitness will be less obese, have minimal systemic inflammatory processes, and have a properly functioning hpa axis . Overall, educating sedentary patients to improve fitness levels by promoting regular, moderate, aerobic, or high - intensity activities will be critical for improving the health of all men and women, but in particular aa women . Although many studies are positive, the diabetes prevention program research group demonstrated that modest aerobic exercise (brisk walking 30 min, 5 times a week) reduced the risk for developing t2 dm by 58% . Health care providers should inform and counsel patients to engage in regular physical activity with a goal of enhancing aerobic fitness and lowering obesity . Aerobic fitness and physical activity should always be assessed, as inactivity could be the primary criteria for early identification of those at risk for t2 dm and cvd in nondiabetic, overweight, and obese populations both in aa and ca populations . Approximately 30% of ca adults are obese compared to 45% of aa adults . Despite this trend, the most widely used treatments for overweight and obesity in u.s.a are largely ineffective . The current paper focused on ethnic variations associated with ms indicators, regulation of the hpa axis, ir, gr, and physical activity all of which are associated with obesity and chronic diseases . Ethnic - specific criteria should be established if ms is to be used as an early indicator, but factors such as aerobic fitness and crp may be far more meaningful than conventional indicators when early intervention is the goal . The literature is clear in that a dysregulated hpa axis may lead to obesity and is a risk factor for cvd . Health care providers must asses levels of physical activity and aerobic fitness; they should encourage sedentary patients to increase physical activity regardless of their body weight and diabetic status.
|
Its prevalence varies among ethnic groups, but it affects approximately 13% of the population in industrialised countries . Its clinical course can vary greatly in terms of morphology, distribution, and severity of the disease . In its commonest subtype it is characterised by the formation of discrete, pink, scaly plaques occurring at various sites on the body . Although it can present at any age, it occurs most frequently between the ages of 15 and 20 and again between 50 and 60 years of age . Psoriasis has very significant psychosocial morbidity which appears independent of objective disease severity [3, 4]. It is also associated with an increased risk of cardiovascular disease and mortality [58]. Indeed patients with psoriasis have almost twice the risk of cardiovascular disease when compared with normal controls [9, 10]. A number of studies have shown significantly increased rates of hypertension, dyslipidemia, diabetes mellitus, smoking, and excessive alcohol consumption in patients with psoriasis . Found that patients with severe psoriasis had an increased risk of diabetes (odds ratio (or), 1.62; 95% confidence interval (ci), 1.32.01), obesity (or, 1.79; 95% ci, 1.552.05), and smoking (or, 1.31; 95% ci, 1.171.47) compared with controls . Prodanovich et al . Also found a higher prevalence of these risk factors in patients with psoriasis . However, even after controlling for these variables, they found a higher prevalence of ischemic heart disease (or 1.78; 95% ci, 1.512.11), cerebrovascular disease (or, 1.70; 95% ci, 1.332.17), and peripheral vascular disease (or, 1.98; 95% ci, 1.322.82) when compared with controls . Indeed they also found psoriasis to be an independent risk factor for mortality (or, 1.86; 95% ci, 1.562.21) as a result of the association with atherosclerosis . These risk factors in combination with the chronic inflammatory process are thought to be significant components in the development of vascular disease [9, 14]. Links with alterations in levels of folate and homocysteine in patients with psoriasis have also been implicated in contributing to the propagation of atherosclerosis and atherothrombotic events [9, 15, 16]. Recent case control studies have demonstrated that patients with psoriasis have lower levels of folate in comparison to normal controls [9, 15, 17, 18]. Postulated mechanisms include alterations in gut absorption of folate due to microscopic inflammatory changes seen in the bowel mucosa of patients with active psoriasis and psoriatic arthritis . A more likely explanation however probably relates to the accelerated keratinocyte turnover seen in patients with psoriasis . This action results in excessive consumption of folate used to methylate dna in these actively dividing cells thus lowering folate levels . Conversely homocysteine levels are elevated in psoriasis patients [9, 15, 17]. In one case - controlled study this was found to directly correlate with disease severity and to be inversely related to plasma folate levels . Plasma homocysteine is an independent risk factor for cardiovascular disease [19, 20], peripheral vascular disease, cerebrovascular disease and possibly alzheimer's diseases . The magnitude of this risk is equivalent to the risk of smoking or dyslipidemia . Umol / l) is thought to favour atherosclerosis and vascular thrombosis by a number of mechanisms . These include damaging endothelial cells, promoting clot formation, decreasing flexibility of blood vessels leading to aortic stiffness, and reducing blood flow velocity . The endothelial dysfunction is thought to result from the accumulation of asymmetrical dimethylarginine (adma) which is a natural inhibitor of nitric oxide synthase . As a result there is a reduction in the production of the vasodilator nitric oxide which protects the vessel wall against the pathogenesis of atherosclerosis and thrombosis (figure 1). It has been suggested that hyperhomocysteinemia in addition to other factors may be caused by reduced levels of folate in these patients [9, 15]. Coenzymes methylene tetra - hydrofolate, methylcobalamin, and pyridoxal phosphate are essential for three of the enzymes involved in the metabolism of homocysteine and are dependent on folate, vitamin b12 and b6, respectively . Hence in patients with severe psoriasis who have large areas of rapid skin turnover and increased keratinocyte activity this in turn results in reduced breakdown and elevated serum levels of homocysteine with all of its adverse effects . It appears from this paper that patients with psoriasis have lower levels of folate and higher levels of homocysteine than normal controls . As psoriasis is associated with an increased risk of cardiovascular morbidity and mortality and homocysteine is an independent risk factor for cardiovascular disease, it may seem intuitive that managing this risk factor would have beneficial effects in terms of cardiovascular mortality and morbidity . Folate has long been used in combination with methotrexate in the management of psoriasis, psoriatic arthritis, and rheumatoid arthritis . Here it is effective in reducing gastrointestinal side effects and liver function test abnormalities . In a retrospective cohort study looking at over seven thousand patients with psoriasis, methotrexate was found to reduce the incidence of vascular disease and this reduction was further enhanced when folic acid was added (figure 2). However, homocysteine levels can be elevated for other reasons including obesity, hypertension, smoking, and excessive alcohol consumption all of which have significant prevalence in the psoriatic patient population . Armitage et al . In a double - blinded randomised control trial assessed the beneficial effects of lowering homocysteine levels in over 12,000 post - mi patients . They failed to demonstrate any benefit in vascular outcomes from long - term reductions in homocysteine with folate and vitamin b12 supplementation . It is possible that hyperhomocysteinemia in psoriasis is independent of folate deficiency and instead is linked with other risk factors such as hypertension and obesity . The psoriatic march, a concept of how severe psoriasis may drive cardiovascular morbidity, suggests a process of genetic susceptibility triggered by environmental factors and immune responses . It is this chronic inflammatory burden and state of insulin resistance which can result in endothelial dysfunction and ultimately atherosclerosis . It is well established that folic acid supplementation has a role in the treatment of psoriasis in conjunction with methotrexate treatment . However, the evidence to support its use in reducing the risk of cardiovascular disease by directly impacting on plasma homocysteine levels is lacking . Now management of associated risk factors such as obesity, dyslipidemia, and hypertension as well as encouraging smoking cessation are paramount for these patients as is, of course, the management of their skin.
|
Patients with signs and symptoms of temporomandibular disorders (tmds) are commonly treated with occlusal splint therapy . Occlusal appliances are commonly used in the treatment of patients with tmds and their effectiveness in reducing symptoms has been reported to vary between 70% and 90% . On the other hand, investigations have shown that soft appliances are effective for the reduction of muscle pain, temporomandibular joint (tmj) clicking, and headache . Hydrostatic appliance was designed by lerman over 30 years ago . In its original form, it consisted of bilateral water - filled plastic chambers attached to an acrylic palatal appliance, and the patient's posterior teeth would occlude with these chambers . Later, this was modified to become a device that could be retained under the upper lip, whereas the fluid chambers could be positioned between maxillary and mandibular posterior teeth . Most of the occlusal splints currently in use are either the hard or soft splints . Hard splints have an advantage of having an occluding surface that is hard enough that does not lose; it is fit and thereby lasts longer . Soft splints are simple to fabricate and have a soft occlusal surface that can be easily adjusted to adequate contact pattern . Soft splints can aggravate bruxism, may be due to premature posterior contacts related to the fact that these splints cannot be balanced . However, the hydrostatic occlusal splints have a flexible fluid layer that equalizes all bite forces by preventing tooth to tooth contact . It has a unique water system that immediately optimizes biomechanics, supports the jaw in a comfortable position, removes the teeth from dominance, placing bite and body in harmony, straightens the bite to maximize other structures, enables systemic function and balance, allows the body to naturally balance itself, and finds perfect occlusal balance after starting the treatment immediately . There are conflict of reports regarding the efficacy of different kinds of splints; it is difficult for clinicians to make evidence - based decisions regarding splint therapy because few randomized controlled clinical trials have compared different occlusal splint designs, including a placebo splint . In this context, this study was carried out to study the efficacy of hard, liquid, and soft splints in the management of myofascial pain dysfunction syndrome . This study was conducted in the department of prosthodontics and department of oral medicine and radiology . Study sample consisted of 45 patients diagnosed with myofascial pain from the department of oral medicine . Sample selection was based on a standardized and complete clinical examination based on the research diagnostic criteria (rdc - tmds). Age: 1865-year - oldshould have at least six natural teeth in each quadrant . Previous experience with occlusal splint therapyany obvious dental decay or periodontal disease to which fascial pain could be attributedhistory of trauma in the pain area in <30 daysany systemic condition associated with widespread pain (e.g., fibromyalgia)medical history of current drug addictionany other disorders such as tmj osteoarthritis or capsulitispatient with psychiatric disordersubject not willing to accept treatment . Previous experience with occlusal splint therapy any obvious dental decay or periodontal disease to which fascial pain could be attributed history of trauma in the pain area in <30 days any systemic condition associated with widespread pain (e.g., fibromyalgia) medical history of current drug addiction any other disorders such as tmj osteoarthritis or capsulitis patient with psychiatric disorder subject not willing to accept treatment . Patients were randomly assigned using randomization table and categorized into three groups, with 15 patients in each group: group 1: hard splintgroup 2: soft splintgroup 3: liquid splint . The splints were fabricated with 3 mm thickness of acrylic between the maxillary and mandibular posterior teeth . The splints were adjusted to create uniform occlusal contact of the centric cusps against the splint on all occluding posterior teeth, anterior teeth was in contact with the splint and provided a mutually protected occlusion [figure 1]. Hard splint inserted in patient's mouth a soft occlusal splint was fabricated from a 3 mm thick, soft polyvinyl sheet . The fabrication was done in a vacuum former, pressure - molding device (biostar scheu - dental gmbh, iserlohn, germany) with a thermally controlled infrared heater over the mandibular cast and occlusal contacts were neutralized [figure 2]. Soft splint inserted in patient's mouth readily available liquid occlusal splints (aqualizer, bvm meditech pvt . Ltd ., new delhi, india) were given to these subjects [figure 3]. Liquid supported splint inserted in patient's mouth study period was for 3 months with evaluation at 7 days, 1 month, 2 months, and 3 months after splint insertion . Patients were instructed to wear splint for 24 h a day for 7 days and taken out during meals . Subjective pain analysis was done using modified symptom severity index (mod - ssi). This scale has 28, characters for each of the three variables: intensity, frequency, and pain duration . An average of the three variables was obtained, and final scores ranged from 0.035 to 1objective pain report analysis of muscular palpation (masseter, temporalis, and pterygoid muscles) was performed bilaterally with tight and constant pressure of approximately 1.500 g and were classified on a scale from 0 to 3 (0 - no pain; 1 - verbally reported pain; 2- pain or discomfort followed by fascial musculature contraction, and 3- when the patient backed away or showed lacrimation). Subjective pain analysis was done using modified symptom severity index (mod - ssi). This scale has 28, characters for each of the three variables: intensity, frequency, and pain duration . An average of the three variables was obtained, and final scores ranged from 0.035 to 1 objective pain report analysis of muscular palpation (masseter, temporalis, and pterygoid muscles) was performed bilaterally with tight and constant pressure of approximately 1.500 g and were classified on a scale from 0 to 3 (0 - no pain; 1 - verbally reported pain; 2- pain or discomfort followed by fascial musculature contraction, and 3- when the patient backed away or showed lacrimation). The obtained data were subjected to statistical analysis using ibm spss software (version 20.0, chicago, il, usa). Tukey test was used to compare the values of the mod - ssi between three groups at all times . Wallis h - test was used to analyze the scores of digital palpation, both between groups and each groups at all times . Age: 1865-year - oldshould have at least six natural teeth in each quadrant . Previous experience with occlusal splint therapyany obvious dental decay or periodontal disease to which fascial pain could be attributedhistory of trauma in the pain area in <30 daysany systemic condition associated with widespread pain (e.g., fibromyalgia)medical history of current drug addictionany other disorders such as tmj osteoarthritis or capsulitispatient with psychiatric disordersubject not willing to accept treatment . Previous experience with occlusal splint therapy any obvious dental decay or periodontal disease to which fascial pain could be attributed history of trauma in the pain area in <30 days any systemic condition associated with widespread pain (e.g., fibromyalgia) medical history of current drug addiction any other disorders such as tmj osteoarthritis or capsulitis patient with psychiatric disorder subject not willing to accept treatment . Patients were randomly assigned using randomization table and categorized into three groups, with 15 patients in each group: group 1: hard splintgroup 2: soft splintgroup 3: liquid splint . The splints were fabricated with 3 mm thickness of acrylic between the maxillary and mandibular posterior teeth . These were stabilization type of splints . The splints were adjusted to create uniform occlusal contact of the centric cusps against the splint on all occluding posterior teeth, anterior teeth was in contact with the splint and provided a mutually protected occlusion [figure 1]. A soft occlusal splint was fabricated from a 3 mm thick, soft polyvinyl sheet . The fabrication was done in a vacuum former, pressure - molding device (biostar scheu - dental gmbh, iserlohn, germany) with a thermally controlled infrared heater over the mandibular cast and occlusal contacts were neutralized [figure 2]., new delhi, india) were given to these subjects [figure 3]. Liquid supported splint inserted in patient's mouth study period was for 3 months with evaluation at 7 days, 1 month, 2 months, and 3 months after splint insertion . Patients were instructed to wear splint for 24 h a day for 7 days and taken out during meals . Subjective pain analysis was done using modified symptom severity index (mod - ssi). This scale has 28, characters for each of the three variables: intensity, frequency, and pain duration . An average of the three variables was obtained, and final scores ranged from 0.035 to 1objective pain report analysis of muscular palpation (masseter, temporalis, and pterygoid muscles) was performed bilaterally with tight and constant pressure of approximately 1.500 g and were classified on a scale from 0 to 3 (0 - no pain; 1 - verbally reported pain; 2- pain or discomfort followed by fascial musculature contraction, and 3- when the patient backed away or showed lacrimation). Subjective pain analysis was done using modified symptom severity index (mod - ssi). This scale has 28, characters for each of the three variables: intensity, frequency, and pain duration . An average of the three variables was obtained, and final scores ranged from 0.035 to 1 objective pain report analysis of muscular palpation (masseter, temporalis, and pterygoid muscles) was performed bilaterally with tight and constant pressure of approximately 1.500 g and were classified on a scale from 0 to 3 (0 - no pain; 1 - verbally reported pain; 2- pain or discomfort followed by fascial musculature contraction, and 3- when the patient backed away or showed lacrimation). The obtained data were subjected to statistical analysis using ibm spss software (version 20.0, chicago, il, usa). Tukey test was used to compare the values of the mod - ssi between three groups at all times . Wallis h - test was used to analyze the scores of digital palpation, both between groups and each groups at all times . The sample included 45 subjects (15 in each group) the mod - ssi score showed statistically significant reduction for all three groups reflecting patients improvement in muscle pain with hard, soft, and liquid supported splints . The hard splints proved to be very effective in a shorter period of time . From baseline to 7-day interval the curve for the hard splints showed a steep change . Whereas the soft and liquid splints showed much more gradual change from baseline to the 7-day interval and was rhythmic thereafter . However, from baseline to 90-day interval, all the three groups showed a considerable and comparable decrease in mod - ssi scores . The results for objective palpation also showed statistically significant difference between baseline and 90 days for all three groups, i.e., hard, soft, and liquid splints . However, hard splints were more effective in shorter duration of time followed by liquid splints and lastly soft splints [tables 1 and 2, figures 4 and 5]. Means and sd for mod - ssi and digital palpation for the three groups p values for mod - ssi and digital palpation scores between hard, liquid and soft splint groups at different intervals mean of modified symptom severity index scores at each intervals for all three groups mean of digital palpation scores of all muscles at each intervals for all three groups treatments for tmds are wide ranging and are directed primarily toward relief from persistent orofacial pain . Due to difficulty in determining the etiology and the possibility that the symptoms are secondary to some other disorders of the tmj or muscles of mastication initial treatment given should be reversible . When a splint is inserted, there is an adaptation of the jaws to a new resting postural position . Occlusal splints that increase the occlusal vertical dimension beyond the freeway space cause an immediate adaptation to the new freeway space at an increased vertical dimension . Thus, an occlusal splint allows a muscle to function more efficiently during contact and be less active during postural functions . Hence, as the vertical dimension increases from the occlusal contact on the insertion of the occlusal splint, muscular effort decreases resulting in the relaxing of the muscles and hence, tmj . Acrylic resin interocclusal appliances have been used in dentistry for the management and treatment of tmds . (1989) had done a study on hard splints and found that it is ineffective in reducing muscle pain, which is in contrast with our study . In the present study, hard splints were more effective compared to soft and liquid splints, showing significant difference throughout the study period . In 1988, a study done by harkins et al . Concluded that soft splints had a reduction in facial myalgia . In our study, soft splints were effective, but when compared to liquid and hard splints it was less effective . For soft splints, the change was much more gradual from baseline to 7-day interval and was rhythmic thereafter . A study done by nevarro et al . (1985) had concluded that soft splints are ineffective, and in another study done by okeson (1987) on nocturnal electromyogram comparison of hard and soft reported significantly less effect with soft splints, but our study found that soft splints are effective in reducing the symptoms of myofascial pain although the time taken by them was slightly longer as compared to the hard and liquid splints . 2006) did a randomized trial in which they found that all the patients improved irrespective of splint design, which is in accordance with our study, where there was both subjective and objective reduction in pain . Davies and gray (1997) did an investigation on the pattern of splint usage found no advantage of any particular pattern of splint use . Whereas in our study, we had an advantage of liquid and hard splints when compared to soft splints . A study done by pettengill et al . However, in the current study, hard splint was more effective in comparison with the soft and liquid splints, though soft splints also showed a significant reduction in pain . Soft splints have been used as an interim appliance until acrylic - resin splints could be provided . These appliances have also been suggested as prognostic tool to evaluate whether an acrylic - resin splint would be advantageous . It has been postulated that the soft occlusal surface of soft splint may contribute to occlusal changes . Liquid supported splints have been advocated for patients with tmds . However, there are few trials that have evaluated efficacy and outcomes have been variable . Aqualizer works by allowing the muscles to automatically reposition the jaw . For relieving tmj pain, restoring aqualizer is a new application of a basic physical law of nature called pascal's law, which states that an enclosed fluid will apply equalized fluid pressure regardless of where the pressure is applied to the fluid . In other words, when a patient bites on the aqualizer, the fluid within it distributes bite forces evenly across the bite, reducing tmj pressure and pain, and hence ensuring relief . Macedo and mello (2002) evaluated the efficacy of the hydrostatic splint aqualizer, microcurrent electrical nerve stimulation (mens) and transcutaneous electrical neural stimulation (tens) therapies in patients with tmd in acute situations and concluded that the mens and the hydrostatic splint were more effective than tens, which is consistent with our study, where liquid supported splint was more effective compare to the soft splints . Research on tmd recommended the evaluation of pain in the masticatory muscle through subjective pain and digital palpation . The mod - ssi is more complete than visual analog scale because it takes into consideration, pain frequency and duration along with its intensity . Sample selection was based on a standardized and complete clinical examination based on the rdcs - tmd . Although the present study supports the use of hard, soft, and liquid splints in the management of myofascial pain dysfunction syndrome, further research is necessary to investigate the most appropriate usage regime of different types of splints, the different design of splints and also the emg activity following the splint usage . This study advocates the use of occlusal splint therapy for the management of myofascial pain . It is simple, with fewer side effects, cost effective, noninvasive, and better patient compliance . The results showed that all three types, i.e. Hard, soft, and liquid occlusal splints reduced the mod - ssi scores and digital palpation scores thereby proving that the type of splint did not have an effect on the overall results among the three groups . The findings from this study suggest the clinicians to consider occlusal splints as a therapeutic protocol when managing patients with myofascial pain dysfunction.
|
Synchronous primary lung cancers are uncommon and the occurrence of synchronous non small cell lung carcinomas (nsclc) with different histological morphologies within the same lobe is rare . The aim of this report is to discuss surgical and oncological management of this entity together with a review of the current literature . A 61-year - old female smoker (42 pack years) was found to have left upper zone shadowing on chest x ray following a history of weight loss . Her performance status was 1 with a forced expiratory volume in one second (fev1) of 1.94 litres . Positron emission tomography in conjunction with computed tomography (pet - ct) showed two nodules in the left upper lobe . There was a 2 cm spiculated lesion in the anterior segment with a standardised uptake value (suv) max of 5.6 units (fig 1a) and a 2.2 cm cavitating lesion in the apico - posterior segment with a suv max of 2.7 units (fig 1b). Coronal view pet ct scan of a high uptake nodule in the left upper lobe coronal view of pet ct scan of moderate uptake synchronous nodule in the left upper lobe a left thoracotomy with upper lobectomy was performed and the intra - operative findings were consistent with the radiological appearances seen in the pet - ct scan . There was a 2 cm infiltrating squamous cell carcinoma in the anterior segment with vascular invasion (pathological (p) t2a according to tnm 7th edition) and a separate 2.4 cm adenocarcinoma with associated broncho - alveolar spread in the apical segment (pt1b). An adjacent peribronchial lymph node was positive for tumour (pn1), however due to the poorly differentiated morphology of the tumour cells it was unclear as to which tumour it originated . Cisplatin and vinorelbine adjuvant chemotherapy was administered with no overt complications; however recurrence of the disease was discovered radiologically one year post - operatively in the form of a left hilar soft tissue mass with bony metastases . The patient subsequently received radiotherapy without complications but a staging ct chest / abdomen / pelvis performed three months later showed progressive disease with left lung collapse in addition to multiple, palpable subcutaneous nodules . These features were in keeping with the patient s symptoms of worsening breathlessness and wheeze . Further investigations including bronchoscopy showed a tumour in left main bronchus which was treated with laser ablation and placement of a tracheo - bronchial stent . Histological assessment of this nodule showed a poorly differentiated adenocarcinoma, which was confirmed with immunohistochemical techniques . Specialist genetic analysis of the subcutaneous lesion was performed, which showed the presence of an egfr mutation sensitive to anti - egfr tyrosine kinase inhibitors . This enabled the oncologists to use monoclonal antibody therapies (anti egfr tki) and the patient was treated for one month with iressa (gefitinib). She responded well with significant decrease in the size of the metastatic subcutaneous nodules . Due to her progressive disease the patient deceased with an overall survival (from the time of the lung resection) of 20 months synchronous primary lung cancers (splc) were first described in 1924 by beyreuther h (1). The true incidence of these remains uncertain but evidence has shown figures between 1% to 8% (2). The criteria for the diagnosis of splc, which was proposed by martini and melamed in 1975, are that synchronous tumours are physically distinct and that the histology is different surgical resection of splc was recommended by many of the reviewed authors (3 - 5). Rostad et al studied the outcome and characteristics of synchronous primary lung cancers . In 15,308 lung cancer resection cases, 94 patients were found to have synchronous non small cell lung cancers, nsclc, 9 patients had synchronous lung cancers with different histological morphologies and only 2 (0.01%) patients had synchronous lung cancers with different histological morphologies within the same lobe . The relative survival rate in patients with different histological morphologies (n=9) was 12.7% . Patients with similar morphologies had a better outcome with a relative survival rate of 29.2%, although the difference was not statistically significant (p=0.24). The authors concluded that surgical resection should be offered to patients with synchronous lung cancers who are operable with respectable tumour . (3) lymph node metastases were found to be a statistically significant prognostic factor . In a study of 92 patients who had surgical resections for multiple synchronous primary lung cancers, the results showed the 5-year survival were 52.5% and 15.5% for patients without and with lymph node metastasis respectively (p = 0.001). (3) skin metastases from lung cancers are not uncommon, and their presence is a poor prognosis factor (6). Recently anti - egfr tki molecules have been introduced for the treatment of advanced malignancies including lung cancers, however long term treatment resistance remains a therapeutic challenge . Our patient had a disease free survival of 12 months post surgery despite a positive lymph node, but she later developed recurrence with distant subcutaneous metastases and an endobronchial mass, which was treated palliatively . She received anti egfr1-tk inhibitors based on genetic analysis from the metastatic adenocarcinoma subcutaneous lesions and her initial response was good . Patients with resectable synchronous primary non small cell lung carcinomas within the same lobe should be offered surgical resection after careful pre - operative staging . Skin metastases should be biopsied and egfr testing should be requested in order to determine the originating the tumour and to assess the patients suitability for anti egfr - tki treatment.
|
This historical cohort study included 12,589 patients at two department of veterans affairs (va) medical centers who had at least one hba1c test <6.5% between 1 january 2000 through 31 december 2001 (baseline hba1c). Patient records were evaluated for 12 months before the baseline hba1c to assure that they had at least one ambulatory care visit, no hba1c 6.5%, and no diagnosis of diabetes . A diagnosis of diabetes was defined as: at least one inpatient diagnosis of diabetes mellitus (icd-9 code of 250 . *), two or more outpatient diagnoses of diabetes mellitus (icd-9 code of 250 . *), or a prescription for any medication used in diabetic treatment . This method to ascertain diabetic status has been previously validated within the va (10). Participants with more than one hba1c test during the baseline period had their first test result used as the baseline . Patients were required to have at least one ambulatory care visit at any time during the follow - up period (from date of baseline hba1c test to 31 december 2008) or until a diabetic diagnosis was made . After the above exclusion criteria were applied, 12,375 patients were classified without diabetes and were entered into the follow - up period . The period of follow - up for a given patient varied based on when they had clinic visits during the follow - up period . Information was collected on outpatient clinic visits, admissions, vital signs, outpatient prescriptions, comorbid diagnoses, patient demographics, and laboratory tests . Because our clinical laboratories do not label glucose measurements as fasting or nonfasting, we recorded glucose levels obtained between 0600 and 1100 h as a proxy for the fasting state . The icd-9 codes used for the comorbid conditions were as follows: cardiovascular disease (cvd) (coronary heart disease 410414; stroke 430438; heart failure 428; cardiac arrest 427.1, 472.4, 427.5; inflammatory heart disease 429.01, 429.1, 420425; and hypertension 401405). The study end point was whether a diagnosis of diabetes mellitus occurred during the follow - up period . During the study, both facilities used the same methodology for measuring hba1c levels, which used a nonporous ion - exchange high - performance liquid chromatography to separate hba1c from other hemoglobin fractions and is certified by the national glycohemoglobin standardization program . This method is fairly immune to the presence of hemoglobinopathies or carbamylated hemoglobin as a result of high urea concentrations . The distribution of baseline hba1c levels was analyzed using univariate procedures and stratified into five groups (1): <4.5% (2), 4.54.9% (3), 5.05.4% (4), 5.55.9% (5), and 6.06.4% . The lowest group, with hba1c <4.5%, was treated as the reference group and all other groups were compared with the reference for risk calculation . Descriptive statistics were conducted on the study sample at baseline: median and range were calculated for the continuous variables; frequency and proportion were calculated for the categorical variables . The study also compared the baseline characteristics for patients who developed diabetes with patients who did not develop diabetes in the follow - up period . The nonparametric wilcoxon rank - sum test was used to compare medians for continuous variables, and the test was used to compare proportions for categorical variables . Given the large sample size, a p value of <0.0001 was used to determine statistical significance . Both logistic regression models and cox proportional hazards models were used to compare the risk of developing diabetes with baseline hba1c level as the main effect (hba1c <4.5% as reference group). Based on the univariate results, a stepwise selection method (using an of 0.05) both unadjusted odds ratio / hazard ratio and multivariable adjusted odds ratio / hazard ratio were calculated . The kaplan meier method was used to calculate survival probability with time, and a diabetic event probability versus time plot was developed and stratified by baseline hba1c groups . All of the 12,375 patients were included in the survival analysis, including those patients who died before developing diabetes . For those patients who died, we used their date of death as the censor time . For patients who survived without developing diabetes, we used the last visit date as their censor time in the survival analysis calculation . We also developed a risk model for predicting the 5-year incidence of diabetes using logistic regression . The 5-year incidence of diabetes was defined as any patient who developed diabetes within the 5-year period after the baseline hba1c test, and patients who did not develop diabetes within the 5-year period were used as the control group . A simple risk model was developed using baseline hba1c as the only predictor, and a multivariable model was developed using baseline hba1c, age, bmi, and systolic blood pressure (sbp) as predictors . The areas under the two receiver operating characteristic (roc) curves (indicated by c statistic) were compared using nonparametric approaches (11). This historical cohort study included 12,589 patients at two department of veterans affairs (va) medical centers who had at least one hba1c test <6.5% between 1 january 2000 through 31 december 2001 (baseline hba1c). Patient records were evaluated for 12 months before the baseline hba1c to assure that they had at least one ambulatory care visit, no hba1c 6.5%, and no diagnosis of diabetes . A diagnosis of diabetes was defined as: at least one inpatient diagnosis of diabetes mellitus (icd-9 code of 250 . *), two or more outpatient diagnoses of diabetes mellitus (icd-9 code of 250 . *), or a prescription for any medication used in diabetic treatment . This method to ascertain diabetic status has been previously validated within the va (10). Participants with more than one hba1c test during the baseline period had their first test result used as the baseline . Patients were required to have at least one ambulatory care visit at any time during the follow - up period (from date of baseline hba1c test to 31 december 2008) or until a diabetic diagnosis was made . After the above exclusion criteria were applied, 12,375 patients were classified without diabetes and were entered into the follow - up period . The period of follow - up for a given patient varied based on when they had clinic visits during the follow - up period . Information was collected on outpatient clinic visits, admissions, vital signs, outpatient prescriptions, comorbid diagnoses, patient demographics, and laboratory tests . Because our clinical laboratories do not label glucose measurements as fasting or nonfasting, we recorded glucose levels obtained between 0600 and 1100 h as a proxy for the fasting state . The icd-9 codes used for the comorbid conditions were as follows: cardiovascular disease (cvd) (coronary heart disease 410414; stroke 430438; heart failure 428; cardiac arrest 427.1, 472.4, 427.5; inflammatory heart disease 429.01, 429.1, 420425; and hypertension 401405). The study end point was whether a diagnosis of diabetes mellitus occurred during the follow - up period . During the study, both facilities used the same methodology for measuring hba1c levels, which used a nonporous ion - exchange high - performance liquid chromatography to separate hba1c from other hemoglobin fractions and is certified by the national glycohemoglobin standardization program . This method is fairly immune to the presence of hemoglobinopathies or carbamylated hemoglobin as a result of high urea concentrations . The distribution of baseline hba1c levels was analyzed using univariate procedures and stratified into five groups (1): <4.5% (2), 4.54.9% (3), 5.05.4% (4), 5.55.9% (5), and 6.06.4% . The lowest group, with hba1c <4.5%, was treated as the reference group and all other groups were compared with the reference for risk calculation . Descriptive statistics were conducted on the study sample at baseline: median and range were calculated for the continuous variables; frequency and proportion were calculated for the categorical variables . The study also compared the baseline characteristics for patients who developed diabetes with patients who did not develop diabetes in the follow - up period . The nonparametric wilcoxon rank - sum test was used to compare medians for continuous variables, and the test was used to compare proportions for categorical variables . Given the large sample size, a p value of <0.0001 was used to determine statistical significance . Both logistic regression models and cox proportional hazards models were used to compare the risk of developing diabetes with baseline hba1c level as the main effect (hba1c <4.5% as reference group). Based on the univariate results, a stepwise selection method (using an of 0.05) was used for further evaluation of the confounders in the multiple logistic regression . Both unadjusted odds ratio / hazard ratio and multivariable adjusted odds ratio / hazard ratio were calculated . The kaplan meier method was used to calculate survival probability with time, and a diabetic event probability versus time plot was developed and stratified by baseline hba1c groups . All of the 12,375 patients were included in the survival analysis, including those patients who died before developing diabetes . For those patients who died, we used their date of death as the censor time . For patients who survived without developing diabetes, we used the last visit date as their censor time in the survival analysis calculation . We also developed a risk model for predicting the 5-year incidence of diabetes using logistic regression . The 5-year incidence of diabetes was defined as any patient who developed diabetes within the 5-year period after the baseline hba1c test, and patients who did not develop diabetes within the 5-year period were used as the control group . A simple risk model was developed using baseline hba1c as the only predictor, and a multivariable model was developed using baseline hba1c, age, bmi, and systolic blood pressure (sbp) as predictors . The areas under the two receiver operating characteristic (roc) curves (indicated by c statistic) were compared using nonparametric approaches (11). There were 12,589 individuals eligible for the study by having an hba1c test during the baseline period; 214 (1.7%) did not visit the clinic during the follow - up period and were lost to follow - up, leaving 12,375 in the study population . Baseline characteristics of the 12,375 individuals, including those who developed diabetes during follow - up, are shown in table 1 . Individuals were predominantly white men (95.4% men and 67.5% whites) with a median age of 65.9 (range 18.5 101.5 . Comparison of the demographics and clinical characteristics between these individuals and the 214 who had no follow - up visits showed no significant differences with regard to hba1c, age, glucose, sbp, creatinine, estimated glomerular filtration rate (egfr), sex, and presence of cardiovascular disease . Those lost to follow - up were significantly lower with regard to diastolic blood pressure, albumin, bmi, and presence of hypertension . Baseline characteristics of study sample data are median (range) or n (%) unless otherwise indicated . P value and odds ratio compare both diabetic and nondiabetic groups . During an average follow - up of 4.4 years and with an average of 140 (sd 194) ambulatory care visits, 26.9% developed diabetes . The criteria by which diabetes was diagnosed were: outpatient codes (59.9%), inpatient codes (5.2%), and new diabetes medication (34.9%). Blood pressure, bmi, glucose, serum creatinine, prevalent cardiovascular disease, and hypertension were significantly higher (p <0.0001) in patients who developed diabetes . During the study period, there was a progressive decline in the number and percentage of individuals with clinic encounters in a given year . In the last year of the study period, 6,997 (56.5%) individuals had one or more clinic visits, 2,671 (21.6%) patients had no clinic visits, and 2,707 (21.9%) had died during the 8 years of follow - up . Logistic regression was used to compare the groups for risk of developing diabetes in the follow - up period (table 2). When compared with the reference hba1c group (<4.5%), the group with hba1c 4.54.9% was not significantly different, whereas risk of developing diabetes increased steadily for the higher hba1c groups (5.0%). The point estimates for unadjusted odds ratios were 1.57 for hba1c 5.05.4%, 4.54 for hba1c 5.55.9%, and 14.93 for hba1c 6.06.4% compared with hba1c <4.5% (p <0.0001). The adjusted odds ratios by multivariable logistic regression showed similar trends with slight differences in point estimates and 95% confidence intervals . We assessed whether the number of outpatient visits affected a diagnosis of diabetes but found that it did not contribute to model fit and was not included in the logistic regression model . Risk comparison by logistic regression for developing diabetes according to baseline hba1c groups * adjusted for age, sex, ethnicity (hispanic or latino, not hispanic or latino, or unknown), race (black, white, other, or unknown), bmi, and systolic blood pressure . Estimates of hazard ratios showed similar patterns as the odds ratio estimates with logistic regression . No significant difference was detected between the reference group and those with hba1c 4.54.9%, but the hazard ratio increased significantly beginning with hba1c 5.0% (p <0.0001), with higher baseline hba1c associated with higher risk . Risk comparison by cox proportional hazards model for developing diabetes according to baseline hba1c groups * adjusted for age, sex, ethnicity, race, bmi, and systolic blood pressure . We analyzed event probability (i.e., developing diabetes) during the follow - up period (fig . 1) differentiated by baseline hba1c . Similar to our other analyses, the curves for incident diabetes in those with baseline hba1c <5.0% intertwined, whereas those with higher baseline hba1c had significantly higher probability of developing diabetes during follow - up (log - rank p value <0.0001). Plot of diabetes event probability against follow - up time, differentiated by baseline hba1c . The curves for the two lowest groups substantially overlap, but groups with hba1c 5.0% have significantly higher probability of developing diabetes during the 8-year study period (log - rank p value <0.0001). Risk models for a diabetic diagnosis over 5 years were developed using hba1c alone (model 1) and a multivariable model using hba1c, age, bmi, and sbp as predictors (model 2). For each predictor, a quadratic term was added into the model because of nonlinear association with risk (indicated by significant p values associated with quadratic terms). For model 1, the area under the roc curve = 0.7543 (95% confidence interval 0.74290.7657), and for model 2, the area under the roc curve = 0.7791 (95% confidence interval: 0.76870.7896). Comparison of the two areas showed significant improvement of the predictability of model 2 (p value <0.0001). The hosmer and lemeshow goodness - of - fit p value was 0.2827 for model 2, indicating good model fit . Based on the above findings, we developed risk calculating equations as follows: where hba1c is hemoglobin hba1c in percentage, age is indicated in years, bmi is measured in kg / m, and sbp is indicated in mmhg . The first equation calculates the odds of developing diabetes mellitus in 5 years, and the second equation calculates the probability of developing diabetes mellitus in 5 years using the result from eq . 1 . We found that baseline hba1c was significantly predictive of the subsequent development of a diagnosis of diabetes over an 8-year period . The risk of developing diabetes increased progressively at hba1c levels 5.0%, with an odds ratio exceeding 16 in those with hba1c 6.06.4% . Not surprisingly, significant predictors for diabetes incidence included clinical parameters, such as blood pressure, bmi, serum creatinine, prevalent cardiovascular disease, and hypertension . From these data, we also developed risk calculating equations for determining the probability of developing a diabetic diagnosis within 5 years . We believe that these data will inform clinicians on how to risk stratify individuals who are screened for diabetes using hba1c but whose levels do not reach the recommended diagnostic threshold of 6.5% . Several studies have evaluated hba1c as a predictor of subsequent diabetes or as a tool to diagnose treatment - requiring diabetes (37,1219). A number of threshold values have been previously proposed for diagnosing diabetes, such as 7.0% (4,7), 6.5% (13),> 2 sd above the normal mean (i.e.,> 6.1%) (6). In addition, a number of hba1c levels have also been proposed to identify individuals at risk for diabetes (i.e., prediabetes), such as 6.16.9%, 6.06.4%, or 5.76.4% . The implementation of new guidelines for diagnosing diabetes using hba1c will help standardize the way in which clinicians apply results from this test . However, there remains uncertainty on how to classify and whether to intervene in individuals whose levels fall below this threshold . There is growing evidence that hba1c may not only predict diabetes but also cardiovascular disease and death (1219). Among women without diabetes, hba1c levels were significantly associated with both, although the presence of other cardiovascular risk factors may contribute additionally to this risk (15,16). (18) showed that, in a community - based population, hba1c was significantly associated with risk of developing both diabetes and cardiovascular disease independent of fasting glucose levels . Therefore, these results strongly suggest that individuals with hba1c levels 6.0% should be targeted for prevention strategies to reduce not only incident diabetes but possibly also cardiovascular disease . Many clinicians have been attracted to using hba1c as a screening test for diabetes since the test reflects longer - term glucose control, does not require fasting, has less day - to - day biologic variability, and is a well - accepted marker of risk of long - term microvascular complications (20). Such usage is evidenced by our large cohort of patients in whom hba1c levels were obtained in patients without a diagnosis of diabetes . However, prior guidelines discouraged use of hba1c for diagnosing diabetes, largely as a result of standardization and reproducibility issues that precluded its use in such broad settings . Current instrumentation and standardization methods (21) aligned with the diabetes control and complications trial have abrogated most of these issues . Such evidence was cited by the international expert committee (8) and affirmed by the american diabetes association (9) in their acceptance of hba1c for screening and diagnosis . Significant strengths of this study are its large population drawn from two va medical centers in different geographic regions, the ability to query a robust electronic medical record for clinical and demographic factors, and patient follow - up for up to 8 years . Important limitations include the largely white men and older population, the reliability of the administrative data set, and the selection and ascertainment bias related to patients for whom hba1c testing was performed . It is possible that hba1c tests were performed in patients who were preselected for the presence of other known risk factors for diabetes . Such selective screening might bias our results toward showing a higher risk of developing diabetes for a given hba1c level . To address this, our analyses controlled for many known risk factors and helped identify those factors that contribute significantly with hba1c in predicting risk of diabetes . To account for the possible confounding effects from sex and age, we incorporated these two variables with several other known risk factors in our 8-year follow - up models to calculate odds ratios (hazard ratios) based on baseline hba1c . In our 5-year risk prediction model, the sex variable did not provide significant contribution to the predictability of the model . We did not have information on the prevalence of smoking in the study population, which is a known risk factor for diabetes (22) and is higher among veterans than nonveterans (23). Another potential bias is that a greater amount of medical care (e.g., increased number of clinic visits) might associate with a higher number of diabetes cases ascertained during the study . We adjusted for the number of medical visits but did not find that this significantly influenced the models . In addition, there were 214 individuals initially eligible to participate but who were lost to follow - up . Demographic and clinical characteristics of these individuals were quite similar to the remaining cohort . Those areas in which they differed were in a direction that paralleled those who did not develop diabetes during follow - up . Although their absence from the cohort can introduce bias in the ascertainment of both exposure and outcome, we believe that both the small number as well as the characteristics of these individuals makes it unlikely that this substantially affected the reported incidence of diabetes . Finally, we identified diagnosed diabetes based on medical record evidence that the patient was actually diagnosed by a clinician and/or treated with a diabetes medication . These criteria would exclude patients who have an unrecorded but true diagnosis of diabetes for whom no medications were prescribed, which could lead to an underestimation of the true risk of developing diabetes . However, the number of such patients should be small because of our use of multiple variables to establish a diagnosis of diabetes and the extended follow - up period . In summary, we have characterized the risk of developing diabetes in patients without a diagnosis of diabetes who had a baseline hba1c levels <6.5% . These data show a progressive risk for developing diabetes when hba1c is 5.0%, with nominal risk below that level . We generated a risk calculator using hba1c and other clinical data that estimate the 5-year risk of developing diabetes . Because clinicians implement hba1c testing to screen for diabetes, these data may be used to help identify the risk of incident diabetes among individuals with hba1c levels <6.5%.
|
The patient was a 5-year - old japanese female who was the second child of healthy parents . The patient had no familial history of movement disorder, but the patient s mother experienced transient hyperthyroidism during pregnancy, the patient s aunt had hyperthyroidism, and the patient s grandmother had hypothyroidism . The patient was born at term by cesarean section and was admitted to a neonatal intensive care unit at 3 days of age for severe respiratory distress syndrome that required mechanical ventilation therapy . Congenital hypothyroidism was diagnosed at 6 days of age (thyroid - stimulating hormone 100.0 u / ml, ft4 1.0 ng / dl), and treatment with levothyroxine was initiated . The size of the thyroid was normal on thyroid echography, and thyroid hormone levels were successfully restored with levothyroxine therapy . Lung computed tomography showed cystic fibrosis and ground - glass opacities characteristic of interstitial lung disease (figure 1), and serum kl-6 was elevated; however, bronchoalveolar lavage was not performed and sp - c (the causative gene for idiopathic interstitial pneumonitis) sequencing did not reveal a relevant alteration . The patient was accordingly treated with mechanical ventilation and hydrocortisone and prednisolone for 1 month and did not receive phototherapy at any point during hospitalization . Bilateral diffuse ground - glass opacities in the lower lobe of the lung observed on computed tomography at 4 years of age . The patient was discharged from the neonatal intensive care unit at 2 months of age with home oxygen therapy to treat chronic lung disease . The family relocated near the hospital and introduced the patient to us for developmental follow - up . The patient s height, weight, and general examination results were all within the normal range . Deep tendon reflex and babinski reflex were normal, but the patient presented with hypotonia and ataxia . At 12 months of age, she was diagnosed with ataxic cerebral palsy, due to the presentation of remarkable degree of incoordination and a high frequency of falls; thus, physical therapy was initiated . Motor developmental delays were observed: roll over was observed at 9 months, sitting was observed at 15 months, walking was observed at 24 months, and speech (her first word) was noted at 30 months . As the patient matured, she exhibited choreic movement of the limbs, face, and tongue at rest and dystonic posture of the upper limbs while walking . Choreic movement and small jerking movements of the trunk, limbs, and face were apparent at rest; these movements were not exaggerated by finger - to - nose test (supplementary videos 1 - 3). To exclude other chorea - related diseases, the authors examined metabolic parameters in blood (ammonia, amino acid, lactate, pyruvate, copper, ceruloplasmin, and -fetoprotein) and urine (organic acid). Brain magnetic resonance imaging showed perivascular space in the lower part of the right basal ganglia, but no other abnormalities related to clinical symptoms were observed (figure 2a, b). A, axial t2-weighted and (b) sagittal t1-weighted brain magnetic resonance imaging of the patient at 4 years of age . Imaging did not reveal any abnormalities except for perivascular space in the lower part of the right basal ganglia . A symptom triad of congenital hypothyroidism, respiratory distress, and chorea was strongly suggestive of nkx2 - 1 mutation . Sanger sequencing of the nkx2 - 1 gene led to the detection of a novel heterozygous insertion (c.915_916insc) in exon 3 that resulted in a frameshift starting at amino acid position 303 and a premature stop codon (p.ala303argfsx132) that rendered the protein nonfunctional . This mutation was not detected in leukocyte dna from the patient s mother, who had a familial history of thyroid dysfunction . The patient s father, who had no clinical manifestation of respiratory problems, thyroid dysfunction, or chorea, did not consent to dna analysis . At the time of this report, the patient s chorea is nonprogressive and she continues to demonstrate normal intelligence; the kyoto scale of psychological development 2001 at 5 years and 11 months indicated a development age of 4 years and 10 months (overall developmental quotient 81; postural motor developmental quotient 51; cognitive adaptive developmental quotient 76; language social developmental quotient 88). However, the patient does have subtle difficulties with handwriting and speech due to choreic movement and accordingly requires educational support and rehabilitation . Genomic dna from the patient and the patient s mother was extracted from leukocytes using a dna isolation kit (wako, japan). Primers were designed corresponding to the intronic sequences flanking the 3 exons of the nkx2 - 1 gene using primer3 software . Polymerase chain reaction (pcr) was performed using gotaq (promega, madison, wisconsin) under standard conditions, pcr products were purified using exosap (usb, cleveland, ohio), and products were sequenced for both forward and reverse strands using the bigdye terminator chemistry kit (version 3; applied biosystems, foster city, california) according to the standard protocol . Sequences were obtained with the abi genetic analyzer 3100 (applied biosystems) and the sequence analysis software program genetyx (version 9; genetyx, japan). Genomic copy numbers were analyzed using the human genome comparative genomic hybridization microarray 105k (agilent technologies, santa clara, california) as described previously . Genomic dna from the patient and the patient s mother was extracted from leukocytes using a dna isolation kit (wako, japan). Primers were designed corresponding to the intronic sequences flanking the 3 exons of the nkx2 - 1 gene using primer3 software . Polymerase chain reaction (pcr) was performed using gotaq (promega, madison, wisconsin) under standard conditions, pcr products were purified using exosap (usb, cleveland, ohio), and products were sequenced for both forward and reverse strands using the bigdye terminator chemistry kit (version 3; applied biosystems, foster city, california) according to the standard protocol . Sequences were obtained with the abi genetic analyzer 3100 (applied biosystems) and the sequence analysis software program genetyx (version 9; genetyx, japan). Genomic copy numbers were analyzed using the human genome comparative genomic hybridization microarray 105k (agilent technologies, santa clara, california) as described previously . In general, the phenotype of benign hereditary chorea is consistent with mutation of nkx2 - 1 . Large nkx2 - 1 deletions have been reported in infants with respiratory distress, congenital hypothyroidism, delayed motor milestones, and ataxia, whereas missense mutations and late protein truncations of nkx2 - 1 have been associated with milder but similar phenotypes . In this case report, the authors anticipated the observation of a large nkx2 - 1 deletion given severe respiratory distress and congenital hypothyroidism in the neonatal period and the later development of chorea . However, the comparative genomic hybridization array showed no abnormalities in chromosome 14 and the authors instead identified a novel heterozygous loss - of - function mutation, c.915_916insc (p.ala303argfsx132), by sanger sequencing analysis . The authors suspect that this mutation rendered the protein dysfunctional: ala303 is in close proximity to the nk2-specific domain, which is thought to function as an accessory dna - binding domain or as an interface for protein a lack of functional nkx2 - 1 protein in neurons is known to impair developmental differentiation and organization of basal ganglia and basal forebrain and causes aberrant trajectory of the dopaminergic pathway in the developing hypothalamus of mice . This can explain the symptomatic manifestations of nkx2 - 1 mutation in this patient . Table 1 shows nkx2 - 1 mutations that produced severe neonatal respiratory distress requiring mechanical ventilation, congenital hypothyroidism, and chorea similar to this case . In 11 severe neonatal respiratory distress cases, there were 4 deletion mutations (2 macrodeletions and 2 microdeletions), 4 point mutations (2 splice site mutations and 2 nonsense mutations), and 3 insertion mutations . The heterogeneity of mutation type and location in these cases of brain lung thyroid syndrome with severe neonatal respiratory distress suggests that there is no correlation between the severity of respiratory problems in benign hereditary chorea and specific mutations of nkx2 - 1 . It remains unclear why some cases present with irreversible lung failure from birth, whereas others only demonstrate transient and mild respiratory complications . Additionally, some cases of nkx2 - 1 mutation that present with mild chorea improve with age, whereas other cases have severe chorea and myoclonus into adulthood . Other factors, such as modifying genes, hormonal factors, and environmental factors, can underlie phenotypic heterogeneity in benign hereditary chorea . Reported severe neonatal respiratory distress requiring mechanical ventilation produced by nkx2 - 1 mutations . Other accompanying features include gait disturbance, dystonia, ataxia, myoclonus, and dysarthria . Prior to the onset of chorea, motor symptoms such as motor delay, ataxia, and gait disturbance can lead to the misdiagnosis of benign hereditary chorea as ataxic cerebral palsy . Mcmichael and colleagues reported the misdiagnosis of a father and his 2 children with ataxic dyskinetic cerebral palsy; in fact, a 7-base pair deletion within exon 1 of nkx2 - 1 . In this study, motor delay and ataxia were observed in early infancy, whereas choreiform movements and unsteady gait appeared in a later stage of development . Doyle and colleagues reported a case study of siblings with an nkx2 - 1 splice mutation that first exhibited congenital hypothyroidism, motor delay, and ataxia and later presented chorea and dysarthria . The mother of the siblings had been diagnosed with cerebral palsy . In the present case, a wide - based gait was initially diagnosed as ataxic gait; however, the authors hypothesize that it can have been a compensatory movement for the truncal jerking rather than cerebellar ataxia . Consistent with this hypothesis, no cerebellar abnormalities were observed in brain images (figure 2b). These studies underscore the need for careful evaluation of cases of ataxia and motor delay in young children who meet the criteria for ataxic cerebral palsy . After appearance of dyskinetic movements, such as chorea, myoclonus, and dystonia, it can be difficult to distinguish benign hereditary chorea from myoclonus dystonia syndrome resultant from epsilon sarcoglycan (sgce) gene mutations . Both diseases share a similar age of onset, dominant inheritance, minimal progression, and additional dystonia with no other neurological abnormalities . Dystonia showed lightning - like myoclonic jerks during the finger - to - nose test, whereas patients with benign hereditary chorea showed small jerking movements but no exacerbation of choreic movement . Salvatore and colleagues also reported the case of 3 patients from an italian family with the s145x nkx2 - 1 mutation and indicated that while dyskinetic movements, chorea, myoclonus, and jerky dystonia were apparent walking and sitting, dyskinetic movements seemed to improve during the finger - to - nose test . In the present study, the patient exhibited small jerks of the arm during the finger - to - nose test, but no exacerbation of choreic movement; rather, chorea was less noticeable during intentional tasks than at rest (supplementary videos 1 and 3). Myoclonus dystonia presents with action myoclonus when the arms are held outstretched and during the finger - to - nose test; alternatively, benign hereditary chorea presents with small jerking movements that are spontaneous, unrelated to intentional movement, and slower than myoclonus . These clinical features of benign hereditary chorea are notable and can thus be used to distinguish benign hereditary chorea from myoclonus dystonia syndrome in patients . In general, the first - line therapy for chorea is levodopa, but few cases of benign hereditary chorea report significant benefit . In contrast, chen and colleagues reported the improvement of symptoms with a low dose of tetrabenazine, an agent used to treat huntington s disease . Gras and colleagues similarly reported a moderate - to - marked beneficial effect of low - dose tetrabenazine on chorea and motor function in children and adults with benign hereditary chorea . Tetrabenazine acts in the central nervous system to deplete monoamines such as serotonin from nerve terminals by inhibiting their incorporation into presynaptic vesicles . Other agents such as trihexyphenidyl, corticosteroids, sodium valproate, and propranolol have also been used for the treatment of chorea and involuntary movement in benign hereditary chorea, but there is no consensus regarding a first - line therapy . However, prior to the discovery of nkx2 - 1, several reports observed reduced intelligent quotient in individuals with clinical benign hereditary chorea syndrome . Gras and colleagues reported nkx2 - 1 mutations in 28 patients with benign hereditary chorea from 13 families; of these patients, 71% experienced learning difficulties . A quantitative evaluation of cognitive performance in 14 children from this study identified mental retardation in 2 children and borderline mental retardation in 2 additional children . Our patient s postural motor developmental quotient was low because of choreic movement, but no apparent cognitive impairment has been observed . The patient did, however, exhibit dysgraphia and learning disability due to choreic movement and thus continues to require educational support . Congenital hypothyroidism for itself could be a cause of cognitive impairment and learning disability if not diagnosed and treated . But this patient had been diagnosed with early neonatal period and treated with levothyroxine . If benign hereditary chorea is pathologically related to dysfunction of the motor and associative striatal networks, chorea and adhd are logical symptomatic manifestations . However, it remains unclear whether functional deficits reflect an integral part of the benign hereditary chorea phenotype or a secondary consequence of social embarrassment and isolation . If an attending physician is aware of brain lung thyroid syndrome and its characteristic clinical features (respiratory distress despite birth at term, congenital hypothyroidism, and chorea), it can be diagnosed without difficulty in most cases . However, in early childhood, benign hereditary chorea can be difficult to identify prior to the appearance of chorea and can be mistaken for ataxic or dyskinetic cerebral palsy . Ataxic cerebral palsy is one of the least common types of cerebral palsy and is frequently associated with hypotonia and cerebellar features including tremor, gait disturbance, and poor coordination . Accordingly, benign hereditary chorea can be misdiagnosed as ataxic cerebral palsy in early childhood based on the observable symptoms . Phenotyping should therefore be performed with care in order to avoid the confusion of chorea and myoclonus with ataxia . It should be noted that, in this patient, despite severe respiratory distress in the neonatal period that may have caused hypoxic damage, a diagnosis of ataxic and/or dyskinetic cerebral palsy in the absence of abnormal imaging findings should be regarded as putative and thus continuously reevaluated according to the development of the clinical course.
|
The current study was nested in an ongoing community - based cohort study that investigated associations between glucose dysregulation and cardiovascular complications . The study design and protocol of baseline data collection of the cohort study were described previously (15). Briefly, all the permanent residents aged 40 years or older in songnan community in shanghai were invited to participate in a screening examination for cardiometabolic diseases . Among 10,185 participants, we randomly selected 5,250 subjects using a ratio of 1.0 [diabetes diagnosed previously or fasting plasma glucose (fpg) 7.0 mmol / l] to 1.2 (no previous diabetes and 5.6 fpg <7.0 mmol / l) to 1.44 (no previous diabetes and fpg <5.6 mmol / l) and oversampling people with lower glucose levels because they might have a lower participation rate than those with higher glucose levels, to undergo a much detailed and comprehensive evaluation including a standard 75-g oral glucose tolerance test . We then reclassified the participating 4,012 subjects (attendance rate, 76.4%) into ngr, prediabetes, and diabetes groups based on their diabetes history and fpg and 2-h postload plasma glucose (ppg) levels according to the 1999 world health organization criteria . There was no significant difference in age and sex distribution between those included and those not included in the cohort . For the current study, we randomly selected 150 individuals from the diabetes group and 150 ngr group and 150 prediabetes group individuals matched for age and sex of diabetic participants, respectively, after excluding subjects with the following characteristics: 1) age older than 60 years; 2) having symptoms of cad (chest pain or shortness of breath); 3) having a history of cardiovascular diseases (myocardial infarction, unstable angina, percutaneous coronary intervention, or stroke); 4) having abnormal q waves on resting electrocardiogram (ecg); 5) having a previous diagnosis of diabetes for> 5 years; 6) having impaired liver or renal function [alanine aminotransferease more than twice the upper limit of the normal range, serum creatinine level> 133 mol / l (1.5 mg / dl), or glomerular filtration rate <60 ml / min]; 7) being pregnant or having significant medical comorbidities; 8) having x - ray examination or ct scan within 1 year; 9) having tachycardia (a heart rate> 90 bpm) or arrhythmia such as atrial fibrillation on ecg that causes coronary artifacts during cta examinations; and 10) having a history of allergic reaction to iodine - containing contrast agent . Finally, a total of 420 individuals (attendance rate, 93%) participated in the current study . The study protocol was approved by the institutional review board of rui - jin hospital and written informed consent was obtained from each participant after providing a full explanation of the protocol and procedure . During july 2009 and august 2010, all participants underwent a comprehensive examination including a detailed questionnaire, anthropometric measurements, biochemical evaluation, and cta examination . Family history of cad in first - degree relatives was recorded and history of chronic diseases and current use of medication were acquired . Smoking status was defined as current if a subject smoked cigarettes regularly in the past 6 months . The bmi was calculated as body weight in kilograms divided by body height in meters squared (kg / m). Blood pressure was measured in the nondominant arm in a seated position three times consecutively at 1-min intervals after at least 5 min of rest using an automated electronic device (omron model hem-752; omron company, dalian, china). All participants were told to fast for at least 10 h before blood samples were collected . Plasma glucose, serum triglycerides, total cholesterol, hdl cholesterol, and ldl cholesterol were measured using an autoanalyser (beckman cx-7 biochemical autoanalyser, beckman coulter, brea, ca). Hemoglobin a1c (hba1c) was determined by high - performance liquid chromatography using the variant ii hemoglobin testing system (bio - rad laboratories, berkeley, ca) in a national glycohemoglobin standardization program certified laboratory of shanghai institute of endocrine and metabolic diseases . Fasting serum insulin was measured by an electrochemiluminescence assay (roche diagnostics). The single - void first morning urinary albumin concentrations were determined by immunoturbidimetry and urinary creatinine concentrations were measured by a modified jaffe method on an automatic analyzer (beckman lx-20). Diabetes was defined either by a previous diagnosis and contemporary antidiabetic medication or by levels of plasma glucose during oral glucose tolerance test according to the 1999 world health organization criteria . Prediabetes was defined as fpg 6.1 mmol / l and <7.0 mmol / l plus ppg <7.8 mmol / l or fpg <7.0 mmol / l plus ppg 7.8 mmol / l and <11.1 the indexes of homeostasis model assessment (homa) of insulin resistance and homa -cell function were calculated according to the formulas: homa of insulin resistance = fasting insulin concentration (miu / l) fpg (mmol / l) / 22.5; homa -cell function = 20 fasting insulin concentration (miu / l)/[fpg (mmol / l) 3.5]. Urinary albumin - to - creatinine ratio was calculated by dividing the urinary albumin concentrations by the urinary creatinine concentrations and expressed in milligrams per gram . Microalbuminuria was defined as an albumin - to - creatinine ratio between 30 and 300 mg / g . All examinations were performed on a dual - source ct scanner (somatom definition; siemens medical solutions, forchheim, germany). A standard retrospectively ecg - gated scanning protocol was applied, with 0.6-mm slice collimation, 330-ms gantry rotation time, 120-kv tube voltage, and a maximum tube current of 400 mas / tube . A bolus of 70 ml iohexol injection (350 mg / ml iodine; omnipaque; ge healthcare shanghai, shanghai, china) was intravenously injected (4 ml / s) via an 18-gauge catheter placed in the antecubital vein, followed by a 40-ml saline chaser . The cta images were interpreted independently by an experienced senior radiologist, who was unaware of the clinical information of study participants, with an offline three - dimensional workstation (adw 4.4; ge healthcare, waukesha, wi). Coronary arteries were divided into 15 segments according to the american heart association classification (16). The presence of atherosclerotic plaques and luminal narrowing were evaluated using axial images and curved multiplanar reconstructions . Coronary plaques were considered when structures> 1 mm were detected within or adjacent to the coronary artery lumen, which could be clearly distinguished from vessel lumen and the surrounding pericardial tissue . Given an estimated response rate of 75%, we calculated that a sample size of 140 participants for each of the 3 groups was needed to provide 90% power at a 2-tailed significance level of 0.05 to detect a difference from 5 to 20% of estimated prevalence of significant coronary stenosis assessed by cta among the groups . All data were analyzed using sas 9.2 (sas institute, cary, nc). Continuous variables were presented as means sd or medians (interquartile ranges) for skewed variables . Demographic and metabolic features and characteristics of coronary arteries in ngr, prediabetes, and diabetes were described and compared using anova for continuous variables and logistic regression analysis for categorical variables, adjusted for age and sex . The study population was reclassified into groups of increasing coronary stenosis (participants without coronary stenosis, with <50% coronary stenosis, and with significant coronary stenosis). Fpg, ppg, and hba1c levels and diabetes prevalence were then compared among these three groups . To investigate the associations of various cardiometabolic factors with significant coronary stenosis, logistic regression models were used to assess the crude and multivariate - adjusted odds ratios (or) of advanced age (age 53 years or older, representing the median cut - off value), female sex, family history of cad, ace inhibitor / angiotensin receptor blocker medication, current smoking, high school education or more, overweight / obesity, hypertension, high ldl, low hdl, prediabetes, and diabetes for risks of significant coronary stenosis . Variables for multivariate regression analysis were chosen as confounding factors depending on their clinical plausibility and external evidence such as previous research and previous beliefs, i.e., their well - recognized clinical relevance to both dysglycemia (the independent variable of interest) and cardiovascular diseases (significant coronary stenosis, the dependent variable) in this study, rather than internal statistical evidence from the data . To further elucidate the differences between diabetic participants with or without significant coronary stenosis, general characteristics were compared . Crude and multivariate - adjusted ors for significant coronary stenosis in diabetes by each 1-sd increase in levels of different glucose evaluations were calculated using logistic regression procedures . The current study was nested in an ongoing community - based cohort study that investigated associations between glucose dysregulation and cardiovascular complications . The study design and protocol of baseline data collection of the cohort study were described previously (15). Briefly, all the permanent residents aged 40 years or older in songnan community in shanghai were invited to participate in a screening examination for cardiometabolic diseases . Among 10,185 participants, we randomly selected 5,250 subjects using a ratio of 1.0 [diabetes diagnosed previously or fasting plasma glucose (fpg) 7.0 mmol / l] to 1.2 (no previous diabetes and 5.6 fpg <7.0 mmol / l) to 1.44 (no previous diabetes and fpg <5.6 mmol / l) and oversampling people with lower glucose levels because they might have a lower participation rate than those with higher glucose levels, to undergo a much detailed and comprehensive evaluation including a standard 75-g oral glucose tolerance test . We then reclassified the participating 4,012 subjects (attendance rate, 76.4%) into ngr, prediabetes, and diabetes groups based on their diabetes history and fpg and 2-h postload plasma glucose (ppg) levels according to the 1999 world health organization criteria . There was no significant difference in age and sex distribution between those included and those not included in the cohort . For the current study, we randomly selected 150 individuals from the diabetes group and 150 ngr group and 150 prediabetes group individuals matched for age and sex of diabetic participants, respectively, after excluding subjects with the following characteristics: 1) age older than 60 years; 2) having symptoms of cad (chest pain or shortness of breath); 3) having a history of cardiovascular diseases (myocardial infarction, unstable angina, percutaneous coronary intervention, or stroke); 4) having abnormal q waves on resting electrocardiogram (ecg); 5) having a previous diagnosis of diabetes for> 5 years; 6) having impaired liver or renal function [alanine aminotransferease more than twice the upper limit of the normal range, serum creatinine level> 133 mol / l (1.5 mg / dl), or glomerular filtration rate <60 ml / min]; 7) being pregnant or having significant medical comorbidities; 8) having x - ray examination or ct scan within 1 year; 9) having tachycardia (a heart rate> 90 bpm) or arrhythmia such as atrial fibrillation on ecg that causes coronary artifacts during cta examinations; and 10) having a history of allergic reaction to iodine - containing contrast agent . Finally, a total of 420 individuals (attendance rate, 93%) participated in the current study . The study protocol was approved by the institutional review board of rui - jin hospital and written informed consent was obtained from each participant after providing a full explanation of the protocol and procedure . During july 2009 and august 2010, all participants underwent a comprehensive examination including a detailed questionnaire, anthropometric measurements, biochemical evaluation, and cta examination . Family history of cad in first - degree relatives was recorded and history of chronic diseases and current use of medication were acquired . Smoking status was defined as current if a subject smoked cigarettes regularly in the past 6 months . The bmi was calculated as body weight in kilograms divided by body height in meters squared (kg / m). Blood pressure was measured in the nondominant arm in a seated position three times consecutively at 1-min intervals after at least 5 min of rest using an automated electronic device (omron model hem-752; omron company, dalian, china). All participants were told to fast for at least 10 h before blood samples were collected . Plasma glucose, serum triglycerides, total cholesterol, hdl cholesterol, and ldl cholesterol were measured using an autoanalyser (beckman cx-7 biochemical autoanalyser, beckman coulter, brea, ca). Hemoglobin a1c (hba1c) was determined by high - performance liquid chromatography using the variant ii hemoglobin testing system (bio - rad laboratories, berkeley, ca) in a national glycohemoglobin standardization program certified laboratory of shanghai institute of endocrine and metabolic diseases . The single - void first morning urine samples were collected to assess urinary albumin - to - creatinine ratio . Urinary albumin concentrations were determined by immunoturbidimetry and urinary creatinine concentrations were measured by a modified jaffe method on an automatic analyzer (beckman lx-20). Diabetes was defined either by a previous diagnosis and contemporary antidiabetic medication or by levels of plasma glucose during oral glucose tolerance test according to the 1999 world health organization criteria . Prediabetes was defined as fpg 6.1 mmol / l and <7.0 mmol / l plus ppg <7.8 mmol / l or fpg <7.0 mmol / l plus ppg 7.8 mmol / l and <11.1 the indexes of homeostasis model assessment (homa) of insulin resistance and homa -cell function were calculated according to the formulas: homa of insulin resistance = fasting insulin concentration (miu / l) fpg (mmol / l) / 22.5; homa -cell function = 20 fasting insulin concentration (miu / l)/[fpg (mmol / l) urinary albumin - to - creatinine ratio was calculated by dividing the urinary albumin concentrations by the urinary creatinine concentrations and expressed in milligrams per gram . Microalbuminuria was defined as an albumin - to - creatinine ratio between 30 and 300 mg / g . All examinations were performed on a dual - source ct scanner (somatom definition; siemens medical solutions, forchheim, germany). A standard retrospectively ecg - gated scanning protocol was applied, with 0.6-mm slice collimation, 330-ms gantry rotation time, 120-kv tube voltage, and a maximum tube current of 400 mas / tube . All scans were performed using ecg - controlled tube current modulation . A bolus of 70 ml iohexol injection (350 mg / ml iodine; omnipaque; ge healthcare shanghai, shanghai, china) was intravenously injected (4 ml / s) via an 18-gauge catheter placed in the antecubital vein, followed by a 40-ml saline chaser . The cta images were interpreted independently by an experienced senior radiologist, who was unaware of the clinical information of study participants, with an offline three - dimensional workstation (adw 4.4; ge healthcare, waukesha, wi). Coronary arteries were divided into 15 segments according to the american heart association classification (16). The presence of atherosclerotic plaques and luminal narrowing were evaluated using axial images and curved multiplanar reconstructions . Coronary plaques were considered when structures> 1 mm were detected within or adjacent to the coronary artery lumen, which could be clearly distinguished from vessel lumen and the surrounding pericardial tissue . Given an estimated response rate of 75%, we calculated that a sample size of 140 participants for each of the 3 groups was needed to provide 90% power at a 2-tailed significance level of 0.05 to detect a difference from 5 to 20% of estimated prevalence of significant coronary stenosis assessed by cta among the groups . All data were analyzed using sas 9.2 (sas institute, cary, nc). Continuous variables were presented as means sd or medians (interquartile ranges) for skewed variables . Demographic and metabolic features and characteristics of coronary arteries in ngr, prediabetes, and diabetes were described and compared using anova for continuous variables and logistic regression analysis for categorical variables, adjusted for age and sex . The study population was reclassified into groups of increasing coronary stenosis (participants without coronary stenosis, with <50% coronary stenosis, and with significant coronary stenosis). Fpg, ppg, and hba1c levels and diabetes prevalence were then compared among these three groups . To investigate the associations of various cardiometabolic factors with significant coronary stenosis, logistic regression models were used to assess the crude and multivariate - adjusted odds ratios (or) of advanced age (age 53 years or older, representing the median cut - off value), female sex, family history of cad, ace inhibitor / angiotensin receptor blocker medication, current smoking, high school education or more, overweight / obesity, hypertension, high ldl, low hdl, prediabetes, and diabetes for risks of significant coronary stenosis . Variables for multivariate regression analysis were chosen as confounding factors depending on their clinical plausibility and external evidence such as previous research and previous beliefs, i.e., their well - recognized clinical relevance to both dysglycemia (the independent variable of interest) and cardiovascular diseases (significant coronary stenosis, the dependent variable) in this study, rather than internal statistical evidence from the data . To further elucidate the differences between diabetic participants with or without significant coronary stenosis, crude and multivariate - adjusted ors for significant coronary stenosis in diabetes by each 1-sd increase in levels of different glucose evaluations were calculated using logistic regression procedures . Nineteen (4.5%) patients with uninterpretable segments attributable to motion artifacts were excluded from analysis, which resulted in 135 with ngr, 132 with prediabetes, and 134 with diabetes participating for data analysis . Generally, percentages of individuals with a family history of cad, educated beyond high school level, or currently smoking were not significantly different among groups, whereas bmi, blood pressure, and triglycerides were elevated and hdl was decreased substantially in parallel with deteriorations in glucose levels, insulin sensitivity, and -cell function . Medications using ace inhibitors or angiotensin receptor blockers were basically similar among the three groups . One was in the ngr group and the other was in the prediabetes group, and both were without coronary plaques . Statins were used by only one individual who was diabetic and had nonsignificant coronary stenosis . General characteristics of study population by glycemic status as shown in table 1, coronary plaques were found in 58 (43.0%) participants with ngr, in 77 (58.3%) with prediabetes, and in 74 (55.2%) with diabetes (p for trend = 0.081), whereas plaques causing significant coronary stenosis were detected in 10 (7.4%), 10 (7.6%), and 22 (16.4%) individuals with ngr, prediabetes, and diabetes, respectively (p for trend = 0.029). Therefore, a similar prevalence of cad detected by cta was found in prediabetes and diabetes, and it was relatively higher than that in ngr; however, prevalence of significant coronary stenosis was doubled in diabetes and was substantially increased compared with that in ngr or prediabetes . When participants were reclassified according to existence or extent of coronary stenosis, individuals with significant stenosis had dramatically elevated levels of fpg, ppg, and hba1c and had a significantly increased prevalence of diabetes compared with both groups of participants with <50% coronary stenosis and without stenosis after controlling for a variety of confounding factors (all p <0.05; fig . Percentages of diabetes and levels of glucose evaluations in participants 1) without coronary stenosis, 2) with <50% coronary stenosis, and 3) with significant coronary stenosis, respectively . P values were adjusted for age, sex, family history of cad, history of ace inhibitor / angiotensin receptor blocker medication, smoking status, educational attainment, overweight or obesity, hypertension, high ldl cholesterol, and low hdl cholesterol . Women were strongly protected from significant coronary stenosis [or, 0.33 (95% ci, 0.140.81); p = 0.016; table 2]. Prediabetes was not associated with an increased risk of significant coronary stenosis, whereas diabetes was associated with a significant 2.34-fold elevated risk [2.34 (1.015.43); p = 0.047] compared with ngr . Correlations of cardiometabolic factors with risks of significant coronary stenosis among 134 patients with diabetes for <5 years, 22 (16.4%) had significant coronary stenosis . Diabetic individuals with significant coronary stenosis were more likely to be males and had more deteriorated glucose metabolism and -cell function compared with those without significant coronary stenosis (table 3). The presence of microalbuminuria tended to be increased in those with significant stenosis, but it failed to reach statistical significance . Among all cases of diabetes, 104 (77.6%) were newly diagnosed, but 14 (13.5%) already had significant coronary stenosis . Although newly diagnosed diabetes tended to occur more often in the group without significant coronary stenosis, the difference was not statistically significant . Comparison of characteristics by coronary artery status in diabetes in univariate and multivariate logistic analyses, glucose evaluation levels were independently and significantly associated with risks of significant coronary stenosis in diabetes . Each 1-sd increase in fpg, ppg, or hba1c conveyed 2.11-fold, 1.73-fold, or 1.81-fold higher risks of significant coronary stenosis, respectively, after controlling for other conventional cardiovascular risk factors (all p <0.05; table 4). Characteristics of the study population according to glycemic status are shown in table 1 . Generally, percentages of individuals with a family history of cad, educated beyond high school level, or currently smoking were not significantly different among groups, whereas bmi, blood pressure, and triglycerides were elevated and hdl was decreased substantially in parallel with deteriorations in glucose levels, insulin sensitivity, and -cell function . Medications using ace inhibitors or angiotensin receptor blockers were basically similar among the three groups . One was in the ngr group and the other was in the prediabetes group, and both were without coronary plaques . Statins were used by only one individual who was diabetic and had nonsignificant coronary stenosis . As shown in table 1, coronary plaques were found in 58 (43.0%) participants with ngr, in 77 (58.3%) with prediabetes, and in 74 (55.2%) with diabetes (p for trend = 0.081), whereas plaques causing significant coronary stenosis were detected in 10 (7.4%), 10 (7.6%), and 22 (16.4%) individuals with ngr, prediabetes, and diabetes, respectively (p for trend = 0.029). Therefore, a similar prevalence of cad detected by cta was found in prediabetes and diabetes, and it was relatively higher than that in ngr; however, prevalence of significant coronary stenosis was doubled in diabetes and was substantially increased compared with that in ngr or prediabetes . When participants were reclassified according to existence or extent of coronary stenosis, individuals with significant stenosis had dramatically elevated levels of fpg, ppg, and hba1c and had a significantly increased prevalence of diabetes compared with both groups of participants with <50% coronary stenosis and without stenosis after controlling for a variety of confounding factors (all p <0.05; fig . Percentages of diabetes and levels of glucose evaluations in participants 1) without coronary stenosis, 2) with <50% coronary stenosis, and 3) with significant coronary stenosis, respectively . P values were adjusted for age, sex, family history of cad, history of ace inhibitor / angiotensin receptor blocker medication, smoking status, educational attainment, overweight or obesity, hypertension, high ldl cholesterol, and low hdl cholesterol . Women were strongly protected from significant coronary stenosis [or, 0.33 (95% ci, 0.140.81); p = 0.016; table 2]. Prediabetes was not associated with an increased risk of significant coronary stenosis, whereas diabetes was associated with a significant 2.34-fold elevated risk [2.34 (1.015.43); p = 0.047] compared with ngr . Among 134 patients with diabetes for <5 years, 22 (16.4%) had significant coronary stenosis . Diabetic individuals with significant coronary stenosis were more likely to be males and had more deteriorated glucose metabolism and -cell function compared with those without significant coronary stenosis (table 3). The presence of microalbuminuria tended to be increased in those with significant stenosis, but it failed to reach statistical significance . Among all cases of diabetes, 104 (77.6%) were newly diagnosed, but 14 (13.5%) already had significant coronary stenosis . Although newly diagnosed diabetes tended to occur more often in the group without significant coronary stenosis, the difference was not statistically significant . Comparison of characteristics by coronary artery status in diabetes in univariate and multivariate logistic analyses, glucose evaluation levels were independently and significantly associated with risks of significant coronary stenosis in diabetes . Each 1-sd increase in fpg, ppg, or hba1c conveyed 2.11-fold, 1.73-fold, or 1.81-fold higher risks of significant coronary stenosis, respectively, after controlling for other conventional cardiovascular risk factors (all p <0.05; table 4). We used cta, which is a noninvasive diagnostic modality, to characterize subclinical cad in a community - dwelling adult population with ngr, prediabetes, or diabetes matched for age and sex . We found that although coronary stenosis was more prevalent in both prediabetes and diabetes, the risk of significant stenosis was only elevated in diabetes, despite a short duration of <5 years . Additionally, glucose evaluation levels were significantly and independently associated with risks of significant coronary stenosis in diabetic participants 17) reported that the presence of diabetes alone confers risk of cardiovascular mortality similar to that in nondiabetic individuals with a previous myocardial infarction . Heart disease is the most frequent cause of death in those with diabetes, accounting for 70% of all deaths (18). Coronary artery lesions in those with diabetes were often characterized as diffuse and multivessel, yet asymptomatic (19). In fact, silent myocardial ischemia is highly prevalent in the diabetic population . In the current study, 74 out of 134 diabetic patients (55.2%) were found to have a certain degree of coronary stenosis and 22 (16.4%) had significant stenosis, which was relatively low compared with most studies using cta in asymptomatic patients with diabetes (1114). The variation in prevalence may well reflect the important differences regarding population characteristics between studies . Those with higher prevalence rates of significant stenosis, ranging between 26 and 41%, generally included a higher proportion of male participants (for example, 66% in the study by rivera et al . (11) compared with 50% in the current study) and more patients with hypertension (69 vs. 59%), dyslipidemia (78 vs. 32%), or family history of cad (30 vs. 19%). Another important and more relevant fact is that participants in those studies had much longer duration of diabetes (mean duration, 710 years). Diabetes duration is thought to contribute significantly to cad risks, and the framingham heart study reported a 1.38-fold increased risk for cad and a 1.86-fold higher risk for cad death for each 10-year increase in diabetes duration (20). In previous studies, much attention has been given to diabetes of an advanced stage and cad evaluation . There is a paucity of data on coronary artery stenosis in asymptomatic diabetes that has been diagnosed for a relatively short period of time . In the current study, a majority of individuals in the diabetes group were newly diagnosed (104/134; 77.6%) and the rest of diabetes cases were detected within 5 years . These diabetic participants were found to have a 2.34-fold increased risk for significant coronary stenosis compared with ngr . It is well - known that diabetes exists for several years before diagnosis, and thus an elevated risk of significant coronary stenosis in recently diagnosed diabetes may reflect a cumulative effect of glucose dysregulation that has been going on for years and for much longer than it had been noticed . Moreover, among diabetic patients in this study, those with significant coronary stenosis had much higher glucose and hba1c levels than those without . Glucose evaluations, i.e., fpg, ppg, and hba1c, were found to be significant and independent risk markers for significant coronary stenosis in diabetes . Although deterioration of glucose homeostasis is reflective of a longer diabetes duration, elevated levels of glucose evaluations still were directly associated with increased risks of significant coronary stenosis in diabetes in that each 1-sd increment of glucose or hba1c level independently conveyed 1.73-fold to 2.11-fold elevated risks, adequately controlled for newly diagnosed cases and antidiabetic medications . Participants with prediabetes were found to have a significantly higher prevalence of any coronary plaques but a similar prevalence of plaques causing> 50% stenosis compared with ngr in this study . The close relationship between prediabetes and future cad has been demonstrated by several studies (2123). The enhanced atherogenic risk profile in prediabetes, such as higher bmi values and greater prevalence of hypertension, together with a prediabetes glucose level, may contribute to the elevated cad prevalence in this study . Nevertheless, significant coronary stenosis as measured by cta was not increased in prediabetes and was statistically lower than that in diabetes (7.6 vs. 16.4%; p = 0.027). Studies investigating biomarker trajectories leading to diabetes diagnosis reported modest changes in insulin sensitivity and secretion from normal to impaired glucose tolerance but substantial decreases during further progression to diabetes, leading to abrupt and steep increases in fpg and ppg levels during the few years (most likely 3 years) immediately before diagnosis (24,25). This is consistent with our findings on homa of insulin resistance and homa -cell function in three groups with different glycemic status, and it might be one of the potential mechanisms of a markedly elevated risk for significant coronary stenosis in early diabetes compared with prediabetes . In fact, despite glucose levels being within a normal range, 42% of these individuals were overweight or obese, 31% had hypertension, and 30% had dyslipidemia . Nearly 80% of these subjects had at least one cardiovascular risk factor, such as cigarette smoking, family history of cad, overweight / obesity, hypertension, or dyslipidemia, indicating a noteworthy overall cad risk profile in middle - aged chinese adults residing in metropolitan cities such as shanghai . Multislice ct coronary angiography recently has emerged as a powerful imaging modality for noninvasive assessment of cad . Except for high diagnostic accuracy for detecting cad compared with the gold standard invasive coronary angiography (2628), with a sensitivity of 89%, a specificity of 96%, and positive and negative predictive values of 78 and 98%, respectively (29), cta also has been found useful in predicting future cardiac events in patients with known or suspected cad (3035). However, consistency is lacking regarding the use of cta in asymptomatic subjects, particularly in high - risk asymptomatic patients, such as those with diabetes . The major concerns are unnecessary ct scan radiation, increased health care spending, and subsequent invasive cardiac procedures (36). However, recent advances in cta technology, such as dual - source ct coronary artery angiography, have dramatically improved spatial and temporal resolution, leading to substantial improvement in image quality and significant reduction in radiation dose and use of contrast material (37) currently, the american diabetes association consensus guidelines recommend cad screening in diabetic individuals with cardiovascular symptoms (38). However, our study showed a markedly increased risk for the presence of significant coronary stenosis in asymptomatic patients with diabetes, even soon after its diagnosis . Furthermore, evidence has indicated similar frequencies of cad in diabetic patients with and without angina symptoms (39,40). Therefore, cardiac testing for asymptomatic patients with diabetes should not be considered unwarranted, especially with the latest development in ct technology, which provides satisfactory cardiac images while reducing radiation dose . Meanwhile, although it could be somewhat premature to suggest that cta examination should be routinely used for patients with recently diagnosed diabetes, our findings may imply that cta screening should be used more aggressively in patients with diabetes, especially in those with additional risk factors such as dyslipidemia and hypertension . Nevertheless, whereas clinical effectiveness proves promising, cost - effectiveness is another important issue of concern before integrating cta into patient care algorithms . It is possible that clinical outcomes may be sufficiently improved by use of cta, justifying the substantially higher spending associated with its use . Therefore, longitudinal studies comparing costs of downstream testing and treatment of cardiovascular complications in diabetes with and without use of cta are urgently needed . Despite the findings and the clinical implications for cad management in asymptomatic early diabetes, this study suffers from several important limitations . First, the sample size was relatively small and thus did not allow separate analyses by sex or stratification by cardiovascular risks and did not take menopause status into account . Second, although participants with ngr, prediabetes, or diabetes were well - matched for age and sex, other important cad risk factors such as hypertension were not considered when selecting participants . Third, coronary stenosis was classified as <50% or 50% in this study, rather than a much detailed quantification to include severe stenosis, which is 75% narrowing of coronary artery lumen and is highly urgent for clinical intervention . Moreover, although many other cardiovascular risk factors such as smoking, hypertension, and dyslipidemia were adjusted for correlation of glucose dysregulation with significant coronary stenosis in the multivariate analyses, some residual or undetected confounding could not be ruled out . In conclusion, we used noninvasive diagnostic cta to anatomically assess subclinical cad in middle - aged community - dwelling chinese adults with different glycemic status . We detected a markedly increased risk of significant coronary stenosis in early diabetes without clinical manifestation of myocardial ischemia . Although prediabetes is thought to be predictive of future cardiac events, it was not found to be associated with a higher risk of significant coronary stenosis . Follow - up studies are needed to investigate the long - term cardiovascular outcomes of cta - confirmed subclinical atherosclerosis in patients with diabetes.
|
Physical inactivity is one of the main risk factors for chronic diseases, which are responsible for 5.3 million deaths annually worldwide (1). In 2009, the global prevalence of inactivity was 17% (2), with more than 30% of the world population not meeting the minimum recommendations for physical activity (3). In iran, 40% of adults (31.6% men and 48.6% women) are reported to be in the low physical activity category, while about 15% (4.7 million people) do not have any physical activity (4). To reduce the burden of diseases like cardiovascular disease, cancer, diabetes mellitus, obesity, and depression, it is strongly recommended that individuals engage in regular physical activity of at least moderate intensity (5). Given this scenario, information on changes in health - risk behaviors like physical activity over time is vital to planning effective programs and policies . Hence, evaluating changes in physical activity levels of populations over time has become a public health priority (3, 6). Since no data are available on time changes of physical activity in iran, this study was designed to evaluate changes in physical activity levels over a median follow up of 6.5 yr among tehranian adults . The tehran lipid and glucose study (tlgs) is a prospective population based study performed on a representative sample of tehran s population, with the aim of determining the prevalence of noncommunicable disease (ncd) risk factors and developing a healthy lifestyle to improve them (7). The baseline survey was performed from 1999 to 2001 and 4751 families, which included over 15000 residents of district 13 of tehran, aged 3 yr were selected by cluster random sampling method . After this cross - sectional prevalence study of ncd risk factors, subjects were enrolled into a cohort and a prospective interventional study and were followed every three years . Data on the physical activity status of subjects were collected using the modifiable activity questionnaire (maq) from phase ii of tlgs . Considering the high numbers of missing physical activity data in phase iii, in the current paper, subjects were examined just for phases ii (from 2002 to 2005) and iv (from 2008 to 2011) i.e. A 6.5 yr - follow up . The study was approved by the ethics committee of the research institute for endocrine sciences of shahid beheshti university of medical sciences . All participants provided written informed consent . From 7268 subjects who completed the follow up period, participants who took part in lifestyle intervention through community education (n=3753) were excluded; the data of 3515 subjects aged 20 yr (2100 females and 1415 males) were analyzed to determine the changes of physical activity levels over a median follow up of 6.5 yr . High reliability (98%) and moderate validity (47%) were found for the maq translated into persian (9). Intra - class correlation coefficients between the two pretest and post - test maqs for all activity domains in the past year, including leisure time, occupational, and total (leisure and occupational combined) physical activity were 0.94, 0.98, and 0.97, respectively . Participants were asked to report the activities that they had participated in (at least 10 sessions) during the past 12 mo in their leisure time and then identified the frequency and duration for each ltpa . Total numbers of minper year, calculated for every physical activity were summed up and then divided by 52 to estimate the minper wk of total leisure time physical activity . The calculation of met - min / wk is summarized as met - min / wk = (met mo per year sessions per mo minper session) / 52 . Met - min / wk of leisure time activity was calculated by multiplying the number of minper wk of each leisure time activity to its metabolic equivalent (met). One met is set at 3.5 ml of oxygen consumed per kg of body weight per min and represents the resting metabolic rate . The numbers of mets corresponding to each activity were calculated using the average metabolic cost for each activity (10). Employed persons were asked to indicate how many h a wk they usually worked . According to the questionnaire, individuals had to identify the number of mo and h they participated in physical activity at work (standing, housework, work activities more intense than standing) over the past year . The assessment of occupational activity was based on summing up the number of h per wk of light, moderate and hard intensity activities, multiplying the sum by 60 in order to express minper wk of occupational activity over the past year . Final occupational (met - min / wk) activity was calculated by multiplying the number of minper wk of each of the three categories of occupational activity by met values (10). Total physical activity was expressed in met - min / wk by adding leisure time to occupational activity; scores <600 mets - min / wk were considered as low physical activity (11). Analyses were carried out using spss software version 20.0 (spss, chicago, il, usa). From 7268 subjects who completed the follow up period, participants who took part in lifestyle intervention through community education (n=3753) were excluded; the data of 3515 subjects aged 20 yr (2100 females and 1415 males) were analyzed to determine the changes of physical activity levels over a median follow up of 6.5 yr . High reliability (98%) and moderate validity (47%) were found for the maq translated into persian (9). Intra - class correlation coefficients between the two pretest and post - test maqs for all activity domains in the past year, including leisure time, occupational, and total (leisure and occupational combined) physical activity were 0.94, 0.98, and 0.97, respectively . Data were collected by participants, assisted by trained interviewers when needed . Participants were asked to report the activities that they had participated in (at least 10 sessions) during the past 12 mo in their leisure time and then identified the frequency and duration for each ltpa . Total numbers of minper year, calculated for every physical activity were summed up and then divided by 52 to estimate the minper wk of total leisure time physical activity . The calculation of met - min / wk is summarized as met - min / wk = (met mo per year sessions per mo minper session) / 52 . Met - min / wk of leisure time activity was calculated by multiplying the number of minper wk of each leisure time activity to its metabolic equivalent (met). One met is set at 3.5 ml of oxygen consumed per kg of body weight per min and represents the resting metabolic rate . The numbers of mets corresponding to each activity were calculated using the average metabolic cost for each activity (10). Employed persons were asked to indicate how many h a wk they usually worked . According to the questionnaire, individuals had to identify the number of mo and h they participated in physical activity at work (standing, housework, work activities more intense than standing) over the past year . The assessment of occupational activity was based on summing up the number of h per wk of light, moderate and hard intensity activities, multiplying the sum by 60 in order to express minper wk of occupational activity over the past year . Final occupational (met - min / wk) activity was calculated by multiplying the number of minper wk of each of the three categories of occupational activity by met values (10). Total physical activity was expressed in met - min / wk by adding leisure time to occupational activity; scores <600 mets - min / wk were considered as low physical activity (11). Analyses were carried out using spss software version 20.0 (spss, chicago, il, usa). In both phases, 59.8% of adults were women and the mean sd of age was 44.3 14.6 years in phase ii and 50.9 14.6 yr in phase iv . After 6.5 years, a significant decrease in ltpa was observed among women, but not among men (fig . 1). Levels of physical activity domains (mets - min / wk) in tehranian men and women in phases ii and iv of tehran lipid and glucose study ltpa, leisure time physical activity level; ocpa, occupational physical activity level p<0.05 between phases the prevalence of low physical activity in the total population decreased significantly between phases ii and iv, being 45.9% and 42.6% respectively (p<0.05). As shown in table 1, the decrease was significant in men, but not in women . Regarding age groups, this table also indicated that the prevalence of low physical activity has decreased significantly among older men (60 yr). Prevalence of low physical activity (<600 mets - min / wk) stratified by sex and age groups in phases ii and iv of tlgs tlgs, tehran lipid and glucose study p<0.05 by mcnemar test regarding the activities in ltpa, there was a significant increase in proportion of individuals participating in swimming, aerobics / dancing, martial arts, and football / handball; however, there was a significant decrease in jogging / walking (table 2). Comparison of ltpas in phases ii and iv of tlgs tlgs, tehran lipid and glucose study; ltpa, leisure time physical activity . The results of present study indicated a 3.3% decrease in the prevalence of low physical activity during a 6.5 yr follow up from 45.9% to 42.6%, among a tehranian adult population . Although this is encouraging, it is difficult to estimate the clinical impact of such superficial changes, although, on a population level they may lead to decrease in morbidity; i.e., recently, from the nhanes data it has been indicated that each additional min daily of high intensity activity lod the odds of obesity by 2% for men and by 5% for women similar to our findings, a brazilian and a peri - urban nepalese population showed a high prevalence of low physical activity, i.e. 43.3% and> 50%, respectively (13, 14). Although most studies indicated a decreasing trend in physical activity levels of populations (1517); recently, in an urban swiss population, a significant increasing trend was reported in physical activity levels from 1999 to 2009 (18), indicating a shift from low intensity physical activity towards moderate / high or vigorous physical activity among this population . Surprisingly, this increasing change occurred outside the occupational setting, demonstrating an increase in ltpa . With respect to ocpa, our results indicated a decrease over time, which is a result of the greater automation in the workplace, a problem that most populations are facing (1923). Similar to our findings, in the madrid region, a decrease in ltpa, which mainly resulted in increased physical inactivity, was accompanied by a decreased ocpa (17), demonstrating a shift from occupations that need moderate to high intensity physical activity to occupations that mostly include sedentary and sitting behavior . The same results were reported in the peri - urban nepalese population (13) i.e., the reduction in ocpa was not compensated by an increase in ltpa . Hence, concerns for public health are increasing since ocpa is a major portion of total physical activity and as long as the decrease in ocpa is not counteracted by increase in ltpa, the approach mentioned above that led to increase physical activity levels in the swiss population (18), overall physical activity will most likely declined . Another main finding of the current study was that ltpa decreased significantly among women during a median follow up of 6.5 years . The association between employment and ltpa reports earlier in this population (24) indicated higher prevalence of inactivity among those employed compared to the unemployed . These findings can be easily explained by less leisure time among employed individuals, which is often reported as a barrier to participate in physical activity and exercise programs . Again, in another study of this population (25), the prevalences of obesity and abdominal obesity in women were found to be higher than in men, indicting the urgent need to target women for prevention and implementation of public educational programs to promote physical activity levels and ltpa in particular, to curtail the rising trend in obesity and abdominal obesity . Physical inactivity is responsible for 610% of the major non - communicable diseases includes coronary heart disease and type 2 diabetes (1). Taking into account that, regarding coronary heart disease risk, about one - quarter of tehranian adults are reported to be eligible for therapeutic interventions (therapeutic lifestyle changes and/or additional drug therapy) (26) and also there is a marked increase in the prevalence of obesity and abdominal obesity (25), significant importance given to our results . Furthermore, since the increasing trend in abdominal and general obesity is also observed in tehranian adolescents (27) and taking into account that sports activities in iran is publicized mostly through physical education classes, schools can play an important role in promoting physical activity in younger groups of this society . The increasing proportion of individuals participating in physical activities such as swimming and aerobics / dancing reflects a preference shift in the type of physical activity performed by adults in leisure time . Such preferences in types of activities may be a result of the growing number of gyms, increased knowledge and awareness about the beneficial effects of physical activity for health, and achieving a desirable body shape . Similar to our findings, in the madrid region (17), activities which mostly contribute to the decrease in ltpa were those of low and moderate intensity, like walking and jogging, implying that a significant decrease in the proportion of subjects participating in these activities can be responsible for decrease in overall ltpa in this population . The major strength of current study is its large and diverse population, which is representative of tehranian adults . Moreover, the reliability and convergent validity of the persian version of questionnaire used for physical activity assessment (maq) had been confirmed before . However, some limitations may have affected accuracy; first, the questionnaire we used (maq) differs to those of other studies, and any comparison of physical activity domains between studies using different methods is problematic; second, it cannot be overlooked that health promotion publicity induces a tendency for socially desirable answers; lastly, in such studies, the clinical significance of the changes in physical activity is difficult to determine . Our analysis of physical activity level changes over a 6.5 yr follow up in tehranian adults indicates a decreasing trend in the proportion of subjects with low physical activity . However despite these encouraging findings, approximately fifty percent of tehranian adults still have physical activity levels below the minimum level recommended, emphasizing the necessity to implement programs encouraging increase in physical activity levels of tehranian adults . Moreover, significant decrease in ltpa among women indicates the urgent need to target women for prevention and implementation of public educational programs to promote physical activity levels and ltpa in particular, to compensate the reduction in ocpa . Ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc .) Have been completely observed by the authors.
|
In a book titled doing psychiatry wrong the author (a psychiatrist) describes several cases treated with medications, mostly inappropriately (e.g., patient with borderline personality treated as bipolar disorder, multi drug abuser treated as schizophrenia), without much gain . Another author (a sociologist) argues that in spite of the evidence that psychiatric conditions improve best with a combination of medicines and therapy, psychotherapy is rarely offered . That author's research has shown that such patients who were treated only with medicines do less well, are readmitted more quickly, diagnosed more inaccurately and medicated more randomly . This case was inaccurately diagnosed, randomly medicated and did less well . Properly conducted psychotherapy has improved the patient . Are we in to a second wave of anti - psychiatry movement against biological imperialism? A 23-year - old male, b.e student from upper middle socio - economic status, accompanied by his parents, presented with the complaints of anger outbursts, inability to mingle with people and inability to study . For the past 2 years, he attended neither the classes nor the home tuitions . Most of the activities, which he tried to do ended in failure and angry frustrations . Most of the time was spent in sleep, which was aided by sedative psychotropic medicines . He could not tolerate inactivity as it led to boredom and this was intolerable . Remaining small time in the day he displayed a typical pattern of behavior as noted by pedesky and beck they may discontinue a task or fail to initiate a task they had planned to do . They may turn on the television, pick up some things to read, reach for food or a cigarette, get up and walk around and henceforth . Mental status of the patient was that of an inhibited plump person with expressionless serious face, answering questions minimally, but relevantly without any psychotic symptoms . Most of his anger outbursts erupted and lasted only for a few seconds and a few are followed by grumbling and shouting for about 10 min . Anger resulted in yelling with angry gestures, banging, crumpling, throwing, tearing and breaking of objects (one per attack) such as shirt, pen, pencils, spectacles, remote control and rarely, mobile phones or computers . He avoided close relatives, strangers and crowds as it induced severe fear and inhibition . He was scared to talk to house maid, lift operator in the apartment, traffic police and ladies . He looked away from them and walked away at the prospect of an approaching lady . In fact his first visit to a psychiatrist was due to fear of lead contamination from wall paints . His other fears were, being cursed by god, old and sick people if they are disturbed by mistake, fear of going deaf or blind by strong sounds or bright light . He had magical beliefs that bad words if heard in the early morning, they will spoil the whole day and if any bad word is heard while praying, god will curse . He had rigid moral values in the matters of sexuality, religion and right or wrongs . He got infuriated with matters such as muslim religion, female gender, britons, beef eaters and rule breakers . He was pre - occupied with his sagging chest and wore tight banyans to hide it . He wanted to understand the subject perfectly and this inability led to a sense of failure and frustration and anger . Patient was born to a highly educated and well - employed couple hailing from upper socio - economic status . His first degree paternal uncle has a chronic psychotic illness, but he is too highly educated and regularly employed . However, he was noted to be angry and used to kick walls if he got angry . In 10 and 11 standard he was bullied frequently by few students and ridiculed for his sagging chest by touching it . Patient remained un - assertive and the abuse and emotional trauma inflicted was significant . During adolescence his past psychological assessments revealed above average intelligence and presence of several personalities disorder traits and low self - esteem . He was treated by more than a dozen of psychiatrist, exhausting all psychotropic medicines, including clozapine at 200 mg / day . When he visited the author he was on lithium 400 mg, fluoxetine 60 mg, amisulpride 100 mg, quetiapine 50 mg, clonazepam 3 mg, pregabalin 300 mg . Cognitive therapy espoused by pedesky and beck was administered over 1 years, weekly 1 - 2 sessions of 1 - 2 h duration . Schema modifications, behavioral experiments, cognitive restructuring, brief repeated exposures (real and imaginary), exposure and/or response prevention for obsessional symptoms were used during therapy . Psychiatrist trained to diagnose patients using atheoretical diagnostic systems and to treat by medicines is likely to over diagnose a psychotic illness in this case and treat accordingly . A flat affect, minimal talk, inhibited appearance, apparently unexplained anger outbursts and psychotic illness in a family member can lead to a misdiagnosis . A closer look reveals his warm affect, a keen interest to socialize and reasons for his anger . In fact historically this patient might have diagnosed as pseudo - neurotic schizophrenia, because of pervasive fears, phobias and strange sexual fantasies . In the past, severe avoidant cases were clubbed with schizoid personality . Knowledge of cognitive theory and therapy helped the author to arrive at a right understanding (diagnosis) of the patient and consequently the proper treatment . Patient warrants an additional diagnosis of intermittent explosive disorder because aggression is not a part of avoidant personality . He also had anankastic personality disorder, obsessive - compulsive disorder, body dysmorphic disorder and paraphilia and even a dhat syndrome . One of the foundations of cognitive theory and therapy is that the appraisal (personal meaning given) of the events results in the emotions . Similarly, his social fear can be explained by extreme sense of shame, sensitivity to insult and appraisal of people as dangerous and paranoid (patient's own word) about him . The typical pattern of behavior noted earlier, can be explained by the cognitive and emotional avoidances . Patient was intolerant of dysphoria and to reduce it, he repeatedly attempted to do one or the other activity . This case proves that psychotherapy can be a mainstay of treatment even in severe personality disorders.
|
A surgically resectable tumor in an anatomically or functionally solitary kidney is an absolute indication for nephron - sparing surgery (nss). The aim of nss is to maximize local tumor extirpation with optimal preservation of renal function to prevent the development of end - stage renal disease . Partial nephrectomy (pn) remains the treatment of choice, and encouraging oncologic outcomes in solitary kidney patients have been reported by several groups [1 - 3]. However, because it requires renal hilar clamping with possible renal ischemia, pn can have deleterious effects on renal function . Cryoablation offers nephron - sparing management of renal masses with fewer technical challenges, favorable oncologic results, shorter convalescence, and minimal parenchymal loss [4 - 6]. The feasibility and efficacy of cryoablative therapy for the management of tumors in solitary kidney patients has been reported earlier . However, sufficient data are lacking comparing the impact on renal function and oncologic outcomes of the two procedures in this specific subset of patients . In this article, we present the experience of a single institution with patients undergoing cryoablation and compare renal functional outcomes with those who underwent pn for such tumors . After obtaining the approval of the institutional review board, we examined the medical records between 1997 and 2007 of all patients who underwent nss for small, localized renal masses in a solitary kidney . The choice of procedure (cryoablation or pn) and the approach (open, laparoscopic, or percutaneous) were chosen on the basis of tumor characteristics, co - morbid conditions, patient preference, and surgeon discretion . The technique used for cryoablation has been described earlier by many authors [8 - 10]. An intraoperative ultrasound was used for laparoscopic and open cryoablation for tumor confirmation and monitoring of ice ball formation . Both open and laparoscopic partial nephrectomy (opn and lpn) were also performed by using standard techniques as described previously . The clinical features studied included patient demographics, perioperative details, and follow - up data . Radiology and pathology reports were reviewed for tumor size, location, and histological subtype . Renal function was assessed by calculating the glomerular filtration rate (gfr) from serum creatinine (scr) values by using the modification of diet in renal disease (mdrd) formula . Chronic kidney disease (ckd) was defined as a gfr of less than 60 ml / min/1.73 m. de novo ckd developed when the baseline renal function was normal but postsurgical gfr was abnormal on two separate occasions . All complications that occurred during the surgery or up to 30 days after the surgery were identified from the patients' medical records and evaluated . The patients were periodically followed up to look for any evidence of recurrence on ct imaging . Mann - whitney u tests were used to compare means and fisher's exact test was used to compare proportions . Kaplan meier survival curves were drawn for each group, and the log rank test was used to test for differences in survival . Of the 38 patients with a renal mass in their solitary kidney, 23 underwent cryoablation and 15 underwent pn . The patient demographics, tumor characteristics, and perioperative details are listed in table 1 . No significant differences between the two groups were found with respect to age, gender, and tumor laterality . However, patients who underwent pn had a higher mean tumor size than did the cryoablation group (3.4 cm vs. 2.5 cm, p=0.01). The mean estimated blood loss (ebl) during the procedure was also significantly higher in the pn group (316 cc vs. 87 cc respectively, p<0.001). Blood transfusion was not required in any patient who underwent cryoablation and was required in only 1 patient undergoing pn . Patients who underwent pn had a longer mean duration of hospital stay (5.8 days vs. 1.8 days, p<0.001) and a higher rate of perioperative complications (53.3% and 8.7% patients, respectively, p=0.03). Two patients (13.3%) in the pn group reported more than one complication . In the comparison of the effect on the gfr (as shown in table 2), there was a trend toward a better preserved gfr in the cryoablation group; however, this effect did not reach statistical significance between the two groups for percentage changes in the values on postoperative day 1 (p=0.1) or on follow - up (p=0.07). Chronic kidney disease was seen in 15 patients (65.2%) in the cryoablation group and in 9 patients (60%) in the pn group at the time of surgery . Table 3 outlines the morbidities associated with ckd, including hypertension, hyperlipidemia, diabetes, and heart disease . De novo development of ckd was seen in 3 of 8 patients (37.5%) in the cryoablation group and 2 of 6 patients (33.3%) in the pn group . The patients were followed up for a mean period of 31.2 months in the cryoablation group and 30.8 months in the pn group . No statistical difference was observed in the rates of progression of disease, which was seen in 3 patients each (13% and 20% in the cryoablation and pn groups, respectively, p=0.7). The mean tumor size in patients who showed disease progression on follow - up was 2.1 cm in the cryoablation group and 4.5 cm in the pn group . In the cryoablation group, 2 of the patients developed distant metastases to the pancreas and the lungs . One of these patients received therapy with il-2 and temsirolimus, but ultimately succumbed to the disease 2 years later . The second patient was put on sunitinib therapy, but was lost to follow - up . The third patient had a local recurrence of the tumor at the cryoablation site and underwent a radical nephrectomy . Local recurrence of disease occurred in 2 patients in the pn group, both of whom were treated with subsequent cryoablation . The third patient had multiple metastatic pulmonary nodules and died shortly thereafter following a rapid deterioration of pulmonary function . Hemodialysis was required in 2 patients (13.3%) in this group after the procedure . One of them developed end - stage renal disease immediately after pn on removal of a large tumor burden . The second patient required hemodialysis after a nephroureterectomy done for a transitional cell carcinoma 1 year after the initial procedure . The estimated mean overall survival of 88.9 months in the cryoablation group was similar to the 86.9 months in the pn group (p=0.8 on log rank test) (fig . Numerous studies have shown promising cancer - specific outcomes along with adequate preservation of renal function in solitary kidney tumors treated with pn [1 - 3]. Lpn and opn were shown to be similar with regard to oncologic efficacy and renal functional outcomes in a multicenter study . The duration of renal ischemia required during pn is the single most important modifiable risk factor for renal function maintenance . Beri et al used a combination of creatinine clearance time, peak concentration time, and mag 3 isotope clearance to accurately evaluate postoperative parenchymal function . Another group used the duration of renal parenchymal retention of mag 3 as a tool to assess ischemic renal damage . It is in keeping with these concerns that pn without ischemia has been gaining popularity in recent years . Cryoablation, which completely avoids hilar clamping and the subsequent renal ischemic insult, is indicated in patients who are elderly, in patients who are poor surgical candidates owing to comorbidities or previous surgical history, and in patients with solitary kidneys . Many studies have shown that cryoablation is technically less challenging, has a shorter duration of hospital stay, and is associated with improved convalescence [4 - 6]. Therefore, cryoablation is now being offered to many patients with cortical tumors less than 3 cm in size . However, there is a dearth of literature comparing preservation of renal function and oncologic outcomes between pn and cryoablation in the specific cohort of patients with solitary kidney tumors . Because a solitary kidney is the most significant risk factor for acute renal failure after nss, preservation of renal function assumes a very important role while choosing the preferred mode of treatment . Deteriorating renal function is associated with an increased risk of cardiovascular morbidity, hospital admissions, and mortality . Other equations like the cockcroft - gault and ckd - epi have also been shown to accurately estimate the gfr [24 - 26]. In two different studies, desai et al and o'malley et al compared the percentage changes in renal function between pn and cryoablation and found that they were similar . A study by turna et al showed that in solitary kidneys, the decrease in gfr caused by pn was significantly more than that caused by ablative techniques . Compared with this, we found that the percentage change in the gfr postoperatively and on follow - up was not statistically different in the two groups . Although the mean values indicate a trend toward lesser impact on the gfr for cryoablation, the p - value (0.07) did not reach significance, likely representing inadequate power to detect a difference with such small sample sizes . At the time of the last follow - up after surgery, 3 patients from the cryoablation cohort (37.5%) and 2 patients from the pn group (33.3%) had developed ckd de novo . However, these patients had a relatively lower gfr at the time of surgery than did the patients who had a normal gfr on follow - up (65 vs. 78 ml / min/1.73 m). In the follow - up of patients who had ckd at the time of treatment, only one cryoablation patient developed additional complications of ckd in the form of hypertension and diabetes, whereas two patients from the pn group had progression, one in the form of coronary artery disease with hyperlipidemia and another in the form of hypertension . All complications that occurred during surgery or up to 30 days after the procedure were defined as perioperative complications . With the widespread use of cryoablation two complications in the form of atrial flutter (n=1) and sinus tachycardia (n=1) occurred in the cryoablation patients . In the pn group, these included cardiac complications (atrial flutter [n=2], myocardial infarction [n=1]), respiratory complications (pulmonary edema [n=1], pneumonia [n=1]), ureteral transection (n=1), wound infection (n=1), flank cellulitis (n=1), jaundice (n=1), and bullous impetigo (n=1). Ghavamian et al and saranchuk et al reported complication rates of 23.8% and 26%, respectively, in patients undergoing pn for solitary kidney tumors . We observed a relatively higher complication rate in the pn group, with 53.3% of the patients having at least one complication . By comparison with the cryoablation group (8.7%), complications in patients treated with pn our results are in line with the data published by desai et al, who reported a higher incidence of complications in the pn group than in the cryoablation group . Turna et al published similar results with as many as 26 perioperative complications in 36 patients undergoing pn as compared with 5 complications in the cryoablation group . We also found that the pn group had more blood loss and a longer duration of hospital stay than did the cryoablation group . Thus, cryoablation offers the option of a nephron - sparing modality with lower morbidity, which could be of particular importance in older patients who are considered to be poor surgical candidates . Meticulous radiological studies were done at regular intervals postoperatively to look for any evidence of local or distant recurrence for an average follow - up of 30 months . Recurrence was seen in 3 patients each (13.3% and 20% in the cryoablation and pn groups, respectively). However, we found no statistically significant difference in disease recurrence or overall survival between the two groups . The three patients who witnessed a progression of disease in the pn cohort had a relatively larger mean tumor size of 4.5 cm as compared to their group (pn) mean size of 3.5 cm . The tumors in these patients were located near the hilum, making complete extirpation especially difficult . Intraoperatively, these three patients experienced a relatively higher amount of ebl (mean 1,400 cc) compared with the group's mean ebl of 316 cc . It is quite feasible that the large tumor size and the relatively inaccessible location of these tumors contributed to the higher intraoperative blood loss and an incomplete tumor excision . In the cryoablation cohort, the three patients who experienced disease recurrence had a mean tumor size of 2.1 cm, which was smaller than the group's mean tumor size of 2.5 cm . However, it is noteworthy that the tumors in these patients were also located near the hilum and were endophytic, located deep inside the kidney, making a thorough ablation relatively challenging . Collectively, we have presented a comparative analysis of renal function and oncologic status after pn or cryoablation in this uncommon cohort of patients with solitary kidney tumors . Our study demonstrates that in appropriately selected patients, renal cryoablation has distinct advantages over pn, including a shorter hospital stay, a lower complication rate, and less blood loss . Also, cryoablation was successful in maintaining optimal renal function and had oncologic outcomes comparable to pn on intermediate follow - up . Thus, cryoablation offers the option of a nephron - sparing modality with impressive renal function and oncologic outcomes and lower morbidity . We acknowledge the limitations of our study, which include among other things, the disparate tumor and sample sizes in the two groups . This could have been due to an inherent selection bias associated with the retrospective, nonrandomized design of our study . Our study was also limited by its relatively small sample size and an intermediate follow - up interval, which precludes a more comprehensive multivariate statistical analysis . This is also the reason the effect of the different approaches (open, laparoscopic, and percutaneous) in the two groups could not be accounted for in the analysis . Therefore, pooled analyses from additional reports comprising larger datasets with longer clinical follow - ups (5 and 10 years) are warranted to better delineate the role of these two procedures in maintaining renal function in this high - risk cohort . In conclusion, the increasing frequency of detection of tumors in solitary kidneys demands a better outlined strategy with stricter selection criteria for effective treatment choice along with long - term renal function maintenance . Both cryoablation and pn are viable techniques with acceptable preservation of renal function for tumors of a small renal mass . Although pn remains the standard of care, cryoablation offers an excellent treatment alternative, with less morbidity, comparable oncologic and functional outcomes, and faster recovery.
|
Dr clark is the director of the centre for gambling research at ubc, which is supported by funding from the province of british columbia and the british columbia lottery corporation (bclc). Dr clark has provided paid consultancy to cambridge cognition ltd . On issues relating to neurocognitive assessment . Dr clark has not received any other direct or indirect payments from the gambling industry or any other groups substantially funded by gambling to conduct research or to speak at conferences or events.
|
Persistence of virulent microorganisms and their byproducts in the root canal system, or the surrounding tissues, following initial endodontic treatment, is the main cause for treatment failure . It has been reported that failing root canal treatments associated with periradicular pathosis frequently demonstrated fungi colonization . The exact factors affecting the colonization of the root canal by fungi are not completely understood . It seems that the most common predisposing factors are certain intracanal medicaments, local and systemic antibiotics, and previous unsuccessful endodontic treatment . It has been hypothesized that the reduction of specific strains of bacteria in the root canal during endodontic treatment may allow fungi overgrowth in the low nutrient environment . In addition, it is possible that fungi, such as c. albicans, may gain access to the root canal from the oral cavity as a result of poor asepsis during endodontic treatment procedures or because of coronal leakage . In fact, the presence of c. albicans in root canals was found to be directly associated with its presence in the oral cavity of the same patients . Failure of initial endodontic treatment can often be treated successfully by retreatment or endodontic surgery . Elimination of the microbial flora and infected tissues as well as a complete seal of the root canal system, to prevent future recontamination, will enhance treatment success . In this regard, mineral trioxide aggregate (mta) has become a popular material to seal off communications between the root canal system and the external surface of the root . It has been mostly used as a retrograde filling material during apical surgery and as a sealant of root perforations . However, mta is difficult to use because of its longer setting time and poor handling properties . Mta (proroot mta, dentsply / tulsa dental, tulsa, ok) is marketed in gray - colored and white - colored preparations; both are 75% portland cement, 20% bismuth oxide, and 5% gypsum by weight . Recently, the use of the white - colored preparation became more popular because of esthetic considerations . Mta is a powder that consists of fine hydrophilic particles that in the presence of water, or moisture, forms a colloidal gel that solidifies to form hard cement within approximately 4 h. the principal constituents of the gray - colored formula are tricalcium oxide, tricalcium silicate, bismuth oxide, dicalcium silicate, tricalcium aluminate, tetracalcium aluminoferrite, and calcium sulfate dihydrate . It has been reported that a fixed concentration of white - colored mta was effective against c. albicans in vitro for periods of up to 3 days . It has also been shown that white - colored mta in concentration of 50 mg / ml was effective in killing c. albicans for periods of up to 3 days, whereas lower concentrations were not as effective . In another study, fungal growth occurred during 1-h incubation in both freshly mixed and 24-h set gray and white mta cements, whereas by increasing the incubation time, no fungal growth was observed in 24 and 72 h. it was also reported that both gray - colored and white - colored mta in concentrations of 50 mg / ml and 25 mg / ml were effective in killing c. albicans for periods up to 1 week . Lower concentrations of gray - colored mta might still be effective, whereas lower concentrations of white - colored mta might not . However, the antifungal effect of mta on multiple strains of c. albicans over longer periods of time is less investigated . The purpose of this study was to evaluate the antifungal action of various concentrations of white - colored mta against seven different c. albicans strains for various exposure periods using the tube dilution test . Fresh mix of white - colored mta (pro - root mta, batch #05002015, dentsply / tulsa dental, tulsa, ok) was prepared at concentrations of 100, 50, 25, and 12.5 mg / ml by dilution with distilled water . Each freshly prepared mix was added to a broth tube containing sabouraud's liquid medium (hi - media laboratories, mumbai, india). A total of 1287 broth tubes were prepared and divided into seven sets with experimental groups of 11 tubes each and control groups . Broth tubes without mta served as positive control and tubes without c. albicans served as negative control . The antifungal action of mta against c. albicans was evaluated by using the tube dilution test . Stock cultures of six strains of c. albicans clinically isolated from the oral cavity (strains 1 - 6: c-89, e-175, c-304, e-259, c-176, and e-170) and a standard strain (strain 7: microbial type culture collection (mtcc) 3017) were obtained . Fresh inoculate of the microorganism was prepared by growing overnight cultures until a complete suspension of growth was achieved . The cultures were diluted in a pre - warmed broth to achieve a final density of 10 colony - forming unit (cfu)/spot . One - milliliter aliquots of the test c. albicans were taken from the broth culture and added to sterile capped test tubes of each experimental and positive control group . All experimental and control groups were then incubated at 37c and evaluated at 1-, 24-, 72-, and 168-h (7 days) time periods . At each time period, 0.1 ml of samples from each tube was subcultured on sabouraud's dextrose agar plates (hi - media laboratories, mumbai, india). Differences among the groups were statistically analyzed using kruskal wallis and mann whitney u tests (statistical package for the social sciences (spss) 7 software). If the calculated critical values were at least as large as the respective critical values at the 0.05 level, then calculations were considered statistically significant in this study . Generally, a direct correlation was found between mta concentrations and its inhibition effect on c. albicans growth . Differing behavior patterns of various strains of c. albicans were also seen [table 1]. Strains 5, 6, and 7 displayed intermediate resistance to mta, whereas strains 3 and 4 appeared to be most resistant . The negative controls showed no fungal growth at any time period, whereas the positive controls always showed fungal growth . Effect of various mta concentrations on growth of c. albicans strains statistically, a significant difference was found between tubes containing either strain 3 or 4 and tubes containing other strains at all mta concentrations at the 1-day time period (p <0.001). A significant difference was found between tubes containing strain 3 and tubes containing other strains at mta concentrations of 12.5 and 25 mg / ml at the 3-days time period (p <0.001). A significant difference was also found between tubes containing 12.5 mg / ml and tubes containing higher concentrations of mta at the 7-days time period (p <0.001). The method used in the present study was the tube dilution test, which is an effective method to evaluate the antifungal and antibacterial properties of any filling material or solution . It was considered appropriate for evaluating the antifungal activity of mta, which has a low solubility and diffusibility . C. albicans was the test organism in this study . It is a frequent colonizer of the oral cavity, and multiple unrelated strains showing varying pathogenicity may be present in the same individual . Mechanisms of pathogenicity of different strains may include the power of adaptability to a variety of environmental conditions attributable to the switching of gene expression dictated by environmental changes and evasion and immunomodulation of the host defenses by different mechanisms . C. albicans is frequently associated with failing root canal treatments and has the ability to form biofilms on different surfaces, which makes it more pathogenic than species that are less able to form biofilms . As it may be involved in cases of persistent and secondary infections, usually present during apical surgery and perforation repair procedures, the antifungal action of the repair material may further assist in the management of such infections and enhance tissue healing . In this study, growth of seven strains of c. albicans in four concentrations of mta was assessed for periods of up to 7 days . Only one strain showed relative resistance even after 3 days at the lower mta concentration of 12.5 and 25 mg / ml . Recurrence of fungal growth was seen with three strains at mta concentration of 12.5 mg / ml after 7 days . It might indicate that mta only exhibits a fungistatic action at lower concentrations, whereas a fungicidal action may be expected at higher concentrations . This highlights the significance of using seven strains in the present study . In a previous study, al - nazhan and al - judai reported white - colored mta to be effective in killing c. albicans in vitro for a period of up to 3 days . However, in that study a higher concentration of mta was used . In another in vitro study, al - hezaimi et al ., assessed the antifungal action of different concentrations of white - colored mta against c. albicans in vitro . White - colored mta in concentration of 50 mg / ml was effective in inhibiting c. albicans for periods of up to 3 days, whereas lower concentrations were not effective . Mohammadi et al ., observed fungal growth during 1-h incubation in both freshly mixed and 24-h set gray and white mta cements, whereas by increasing the incubation time, no fungal growth was observed in 24 and 72 h. another study also showed that both white and gray mta in concentrations of 50 and 25 mg / ml were effective in inhibiting c. albicans for periods of up to 7 days . Kangarlou et al ., assessed the antifungal activity of proroot mta and mta - angelus and concluded that mta - angelus was a more effective antifungal agent compared with proroot mta at concentrations of 50 and 100 mg / ml . Regarding the antifungal action of mta, it has been shown that the mechanism of action of mta involves the dissolution of calcium oxide, which increases ph by releasing ca and oh . The high ph increases the permeability of cell membranes with leakage of intracellular components . Waltimo et al ., reported that c. albicans survived incubation in calcium hydroxide solution for 1 and 6 h and was killed after 6 h of incubation . Ferguson et al ., found that an aqueous solution of calcium hydroxide had no activity against c. albicans . However, when maintained in direct contact with c. albicans cells, calcium hydroxide paste was very effective in killing this fungus . Therefore, it can be assumed that mta is more effective in killing c. albicans when placed in direct contact with the fungus . In addition, samiei et al ., reported that adding silver nanoparticles to mta improved its antimicrobial efficacy . In conclusion, it appears that under the conditions of this study, white - colored mta in concentrations of 100 and 50 mg / ml is effective in inhibiting the seven tested strains of c. albicans for periods up to 1 week . However, the antifungal activity of white - colored and gray - colored mta over longer periods of time on more number of strains merits further investigation.
|
Primary stability of implants is commonly considered as a key factor for achieving successful osteointegration . Micro - motions higher than the threshold of 50 to 100 m can lead to formation of fibrous tissue at the bone - to - implant interface . Hence, when immediate loading of the dental implant is preferred as the treatment strategy, primary stability of the implant must be taken into account as a determining criterion [46]. Numerous techniques have been introduced to evaluate implant stability, among which the technique developed by meredith in 19987 is a straightforward and noninvasive method . Meredith technique can reproducibly assess the bone - to - implant contact through direct attachment of a transducer to the abutment or the implant body with the application of osstell mentor device . This technique expresses the implant stability by reading the implant stability quotient (isq), obtained through the resonance frequency analysis (rfa). The isq values range from 1 to 100 with higher values of the isq indicating higher implant stability [8, 9]. It has been discussed by several researchers that some factors, such as bone quantity and quality, the surgical technique of implant placement and geometrical factors of the implant including shape, length and diameter may influence the primary stability of the implant [3, 10, 11]. As bone density affects the amount of bone - to - implant contact, high bone density on the implant side can positively influence the primary implant stability . However; there is no unanimous agreement on the impacts of the implant type or geometry as to how effectively they improve the primary stability in different bone types . Argued that length and diameter of the implants have no significant effects on the isq . It should be noted, however, that in the clinical study conducted by bischof et al . Patients with bone type iv were excluded . According to a study performed by ostman et al . Placement of wide platform implants in the posterior regions can lead to achieving higher isq values . On the other hand, lower isq values were reported when the length of the implants increased . A finite element analysis by winter et al . Demonstrated that in a higher level of bone stiffness, the implant length did not have any significant effect on isq values . However, in simulation of the lower level of bone stiffness a positive correlation was observed between the implant length and stability . Compared the effect of different implant diameters of 3.8 mm and 4.6 mm on the primary stability in cancellous bone and found no statistically significant differences in isq values . Studied the effect of implant diameter on implant primary stability in d1 and d2 bone types and postulated that the implant diameter did not have any significant effects on primary implant stability . The aim of this study was to evaluate the influence of implant length and diameter on primary implant stability in different bone types, based on resonance frequency analysis through in vitro conditions to avoid the effects of other intermediary factors . To evaluate the effects of the quality of the bone bed on the primary stability of the implant, two different artificial bone blocks similar to d1 and d3 bone types were prepared . A number of 60 nobel biocare replace select tiunit tapered implants with two different lengths (10 mm and 13 mm) and three different widths of 3.4 mm (narrow platform (np)), 4.3 mm (regular platform (rp)) and 5 mm (wide platform (wp)) were utilized for primary stability evaluation . Two main groups, based on the bone type, and 6 subgroups, based on the geometrical features of the implants, were formed, as presented in table 1 . In each case the surgical protocol was performed based on the instruction provided by the manufacturer . Immediately after implant placement, primary implant stability was measured based on the rfa using the osstell mentor device (osstell tm mentor; integration diagnostics ab, sweden) and the isq values were recorded . The mean isq values of each of the twelve groups studied in the current research were statistically compared using t - test and tukey s hsd post - hoc test . Mean values and standard deviations of the isq values for each of the twelve groups, studied in this paper, are presented in table 2 . Univariate analysis of variance was performed to assess if the variables, i.e. Bone type and implant length and diameter, had any interactions . Since the tests indicated that significant interactions existed between the variables, t - test with p value adjustment, using bonferroni correction method, was performed to evaluate the effect of bone type together with implant length on the isq values . Tukey s hsd post - hoc test was also conducted to evaluate the effect of the implant diameter on the isq values . The isq values measured for implants placed in d1 bone type were significantly higher than those measured for implants with the same length and diameter, but placed in d3 bone type (p 0.001). When utilizing wp implants of 13 mm and 10 mm length, the comparison of isq values for implant placements in d1 bone type indicated that the isq values were significantly higher for longer implants, with the mean difference of 2.60 (p = 0.000). More importantly, for implants of 13 mm in length, the isq values were even higher when placing the implant in d3 bone type, with the mean difference of 9.60 (p = 0.005). Rp implants of 13 mm in length presented significantly higher isq values compared to their 10 mm counterparts when placed in d3 bone type (p = 0.011). However, different lengths of rp implants did not make any significant differences in isq values measured for implants placed in d1 bone type (p = 0.785). In np implants, there was a positive correlation between the implant length and the isq values in d3 bone type (p = 0.001), while in d1 bone type no statistically significant difference was observed in the isq values when np implants of different lengths were applied (p = 0.065). Analysis of the experimental data revealed that narrow platform implants presented significantly lower isqs in comparison with regular and wide platform implants (p 0.007). However, no significant differences were observed when comparing the isq values for regular and wide platform implants (p 0.215) the isq values measured for implants placed in d1 bone type were significantly higher than those measured for implants with the same length and diameter, but placed in d3 bone type (p 0.001). When utilizing wp implants of 13 mm and 10 mm length, the comparison of isq values for implant placements in d1 bone type indicated that the isq values were significantly higher for longer implants, with the mean difference of 2.60 (p = 0.000). More importantly, for implants of 13 mm in length, the isq values were even higher when placing the implant in d3 bone type, with the mean difference of 9.60 (p = 0.005). Rp implants of 13 mm in length presented significantly higher isq values compared to their 10 mm counterparts when placed in d3 bone type (p = 0.011). However, different lengths of rp implants did not make any significant differences in isq values measured for implants placed in d1 bone type (p = 0.785). In np implants, there was a positive correlation between the implant length and the isq values in d3 bone type (p = 0.001), while in d1 bone type no statistically significant difference was observed in the isq values when np implants of different lengths were applied (p = 0.065). Analysis of the experimental data revealed that narrow platform implants presented significantly lower isqs in comparison with regular and wide platform implants (p 0.007). However, no significant differences were observed when comparing the isq values for regular and wide platform implants (p 0.215) primary stability of the implant can be defined as the absence of implant movement, including micro - motions, immediately after insertion of the implant into the bone bed [2, 3]. The bone quality and implant length and diameter have been assumed to be influential on the bone - to - implant contact and consequently on implant primary stability . In this paper, pointed out that the presence of cortical bone, which is 10 to 20 times more rigid than cancellous bone, can be the cause of high primary stability in high bone quality . Based on the results of this study, which is in accordance with the result of numerous clinical studies [8, 13, 16, 17], the authors recommend avoiding the immediate loading protocol after implant placement in low quality bones . The same approach was also proposed by trisi et al . Who measured primary stability based on insertion torque in different bone densities . Moreover, neugebauer et al . Concluded, from their animal study, that implant immediate loading can be applicable only when a high primary stability is achieved . According to a study carried out by javed et al ., immediate loading in the anterior region of the mandible showed a high success rate due to good bone quality in this region . However, recently degidi et al . Noted that the different implant geometry, used in their study may be one of the reasons of inconsistency between their results and results from other studies [6, 11]. The implant used by degidi et al . Was xive implant, which has a cylindrical core with an increasing thread depth from the crestal to apical region in a way that the thread pitch remains equal . Degidi et al . Also denied the importance of implant length and diameter on the isq values, since they only found a weak correlation between rfa and implant length and diameter . Did not analyze the interaction between the effects of implant geometrical factors (length and diameter) and different bone qualities on rfa . On the contrary, the current study showed that there is an interaction between the effects of implant geometrical variables and the quality of bone bed on rfa . Based on the results of the current study, implant length did not have any significant influence on primary stability when there was a high bone quality on the implant side . However, in cases of insufficient bone quality, an increase in implant length resulted in an increase in implant primary stability . In addition, in accordance with the results of the current study, a finite element analysis by winter et al . Reported a positive correlation between implant stability and implant length in low levels of bone stiffness . Moreover, lachman et al . Conducted an in vitro study to compare implants with different lengths of 11 mm, 13 mm, 15 mm and 18 mm . They concluded that only11 mm - long implants, which were placed in soft bone blocks, presented significantly lower isq values . On the contrary, ostman et al . Reported that the use of longer implants resulted in lower primary stability . They compared primary stability of implants with the lengths of 7 mm, 8.5 mm, 10 mm, 11.5 mm, 13 mm, 15 mm and 18 mm and found that by increasing the implant length from 8.5 mm to 10 mm, primary stability increases and for a range of implant lengths from 10 mm to 13 mm, primary stability is almost constant . They also found that implants 15 mm and 18 mm in length resulted in lower primary stability compared to implants 13 mm in length, mainly because they were more heat generated due to the longer bone drilling . Therefore, in low quality bone types with an inadequate bone height, and bone augmentation should be performed instead of applying short implants . Moreover, implant site preparation with osteotome technique is preferred since it can improve the bone density [19, 20]. Furthermore, application of implants that are specially designed to achieve more primary stability should be given consideration . It is also believed by some researchers that the cortical bone is more rigid than cancellous bone and the highest load concentrates in the cortical zone and thus, the role of implant width is more important than the role of implant length to achieve high primary stability [3, 15]. In the current study, np implants presented a significantly lower isq when compared with rp and wp implants, especially in d3 bone type . Conducted a study on nobel biocare replace select tiunit tapered implants and found that rp (4.3 mm) implants presented a significantly higher primary stability compared to np (3.5 mm) implants . On the other hand, ostman et al . Compared implants of 3.75 mm, 4 mm and 5 mm diameters and found that wp (5 mm) implants had significantly higher primary stability; however, he did not observe any difference between rp and np implants . The reason for this similarity in primary stability of rp and np implants can be ascribed to the slight difference between implant diameter in rp and np implants in ostman s study . Accordingly, in cases of low bone quality, it is recommended to avoid using np implants and it is preferred to perform bone reconstruction methods such as guided bone regeneration . Basically, application of np implants must be limited to the anterior region of the mandible and premolar sides in the maxilla where implants are imposed to mild occlusal loads . Since isq values in rp and wp implants did not show any significant differences, the authors suggest using rp implants to preserve further thickness of bony walls . In general, in order to achieve high primary stability of implants placed in low bone quality, optimum increase in length and diameter of implants should be seriously considered . Within the limitation of the current in vitro study it can be concluded that implant primary stability was higher in high bone quality . Implant length was a determining factor to achieve more primary stability in low bone quality . The difference in primary stability, which was affected by application of narrow platform implants, was more prominent in low bone quality rp and wp implants did not have any significant influence in terms of implant primary stability.
|
Glutathione (gsh) is a ubiquitous tripeptide (l - y - glutamyl - l - cysteinylglycine) known to function in many aspects of cellular activity including protein and dna synthesis, transport, detoxification of xenobiotics and carcinogens, metabolism, and defense against free radicals and oxidative stress . It is synthesized in the -glutamyl cycle (fig 1) by glutathione synthetase (gs). Gsh is most commonly found in its reduced form with only 15% existing in the oxidized form glutathione - disulphide (gssg). Gssg is reduced back to gsh by glutathione - disulphide reductase requiring one equivalent of nadph . Biallelic, pathogenic variants in the gss generesult in glutathione synthetase deficiency (gssd). Gssd (mim:266130) is a rare autosomal recessive disorder that has only been described in approximately 70 individuals worldwide . Erythrocytes have some of the highest concentrations of gsh in the body, and thus, erythrocytes from patients with gssd are more susceptible to oxidative stress . As a result, patients with the mild gssd present with isolated hemolytic anemia as their only clinical symptom whereas patients with the moderate form of the disorder present with hemolytic anemia and metabolic acidosis in the neonatal period . In addition to these symptoms, patients with severe disease have neurologic findings including motor disturbances and developmental delay . Some severely affected patients show an increased susceptibility to bacterial infections, thought to be due to defective granulocyte function . To date there is only one severely affected individual who was treated with antioxidants from birth and long - term follow - up on that individual is not reported . 5-oxoprolinuria can also be found in all patients with gssd due to the accumulation of -glutamylcysteine being hydrolyzed to 5-oxoproline and cysteine by -glutamyl cyclotransferase (gct). This accumulation of 5-oxoproline exceeds the capacity of 5-oxoprolinase leading to high urinary excretion of 5-oxoproline that is detectable on urine organic acid analysis . In the present report, we describe long - term follow - up of a case of severe gssd . The proband is a female born at 40 weeks gestation by cesarean section weighing 2.970 kg (16 centile) with apgar scores of 8 and 9 at 1 and 5 minutes respectively . At approximately 18 hours of life, arterial blood gas showed ph of 7.32 (normal ph 7.357.45), paco2 17 mmhg (normal 3545 mmhg), pao2 94 mmhg (normal 80100 mmhg) and bicarbonate of 8 mmol / l (normal 2132 mmol / l). She was given 5 mg intravenous (iv) sodium bicarbonate and transferred to a tertiary hospital at which time her metabolic acidosis had worsened to ph 7.15, paco2 12 mmhg, pao2 94 mmhg and bicarbonate of 4 mmol / l . She was treated with iv fluids and iv sodium bicarbonate of 1.1 meq / kg / hr . Urine organic acid analysis performed at this time revealed high levels of 5-oxoproline, suggesting a defect in glutathione metabolism . Once stabilized, she was started on a combination of citric acid, potassium citrate, and sodium citrate (~30 meq / kg / day)for metabolic acidosis and vitamin c (250 mg per day)and e (30 international units per day) to help prevent oxidative stress . Mother was also advised to avoid foods and medications that cause oxidative stress (from a list standardly used for individuals with glucose-6-phosphate dehydrogenase (g6pd) deficiency). She was then discharged home with follow - up in the metabolic clinic . At her 2-month follow - up visit at the metabolic clinic, she had remained stable with no intercurrent illnesses or hospitalizations since her discharge . She was tolerating her medications and growth was satisfactory (40 centile for weight, 10 centile for height). Laboratory results showed mild macrocytic anemia with reticulocytosis and hyperkalemia, which became her baseline for most of her life, and a normal bicarbonate of 23 mmol / l . At 6 months her red blood cell gsh and gs activity levels were analyzed, and she had a marked decrease in both gsh level and gs activity (see table 1), consistent with the diagnosis of gssd . Her mother also had a decrease in gs activity, although normal gsh levels, suggesting heterozygosity . Throughout early childhood, she had multiple episodes of otitis media which resolved with antibiotics and multiple minor viral respiratory infections . She also developed an episode of tachypnea and acidosis in the setting of 4 days of fever, watery diarrhea, congestion and spitting up . She was hospitalized again at 18 years of age when a viral gastroenteritis caused decreased oral intake and inability to take medications; her serum bicarbonate level at that time was 7 numerous dental caries were reported, and her dental healthy improved after a switch to a sugar free combination of citric acid, potassium citrate, and sodium citrate . At approximately 2 years 3 months of age, there was concern that increased oxidative stress might hinder her developmental progress, so she was started on selenium one crushed tablet daily . A formal developmental evaluation was performed at approximately 2 years 6 months and was reported as normal . However, by 3 years 9 months developmental delays were evident . She was unable to copy a circle or ride a tricycle and her language was unintelligible . Speech therapy led to significant improvement, and by 4.5 years her speech had greatly improved and her other developmental concerns resolved . At her 33 months of age, weight loss and deceleration of head growth were noted, and these findings were attributed to chronic metabolic acidosis (bicarbonate of 1518 mol / l), her citric acid, potassium citrate, and sodium citrate was adjusted to ~18 meq / kg / day and by three years of age, her acidosis and weight gain had improved . Ophthalmology evaluation for pigmentary retinopathy, a known finding in patients with gssd, was normal . At the age of 5 years, she underwent a tonsillectomy and adenoidectomy for chronic snoring and mouth breathing without any complications . She was diagnosed with attention deficit disorder at approximately 7 years of age and was successfully treated with guanfacine . At approximately 10 years, she was diagnosed with a seizure disorder and treated with the antiepileptic lamotrigine . However, by age 15 years, she had no additional seizures, and her eeg was normal so she was successfully weaned off lamotrigine . At age 16 years, her psychologist diagnosed her with asperger syndrome . She is currently prescribed guanfacine for adhd, citric acid, potassium citrate, and sodium citrate syrup (11 meq / kg / day) for metabolic acidosis, and vitamin c (14.5 mg / kg / day), vitamin e (150 international units per day) and selenium (50 mcg per day) to prevent oxidative stress . However, high doses of vitamins c and e are recommended in all patients for protection against oxidative stress . Avoidance of foods and drugs known to cause hemolytic crisis in g6pd deficiency is also important as these same triggers can cause hemolytic crisis in gssd . Vitamin e is also used to prevent granulocyte dysfunction, which could cause recurrent infections . Patients with moderate and severe phenotypes typically require treatment for metabolic acidosis . In acute crisis, iv bicarbonate is given for immediate correction, and for long term management, citrate or trometamol (tham) are given . Our patient has required very high doses of citric acid, potassium citrate, and sodium citrate (ranging from ~30 meq / kg / day in early infancy to approximately 1020 meq / kg / day throughout most of her childhood and adolescent years) to maintain a normal serum bicarbonate level (figure 2). Moreover, her persistently elevated reticulocyte count (figure 2) reflects ongoing but compensated hemolytic anemia . Previously, cysteine delivery compounds, specifically n - acetylcysteine (nac), were given because they increase gsh levels in healthy patients . However, nac has been found to increase intracellular levels of cysteine, which are already elevated in gssd and these high levels of cysteine are known to be neurotoxic . Selenium was another agent used in our patient to prevent oxidative stress, and to our knowledge this is the first reported use of selenium in a patient with gssd . Selenium is found to have strong antioxidant properties through formation of selenoproteins, which are thought to protect against reactive oxygen species . If a neonate presents with hemolytic anemia and metabolic acidosis, it is important to consider gssd, as rapidly fatal gssd in newborns has been described . Advanced diagnostic techniques such as antenatal diagnosis can be made by measuring 5-oxoproline in amniotic fluid or a presumptive diagnosis can be made by detecting elevation of 5-oxoproline in newborn screen blood spots using tandem mass - spectrometry . Our patient s diagnosis was made within a few days of life, based on urine organic acid analysis and thus, treatment with vitamin c and e were started early . Njalsson et al (2005) demonstrated that early initiation of vitamins c and e could prevent the moderate phenotype from progressing to severe phenotype . In a study of 41 patients with gssd, only 1/18 of the severely affected patients were started on vitamin therapy early in life as compared to 6/17 moderately affected patients . With this data and the fact that there is no significant difference between enzymatic activity in the moderate and severe phenotypes, they posit that early initiation of vitamins could prevent or slow down progression of the disease . There were no specific comments on length of follow - up of this cohort however . Our patient was diagnosed at birth with moderate disease because of her acidosis and hemolytic anemia . However, at the age of 10 years, she developed seizures, a finding which is more consistent with the severe phenotype . Her seizures were very mild and anti - epileptics were eventually discontinued once she was seizure free for 5 years . We posit that early initiation of vitamins and combination citric acid, potassium citrate, and sodium citrate coupled with good compliance explains her relatively mild course . Our patient had an episode of weight loss and deceleration in head growth in early childhood that was attributed to chronic metabolic acidosis and that improved with increased doses of her medications . Currently, she has short stature (<5 percentile, z - score 2.1) with appropriate weight and head circumference . Developmentally, our patient experienced minor speech delays early in life that required speech therapy and there were concerns for mild fine motor delay that resolved without intervention . However, she is currently performing satisfactorily in high school with plans for attending college . In conclusion, we describe long - term follow - up of a patient with severe gssd and good outcome . We attribute early recognition of the disease, initiation of appropriate vitamin therapy and acidosis correction along with excellent compliance from our patient for her success to date . We also believe the use of selenium as an additional antioxidant has contributed to her relatively mild course . Finally, we recommend testing for gssd in patients with metabolic acidosis or hemolytic anemia in the newborn period as early recognition and initiation of therapy appears to correlate with better outcomes.
|
Thyroid incidentalomas are newly detected thyroid nodules, discovered during a computed tomography (ct), magnetic resonance imaging (mri), ultrasound (us), or positron emission tomography (pet) exam for nonthyroid diseases . Focal type is defined as an area of uptake of 2-fluorodeoxyglucose (fdg) in less than one lobe, whereas diffuse type is fdg uptake in the entire thyroid gland . There are guidelines for managing palpable thyroid nodules but there are no such guidelines for nonpalpable nodules . Fdg - pet has a reported sensitivity of 7590% and a specificity of 90% for detecting thyroid malignancies . Whole body fdg - pet / ct combines the technology of whole body ct scan with fdg uptake localization to assess glucose utilization rates . The vast majority of solid tumors have an enhanced glycolytic rate, and they are therefore amenable of being imaged with fdg - pet . Fdg - pet has become the standard of practice for staging, restaging, and assessment of therapy response in a variety of malignant solid tumors . Normal thyroid gland shows very low fdg uptake, and some data suggest that a moderate diffuse uptake can represent a normal variant . However, fdg has high accumulation in tumors because of increased glucose transport and glycolysis . Benign diseases of the thyroid such as graves' disease and thyroiditis can have high standard uptake values (suv) because of increased blood flow, increased glucose metabolism, autoimmune response by lymphocytes, and inflammation . Therefore, areas with high fdg uptake can be nonspecific and can range from benign processes to malignant neoplasms . Thyroid nodules have different morphologic appearances, and they can be seen as diffuse or focused lesions on the pet / ct . Us is usually the first step in the management of thyroid abnormalities found in physical and laboratory exams . The overall incidence of thyroid incidentalomas is on the rise with 2.4 times increase in the last decade . Based on the pet / ct alone, definite diagnosis cannot be made since there are no established criteria to diagnose benign or malignant incidentalomas . Current practice is to biopsy all nodules> 1 cm and all nodules <1 cm with risk factors for malignancy . This study also aims to assess the value of pet / ct as a screening tool to screen for thyroid incidentalomas . There is no report on the prevalence of pet / ct thyroid incidentalomas in the general population . Yet, thyroid incidentalomas are quite common, and their prevalence depends on the population studied, region or country where the study was conducted, and the diagnostic methods used . We performed a retrospective analysis of all whole body fdg - pet / ct done at our institution between 1/1/2000 and 8/20/2008 (figure 1). An inclusion criteria was that the lesions were not visible or palpable during routine clinical exam . We excluded from our study all cases with prior histories of thyroid neoplasms and thyroidectomies or any type of thyroid surgery . A total of 156 patients who had abnormal fdg uptake were further selected for secondary analysis . From this group, univariate one - way analysis of variance (anova) was performed to characterize differences between segments classified according to the results of diagnostic methods . A p value of <0.05 was considered statistically significant . In this study, only 40 out of 156 incidentalomas (25.6%) had biopsies . There are lesions with low, hyperdense, and a mixture of low and high attenuations . Our results show no correlation between the size of the lesion and the suv value . There are papers that show the correlation between size and suv value while some show no correlation . In the group of cancerous incidentalomas, there are 6 males (mean age 53.3, age range 3378) and 9 females (mean age 56.2, age range 3873). For the group with benign incidentalomas, there are 4 males (mean age 76.5, age range 6983) and 21 females (mean age 59.1, age range 3986). 14 out of 15 (93%) malignant lesions have focal uptake, and the remaining 1 has diffuse uptake . This finding agrees with prior studies in that focal uptake lesions have> 30% increase in malignancy rates compared to diffuse uptake lesions . Among cancerous nodules, the highest suv value is 9.9 and the lowest is 2.5 (figure 2), and for the benign lesions, the highest is 46 and the lowest is 0.8 . From the 40 biopsy reports, there are 15 cancerous nodules, corresponding to about 38% . There are 11 papillary carcinomas (6 right lobe, 3 left lobe, 2 both lobes), 1 follicular carcinoma (left lobe), 1 anaplastic carcinoma (right lobe), 1 adenocarcinoma from the breast primary (left lobe), and 1 metastasis of unknown origin (right lobe). Among benign incidentalomas, there are 2 nonspecific benign lesions (1 left lobe and 1 right lobe), 18 colloid lesions (10 left lobe and 8 right lobe), 1 chronic lymphocytic thyroiditis (right lobe), 3 follicular adenomas (2 left lobe and 1 right lobe), and 1 hurthle cell lesion (right lobe). Generally, incidentalomas found in males at young age carry a risk of being malignant . We also find that incidentalomas are far more common in females than males, and this is in agreement with other studies . Framingham population - based study reported the thyroid nodule prevalence of 6.4% for females versus 1.6% for males . The average size of nodules in the malignant group (n = 12) is 2.0 cm (range 0.66 cm), and that in the benign group (n = 20) is 1.4 cm (range 0.22.4 cm). The graphical representation of sizes in figure 3 shows that the majority of sizes of benign and malignant incidentalomas are in the same range (i.e., below 3 cm) with only 2 of the lesions in the malignant group (5 cm and 6 cm, resp .) Outside this range . We cannot safely conclude that small nodules are benign and large nodules are cancerous, and there is no agreement yet on the cutoff value of the size of the lesion to warrant further workup . Adequate samples are difficult to obtain from lesions <8 mm . On the other hand, studies have shown that cancer prevalence for lesions <1 and> 1 cm is the same, and so we must take the other risk factors into consideration besides the size . Moreover, papini et al . Who examined us features of thyroid nodules also found no correlation between malignancy and the dimensions of the lesion or the multinodularity . Attenuation is a feature of ct, and low attenuation means that a particular area is less intense than the surrounding . All of the malignant nodules confirmed by biopsy have low attenuation, with the exception of two which have a mixture of high and low attenuation . For this study, some studies associate low attenuation with malignancy while some suggest high attenuation for malignancy [4, 6]. Diffuse fdg uptake is often due to hypothyroidism or thyroiditis, especially hashimoto's or autoimmune thyroiditis . One study reported higher likelihood of cancer when the average suv is greater than 5.69 but most other studies cannot find a correlation between suv and malignancy [9, 19]. In our study, the average suv value of the malignant group (n = 15) is 5.5, and that of the benign group (n = 25) is 7.2 . As a result, considering these information, we cannot set a threshold suv value for malignant lesions . Our prevalence of 38% falls within the range of 1850% reported in the literature for prevalence of malignancy among incidentalomas . In our study, most of the malignant incidentalomas are primary thyroid malignancies, and this is similar to other studies where most cancerous incidentalomas are thyroid primary . Papillary carcinomas are most common, and the majority of them occur as right lobe lesions . Prevalence of incidentalomas found by pet / ct at our institution is 1.84%, which is within the range of 1.24.3% reported by liu . The results of our study are compared to studies done on incidentalomas at other institutions (table 3).the investigations at various hospitals mentioned in table 3 reflect different study designs, patient populations, and institutional practices . The prevalence of incidentalomas range from 1.1% to 7% and the prevalence of malignancy among incidentalomas ranges from 14% to 66% . A new thyroid nodule appears at the rate of 0.08% per year in the general population, and the incidence of thyroid malignancy is 0.0040.1% per year . Not surprisingly, the incidence and prevalence of thyroid cancer in the patient population we see at the nuclear medicine department at ucsf is much higher than the general population . If we perform pet / ct scans to screen for incidentalomas in the general population, it will increase the health care costs with little benefit to the patients . The cost of pet / ct far outweighs the reduced mortality associated with early diagnosis of thyroid cancers . A paper by ohba et al ., who prospectively followed patients for 3 years, mentioned that repeated fdg - pet to follow up patients with thyroid nodules is ineffective . Diffuse - type incidentalomas with the absence of risk factors can be managed by physician visits, lab tests, and monitoring with us . Yet, we cannot rule out malignancy based on the diffuse pattern alone because the diffuse uptake can mask the focal lesions . One of the limitations of our study is that out of 156 abnormal pet / ct thyroid exams, only 40 had biopsy reports . The prevalence of malignancy among incidentalomas may be different from the current one if all underwent biopsy . We must be aware that pet / ct cannot detect small lesions such as micropapillary thyroid carcinomas which can have normal or low suv uptake . Our data suggest that focal fdg uptake representing incidentalomas should be followed by pathologic diagnosis, especially in those with chronic conditions or known diagnosis of a solid tumor . This study did not support the idea for using pet / ct to screen the general population for incidentalomas.
|
Mapping nucleobases in dna or rna modified by alkylation or oxidation often relies on the ability to specifically cleave the oligonucleotide at the site of modification, followed by gel electrophoresis to develop the map . A method established by maxam and gilbert utilizes base - specific alkylation followed by hot piperidine cleavage as the key steps in sequencing the dna bases . The success of this method lies in the ability to specifically alkylate the dna bases, as well as having high yields in the hot piperidine cleavage step . A similar base - specific labeling protocol followed by chemical cleavage has been advanced for sequencing rna; however, the inherent alkaline instability of rna requires substitution of piperidine at ph 11 with aniline at ph 4.5 . These methodologies developed a framework for identifying other labile sites of modification in dna and rna, for example, those resulting from base oxidation . Site - specific oxidation of nucleobases, if left unrepaired, can lead to mutations that are proposed to cause a wide range of diseases such as alzheimer s disease, melanoma, prostate cancer, and diabetes . Many research efforts have focused on understanding the mechanisms and products that result from oxidation of the nucleobases in dna oligomers to develop hypotheses that explain cellular mutagenesis . The nucleobase guanine stands out as a prominent site of oxidation because it has the lowest standard reduction potential . In dna, oxidation of 2-deoxyguanosine (dg) is modulated by the context of the reaction (i.e., single - stranded, duplex, or g - quadruplex contexts). Moreover, in duplex dna, the reactivity of dg toward oxidation shows a subtle sequence context effect . Locating site - specific oxidation at dg nucleotides in dna oligomers is determined by hot piperidine work up followed by gel electrophoresis to read the damaged sites in comparison to a control ladder . Another approach to identifying damage sites utilizes the base excision repair enzyme fpg for cleavage at the lesion . In rna, oxidation of guanosine (rg) has been studied, but to a lesser degree than dna oxidation . For the chemical cleavage methods to be successful at mapping oxidation sites, it is critical to know the rates and extent to which scission of the strand occur for different oxidation products of dg and rg . Oxidation of the guanine heterocycle yields classes of products based on the extent of oxidation . Two - electron oxidation of dg yields 8-oxo-7,8-dihydro-2-deoxyguanosine (dog) and 5-carboxamido-5-formamido-2-iminohydantoin-2-deoxyribonucleoside (d2ih), while four - electron oxidation of dg yields two diastereomers of spiroiminodihydantoin-2-deoxyribonucleoside (dsp), two diastereomers of 5-guanidinohydantoin-2-deoxyribonucleoside (dgh), and 2,5-diaminoimidazolone-2-deoxyribonucleoside (diz) that readily hydrolyzes to 2,2,4-triamino-2h - oxazol-5-one-2-deoxyribonucleoside (dz). The six - electron oxidation of dg yields dehydroguanidinohydantoin-2-deoxynucleoside (dgh), a compound of limited stability that ultimately decomposes to a ribosyl - urea lesion . Another product that is formed during certain oxidations, but is not formally oxidized, is 2,6-diamino-4-hydroxy-5-formamidopyrimidine-2-deoxyribonucleoside (fapydg), a ring - opened hydrolysis product of dg (scheme 1). Many of these lesions resulting from dg oxidation have been observed in genomic samples of biological origin, except d2ih and dgh, whose cellular existence is awaiting confirmation . In cellular rna samples, rog is the only lesion that has been characterized thus far . Detection of these lesions in cellular samples typically occurs by nuclease digestion that leads to complete loss of sequence information, followed by mass spectrometry . Therefore, in vitro model studies with dna or rna oligomers allow understanding the sequence context effects on lesion formation . The standard approach for identification of lesion sites requires strand cleavage by piperidine (dna) or aniline (rna) followed by gel electrophoresis; another method utilizes esi - ms, but this approach is limited to short oligomers with only one possible site of modification . Oxidations conducted on dna oligomers of known sequences have yielded a wealth of information concerning the sequence context - dependent reactivity of dg toward oxidants and the products . For interpretation of these data from the point of view of biology, the oxidation reactions are conducted under low product conversion (i.e., single - hit chemistry). Typically, the reaction yield is determined by hot piperidine cleavage of the oligomers using a standard protocol (0.21 m piperidine, 30 min, 90 c); however, the yields and rates for cleavage of the consistently observed dg oxidation products have not been established under identical reaction conditions . Ligation - mediated pcr is an approach for determining lesion sites in genomic samples that relies on the ability to site - specifically cleave the lesion for ligation on a primer sequence used to identify the original lesion location . In this method, chemical cleavage is one approach for effecting strand scission at the lesion site . In rna, cleavage at sites of base modification sites employs aniline following a standard procedure (0.21 m, ph 4.5, 60 c, 20 min). As for dna, strand cleavage efficiencies are not known for rg oxidation products in rna oligomers . Because rates of chemical strand scission for dg and rg lesions have not been established, the extent of an oxidation reaction on dna or rna polymers is inaccurately determined . In the present study, the rates of cleavage for og, sp, gh, 2ih, and z were determined in dna oligomers treated with piperidine, and in rna oligomers treated with aniline under standard reaction conditions . These data determined that lesion cleavage rates using typical reaction conditions are quite variable, and some are not significantly cleaved, even at long incubation times . These data will be paramount for researchers that oxidize oligomers of dna or rna with known sequences and then interpret the reaction yields from a biological prospective, for which oxidation events on these polymers are a rare occurrence . The dg oxidation products dog, (s)-dsp, (r)-dsp, and a mixture of the dgh diastereomers were synthesized in the 18-mer dna oligomer 5-tca tgg gtc xtc ggt ata-3 (where x = lesion site). The dna oligomer was synthesized via solid - phase synthesis by the dna / peptide core facility at the university of utah with a dog phosphoramidite (glen research, sterling, va) at the position of x following standard methods . Analytical ion - exchange hplc - purified samples provided the dog - containing dna oligomer for the piperidine studies . Syntheses of the dsp diastereomers and dgh diastereomers were conducted following literature protocols . The dsp diastereomers were individually purified while the diastereomers of dgh were studied as a mixture because they readily interconvert . Syntheses of dz and the (r)- and (s)-d2ih diastereomers were achieved in a 15-mer dna oligomer containing a single dg site (5-aat cca cga cac ctc-3) following literature methods . All product oligomers were purified using an analytical ion - exchange hplc column that resolves diastereomeric products (figure s1). The dna oligomer products were characterized by esi - ms (dog: calcd 5560.6, found 5560.8; mixture of the dsp diastereomers: calcd 5576.6, found 5576.4; mixture of the dgh diastereomers: calcd 5550.6, found 5550.2), or maldi - ms was used to characterize a mixture of the d2ih diastereomers as well as for dz (d2ih: calcd 4498.8, found 4498.2; dz: calcd 4445.8, found 4445.3). The dz - containing oligomer was found to have limited stability due to further decomposition of dz to guanidinoformimine-2-deoxyribonucleoside (dgf). The dgf - containing strand was characterized by maldi - ms (dgf: calcd 4398.8; found 4398.2). Because of the limited stability of dz in the dna oligomer leading to dgf, the dz sample was studied immediately after purification and a 2 h dialysis to remove the purification salts . The rg - oxidation products rog, (r)-rsp, (s)-rsp, rz, (r)-r2ih, (s)-r2ih, and rgh diastereomers were synthesized in the 7-mer rna oligomer 5uuuguuu-3. Selective conversion of the single rg to the rsp diastereomers, rgh diastereomers, r2ih diastereomers, and dz was achieved via literature protocols . These strands were then purified by the same method as stated for the lesions in the dna oligomers (figure s2). The 7-mer containing rog was synthesized using the phosphoramidite of rog (chem genes, wilmington, ma) following the manufacturer s protocol . The rna oligomer products were characterized by maldi - ms (rog: calcd 2136.3, found 2136.7; (s)-rsp and (r)-rsp diastereomer mixture: calcd 2152.3, found 2153.0; rgh diastereomer mixture: calcd 2126.3, found 2126.1; rz: calcd 2099.3, found 2099.4; (r)-r2ih and (s)-r2ih diastereomer mixture: calcd 2154.4, found 2154.3). To monitor the cleavage reactions, the lesion - containing dna oligomers were 5-labeled with p for visualization by gel electrophoresis followed by phosphorimager autoradiography . Piperidine cleavage reactions for lesion - containing dna oligomers were conducted by mixing 100 pmol of unlabeled strand with 20 000 cpm of p - labeled strand in 50 l of ddh2o . For each lesion, nine tubes were prepared for taking time points at 0, 5, 10, 20, 30, 45, 60, 90, and 120 min . To these samples were added 50 l of freshly prepared aqueous piperidine (2 m) and -mercaptoethanol (bme, 0.5 m) to give a final concentration of 1 m dna with 1 m piperidine and 0.25 m bme . Next, the samples were incubated at 90 c, and at each time point, a sample was removed from the heat source . The samples were lyophilized to dryness and resuspended in 10 l of gel loading dye (0.25% xylene cyanol, 0.25% bromophenol blue in an aqueous 30% v / v glycerol solution). To a 20% denaturing polyacrylamide gel electrophoresis (page), 5 l of sample in loading dye was added . The samples were electrophoresed at 45 w for 2 h, after which the gel was loaded into a phosphor screen and visualized by phosphorimager autoradiography after an 18 h exposure . One reaction was also monitored by ion - exchange hplc on a sample that was not p labeled that allowed observation of all species in solution during the reaction . The method utilized was the same as the one used for product purification . To monitor the cleavage reactions, the lesion - containing rna oligomers were 5-labeled with p for visualization by gel electrophoresis followed by phosphorimager autoradiography . Aniline cleavage reactions for lesion - containing rna oligomers were conducted by mixing 100 pmol of unlabeled strand with 20 000 cpm of p - labeled strand in 50 l of ddh2o . For each lesion, seven tubes were prepared for taking time points at 0, 10, 20, 30, 40, 60, and 90 min . To these samples was added 50 l of freshly prepared aniline (ph 4.5, 2 m) to furnish a final concentration of 1 m rna and 1 m aniline . The aniline at ph 4.5 was prepared by taking a solution of reagent grade aniline and adjusting the ph to 4.5 with glacial acetic acid to obtain a 2 m stock solution . Next, the samples were incubated at 60 c in the dark, and at each time point, a sample was removed from the heat source . After completion of the reactions, the samples were processed in an identical fashion as previously described for the lesion - containing dna oligomers . Scheme 2a shows the proposed mechanism for piperidine - induced strand scission at the site of a dna lesion . In the first step, this intermediate increases the acidity of h2 that accelerates -elimination of the 3-phosphate terminus in the second step . In the third step, -elimination of the 5-phosphate terminus occurs to yield complete cleavage at the site of the lesion . The correct kinetic rate equation for this complex reaction should take into account all intermediate reactions; however, in practice, piperidine cleavage reactions are followed by page, in which only the intact strand and the cleaved strand are observed . By monitoring loss of the starting material and the appearance of the product, the rate equation can be simplified to a pseudo - first - order rate equation . The assumptions in this simplified approach are that the piperidine concentration does not change, as is validated by the large excess of piperidine used to effect strand scission (10-fold excess). The second simplifying assumption was the use of the steady - state approximation, in which the concentrations of the reaction intermediates were assumed to reach a steady state, and their concentrations were limited by the rate of the first reaction step . Verification of this assumption was applied to a test reaction that monitored the piperidine cleavage of a (r)-dsp - containing dna oligomer by hplc . This approach allowed inspection of all species in solution, which is not possible by page . In this analysis, the intact strand, cleavage products (5 and 3 fragments), and intermediates the intermediates were not characterized due to their instability . In support of the steady - state approximation, the intermediate peak ratios did not significantly change during the 90 min reaction profile, while the starting material decreased and the product strands increased (figure s3). To monitor cleavage reactions for all lesions, the traditional approach of monitoring these reactions by page was selected, and a pseudo - first - order reaction rate equation was applied for mathematically modeling . The rate - limiting step was assumed to be formation of the schiff - base intermediate . Thus, plots of the ln [intact strand] vs reaction time provided linear data points that were described by eq 1, where the slope of the line provides the reaction rate constant.1 monitoring the time - course cleavage for the guanine oxidation products studied in a dna oligomer by page gave a wide distribution of reaction course profiles (figure 1a and figure s4). For example, the dog lesion was not significantly cleaved (<1%) by hot piperidine during the 2 h reaction (figure 1a). Next, the dsp diastereomers were found to have undergone slow hot piperidine strand scission (figure 1a), with rate constants for the isomers to be nearly identical values of (9.5 1.5) 10 and (9.5 1.2) 10 min for the r and s isomers, respectively (table 1). For dgh, strand scission by hot piperidine was faster, with a cleavage rate constant of (22 3) 10 min (table 1), while the dz lesions were cleaved with a rate constant of (51 5) 10 min (table 1). Finally, the diastereomers of d2ih cleaved the quickest with rate constants of (100 10) 10 and (110 11) 10 min for the r and s isomers, respectively (table 1). These studies demonstrate that the dg oxidation products, other than dog, cleave with hot piperidine at variable rates, and near - quantitative reaction yields can be obtained if the reactions are allowed to progress long enough . The pseudo - first - order kinetic rate constants allow determination of the half - life (t1/2) of each lesion under the reaction conditions . These values will allow researchers to calculate the reaction time required to achieve the yield they would desire . The dog lesion in dna cannot be found by piperidine cleavage reactions, and this point is further discussed below . As for the other dg lesions in dna, the t1/2 values were 73 min for the dsp diastereomers, 32 min for the dgh diastereomers, 14 min for dz, 6.8 min for (r)-d2ih, and 6.0 min for (s)-d2ih (table 1). On the basis of these results, all dg - lesions, besides dog, can be cleaved in high yield as long as the reactions are allowed to progress for a sufficiently long time period . Plots of ln[intact strand] vs time for dg oxidation products in dna (a) and rg oxidation products in rna (b) oligomers . (a) time - dependent cleavage yields for the dna lesions, dog, (r)-dsp, (s)-dsp, dgh, dz, (r)-d2ih, and (s)-d2ih are plotted . The reaction yields were monitored on 5-p - labeled strands that were separated by page and quantified by phosphorimager autoradiography . Cleavage reactions for these damaged dna oligomers were conducted with fresh, aqueous piperidine (1 m) and bme (0.25 m) at 90 c . (b) time - dependent cleavage yield for the rna lesions, rog, (r)-rsp, (s)-rsp, rgh, rz, (r)-r2ih, and (s)-r2ih are plotted . The reactions were monitored in the same fashion as those for the dna oligomers . Cleavage reactions for the damaged rna oligomers were conducted with fresh, aqueous aniline (ph 4.5) at 60 c in the dark . Scheme 2b shows the proposed mechanism for aniline - induced strand scission at the site of a rna lesion . This intermediate increases the acidity of h2 that accelerates -elimination of the 3-phosphate terminus . Finally, -elimination of the 5-phosphate terminus occurs to yield complete cleavage at the site of the lesion . Because of the similarity in the cleavage mechanisms, and the fact that the same concentration of aniline was used to cleave rna lesions as was used for piperidine to cleave dna lesions, the same assumptions were used to fit the data to determine the pseudo - first - order reaction rate constants (eq 1). The rates of aniline - mediated cleavage (1 m, ph 4.5, 60 c) for all rg lesions were assayed by following strand cleavage via page analysis . Under these reaction conditions, rog showed <1% cleavage after a 90 min reaction (figure 1b). The diastereomers of rsp gave slightly more cleavage than rog with <10% strand scission . The fitting equations to these data determined the cleavage rate constants for the r and s diastereomers of rsp to be (1.2 0.2) 10 and (1.4 0.2) 10 min, respectively (table 1). Next, the diastereomers of rgh were found to give more strand scission under these conditions (figure 1b). For rgh, the aniline cleavage rate constant was (6.9 0.5) 10 min (table 1). The rz reaction preceded more quickly giving a reaction rate constant of (51 6) 10 min (table 1). Finally, the r2ih diastereomers cleaved the fastest (figure 1b) with the r and s isomers having reaction rate constants of (61 7) 10 and (63 7) 10 min, respectively (table 1). These studies demonstrate that site specific cleavage of rg oxidation lesions in rna were slower than the analogous cleavage reactions in dna with piperidine . The pseudo - first - order reaction rate constants for cleavage of rg lesions in rna allow calculation of the t1/2 values for these lesions by aniline . On the basis of these rate constants, the t1/2 for the rsp diastereomers were calculated to be 580 and 500 min for the r and s isomers, respectively (table 1). These lifetimes are too long to be usable for detection of rsp lesions in rna, and therefore, support rsp as not being cleavable by aniline in any practical applications . For the other rg lesions, the t1/2 values were determined to be 100 min for rgh, 14 min for rz, and 11 min for the diastereomers of r2ih . In the current studies, dog was not piperidine labile, an observation that has stirred some debate in the literature . The bme added during the reaction is hypothesized to quench unwanted oxidation of dog . After a 2 h incubation (1 m piperidine, 90 c), 10% of the dog - containing strand cleaved based on page analysis, thus supporting the hypothesis that dog can be oxidized during the cleavage reaction, albeit slowly . Analysis of a test reaction by hplc confirmed dog to slowly oxidize to dsp under the hot piperidine reaction conditions (figure s5), an expected result because at high ph oxidation of dog yields dsp . Because dog is poorly cleaved by hot piperidine, one approach for sequencing this lesion via chemical cleavage has been developed by our laboratory . In this method, dog is further oxidized to dgh with a selective and mild one - electron oxidant, such as na2ircl6, and the corresponding secondary hydantoin lesion is much more labile to piperidine (figures 1a and table 1). The hydantoins dsp and dgh were both found to be labile to piperidine, but with considerably different rate constants (dsp = 9.5 10 min; dgh = 22 10 min; table 1). Further, the r and s isomers of dsp were studied individually and shown to give similar cleavage rates with hot piperidine (figure 1a and table 1). The standard piperidine reaction time is 30 min that will give 25% and 48% strand scission for the diastereomers of dsp and dgh, respectively (figure 2a); the incomplete strand scission currently observed for these lesions after a 30 min piperidine reaction is consistent with previous literature reports . To effect strand scission in a yield> 90% for the hydantoins, the piperidine reaction times must be> 242 min and> 106 min for dsp and dgh, respectively (figure 3a). As for dz after a 30 min reaction, the cleavage yield would be 78%, an observation that is nearly identical to literature reports (80% cleavage after 30 min). Furthermore, extension of the reaction time to> 46 min will lead to> 90% cleavage at dz sites (figure 3a). (figure 2a), and the standard 30 min piperidine cleavage reaction time will sufficiently identify d2ih sites (figure 3a). Only one sequence context was studied for each g lesion in dna, and sequence context effects on reaction rates may exist; however, it is anticipated that these effects will be minimal at ph 11 and 90 c . Other reagents that cleave dna lesions at lower ph have been utilized, such as n, n-dimethylethylenediamine (0.1 m) at ph 7.4 and 37, 65, or 90 c . Studies were conducted to determine if this reagent could cleave dog, dsp, or dgh under the reported conditions (0.1 m, ph 7.4, and 65 c). These reactions were monitored by hplc and found that n, n-dimethylethylenediamine was incapable of cleaving all three lesions at reaction times <2 h (figure s6). Cleavage efficiency for dg lesions (a) and rg lesions (b) following the standard chemical cleavage protocols . (a) the dg lesion - containing oligomers were incubated following the standard cleavage recipe that includes fresh, aqueous 1 m piperidine and 0.25 m bme at 90 c for 30 min . (b) the rg lesion - containing oligomers were incubated following the standard cleavage recipe that includes fresh, aqueous 1 m aniline (ph 4.5) at 60 c for 20 min in the dark . Chemical cleavage of rg lesions in rna was determined to have lower rate constants for all lesions compared to dg lesions in dna . For example, the reaction rate for aniline cleavage of the rsp diastereomers was very slow, and almost undetectable; these reaction rate constants determined that after a standard 20 min aniline reaction time, 3% of the rsp diastereomers would cleave (figure 2b). The aniline reaction time would need to be increased to 1900 and 1700 min for the r and s diastereomers of rsp, respectively, to effect> 90% strand scission (figure 3b). These reaction times are not practically useful, effectively causing the rsp isomers to be resistant to aniline treatment . In comparison, the rgh isomers will cleave 13% after the standard aniline reaction time (figure 2b), and the reaction time would need to be extended to> 330 min to effect> 90% strand scission yield at these sites (figure 3b). Next, rz would cleave by 64% under the standard reaction time (figure 2b), and extension of the cleavage reaction to> 47 min will facilitate> 90% cleavage at these sites (figure 3b). Finally, 71% of the r2ih diastereomers will cleave under the standard reaction time (figure 2b); for these lesions, extending the reaction time to> 37 min will lead to> 90% strand scission at these sites (figure 3b). In contrast, rog did not give detectable amounts of strand scission with aniline, even after a 90 min reaction . The only rg lesion detected in vivo so far is rog, an observation that validates rg oxidation occurrence in the cell . This is particularly striking because there exists 3 times more rna in a cell than dna, and rna takes on a variety of secondary structures that expose the bases to solvent . These exposed bases will be much more prone to oxidation than those in duplex structures, and a major rg oxidation product will likely be rsp . However, the use of rna oligomers and chemical cleavage agents will not provide an accurate measure of the sites and yields for the rg oxidation product rog, and likely rsp . Therefore, to study the formation of these lesions in rna will require a different assay . Either a polymerase stop assay can be performed, as has been previously shown, or rog can be further oxidized with a mild oxidant (na2ircl6) to yield a hydantoin (e.g., rgh) that is labile to aniline, an approach that has also been previously proposed . These studies in rna were only conducted in one sequence context; however, at ph 4.5 and 65 c, it is anticipated that sequence context effects will be minimal . Minimum reaction time to achieve> 90% cleavage at dg lesions in dna oligomers (a) and at rg lesions in rna oligomers (b). The chemical cleavage of lesion - bearing dna oligomers was conducted with freshly prepared, aqueous piperidine (1 m) and bme (0.25 m) at 90 c, and the lesion - bearing rna oligomers was conducted with freshly prepared, aqueous aniline (ph 4.5) at 60 c in the dark . The reaction times were computed from the strand scission rate constants values for these lesions provided in table 1 . * these lesions the current research determined the chemical cleavage rate constants for many lesions derived from oxidation of the nucleobase guanine in dna and rna oligomers . In these studies, the base damages og, sp, gh, z, and 2ih were site - specifically synthesized in dna and rna oligomers . These strands were then subjected to the standard chemical cleavage reagents for dna (1 m piperidine, ph 11, 90 c) or rna (1 m aniline, ph 4.5, 60 c), and the pseudo - first - order rate constants for stand scission were determined . In dna, dog was not cleaved, and the rate for the dsp diastereomers was found to be slow (table 1). The rates of strand scission for dz, dgh, and d2ih were faster, and only d2ih will cleave in a> 95% yield after the standard reaction time of 30 min (figure 2a). The current results highlight that most lesions may require long reaction times to obtain high strand scission yields (figure 3a). These results taken together identify a great variability in the cleavage yield for the dg lesions . The alternative approach for identifying lesion sites in dna utilizes a base excision repair enzyme, such as fpg; however, fpg cleavage also suffers from lesion - dependent cleavage kinetic differences . In addition, in rna strands, all lesions were found to cleave at a slower rate than their analogous dna counterparts (table 1). More specifically, rog and rsp gave insignificant cleavage with aniline at long reaction times (figure 1b), while rgh and rz could be cleaved at very long reaction times (figure 3b). Finally, these results identify og and sp lesions in dna and rna strands to be the most stable (table 1), and they support a hypothesis that these lesions in the genome or transcriptome would be much longer lived than the other guanine base lesions.
|
Gastric volvulus, an entity seen in both pediatric and adult patients, occurs when the stomach twists upon itself . This event may be transient, producing few if any symptoms, or may lead to obstruction or even ischemia and necrosis . Pare described the first case of gastric volvulus in 1579 in a patient with diaphragmatic injury from a sword wound . Acute gastric volvulus in pediatric and adult patients has been reported but chronic organo - axial gastric volvulus with diaphragmatic eventration has not been reported . This report describes a rare case of gastric volvulus with a review of the literature . He had a feeling of fullness and discomfort in the upper abdomen for 2 months . Twenty years ago, he was told that his chest radiography, performed due to routine survey for military service, was abnormal . But he had received no specific measures since then and he had been relatively healthy . A thorough review of symptoms was performed but did not disclose any respiratory or cardiovascular symptoms . On clinical examination, he appeared not acutely ill looking and his body temperature was 36c, blood pressure 120/70 mmhg, respiratory rate 24/min . Auscultation of the chest showed diminished breathing sound at the left lung base and heart sound was normal . No abnormalities were observed in cbc, liver chemistry, serum amylase and stool examination . The chest radiograph showed the left hemidiaphragm to be located at an unusually high intercostal space with large air - fluid level (figure 1). Upper gastrointestinal series demonstrated the typical appearance of an organo - axial volvulus of the stomach (figure 2). The barium - filled stomach was twisted on an axis from the pylorus to the esophagus . The patient refused surgical or endoscopic correction of the volvulus and has been followed in the outpatient clinic for more than 6 months without symptom aggravation . The stomach is relatively fixed at the esophageal hiatus and the pylorus by the four gastric ligaments . The gastrophrenic ligament and the retroperitoneal attachment of the second part of the duodenum provide the superior and inferior fixation . The gastrohepatic ligament tethers the lesser curve, the gastrocolic ligament connects the stomach to the transverse colon, and the gastrosplenic ligament tethers the greater curve . The clinical symptoms depend upon the extent or degree of rotation, obstruction and associated defect . Borchard s triad of pain, violent retching and inability to pass a nasogastric tube6 should lead to a strong clinical suspicion of acute gastric volvulus . An acute volvulus is an emergency situation, with either obstruction or strangulation of the stomach and requires expeditious surgery . In comparison, this explains why the diagnosis is often delayed in the elderly or after complication has occurred . . Gastro - esophageal reflux may give rise to epigastric pain, which is intermittent during the periods of engorgement or gastric emptying . Gastric ulceration is caused by localized ischemia and acidity within the herniated stomach or mucosal congestion due to venous obstruction of the herniated stomach . Angina - like pain and electrocardiographic abnormalities may make the differential diagnosis difficult in the elderly . The diagnosis is confirmed by the presence of a large, unusual gas - filled viscus in the chest or abdomen on plain radiographs . If necessary, a barium swallow study can define the anatomic changes more exactly . On barium examination, the characteristic findings are 1) esophagogastric junction lying lower than normal, 2) reversal of the greater and lesser curvatures, 3) pylorus pointing downward, 4) greater curvature crossing the esophagus, 5) two air - fluid levels and lowering of the gastric fundus . Secondary gastric volvulus is more frequent than idiopathic volvulus, therefore the diagnosis of gastric volvulus can be made after a thorough search for possible causative factors . These conditions have been reported as follows; para - esophageal hernia, traumatic rupture of the diaphragm, eventration of the diaphragm and phrenic nerve injury . In infants and children, 15 (33%) among 46 patients with gastric volvulus had diaphragmatic eventration . Organo - axial volvulus is commonly associated with diaphragmatic hernia and usually manifests as an acute event . Diaphragmatic eventration is suggested when a part or all of the hemidiaphragm is located at an unusually high level in the thorax . It does not refer to a defect or hole in the diaphragm with discrete edges, but rather to a diffuse or localized bulging of the diaphragm itself . Acquired lesions are usually related to phrenic nerve injury, which may be diverse in origin . Radiological investigation combined with fluoroscopy of the diaphragm our patient showed no diaphragmatic movement during respiration, so he had complete left hemidiaphragmatic eventration . Acute gastric volvulus and symptomatic chronic gastric volvulus require operative treatment . If the volvulus is secondary, definitive treatment must include correction of the associated abnormalities . This is done by advancing the endoscope just past the point of torsion, turning and locking the tip of the instrument, and rotating it 180 degrees . With rapid diagnosis and modern treatment, the rate of mortality from acute gastric volvulus is now about 15% to 20% . If the stomach is strangulated, the mortality rate of emergency surgery is 4060% . In recognized cases of chronic gastric volvulus
|
Neuroblastoma (nb) is the most common extracranial solid tumor in children, with an incidence of 1 case per 100.000 children per year, and causes 15% of cancer deaths in pediatric age . Nb originates from the sympathetic nervous system, most frequently in the adrenal medulla or the paraspinal ganglia . The causes are unknown, although 1 - 2% of nb may have a hereditary basis . Different genetic alterations have been characterized in nb, that is, gain - of - function of alk gene, losses of 11q and 1p, gain of 17q, and amplification of the mycn gene . Nb is heterogeneous, as it may undergo spontaneous remission or evolve to progressive metastatic disease, with dissemination to lymph nodes, bone, bone marrow, liver, skin, and other organs . In particular the international neuroblastoma risk group staging system takes into account genetic alterations, dna ploidy, histological features, and clinical data, as criteria for defining the risk classes . The prognosis of low / intermediate risk nb patients is favorable, and tumors can be cured by surgery alone or minimal chemotherapy . In contrast, high - risk nb patients' prognosis is poor, in spite of aggressive treatment based on surgery, chemotherapy, radiation therapy, hematopoietic stem cell transplantation, and adjuvant therapy with retinoic acid . In fact, survival rates of these patients at 5 years are less than 50% . In the last years, the role of hla - class ib molecules in the progression of nb has been characterized by our group [47] and by others . Hla - ib family includes hla - g, hla - e, hla - f, and hla - h . In contrast with high polymorphic hla - ia molecules (hla - a, hla - b, and hla - c) all these molecules display a limited polymorphism, with few alleles encoding a limited number of functional proteins . Moreover, although hla - class ib molecules can bind small peptides and present them to specific cd8 t cell subsets (similarly to hla - class ia counterparts), their main function is the modulation of the immune response in both physiological and pathological conditions . Hla - g and hla - e are the best characterized among hla - ib molecules . Hla - g has seven different isoforms, four membrane bound (namely, hla - g1, hla - g2, hla - g3, and hla - g4) and three soluble (namely, hla - g5, hla - g6, and hla - g7), that are generated by alternative splicing from the same primary transcript . Hla - g can interact with at least four receptors, namely, immunoglobulin - like transcript (ilt)2, ilt4, kir2dl4, and cd160, thus affecting the function of different immune effector cells (t and b lymphocytes, natural killer nk cells, dendritic cells, granulocytes, and monocytes). In contrast, hla - e can be expressed as membrane bound or soluble isoform (generated through metalloproteases cleavage) and can inhibit cd8 t cells or nk cells though interaction with cd94/nkg2a heterodimeric receptor . However, hla - e can also interact with the activating receptor cd94/nkg2c, thus leading to nk cell activation . These interactions are crucial during trophoblast implantation to abrogate nk cell lysis of semiallogeneic fetal tissue and, on the other hand, to activate nk cell functions in the process of tissue remodeling . We have previously demonstrated that soluble (s)hla - g concentration is higher in plasma samples from nb patients than in controls, and shla - g can be released by nb cells themselves, or by monocytes (stimulated by soluble factors secreted by tumor cells). Moreover, high shla - g plasma levels correlated with nb patients' relapse . Finally, we have assessed that hla - g is expressed by metastatic nb cells in the bone marrow from nb patients . Also soluble hla - e levels are higher in nb patients than in healthy controls . However, we have demonstrated that high plasma levels of shla - e at diagnosis correlated with a better overall survival (os) of nb patients at follow - up, in contrast with shla - g . Here, we demonstrated for the first time that shla - g and shla - e are present also in bm plasma samples derived from either nb patients at diagnosis or healthy donors . Moreover, we have assessed that shla - g and shla - e levels in bm plasma samples are related to the stage of the disease . Analysis of these patients at follow - up will reveal whether shla - g and shla - e concentration in bm plasma may predict the clinical outcome of nb patients . The study was approved by the ethics committee of the g. gaslini institute, genoa, italy . Bone marrow (bm) samples were collected at diagnosis and centralized at istituto giannina gaslini in genoa, italy . Patients' characteristics, that is, age at diagnosis, sex, mycn amplification (single copy or amplified), bm infiltration, and stage, are summarized in table 1 . As controls, bm aspirates were obtained from 13 healthy donors, selected according to the transplant unit clinical protocol of ematologia 2 at the irccs san martino - ist in genoa, following a written informed consent at the time of donation . Samples were processed as described in, and an aliquot was taken at the end of processing to perform quality control tests, such as cd34 cell count, in vitro progenitors' cell growth, and sterility . The remaining bm blood sample from this aliquot was subjected to centrifugation (3000 g 10) to obtain bm plasma . Enzyme - linked immunosorbent assay (elisa) for shla - g and shla - e was performed as previously described . Briefly, maxisorp nunc - immuno 96-microwell plates (nunc a / s, roskilde, denmark) were coated overnight at 4c with 1 g / ml of mem - g9, specific for hla - g hc (exbio, prague), that recognizes shla - g1/g5, or 3d12 mab, specific for hla - e hc (ebioscience, science center drive, san diego, ca, usa). After three washes with pbs 0.05% tween 20 (washing buffer), plates were saturated with 200 l / w of pbs 2% bsa (sigma, st . One hundred l of bm plasma samples and serial dilutions of 721.221.g1 cell line supernatant (for hla - g) or total extract from normal peripheral blood mononuclear cells (standard) were added to each well and incubated at rt for 1 hour . After three washes, 100 l of detection reagent (hrp - conjugated anti-2 microglobulin mab, exbio, vestec, cz) was added, and plates were incubated for 1 hour at rt . After three washes, 100 l of tmb (substrate for hrp, sigma) was added, and reaction was stopped after approximately 30 minutes by adding h2so4 5 n. absorbance at 450 nm was measured using infinite 200 pro spectrometer (tecan group ltd ., results are expressed as ng / ml shla - g and arbitrary units / ml shla - e (1 unit = quantity of shla - e in 1 g of total extract). Normal distribution of data was tested using kolmogorov - smirnov test, using prism software (graphpad software inc ., la jolla, ca). Since data distribution was not normal, differences in plasma levels between (i) patients and controls or (ii) different groups of patients were compared by mann - whitney test, using prism software . Correlations between plasma levels of shla - g and shla - e were calculated by spearman's test using prism software . A p value 0.05 significance ranges are the following: p <0.05; p <0.01; and p <0.001 . First, we have tested shla - g and shla - e concentration in bm plasma samples from nb patients and healthy donors . As shown in figure 1(a), shla - g concentration was similar between nb patients (median se: 24.69 8.45 ng / ml) and controls (25.16 7.38 ng / ml). In contrast, shla - e levels were lower in nb patients (3.72 7.89 u / ml) than in controls (48.01 10.93 u / ml). However, such difference was not statistically significant, likely due to the wide distribution of the results in both groups (figure 1(b)). Finally, shla - g and shla - e levels in bm plasma samples from nb patients (r = 0.96, p <0.0001, figure 1(c)) and healthy donors (r = 0.92, p <0.0001, figure 1(d)) strongly correlated with each other . We have next analyzed possible correlation between shla - g and shla - e levels in bm plasma samples and patient's characteristics or clinical parameters . Accordingly, nb patients were divided into two groups on the basis of (i) mycn amplification (single copy versus amplified), (ii) bm infiltration (not infiltrated versus infiltrated), (iii) age at diagnosis (<18 months versus> 18 months), (iv) stage of the disease (stages 1 - 2 versus stages 3 - 4), and (v) sex (male versus female). Next, differences in shla - g and shla - e levels between these groups of nb patients have been evaluated . No significant differences in shla - g levels have been detected between nb patients (i) carrying amplified (29.96 13.57 ng / ml) or single - copy (23.65 9.6 ng / ml) mycn gene (figure 2(a)) and (ii) presenting (31.17 11.99 ng / ml) or not (21.05 9.91 ng / ml) nb cells infiltrating the bm (figure 2(b)). In contrast, shla - e levels were higher in (i) patients with single - copy mycn (7.56 9.17 u / ml) than in those with amplified mycn (1.03 16.2 u / ml) (figure 2(a)) and in (ii) patients with infiltrated bm (6.45 10.29 u / ml) than in those without bm infiltration (1.86 12.43 u / ml) (figure 2(b)). However, such differences were not statistically significant . Furthermore, both shla - g and shla - e levels were similar between patients with an age below (21.05 9.92 ng / ml shla - g and 6.45 10.29 u / ml shla - e) or above (28.77 11.36 ng / ml shla - g and 2.37 12.81 u / ml shla - e) 18 months at diagnosis (figure 3(a)). Notably, no correlation was found between age and shla - g or shla - e levels in bm plasma samples in healthy donors (data not shown). Both shla - g and shla - e levels were significantly higher in patients with disease stages 3 - 4 (32.34 8.08 ng / ml shla - g and 13.87 9.42 u / ml shla - e) than in those with disease stages 1 - 2 (0 4.32 ng / ml shla - g and 0 3.27 u / ml shla - e, p = 0.01 and 0.03, resp .) Surprisingly, both shla - g and shla - e levels were found to be higher in male (45.87 12.5 ng / ml shla - g and 34.19 14.83 u / ml shla - e) than in female (2.52 8.81 ng / ml shla - g and 0 8.18 u / ml shla - e, p = 0.05 and 0.03, resp .) In contrast, healthy donors showed higher levels of shla - g and shla - e in female (50.74 14.1 ng / ml shla - g and 52.25 14.8 u / ml shla - e) than in male (12.35 10.98 ng / ml shla - g and 11.33 13.25 u / ml shla - e) subjects . However, such differences were not statistically significant (figure 3(d)). To the best of our knowledge, this is the first demonstration of the presence of shla - class ib molecules hla - g and hla - e in bm plasma samples . Previous studies have demonstrated that shla - g can be released by some cell populations that are present in the bm environment, such as erythroblasts and mesenchymal stromal cells [1619]. In contrast, no information is available regarding hla - e expression and release in the bm . The strong correlation observed between the levels of these two molecules in bm samples either from nb patients or controls suggested that both molecules may be released by the same cell populations, or at least induced by similar stimuli . We have previously demonstrated that metastatic nb cells in the bm expressed high levels of hla - g on their surface, in contrast with primary tumors, that tested negative for hla - g . Here, we have demonstrated that both shla - g and shla - e are present at similar levels in nb patients and healthy donors, thus suggesting that malignant metastatic nb cells are unlikely involved in their release . This observation is further confirmed by the finding that bm infiltration by metastatic nb cells did not affect shla - g or shla - e levels in bm plasma samples . Moreover, mycn amplification and age at diagnosis that represent important prognostic factors were not related to shla - g and shla - e levels in bm, thus further suggesting that these molecules might be released by bm stromal cells or bm resident cell populations instead of nb cells themselves, and may be present in the bm environment in physiological conditions . However, the increased tumor burden might be correlated to a higher release of tumor - derived factor(s) that, in turn, can upregulate hla - g and hla - e production by bm stromal cells . The finding that shla - g and shla - e bm plasma levels are higher in male than in female patients is in line with a previous study on multiple sclerosis, where the authors demonstrated that shla - g levels in plasma samples were higher in male than in female patients . However, this study has been carried out using peripheral blood plasma samples, and this is the first demonstration of this difference between male and female subjects in bone marrow plasma samples . Notably, such difference may be a prerogative of nb patients, since shla - g and shla - e levels were higher in female than in male normal subjects . The most important finding of our study is the demonstration that shla - g and shla - e levels were significantly higher in bm plasma samples from patients with metastatic disease than in patients with localized nb . This data may suggest that the levels of these molecules in the bm at diagnosis might be associated with disease progression and might be predictive of the clinical course of nb patients . However, this hypothesis can be confirmed only by analyzing the clinical parameters of these patients at follow - up . In conclusion, we demonstrated for the first time that soluble hla - ib molecules hla - g and hla - e are present in bm plasma samples in physiological and pathological conditions, and their concentration correlated with stage disease in nb patients . The prognostic value of shla - g and shla - e concentration in bm plasma samples from nb patients at diagnosis has to be confirmed in future studies.
|
We adopt a simple cvd process, the salient stages of which are outlined in figure 1: (1) the preannealing stage, during which the samples are gradually heated in h2 or nh3, (2) the vacuum stage preceding borazine exposure, (3) the borazine exposure period, and (4) the vacuum cooling stage . Figure 2 compares the morphology of the h - bn grown on as - received fe foils (100 m) at 900 c and 6 10 mbar borazine partial pressure following preannealing in 4 mbar of either h2 or nh3 (stage 1) as a function of borazine exposure time (stage 3). We observe a strong effect of the preannealing gas on the growth . In particular, for the nh3 preannealing, an increase in growth time results in a gradual closure of the monolayer h - bn film, without multilayer formation . Conversely, the h2 preannealing does not lead to a continuous, homogeneous h - bn film even for extended growth times, and primarily results in thicker, few - layer h - bn domains . The shaded areas correspond to the preannealing stage in either h2 or nh3 (1), the vacuum stage prior to borazine exposure (2), the borazine exposure period (3), and the vacuum cooling stage (4). Sem images of h - bn domains grown at 900 c and 6 10 mbar borazine exposure on (a, b, c) h2-preannealed fe foil for 45 s, 90 s, and 480 s borazine exposure time, respectively, and on (d, e, f) nh3-preannealed fe foil for 45 s, 90 s, and 480 s borazine exposure time, respectively . The preannealing pressure in both h2 and nh3 is 4 mbar for all of the above growths . The insets in (a, d) correspond to 14 s exposures and illustrate the longer incubation time preceding nucleation of h - bn on the h2-preannealed sample . The average domain size for the h2-preannealed sample is <1 m after 45 s borazine exposure (figure 2a). For a 14 s borazine exposure following a h2 preannealing (figure 2a inset) no h - bn was observed on the fe surface, indicative of an incubation period preceding crystal nucleation of at least 14 s. we note that the h - bn domain orientation, size, and density varies across the sample surface, suggesting fe grain orientation - dependent growth kinetics, a feature that is also observed for the nh3-preannealed foil (see supporting information figure s1). After 90 s, the domains grow to average lateral sizes of 3 m (figure 2b), and the change in contrast from the edges to the center of the triangular domains also indicates a notable thickness variation, with brighter regions corresponding to multilayers and conversely dark gray regions indicating monolayer h - bn . Extended growth times (480 s, figure 2c) lead to slightly higher h - bn coverages; however, growth appears to primarily proceed through the formation of additional h - bn layers, rather than lateral expansion of the existing layers . Additionally, we observe a bimodal domain size distribution across the entire sample surface (figure s2), with large domains (side length 15 m) coexisting with domains about ten times smaller . This observation suggests that two distinct growth regimes appear to exist under these growth conditions for the h2 preannealing . For the nh3-preannealed fe foil, the most notable difference after 45 s borazine exposure is the formation of a lower density of triangular domains with lateral dimensions of 20 m (figure 2d), which are more than ten times the size of the domains grown on the h2-preannealed fe foil for the same exposure time . We also note that the edges are sawtoothed, a feature that we have observed previously for large h - bn domains grown on fe / sio2/si substrates, and which can be ascribed to diffusion instabilities . The incubation time for the nh3-preannealed catalyst is shorter, highlighted by the appearance of triangular domains after just 14 s of borazine exposure (figure 2d inset). The h - bn domains are largely merged after 90 s exposure (figure 2e), with only a few gaps left in the film . After 480 s of borazine exposure, we achieve full coverage of the fe catalyst with monolayer h - bn (figure 2f). The remaining variations in sem contrast over the surface of the sample are due to channeling contrast from the polycrystalline fe foil . Figure 3 summarizes the effect of three different cooling rates (stage 4 in figure 1) on the h - bn morphology after growth on the differently preannealed foils (h2 or nh3, 4 mbar), followed by a fixed borazine exposure (6 10 mbar, 900 c, 5 min). We note that the cooling rates refer to the initial cooling period from 900 c down to 500 c, after which the rate slows down and is comparable for all three cases . Figure 3a corresponds to immediate quenching (300 c / min) for a h2-preannealed foil, with the heater turned off immediately after borazine exposure, and shows a bimodal domain size distribution of few - layer h - bn . For an intermediate cooling rate of 100 c / min, the h - bn domains appear larger and more multilayered, and the density of the smaller domains is considerably higher (figure 3b), indicating that some portion of the h - bn is formed on cooling . For slow cooling rates of 50 c / min, the domains are similar in size and thickness to the intermediate cooling rate; however, the shapes are no longer strictly triangular and the edges become less sharp (figure 3c inset). The same cooling experiments were performed on the nh3-preannealed foils (figure 3d f). The h - bn domains remain as monolayers, with no multilayers observed for any of the cooling rates used here . This illustrates that, contrary to the h2-preannealing case, additional layer formation and nucleation do not occur on cooling, both of which are effects that can be linked to precipitation of b and n species from the catalyst bulk . Morphology of h - bn on h2-preannealed fe foil after (a) fast cooling (300 c), (b) medium cooling (100 c / min), and (c) slow cooling (50 c / min). Inset: detail of multilayered domains with irregular edges, indicating possible dissolution of material back into the bulk during slow cooling . Morphology of h - bn on nh3-preannealed fe foil after (d) fast cooling, (e) medium cooling, and (f) slow cooling . All growths were performed at 900 c and 6 10 mbar borazine exposure for 5 min . To further understand this difference in behavior with preannealing atmosphere, we performed a growth where we first preannealed the sample in nh3, and then held it at the growth temperature in vacuum for 30 min after the nh3 preanneal but prior to borazine exposure (i.e., extended stage 2 in figure 1). Figure s3 shows the postgrowth surface of this sample, which consists of a h - bn domain size distribution tending to be more bimodal than for the standard growth with the nh3 preannealing . Furthermore, most of the domains are smaller and multilayered, and are reminiscent of those grown on the h2-preanneled sample, despite the lower nucleation density . The structure and crystallinity of the h - bn formed by the optimized process using nh3 preanneling was determined by transmission electron microscopy (tem)/scanning transmission electron microscopy (stem). Figure 4a shows a scanning electron microscopy (sem) image of a holey carbon / copper tem grid after transferring isolated h - bn domains corresponding to figure 2d . Figure 4b shows a dark - field tem (df - tem) image of one such h - bn island, confirming the single - crystalline nature of the domain . The corresponding selected area electron diffraction pattern (upper right inset) shows one hexagonal set of diffraction spots also consistent with single - crystalline h - bn . The green circle indicates the diffraction spot used to produce the df - tem image . The lower right inset shows a defocused bright - field (bf) tem image confirming that the domain is indeed isolated, and a second differently oriented h - bn domain can be partially seen in the lower left corner . We further analyze the film by high angle annular dark field (haadf) stem, which reveals atomic and element specific contrast . The high - resolution stem image in figure 4c displays the hexagonal lattice of h - bn and the intensity profile along the marked yellow line exhibits a clear distinction of the b and n atoms (figure 4c inset), where the extracted intensity ratio is consistent with monolayer h - bn . Further confirmation of the monolayer nature of the h - bn was obtained by electron beam induced sputtering in supporting information figure s4, which exhibits direct, step - free sputtering of the layer to vacuum consistent with single - layer h - bn (whereas multilayer h - bn is sputtered in a layer - by - layer fashion).figure 4d shows a bright - field tem image of a suspended closed h - bn film, with the corresponding diffraction pattern and film edge close - up (top left and bottom right insets, respectively), demonstrating the crystalline and monolayer properties of the material . An optical image of h - bn domains transferred onto a sio2(300 nm)/si wafer, acquired with a green filter to enhance the contrast with the substrate, is shown in figure 4e . The raman spectrum from the area indicated by the red dot (figure 4f) exhibits a peak at 1369 cm, in agreement with literature values for cvd h - bn . (a) sem image of h - bn domains from figure 2d transferred onto a holey carbon / copper tem grid . (b) df - tem image of a triangular h - bn domain, confirming its single - crystalline nature . The upper right inset shows the corresponding hexagonal diffraction pattern with the diffraction spot, from which the df - tem image was acquired, indicated by the green circle . The lower right inset shows a defocused bf - tem image demonstrating that the island is indeed isolated . (c) high - angle annular dark field (haadf) stem image from the center of a triangular domain showing atomic and element specific contrast . The image was processed following krivanek et al . To reduce contributions from probe - tails . The intensity profile along the indicated yellow line (inset) exhibits an n / b intensity ratio consistent with monolayer h - bn . (d) bright - field tem image of a suspended h - bn film from the sample in figure 2f . The top left inset shows the diffraction pattern from this region and the bottom right inset is an edge - analysis, confirming the monolayer nature of the film . (e) optical image after transfer of the sample in figure 1e, showing coalescing h - bn domains . We note that the image was acquired using a green filter to enhance the contrast . (f) raman spectrum acquired in the region indicated by the red dot in panel (e), displaying the characteristic h - bn peak at 1369 cm and the si - related peak . In order to elucidate the growth mechanisms responsible for the differing h - bn morphologies observed in figure 2, we employ in situ xrd and in situ xps during growth . Figure 5 compares in situ xrd patterns acquired during h2- (a) and nh3-preannealed (b) h - bn growth . The as - loaded catalyst foils display reflections corresponding to body - centered - cubic (bcc) -fe . For the h2 preannealing (figure 5a), the foils transform to face - centered - cubic (fcc) -fe upon heating and concurrently grain growth occurs (shown via a sharpening of the reflections). We note that in our experimental setup the large fe grain sizes (80 m estimated from sem) result in the diffraction pattern originating from only a few grains, and hence, the measured apparent texture directions are not necessarily representative of the texture of the entire foil . Upon introduction of borazine following the h2 preannealing, a strong reflection at 18 is observed, which is ascribed to isothermal growth of few - layer h - bn, accompanied by multiple sharp reflections at higher angles that can be attributed to the formation of a small amount of fe - borides (fe2b, and possibly feb). Furthermore, the majority catalyst phase changes from -fe to -fe (with possibly small amounts of -fe remaining). After cooling to room temperature, the catalyst state consists of mostly -fe with some minor fe - borides and possibly some -fe . The observed fe - boride formation and the isothermal -fe -fe transition are both indicative of b uptake into the fe and are consistent with our recent work on h2-preannealed h - bn growth on fe films . In situ xrd patterns of fe foil catalyzed h - bn growth during the salient stages of the cvd process, comparing the h2 preannealing (a) and the nh3 preannealing (b). We note that intensity is plotted here on a log scale to emphasize minority phases . The catalyst was annealed in 2.6 mbar of h2 (a) and 4 mbar of nh3 (b), and the growth was performed at 900 c (estimated uncertainty in temperature 25 c) in 6 10 mbar borazine partial pressure for 10 min . In contrast, the nh3 preannealing (figure 5b) leads to a very different evolution: upon preannealing in nh3 the majority catalyst phase changes from -fe to -fe (as with h2 preannealing). However, although the xrd pattern for nh3 preannealing can be fully assigned to -fe, we note that the comparably broad peak at 30 could also correspond to the highest intensity reflection of -fe3n (not observed for h2 preannealing), which requires n uptake into the fe foil . We corroborate such n uptake by control experiments on fe(250 nm)/sio2(300 nm)/si which are exposed to the same nh3 preannealing annealing conditions . Although trace levels of diffused si have to be considered for these films, the lower degree of texture allows rietveld refinement of lattice constants . Upon nh3 exposure over a vacuum baseline, we find a lattice expansion corresponding to 0.6 atom% n uptake into the bulk of the -fe films (figure s5), confirming that n dissolves into the fe under our nh3 preannealing conditions . When borazine is introduced, the preceding nh3 preannealing impacts on the further catalyst evolution: in contrast to the h2-preannealed foil, -fe remains the majority catalyst phase and neither fe - borides are formed nor is a -fe -fe transition observed during borazine exposure . Additionally, we do not observe the emergence of a few - layer h - bn related reflection at 18. this lack of a signal corresponding to thick h - bn films is consistent with the exclusive monolayer h - bn growth observed in our ex situ characterization above (we note that our xrd setup cannot detect monolayer h - bn; however, monolayer h - bn growth was confirmed by ex situ sem for the nh3-preannealed sample grown during in situ xrd). During cooling to room temperature, after borazine exposure, a -fe -fe transition occurs . The additional emergence of fe4n reflections upon cooling (which were not observed in the h2 preannealing data) further corroborates n uptake into the catalyst during the nh3-preannealed h - bn cvd . Fe4n is only thermodynamically stable below 680 c, which explains why fe4n only nucleates upon cooling . In situ xps data summarized in figure 6 provides complementary, surface - sensitive information on the h - bn nucleation and growth processes . We note that the assignment of all the xps peaks and their corresponding shifts is not trivial for such a complex multicomponent system . Hence, we focus here on the main signatures of h - bn cvd and the major differences arising from the two preannealings . Figure 6a d show the time - resolved evolution of the xp b1s and n1s core level spectra for a fe foil preannealed at 900 c in either h2 (a, c) or nh3 (b, d) and subsequently exposed to borazine . The borazine exposure time (measured from the time at which the desired exposure pressure is reached) for each scan prior to cooling is indicated in the top left corner of each frame . Two main peak pairs are observed for both preannealings and are related to the h - bn structure, where the b atom is bonded to three n atoms in a planar hexagonal configuration . The b / n peak pair with higher binding energies (be) both pairs can relate to monolayer h - bn and can arise due to differences in coupling between the h - bn layer and the catalyst . In particular, effects such as different grain orientations, intercalation, and rippling / restructuring of the surface are known to change the interaction of the overlying 2d film with the substrate . The increase in the intensity of the lower be pair is then tentatively attributed to few - layer h - bn: on the basis of our previous work on cu - catalyzed h - bn cvd, we note that although the first h - bn layer in direct contact with the catalyst does show such coupling effects, for few - layer h - bn this interaction can be screened . Hence, an increasing peak intensity of the lower be pair here can be indicative of the presence of few - layer h - bn . For the h2-preannealed foil during the initial exposure to borazine, the peaks of both the high and low be pairs appear concurrently, with the low be pair dominating . This is consistent with our sem images showing the growth of multilayered islands for very short exposure times . With continuing borazine exposure, the relative peak intensity of the lower be pair continues to rise, indicative of a thickening of the h - bn domains, and then increases even further upon cooling . In contrast, for the nh3-preannealed foil, the high be pair is more intense in the early stages of growth, relative to the h2 preannealing, indicating isothermal growth of predominantly monolayer h - bn on fe . The fact that the low be pair dominates for the rest of the growth suggests that few - layer h - bn evolves with extended exposure time, which is indeed observed in the ex situ sem image of this sample taken after growth and cooling (figure s6b). Though the xps growth results differ somewhat from those of our well - calibrated reactor and the optimized growths in figure 2d f, we attribute this to the different conditions in the xps chamber, notably the much lower base pressure [10 mbar] and the lower permissible pressure of nh3 [0.5 mbar]. Effectively, the in situ xps experiments resemble the cvd growth with an extended stage 2, where insufficient n - enrichment of the catalyst bulk causes the formation of h - bn multilayers . We note that the slightly different ratio of the two be components between the b1s and the corresponding n1s (bottom two frames, figure 6) for the nh3 case can be attributed to the fact that the n1s spectra were always acquired after the b1s spectra, that is, with a typical time lag of 1 min, the n1s spectra thus correspond to a later stage of the growth . The increase in the intensity of the two peak pairs with extended borazine exposure (as apparent from the different intensity scalings of each frame) confirms that, regardless of the preannealing, h - bn formation occurs isothermally, which is in agreement with our in situ xrd measurements and ex situ sem observations . We further note that for the h2 preannealing case, a minor contribution from bulk precipitation upon cooling is also observed (figure 3a c). Conversely, for the nh3 preannealing case no signatures of precipitation are observed, even for very slow cooling rates (figure 3d f). Time - resolved in situ xps of the b1s core level for (a) h2- and (b) nh3-preannealed fe foil and the n1s core level for (c) h2- and (d) nh3-preannealed fe foil during borazine exposure and initial cooling stage (900 c, 1 10 mbar for nh3-preannealed sample and 1 10 mbar for h2-preannealed sample, 10 min). Spectra are collected in normal emission geometry at photon energies of 620 ev (escape 13). Figure 7a, b show depth - resolved b1s core level spectra taken after borazine exposure and cooling for h2- and nh3-preannealed foils, respectively . By changing the incident x - ray energies, and hence the inelastic mean free path of the photoelectrons (escape), the information depth can be varied, giving a more surface sensitive spectrum (escape 10) for hv = 400 ev and a more bulk sensitive (escape 13) spectrum for hv = 640 ev . For the h2-preannealed foil, we assign the component at 188 ev to b - related species (dissolved b or borides) and note that this component does not have a corresponding pair in the n1s spectrum, as would be expected . Comparison of the relative intensities of this component shows that the b - related species are stronger in the more bulk sensitive spectra (figure 7a inset), which is consistent with our xrd analysis, where we demonstrate that species present at higher concentrations in the catalyst bulk can segregate toward the surface during cooling, leading to the formation of additional phases (i.e., borides for h2 preannealing and nitrides for nh3 preannealing). Interestingly, the component at 188 ev is not detected for the nh3-preannealed foil, neither in the surface sensitive scan nor in the more bulk sensitive scan (figure 7b). This is consistent with the b uptake being significantly reduced by the presence of n dissolved in the fe bulk from the nh3 preannealing step . Both preannealings lead to the appearance of a small peak at 200 ev, which has been attributed to the * plasmon shake up satellite corresponding to sp bonded hexagonal boron nitride . The sharper satellite peak for the nh3-preannealed sample attests to the higher quality of the h - bn grown (figure 7b inset). Depth - resolved in situ xps b1s core level lines measured at room temperature in vacuum for (a) h2- and (b) nh3-preannealed fe foil after borazine exposure and cooling (growth parameters: 900 c, 1 10 mbar for nh3-preannealed sample and 1 10 mbar for h2-preannealed sample, 10 min borazine exposure time). The spectra are collected at photon energies of 400 ev (surface sensitive; escape 10) and 640 ev (bulk sensitive; escape 13). We also find that the onset of h - bn growth is strongly dependent on the borazine partial pressure, as summarized in figure s7 . For a given constant temperature, upon introducing 1 10 mbar of borazine, the n1s and b1s scans for both types of preannealed foils exhibit flat lines, indicating the absence of h - bn growth . After 9 min at 1 10 mbar of borazine, the n1s and b1s scans for the h2-preannealed sample remain flat . Small n1s and b1s peaks only start appearing for an increased borazine partial pressure of 7 10 mbar . For the h2-preannealed sample, significant h - bn growth is achieved by further increasing the borazine pressure to 1 10 mbar . We note that the sample was exposed to 7 10 mbar of borazine for 15 min before increasing the pressure to 1 10 mbar, and then exposed to this pressure for a further 20 min . In contrast, after 3 min of borazine exposure at 1 10 mbar of borazine, a notable peak appears in both n1s and b1s scans for the nh3-preannealed sample, which corresponds to h - bn nucleation . Upon continued exposure (6 min) at this same pressure, the peaks increase in intensity as the h - bn domains grow further . Combined, our in situ xrd and in situ xps characterizations suggest a strong influence of nh3 preannealing not only on h - bn growth but also on the underlying catalyst phase evolution and b / n uptake mechanisms . Our data reveals that the contribution of the fe bulk reservoir is critical in determining the h - bn growth behavior . Although other catalysts offer much lower b and n solubilities such that precipitation - driven growth is minimized, our focus here on fe substrates is motivated by our previous work, which shows fe to be an excellent catalyst for high - quality h - bn growth . In fact, we are able to use the bulk solubility of b and n in fe as a key advantage, and hence achieve better growth control using a bulk prefilling method, as discussed below . Similarly, a finite carbon solubility has previously been shown to substantially improve graphene growth uniformity on polycrystalline ni and co substrates, where the catalyst bulk acts as a mediating carbon sink that moderates variations in growth across different catalyst grains . Figure 8 schematically summarizes the processes taking place during the salient stages of the cvd process on fe foils, comparing the effects of the h2 and nh3 preannealing on the catalyst chemistry and on the growth of h - bn (figure 8a, b), and interpreting them in the context of ternary phase diagram considerations (figure 9). The two suggested growth pathways are summarized in the fe - rich corner of the fe n phase diagram at 950 c, and are marked by the red and blue arrows, denoting the h2 and nh3 preannealing routes, respectively . The phase diagram is applicable to our experiments given that the changes in the phase boundaries between the sections at 950 and 1050 c are minimal, and temperature uncertainties of 50 c in commercial cvd reactors are not untypical . In the framework of our discussion, we assume that the b and n diffusivity does not vary significantly depending on their relative proportions but is only dependent on temperature . Schematic illustrating the near - surface region of the fe catalyst and the proposed mechanisms involved in the growth of h - bn, comparing the (a) h2- and (b) nh3-preannealed foils respectively during different cvd stages . The downward and upward arrows indicate species diffusing into and out of the catalyst, respectively .,,, and represent the state of the catalyst during preannealing, short borazine exposure, long borazine exposure, and vacuum cooling, respectively . The red and blue arrows represent the reaction pathway for the h2- and nh3-preannealed foils respectively with the corresponding postgrowth sem images of the sample surface . Cvd of a compound material, such as h - bn, requires simultaneous feeding of b and n species into the growing stoichiometric crystal, which presents a more complex scenario compared to graphene growth, where only c atoms need to be incorporated into the graphitic lattice . First, we outline the b and n fluxes necessary for h - bn cvd, involving precursor dissociation and the formation of h - bn domains . The impingement of borazine molecules and their dissociation provides a flux of b and n species at the catalyst surface . Concurrently, a flux of b and n dissolving into the catalyst bulk will deplete the surface . A net flux is required for h - bn growth, which is equal to the difference between the flux reaching the surface and that diffusing into the catalyst . During the initial stages of borazine dosing these two fluxes will be matched and no h - bn nucleation occurs (i.e., incubation period). However, the concentration of b and n at the surface will start to increase gradually until a critical supersaturation at the fe surface is reached, giving rise to the first h - bn nucleation events . Following nucleation, growth of h - bn islands will proceed, fed by the net flux resulting from the precursor dissociation at the surface and the diffusion into the catalyst bulk . We briefly outline the importance of the bulk reservoir to cvd growth on catalysts with finite solubilities of the precursor species . For h - bn cvd on catalysts with significant b and n solubilities (such as fe), the supply of b and n to the catalyst surface to feed h - bn growth is mediated by their diffusion into the catalyst bulk . This allows uniform h - bn to be formed over the catalyst by locally saturating the catalyst close to the surface, while the bulk continues to provide a sink for b and n species . However, if the catalyst becomes saturated with these species throughout its bulk during the growth process, then the bulk of the catalyst no longer acts as a mediating sink and inhomogeneous few - layer h - bn can readily form isothermally, as well as by the precipitation of b and n to the surface upon cool - down . How quickly the catalyst becomes saturated throughout its thickness is ultimately dictated by its permeability (i.e., the product of solubility and diffusivity). Thus, low permeabilities, relative to the rate of b and n delivery to the catalyst surface, favor a broad processing window for monolayer h - bn formation, and hence, it is highly desirable to be able to control this property . Preannealing up to 900 c (figure 8a), the fe surface undergoes a reduction reaction of fe oxides, formed from ambient air storage of the foils, accompanied by the phase transformation -fe -fe, as confirmed by the xrd data in figure 5a . On precursor exposure, uptake of both b and n from the dissociation of borazine is confirmed by xrd, which shows the formation of borides and an expansion of the fe lattice due to n dissolution for similar growth conditions . The catalyst reservoir is therefore partially filled by the constituent species, illustrated by the downward red and blue arrows in figure 8a, and results in a longer incubation time for crystal growth because the critical supersaturation required for nucleation will take longer to achieve . N phase diagram, in which the curve in the solvus for intermediate b: n ratios is crossed at relatively high solubilies, and hence high permeabilities, of both species . At such intermediate b: n ratios, it is not clear which of the species it is whose supply is the limiting factor that controls the growth . After 10 min of borazine exposure, the xrd data shows the appearance of the few - layer h - bn reflection at 18, demonstrating that growth of additional h - bn layers occurs isothermally (small triangular domains in figure 8a). This is further confirmed by in situ xps, which additionally reveals the concurrent appearance of h - bn mono- and multilayers (figure 6a, c). Upon borazine removal and cooling (figure 3b, c), we show that further nucleation of new domains and thickening of existing domains occurs for relatively slow cooling rates on h2-preannealed foils . The fact that the h - bn domain edges appear to be dissolving for a cooling rate of 50 c / min (figure 3c inset) could be explained by considering that while the catalyst surface is saturated with b and n during growth, the catalyst is not saturated throughout the bulk (i.e., the b and n concentration is not uniform across the depth). Thus, the slow cooling rate is effectively equivalent to a postanneal, which can result in the increased diffusion of b and n into the catalyst bulk, decreasing the concentration of b and n near the catalyst surface and leading to the partial dissolution of the existing domains . For rapid quenching, a smaller contribution to growth from bulk precipitation we note that although we show clear evidence of b and n uptake in the fe bulk, this does not necessarily imply precipitation into h - bn upon cooling because the assembly of an h - bn domain requires a stoichiometric arrangement of atoms in the hexagonal lattice . For monoelemental systems such as c, growth of a graphic lattice by precipitation on cooling is simpler because only one element needs to be incorporated at the growth front . For compound materials like h - bn, however, our ex situ sem indicates that precipitation - driven growth does in fact occur . This can be explained by noting that the high permeabilities of b and n that saturate the catalyst surface (evidenced by boride formation and n - induced fe lattice expansion in xrd, as well as dissolved b / borides in the fe subsurface observed by in situ xps) can lead to inhomogeneous isothermal multilayer growth, as well as further multilayer formation upon cooling . The compositional trajectory for the h2-preannealed foil can thus be summarized by the red arrow in figure 9, which starting in the -fe phase field crosses the boundary -fe -fe + h - bn + fe2b during simultaneous feeding of n and b from borazine dissociation . In terms of growth modes, the formation of h - bn is predominantly isothermal, with a small contribution from bulk precipitation on immediate cooling, which is relatively minor given that the diffusivity of species rapidly decreases with temperature . The chemical and structural changes in the fe catalyst for the nh3-preannealed foil are markedly different . For the preannealed sample, we measure a lattice expansion of the fe due to n uptake corresponding to 0.6 atom% n (downward blue arrows in figure 8b), and possibly due to the additional formation of -fe3n as a minority phase . N phase diagram does not predict the presence of -fe3n at the temperatures and n content of our experiment; therefore, the contribution to the small reflection at 30 in figure 5b during preannealing is most likely due to -fe . The presence of n in fe has been shown to significantly reduce the -fe -fe transformation temperature, from 912 c for phase - pure fe to 875 c for fe-0.6 atom% n, which is therefore in line with our xrd phase assignment . As a consequence of the n enrichment before borazine dosing, the b: n ratio in the bulk of the foil is close to zero during dosing due to the shape of the solvus, crossing it to form h - bn only requires a small fraction of added b (0.002 at% b). The fact that borides are not detected effectively means that the solubility, and thus permeability, of b is greatly reduced when the catalyst is saturated throughout with n. with lower b permeability, the supersaturation required to form an h - bn nucleus therefore will be reached for lower borazine partial pressures compared to the h2-preannealed foil or for shorter exposure times at the same borazine exposure pressure . The first case is clearly supported by the in situ xps measurements . As shown in figure s7, the borazine partial pressure required for h - bn growth at 900 c depends on the preannealing performed and is found to be 1 10 mbar for the h2-preannealed foil, upon which growth is immediately observed, and 1 10 mbar for the nh3-preannealed foil where h - bn begins to form after 3 min of borazine exposure . We also confirm a shorter incubation time under the same borazine exposure pressure for nh3-preannealed foils compared to h2-preannealed foils through sem characterization (figure 2a, d insets). The ex situ sem images show that as we start dosing borazine, the nucleation of h - bn domains occurs much more rapidly on the nh3-preannealed foil, with triangular - shaped islands already observed after 14 s exposure (figure 8b). For longer exposure times the domains continue to grow laterally and neighboring domains start to coalesce . Contrary to the h2 preannealing case, we do not detect the isothermal -fe phase transformation to -fe . This, together with the lack of borides, both of which are processes linked to b uptake, reinforces the conclusion that the nh3-preannealing and the corresponding n uptake into the catalyst limits b diffusion into the fe during subsequent borazine exposure, that is, given that the b and n solubilities are interdependent, the high n concentration in -fe makes the corresponding b permeability very low . Additionally, the limited amount of b in the bulk reduces the likelihood of multilayer formation on cooling, as only n can escape from the sample surface . We note that during dosing at temperature, and in the absence of nh3 to replenish the bulk with n, these species can diffuse back out to the surface where they can leave as n2 (upward blue arrows in figure 8b). Indeed, figure s3 provides evidence demonstrating that the beneficial effect of the bulk n can be lost by long annealing times in vacuum prior to borazine exposure . Hence, to achieve uniform monolayer films, stage 2 (figure 1) should be kept as short as possible . The xrd data in figure 5b illustrates that during growth, and subsequently during simultaneous cooling and borazine removal, we do not detect the appearance of the reflection at 18 for the nh3 preannealing case, confirming that the h - bn thickness does not increase significantly . This is corroborated by ex situ sem (figure 2d f), which shows that the h - bn remains as monolayers for these growth conditions . Indeed, the sem images in figure 3d f are further proof that multilayers do not appear during cooling, even for slow cooling rates, which typically allow sufficient time for species to segregate at the surface, indicating a lack of b atoms that can be supplied from the bulk . The main difference compared with the h2-preannealing case is that b is now the limiting factor that governs the growth, which thus becomes kinetically controlled . In this regime, it therefore is possible to exclusively grow monolayer h - bn on a n - prefilled catalyst, as long as the extent of b diffusion into the catalyst bulk remains limited (figure 8b). On the basis of our experimental evidence, we propose that for the nh3-preannealed foil, the compositional pathway runs along the fe n edge in the -fe phase field during annealing (figure 9, long blue arrow), because no b is supplied at this stage and only n dissolves into the fe . Upon subsequent borazine dosing, the trajectory crosses the boundary into the -fe + h - bn + n2 phase field (short blue arrow) based on the isothermal growth of h - bn monolayers observed in combined sem, xrd, and xps . Our current work shows that the general bulk - mediated growth model reported here is also applicable to graphene cvd on fe foils using a c2h2 carbon source . Analogously to the h - bn growth, we observe reduced incubation times for nh3-preannealed foils (i.e., n - filled bulk reservoir) compared to vacuum - annealed foils (unfilled bulk reservoir). The data will be presented in future work; however, it demonstrates the robustness and wide applicability of our model, which is anticipated to be relevant for the fabrication of heterostructures . Indeed, the use of a catalyst prefilling method for growth control has previously been used in other material systems . A notable example is the catalytic growth of si / ge heterostructure nanowires (nws) with compositionally abrupt interfaces, which requires the minimization of the solubility of si and ge in the liquid au catalyst in order to reduce the catalyst bulk reservoir effect . In summary, a significant level of improvement in the growth of h - bn is achieved through a bulk reservoir filling effect in an as - received fe foil by predosing n, one of the constituent species, in the form of nh3 during preannealing . Using in situ xrd and xps, we demonstrate how n - induced changes to the fe catalyst phase evolution and composition directly impact the h - bn incubation time and the uptake of b and n species during dosing . These critical parameters then determine structural h - bn features, such as number of layers, domain size, and nucleation density . When the catalyst bulk is enriched with n from the high temperature and high pressure preannealing in nh3, the diffusion of b and n species in the fe subsurface during subsequent borazine exposure is limited . This effectively prevents significant additional h - bn layer formation that typically occurs by precipitation upon cooling, and which is indeed observed for h2-preannealed fe foils (i.e., unfilled bulk reservoir). Bulk filling also leads to shorter incubation times and lower borazine partial pressures required to nucleate h - bn, which reduces the probability of multilayer formation and a large domain size distribution . Preannealing the catalyst with nh3 allows us to control the subsequent uptake of precursor species during dosing . Importantly, given the interdependency of the b and n solubilities, it allows us to lower the permeability of b, which leads to uniform h - bn monolayer growth . The catalyst bulk prefilling method presented here therefore provides an elegant alternative to using different catalysts or using catalyst alloying to control the permeability of the growth species . The general model that we derive, based on complementary ex situ and in situ data, in combination with phase diagram considerations, forms a coherent picture of the key bulk contributions to growth control and, importantly, is applicable to other catalytically grown 2d materials . As - received fe foil (0.1 mm, alfa aesar, 99.99% purity) is used for all experiments . The h - bn domains and films are grown in a customized aixtron bm3 cold - wall reactor (base pressure 1 10 mbar). Cvd growth of h - bn is performed using a borazine (hbnh)3 precursor at a temperature of 900 c and a total pressure of 6 10 mbar . The samples are typically heated in 4 mbar of nh3 or h2 at 100 c / min up to 750 c and then at 50 c / min up to 900 c . The estimated uncertainty in the temperature measurement is 25 c . Immediately after reaching 900 c borazine is dosed into the chamber through a leak valve (from a liquid reservoir) and after growth (growth times varied from 14 s to 8 min) the borazine leak valve is closed and the heater is turned off . Samples are cooled in vacuum . For raman spectroscopy, optical microscopy, and df - tem / stem, we perform the transfer by spin coating a support layer of poly(methyl methacrylate) (pmma) at 5000 rpm for 40 s onto the h - bn . The sample is placed in a naoh bath (1 m) and during electrolysis h2 bubbles evolve at the h - bn / fe interface, lifting the film from the substrate . The pmma / h - bn film is rinsed in deionized (di) water and scooped onto a sio2(300 nm)/si wafer where it is left to dry . The pmma is removed by immersing the sample in acetone for 12 h, followed by a rinse in ipa . For the ex situ characterization of the h - bn on the catalyst, we use scanning electron microscopy (sem, zeiss sigmavp, 2 kv). Optical images are acquired using a nikon eclipse me600l microscope and a green filter was introduced for enhanced contrast . Raman spectroscopy is performed with a renishaw raman invia microscope using a 50 objective lens and a 532 nm laser excitation . A philips cm200 was used for bright - field (bf-) and dark - field transmission electron microscopy (df - tem) and selected area electron diffraction (saed) at 80 kv . A nion ultrastem 100 was employed for scanning transmission electron microscopy (stem), using an electron acceleration voltage of 60 kv and a high angle annular dark field (haadf) detector . Atomic - resolution stem data was processed to reduce contributions from probe - tails (an unprocessed stem image is shown in figure s4). The intensity profile was extracted from the processed image data by subtracting the remaining averaged intensity at intensity minima between atoms from the profile followed by normalizing the intensity at b sites to 1 . As grown h - bn films were transferred from the catalyst for s(tem) via the bubbling method onto holey carbon tem grids with regular hole arrays . In situ x - ray diffraction (xrd) was measured at the european synchrotron research facility (beamline bm20/robl) using a x - ray wavelength of 1.078 in a previously described setup . Measurements were acquired in symmetric theta-2theta geometry (information depth in m range) with the fe foils clamped on one side by alumina spacers . The intensity step at 16 in all measurements is due to the arrangement of detector and x - ray entrance / exit slits into the reaction chamber . We note that reflection positions shift between room temperature and cvd temperature scans due to thermal expansion . For phase identification the inorganic crystal structure database (icsd) (-fe, 53451; -fe, 44862; fe2b, 391330; feb, 391331; -fe3n, 80930; -fe4n, 79980; h - bn, 167799) and the international center for diffraction data (icdd) database (fe2b, 361332; feb, 320463) were used . Rietveld refinement of data was done using xpert plus software . Quoted in situ xrd temperatures (900 c) may be underestimated by up to 25 c, thus explaining the observed -fe -fe transition for the h2 pretreatment (figure 5a), which would be thermodynamically only expected for> 912 c for pure fe . In situ high - pressure xps measurements during preannealing, growth, and cooling were performed at the isiss end station of the fhi - mpg at the bessy ii synchrotron . We employ a high - pressure setup that consists of a cell (base pressure 10 mbar) that is attached to a set of differentially pumped electrostatic lenses and a differentially pumped analyzer (phoibos 150, specs gmbh). All the spectra are acquired in normal emission geometry, using a spot size of 80 m 150 m and with a spectral resolution of 0.3 ev . To perform depth - resolution experiments, the photon energy (ephoton) is varied in order to change the kinetic energy of the emitted photoelectrons, thus changing the inelastic mean free paths, escape.
|
Cardiovascular disease (cvd) affects the heart and blood vessels and is the principal cause of death worldwide . Considered the primary risk factor for cvd, high blood pressure, or hypertension, consists of a sustained increase in blood pressure levels . In 2000,> 25% of the population worldwide (approximately 1 billion) suffered from hypertension, a figure predicted to increase to 1.56 billion by 2025 . Angiotensin i - converting enzyme (peptidyl carboxy peptidase, ec 3.4.15.1, ace) belongs to the class of zinc proteases that require zinc and chloride for activation . Ace plays an important role in blood pressure regulation via the renin - angiotensin system (ras) and the kallikrein - kinnin system (kks). In the kks, ace inactivates the vasodilator bradykinin, while in the ras, ace acts as an exopeptidase cleaving his - leu from the c - terminal of decapeptide angiotensin i and producing the potent vasoconstrictor octapeptide angiotensin ii . Use of enzyme technologies for protein recovery and modification has led to production of a broad spectrum of food ingredients and industrial products . Hydrolysis selectivity is commonly manipulated by employing proteases from different sources due to their specificity for peptide bonds adjacent to certain amino acids . Ace - i inhibitory peptides derived from food proteins have attracted particular attention for their ability to prevent hypertension . Compared with chemosynthetic drugs, peptides from food proteins they therefore hold promise as potent functional food additives and represent a healthier and more natural alternative to ace inhibitor drugs . Dietary ace - i inhibitory peptides may be bioavailable . Some ace - i inhibitory peptides resist digestion, can be absorbed in the intestine, and are stable in the blood, suggesting they may produce an acute blood pressure - lowering effect after oral administration . The first ace - i inhibitory peptide was isolated from snake venom, and since then many others have been discovered in enzymatic hydrolysates of different food proteins . Food protein sources used to date include casein, whey protein, fish protein, chicken eggs, and wheat germ . . The degree of hydrolysis and inhibitory activities of angiotensin i - converting enzyme (ace) increased with increasing proteolysis time . Population growth and shrinking food resources are ongoing challenges in developing countries, while excessive animal protein intake and associated unhealthy levels of saturated fats exposure are increasingly common in developed countries . In response, research interest has steadily grown in the search for new sources of proteins from nonconventional raw materials . The genus salvia l. belongs to the lamiaceae family and includes about 900 species found worldwide, most mainly in the mediterranean, southeast africa, and central and south america . Cultivated for culinary, medicinal, and ornamental uses, salvia species form part of ethnopharmacological traditions and are an important crop, especially for small farmers . Recent research has addressed the chemical composition, biological properties, and possible applications of its essential oils, which may be sources for economically promising natural products with uses in the food, pharmaceutical, and cosmetic industries . The present study objective was to identify and quantify ace - i inhibitory activity in protein hydrolysates from a salvia hispanica protein rich fraction hydrolyzed with an alcalase - flavourzyme sequential system, and in ultrafiltered fractions from this hydrolysate . Chia (s. hispanica, l.) seeds were obtained in yucatan state, mexico . Reagents were of analytical grade and purchased from j. t. baker (phillipsburg, nj, usa), sigma (sigma chemical co., st . Louis, mo, usa), merck (darmstadt, germany), and bio - rad (bio - rad laboratories inc . Angiotensin - converting enzyme from rabbit lung (2 units / mg protein) was purchased by sigma (a6778 sigma chemical co., st . The alcalase 2.4 l and flavourzyme 500 mg enzymes were purchased from novo laboratories (copenhagen, denmark). Flour was produced from 6 kg chia seed by first removing all impurities and damaged seeds, crushing the remaining sound seeds (moulinex dpa139, zapopan, mexico), and milling them (krups 203 mill, mexico city, mexico). Oil extraction from the milled seeds was done with hexane in a soxhlet system for 2 h. the remaining fraction was milled with 0.5 mm screen (thomas model 4 wiley, swedesboro, nj, usa). The defatted chia flour was dried in a labline stove at 60c for 24 h. extraction of the protein - rich fraction was done by dry fractionation of the defatted flour according to vzquez - ovando et al . . Briefly, 500 g flour was sifted for 20 min using a tyler 100 mesh (140 m screen) and a ro - tap agitation system . The chia protein - rich fraction was hydrolyzed in batches by sequential treatment with alcalase and flavourzyme . A predigestion with alcalase for 60 min hydrolysis conditions were substrate concentration, 2 g/100 g; enzyme / substrate ratio, 0.3 au g for alcalase and 50 lapu g for flavourzyme; ph, 7 for alcalase and 8 for flavourzyme and temperature, 50c . Hydrolysis was done in a reaction vessel equipped with a stirrer, thermometer, and ph electrode . In all treatments, the reaction was stopped by heating to 85c for 15 min, followed by centrifuging at 9880 g for 20 min to remove the insoluble portion . Degree of hydrolysis (dh) was calculated by determining free amino groups with o - phthaldialdehyde following the methodology described by nielsen et al . As follows: dh = h / htot100, where htot is the total number of peptide bonds per protein equivalent, and h is the number of hydrolyzed bonds . The htot factor is dependent on the raw material amino acid composition and was determined by reverse - phase high performance liquid chromatography (rp - hplc). Samples (24 mg protein) were treated with 4 ml of 6 mol equivalent to l hcl, placed in hydrolysis tubes, and gassed with nitrogen at 110c for 24 h. they were then dried in a rotavapor and suspended in 1 mol l sodium borate buffer at ph 9.0 . Amino acids were separated using hplc with a reversed - phase column (300 3.9 mm, nova - pak c18, 4 mm; waters) and a binary gradient system with 25 mmol l sodium acetate containing (a) 0.02 g l sodium azide at ph 6.0 and (b) acetonitrile as solvent . Flow rate was 0.9 ml min, and elution gradient was time 0.03.0 min, linear gradient a: b (91: 9) to a b (86: 14); time 3.013.0 min, elution with a b (8614); time 13.030.0 min, linear gradient a b (86: 14) to a b (69: 31); time 30.035.0 min, elution with a b (69: 31). The hydrolysate was fractionated by ultrafiltration, using a high performance ultrafiltration cell (model 2000, millipore). Five fractions were prepared using four molecular weight cut - off (mwco) membranes: 1 kda, 3 kda, 5 kda, and 10 kda . Soluble fractions prepared by centrifugation (9880 g for 20 min) were passed through the membrane starting with the largest mwco membrane cartridge (10 kda). Retentate and permeate were collected separately, and the retentate recirculated into the feed until maximum permeate yield was reached at this size, as indicated by a decrease in permeate flow rate . Permeate from the 10 kda membrane was then filtered through the 5 kda membrane with recirculation until maximum permeate yield was reached . The 5 kda permeate was then recirculated through the 3 kda membrane and the 3 kda permeate through the 1 kda membrane . This process minimized contamination of the larger molecular weight fractions with smaller molecular weight fractions, while producing enough retentates and permeates for the following analyses . The five ultrafiltered peptide fractions (upf) were prepared and designated as> 10 kda (10 kda retentate); 510 kda (10 kda permeate5 kda retentate); 35 kda (5 kda permeate3 kda retentate); 13 kda (3 kda permeate1 kda retentate); <1 kda (1 kda permeate). Ace - i inhibitory activity in the hydrolysate and its upf was analyzed following the method of hayakari et al . Which is based on the fact that ace - i hydrolyzes hippuryl - l - histidyl - l - leucine (hhl) yielding hippuric acid and l - histidyl - l - leucine . This method relies on the colorimetric reaction of hippuric acid with 2,4,6-trichloro - s - triazine (tt) in a 0.5 ml incubation mixture containing 40 mol potassium phosphate buffer (ph 8.3), 300 mol sodium chloride, 40 mol 3% hhl in potassium phosphate buffer (ph 8.3), and 100 mu / ml ace - i . This mixture was incubated at 37c/45 min and the reaction terminated by addition of tt (3% v / v) in dioxane and 3 ml 0.2 m potassium phosphate buffer (ph 8.3). After centrifuging the reaction mixture at 10,000 g for 10 min, enzymatic activity was determined in the supernatant by measuring absorbance at 382 nm . All runs were done in triplicate . Ace - i inhibitory activity was quantified by a regression analysis of ace - i inhibitory activity (%) versus peptide concentration, and ic50 values (i.e., the peptide concentration in g protein / ml required to produce 50% ace - i inhibition under the described conditions) was defined and calculated as follows: (1)ace - i inhibitory activity (%) = (ab)(ac)100, where a represents absorbance in the presence of ace - i sample, b absorbance of the control, and c absorbance of the reaction blank . Consider the following: (2)ic50=(50b)m, where b is the intersection and m is the slope . Amino acid composition was determined in the upf with the highest biological activity, according to the method of alaiz et al . Samples (24 mg protein) were treated with 4 ml of 6 mol equivalent l hcl, placed in hydrolysis tubes, and gassed with nitrogen at 110c for 24 h. they were then dried in a rotavapor and suspended in 1 mol l sodium borate buffer at ph 9.0 . Amino acids were separated using hplc with a reversed - phase column (300 3.9 mm, nova - pak c18, 4 mm; waters) and a binary gradient system with 25 mmol l sodium acetate containing (a) 0.02 g l sodium azide at ph 6.0 and (b) acetonitrile as solvent . Flow rate was 0.9 ml min, and the elution gradient was time 0.03.0 min, linear gradient a: b (91: 9) to a b (86: 14); time 3.013.0 min, elution with a b (8614); time 13.030.0 min, linear gradient a b (86: 14) to a b (69: 31); time 30.035.0 min, elution with a b (69: 31). After filtration through 10, 5, 3, and 1 kda membranes in a high performance ultrafiltration cell, 10 ml of the fraction with highest ace - i inhibitory activity was injected into a sephadex g-50 gel filtration column (3 cm 79 cm) at a flow rate of 25 ml / h of 50 mm ammonium bicarbonate (ph 9.1). Peptide molecular masses were determined by referring to a calibration curve running molecular mass markers on the sephadex g-50 under identical conditions and those used for the test samples . Molecular mass standards were thyroglobulin (670 kda), bovine gamma globulin (158 kda), equine myoglobin (17 kda), vitamin b12 (1.35 kda), and thr - gln (0.25 kda). Fractions selected for further peptide purification were pooled and lyophilized before rp - hplc . One - way anovas were run to evaluate in vitro ace - i inhibitory activity and a duncan multiple range done to identify differences between treatments . All analyses were done according to montgomery and processed using the statgraphics plus version 5.1 software . With a protein content of 46.7%, the chia protein - rich fraction proved to be good starter material for hydrolysis . Production of extensive (i.e.,> 50% dh) hydrolysates requires use of more than one protease because a single enzyme cannot achieve such high dhs within a reasonable time period . For this reason, an alcalase - flavourzyme sequential system was used in the present study to produce an extensive hydrolysate . Alcalase (ec 3.4.21.62) is a proteinase from bacillus licheniformis and the flavourzyme (ec 3.4.11.1) is a fungal protease from aspergillus oryzae with both endo- and exopeptidase activities . The bacterial endoprotease alcalase is limited by its specificity, resulting in dhs no higher than 20%25%, depending on substrate, but can attain these dhs in a relatively short time under moderate conditions . When alcalase is used to hydrolyze protein it tends to produce peptides whose c - terminals are amino acids with large side chains and no charge (aromatic and aliphatic amino acids), such as ile, leu, val, met, phe, tyr, and trp . The fungal protease flavourzyme has broader specificity, which, when combined with its exopeptidase activity, can generate dh values as high as 50% . Flavourzyme is recommended for production of hydrolysates or peptides with biological activity and low bitterness . Both alcalase and flavourzyme tend to generate peptides with hydrophobic amino acid c - terminals, and qsar analyses of ace - i inhibitory peptides have shown that peptides with hydrophobic amino acid c - terminals exhibit potentially strong ace - i inhibition . Therefore, alcalase and flavourzyme are probably suitable for preparing high - activity ace - i inhibiting peptides . In addition, both proteases are suitable for industrial applications and are microbial enzymes, meaning that they are easily obtained and relatively low cost compared to other enzymes such as proteinase k and chymotrypsin c . Chia hydrolysates obtained with the alcalase - flavourzyme sequential system had clear biological activity and are promising prospects for use in new product development . The highest dh in the present study (51.6%) was attained with flavourzyme at 150 min . However, it was made possible by alcalase predigestion, which increases the number of n - terminal sites, thus facilitating hydrolysis by flavourzyme . This dh was higher than reported for hard - to - cook bean hydrolyzed with alcalase - flavourzyme (43%) or pepsin - pancreatin (26.2%) for 90 min . But it was lower than reported for chickpea (65%) hydrolyzed with alcalase - flavourzyme at 150 min and for rapeseed (60%) hydrolyzed with the same system at 3 h . When hydrolyzed sequentially with alcalase and flavourzyme, chia s. hispanica is an appropriate substrate for producing extensive hydrolysates (dh higher than 10%), and a natural source of peptides with potential bioactivity . A number of natural ace - i inhibitors have been isolated from different organism proteins, including peptides extracted by enzymatic hydrolysis . When added to food systems, enzymatic hydrolysates have exhibited advantages such as improved water - binding capacity, emulsifying stability, protein solubility, and nutritional quality . Hydrophobicity of amino - acid side chains is normally due to relatively small peptides with molecular weights between 1,000 and 6,000 da . Enzymatic hydrolysis is an effective way of producing bioactive peptides, which are short peptides released from food proteins by hydrolysis and with biological activities that may be beneficial to the organism . Bioactive peptides usually contain 320 amino acids per molecule and are inactive within the parent protein molecule sequence . The protein hydrolysate obtained from the chia protein - rich fraction had 58.5% ace - i inhibition . This is lower than the 79.5% reported for yak milk casein hydrolyzed with alcalase for 240 min, but higher than the 5%50% obtained for protein hydrolysates from amaranth (amaranthus hypochondriacus) albumin 1 and globulin . Ace - i inhibition ranged from 53.8% to 69.3% (figure 1), with clear increases (p <0.05) in activity in progressively smaller fractions; that is, the> 10 kda fraction had the lowest activity and the <1 kda had the highest . This coincides with a study in which the <1 kda fraction of an alaska pollock (theragra chalcogramma) frame protein hydrolysate had the highest ace - inhibitory activity (87.6%, ic50 = 457 g / ml). In another study, protein hydrolysate from chinese soft - shelled turtle had a lower ace - i inhibitory effect (ic50 = 280 g / ml) than its corresponding <5000 kda ultrafiltered fraction (ic50 = 190 5 g / ml). A greater inhibitory effect at smaller molecular weights was also observed in a yak casein hydrolysate fraction, in which the <6 kda fraction was the most effective (85.4%). When taken in conjunction, these results support the suggestion that ultrafiltration is an effective way of enriching ace - i inhibitory peptides from chia proteins . An amino acid profile was generated for the <1 kda upf because it had the highest ace - i inhibitory activity . During hydrolysis, asparagine and glutamine partially converted to aspartic acid and glutamic acid, respectively; the data for asparagine and/or aspartic acid were therefore reported as asx while those for glutamine and/or glutamic acid were reported as glx . The high inhibitory activity (69.3%) exhibited by the <1 kda upf was probably due to its high concentration of hydrophobic amino acids (41.68 g/100 g), including pro (6.11 g/100 g), phe (11.03 g/100 g), leu (10.23 g/100 g), and ile (6.57 g/100 g) (table 1). Amino acid c - terminal hydrophobicity has the greatest influence on ace - i inhibitory activity, and the higher the hydrophobicity the higher the inhibitory activity . Found that dipeptides could have high ace - i inhibitory activity if c - terminals were aromatic amino acids and proline, and n - terminals were aliphatic amino acid branches . Measured cheung's peptides samples in the same laboratory, modeled the results with a qsar, and found that dipeptides with positively - charged amino acids at the n - terminal and bulky hydrophobic amino acids at the c - terminal had stronger ace - i inhibitory activity . Using z descriptors to investigate the quantitative structure - activity relationship of ace - inhibitory dipeptides, wu et al . Found that ace - inhibitory activity was strongly affected by the three - dimensional chemical properties and hydrophobicity of c - terminal amino acids; that is, the higher the volume and the greater the hydrophobicity of the amino acids, the higher the ace - i inhibitory activity . Therefore, some dipeptides with hydrophobic amino acids at the c - terminal, such as phenylalanine, tryptophan, and tyrosine, will have high ace - i inhibitory activity . Wu et al . Also reported that strongly hydrophobic and small n - terminal amino acids, such as valine, leucine, and isoleucine, were more suitable for high - activity tripeptides . Low charge, large size, and weak hydrophobicity in the second amino acid from the n - terminal were more suitable high activity . Finally, for the c - terminal, high charge, large volume, and strongly hydrophobic residues (e.g., aromatic amino acids) were more suitable . In an analysis of ace - i inhibitory . Found that for peptides with 6 amino acids at the c - terminal, ace - i inhibition was strongly affected by hydrophobicity, positive charge, and volume of amino acids adjacent to the c - terminal, whereas the n - terminal amino acid had no direct relationship . Hydrophobicity and c - terminal amino acid size are apparently the principal aspects affecting ace - inhibitory activity . It stands to reason that hydrophobic amino acids, aromatic amino acids, and branched - chain amino acids are important components in high - activity peptides, and that proteins with high contents of these amino acid types (especially aromatic amino acids) have more potential to produce high activity ace - i inhibitory peptides . Therefore, digestion of proteins to produce peptides with hydrophobic amino acids at the c - terminal will tend to increase ace - i inhibitory activity in any derived hydrolysates . Although the structure - activity relationship of ace - i inhibitory peptides has not yet been established, these peptides show some common features . Studies of the structure - activity relationships in different ace - i inhibitory peptides indicate that binding to ace is strongly influenced by substrate c - terminal tripeptide sequence . Ondetti and cushman proposed a binding model for interactions between the substrate and active ace site . C - terminal tripeptide residues may interact with the s1, s1, and s2 subsites at the active ace site . Ace appears to prefer substrates or competitive inhibitors that contain hydrophobic amino acid residues at the three c - terminal positions . Captopril owes its potency and selectivity to chemical design guided by a hypothetical active site model based on the observed properties of ace and on an analogy to the known active site of a related zinc - containing peptidase . Ace's zinc ion is appropriately located between s1 and s1, allowing it to participate in hydrolytic cleavage of the substrate peptide bond and resulting in release of a dipeptide product . Studies of the structure - activity relationship in ace - i inhibitory peptides have shown that those with potent inhibitory activity have proline, phenylalanine, or tyrosine at the c - terminal, as well as hydrophobic amino acids in their sequence . In light of this previous research, the ace - i inhibitory activity observed here in the chia hydrolysate was probably due to amino acid composition (figure 2). Of the upfs, the <1 kda fraction exhibited the highest ace - i inhibitory activity and was selected for further fractionation . A molecular weight profile was generated of this upf using gel filtration chromatography (sephadex g-50 column). This profile was typical of a protein hydrolysate formed by a pool of peptides, with gradually decreasing molecular masses . Elution volumes between 406 and 518 ml included free amino acids and peptides with molecular masses ranging from 0.4 to 3.6 kda . This range was fractionated into three fractions and ace - i inhibitory activity determined for each . Fractions with elution volumes smaller than 406 ml and greater than 518 ml were not analyzed because they largely included peptides with high molecular weights, as well as free amino acids . Ace - i inhibitory activity (%) in the <1 kda upf ranged from 48.4% to 62.6% (figure 3). The highest ace - i inhibitory activity was observed in fraction f1 (62.6%; 427455 ml elution volume). Its molecular mass was approximately 1.52.5 kda, indicative of 712 amino acid residues . The ic50 value for f1 (3.97 g / ml) was lower than those of gel filtration (sephadex g-25) peptide fractions from tuna broth hydrolysate (210 to 25,260 g / ml) or from buckwheat fagopyrum esculentum moench (sephadex c-25 = 25,715.1 g / ml; sephadex g-10 = 21,315.1 g / ml). Other studies suggest that ace inhibition by hydrolysates depends on the source species and hydrolysate purity level . For instance, production of a 13 kda protein hydrolysate from alaska pollock frame using an ultrafiltration membrane bioreactor system resulted in high ace - i inhibition (ic50 = 110 g / ml), but further purification using consecutive chromatographic methods in a sp - sephadex c-25 column and hplc in an octadecylsilane column resulted in a still stronger effect (ic50 = 14.7 g / ml). In another example, an enzymatic hydrolysate from cuttlefish (sepia officinalis) muscle protein was found to have high ace - i inhibitory activity (87.1 0.9% at 200 g / ml). However, size exclusion chromatography with a sephadex g-25 produced a fraction (p6) with yet higher ace - i inhibition (ic50 = 11.6 mol / l), which, when fractionated by reverse - phase (rp)-hplc, was found to contain ala - his - ser - tyr, gly - asp - ala - pro, ala - gly - ser - pro, and asp - phe - gly . In ace - i inhibitory peptides from chia, the protein - rich fractions are not as potent as hypertension treatment drugs but hold promise as a safe, natural therapeutic agent without adverse side effects . The potential of chia protein - derived peptides as antihypertension agents depends on the ability of these peptides to reach their target site without suffering degradation and consequent inactivation by intestinal or plasma peptidases . Resistance to peptidase degradation is a probable prerequisite for any ace inhibitory hydrolysates / peptides to exercise an antihypertensive effect after oral or intravenous administration . Proline - containing peptides are generally resistant to degradation by digestive enzymes, and tripeptides containing a pro - pro c - terminal are resistant to proline - specific peptidases . However, peptide degradation or fragmentation results in smaller peptides and therefore in potentially more potent ace - i inhibitory activity . Clearly, in vivo studies are needed to confirm the effect of these peptides since it is both difficult and unwise to extrapolate directly from in vitro to in vivo activity . The main challenges in doing this are determining bioavailability of ace - i inhibitory peptides after oral administration and the fact that peptides may influence blood pressure by mechanisms other than ace - i inhibition . To exert an antihypertensive effect after oral ingestion, ace - i inhibitory peptides must reach the cardiovascular system in an active form, meaning that they need to remain active during digestion by human proteases and transport through the intestinal wall into the blood . Bioavailability has been studied for some ace - i inhibitory peptides, and it is known that proline - containing peptides are generally resistant to degradation by digestive enzymes . Peptides can be absorbed intact through the intestine by paracellular and transcellular routes, although postabsorption bioactivity potency is inversely correlated to chain length . All the chia derivatives studied here had ic50 values far higher than the 0.0013 g / ml of captopril, a synthetic ace - i inhibitor . Nonetheless, the chia purified peptides have biological potential, and the f <1 kda fraction had high ace - i inhibitory activity, suggesting that ace - i inhibitory peptides are rich in hydrophobic amino acids (aromatic or branched chains) and in proline . Ace - i inhibiting peptides from food sources have garnered increasing attention in recent years as promising natural biofunctional alternatives to synthetic drugs . Many of these peptides have been discovered in enzymatic hydrolysates of different food - source proteins and subsequently applied in the prevention of hypertension and initial treatment of mildly hypertensive individuals . The ongoing search for natural ace inhibitors may eventually help to create safer and less costly alternatives to synthetic pharmaceutical treatments . Chia proteins hydrolyzed with the alcalase - flavourzyme sequential enzyme system resulted in hydrolysates with ace - i inhibitory activity . Ultrafiltration produced a very low molecular weight fraction (<1 kda) which had the highest activity . Enzymatic hydrolysis and ultrafiltration are promising bioprocesses for production of new bioactive food ingredients such as ace inhibitory peptides purified from chia hydrolysate.
|
Septicemia in neonates refers to generalized bacterial infection documented by positive blood culture in the first four weeks of life and is one of the four leading causes of neonatal mortality and morbidity in india . Neonatal septicemia continues to be a major problem for neonates in neonatal intensive care units around the world . Neonatal mortality rate is one of the indicators for measuring the health status of a nation . There could be various reasons for neonatal mortality but septicemia continues to be a major cause of neonatal mortality and morbidity worldwide . Incidence varies from country to country, but it is much higher in developing countries than in developed nations . According to world health organization (who) estimates, there are about 5 million neonatal deaths a year, with 98% occurring in developing countries . Neonatal sepsis is broadly divided into two types according to age of onset: early - onset sepsis (<72 hrs) and late - onset sepsis (72 hrs-28 days). Early - onset sepsis is acquired during fetal life, delivery, or at the nursery . Neonatal sepsis is caused by a variety of gram - positive as well as gram - negative bacteria, and sometimes yeasts . The spectrum of organisms that causes neonatal sepsis changes over times and varies from region to region . Periodic evaluation of organisms responsible for neonatal sepsis is essential for the appropriate management of neonates . Therefore, this study was undertaken to determine the profile and antibiotic sensitivity patterns of aerobic isolates from blood cultures of neonates in a tertiary care hospital in bijapur, india . An analysis was conducted on all blood culture reports obtained between january 2008 and december 2010 from newborns admitted to the department of pediatrics and the neonatal intensive care unit (nicu) at shri b m patil medical college, bijapur . Blood culture sample included a single sample collected from a peripheral vein or artery under aseptic conditions . The local site was cleansed with 70% alcohol and povidone iodine (1%), followed by 70% alcohol again . Blood cultures were done in a brain heart infusion biphasic medium . Approximately, 3 ml of blood was inoculated into the brain heart infusion broth and incubated at 37c . Subcultures were done on sheep blood agar and macconkey agar at the earliest visual detection of turbidity or blindly on days 1, 4, and 7 if the bottles did not show turbidity . Isolate was identified by their characteristic appearance on their respective media, gram staining and confirmed by the pattern of biochemical reactions using the standard method . Members of the family enterobacteriaceae were identified by indole production, h2s production, citrate utilization, motility test, urease test, oxidase, carbohydrate utilization tests, and other tests . For gram - positive bacteria, coagulase, catalase, blood culture broth that showed no microbial growth within seven days was reported as culture negative, only after result of routine subculture on blood, macconkey, and chocolate agar . Bauer disc diffusion method as recommended in the national committee for clinical laboratory standards (nccls) guidelines . The drugs for disc diffusion testing were in the following concentrations: ampicillin (10 g), cloxacillin (1 g), lomefloxacin (10 g), amoxiclav (20/10 g), cephalexin (30 g), cefuroxime (30 g), ciprofloxacin (5 g), erythromycin (15 g), gentamicin (10 g), (30 g), penicillin (10 units), tetracycline (30 g), co - trimoxazole (125 g trimethoprim/2375 g sulfamethoxazole), amikacin (30 g), ofloxacin (5 g), sparfloxacin (5 g), pefloxacin (5 g), cefoperazone (75 g), netilmicin (30 g), imipenem (10 g), piperacillin / tazobactam (100/10 g), azithromycin (15 g), and linezolid (30 g). The discs were obtained from himedia (india) laboratories . Data analysis was done using statistical package for social sciences (spss) software version 14.0 . During the study period, a total of 683 newborns with clinical sepsis were admitted . Positive cases, there were 86 (65.5%) male and 45 (34.5%) female neonates with the male - to - female ratio of 1.9:1 . Early - onset sepsis cases were found to be three times higher than late - onset sepsis . Out of 131 cases, 98 (74.8%) had early - onset sepsis and 33 (25.2%) had late - onset sepsis . These included gram - negative bacilli (73/131, 55.7%) and gram - positive cocci (58/131, 44.3%). Distribution of isolated organisms tables 2 and 3 show the antibiotic susceptibility pattern in gram - negative and gram - positive isolates . Best overall sensitivity among gram - negative isolates was to imipenem (93%), followed by amikacin (52%) and netilmicin (41%). Gram - positive isolates had sensitivity of 91% to linezolid, 68% to tetracycline, 64% to piperacillin / tazobactam erythromycin, and 52% to ciprofloxacin . The uncertainty surrounding the clinical approach to treatment of neonatal septicemia can be minimized by periodic epidemiological surveys of aetiological agents and their antibiotic sensitivity patterns leading to recognition of the most frequently encountered pathogens in a particular geographical area . For effectual management of septicemia cases, study of bacteriological profile along with the antimicrobial sensitivity pattern plays a noteworthy role . Out of the 683 clinically suspected cases of sepsis in our study, the incidence of gram - negative and gram - positive organisms was 55.7% and 44.3%, respectively . There were 98 (74.8%) isolates from early onset septicemia cases, while 33 (25.2%) were from late - onset illness . In this study, a male predominance with male - to - female ratio of 1.9:1 this might be because of the importance given to the male infants and also because of more number of male infants born compared to female infants born . Culture - positivity for aerobic organisms in neonates vary from 25% to 60% . In this study, however, a high blood culture - positivity rate in septicemic children (56%) had been reported by sharma et al . And jain et al . A low blood culture isolation rate could be due to administration of antibiotic before blood collection from the primary centers or the possibility of infection with anaerobes . A negative blood culture does not exclude sepsis and about 26% of all neonatal sepsis could be due to anaerobes . The pathogens most often implicated in neonatal sepsis in developing countries differ from those seen in developed countries . Overall, gram - negative organisms are more common and are mainly represented by klebsiella, escherichia coli, pseudomonas, and salmonella . Of the gram - positive organisms, staphylococcus aureus, cons, streptococcus pneumonia, and s. gram - negative and gram - positive septicemia was encountered in 56% and 44% of the culture - positive cases in this study, which is comparable to a study conducted by agnihotri et al ., which reported that gram - negative and gram - positive organisms were responsible for 59% and 41% of the septicemia cases, respectively . The report of the national neonatal - perinatal database showed klebsiella as the predominant (29%) pathogen . Klebsiella spp. (31%) was the predominant gram - negative species isolated in this study, which agrees with previous reports . Of the total 131 cases of neonatal sepsis, 98 (74.8%) were early - onset sepsis in this study, which is comparable to previous studies . The wide availability of over - the - counter antibiotics and the inappropriate use of broad - spectrum antibiotics in the community may explain this situation . It is difficult to compare antibiotic resistance between countries because the epidemiology of neonatal sepsis is extremely variable . The analysis of drug resistance pattern showed that, among gram - negative isolates, maximum numbers (97%) were resistant to ampicillin and lowest to imipenem (7%). Resistance was observed to be against commonly used antibiotics such as ampicillin, amoxiclav, cephalexin, and co - trimoxazole . Among gram - positive isolates, high resistance was seen to penicillin (90%), cloxacillin (84%), and amoxiclav (76%). Least resistance was seen to linezolid (9%), followed by tetracycline (32%), and piperacillin / tazobactam (36%). The greater prevalence of resistance to commonly used antibiotics has also been reported by other studies . Among aminoglycosides, amikacin was found to have an edge over netilmicin and gentamicin in gram - negative septicemia, with sensitivity of 52%, 41%, and 33%, respectively . Sensitivity to imipenem and linezolid was much higher than that to other antibiotics and the difference was statistically significant (p <0.05), but these two drugs should not be used indiscriminately and be kept as a reserve drugs, otherwise resistance to these drugs may develop, thereby threatening the treatment . It is evident from this study that gram - negative organisms (klebsiella, acinetobacter), cons, and s. aureus are the leading cause of neonatal sepsis in this study, and most of them are resistant to multiple antibiotics . Therefore, the authors suggest that surveillance of antimicrobial resistance is necessary . Furthermore, we advise that health education be provided to the public on the dangers of indiscriminate use of antibiotics, which is currently considered to be a menace in our society and which has been responsible for the ineffectiveness of most commonly used antibiotics such as penicillin and ampicillin, as observed in our study.
|
In the last 30 years the number of people in the world aged 60 or above has doubled from 378 million in 1980 to 759 million in 2010 . It is projected to more than double again in the next 40 years, rising to two billion by 2050 . In addition, the older population is itself ageing; currently, the oldest old, those aged 80 and above, represent 13% of the global population aged 60 and over; yet projections indicate that by 2050 that proportion will have grown to 20% . Long - term conditions (ltcs) are more prevalent in older populations (58 percent of people over 60 compared to 14 percent under 40) and in more deprived groups (people in the poorest social class have a 60 percent higher prevalence than those in the richest social class and 30 percent more severity of disease). In the united kingdom (uk), the number of people with more than one ltc is expected to rise from 1.9 million in 2008 to 2.9 million in 2018 and this increasing prevalence is considered to be one of the biggest challenges facing the national health service (nhs). In the light of the increasing pressures on health and social care created by an ageing population, the uk house of lords recently called for an urgent revision of how care is delivered, arguing for a move toward more integrated, person - centred care . Diabetes is an example of a ltc and the number of adults across the globe living with it has quadrupled since 1980 to 420 million people . In the uk it is the fourth most prevalent ltc and has increased by 25 percent from 1,962,000 people in 2007 to 2,456,000 people in 2011 . Factors driving this increase are largely lifestyle related, that is, obesity because of poor nutrition and a lack of physical activity . Good clinical management of diabetes is critical as poor control can result in complications such as blindness, renal failure, neuropathy leading to impotence, and foot disorders that can result in amputation, stroke, and heart disease . It may be that inadequate health literacy is a significant factor in the disproportionate burden of diabetes and diabetes - related complications in more socioeconomically disadvantaged populations . Moreover, those with low health literacy have lower levels of good self - management of chronic disease, including poorer diabetes self - management [6, 7]. Health literacy can be defined as the personal characteristics and social resources needed for individuals and communities to access, understand, appraise and use information and services to make decisions about health . As a part of the response to the growing number of people living with long - term conditions, a number of which relate to health behaviours, many countries have developed the role of health - related lifestyle advisors (hrlas). In the uk the term lay health trainer (lht) has been adopted . Lhts are people living in the local community, intended to be demographically similar to those with whom they work, offering support from next door rather than they are trained to a minimum of uk national qualification framework (nqf) level three in using techniques based on psychological and behavioural theories to help change behaviours (https://www.gov.uk/what-different-qualification-levels-mean/overview). The role emerged as a result of the uk department of health's choosing health public health white paper, which had as its aim the reduction of health inequalities by targeting disadvantaged groups in order to increase healthy behaviours and create opportunities for employment and training . Lhts have been found to be effective in engaging with less heard groups and supporting them to make and maintain lifestyle changes . However, they were not designed to work with specific health conditions and little work has explored their efficacy in chronic long - term condition management, such as diabetes [11, 12]. Nonetheless, pennington and colleagues, in their systematic review of the effectiveness, cost - effectiveness, equity, and acceptability of different types of hrla role, identified some evidence that lay - led self - management interventions can be both effectual and cost - effective . Given that self - management of type 2 diabetes is dependent on healthy lifestyle choices, the study of health trainer improved patient self - management (ships) was a randomised controlled feasibility pilot trial (rct) to develop and then compare a lht intervention to improve patient self - management with usual care for those with low health literacy and poorly controlled type 2 diabetes mellitus (t2 dm). Patients with hba1c> 7.5 or 58 mmol / mol in at least the last two measures were eligible to be recruited from a socioeconomic disadvantaged population (see protheroe, rathod, bartlam, rowlands, richardson, and reeves, this issue). The feasibility, pilot rct took place in a uk local government council authority funded health promotion service . This local service employed four lhts to offer information and support to help individuals improve their lifestyle and general health, and it was overseen by two service managers . The service was located in a victorian gate - lodge to a large public park, two miles from the town centre, with a bus every half an hour . The aim of not being located in an obvious health built environment, such as a clinic, was to emphasise supporting health and well - being from within the community . However, patients could be seen elsewhere if other venues were more convenient to them, including their local primary care centre or their own home . The ships study was a complex intervention and, in line with medical research council guidance, the process evaluation reported here had three research objectives: to explore if the intervention was considered acceptable to patients, health care practitioners (service managers and practice nurses), and lhtsto explore whether patients, health care practitioners, managers, and lhts considered the intervention likely to change health behavioursto consider the implications of findings for any future rctships was reviewed and approved in the uk by the national research ethics service committee east midlands - derby 2: 11/em/0294 . To explore if the intervention was considered acceptable to patients, health care practitioners (service managers and practice nurses), and lhts to explore whether patients, health care practitioners, managers, and lhts considered the intervention likely to change health behaviours to consider the implications of findings for any future rct the qualitative methods reported here form part of a mixed methods approach to pilot rcts, and both sampling and analysis were integrated with some of the baseline data from the pilot rct (table 1). Semistructured interviews (in person or by telephone) were carried out with patients in the intervention arm, the lhts delivering the intervention, and the service managers and practice nurses (practice nurse is the term applied to nurses working as part of a primary care team within a family physician / general practice setting in the uk) recruiting patients to the study . The intervention consisted of a structured interview with the lht, development of an individualised self - management plan with the identification of specific agreed goals, and up to three support telephone calls from the lht for a maximum of six months . In addition, a self - management pamphlet on t2 dm was developed which the lhts gave to patients . This differed from usual lht care, in which the lhts in the study would normally work on a one - to - one basis with patients for up to 12 months . It also differed from usual practice in the uk, where lhts generally work with patients over a six - to - twelve - week period . Recruitment of patients took place once the study team research nurse had completed the seven - month trial follow - up . This was to ensure sufficient time had lapsed for them to have had experience of the intervention . As part of this follow - up, they were asked to consent to further contact for the purposes of an interview exploring their views about the lht service (figure 1). As previously mentioned, drawn from the baseline demographics in the pilot trial, a purposive sampling strategy based on an iterative analysis concurrent with data collection was used to ensure balance for factors likely to influence outcome such as diabetic control, length of time since diagnosis, age, and gender . Health literacy levels were also taken into account, using the newest vital sign (nvs)uk . The nvs asks six questions based on a food label: a score of less than four is taken as indicating less than adequate health literacy . In addition, scores on the warwick - edinburgh mental well - being scale (wemwbs) were considered . The wemwbs scores range from a minimum of 14 to a maximum of 70, with higher scores indicating better mental well - being . The wemwbs population mean for england in 2012 was 52.4, with men scoring slightly higher than women . The lhts, service managers, and practice nurses involved in the trial were also invited to interview . Follow - up interviews also took place with the lhts and service managers toward the end of the pilot trial, with the aim of checking if their views or experiences had changed since the initial interview . The practice nurses were interviewed after referring patients to the lht service, so a follow - up interview was not deemed necessary . Since patients were interviewed once the seven - month follow - up with the research team nurse had taken place, this was considered sufficient time to capture change in views within that group . All participants were offered a choice of interview format and, in case of a face - to - face interview, a choice of location . Patients received a patient information leaflet (pil) at the time of their seven - month follow - up, ahead of deciding whether to consent to contact about a possible interview . Having been contacted by the qualitative researcher, and agreeing to be interviewed, they were sent a further copy of the pil ahead of the interview as an additional reminder and explanation . The information encouraged them to discuss the study with family or friends ahead of deciding whether or not to continue . The written information was developed in collaboration with the patient and public involvement research user group within the research institute for primary care and health sciences at keele university . This two - arm approach to informed consent was not considered necessary for the health professionals collaborating in the trial, who were familiar with the pil / purpose of the interview study in order to answer any questions patients might have and who consequently received one set of information prior to their interview . Information to all interview participants emphasised that any quotes that might be used in publications would be anonymised, and names and personal details would not be used in such publications . Those for patients explored their overall health, the history of their diabetes, and their experience of the information they had received since diagnosis in terms of enabling them to understand and manage their condition . The interviews also explored their expectations, experiences, and views of working with their lht and the extent to which they had changed their self - management as a result . The guide evolved in the light of emerging findings, which also informed the continuing sampling strategy . The questions to the lhts, practice nurses, and service managers focused on their experiences of working with this particular patient population and what they considered the challenges and opportunities . They also explored aspects of practice and service provision including the intervention seen as useful, or not, in supporting behaviour change . The steps outlined in the pil on data anonymity and participant confidentiality were highlighted again before beginning the interview, and consent checked both at the start and end of the interview . The written information and consent forms for the lhts, service managers, and practice nurses also highlighted both of these issues, providing a framework for discussion and checking . Interviews lasted approximately half an hour, except for the dyadic interview with the two service managers which lasted one and a half hours, because of the more co - constructed nature of the discussion . Data collection with all four sets of participants took place between april and october, 2013 . An exploratory thematic framework was adopted for the analysis, with emergent findings checked out in subsequent interviews across all four groups of participants in an iterative cycle . To maximize the benefits of being an interdisciplinary team, the two coders brought differing perspectives to bear on the data (bernadette bartlam, social science; joanne protheroe, family medicine). To ensure intercoder reliability, each independently coded a random selection of interviews as part of reaching agreement on the coding frame, which was then applied across the whole data set by bernadette bartlam, checking for consistencies and confounding cases [1921]. In total, 24 participants were interviewed: 14 patients with poorly controlled t2 dm, two service managers, four lhts, and four practice nurses . Follow - up interviews also took place with three of the lhts and one service manager, giving sufficient data to ensure that no issues had been overlooked . One person had been in post six years, two for five years, and one for three years . All had undertaken the royal institute of public health understanding health improvement course, nqf level two qualification, together with the city & guilds health trainer course, nqf level three . In addition, they had all undertaken a variety of short courses on motivational interviewing and they all came from the local area . Three had previous backgrounds in health and fitness, and one had been a delivery driver . One manager was a nurse with degree level education in public health who had been responsible for originally commissioning the lht service . The other had been the day - to - day manager of the service since its inception in 2007 and had degree level education in nutrition, health, and exercise, and in voluntary and third sector management . The four practice nurses recruiting patients to the study had been trained and working as primary care nurse specialists in diabetes for between six and eight years . Seventy - six patients were randomised into the pilot trial, 39 to the intervention arm . There was a follow - up rate at seven months of just under 70%, resulting in 27 patients available for invitation to interview . The reasons for refusing were poor health of self or partner and having other commitments . Based on the sampling strategy, contact was attempted with 18 participants, three of whom were noncontactable one person's phone number was invalid and it was not possible to contact the other two people, despite five attempts at different times on different days . One person that was contacted declined participation because of a recent bereavement (figure 1). Ten participants were aged over 60 years; the age range was 4386 years, with men being generally younger than the women (mean of 59 compared to 73 years). From the baseline data in the pilot rct, although there was a considerable range in this (from one year to 25 years), the majority of participants (11) had lived with the condition for ten years or more . All participants were also living with at least one additional ltc, and the majority rated their own health as fair or good . However, with a mean score on the wemwbs of 23, participants' mental well - being was very much lower than the uk population norm of 52.4, with men scoring slightly higher overall . There was a spread of scores across the nvs, with the mean for women (2.5) being slightly lower than that for men (3) (table 2). It is also worth noting that a number of participants who had low scores on the nvs self - reported their health as good or excellent . Three key interrelated themes emerged from the analysis: health literacy and understanding of diabetes, responses and coping strategies, and motivation to change . In what follows we present details of these using illustrative quotations, before turning to look at the implications . The relationship between health literacy and people's understanding of their condition was immediately apparent, as this excerpt from the interview with beth illustrates; she was an 86-year - old lady, diagnosed with diabetes for 12 years and with a low nvs score of two: i really don't feel it's as serious as they try to make out the younger sister, she's abandoned all pills [for t2 dm]. I really don't feel it's as serious as they try to make out the younger sister, she's abandoned all pills [for t2 dm]. She does eat well but she does drink a little bit too [laughs] we can tell when she falls over that she's had a little bit too much [laughs], and she smokes similarly, fred, a 72-year - old man with a low nvs score of one, who had lived with diabetes for 18 years, found it difficult to accept even general advice on health, as this excerpt shows: it says giving up smoking is one of the most positive things you can do to improve your health, right? Well, when i stopped smoking, just over two years ago, my diabetes became uncontrollable, so i disagree . It says giving up smoking is one of the most positive things you can do to improve your health, right? Well, when i stopped smoking, just over two years ago, my diabetes became uncontrollable, so i disagree . This lack of health literacy was reflected in the interviews with the lhts, as this account by lht3 illustrates: one client was told by someone at the gym that he needed to be on a higher protein diet and cut out his carbohydrates, lose weight, and when i explained the eat well plate to him, he wouldn't have it . One client was told by someone at the gym that he needed to be on a higher protein diet and cut out his carbohydrates, lose weight, and when i explained the eat well plate to him, he wouldn't have it . Linked to this lack of clarity was a lack of understanding about the role of lhts in supporting self - management of the condition, as these excerpts from the interview with lht1 illustrates when reflecting on the people seen in the trial: they don't know who we are they've had the condition for so many years and why haven't they addressed it before they've come to us? And they're so set in their ways now that they don't want to, there's quite a lot of resistance . They don't know who we are they've had the condition for so many years and why haven't they addressed it before they've come to us? And they're so set in their ways now that they don't want to, there's quite a lot of resistance . Such lack of clarity could result in unrealistic expectations on the part of patients and of other health professions of what the service might offer, given the level of training and expertise amongst lhts, as this interview with practice nurse 1 indicates: i think a health trainer would look at more like the whole person and the whole thing, whereas when we refer them to different services . They're either just looking at the weight loss, or they're just looking at smoking cessation, or they're just looking at alcohol, whereas there's a lot of other factors that come into the whole person . I think a health trainer would look at more like the whole person and the whole thing, whereas when we refer them to different services . They're either just looking at the weight loss, or they're just looking at smoking cessation, or they're just looking at alcohol, whereas there's a lot of other factors that come into the whole person . It was also apparent that the ships pilot trial was recruiting patients who would not generally fall within the age range targeted by the service employing the lhts, as service manager 2 clarifies: just one thing that i noticed from this group from the ships study that we don't tend to have with the people that we regularly support, is the age group . Do you tend to deal with over 65? And it might be so many, but after that we don't tend to have those older age groups and so straightaway you've got issues around the fact that they've obviously had the condition for a long time the behaviour's so engrained and it's a group that, although we deal with that group, it's not a large age category that's supported by health trainers usually . Just one thing that i noticed from this group from the ships study that we don't tend to have with the people that we regularly support, is the age group . Do you tend to deal with over 65? And it might be so many, but after that we don't tend to have those older age groups and so straightaway you've got issues around the fact that they've obviously had the condition for a long time the behaviour's so engrained and it's a group that, although we deal with that group, it's not a large age category that's supported by health trainers usually . Patients' lack of understanding of their condition was reflected in their self - management, as beth's description of her diet illustrates: when you're old you can't possibly eat five portions of fruit and vegetables a day . When you're old you can't possibly eat five portions of fruit and vegetables a day . Despite a score on the wemwbs of 23.21, and even though she had multiple coexisting chronic health problems, beth reported her own health as good . The sense of already doing what was necessary to live well with diabetes was reflected throughout the interviews, as this excerpt from the interview with tom, a 54-year - old man, diagnosed with diabetes for 14 years and with an nvs score of three, shows: the things they've got in the book [pamphlet] i eat anyway . I don't like macdonald's, i can't be doing with that kind of rubbish . I don't like macdonald's, i can't be doing with that kind of rubbish . Jane, a 62-year - old woman living with diabetes for 20 years, with an nvs score of four, also felt she was managing well despite poor glycemic control: i do consider myself a bit of an expert because i've been diabetic for quite a while . I do consider myself a bit of an expert because i've been diabetic for quite a while . However, this was not exclusively the case, as john, a 67-year - old man with a low nvs score of one and who had lived with diabetes for 15 years, illustrates when he responds to the question on the ways in which he found the lht helpful: first of all i think what [lht] done really, i started looking at what i eat because [lht] explained everything was very not complicated, if you know what i mean? First of all i think what [lht] done really, i started looking at what i eat because [lht] explained everything was very not complicated, if you know what i mean? Straightforward and just said if you want to control it you've got to do this . Without doing this, fred, too, despite his earlier scepticism over health advice, reported finding the consultation with the lht helpful: [lht] completely changed and broadened, in effect, what i was eating . [lht] completely changed and broadened, in effect, what i was eating . And i feel a lot better as a result of that . The degree to which participants found the intervention helpful appears to be directly related to communication within the consultation . Motivation and capacity for change also emerged as an important factor, as beth indicates when asked what she first though when the lht was suggested: i think it was a bit of a waste of time, at my age, when i've had it for so long i've had no problems . I think it was a bit of a waste of time, at my age, when i've had it for so long i've had no problems . However, despite this she did feel that the intervention had brought some benefit: do you recall setting goals with him? Yes, when i knew i had to record what i was eating, it did make me eat better, because i had to put it down what i'd had, you know? I couldn't just say, oh, a couple of biscuits, or something, you know, for a meal [yeah]. I did make the effort to eat properly while i was recording, you know . Yes, when i knew i had to record what i was eating, it did make me eat better, because i had to put it down what i'd had, you know? I couldn't just say, oh, a couple of biscuits, or something, you know, for a meal [yeah]. I did make the effort to eat properly while i was recording, you know . Whilst jane recognised that her glycaemic control was poor, she reported not finding the lht consultation helpful, echoing issues around long - established conditions and coping strategies and age: [lht was] on about me doing more exercise than i do, and i do exercise [laughs] everyday . Then [lht] was on about the food i was eating . Well, you go through this so many times . That's all they seem to think; because you've got a little bit of weight on, you need to lose weight . I thought; i'm 63, what do i want to do with cookery lessons at my age? [lht was] on about me doing more exercise than i do, and i do exercise [laughs] everyday . Then [lht] was on about the food i was eating . Well, you go through this so many times . That's all they seem to think; because you've got a little bit of weight on, you need to lose weight . I thought; i'm 63, what do i want to do with cookery lessons at my age? Jane's reluctance to engage with the lht reflects her sense of herself as expert and also may be a reflection of her self - reported poor health status and her low score on the wemwbs of 15.32 . She clearly had complex health needs: throughout the interview she also spoke of her chronic heart condition, and the high impact that was having on her life . Shortly before participating in the trial she lost her mother and nonetheless, she spoke positively of the lht as an individual: he was excellent, really, it just didn't suit me however, john, clearer in his understanding of his condition as a result of meeting with the lht, reported being highly motivated to change, as this response to being asked whether he had identified particular goals shows: lose weight [laughing]. So i thought yes, i'll just follow what [lht] said . And i put my control over diabetes into motion, really, and i figure that it's thanks to that lht . That's the number one so i thought yes, i'll just follow what [lht] said . And i put my control over diabetes into motion, really, and i figure that it's thanks to that lht . John had one of the highest scores amongst participants on the wemwbs, 28.13, and reported his health as good even though he too had coexisting ltcs . Other factors, such as working conditions appeared to play a part in capacity for change, again illustrated by this response from tom, a long - distance lorry driver, when asked about his views on the lht service: he was nice lad everything, explaining like, this might help and that might help it's all right now because i'm not working, i said, but as soon as i go back to work everything just goes out the window again i sometimes miss my dinner - time tablets, because you start at daft times . He was nice lad everything, explaining like, this might help and that might help it's all right now because i'm not working, i said, but as soon as i go back to work everything just goes out the window again i sometimes miss my dinner - time tablets, because you start at daft times . Yes, because [lht] said due to your lifestyle there's not a lot you can do about it really, you know? It was managing the medication and the complexity of his other health conditions that he found challenging, and which left him with a sense of being overwhelmed and unable to change . This was reflected in his wemwbs score of 13.33, the lowest of all participants, and he reported his health as poor . Fred highlights the importance of timing and early intervention after diagnosis, and the usefulness of the pamphlet the lhts used to explain how to live well with diabetes so you've been living with it for a long time? So are you coming across stuff in [the pamphlet] that's new? [pause] no, i mean, i am aware of everything that's there? If i'd have been given something like this in the early days, it would have been a much greater help than that which i received . Yeah, because people tend to get a bit bored of lots of words, particularly if they're not presented well . Again, this was reflected in the interviews with the lhts, as this quote from lht4 indicates: i think the kind of patients that need to come in need to be people who want to change and are ready to change . I think the kind of patients that need to come in need to be people who want to change and are ready to change . The challenge in how best to address the complexity with which patients could present, and their prioritisation of their needs, was also evident in the interviews with the lhts: so when i was dealing with [patient name], there was lots of issues and a lot of the time was spent just listening and trying to help her deal with these issues because her health and lifestyle was really poor it was very hard to get her to engage in the topic of what we were looking at without her going off on a tangent . She kept apologising for the fact that she was doing all this talking, and she was talking about all this other stuff, which, it had some relevance, but it's not relevant for what we're trying to achieve . So she was aware that, you know, the study was to help with managing diabetes, and she wasn't allowing me to do that, and she wasn't able to engage in that . (lht3) so when i was dealing with [patient name], there was lots of issues and a lot of the time was spent just listening and trying to help her deal with these issues because her health and lifestyle was really poor it was very hard to get her to engage in the topic of what we were looking at without her going off on a tangent . She kept apologising for the fact that she was doing all this talking, and she was talking about all this other stuff, which, it had some relevance, but it's not relevant for what we're trying to achieve . So she was aware that, you know, the study was to help with managing diabetes, and she wasn't allowing me to do that, and she wasn't able to engage in that . (lht3) finally, location of the lht service was also something that emerged as an influencing factor on people's motivation and capacity to engage: i think location can make a difference to people, if it's hard for them to get to the service . That does sometimes figure as a factor in them not turning up for appointments . (lht4) i think location can make a difference to people, if it's hard for them to get to the service . That does sometimes figure as a factor in them not turning up for appointments . People with long - term conditions can and do self - manage complex medical regimes every day including medicine taking, self - injecting, and dressing wounds as well as dealing with their many challenges of everyday living . They need help to have the confidence and knowledge to know what they can do effectively and safely for themselves and when to seek professional help . (dr . Patricia wilkie, president and chairman national association for patient participation . )this study had three research objectives: first, to explore if the intervention was considered acceptable to patients with low health literacy and t2 dm and to practitioners; second, to explore whether they considered if the intervention was likely to change health behaviours; and finally to consider any implications for a future main trial . People with long - term conditions can and do self - manage complex medical regimes every day including medicine taking, self - injecting, and dressing wounds as well as dealing with their many challenges of everyday living . They need help to have the confidence and knowledge to know what they can do effectively and safely for themselves and when to seek professional help . (dr . Patricia wilkie, president and chairman national association for patient participation .) Given the complex characteristics of the study population, it is important to note that the ships pilot trial results indicate that the intervention is feasible and should be carried forward into a main trial . Moreover, the pilot trial results indicate that the lhts had a positive impact on the mental health of participants in the intervention arm compared to those in the control arm (see protheroe et al ., this issue, for full details of the pilot trial results). However, as the qualitative findings here indicate, and as to be expected, the picture is more nuanced than the trial findings alone suggest, with patients experiencing a range of responses in terms of the acceptability of the intervention and the likelihood of it resulting in behaviour change . Findings must be interpreted with caution given that participants were drawn from a small pilot study located in one specific area in the uk . Moreover, the data is cross - sectional and does not allow for follow - up over time . Finally, participants in this study all scored well below the population mean of the wemebs of 52.4, with a range of 13.3335, despite most self - reporting their health as good . However, it is worth noting that older people have been found to be significantly less likely to partially or not respond to the tool, and more work is needed to establish its reliability in older populations and amongst those with low health literacy . Nonetheless, despite these limitations, these findings suggest that a full rct intervention could be enhanced if attention were paid to a number of issues . First, in keeping with the work of authors such as carollo, relationships and communication emerged as critical . Even those patients who did not find the intervention helpful spoke of their experience of engaging with the lht in positive terms, which itself is important in terms of likelihood of accessing support in the future . Whilst the lhts and health care professionals in the study found the intervention acceptable, not all patients did so key to engaging patients in behaviour change is clarity around roles and responsibilities . Whilst the lhts and managers interviewed were very clear about the role, other health care professionals, and in particular patients, appeared less so, leading to unclear expectations for some patients . Greater promotion of the lht service would improve patient and public understanding of what it can, and cannot, offer . With its emphasis on reaching those patients less likely to access services, careful thought needs to be given as to the ways in which such information is delivered . In addition, there may be something in the title health trainer that may hold less appeal to older people who have been living with their condition(s) for protracted lengths of time . It may also be that, given the training and qualifications outlined here, the term lay health trainer no longer reflects the original emphasis on amateur peer support from next door . It was also clear from this work that lhts appear to be effective for those patients who are already motivated to change health behaviours . However, they may be less effective with those patients who have a more established view of their condition, and those with complex health needs, for example, multiple ltcs, and those who are older . Moreover, there remains a dearth of evidence around the relationship between adherence and older adults with low health literacy . The lht service in this study tended not to work with those over 65 years . In addition, most lht services aim to recruit a high proportion of their staff from similar backgrounds to their clients and it may be that the disparity in the average age of the lhts in this study compared to participants (30 versus 64 years) had an impact on the potential therapeutic impact of the intervention . Given the ageing population and the concomitant increase in those growing older with more than one ltc, having the skills needed to engage such individuals will become more necessary . Such skills need to include an understanding of developmental ageing, in particular the challenges of later life [27, 28], as the individual psychosocial context within which any intervention is delivered . Finally, location also emerged as an issue in this feasibility pilot rct, with some lhts and patients reporting challenges in accessibility, despite the efforts to offer a range of settings . Emphasising the message that a variety of consultation settings are available is something a future rct should take account of . In addition, whilst this service was located in an urban environment, thought should also be given to how to best reach people living in rural areas, which are experiencing the fastest growth amongst older populations . This work suggests that lhts appear to be effective for those patients already motivated to change health behaviours but that they may be less effective with those who are older and have a more established view of their condition and how best to self - manage . However, recent systematic reviews indicate that, whilst interventions are potentially effective, there remains a paucity of evidence on this topic [25, 29, 30]. Further research is needed on the association between health literacy and general health behaviour and on the effectiveness of interventions such as those in the ships pilot rct . In particular, work is needed that can take into account the complexity of diverse populations, including issues such as environment, culture, gender, and life - course perspectives and which can allow for a longitudinal follow - up to evaluate the effectiveness of interventions . These qualitative findings highlight the importance of expanding lht practice to develop skills around working with older populations . They also contribute to the argument for the inclusion of mixed methods, qualitative research in rcts.
|
Imidazole / benzimidazole and their derivatives are important class of organic compounds in coordination chemistry, photophysics, photochemistry, bioinorganic chemistry, and bioorganic chemistry [17]. 2-(2-hydroxyphenyl) benzimidazole (hpbm) is an important benzimidazole - derived n, o - donor ligand . Hydroxyl benzimidazole shows excited - state intramolecular transfer (esipt) properties due to acidic protons of phenol and imidazole nitrogen (tautomerism phenomenon). It has been reported that hydroxyl benzimidazole and benzoxazole behave as a structural mimic of dna base pair for which tautomerism may be initiated at a definite time and position within duplex dna . Structurally similar natural product bis(benzoxazole) uk-1 has been reported to posses anticancer activity, and the metal - binding studies of uk-1 indicates that benzoxazole - like compound are capable of binding a variety of biologically important metal ions . Benzimidazole derivatives exhibit significant activity against several viruses such as hiv, human cytomegalovirus (hcmv) [11, 12], herpes (hsv-1), and influenza . Benzimidazole derivatives are unique and broad - spectrum class of antirhino / enteroviral agents such as antihistaminic, antipyretic, antiulcerative, antihypertensive, antiviral [18, 19], antitumor [2024], antihistaminic, and antiallergic and are also efficient selective neuropeptide y y1 receptor antagonists . Several derivatives are reported in literature for the synthesis of benzimidazole - metal complexes for fluorescent probes and bioorganic application, but antimicrobial activities of these classes of compounds have received little attention . In the literature, there are no reports available describing synthesis and antimicrobial activities of metal complexes of 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol . In this paper, we have synthesized novel ligand 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol and its metal complexes and studied their antimicrobial activities . The synthetic route for the preparation of 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol metal complexes is shown in scheme 1 . M-(n, n - diethylamino)phenol (1) on formylation by using vilsmeier - haack reaction with dmf: pocl3 at 60c yielded p - n, n - diethyl amino salicylaldehyde (2). The p - n, n - diethyl amino salicylaldehyde on further reaction with 4-nitrobenzene-1,2-diamine in ethanol and pcl3 at 60c yielded the 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol (3). The 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol was reacted with different metal salt in methanol in the presence of catalytic amount of triethyl amine at room temperature to form 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol - metal complexes . The purity of the compounds was confirmed by tlc using precoated silica gel as stationary phase, using appropriate solvent system as mobile phase and visualized under uv - light . Structures of the title compounds were confirmed by ft - ir, h - nmr analysis, and metal ligand complex formation is confirmed by atomic absorption spectroscopy . Intermediate p - n, n - diethyl amino salicylaldehyde formation was confirmed by its melting point . Ft - ir and h nmr spectrum of 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol showed absence of absorption band at 1670 cm confirming the absence of aldehydic functional group by way of conversion into the corresponding benzimidazole, and absence of peak at 9.90 ppm confirmed the conversion of formyl functional group into 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol . The ft - ir spectra of compounds 4a4d are also in complete agreement with their structure . There was sharp modification between the ft - ir spectra of the metal complexes 4a4d and the ligand 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol (3), most of the bands change their pattern in the region due to coordination of the phenolic oxygen atom of oh group and nitrogen atom of imidazole ring to the metal ions . The complexation of biologically important metals with 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol was further explored with the evaluation of their antimicrobial activity . All compounds were evaluated for in vitro antibacterial activities against e. coil and s. aureus strains and in vitro antifungal activity tested against c. albicans and a. niger strains by using serial dilution method . Incubator at 35 and 37c; pipettes of various sizes (gilson); sterile tips, 100, 200, 500, and 1000 l; sterile normal saline; sterile isosensitest agar (southern group laboratory, sgl); antibiotic solutions (sigma - aldrich); sterile solution of 10% (v / v) dmso in water (sigma - aldrich). Although nccls recommends the use of mueller hinton medium for susceptibility testing, the isosensitest medium had comparable results for most of the tested bacterial strains . A volume of 100 l of test material in 10% (v / v) dmso (usually a stock concentration of 4 mg / ml) was pipetted into the first row of the plate . To all other wells, 50 l of nutrient broth was added . Tips were discarded after use such that each well had 50 l of the test material in serially descending concentrations . To each well, 10 l of resazurin indicator solution was added . Using a pipette, 30 l of 3.3x strength isosensitised broth added to each well to ensure that the final volume was single strength of the nutrient broth . Finally, 10 l of bacterial suspension (5 10 cfu / ml) was added to each well to achieve a concentration of 5 10 cfu / ml . Each plate was wrapped loosely with cling film to ensure that bacteria did not become dehydrated . Each plate had a set of controls: a column with a broad - spectrum antibiotic as positive control, a column with all solutions with the exception of the test compound, and a column with all solutions with the exception of the bacterial solution, adding 10 l of nutrient broth instead . The plates were prepared in triplicate and placed in an incubator set at 37c for 1824 h. the colour change was then assessed visually . The average of three values was calculated and that was the mic for the test material and bacterial or fungal strain . The new ligand and their metal complexes were evaluated for their in vitro antibacterial activity against e. coli and s. aureus strains and in vitro antifungal activity against c. albicans and a. niger strains by using serial dilution method . The minimum inhibitory concentration (mic) measurement determined for compounds showed significant growth inhibition zones using serial dilution method . The results mentioned in figure 1 indicate that most of the tested compounds displayed variable inhibitory effects on growth of tested against bacterial strain and antifungal strain . The metal complex 4d showed excellent antibacterial activity against e. coli and s. aureus strains but metal - complexes 4a4c showed moderate activity against tested antibacterial strains . The inhibitory growth of metal - complexes 4a4d are almost double than novel synthesized ligand 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol (3), and the compound 4b is less active against s. aureus antibacterial strain . Regarding the structure - activity relationship of the novel compound 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol complex with cobalt(ii) metal showed better activity than fe, ni, and cu metals . The results mentioned in figure 1 showed that ligand as well as metal complexes show good inhibitory growth in case c. albicans as well as a. niger strains . These results indicate that after coordination of biologically important transition, metal with 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol has substantial effect on the antimicrobial activity against tested microorganism . In general, most of the tested compounds revealed better activity against the antibacterial strain (e. coli, s. aureus) and antifungal strain (c. albicans, a. niger). It was also noticed that ligand (3) and metal complexes 4a4d showed stronger antibacterial activity than antifungal one . In order to examine the thermal stability of these complexes, thermal gravimetric (tg) and differential scanning colorimeter (dsc) analysis the tg results indicate that the skeleton of the synthesized ligand and its transition metal complexes are stable up to 250c . Above 250c, the comparisons of the td (decomposition temperature) showed that the thermal stability of the 3 and 4a4d decreases in the order 4b> 4c> 4d> 4a> 3 . Metal complexes and ligand do not decompose completely even up to temperature 600c . In the dsc curve metal complexes 4a4d shows exothermic peak 310, 395, 290, and 385, respectively, as shown in figure 3 . The electrochemical behavior of all the complexes and ligand were studied by using cyclic voltametry (cv) in dimethyl methyl sulphoxide (0.1 m net4clo4) in the potential range 1.6 to + 1.2 v by using platinum auxiliary electrode and pt disc - working electrode at ambient temperature (300 k) with no trace of decomposition as reflected in smooth curve . Cyclic voltammetric studies of the ligand 3 and complexe 4a4d in dimethyl formamide solution under nitrogen atmosphere are irreversible . The result of cyclic voltammetry of ligand closely resembles with that of metal complexes compounds, which serve as further evidences for similar structural and electronic properties figure 4 . The uv - vis absorption and emission spectra of ligand 3 and its metal complexes 4a4d were recorded in dmf at room temperature, and the compound concentrations are 1 10 m. the max (absorbance) values of ligand 3 is 294 (0.484), and metal complexes 4a4d were obtained as 387 (0.629), 366 (1.00), 354 (0.818), and 339 (1.043) nm, respectively . As can be seen, the absorption characteristics of metal complexes 4a4d are nearly same, while ligand absorbs in blue region as compared to complexes . In conclusion, we have synthesized new ligand 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol and their metal complexes . These novel compounds were evaluated for in vitro antibacterial activity against e. coli and s. aureus strains as well as for antifungal activity against c. albicans and a. niger strains using serial dilution technique . All synthesized compounds are confirmed by ft - ir, h - nmr, and atomic absorption spectroscopy . The reaction was monitored by tlc using on 0.25 mm e - merck silica gel 60 f254 precoated plates, which were visualized with uv light . Melting points were measured on standard melting point apparatus from sunder industrial product mumbai and are uncorrected . The ft - ir spectra were recorded on perkins - elmer 257 spectrometer using kbr discs . H - nmr spectra were recorded on vxr 300-mhz instrument using tms as an internal standard . Phosphorous oxychloride (pocl3) (2.75 ml, 0.03 mole) was slowly added to dimethylformamide (dmf) (3.65 ml, 0.05 mole) at 510c under the stirring . To this cooled reagent, 3-(diethyl amino)phenol (0.01 mole) was added by dissolving it into dmf (6 ml) under the stirring, and the resulting mixture was heated at 75c for 4 hrs . The reaction mixture was cooled to room temperature and then poured into ice water (60 ml). Reaction mass was neutralised with sodium carbonate, brown colored solid separated out, filtered the separated product washed with cold water, dried, and crystallised from ethanol to get pure product (m.p . Phosphorus trichloride (0.33 mol) was added dropwise to a solution of the p - n, n - diethyl salicylaldehyde (0.33 mol) n, n - diethyl m - amino phenol (0.33 mol) in ethanol (50 ml), maintaining the temperature at 4045c . The mixture was heated at 60c for 4 h, after the completion of reaction (monitored by tlc) cooled the reaction mass at room temperature and made alkaline to ph 8 with aqueous sodium bicarbonate solution (20% w / v). Reaction mass was concentrated under vacuum, and the solid which obtained was collected and crystallized from isopropyl alcohol . Ir (kbr, cm): 2991, 1620, 1520, 1338, 1149, 946, 817, 733 . H - nmr (300 mhz): 1.21 (t, 6h, ch3), 3.42 (q, 4h, ch2), 6.27 (d, 1h, aromatic h), 7.26 (d, 1h, aromatic h), 7.29 (m, 2h, aromatic h), 7.88 (d, 1h, aromatic h), 7.85 (d, 1h, aromatic h), 8.17 (s, 1h, nh), 12.43 (s, 1h, oh). 4a: ir (kbr, cm): 2972, 1606, 1498, 1262, 1144, 1074, 821, 726 . 4b: ir (kbr, cm): 2974, 1608, 1498, 1336, 1258, 1148, 1063, 1019, 818 . 4c: ir (kbr, cm): 2973, 1607, 1497, 1336, 1258, 1149, 1019, 820 . 4c: ir (kbr, cm): 2974, 1607, 1498, 1339, 1261, 1069, 827, 732 . To a solution of ligand, 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol (3) (0.1 mole) in methanol (15 ml) was added a few drops of triethyl amine and solution of metal salts (0.05 mole) in methanol (2 ml). The product, thus, separated was filtered, washed with water followed by methanol, and dried to give 4a4d . Atomic absorption spectra were recorded using atomic absorption spectrometer model gbc 932 (gbc scientific equipment, australia). Exactly weighed dye samples were dissolved in 20 ml of dimethyl sulphoxide and diluted to 100 ml with distilled water and analyzed by gbc 932 plus atomic absorption spectrometer (aas). Acetylene was used as fuel, and air was used as carrier gas . Certified 1000 mg / l standard solution of iron (merck, mumbai) was used to perform calibration using hallow cathode lamp for iron at 248.3 nm wavelength . The samples were prepared in such a manner that they will result in 2 mg / l solution containing 1: 2 complexes . Table 1 compares the experimental results of aas analysis and with one calculated on the theoretical basis . The results of aas analysis are in well agreement with the predicted results within the limitations of the experimental error, which confirms the proposed 1: 2 metal complex stoichiometric between metal and ligand.
|
Klippel trenaunay syndrome (kts) is a rare congenital anomaly characterized by venous and lymphatic abnormalities, cutaneous capillary malformations, and hypertrophy of soft tissue and bone . Kts is usually isolated to one extremity; however any part of the body may be affected . Deep venous thrombosis (dvt) with a pulmonary embolism (pe) has been described in these patients,1 though children have a lower rate of thromboembolic episodes . We report a life - threatening pe and its management in a child after radical resection of a kts vascular malformation involving the right calf and adjacent tissues . A 3-year - old boy (weight 22 kg), with kts involving the right lower extremity, pelvis and genitalia was admitted to the medical - surgical intensive care unit (msicu) after resection of his right lower extremity malformation . Except of his malformation he was a healthy child with normal kidney and liver function tests and no underlying pathology in hemostasis . The operation lasted 8.5 hours and was complicated by major blood loss (estimated blood loss 3400 ml). The patient remained intubated because of extensive volume resuscitation with blood products (2265 ml packed red blood cells, 436 ml fresh frozen plasma, and 6 units of platelets) and crystalloid (8900 ml). Patient was anticoagulated with low molecular weight heparin (lmwh), enoxaparin, for a few weeks prior to surgery . The drug was discontinued the day before the operation, and restarted on postoperative day (pod) 1 at a dose of 10 mg subcutaneously twice per day . On pod 2 a peripherally inserted central catheter was placed, active diuresis was initiated, and he began to wean from mechanical ventilation . On pod 3 the patient experienced a sudden episode of agitation, oxygen desaturation to 55%, and drop of end tidal partial pressure of carbon dioxide (petco2) from 40 s to 24 mmhg . Oxygenation slowly improved to 90% after administration of 100% oxygen and an increase in positive end - expiratory pressure (peep) from 5 mmhg to 16 mmhg . An arterial blood gas revealed a partial pressure of carbon dioxide (paco2) of 75 mmhg and partial pressure of oxygen (pao2) of 62 mmhg on a fio2 of 1.0 . Pe was suspected and immediate systemic anticoagulation with unfractionated heparin (ufh) was initiated using a bolus dose of 80 units / kg followed by an infusion at a rate of 18 units / kg / hour . Urgent pulmonary ct angiography showed a large filling defect in the right main pulmonary artery (pa) (figure 1) and a number of smaller defects in the peripheral pa branches . In light of respiratory instability pulmonary arteriogram via the right internal jugular vein confirmed a large thrombus in the right pa . A successful transcatheter pulmonary embolectomy was followed by thrombolysis with 2 mg of recombinant tissue plasminogen activator (tpa), alteplase, injected through the pa catheter . The same dose of alteplase was repeated once and an inferior vena cava (ivc) filter was placed . Post - procedure angiogram revealed recanalized right pa with persistent smaller defects in upper and lower lobe branches . Heparinization was monitored by measuring heparin levels with a goal of 0.30.5 units / ml . Trans - thoracic echocardiogram (tte) confirmed a dilated right ventricle (rv) with elevated pa pressure (mean pa pressure equaled of mean systemic pressure) and dopamine 5 g / kg / min was initiated to support rv function . Treatment with inhaled nitric oxide (ino) started for pulmonary hypertension as well as for ventilation perfusion (vq) mismatch . Within 24 hours, the patient experienced an episode of hypotension and deterioration of gas exchange (table 1); however, follow - up pulmonary angiography showed no new emboli, unchanged angiogram, and a still elevated pa pressure of 32/22 mmhg . Patient was resuscitated with intravenous crystalloids and dopamine and epinephrine was added for a short period of time . Three days after the embolectomy, the patient bled extensively and required multiple blood transfusions . Patient was weaned off dopamine, ino, and extubated within the next few days . Subsequent lung perfusion scan showed good perfusion of all lung units (figure 2). The patient s ivc filter was removed in the interventional radiology suite prior to discharge from hospital one month after its insertion . All patients with kts are at high risk of dvt and pe and may require long term thromboprophylaxis.1,2 anticoagulation is of vital importance in the perioperative setting or in trauma victims with this condition . Our patient developed massive pe despite the use of prophylactic anticoagulation with lmwh, which was interrupted only for 24 hours in the immediate peri - operative period . The fibrinolytic system in the pediatric population differs from the one in adults;3 however controlled studies are unavailable and treatment regimens of pe are usually extrapolated from the adult guidelines,4 which are outlined here . The mainstay of pe therapy is anticoagulation with either ufh or lmwh . In massive pe or where subcutaneous absorption is a concern, an intravenous ufh is a preferred antithrombotic agent . The recommended initial dose is an intravenous bolus of 80 units / kg followed by intravenous infusion 18 units / kg / h . Therapeutic effect is monitored by measuring either activated partial thromboplastin time or heparin levels . Subcutaneously administered lmwh is an effective alternative of ufh for treatment of pe in patients without hemodynamic compromise . While antithrombotic agents or anticoagulants prevent further thrombus formation; clot dissolution is achieved by thrombolysis . Thrombolytic agents, also known as plasminogen activators, initiate fibrinolysis by conversion of plasminogen to plasmin, the main fibrinolytic enzyme . Since plasmin breaks fibrin in occlusive as well as hemostatic plug, bleeding may result . Because of risk of hemorrhage and no mortality benefit when compared to treatment with anticoagulants alone,4 thrombolysis is recommended only in hemodynamically unstable pe with high risk of death and low risk of bleeding and in patients with poor prognosis due to right ventricular failure or severely compromised oxygenation.4 agents with a short half - life, like tpa (t = 5 min), are usually administered by infusion, while derivatives of tpa with longer half - life such as reteplase or tnk - t - pa (t = 15 and 20 minutes, respectively) may be given as a bolus.5 management of pe by means of interventional catheterization techniques as well as surgical embolectomy should be restricted only to highly compromised patients who cannot receive thrombolytics.4 there are few case reports and case series depicting treatment of acute pe in children with thrombolytic agents6,7 and surgical embolectomy.8 cannizzaro and colleagues described an administration of tpa by catheter into two branches of pa in a 10-year - old girl with a massive pe and echocardiographic evidence of reduced right ventricular function.6 they used two boluses of tpa followed by a continuous infusion . Maeda and colleagues administered thrombolytics successfully through the central venous catheter in a hemodynamically stable 4-year - old boy with poor oxygenation shortly after total cavopulmonary shunt.7 combination of tpa (bolus) and urokinase (infusion) were used together with an infusion of ufh . We present a successful treatment of massive pe in a 3-year - old patient with oxygenation failure employing combination of interventional catheterization technique and regional thrombolysis . As we mentioned above, any embolectomy should be reserved only for highly unstable patients where thrombolytics are being contraindicated or has already failed . Our patient had significantly compromised oxygenation, however thrombolytics were contraindicated because of recent major vascular resection and high risk of bleeding . Catheter extraction technique was chosen over an open approach since expertise was available, pa catheter was in situ (for selective pulmonary angiography) and the possibility of combining two techniques emerged . Risk of hemorrhage was weighed against the risk of significant damage and/or death from progressive hypoxemia and it was decided to proceed with thrombolysis . Availability of tpa, a thrombolytic agent that preferentially activates fibrin - bound plasminogen and not circulating plasminogen supported our treatment alternative . Streptokinase, urokinase, and reteplase do not have this discriminative property and resulting systemic plasminemia causes degradation of fibrinogen and other clotting factors, and more bleeding . Nonetheless, the use of a fibrin - specific agent does not eliminate the risk of bleeding and an intervention should be undertaken only if all means for resuscitation including blood products and antifibrinolytics are immediately available . Procedure in this patient was performed by skilled interventional radiologist in fully equipped radiological suite with on - site critical care specialist and stand - by blood products . In vivo experiments show that flow in pulmonary circulation with embolus in its branches is almost instantly diverted into nonoccluded vessels.9 however fragmentation of the thrombus enables flow into previously occluded pa.9 we believe that repeated administration of tpa contributed to sustained patency of pa based on the evidence that supports prolonged fibrinolysis after tpa administration despite its short half - life10 as well as the fact that the agent was injected into recently opened pa after catheter instrumentation . Study of patients from the international cooperative pulmonary embolism registry (icoper) evaluated the effect of adjunctive therapies, such as thrombolysis and ivc filter placement on the clinical outcome of patients with massive pe.5 while thrombolytic therapy did not reduce mortality or recurrent pe, none of the patients with ivc filter developed recurrent pe and 90-day mortality was reduced . It is unknown whether preoperative placement of the ivc filter in our patient would have prevented the thromboembolic episode and subsequent complications of therapy that we observed . In small children, ivc filter placement is technically challenging and carries the risk of ivc injury and thrombosis.11,12 as with adults, this technique does not provide complete protection against life - threatening pe . Our patient developed rv dysfunction with elevated pa pressures as per tte . Dopamine was started to support failing rv and ino was initiated to unload the rv as well as improve vq matching . Dose of ino was titrated up to 30 ppm to improve oxygenation . Weaning off ino was a lengthy process lasting few days because of frequent episodes of desaturation initially . Forty - eight hours after the embolectomy / thrombolysis, the patient experienced hypotension, hypoxemia, required higher doses of dopamine, and, for a short period of time, epinephrine . The exact nature of hypotension was not clear because the repeat angiography revealed patent right pa and tte showed decreasing pa pressure, and improvement of right ventricular function . Some hypovolemia secondary to third space losses in this sick and very edematous patients might have been contributing factors . Bleeding, which occurred three days after thrombolysis, was almost certainly related to use of anticoagulants, considering the short half life of thrombolytic agents . Discontinuation of ufh, pressure dressing, and blood transfusions were the only measures to treat hemorrhage . We present a unique management of pe in a child with oxygenation failure and recent major vascular surgery using a catheter extraction technique combined with regional thrombolysis followed by ivc filter placement . We believe that prompt diagnosis and expertise of hospital staff as well as availability of resources contributed to a good patient outcome in our case.
|
Dilatation of various lengths and severity of the common bile duct (cbd), entitled choledochal cyst, has been detected in utero and usually presents with icterus in infancy, clinically mimicking biliary atresia and neonatal hepatitis1). Younger children and occasionally infants tend to present with painless jaundice, and older children present with recurrent abdominal pain, which was actually due to acute pancreatitis12). Postnatally, ultrasonography (us) is the initial diagnostic modality of choice, allowing for precise measurements of intra- or extrahepatic duct dilatation and identification of stones and sludge . Magnetic resonance cholangiopancreatography (mrcp) and endoscopic retrograde cholangiopancreatography (ercp) has superseded the use of computed tomography (ct) for preoperative anatomical delineation of the pancreaticobiliary tract . Here we present the biggest choledochal cyst reported in infancy in the literature to our knowledge . A term female baby was born by normal delivery route after consanguineous marriage, at house, in syria as the 6th child of her parents . This family was living the civil war in syria at that time . During 4 months period abdominal distension had increased . Because of restlessness and growing abdominal distension but as a result of investigations they were sent home because of normal laboratory results . After 15 days they admitted to hospital because of jaundice and abdominal cyst she had referred to our hospital (in turkey) with the preliminary diagnosis of abdomianl cyst and hepatitis developed due to compression of cyst . On her admission, us revealed giant abdominal cyst with thin wall and liquid - debris level extending from right upper quadrant to pelvic region . It was suspected that bile duct's dilatation was due to abdominal giant cyst's pressure . She was operated with the differential diagnosis of duplication, omental or mesenteric cyst . At operation, a giant type 1a choledochal cyst, 160 mm in diameter, was surprisingly detected (fig . Serum levels of bilirubin decreased sharply and the patient was discharged without any problems on the tenth postoperative day . A choledochal cyst is a dilation that encloses the intrahepatic or both extra- and intrahepatic portions of the biliary ducts2). Type ia is a cystic dilation of the cbd; type ib is a focal segmental dilation of the distal cbd; type ic is a fusiform dilation of both the common hepatic duct and cbd . In type ii, the cyst forms a diverticulum from the extrahepatic bile duct . Type iii, also known as choledochocele, is a dilation of the distal cbd lying mainly within the duodenal wall . Type iv is essentially type i anatomy with either intrahepatic bile duct cyst (iva) or choledochocele (ivb). Some authors refer to caroli's disease with multiple cystic dilations of the intrahepatic biliary tree as type v23). In our case there was a cystic dilation of the cbd as a type ia choledochal cyst with minimal dilatation of intrahepatic bile ducts . This malformation primarily affects girls (4:1) and about 80% become symptomatic during childhood . Choledochal cysts remain relatively uncommon in western europe and the united states, although they are appreciably more common in asia . Obstructive jaundice is the main presentation symptom in children, but abdominal pain is the commonest symptom in adults . The classical triad of pain, jaundice and a palpable mass is uncommon, occurring in no more than 6% in one uk series4). The complications of congenital cystic dilatation of the bile duct are biliary stone formation, progressive biliary cirrhosis with portal hypertension, and carcinoma . Fusiform lesions are never large enough to be palpable while multiple intrahepatic type 4 lesions cause predisposition to stone formation and sepsis . A wide variety of imaging techniques are available which noninvasively reconstruct biliary anatomy and give an excellent idea of biliary function . Although us is the first described imaging method to determine the cbd cyst, ct, mrcp, and ercp are superior to assess the extention of the cyst and associated pathologies such as cholangitis, pancreatitis and pancreaticobiliary junction anomaly . In our case, we used us and ct to identify the abdominal mass' origin . Because of the diameter of the cyst, cbd cyst was not thought in the differential diagnosis . Cbd malformations should be kept in mind as a differential diagnosis of the cystic mass regardless of size, and patient's age . Surgery is the main choice of treatment but some centres in south american and asia have reported ercp and sphincterotomy alone as definitive treatment for mild fusiform dilatation although their long - term prognosis is not known3). At operation, it was difficult to make the differential diagnosis of our giant cyst . It was important to make the dissection of the cyst carefully in order to avoid the iatrogenic injury . Following the diagnosis of choledochal cyst was confirmed, hepaticojejunostomy was easier depending on the wide common hepatic duct of our huge cyst . Tang et al.5) reported a study that involves 62 children (average age of 2.3 years) who had cysts with the average diameter of 42 mm (range, 12158 mm). There is no information about the child'a age with the 158-mm cyst . As our knowledge we report the biggest choledocal cyst case in infancy in the literature . In conclusion, cbd malformations should be kept in mind as a differential diagnosis at the cystic mass regardless of cyst's size, and patient's age, especially in children presented with abdominal pain, jaundice, and palpable mass.
|
Approximately 6% of patients undergoing dialysis have malignancies, and the number of these patients undergoing chemotherapy continues to increase . Chemotherapy in dialysis patients is challenging because excretion of the drug differs between dialysis and non - dialysis patients . Drugs that are excreted by dialysis may show reduced efficacy, while drugs that are not excreted may cause adverse events . Imatinib is an effective and safe therapy for kit - positive gastrointestinal stromal tumors (gist) [2, 3, 4, 5]. However, the efficacy and safety of imatinib treatment in dialysis patients remain unclear because clinical trials have not been conducted in this population . We report a patient with duodenal gist undergoing dialysis who was treated with regular - dose imatinib and remained stable with no adverse events for 4 months . We also present a literature review of the efficacy and safety of imatinib treatment in patients with gist undergoing dialysis . In a 75-year - old japanese man, a large duodenal tumor was identified on computed tomography (ct) in 2008 and was suspected as gist on endoscopic ultrasonography . However, the patient refused further investigation and underwent a follow - up visit . He also had chronic kidney disease, due to type 2 diabetes, and began a course of hemodialysis three times weekly in 2014 . A large - volume and painless tarry stool with hemorrhagic shock occurred in april 2014 . A complete blood count showed a hemoglobin level of 6.3 g / dl, and the patient required transfusion of 32 units of packed red blood cells to recover a general condition . Contrast agent - enhanced ct scans revealed a duodenal tumor 14 cm in diameter, with multiple liver and bone metastases . The patient underwent an urgent upper endoscopy, which showed a 3.5-cm submucosal tumor with stigmata of recent hemorrhage in the bulb of the duodenum . Endoscopic hemostasis was not conducted due to the large exposed vessel, and an urgent pancreaticoduodenectomy was performed to achieve hemostasis . The resected tumor showed a gist composed of spindle - shaped cells with positive staining for kit and cd34 and negative staining for s-100; the mitotic count was 12 per 50 high - power fields, and the mib-1 labeling index was 10% (fig . The patient was treated with imatinib at a dose of 400 mg daily (orally). Complete blood counts showed a white blood cell count in the range of 3669 10/l, a hemoglobin level range of 8.9 - 10.2 g / dl, and a platelet count range of 162265 10/l . Furthermore, no severe adverse event, including liver functional impairment, intestinal pneumonia, or stevens - johnson syndrome occurred . However, mild adverse events including fatigue and edema were observed, and his metastases remained stable for 4 months . Finally, imatinib treatment was discontinued because of progression of the disease and worsening performance status in january 2015 . The patient died of duodenal perforation induced by residual tumor progression in february 2015, 6 years after the initial diagnosis and 5 months after chemotherapy for gist . We administered regular - dose imatinib in a patient with multiple metastases from gist undergoing dialysis . The patient remained stable without any adverse event associated with imatinib for 4 months . To our knowledge, this is the first literature review and third case report of imatinib treatment for gist in patients undergoing dialysis . Several cytotoxic drug therapies have been reported in patients with major gastrointestinal tract malignancies undergoing dialysis . Dialysis patients with esophageal cancer have been administered 5-fluorouracil and cisplatin [7, 8, 9] and dialysis patients with gastric cancer have been administered tegafur - uracil, docetaxel, and irinotecan [10, 13]. Dialysis patients with colon cancer have been treated with tegafur - uracil, irinotecan, and oxaliplatin [14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24]. In these cases, most cytotoxic drugs were used safely without dose modification, but irinotecan caused severe myelosuppression and resulted in death [15, 18]. However, available data on molecular - specific therapies in dialysis patients are limited, and treatment - related issues are unclear . Pappas et al . Reported a 44-year - old woman with rectal gist and liver metastasis undergoing dialysis for 1 year . Six years later, she was diagnosed with liver metastasis of the resected rectal gist and received regular - dose imatinib for the liver metastasis . No severe adverse events associated with imatinib occurred, and the liver metastasis remained in partial remission for 12 months . Reported a 69-year - old japanese man with peritoneal gist undergoing dialysis for 9 years . The main lesion of the peritoneum gist was resected, and he received regular - dose imatinib for residual disease . No severe adverse event occurred, and the residual disease remained in partial remission for 20 months . In our case and the previous cases, no severe adverse events were associated with imatinib . A previous study evaluated the pharmacokinetics of both imatinib and its metabolite cgp74588 in dialysis patients and reported that the pharmacokinetics of dialysis patients were similar to those of non - dialysis patients . The clinical importance of these data was supported by our results, and we also consider that dialysis patients can be treated safely with imatinib . Heart failure was not observed in our case due to strict water management, but physicians should pay careful attention to patients with these findings . Previous studies reported good clinical outcomes, with no recurrence after 1220 months [25, 26]. Imatinib treatment without dose reduction may contribute to good responses in patients undergoing dialysis as well as those without dialysis . In our case, overall survival was shorter than that in previous reports [25, 26], but the difference may be due to the clinical stage at the time of initiation of imatinib treatment . Our case had poor prognostic factors, such as a lesion size larger than 11.1 cm, wall invasion, and hepatic metastasis . In conclusion, regular - dose imatinib was an effective and safe treatment in a patient with gist undergoing dialysis.
|
Primary ciliary dyskinesia (pcd) is a rare genetic disorder of ciliary function and affects ~1/20,000 live births (1/12,5001/30,000) (1). Cilia serve critical roles throughout the human body and at all stages of life, including during left - right patterning of embryonic organs, the clearing of mucus and dirt from the respiratory tract, and the proper movement of sperm or ova; therefore, symptoms of pcd are diverse and include situs inversus, chronic oto - rhino - pulmonary infection and infertility . A large number (> 200) of genes code ciliary components, and the symptoms of pcd can vary between patients . This heterogeneity makes the diagnosis of pcd challenging, particularly when heterotaxia is absent and other symptoms are mild (2). This challenge is a problem, due to the fact that the later pcd is diagnosed, the worse the prognosis is (3). There is no gold - standard for diagnosis of pcd, and a comprehensive approach using both structural and functional analysis of the cilia and genetic analysis of causative genes is required . In the current study, a case of pcd in which the patient had been treated for intractable atelectasis with unknown origin for nine years the present study highlighted the benefits of diagnostic strategies that include ultrastructural analysis accompanied by extended genetic analysis involving whole - exome sequencing . Chest computed tomography (ct) images were acquired by an aquillion ct scanner (toshiba medical systems corporation, otawara, japan), with automatic exposure control (sd:12) at 120 kvp with 5 mm section thicknesses . A small amount of nasal mucosa was extracted from the patient's inferior turbinate . According to the methodology of rubin (4), greater than 100 cilia from the patient nasal mucosa was also extracted from a subject with no nasal diseases and served as a normal control group . Genetic analysis was approved by the mie university university ethics committee (no . 1363), and written informed consent was obtained from the proband and each parent . Genomic dna was extracted from peripheral blood samples taken from the forearm of each participant . Subsequently, known hot spots were sequenced in two candidate genes, dnah5 and dnai1, due to the fact that a previous study observed mutations in dnah5 or dnai1 in approximately a third of all patients with pcd (5). For the whole - exome sequencing, proband dna was amplified with the ion ampliseq exome rdy kit (life technologies; thermo fisher scientific, inc ., waltham, ma, usa), which targets more than 97% of human consensus coding sequences . After quality control thaws were performed with the bioanalyzer high sensitivity chip (agilent technologies, inc ., santa clara, ca, usa) and emulsion polymerase chain reaction (pcr; ion pi hi - q ot2 200 kit; life technologies; thermo fisher scientific, inc . ), samples were sequenced with a proton pi chip version 3 and the ion proton semiconductor sequencer system (life technologies; thermo fisher scientific, inc . ). Base calling, pre - processing of the reads, short read alignment and variant calling were performed with the torrent suite, the torrent variant caller (version 4.6; thermo fisher scientific, inc . ), and the default parameters recommended for the ampliseq exome panel (low stringency calling of germline variants, version, april 2014). Variant annotation was performed with ion reporter, version 4.6 (life technologies; thermo fisher scientific, inc .) And was data integrated from a variety of public databases . Exome variant analysis was performed by filtering the whole variant list according to three criteria: i) consistent autosomal recessive inheritance patterns, ii) novelty in comparison to human polymorphism databases [including the 1000 genomes (http://www.1000genomes.org/) and dbsnp (http://www.ncbi.nlm.nih.gov/projects/snp/)], and iii) functional significance . These analyses required the presence of at least one homozygous or two heterozygous changes occurring with an estimated frequency of <0.01 . Variants were validated via pcr and sanger sequencing with the 3500 series genetic analyzer (thermo fisher scientific, inc . ). These tests were performed according to standard protocols specifically adapted to preclude technical artifacts and test for segregation . The primers used for the amplification were as follows: dnah5 exon 36 f, 5-cttgtgtgcgtttcatgcca-3; dnah5 exon 36 r, 5-ctgcaaccgagagaactggt-3; dnah5 exon 54 f, 5-gatgataacggtgttgggggat-3, dnah5 exon 54 r, 5-gtagccccggaaaggagtaaat-3. Mutationtaster (http://mutationtaster.org/) and polyphen-2 (http://genetics.bwh.harvard.edu/pph2/) analyses were conducted in order to predict the impact of variants in silico . The proband was a nine - year - old boy who was the offspring from non - consanguineous japanese parents . His close relatives, specifically his parents, grandparents, two younger sisters and one younger brother, did not have a history of significant respiratory illness (fig . The first year of his life was uneventful, except that he experienced mild dyspnea and required low dose oxygen from the second to fourth days after birth . At one year of age, he was hospitalized for five days with a diagnosis of asthmatic bronchitis, with no significant lung infiltrations observed by x - ray (fig . Since then, the patient experienced chronic nasal discharge and productive cough along with multiple episodes of sinusitis and/or otitis media . Atelectasis of the right lower lobe was first noted on a chest x - ray at three years of age, when he was admitted for acute pneumonia (fig . Despite intense physiotherapy including positive expiratory pressure therapy or high frequency chest wall oscillation, the atelectasis remained unresolved (fig . 2c and d). He was also diagnosed with asthma due to a frequent cough, leading to administration of inhaled corticosteroids and other asthma - specific therapies . At nine years of age, bronchiectasis was observed in the same lobe of the right lung by ct scanning (fig . Respiratory function tests at that point demonstrated mild airway obstruction with forced vital capacity of 1.64 l (95.3% predicted), forced expiratory volume1.0 1.18% (80.1% predicted) and maximum midexpiratory flow 0.73 l / sec (37.2% predicted). All standard screening tests for immunodeficiency including serum immunoglobulin levels and t / b lymphocyte counts were normal; consequently, pcd was suspected, and the patient was referred to the department of otorhinolaryngology at our hospital . Otological examination revealed that the light reflex was missing from the right eardrum (fig ., the posterosuperior quadrant was bulging, and the light reflex was missing (fig . Pure - tone audiometry revealed bilateral conductive hearing loss with a hearing level of 35 db in the right and 50 db in the left (fig . Rhinological evaluation demonstrated that the nasal cavities were filled bilaterally with mucopurulent nasal secretions (fig . Nasal x - ray revealed soft - tissue density bilaterally in the maxillary sinuses, suggesting chronic sinusitis and agenesis of the frontal sinuses (fig . Nasal and exhaled nitric oxide (no) was measured via an analyzer cld 88 according to american thoracic society / european respiratory society recommendations (6); the fractional concentration of exhaled no (feno) and nasal no values were 10.0 and 0.2 ppb, respectively (data not shown). Analysis of the specimen collected from the patient demonstrated shortened outer dynein arms (fig . No causative mutations were identified via conventional sanger - based analyses of exons 34, 50, 63, 76 and 77 of dnah5 (8) and exons 1, 13, 16, and 17 of dnai1 (9). Based on these observations, whole - exome sequencing was conducted . The whole - exome analysis of the proband genomic dna identified two novel compound heterozygous mutations in dnah5: nm_001369.2:c.5983c> t, p.arg1995x in exon 36; and nm_001369.2:c.9101delg, p.gly3034valfsx22 in exon 54 . Polyphen-2 analysis indicated that the p.gly3034valfsx22 mutation of dnah5 was likely to be functionally damaging, with a score of 1.000 . Mutationtaster predicted that each of these dnah5 mutations would cause nonsense - mediated mrna decay . Sanger sequencing confirmed the compound heterozygous mutations in dnah5 identified by the whole - exome analysis in the proband (fig . 7a); nm_001369.2:c.5983c> t, p.arg1995x in exon 36 and nm_001369.2:c.9101delg, p.gly3034valfsx22 in exon 54 . The proband was a nine - year - old boy who was the offspring from non - consanguineous japanese parents . His close relatives, specifically his parents, grandparents, two younger sisters and one younger brother, did not have a history of significant respiratory illness (fig . The first year of his life was uneventful, except that he experienced mild dyspnea and required low dose oxygen from the second to fourth days after birth . At one year of age, he was hospitalized for five days with a diagnosis of asthmatic bronchitis, with no significant lung infiltrations observed by x - ray (fig . Since then, the patient experienced chronic nasal discharge and productive cough along with multiple episodes of sinusitis and/or otitis media . Atelectasis of the right lower lobe was first noted on a chest x - ray at three years of age, when he was admitted for acute pneumonia (fig . Despite intense physiotherapy including positive expiratory pressure therapy or high frequency chest wall oscillation, the atelectasis remained unresolved (fig . 2c and d). He was also diagnosed with asthma due to a frequent cough, leading to administration of inhaled corticosteroids and other asthma - specific therapies . At nine years of age, bronchiectasis was observed in the same lobe of the right lung by ct scanning (fig . Respiratory function tests at that point demonstrated mild airway obstruction with forced vital capacity of 1.64 l (95.3% predicted), forced expiratory volume1.0 1.18% (80.1% predicted) and maximum midexpiratory flow 0.73 l / sec (37.2% predicted). All standard screening tests for immunodeficiency including serum immunoglobulin levels and t / b lymphocyte counts were normal; consequently, pcd was suspected, and the patient was referred to the department of otorhinolaryngology at our hospital . Otological examination revealed that the light reflex was missing from the right eardrum (fig ., the posterosuperior quadrant was bulging, and the light reflex was missing (fig . Pure - tone audiometry revealed bilateral conductive hearing loss with a hearing level of 35 db in the right and 50 db in the left (fig . Rhinological evaluation demonstrated that the nasal cavities were filled bilaterally with mucopurulent nasal secretions (fig . Nasal x - ray revealed soft - tissue density bilaterally in the maxillary sinuses, suggesting chronic sinusitis and agenesis of the frontal sinuses (fig . Nasal and exhaled nitric oxide (no) was measured via an analyzer cld 88 according to american thoracic society / european respiratory society recommendations (6); the fractional concentration of exhaled no (feno) and nasal no values were 10.0 and 0.2 ppb, respectively (data not shown). Analysis of the specimen collected from the patient demonstrated shortened outer dynein arms (fig . No causative mutations were identified via conventional sanger - based analyses of exons 34, 50, 63, 76 and 77 of dnah5 (8) and exons 1, 13, 16, and 17 of dnai1 (9). Based on these observations, whole - exome sequencing was conducted . The whole - exome analysis of the proband genomic dna identified two novel compound heterozygous mutations in dnah5: nm_001369.2:c.5983c> t, p.arg1995x in exon 36; and nm_001369.2:c.9101delg, p.gly3034valfsx22 in exon 54 . Polyphen-2 analysis indicated that the p.gly3034valfsx22 mutation of dnah5 was likely to be functionally damaging, with a score of 1.000 . Mutationtaster predicted that each of these dnah5 mutations would cause nonsense - mediated mrna decay . Sanger sequencing confirmed the compound heterozygous mutations in dnah5 identified by the whole - exome analysis in the proband (fig . 7a); nm_001369.2:c.5983c> t, p.arg1995x in exon 36 and nm_001369.2:c.9101delg, p.gly3034valfsx22 in exon 54 . The proband was a nine - year - old boy who was the offspring from non - consanguineous japanese parents . His close relatives, specifically his parents, grandparents, two younger sisters and one younger brother, did not have a history of significant respiratory illness (fig . The first year of his life was uneventful, except that he experienced mild dyspnea and required low dose oxygen from the second to fourth days after birth . At one year of age, he was hospitalized for five days with a diagnosis of asthmatic bronchitis, with no significant lung infiltrations observed by x - ray (fig . Since then, the patient experienced chronic nasal discharge and productive cough along with multiple episodes of sinusitis and/or otitis media . Atelectasis of the right lower lobe was first noted on a chest x - ray at three years of age, when he was admitted for acute pneumonia (fig . Despite intense physiotherapy including positive expiratory pressure therapy or high frequency chest wall oscillation, the atelectasis remained unresolved (fig . 2c and d). He was also diagnosed with asthma due to a frequent cough, leading to administration of inhaled corticosteroids and other asthma - specific therapies . At nine years of age, bronchiectasis was observed in the same lobe of the right lung by ct scanning (fig . Respiratory function tests at that point demonstrated mild airway obstruction with forced vital capacity of 1.64 l (95.3% predicted), forced expiratory volume1.0 1.18% (80.1% predicted) and maximum midexpiratory flow 0.73 l / sec (37.2% predicted). All standard screening tests for immunodeficiency including serum immunoglobulin levels and t / b lymphocyte counts were normal; consequently, pcd was suspected, and the patient was referred to the department of otorhinolaryngology at our hospital . Otological examination revealed that the light reflex was missing from the right eardrum (fig ., the posterosuperior quadrant was bulging, and the light reflex was missing (fig . Pure - tone audiometry revealed bilateral conductive hearing loss with a hearing level of 35 db in the right and 50 db in the left (fig . Rhinological evaluation demonstrated that the nasal cavities were filled bilaterally with mucopurulent nasal secretions (fig . Nasal x - ray revealed soft - tissue density bilaterally in the maxillary sinuses, suggesting chronic sinusitis and agenesis of the frontal sinuses (fig . Nasal and exhaled nitric oxide (no) was measured via an analyzer cld 88 according to american thoracic society / european respiratory society recommendations (6); the fractional concentration of exhaled no (feno) and nasal no values were 10.0 and 0.2 ppb, respectively (data not shown). Analysis of the specimen collected from the patient demonstrated shortened outer dynein arms (fig . 6a - c); and this observation was compatible with pcd . In the normal control group (fig . No causative mutations were identified via conventional sanger - based analyses of exons 34, 50, 63, 76 and 77 of dnah5 (8) and exons 1, 13, 16, and 17 of dnai1 (9). Based on these observations, whole - exome sequencing was conducted . The whole - exome analysis of the proband genomic dna identified two novel compound heterozygous mutations in dnah5: nm_001369.2:c.5983c> t, p.arg1995x in exon 36; and nm_001369.2:c.9101delg, p.gly3034valfsx22 in exon 54 . Polyphen-2 analysis indicated that the p.gly3034valfsx22 mutation of dnah5 was likely to be functionally damaging, with a score of 1.000 . Mutationtaster predicted that each of these dnah5 mutations would cause nonsense - mediated mrna decay . Sanger sequencing confirmed the compound heterozygous mutations in dnah5 identified by the whole - exome analysis in the proband (fig . 7a); nm_001369.2:c.5983c> t, p.arg1995x in exon 36 and nm_001369.2:c.9101delg, p.gly3034valfsx22 in exon 54 . Pediatricians had followed this case for nine years before the diagnosis of pcd was made . Reportedly, the majority of patients with pcd are seen by physicians greater than 50 times prior to a diagnosis of pcd being made, and the mean age at pcd diagnosis is 10.914.4 years (2). In addition, it takes markedly longer to make a diagnosis of pcd when situs inversus is absent . The value of this case is that sequential chest x - rays and ct scans were obtained over a long time period . An abnormality in a chest x - ray was observed at the age of three, however not at the age of one, and at a later date the patient had consolidation only in one lobe (the right middle lobe). For subjects 511 years old, davis et al (10) observed that patients with inner dynein arm and central apparatus defects with microtubular disorganization presented with more lobes with bronchiectasis (median, 5; p=0.0008) and consolidation (median, 3; p=0.0001) than patients with outer dynein arm defects (median, 3 and 2, respectively). The proband in the current study had defects only in the outer dynein arms; therefore, he had a less severe lung pathology, which may partly explain the delay in a diagnosis of pcd . According to santamaria et al (11), the prevalence of lung changes on ct is as follows: bronchiectasis, 80%; peribronchial thickening, 80%; mucous plugging, 75%; parenchyma, 65%; and mosaic perfusion, 45% . The lung ct of the patient in the present study indicated the presence of bronchiectasis and peribronchial thickening, the two most frequently occurring observations . Pcd is a mendelian autosomal recessive and a genetically heterogeneous disorder . In a review article from 2013, knowles et al (12) reported that pcd - causing mutations had been identified in 21 genes . The genes most commonly identified to result in pcd were dnah5 (1521%), dnai1 (29%), dnaaf1 (lrrc50) (45%), ccdc39 (210%), ccdc40 (28%), dnah11 (6%) and lrrc6 (3%). Pathogenic mutations in 28 genes can reportedly lead to pcd, and these 28 genes reportedly account for approximately 70% of individuals affected with pcd (13). In the current study, electron microscopy identified shortened outer dynein arms . Regarding the association between ultrastructural phenotypes and genotypes, mutation of dnah5, dnai1, dnai2, dnal1, ccdc114, txndc3 or armc4, which code for the structural components of the outer dynein arms, results in their loss . In the present study, whole - exome analysis of proband genomic dna identified compound heterozygous mutations in dnah5 . At present, dnah5 is reportedly the gene most often responsible for pcd; for example, mutations on both alleles of dnah5 were identified in 15% of a clinically heterogeneous cohort of patients (14). Dnah5 is a large gene comprising 79 exons and one alternative first exon and it encodes a heavy chain of the the outer dynein arm (8). A 1.5-kb partial cdna representing dnah5 was identified by omran et al (15), and a full - length, 14 kb dnah5 transcript was characterized by olbrich et al (16). Hornef et al (8) used haplotype analyses and/or sequencing to screen 109 caucasian pcd families originating from europe and north america for the presence of dnah5 mutations . They observed clustering of mutations within five exons (exons 34, 50, 63, 76 and 77); and these five exons harbored 27 (52%) of all 52 detected mutant alleles . Based on these observations, direct sequencing was conducted in the current study with these five exons to screen pcd - causing mutations . However, the two mutations in the examined patient were in exons 36 and 54: nm_001369.2:c.5983c> t, p.arg1995x in exon 36 and nm_001369.2:c.9101delg, p.gly3034valfsx22 in exon 54 . Although dnah5 mutations have been reported in pcd patients outside japan, only one report of dnah5 mutation in a japanese patient was identified in pubmed . Tate et al (17) analyzed the case of a male neonate who exhibited three lobes of the left lung, asplenia and complex heart anomalies, who died 6 h subsequent to delivery . A heterozygous single nucleotide change (c.7829a> g) was identified in exon 47 of dnah5, and this mutation resulted in the missense mutation of p.glu2610gly (17). Zhang et al (18) performed exome capture and sequencing with samples from one affected individual and the unaffected parents from a chinese han community . They identified a homozygous mutation, c. 8030g> a (arg2677gln), in dnah5 . This mutation was in exon 49, which is a known hot spot for pcd - causing mutations . Additionally, a patient from germany had a missense mutation in 8029c> t, which resulted in arg2677x (8). To the best of our knowledge, both mutations identified in the current study are novel (5,8,14,16). One is a nonsense mutation (nm_001369.2:c.5983c> t, p.arg1995x) in exon 36; the other is a frame - shift mutation (nm_001369.2:c.9101delg, p.gly3034valfsx22) in exon 54 . Of the pcd - causing mutations analyzed thus far, 85% are loss - of - function variants, and approximately 15% are conservative missense mutations (12). Among the 33 novel dnah5 mutations detected by hornef et al (8), 12 were nonsense mutations; 8 frame - shift mutations; 5 splicing variants and 8 missense mutations . To predict the impact of these two newly identified variants in silico, mutationtaster (19) and polyphen-2 (20) analyses were conducted . Polyphen-2 analysis indicated that the p.gly3034valfsx22 mutation of dnah5 was likely to be functionally damaging, with a score of 1.000 . Mutationtaster assesses whether the predicted mutant proteins will be long- or short - lived and whether nonsense - mediated mrna decay is likely to occur . Mutationtaster predicted that each of these dnah5 mutations would cause nonsense - mediated mrna decay . Dnah5 encodes ciliary dynein axonemal heavy chain 5, a 4624-amino acid protein (16). The n - terminal domain forms the stem domain of the outer dynein arm complex and is involved in interactions with other heavy, intermediate and light chains . The c - terminal region that constitutes the globular head contains six conserved 6 p - loop domains and a conserved microtubule binding site (16). The first p - loop domain is known to bind and hydrolyze adenosine triphosphate (16). Both newly identified mutations were predicted to cause loss of function of the protein; therefore, it is likely that the two novel mutations are causal mutations resulting in pcd . This is, to the best of our knowledge, the first report describing dnah5 mutations in a japanese patient with pcd . Whether there are relatively fewer patients with pcd among the japanese compared with other ethnic groups is unclear, and requires investigation in future studies . The present study reported a boy who had been followed by pediatricians for the first nine years of his life . Whole - exome analysis of the genomic dna identified novel compound heterozygous mutations in dnah5: nm_001369.2:c.5983c> t, p.arg1995x in exon 36; and nm_001369.2:c.9101delg, p.gly3034valfsx22 in exon 54.
|
The key point of hyperbaric treatment is the elevated po2 level both in the plasma and tissues . Normal alveolar po2 level is reached at 1 ata (ata = 760 mmhg, which is the normal atmosphere pressure at the sea level), while higher atmosphere pressures cause an increase of the level of oxygen dissolved in the plasma . Scuba (scuba, self - contained underwater breathing apparatus) diving was shown to induce a significant increase of the total number of leukocytes, particularly neutrophils . Whereas the exact mechanisms that lead to endothelial dysfunction are still incompletely understood, hyperoxia - induced production of reactive oxygen species (ros), reduction in the bioavailability of nitric oxide (no) and direct mechanical damage to the endothelium during decompression, are considered to play an important role (1). Recently, it has been demonstrated that a single air scuba dive induced a significant increase in the number of monocytes expressing cd15 as well as the increase in the small monocyte subpopulation highly expressing cd15s (2). Sialylated fucosylated glycans (sialyl lewis x - type glycans or cd15s) are expressed on circulating granulocytes and monocytes and, upon recognition by endothelial selectins, mediate initial leukocyte endothelium interactions (3). Zen et al showed directly that human leukocyte cd11b is a major membrane protein decorated with cd15s and that cd15s related moieties mediate the binding of cd11b with e - selectin (4). The expression of atherogenic adhesion molecule cd11b was found to be decreased after high frequency and long duration exercise (6). It has also been shown that a competitive marathon race can decrease neutrophile functions (oxidative burst activity and phagocytic activity) in athletes (7). They are flask - shaped membrane invaginations formed from lipid rafts by polymerization of caveolins, which are integral membrane proteins that tightly bind cholesterol and sphingolipids . Caveolae have been found to be partaking in many physiological and pathological processes involving endothelial cells, such as atherosclerosis, hemostasis, and thrombosis . Caveolae of endothelial plasma membranes are rich in neutral glycosphingolipid, globotriaosylceramide, gb3cer or cd77 . Excessive endothelial cd77 accumulation is associated with endothelial dysfunction (8). Hyperbaric oxygen treatment, a method based on 100% oxygen exposure, has a beneficial effect on renal dysfunction in sepsis caused by escherichia coli (9). Cd77 is a receptor for stxs (stxs, shiga toxins) produced by shigella dysenteriae type 1 and enterohemorrhagic e. coli that are most common cause of hus (hus, hemolytic - uremic syndrome). Uschida et al showed that specific antibodies for stxs positively stained pulmonary tissue from a patient who died of hus associated with stx - producing e. coli infection, indicating the deposition of stxs in the lung . Related experiments with normal pulmonary tissue revealed apparent stx binding to both vascular endothelium and to portions of the pulmonary epithelium . In addition, cd77-positive lung carcinoma cell lines, which are derived from lung epithelium, showed reactivity to stx and a high susceptibility to stxs, as determined by mtt assay (10). Glomerular endothelial cells in humans are the primary target of the toxic effects of stxs, but why lesions in stx - associated hus preferentially localize to the renal microvasculature is still unclear (11). Whether stem cells are the source of the epithelial progenitors replacing injured and dying tubule, epithelium is currently an area of intensive investigation . The fundamental unanswered questions in this field include whether renal stem cells exist in adults, if they do, where are they located (interstitium, tubule, cortex, medulla) and what markers can be relied upon for the isolation and purification of these putative renal stem cell (12). Resident stem / progenitor cells of different human adult organs are known to express stem cell markers such as cd34, cd117 and cd133 . As we know, cd34 is a sialomucin - type glycophosphoprotein, traditionally a marker of hematopoietic stem cells and was found on endothelial cells and fibroblasts as well (13). Despite its utility as a stem - cell marker, the function of cd34 has remained remarkably elusive . It is believed that cd34 promotes cell proliferation and / or blocks differentiation of progenitor cells, while other members of cd34 family stimulate the migration of hematopoietic cells, or play a role in cell morphogenesis . It is interesting to point out that members of the cd34 family can stimulate and block cell adhesion (14). Exercise and the improvement of cardiovascular health tend to promote higher levels of circulating cd34 + cells (15). Advanced age and chronic cardiovascular disease tend to decrease both the functionality and the total count of cd34 + cells (16, 17). In many current researches, the bone marrow - derived cd34 + cells have been evaluated as a tool to repair the endothelial damage caused by cardiovascular disease . New evidence supports both a role of transdifferentia - tion of cd34 + cells to cardiomyocytes (18) and their ability to fuse with existing cardiomyocytes (19). In recent review, mackie and losordo showed the preclinical evidence supporting the therapeutic potential of cd34 + cells in ischemic models, and the evidence for the clinical usefulness of cd34 + cells in the treatment of human ischemic disease (20). Muller et al demonstrated that cd34 is hetero - geneously expressed by human pulmonary endothelial cells, and that expression is under influence of different physiological / pathophysiological factors, such as age or pulmonary hypertension (21). Due to the described beneficial effects of hyperbaric treatment on the one hand, and its potential proinflammatory effect on the other hand, the aim of this study was to estimate effects of hyperbaric treatment by determination of cd15s and cd11b leukocyte proinflammatory markers as well as cd77 and cd34 expression on rat renal, pulmonary and cardiac cells . Experiments were performed with male sprague - dawley rats raised under controlled conditions (temperature of 22 1c and a light schedule of 14-hr light/10-h dark) at the split university animal facility . Animals were bred and maintained according to the guide for care and use of laboratory animals and the protocol was approved by the ethics committee of the split university medical school . Four weeks old rats were separated in 2 groups: the examination group (n=9) which underwent the hyperbaric treatment and untreated control group (n=5). Rats were exposed to hyperbaric pressure of air mixture (21% oxygen, 79% nitrogen) which equals the immersion depth of 65 meters (7.5 ata), in duration of 30 min . Decompression stops were 1 min at the depth of 15 m, 7 min at 12 m, 10 min at 9 m, 23 min at 6 m and 47 min at 3 m, according to us navy decompression tables (http://www.usu.edu/scuba /navy_manual6.pdf). The animals were exposed to hyperbaric treatment in a comex hyperbaric chamber (comex, marseilles, france). The oxygen and carbon dioxide concentrations in the chamber were controlled by servomex 570a oxygen analyzer (servomex, houston, tx, usa) and by infrared carbon dioxide gas analyzer (infrared industries inc ., santa barbara, ca, usa). In this study, the method for the preparation of samples for flow cytometry, as well as all antibodies used, were in accordance with our previous study (8). Blood samples for flow cytometry were collected from jugular vein into glass vacuum tubes with edta anticoagulant, one hr after hyperbaric treatments and before sacrifice . One hundred l of whole blood was pre - treated with fcr (fc - receptor)-blocking reagent (miltenyi biotec gmbh, bergisch gladbach, germany) to prevent non - specific binding and it was incubated in the dark for 30 min on ice with 0.5 g of primary anti - cd15s antibody produced in mouse (pharmingen, san diego, ca, usa). After two washes in 0.1 m pbs with 0.1% sodium azide, 0.5 g of secondary fitc - conjugated, affinity chromatography - purified rabbit anti - mouse antibody (pharmingen, san diego, ca, usa) and 1 g of phycoerythrin (pe)-conjugated antibody reactive to cd11b (iq test, beckman coulter, marseille, france) were added to cells and incubated in the dark on ice for 30 min . After red blood cell lysis with red blood cell lysis solution (miltenyi biotec, bergisch gladbach, germany), 10 events were recorded on a coulter epics xl flow cytometer (beckman coulter corporation, miami, usa). Fluorochrome and isotype - matched controls as well as unstained cell samples were used as negative controls . After third hyperbaric treatment, rats were euthanized with prolonged exposure to diethylether and kidneys, lungs and heart were dissected from all animals . Tissues were minced with scissors and incubated in a solution of 0.1 m pbs (phosphate buffer solution) with 0.1% (for kidney) and 0.2% (for heart and lung) collagenase type ia (roche diagnostics gmbh, mannheim, germany) in ratio: 100 mg tissue/5 ml collagenase in pbs . Cell suspensions were incubated for 30 min/1 hr (kidney / heart, lung) at 37c, with gentle stirring . After incubation, cell suspensions were filtrated through a 40-m nylon mesh (cell strainer; bd biosciences, san jose, ca, usa) and suspended at 1.0 10 cells ml in 100 l 0.1 m pbs . Monoclonal anti - cd34 antibody conjugated with phycoerythrin cyanin 5 (pc5, beckman coulter, marseille, france) was used for detection of cd34 positive cells . Monoclonal anti - cd77 antibody conjugated with fitc (bd pharmingen, erembodegem, belgium) was used for detection of cd77 positive cells . Isolated tissue cells were incubated in dark at 4c for 30 min with two antibodies for double cell labeling: 1 g of anti - cd34-pc5 and 1 g of anti - cd77-fitc . After two washes in 0.1 m pbs 10 events were recorded on a coulter epics xl flow cytometer (beckman coulter corporation, miami, usa). Fluorochrome - minus - one controls as well as unstained cell samples were measured and processed as negative controls to set the appropriate regions . Total cd15s+ leukocytes were defined as a sum of% of cd11b - cd15s+ and% of cd11b+cd15s+ leukocytes and total cd11b+ leukocytes as a sum of% of cd11b+cd15s+ and% of cd11b+cd15s- cells . Total cd34 + cells were defined as a sum of% of cd34+cd77- and% of cd34+cd77 + and total cd77 + cells as a sum of% of cd34-cd77 + and% of cd34+cd77 + cells . Due to relatively small sample, nonparametric mann whitney u test was used to test significance of differences between control and experimental group . Coefficient of correlation was calculated between variables: cd11b+ and cd15s+ leukocytes with cd34 + and cd77 + tissue cells . All of the results were considered significant at 95% confidence level (p <0.05) and were obtained by using software statistica 12.0 (statsoft, tulsa usa). Blood samples for flow cytometry were collected from jugular vein into glass vacuum tubes with edta anticoagulant, one hr after hyperbaric treatments and before sacrifice . One hundred l of whole blood was pre - treated with fcr (fc - receptor)-blocking reagent (miltenyi biotec gmbh, bergisch gladbach, germany) to prevent non - specific binding and it was incubated in the dark for 30 min on ice with 0.5 g of primary anti - cd15s antibody produced in mouse (pharmingen, san diego, ca, usa). After two washes in 0.1 m pbs with 0.1% sodium azide, 0.5 g of secondary fitc - conjugated, affinity chromatography - purified rabbit anti - mouse antibody (pharmingen, san diego, ca, usa) and 1 g of phycoerythrin (pe)-conjugated antibody reactive to cd11b (iq test, beckman coulter, marseille, france) were added to cells and incubated in the dark on ice for 30 min . After red blood cell lysis with red blood cell lysis solution (miltenyi biotec, bergisch gladbach, germany), 10 events were recorded on a coulter epics xl flow cytometer (beckman coulter corporation, miami, usa). Fluorochrome and isotype - matched controls as well as unstained cell samples were used as negative controls . After third hyperbaric treatment, rats were euthanized with prolonged exposure to diethylether and kidneys, lungs and heart were dissected from all animals . Tissues were minced with scissors and incubated in a solution of 0.1 m pbs (phosphate buffer solution) with 0.1% (for kidney) and 0.2% (for heart and lung) collagenase type ia (roche diagnostics gmbh, mannheim, germany) in ratio: 100 mg tissue/5 ml collagenase in pbs . Cell suspensions were incubated for 30 min/1 hr (kidney / heart, lung) at 37c, with gentle stirring . After incubation, cell suspensions were filtrated through a 40-m nylon mesh (cell strainer; bd biosciences, san jose, ca, usa) and suspended at 1.0 10 cells ml in 100 l 0.1 m pbs . Monoclonal anti - cd34 antibody conjugated with phycoerythrin cyanin 5 (pc5, beckman coulter, marseille, france) was used for detection of cd34 positive cells . Monoclonal anti - cd77 antibody conjugated with fitc (bd pharmingen, erembodegem, belgium) was used for detection of cd77 positive cells . Isolated tissue cells were incubated in dark at 4c for 30 min with two antibodies for double cell labeling: 1 g of anti - cd34-pc5 and 1 g of anti - cd77-fitc . After two washes in 0.1 m pbs, cells were resuspended in 0.3 ml of 0.1 m pbs . 10 events were recorded on a coulter epics xl flow cytometer (beckman coulter corporation, miami, usa). Fluorochrome - minus - one controls as well as unstained cell samples were measured and processed as negative controls to set the appropriate regions . Total cd15s+ leukocytes were defined as a sum of% of cd11b - cd15s+ and% of cd11b+cd15s+ leukocytes and total cd11b+ leukocytes as a sum of% of cd11b+cd15s+ and% of cd11b+cd15s- cells . Total cd34 + cells were defined as a sum of% of cd34+cd77- and% of cd34+cd77 + and total cd77 + cells as a sum of% of cd34-cd77 + and% of cd34+cd77 + cells . Due to relatively small sample, nonparametric mann whitney u test was used to test significance of differences between control and experimental group . Coefficient of correlation was calculated between variables: cd11b+ and cd15s+ leukocytes with cd34 + and cd77 + tissue cells . All of the results were considered significant at 95% confidence level (p <0.05) and were obtained by using software statistica 12.0 (statsoft, tulsa usa). In this study an effect of 3 repeated hyperbaric treatments on percentage of cd11b+ and cd15s+ leukocytes was investigated . The percentages of cd15s+cd11b- leukocytes were significantly increased (from 1.71 1.11 to 23.42 2.85, p<0.05) and total cd15s+ leukocytes were significantly increased (from 4.51 2.42 to 25.68 3.22, p<0.05) in group that went hyperbaric treatment after the first day . Hyperbaric treatment did not change percentage of total cd11b+ leukocytes (from 7.32 3.98 to 5.25 0.75) (figure 1). The percentages of different cd11b+ and cd15 + leukocyte subpopulations in control rats (n=5) and rats that went hyperbaric treatment (n=9). Significance is obtained by using mann whitney u test, * p<0.05 (vs. control group) the percentage of cd15s+cd11b- leukocytes decreased from second to third day, although not statistically significant . After strong increase of percentage of total cd15s+ leukocytes, following first and second hyperbaric treatment, this percentage slightly decreased following third treatment . Furthermore, the goal of this study was to determine expression of cd77 and cd34 on renal, cardiac and pulmonary rat cells after repeated hyperbaric treatment in comparison to non - treated animals . Hyperbaric treatment significantly decreased sum percentage of cd77+cd34- and cd77+cd34 + renal cells (from 16.355.5 to 4.48 1.28, p <0.05). The percentages of total cd34 + rat renal cells in the group exposed to hyperbaric treatment was also significantly lower compared to the control group, from 23.24 8.38 to 10.76 6.32, p <0.05 (figure 2). The percentages of cd77 positive and cd77 negative endothelial (cd34 +) cells, of cd77 positive non - endothelial cells (cd34-), of total cd77 positive and of total cd34 positive cells in suspensions of total kidney cells of control rats (n=5) and rats that went hyperbaric treatment (n=9). Significance is obtained by using mann whitney u test, * p<0.05 (vs. control group) it is well known that lung epithelium is another target for stxs, and stx - mediated injury to lung epithelial cells is thought to play an important role in the pathogenesis of pulmonary involvement associated with e. coli infection (10). Based on our results, hyperbaric treatment would not have beneficial effect on lung in conditions associated with e. coli infection as percentage of total cd77 + lung cells increased in rats that went hyperbaric treatment, from 3.412.11 to 23.53 13.09, p <0.05 (figure 3). Total cd34 + rat lung cells in the group exposed to hyperbaric treatment was significantly higher compared to the control group (from 3.272.01 to 11.92 6.22, p <0.05). The percentages of cd77 positive and cd77 negative endothelial (cd34 +) cells, of cd77 positive non - endothelial cells (cd34-), of total cd77 positive and of total cd34 positive cells in suspensions of total pulmonary cells of control rats (n=5) and rats that went hyperbaric treatment (n=9). Significance is obtained by using mann whitney u test, * p<0.05 (vs. control group) the percentage of total cd34 + cells was significantly increased in cardiac tissue in group of rats that went hyperbaric treatment, from 4.983.17 to 33.79 14.69, p <0.05 . We found out that percentage of cd77 + cardiac tissue cells were significantly increased due to hyperbaric treatment, from 1.812.15 to 8.19 4.29, p <0.05 (figure 4). The percentages of cd77 positive and cd77 negative endothelial (cd34 +) cells, of cd77 positive non - endothelial cells (cd34-), of total cd77 positive and of total cd34 positive cells in suspensions of total cardiac cells of control rats and rats that went hyperbaric treatment . Significance is obtained by using mann whitney u test, * p<0.05 (vs. control group) the results of correlation analysis between leukocytes markers cd11b and cd15s with tissue antigens cd34 and cd77 in rats that went hyperbaric treatment are presented in table 1 . There is no statistically significant correlation between leukocytes markers and tissue antigens in rats that went hyperbaric treatment . The correlations between the proportion of different leukocytes and cellular subpopulations from the heart, lung and kidney of rats that went hyperbaric treatment correlation analysis results: r = coefficient of correlation, p= significance of correlation coefficient the expression of atherogenic adhesion molecule cd11b was found to be decreased after high frequency and long duration exercise (23). Granulocyte - endothelial cell adhesion tests indicate that cd11b, the major membrane protein decorated with cd15s (4) is decreased after hyperbaric oxygen treatment . However, our results show unchanged cd11b leukocyte expression in rat following hyperbaric treatment . The opposite results obtained in our study could be explained by the different conditions in hyperbaric air treatment versus hyperbaric oxygen treatment . Several fold higher percentage of total cd15s+ than total cd11b+ leukocytes was detected in group that went hyperbaric treatment . That could be the consequence of higher affinity of anti - cd15s antibody to sialyl lewis x - type glycoepitope of cd11b glycoprotein than the affinity of anti - cd11b antibody to peptide epitope mac-1 of cd11b glycoprotein . Result of significantly increased percentage of cd15s+ leukocytes after repeated hyperbaric treatment is in accordance with previously reported results and elucidated role of this protein in the acute inflammatory process (24). Edremitliolu et al showed previously that renal dysfunction in sepsis improved by the use of hyperbaric oxygen was accompanied by an increase of antioxidative defense mechanisms: the superoxide dismutase and catalase activities in the renal cortex, and an increase in the catalase activity in the renal medulla (9). Daily secretion of urine increases for 500 ml during diving with air mixture (up to 3 - 49 ata), although the intake of fluids and velocity of glomerular filtration remains unchanged (25). Furthermore, it has been demonstrated that gb3 is over - expressed in proliferative endothelial cells of growing tumor relative to quiescent cells and it could be a viable alternative target for tumor immunotherapy and angiogenesis inhibition (26). Our study showed that percentage of cd77+cd34 + rat renal cells in the group exposed to hyperbaric treatment was significantly lower than in the control group, as well as the percentage of total cd77 + cells . Considering the fact that cd77 molecules are located next to na / k - atpase in caveoli of kidney epithelial cells (27), our results show the possible role of cd77 in mechanisms responsible for development of hyperbaric diuresis . In adult kidneys, antibodies against cd34 label almost all endothelial cells (28). Podocyte luminal membrane domain contains other sialomucins of the cd34 family: podocalyxin and endoglin whose function is still poorly understood . Acevedo et al reported an increased expression of cd34 on renal glomerular cells of older diabetic animals which reflect involvement of cd34 in the pathogenesis of glomerular alterations related to age and diabetes (29). In addition, putative progenitor cell mobilization is higher with 2.5 versus 2.0 ata of oxygen treatments, and all newly mobilized cells exhibit higher concentrations of an array of regulatory proteins (30). Endothelial progenitor cells and circulating angiogenic cells contribute to endothelial repair, either by integrating in injured endothelium or by secreting angiogenic growth factors (31). The present study, in contrary, showed a clear decrease in percentage of cd34 + renal cells after repetitive hyperbaric treatment . Hyperoxia - induced production of ros, reduction in the bioavailability of nitric oxide (no) and direct mechanical damage to the endothelium during decompression are considered to play an important role to endothelial dysfunction (1). It is speculative that ros induced apoptosis of renal cd34 + is plausible mechanism for the observed decrease . In addition, it has been reported in rats that a decompression trauma acutely increased levels of interleukin-6 (32) and therefore we can speculate that the release of pro - inflammatory cytokines in response to hyperbaric treatment may account for apoptosis in endothelial renal cells . The percentage of total cd34 + cells in lung tissue was also increased and we can assume that hyperbaric conditions induce pulmonary endothelial angiogenesis . Few recent studies showed how blood - derived cd34 + endothelial progenitor cells contribute to pulmonary angiogenesis . It has been concluded that circulating cd34 + endothelial progenitor cells, characterized by active cell division and an amplified transcriptional signature, transit into resident endothelial cells during compensatory lung growth . The authors discuss how therapeutic manipulation of these cells may be beneficial in a variety of lung diseases (33). In this study, we used immersion depth of 65 meters, and assume that changing the hyperbaric conditions and changing depths would change results of measured antigens . Exposure to special environment conditions may induce systemic physiological changes that impact on thermal homeostasis . We analysed expression of cd34 and cd77 on all cardiac cells, which includes cardiomyocytes as well . Different cell - types have been used recently, including bone marrow - derived mononuclear cells and mobilized cd34 + cells, in studies that suggested a potential of cell - based therapies to reduce cardiac scar size and to improve cardiac function in patients with ischemic cardiomyopathy . Recently, in experimental studies direct in vivo reprogramming of cardiac fibroblasts towards cardiomyocytes that are cd34 + has been reported, which may represent novel therapeutic approach for cardiac regeneration (35). Both myocardial ischemia and peripheral ischemia are known to stimulate endogenous cd34 + cell mobilization and upon mobilization, these cells tend to target zones of ischemia where they are thought to promote angiogenesis either through their direct incorporation into newly developing blood vessels or through their secretion of angiogenic growth factors that stimulate local peri - endothelial vascular development (36, 37). Use of cd34 + cells for the treatment of ischemic cardiovascular disease is relatively novel . Few research groups have shown that a single maximal exercise bout elicits an increase in the numbers of circulating endothelial progenitor cells in both healthy subjects and in cardiovascular patients (38, 39). The high vascular oxidative load of a maximal exercise bout causes a temporary decrease in endothelium - dependent vasodilatation, which is followed by a substantial improvement 12 - 24 hrs later . Such an acute period of vascular stress appears to stimulate repair mechanisms, including the mobilization of endothelial progenitor cells, which could be considered as an adequate physiological response . This study is the first reporting the hyperbaric environment effect on cd34 + cardiac cells in rats . We found out a significantly increase in percentage of cd34 + cardiac cells in rats after hyperbaric treatment comparing to non - treated rats which is in accordance to previously reported repair mechanism of injured endothelium caused by hyperoxia . These findings are very interesting and open a broad range of explanations . In this study that would be cd77-cd34 + cells in kidney tissue, cd77-cd34 + cells in pulmonary tissue and cd77+cd34- cells in cardiac tissue . These cells are very interesting because they represent the cells with high ability to adjust extreme conditions and remain unchanged in hyperbaric conditions that we have used in this study . Cd77 is abundant in endothelial lipid rafts (40, 41) that are associated with transendothelial transport of nutrients and ions (42). It has been found that excessive endothelial cd77 gycosphin- golipid accumulation leads to k (ca) channel dysfunction (43). In study of rei - muini et al renal cd34+cd77- cells showed the most sensitivity to elevated calcium (8). That results are significant in the view of recent finding of vascular endothelial - cadherin cleavage caused by ca influx that contribute to the dissolution of adherent junctions during endothelial cell activation and apoptosis (44). We speculate that cd77-cd34 + cells in kidney tissue and cd77-cd34 + cells in pulmonary tissue, due to their poorer lipid raft content, succumb at higher extent to the dissolution of adherent junctions during endothelial cell activation and apoptosis provoked by ca influx . Results of this study recruit cd15s analyses for the majority investigations of leukocyte proinflammatory features and present cd15s+ leukocytes as intelligent cells critical for the regulation of the inflammatory process with ability to adjust to extreme conditions . Based on our findings, we also speculate that positive effects of hyperbaric oxygenation on renal dysfunction in sepsis caused by e. coli are mediated by the decreased percentage of cd77 + cells . Our result of increased percentage of cd34 + pulmonary cells after hyperbaric treatment support the hypothesis that endothelial progenitor cells play a very important role in lung growth in physiological and many pathophysiological conditions . For now, we can only speculate of beneficial effects of hyperbaric treatment in promoting heart angiogenesis as well as use of hyperbaric conditions as possible therapeutic method for ischemic cardiovascular diseases treatment.
|
Among 57 patients with spinal vascular malformations that were documented in a neurointerventional database that contained data that were prospectively collected since 1992, recent 16 patients underwent three - dimensional digital subtraction rotational angiography using the axiom artis dba system (siemens medical solutions, forchheim, germany). In these patients, there were 13 sdavfs and three spinal pial arteriovenus fistulas (spavfs). Sdavfs were the most common form of spinal vascular disease (56%) at our institute . Thirteen sdavf patients (age range: 34 - 77, mean: 57; m: f = 11:2) were out of 32 sdavf which were the most common form of spinal vascular disease (56%) at our institute . Lesions were found at the cervical (n = 1), thoracic (n = 9) and lumbosacral (n = 3) levels . Data collection and analysis for this prospective 3d angiography study of spinal avms were performed according to our local institutional review board guidelines, and written informed consent was obtained from all patients and their families . The rotational angle was 200 and the rotational speed of the c - arm was 45/second . Data were acquired in a 2480 1920 matrix using a 16-inch field - of - view flat panel detector . Two - hundred and seventy mgi / ml of nonionic contrast medium (visipaque, ge healthcare, chalfont, st . Giles, united kingdom) was injected into segmental arteries (flow rate: 1 ml / s; total volume: 5 ml), and the injection started 1 second before the rotational run to achieve complete filling of the selected artery during angiography . Both mask data and contrast data were transferred to a workstation (syngo x - workplace, siemens ag, healthcare sector, erlangen, germany) and reconstructed into 3d images using a maximal intensity projection reconstruction algorithm . Reconstructed images, including maximum intensity projection images, shaded surface - rendered displays, and volume - rendered displays with adjustable transparencies for various structures, in addition to full stereoscopic capabilities, were used to study the anatomic relationships shown in these images . To assess the additional information obtained by 3dra, two neuroradiologists (h.j.b ., d.c.s .) Evaluated pretherapeutic 3dra reconstructed images compared to 2d angiograms with respect to three categories of parameters graded by four scores (1 = poor, 2 = fair, 3 = good, 4 = excellent). The three categories were: (1) the exact anatomic location of the fistula; (2) the precise angioarchitecture, including the number and course of feeding arteries and draining veins; and (3) the contribution of 3dra to additional interventions that facilitated treatment, modified therapeutic strategies or improved safety . Although most sdavfs consisted of a fistula at a single segmental artery level, multiple channels of feeders at each level of the fistula were sometimes observed . The number and course of feeders and draining radicular veins were evaluated because the angioarchitecture, including the course of feeders while traversing the neural foramen, is critical for embolisation and surgical resection . Although radicular branches usually go upward along the radicular root below the pedicle, the feeder arising from the radicular artery or arising separately from the radicular artery took diverse courses in sdavfs . Therefore, directions were categorised as upward, horizontal or downward and demarcated on the dual volume window, which shows vessels and bone (fig the most appropriate image was chosen by controlling the image window and the contrast on the workstation, and 3d images were reviewed at different angles . The most suitable views were chosen to reveal the two most complex areas of the feeder angioarchitecture: (1) the level where the segmental artery branches into intercostal, radicular and dorsal muscular arteries, and (2) the level where the radicular artery makes a sharp medial turn into the intervertebral foramen . Inter - observer agreement on the angiographic findings was assessed, and analyses of the number of feeders and the course of feeders were performed by two neuroradiologists (h.j.b ., d.c.s . ). Data were analysed using cohen's kappa statistics, and calculations were performed with medcalc (medcalc software version 9.0, mariakerke, belgium). The rotational angle was 200 and the rotational speed of the c - arm was 45/second . Data were acquired in a 2480 1920 matrix using a 16-inch field - of - view flat panel detector . Two - hundred and seventy mgi / ml of nonionic contrast medium (visipaque, ge healthcare, chalfont, st . Giles, united kingdom) was injected into segmental arteries (flow rate: 1 ml / s; total volume: 5 ml), and the injection started 1 second before the rotational run to achieve complete filling of the selected artery during angiography . Both mask data and contrast data were transferred to a workstation (syngo x - workplace, siemens ag, healthcare sector, erlangen, germany) and reconstructed into 3d images using a maximal intensity projection reconstruction algorithm . Reconstructed images, including maximum intensity projection images, shaded surface - rendered displays, and volume - rendered displays with adjustable transparencies for various structures, in addition to full stereoscopic capabilities, were used to study the anatomic relationships shown in these images ., d.c.s .) Evaluated pretherapeutic 3dra reconstructed images compared to 2d angiograms with respect to three categories of parameters graded by four scores (1 = poor, 2 = fair, 3 = good, 4 = excellent). The three categories were: (1) the exact anatomic location of the fistula; (2) the precise angioarchitecture, including the number and course of feeding arteries and draining veins; and (3) the contribution of 3dra to additional interventions that facilitated treatment, modified therapeutic strategies or improved safety . Although most sdavfs consisted of a fistula at a single segmental artery level, multiple channels of feeders at each level of the fistula were sometimes observed . The number and course of feeders and draining radicular veins were evaluated because the angioarchitecture, including the course of feeders while traversing the neural foramen, is critical for embolisation and surgical resection . Although radicular branches usually go upward along the radicular root below the pedicle, the feeder arising from the radicular artery or arising separately from the radicular artery took diverse courses in sdavfs . Therefore, directions were categorised as upward, horizontal or downward and demarcated on the dual volume window, which shows vessels and bone (fig the most appropriate image was chosen by controlling the image window and the contrast on the workstation, and 3d images were reviewed at different angles . The most suitable views were chosen to reveal the two most complex areas of the feeder angioarchitecture: (1) the level where the segmental artery branches into intercostal, radicular and dorsal muscular arteries, and (2) the level where the radicular artery makes a sharp medial turn into the intervertebral foramen . Inter - observer agreement on the angiographic findings was assessed, and analyses of the number of feeders and the course of feeders were performed by two neuroradiologists (h.j.b ., d.c.s . ). Data were analysed using cohen's kappa statistics, and calculations were performed with medcalc (medcalc software version 9.0, mariakerke, belgium). The angioarchitectures of 13 sdavf patients are summarized in table 1 . Although the radicular arterial feeder and fistula were expected to have an oblique ascending course, the feeder courses at the neural foramen were diverse: horizontal (n = 5), oblique upward (n = 4), and oblique downward (n = 4) (fig . Such diverse courses of the feeder from the radicular artery were characterised by a variable length of the transdural portion of the fistula before draining into the radicular vein, which depended on the anatomical position of the neural foramen . The number of feeders at the lesion level was one (n = 6), two (n = 5) (figs . 1, 2), or plexiform (n = 2). The dual mode (showing bone and vessels) of 3d images clearly demonstrated such feeder courses relative to the vertebral pedicle (fig . The courses of veins at the level of the fistula showed ascending (n = 10), descending (n = 1) or bidirectional (n = 2) patterns . Draining veins with an ascending pattern regurgitated to perimedullary veins at the level of the spinal cord bidirectionally (n = 6), only downward (n = 3) or upward (n = 1). Both readers gave high scores (mean 3.9), reflecting the overall agreement with excellent presence of additional information obtained by 3dra . Inter - observer evaluations showed good agreement for the number of feeders (= 0.769), the course of feeders (= 0.893) and the course of veins (= 0.809). Two useful views were used to follow the proximal branching pattern of each segmental artery and the distal branching pattern of the radicular artery entering the transverse foramen . The first one (spider view) isolated the feeder by showing the branching pattern of the segmental artery (fig . 1). The angles were the right anterior oblique (rao) view of the feeder's right side and the left anterior oblique (lao) view of the feeder's left side . The second one (tunnel view) showed the feeder course at the neural foramen and the location (ventral vs. dorsal) including sharp medial turn of the feeder . Spider and tunnel views provided better working angles on 3d workstations, especially during embolisation . The best working angle was obtained at rao / lao 30 and caudal 20. this angle was selected through the synchronised rotation of the 3d image on the console during embolisation, and it depended on which vessel segment was analysed and how much the microcatheter advanced . The dual mode showing bone and vessels revealed the locations of feeders and their courses . The angioarchitectures of 13 sdavf patients are summarized in table 1 . Although the radicular arterial feeder and fistula were expected to have an oblique ascending course, the feeder courses at the neural foramen were diverse: horizontal (n = 5), oblique upward (n = 4), and oblique downward (n = 4) (fig . Such diverse courses of the feeder from the radicular artery were characterised by a variable length of the transdural portion of the fistula before draining into the radicular vein, which depended on the anatomical position of the neural foramen . The number of feeders at the lesion level was one (n = 6), two (n = 5) (figs . 1, 2), or plexiform (n = 2). The dual mode (showing bone and vessels) of 3d images clearly demonstrated such feeder courses relative to the vertebral pedicle (fig . The courses of veins at the level of the fistula showed ascending (n = 10), descending (n = 1) or bidirectional (n = 2) patterns . Draining veins with an ascending pattern regurgitated to perimedullary veins at the level of the spinal cord bidirectionally (n = 6), only downward (n = 3) or upward (n = 1). Both readers gave high scores (mean 3.9), reflecting the overall agreement with excellent presence of additional information obtained by 3dra . Inter - observer evaluations showed good agreement for the number of feeders (= 0.769), the course of feeders (= 0.893) and the course of veins (= 0.809). Two useful views were used to follow the proximal branching pattern of each segmental artery and the distal branching pattern of the radicular artery entering the transverse foramen . The first one (spider view) isolated the feeder by showing the branching pattern of the segmental artery (fig . 1). The angles were the right anterior oblique (rao) view of the feeder's right side and the left anterior oblique (lao) view of the feeder's left side . The second one (tunnel view) showed the feeder course at the neural foramen and the location (ventral vs. dorsal) including sharp medial turn of the feeder . Spider and tunnel views provided better working angles on 3d workstations, especially during embolisation . The best working angle was obtained at rao / lao 30 and caudal 20. this angle was selected through the synchronised rotation of the 3d image on the console during embolisation, and it depended on which vessel segment was analysed and how much the microcatheter advanced . The dual mode showing bone and vessels revealed the locations of feeders and their courses . Our study provides additional evidence that spinal 3dra is useful for evaluations of the angioarchitecture of sdavfs including the course of feeders . We also recommend useful and routine angled (spider and tunnel) views for 3d angiographic analysis . The spider view (contralateral - oblique - angled caudal view) and the tunnel view (bottom - up view) are useful for distinguishing the proximal branching vessels of the intercostal artery, the dorsal muscular arteries, and the fistula from the radicular artery with its sharp medial turn from the segmental artery . The dual mode of spinal 3dra reveals the relationship between the fistula and bony landmarks, so these images provide a good roadmap for feeder selection during embolisation as well as for surgical guidance without obtaining further conbeam ct angiography . 3dra with the routine views (spider and tunnel views) proposed in our study allows for the easy selection of working angles and separates the complex and overlapping vascular anatomy of feeders . The radicular artery, which branches from the segmental artery, is known to be associated with the main feeder in sdavfs, and it usually has an obliquely ascending course because the fistula is located in the dura adjacent to the nerve root . However, only one - third of our patients had feeders with an upward oblique course, and the majority of patients had feeders with a transverse or downward course that possibly traveled along the dura between two adjacent nerve roots (intersegmental). This discrepancy may originate from the different anatomical positions of the radicular vein and artery; in contrast to the radicular artery, the radicular vein does not follow the segmental rules . The radicular vein pierces the dura and nerve roots in only 60% of cases; in the remaining 40%, there is a separate foramen in the dura, between the two spinal nerves . Such orientations shown in the dual mode, which reveals bone and vessels, are helpful for embolisation as well as surgical planning . However, the exact angioarchitecture of sdavfs at the lesion level has not been well described . The feeders even formed plexiform channels in which each feeder was not separately identified in two patients . Multiple feeders result in dual arterial supply to sdavfs, which makes it imperative at the time of treatment to reach the venous outlet of the shunt, because otherwise the shunt will continue to be fed by the untreated arterial pedicle . On the other hand, reciprocal communication in each feeder can cause glue injection at the wedged position of one feeder to regurgitate in a retrograde fashion, while making the glue penetrate the fistula and the proximal radicular vein . If there is any intersegmental anastomosis connecting to the anterior spinal artery in the adjacent level, such proximal regurgitation can lead to serious complications caused by glue inadvertently entering the anterior spinal artery . Therefore, 3dra can be helpful for evaluating the anatomy of multiple feeders and provide a good roadmap for further superselection and treatment of the fistula (fig . First, the number of patients is small because sdavfs are a relatively rare occurrence . Second, obtaining 3dra requires complex and high - quality angiographic equipment with three - dimensional and rotational capabilities . Third, the selection of the segmental artery from the aorta and the stable engagement of the catheter to perform 3dra are not always easy because of the relatively acute angle of the segmental artery run - off from the aorta . Differently shaped catheters might be required for different levels of the segmental artery . In conclusion, 3dra provided additional information for evaluating the angioarchithecture of sdavfs including the number and course of feeders, and for choosing appropriate working angles to select feeders and achieve successful embolisation or surgical treatment.
|
Bilateral shoulder dislocation are most commonly posterior type . These are most commonly due to seizure disorder and electrocution . To best of our knowledge there are only few cases of similar kind are reported in literature . We hereby report a interesting case of posttraumatic, bilateral anterior dislocation of shoulder without associated fracture in a 45 old women without any predisposing pathoanatomy . A 45-year - old women presented to casualty with sudden onset of pain and restriction of movement in both shoulders fallowing trauma . Immediately post trauma she had severe pain and restriction of both shoulders . On examination arms were abducted and externally rotated . There was loss of round contour of shoulder with increased vertical diameter of axilla anteriorly . Bilateral traumatic anterior shoulder dislocations are rare and are seen as a result of unique mechanism of injury . In our case patient bilateral shoulder dislocations are usually posterior type and are almost pathognomonic of seizure disorder or electrocution . Though anterior dislocation of shoulder is commonest bilateral simultaneous dislocation is very rare[1 - 7]. We hereby report a case of posttraumatic, bilateral anterior dislocation of shoulder without associated fracture in a 45 old women . A 45-year - old women presented to lok nayak hospital, new delhi, india in august 2010 with sudden onset of pain and restriction of movement in both shoulders fallowing trauma . She had no history of seizure, epilepsy, previous shoulder dislocation or instability in other joints . On examination arms . There was loss of round contour of shoulder with increased vertical diameter of axilla anteriorly . Radiological examination revealed bilateral anterior dislocation of the shoulders without any associated fractures(figure 2). Radiograph showing bilateral shoulder dislocation concentric reduction of bilateral shoulder joint achieved closed reduction done by milch technique after intraraticular lignocaine injection . Majority of the bilateral shoulder dislocations are of posterior type most commonly seen during convulsion, electric shock or hypoglycaemic seizures . Posterior type is common in these conditions due to violent contractions of the muscles of the shoulder girdle [8 - 10]. Unlike bilateral occurrence of anterior shoulder dislocation is rare because of the fact that one extremity takes the brunt of the impact . To best of our knowledge only three cases of bilateral anterior dislocations are reported in literature . In two of the three cases reported were sequential, one sided followed by contra lateral side dislocation . In our case impact is same on both shoulders at the same time . The mechanism of anterior dislocation is forced extension, abduction and external rotation of the arm . In our case mechanism of injury mechanism of injury, systemic disease and associated fractures in various similar cases is depicted in table 1 . Croswell and smith reported a case of bilateral anterior dislocation of the shoulder without any fractures in a bench - pressing athlete . In an unusual mechanism of injury weight on the bar forced his arms into hyperextension in the mid - abducted position . The humeral shaft gradually pivoted on the bench and the humeral heads were slowly dislocated interiorly by the weight of the bar . Sandeep s and sudhir k reported a case of sequential bilateral anterior dislocation in which the left shoulder dislocated first due to trauma followed by atraumatic dislocation of the right shoulder . Sreesobh k v et al reported a case where atraumatic right shoulder dislocation was followed by traumatic dislocation of the left . This type of dislocation involves a unique type of mechanism injury and in our case it was fall on pointed elbow causing forced extension . Traumatic bilateral anterior dislocations without any pathologic lesion are very rare with only few cases reported in literature.
|
Dna lesions can be mutagenic and have been implicated in a variety diseases, most notably cancer, as well as aging . Quantifying nucleic acid damage is a valuable exercise as modified nucleosides, nucleobases, and sugar fragments are potential biomarkers . 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodguo) is a well - studied, mutagenic lesion that is used as a biomarker . This lesion is produced from the most readily oxidized of native nucleotides, dg, by a variety of damaging agents . 8-oxodguo is even more readily oxidized than dg and serves as the precursor to a number of highly mutagenic lesions that are of increasing interest to chemical toxicologists and other scientists . Herein, we describe a method for detecting 8-oxodguo that relies upon its tagging by a reagent and subsequent signal detection using quantitative pcr (qpcr). Mass spectrometry is a sensitive and selective method for detecting a large number of dna lesions . The variety of lesions detectable is expanded when the method is coupled with chemical derivatization techniques . Quantification is greatly facilitated by spiking samples with known quantities of isotopically labeled lesions that require costly independent synthesis . In addition, mass spectrometers are increasingly powerful, and dna lesion detection methods employing them are proportionally more sophisticated, but the instruments are also expensive . The comet assay is a less costly method that is particularly useful for detecting lesions in cellular dna, but its specificity is limited by the selectivity of chemical reagents and enzymes that cleave dna . Sophisticated methods that take advantage of selective dna lesion tagging and enable the detection of the location of individual lesions in single strands of dna are on the horizon . Reagents that also take advantage of distinctive lesion reactivity but utilize more conventional reporting methods are more common . Aldehyde reactive probe(s) equipped with fluorophores or biotin have proven useful for quantifying abasic sites and are still being developed . Turn on sensors that exploit the formation of a fluorophore upon reaction with a functional group within a lesion have also been developed . Recently, molecules that selectively recognize 8-oxodguo and incorporate fluorescence reporting have been developed . There are also efforts reported that utilize aptamers to detect this damaged nucleoside or its respective free base (8-oxogua). It is well known that mild oxidation of 8-oxodguo produces the guanidinohydantoin (gh) and spiroiminohydantoin (sp) lesions that are interesting in their own right . However, oxidation of 8-oxodguo or fapydg in the presence of an amine nucleophile, such as a spermine (rnh2), provides a dna adduct (scheme 1) in competition with gh and sp . We previously reported a method for selectively detecting 8-oxodguo and the mechanistically related fapydg lesion by utilizing this chemistry, first reported by burrows, and 1 as a nucleophile to trap the reactive oxidized species . Fapydg and 8-oxodguo were distinguished from one another by using an oxidant, k3fe(cn)6, which oxidizes the latter but not the former lesion . Following the adsorption of tagged dna to a surface, the biotinylated material is used to capture a streptavidin horseradish peroxidase complex, which yields the signal by oxidizing a profluorescent molecule to a fluorescent one . The fluorescence intensity is proportional to the amount of horseradish peroxidase bound to the surface, which is dependent upon the amount of biotinylated spermine (1) that is covalently bound to the dna . This method was time - consuming due to a lengthy procedure for preparing the plates on which the lesions were quantified . Herein, we have improved the limit of detection to 14 amol and decreased the time required to complete the analysis to 3 h by combining qpcr with a new biotinylated reagent . Taq dna polymerase, onetaq hotstart dna polymerase, and exonuclease i (exo i) were from new england biolabs . Oligonucleotides were synthesized via standard automated dna synthesis on an applied biosystems model 394 instrument . Dna synthesis reagents, including the phosphoramidite for incorporating 8-oxodguo were obtained from glen research . Radiolabeled oligonucleotides were hybridized with 1.5 equiv of complementary oligonucleotides in 10 mm potassium phosphate (ph 7.2) and 100 mm nacl at 90 c for 5 min and cooled to room temperature . Solid - phase peptide synthesis was carried out on 10 obtained from cs bio (0.45 mmol / g). Automated solid - phase peptide synthesis (spps) was performed on a symphony peptide synthesizer . The plasmid containing the p53 gene was obtained as previously described . A solution (500 l) of primers (0.2 m each), template dna (20 fm), dntps (0.4 mm), and onetaq hotstart dna polymerase (2.5 10 u / l) in 1 onetaq standard buffer was partitioned into thin - walled pcr tubes (50 l each). The pcr cycle was performed as follows: (1) 94 c for 30 s, (2) 94 c for 30 s, (3) 60 c for 30 s, (4) 68 c for 1.5 min, (5) steps 24 repeated 45 times, (6) 68 c for 5 min, and (7) held for 4 c . The reactions were pooled, incubated with exo i (4 10 u / l, 1 h, 37 c), and evaporated under vacuum . The dna was purified from the residue by silica spin column chromatography (qiagen pcr purification kit) following the manufacturer s protocol, except that an additional wash with 35% guanidinehcl was performed following adsorption to the spin column . Purity and product length were confirmed on 1% agarose gel/1 tbe precasted with etbr (0.5 mg / l). Typical yields were 20200 nm in 200 l solution, corresponding to 10- to 10-fold amplification . The p53 pcr fragment containing 8-oxodguo was prepared using the forward primer 5-d(gca gtc aga tcc tag cxt cga gc) where x = 8-oxodguo and the reverse primer 5-d(ggg cag tgc tcg ctt agt gc). The p53 pcr fragment without 8-oxodguo was prepared using the same reverse primer and 5-d(gca gtc aga tcc tag cgt cga gc) as the forward primer . The puc19 pcr fragment was prepared using the forward primer 5-d(bggt gat gac ggt gaa aac ctc), where b = biotin linked to the remainder of the primer via tetraethylene glycol . A chilled acetonitrile solution (81 ml) of fmoc - nhs ester (18.1 g, 53.7 mmol) was added to a vigorously stirred solution of carboxylated spermine (5 g, 12.8 mmol) in 10% na2co3 (81 ml) in an ice bath . Acetonitrile was removed under vacuum, and the residue was acidified with a solution of brine and 0.1 m hcl (ph 1, 200 ml). The aqueous solution was extracted with dcm (5 200 ml), and the organic layer was dried over na2so4 and filtered . The resulting dry powder was applied over a silica gel plug and washed with copious amounts of 1:1 hex / ea . Removal of the nonpolar species was monitored by tlc . After removing the nonpolar species, 1% formic acid was added to the eluent to elute product - containing fractions . The eluent was concentrated under vacuum and applied to a silica gel column to further purify the product using 1% formic acid in 1:1 hex / ea . Fractions containing the product were pooled, concentrated under vacuum, and redissolved in ea for a basic wash (5, saturated nahco3/brine), followed by an acidic wash (2, brine 0.1 m hcl, ph 1). The organic layer was concentrated to a sticky white foam (5.4 g, 37% yield) and redissolved in minimal dcm for precipitation into vigorously stirring hexane . The supernatant was removed following centrifugation and the residue lyophilized to a white powder . 2.603.30 (br, m, 9h), 4.13 (br, m, 4h), 4.38 (br, m, 5h), 4.56 (br, s, 4h), 7.247.74 (m, 32h). C nmr (cdcl3): 11.4, 14.2, 18.8, 20.7, 22.6, 25.3, 29.1, 31.6, 34.5, 34.7, 36.1, 47.1, 47.3, 47.36, 47.44, 77.2, 119.9, 120.0, 124.5, 124.6, 124.7, 124.9, 125.1, 127.0, 127.1, 127.7, 127.8, 141.31, 141.34, 141.37, 141.39, 143.8, 143.9, 156.5, 156.6 . Ir (film): 3292 (br), 2927, 1703, 1644, 1553 cm . Hrms calcd for c71h67n4o10 (m + h) 1135.4852; found, 1135.4887 . A thf solution of dmtrcl (1.4 g, 4 mmol, 0.2 m) was added dropwise to a vigorously stirred solution of diamine 4 (48 mmol, 10.6 ml) in thf (20 ml). Additional dmtrcl / thf solution (6.7 g, 20 mmol, 55 ml thf) was added in 5 ml portions for a total of 30 ml . The appropriate amount of dmtrcl added was followed by tlc (30% ea / hex) to maximize monotritylation and minimize ditritylation . Volatiles were removed in vacuo, and the residue diluted with dcm (200 ml), washed with brine (3 200 ml), dried with na2so4, filtered, and concentrated for column chromatography (isocratic elution: 5% tea / dcm), affording 7.7 g (91% yield) of 5 as a clear yellow oil . H nmr (cdcl3): 1.701.80 (m, 7h), 2.2 (t, j = 8 hz, 2h), 2.762.79 (t, j = 8 hz, 2h), 3.493.59 (m, 12h), 3.75 s, 6h), 6.766.79 (m, 4h), 7.13 (m, 1h), 7.217.26 (m, 2h), 7.337.35 (m, 4h), 7.427.45 (m, 2h). C nmr (cdcl3): 30.8, 33.3, 39.8, 41.3, 55.4, 69.7, 70.1, 70.3, 70.37, 70.44, 70.8, 70.9, 113.2, 126.2, 127.9, 128.7, 129.9, 139.0, 147.1, 157.9 . Hrms calcd for c31h42n2o5na (m + na) 545.2986; found, 545.3007 . A mixture of biotin (770 mg, 3.2 mmol), dcc (710 mg, 3.4 mmol), and hobt (465 mg, 3.4 mmol) was dried in vacuo for 30 min prior to suspension in 5% tea / dmf (10 ml) under ar at 40 c . After 30 min, the suspension was added to 5 (1.53 g, 2.94 mmol) (predried by azeotropically drying from pyridine) in 5% tea / dmf (10 ml). The precipitate was again removed by passage through celite, and the filtrate was concentrated for column chromatography (5% tea / dcm to 5% tea/5% meoh / dcm), affording 2.2 g (100%) of 6 . H nmr (cdcl3): 1.201.23 (m, 2h), 1.421.76 (m, 8h), 2.162.19 (m, 4h), 2.802.84 (m, 3h), 3.103.15 (m, 1h), 3.323.34 (m, 1h), 3.523.61 (m, 12h), 3.77 (s, 6h), 4.264.29 (dd, 1h), 4.434.50 (dd, 1h), 5.02 (br, s, 1h), 5.72 (br, s, 1 h), 6.49 (bd s, 1h), 6.77 (d, 4h), 7.157.44 (m, 9h). C nmr (cdcl3): 24.9, 25.58, 25.63, 28.1, 28.2, 28.8, 33.2, 33.9, 35.9, 37.6, 39.6, 40.5, 55.6, 60.1, 61.8, 69.5, 69.9, 70.0, 70.1, 70.5, 76.7, 77.0, 77.2, 77.3, 163.8, 173.1 . Hrms calcd for c41h57n4o7s (m + h) 749.3942; found, 749.3968 . A methanolic solution (10 ml) of 6 (1.3 g, 1.8 mmol) was detritylated within minutes following the addition of 1 m hcl / meoh (20 ml). The reaction was diluted with water (100 ml), washed with dcm (3 100 ml), and the ph adjusted to 4 with 4 m naoh . Dcm washes were repeated, the ph adjusted to 12, and the solvent evaporated to dryness . The product was extracted from the residue triturating with dcm (5), followed by filtration over a glass frit . The filtrate was concentrated to provide 7 as a pale yellow amorphous solid (480 mg, 60% yield). H nmr (cdcl3): 1.421.46 (m, 3h), 1.631.78 (m, 13h), 2.172.20 (t, j = 6 hz, 2h), 2.712.80 (m, 4h), 2.882.93 (dd, j = 6.7, 2 hz, 1h), 3.143.15 (m, 1h), 3.323.36 (q, j = 5.3 hz, 3h), 3.533.65 (m, 14h), 4.31 (m, 1h), 4.48 (m, 1h), 5.21 (s, 1h), 6.09 (s, 1h), 6.79 (m, 1h). Hrms calcd for c20h39n4o5s (m + h) 447.2636; found, 447.2651 . A solution of 7 (482 mg, 1.1 mmol), edci (345 mg, 2.2 mmol), and hobt (300 mg, 2.2 mmol) in dmf (5 ml) under ar was added dropwise to 3 (1.7 g, 1.5 mmol) in dmf (10 ml). After 16 h, the reaction was quenched with a solution of brine and 0.1 m hcl (100 ml) and extracted with dcm (5 100 ml). The organic layer was dried with na2so4, filtered, and concentrated for column chromatography . (the column was packed with 1% hco2h / ea and then eluted with ea to 10% meoh / dcm .) Depending on the purity of the product, an optional second column chromatography was performed (chcl3 to 5% meoh / chcl3). Fractions of pure product were identified by esi, pooled, and concentrated to a white foam (1 g, 55% yield) that was redissolved in dcm and precipitated by dripping the solution into hexanes . The solution was centrifuged, the solvent decanted, and the residue lyophilized over 1 week into a white powder . Some fmoc cleavage was detected in the powder and confirmed by esi / ms (1363.8 m / z = [m - fmoc + na] 1363.6). Hrms calcd for c91h103n8o14nas (m + na) 1563.7309; found, 1563.7302; confirmed the desired product . Fmoc - cleavage of the protected form of 2 (200 mg, 128 mol) was carried out in 50% cyclohexylamine / dcm (15 ml). After 15 min, the reaction was diluted with dcm (20 ml) and water (100 ml). After increasing the ph of the aqueous layer to 12 with naoh (4 m), the aqueous layer was filtered through a 0.2 m syringe filter, concentrated under vacuum, and azeotropically dried with toluene (3 50100 ml), to afford 2 as a pale yellow, amorphous solid that was difficult to completely dry (264 mg,> 100% recovery). H nmr (d2o): 1.33 (br, m, 6h), 1.451.56 (m, 13h), 2.172.19 (t, j = 4 hz, 2h), 2.372.54 (m, 10h), 2.682.71 (d, j = 12 hz, 1h), 2.892.93 (dd, j = 4, 8 hz, 1h), 3.033.07 (t, j = 6 hz, 1h), 3.163.19 (t, j = 6 hz, 4h), 3.203.26 (m, 1h), 3.49 (t, j = 6 hz, 4h), 3.583.61 (m, 8h), 4.324.35 (dd, j = 6, 2 hz, 1h), 4.514.54 (dd, j = 4, 2 hz, 1h). C nmr (d2o): 22.4, 22.6, 25.2, 25.4, 25.7, 25.8, 27.8, 29.1, 29.2, 33.0, 33.6, 36.1, 36.1, 37.2, 42.0, 43.5, 45.68, 52.9, 57.67, 59.2, 59.5, 65.8, 65.9, 66.8, 66.9, 67.01, 67.02, 162.7, 165.9, 173.8, 173.9 . Hrms calcd for c31h62n8o6nas (m + na) 697.4405; found, 697.4429 . For the preparation of 1113, the arginine(s) and lysine residues were added via automated spps from 10 (150 mg, 0.45 mmol / g, 70 mol - scale). All residues were coupled (2 30 min) with amino acid (0.2 m, 5 equiv), hbtu (0.2 m, 5 equiv), and dipea (0.4 m, 10 equiv) in nmp . The remaining portions of the syntheses of 1113 and all of 9 were carried out manually as follows . Alloc group cleavage was performed with me2nhbh3 (6 eq 24 mg, 0.4 mmol) and (ph3p)4pd(0) (0.05 equiv, 4 mg, 3.4 mol, 15 min, 3), where the former was added first to a dmf suspension of resin with ar bubbling . Following washing as described below, fmoc - protected aminocaproic acid (3 equiv) was then preactivated with pybop (3 equiv) and tea (6 equiv) in dmf (0.1 m) for 1 h with ar bubbling . The fmoc group was removed by treating the preswollen resin with 20% piperidine / dmf (5 min, 3). Unreacted amine was acetylated by treatment with 50% ac2o / dcm 3 times for 3, 3, and 7 min, with dcm washing in between each treatment . After each coupling, the resin was washed sequentially with dmf, dcm, meoh, dry dcm, and dry dmf (2). The lysine residue in 9 was coupled as described above for aminocaproic acid prior to the removal of the alloc group . The amino group was quantified indirectly by the released fulvene chromophore (300 7.8 10 mcm). Peptide cleavage / deprotection was performed with a cleavage cocktail (88% tfa, 2% tips, 5% h2o, and 5% phenol). Hplc purification was performed on a c18 semipreparatory column (waters 300 7.8 mm i.d .) Using h2o (solvent a) and acetonitrile (solvent b) with 0.1% tfa in an elution gradient optimized for each probe at 3 ml / min . Probe - containing fractions were lyophilized, redissolved in water and titrated to ph 9 with 4 m naoh, and analyzed by ms in the positive mode . The following gradients (time,% b) esi - ms [m + h]: calcd, 501.2; obsd, 501.2 . 11: 0, 0; 5, 0; 10, 20; 20, 20 . Esi - ms [m + h]: calcd, 770.1; obsd, 770.5 . Esi - ms [m + h]: calcd, 926.3; obsd, 926.6 . Esi - ms [m + h]: calcd, 1082.5; obsd, 1082.8 . Manual spps from 10 (0.3 g, 135 mol) was identical to that described above . Detritylation of the cysteine thiol was carried out with 2% tfa / dcm (12 ml, 12 min) and repeated until the cleavage solution ran clear (10). The cysteine thiol was converted into the disulfide by treating with cystamine2 hcl (5 equiv, 0.51.0 h, 3) and dipea (15 equiv) in dmso (5 ml). The resin was washed with dmf, meoh, dcm, and dmf following each cycle . The penultimate compound was cleaved, purified, and characterized as described above . Instead of evaporating the cleavage cocktail, the precipitate was lyophilized, redissolved in water, and filtered prior to hplc purification . The following gradient (time,% b) was used at 4 ml / min: 0, 0; 5, 0; 90, 16 . Ret . Maldi - tof - ms [m + h]: calcd, 1414.8; obsd, 1416.1 . A solution (5 l) of dna analyte (1 fmol), bpuc19 dna reference (0.1 fmol), probe (0.2 mm), ctdna (4 mg / l), and trishcl (20 mm, ph 9) was added with k3fe(cn)6 (5 l, 2 mm) and allowed to stand for 10 min . The reaction was quenched with carrier dna and detergent (20 l of ctdna (50 mg / l), dtt (10 mm), and 0.05% tween-20). G / l in 0.05% tween-20) and pelleted on a magnetic pcr plate . The beads were washed with wash buffer (100 l, 8 with 10 mm dtt, 10 mm guanidinehcl, and 0.05% tween-20). Pei - beads were resuspended in fresh cleavage / elution buffer (50 l: 0.1 m dtt, 0.1 m trishcl, at ph 10, and 2 m nacl), incubated for 5 min, and pelleted magnetically for an additional 5 min . Magnetic streptavaidin beads (dynabead myone t1) were washed with 0.05% tween-20 (3). A suspension of the beads (25 l, 1 g / l) was then incubated with pei - bead eluent (25 l). After 30 min, the beads were magnetically pelleted and washed sequentially with 1, 0.5, and 0.25 ttbs (100 l 3 at each concentration; 1 ttbs, 0.05% tween 20, 40 mm trishcl at ph 9, and 1 m nacl). Beads were washed a final time with and resuspended in 50 l of 0.05% tween-20 . Each qpcr solution (50 l) contained 10% sample (5 l), 0.2 mm dntp, primers (0.2 m each, table 1), taqman probes (0.1 m each), neb onetaq hotstart buffer (1), and onetaq hotstart dna polymerase (2.5 10 each qpcr experiment was prepared with a single calibration series of analyte and reference dnas (0, 2.58.5 logcn), ideally from the same working solution as the experimental samples . The 96-well plate (biorad mlp 9601) was sealed with optical film (abi). Qpcr was performed on the icycler iq (filter set 4: fam-490 and texas red-575) using the following program: (1) 95 c for 15 s, (2) 66 c for 1 min with optical measurement, (3) repeated 12 50, and (4) held at 25 c . The following taqman probes were used: 5-d(fam - ttg atg ctg tcc ccg gac ga - bhq1) for p53 and 5-d(cal - fluor- ctg aga gtg cac cat atg cgg tgt g - bhq2) for the puc19 internal standard . The threshold position was manually set at the same position in the exponential phases of the two sets of amplification curves (one set for each fluorophore). Cq (cycle at which fluorescence from amplification exceeds the background fluorescence) data were recorded and processed using microsoft excel . Calibration curves (cq vs log cn; cn = copy number) were generated by linear regression for both reference and analyte dnas . The amount (log cn) of dna was calculated by interpolation from the calibration curves . The tagging yield (% yraw) was calculated by1 the normalized tagging yield (% ynorm) was obtained by2where% yrawmax was the% yraw with 100% p53og . The amount of 8-oxodguo detected was calculated by3or by4 dna solutions (10 l, 500 pm p53-g, and 50 pm bpuc19) in tes buffer (20 mm tris hcl, ph 7.5, 2 mm edta, 0.2 m nacl) in a 96-well titer plate were treated for 50 min at 37 c with an equal volume of oxidizing agents (fecl2, h2o2, and ascorbic acid) the oxidizing agents ranged in concentration from 50 m fecl2, 400 m h2o2, and 4 mm ascorbic acid to 1.5 m fecl2, 12.5 m h2o2, and 0.125 mm ascorbic acid and were changed in 2-fold increments . Two control reactions were carried out, one with p53-og and one with p53-g as analytes . The control reactions contained h2o (10 l) in place of the oxidizing reagents . The reactions were quenched with quencher q (10 l of 50 mg / l calf thymus dna and 30 mm l - methionine) and purified by silica spin column chromatography (qiagen qiaquik pcr purification kit), resulting in a final solution (50 l) of dna buffered in 10 mm tris hcl at ph 7.5 . 8-oxodguo was quantified from an aliquot of the dna sample (4 l) mixed with 14 (1 l, 0.5 mm), following the general procedure for tagging and qpcr described above . We previously developed a method for quantifying 8-oxodguo in dna that took advantage of its lower oxidation potential than native nucleotides and many dna lesions, in combination with nucleophilic trapping of a reactive oxidization product of the lesion . Multiple aspects of the previously reported method for detecting 8-oxodguo were modified in the current work . The horseradish peroxidase amplification was replaced with quantitative pcr, procedures for removing excess probe were examined, and new probes were synthesized . The overall procedure (scheme 2) involved tagging, removal of the excess probe, binding tagged dna to streptavidin - coated magnetic beads, removal of untagged dna (nonbiotinylated dna), and finally pcr amplification of the bead bound dna . The ideal probe will selectively form a covalent bond to 8-oxodguo in a large excess of dg and be readily removed from dna when it is noncovalently bound . We postulated that incomplete removal of noncovalently bound 1 contributed to the background signal and prevented us from reaching a lower limit of detection . Consequently, we sought to design probes that would provide good yields of tagged 8-oxodguo but be readily removed . The triethylene glycol group was expected to increase the probe s water solubility, facilitating its removal from dna . In addition, the longer linker separating the dna tagging and reporting domains could enhance probe function . Probe 2 was synthesized by coupling the biotinylated triethylene glycol (7, scheme 3) with the fmoc - protected spermine derivative (3). The carboxylated spermine derivative (3) was prepared via a recently improved procedure, whereas 7 was obtained from the commercially available diamine . Diamine 4 was dimethoxytritylated prior to biotinylation to facilitate purification and handling of the advanced intermediates due to the polar nature of biotin and the lack of a convenient way of visualizing the compounds upon thin layer chromatography analysis . The fmoc - protected probe was purified by column chromatography and deprotected with cyclohexylamine . Probe 2 was purified from this reaction by extracting it into water and washing away less polar entities via extractions . The functionality of 2 was generally established by analyzing its tagging of an independently synthesized oligonucleotide containing 8-oxodguo (5-p-8). The general tagging and washing procedure employed however, instead of using qpcr, tagging was quantified using liquid scintillation counting by measuring the amount of p in the washing solutions and on the bead . At ph 8.0, 2 (0.1 mm) yielded> 60% tagged 8-oxodguo (data not shown). While an amine is necessary to trap the oxidized 8-oxodguo, we considered the possibility that the polycationic probes (e.g., 1 and 2) bind too avidly to dna, resulting in higher backgrounds . Consequently, we tested the biotinylated intermediate (7, scheme 3) and synthesized 9, which has the same net + 1 charge as 7 . Probe 9 was synthesized by solid - phase peptide synthesis starting from wang resin that was preloaded with -fmoc--alloc lysine (10). The orthogonally protected resin enabled us to couple the very polar (and poorly soluble) biotin component last, just prior to peptide cleavage from the solid support . While the synthesis of 9 only required coupling boc - lysine prior to cleavage of the alloc group (pd(0)) and reaction with biotin, this strategy proved useful for synthesizing several other probes (see below). Although noncovalently bound 7 and 9 may be easier to remove from the dna, neither one efficiently tagged dna containing 8-oxodguo . Using 9 as a prototype, we synthesized a series of related probes (1113) containing between 1 and 3 arginine residues, followed by a lysine at the amino terminus . In addition, following pd(0) cleavage of the alloc group, the liberated -amino group was coupled to aminocaproic acid prior to conjugating biotin . This assembly method increased the distance between the tagging and capture (biotin) domains of the probes . Peptide 13, containing 3 arginine residues, possessed the same overall positive charge as 1 . The tagging abilities of the arginine probes (1113) were crudely evaluated with an independently synthesized oligonucleotide containing 5-p-8 and liquid scintillation counting, as described above for 2 . Although the tagging efficiency of 11 was 3-fold greater at the higher concentration used, a much smaller difference was observed with the more highly positively charged probes (12 and 13). The concentration of k3fe(cn)6 (1 and 10 mm) also had little effect on tagging yield . Overall, the probe containing the same overall charge as 1 (13) provided the highest tagging efficiency . Tagging efficiency of 1113 for p-8 as a function of probe and oxidant concentrations . Yields were determined using streptavidin - coated magnetic beads via liquid scintillation counting . Finally, to minimize the effect of probe bound noncovalently to target dna, we synthesized a peptide (14) in which the highly positively charged dna binding domain was separated from biotin (used to capture tagged dna) and the tagging domain by a cleavable disulfide linkage . We rationalized that while a positively charged dna binding domain might assist delivering the tagging agent, it would also hinder removing it after reaction . The cleavable disulfide facilitates removing biotin that is not covalently bound to the dna and reduces the background . Synthesis was carried out on the wang resin containing -fmoc--alloc lysine (10). A s - monomethoxytrityl protected cysteine, which served as the disulfide precursor was incorporated, followed by the aminocaproic acid spacer and the lysine employed as the tagging component . The biotin group was introduced as described above prior to revealing the cysteine thiol that was elaborated further to the disulfide . The thiol was condensed with cystamine by disulfide exchange, and the resulting primary amine was used to introduce the triarginine dna binding domain . All of the peptide probes were purified by reverse - phase hplc following cleavage from the solid phase support and characterized by mass spectrometry (see supporting information). Large excesses of probes relative to dna are used in the reactions to maximize the tagging, which involve trapping a reactive species . Excess probe considering that the probes are typically employed at 100 m, a large majority must be removed . Several techniques to minimize the excess probe after tagging were investigated, including size - exclusion spin column chromatography, silica - spin column chromatography, nacl / etoh precipitation and washing, and polyethylene imine - coated magnetic bead (pei - beads) binding and washing . The former two were extremely limited due to cost, low - throughput, and labor intensiveness . Precipitation was time - consuming and was inefficient at separating the probe(s) from dna . In contrast, purification by pei - beads was fast, facile, and, barring high - ionic strength media, flexible with conditions for probe removal . A variety of washing solutions (10 mm) containing dtt or positively charged small molecules (guanidine, lysine, arginine, and spermidine) were assayed for removing excess probe . Their effectiveness was screened using p-15 (10 pmol) and probe 14 (1 nmol). Pei - beads were added and washed with the additives . When excess probe was present, p-15 could not bind to the streptavidin - coated beads . In contrast, when 14 was removed using any of the wash solutions, biotinylated dna (p-15) bound the beads as effectively as that when 14 had not been added . Given the ease of preparation, both guanidine and dtt were maintained in subsequent experiments . We recognized that these experiments do not guarantee that the probe is completely removed and that any excess probe that is even noncovalently bound to dna would contribute to the background signal . However, we were unable to directly measure low levels of probe remaining bound to the bead . It was conceivable that one could measure the bead - bound probe by radiolabeling the latter, but this was deemed impractical . After eight rounds of washes, dna was eluted using high ionic strength buffer containing dtt to reduce the disulfide (0.1 m trishcl, ph 10, 2 m nacl, 0.1% tween-20, and 0.1 m dtt). After 5 min, the suspension was clarified by magnetic pelletization for 5 min . The eluted dna was then bound to streptavidin beads (15 pmol binding capacity) and quantified by liquid scintillation counting . All probes contained biotin as a means for capturing tagged dna and separating tagged and untagged material from one another . These beads contain a hydrophobic surface, which was desirable, as it should bind more weakly to the charged molecules (dna and probes) employed in these studies . A 916 bp pcr fragment obtained from a plasmid containing the p53 gene was used as analyte dna . The pcr product containing 8-oxodguo (p53-og) an otherwise identical pcr product was prepared without 8-oxodguo (p53-g) using a forward primer that did not contain the lesion . (the pcr fragment without 8-oxodguo was used for optimizing conditions for removing untagged dna .) A 1.2 kb biotinylated pcr fragment of puc19 (bpuc19) was used as an internal standard . Wash buffers varying in ph and ionic strength were screened, and tbs buffer (10 mm tris, ph 9, and 1 m nacl) was found to be the best . Qpcr was chosen because of its large dynamic range, as well as its compatibility with sample preparation methods that shorten the time required to carry out the procedure . The previous procedure required adsorbing the (tagged and untagged) dna to the surface of the microtiter plate well, followed by binding the horseradish peroxidase employing magnetic beads facilitates the removal of untagged (nonbiotinylated) dna by washing . Removal of untagged dna is one crucial parameter for minimizing the limit of detection because untagged dna will be amplified equally as untagged during pcr . However, greater reproducibility was achieved by adding an undamaged dna (a 1.2 kb pcr fragment from puc19) to the mixture prior to tagging as internal standard . The internal standard and target dnas were quantified by multiplex qpcr using taqman probes containing different fluorophores for each nucleic acid substrate . Having optimized the qpcr process and individual assay steps (scheme 2), the performance of probes 2, 9, and 1114 were compared (table 1). The background with no oxidant (k3fe(cn)6) was also measured using p53-g . In general, the data were consistent with the more crude measurements described above for 1113 . Lower charged probes (e.g., 9 and 11) captured the 8-oxodguo containing dna less efficiently . However, the more highly charged probes yielded a higher background signal, as evidenced by the tagging of p53-g . Cleavable probe 14 provided the highest overall tagging yield and greatest selectivity (2,000-fold). Although the tagging yield for 14 was only 40%, this is less significant than the selectivity . Increasing the tagging yield to 100% would only improve the sensitivity 2.5-fold, assuming that the background reaction remained the same . Finally, the limit of detection using 14 was established using a mixture of p53-g and p53-og totaling 0.9 fmol (1 ng). The signal (% yraw) was distinguishable from that for the background (p53-g only, 2.9 0.7) when the sample contained as little as 14 amol of p53-og (6.5 1.0) in a sample containing 0.9 fmol dna strands . This translates to 14 amol 8-oxodguo per 1.65 pmol nt based upon 916 bp per p53-og molecule or <12 8-oxodguo/10 nt . The amount of 8-oxodguo formed in the p53-pcr fragment upon treatment with fecl2, h2o2, and ascorbate was measured using 14 and the above - described process . The 8-oxodguo dependence on the level of oxidative stress was determined over a range of ferrous ion concentrations and compared to the amount determined by cadet using lc / ms to analyze dna exposed to the same conditions (figure 2). The agreement in the amount of 8-oxodguo formed as measured by the two methods was excellent over the range of fecl2 between 0.8 and 12.5 m (figure 2). However, the methods yielded very different results at 25 m fecl2, where we measured 477 113 8-oxodguo nt/10 nt, but one would expect a value of 250 using cadet s lc / ms method . Nonetheless, the two methods agree well with one another over a 15-fold range . Quantity of 8-oxodguo formed from fe oxidation of p53-g as a function of oxidant level . The data are compared to the levels previously established under the same oxidation conditions using lc / ms analysis of enzyme digested dna . Data plotted are the average of 3 independent measurements, and error bars represent the sd of these measurements . Probe 14 enables us to detect as little as 14 amol of 8-oxodguo in 34 h. typical experiments used 1 ng of dna substrate, and the quantitative analysis agrees well with data obtained using enzyme digestion and lc / ms analysis . The amount of dna substrate used in the assay is considerably less than that in a typical experiment using lc / ms . Furthermore, the small quantity of substrate needed will be useful for analyzing valuable nucleic acids that are in short supply, such as telomeric dna . The sensitivity of the experiment is enhanced by the use of qpcr, which can in principle be used to detect very small numbers of molecules, provided the background tagging reaction can be reduced further . Furthermore, the qpcr method only requires that a portion of the target dna sequence be known . Because dna is not digested during the analysis, the method is potentially useful for analyzing for 8-oxodguo at specific sites by taking advantage of known methods for recognizing specific nucleic acid sequences . The advent of a pei - bead washing procedure and a probe (14) that allows us to reductively cleave the highly positively charged dna binding domain from the reporter group reduces the likelihood that the background signal is due to the noncovalently bound probe . Nonspecific tagging of dg by the probe(s) under the oxidative conditions of the reaction is a possible source of background signal . Future improvements will address this potential problem by examining the oxidation conditions in the tagging reaction . Given the large dynamic range of qpcr and its sensitivity, reducing the background signal will decrease the method s limit of detection and increase its value to the scientific community.
|
Maculopapular exanthema (mpe) is the most frequent clinical manifestation of nonimmediate allergic reactions due to drugs and t helper 1 (th1) cytokines and cd4 (+) t cells have been shown to play an important role in its pathogenesis . Pyrazinamide is used in the management of tuberculosis (tb) in combination with other drugs . The common side effects due to pyrazinamide are hyperuricemia (gout), hepatotoxicity, nausea, vomiting, flushing, dysuria, arthralgia, and sideroblastic anemia . We hereby report a case of maculopapular rash due to pyrazinamide in a patient undergoing antitubercular treatment . We also established the causality, severity, and preventability of the suspected adverse drug reactions (adrs). A 19-year - old patient of bersarai area, new delhi, belonging to a lower middle class family, visited a microscopic center situated in delhi government dispensary of bersarai area with complaints of cough with expectoration and fever, loss of appetite, and weight for the past 1 month . The sputa were examined as per revised national tuberculosis control programme (rntcp) by zeihl - neelson (zn) staining for acid - fast bacillus (afb) and were found to be negative . Chest radiograph showed bilateral upper zone infiltration . On the basis of the above clinical examination and observation he was referred to the directly observed treatment (dot) center of his area for the initiation of category i antituberculosis therapy as per national rntcp guidelines according to his weight (40 kg). Category i antituberculosis therapy includes isoniazid 600 mg (2 tablets), rifampicin 450 mg (1 capsule), pyrazinamide 1500 mg (2 tablets), and ethambutol 1200 mg (2 tablets). (after the second day of therapy), the patient visited dot center with generalized maculopapular rashes all over the body and more on both shoulders and upper and lower limbs . A diagnosis of antitubercular drug - induced maculopapular rash was made by the medical officer . Rashes were round in shape, raised from the body surface, appeared reddish in color, and hot on touch . Therefore, pyrazinamide was stopped on the advice of the medical officer and other antituberculosis medicines were continued with the addition of oral antihistaminic and the patient was kept under close observation for evaluation . He tolerated isoniazid, rifampicin, and ethambutol but on inclusion of pyrazinamide, rashes reappeared in the same part of the body . He is on regular follow - up with disappearance of rashes and signs and symptoms of tuberculosis . We carried out the causality assessments as per the naranjo algorithm and preventability and severity assessments as per the hartwig scale . The causality assessment revealed a probable association (naranjo score 7) between the adr and pyrazinamide . Higher rates were found in elderly patients who are likely to be receiving multiple medications for long - term illnesses . Maculopapular rashes consist of macules (distinct flat areas) and papules (raised lesions). The rash is usually bright red in color and the skin may feel hot with burning sensation or itch . The whole of the skin surface may be involved, though the face is often spared . Up to 5% of the patients receiving penicillin, sulfonamides, phenytoin, or gold, erythema multiforme has been reported in one patient following pyrazinamide administration for cutaneous tuberculosis related to a pleural fistula . Daily antituberculosis treatment (att) was initiated in this patient with isoniazid, rifampicin, ethambutol, and pyrazinamide . After 26 days of therapy, maculopapular erythematous lesions appeared, and biopsy results confirmed the diagnosis of erythema multiforme . The rash disappeared with the discontinuation of all drugs, but reappeared when rifampicin and pyrazinamide were reintroduced 5 days later . The patient developed the rash on the third day (after second dosing day of therapy) after initiating antituberculosis therapy and disappeared after few days when the drug (pyrazinamide) was stopped . The causal relationship between the drug and the adr was found to be probable . Generalized maculopapular skin rash was a common adr reported in an investigational trial of ofloxacin (800 mg a day) and pyrazinamide (1500 mg a day). The management of such reactions needed withdrawal of the suspected drug and management of symptoms, if any . In this study, the suspected drug was stopped immediately following the adr and antihistamines were added to manage associated itching due to drug reaction, to which patient responded well . The severity assessment revealed the adr to be moderate (level 3), suggesting that the suspected drug should be withheld, discontinued, otherwise changed, and/or on antidote or other treatment is required . Since this patient did not have any past history of skin reaction due to pyrazinamide or any other drugs, therefore this reaction was unpreventable . Since pyrazinamide is a common drug used in tb management, and tb is also a common problem in countries like india, the dermatological manifestations due to pyrazinamide gain attention . Upon occurrence of dermatological manifestations, the patients may become noncompliant, which is one of the common causes with other anti - tb drugs for treatment failure in tb therapy . Although skin reactions due to pyrazinamide are not well reported, one should be suspicious of maculopapular rashes due to pyrazinamide also . Upon occurrence, the suspected drug/(s) should be stopped immediately and the patient should be managed symptomatically . The patients undergoing treatment on an outpatient basis should be counseled for the early recognition of dermatological manifestations.
|
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.